WO2023026179A1 - Liposomes, compositions comprising the same, uses thereof - Google Patents

Liposomes, compositions comprising the same, uses thereof Download PDF

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Publication number
WO2023026179A1
WO2023026179A1 PCT/IB2022/057879 IB2022057879W WO2023026179A1 WO 2023026179 A1 WO2023026179 A1 WO 2023026179A1 IB 2022057879 W IB2022057879 W IB 2022057879W WO 2023026179 A1 WO2023026179 A1 WO 2023026179A1
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WIPO (PCT)
Prior art keywords
liposomes
phospholipid
solution
imatinib
obtaining
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PCT/IB2022/057879
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French (fr)
Inventor
Silvia ARPICCO
Barbara Rolando
Barbara Stella
Federica MELONI
Laura PANDOLFI
Emanuela COVA
Veronica CODULLO
Rosanna DI PAOLA
Salvatore Cuzzocrea
Original Assignee
Università Degli Studi Di Torino
Fondazione Irccs Policlinico San Matteo
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Application filed by Università Degli Studi Di Torino, Fondazione Irccs Policlinico San Matteo filed Critical Università Degli Studi Di Torino
Priority to EP22769372.8A priority Critical patent/EP4392021A1/en
Publication of WO2023026179A1 publication Critical patent/WO2023026179A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A61K9/1278—Post-loading, e.g. by ion or pH gradient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

Definitions

  • This disclosure relates to liposomes , their use as vehicles for medical applications , and methods for producing them .
  • nanotechnologies in the therapeutic field is extremely innovative and associated with numerous advantages , including : 1 ) increase in the bioavailability of several drugs ; 2 ) possibility of exploiting alternative routes of administration (such as inhalation delivery which allows high doses of the drug to be reached at the level of the target organ cells ) ; 3 ) possibility of directing the treatment , through functionali zation, towards a speci fic cell population ( targeting) .
  • routes of administration such as inhalation delivery which allows high doses of the drug to be reached at the level of the target organ cells
  • targeting a speci fic cell population
  • the aim of this disclosure is to provide biocompatible carriers of active ingredients , which are ef fective in the treatment of diseases for which there is an urgent need for a well-tolerated resolutive therapy, which do not present toxicity, and which are able to optimi ze the quantity of active ingredient at the target organ .
  • This disclosure relates to liposomes coated with hyaluronic acid (HA) having a molecular weight greater than 17000 Daltons , preferably between 20000 Daltons and 60000 Daltons .
  • HA hyaluronic acid
  • the liposomes may contain an active ingredient in the core , in particular a protein kinase enzyme inhibitor, preferably a tyrosine kinase enzyme inhibitor, more preferably selected from the group consisting of imatinib, nintedanib and other derivatives belonging to the tyrosine kinase inhibitor class .
  • a protein kinase enzyme inhibitor preferably a tyrosine kinase enzyme inhibitor, more preferably selected from the group consisting of imatinib, nintedanib and other derivatives belonging to the tyrosine kinase inhibitor class .
  • compositions comprising the liposomes and their uses .
  • the disclosed liposomes and the compositions comprising them may be used in medicine , to deliver active ingredients to speci fic target cells .
  • the disclosed liposomes and the compositions comprising them may be ef fectively used in the treatment of respiratory diseases , pre ferably pulmonary fibrotic diseases .
  • Respiratory diseases may be selected from idiopathic or inflammation-based pulmonary fibrosis , particularly diseases associated with collagen diseases , and idiopathic bronchiolitis obliterans or secondary to bone marrow or lung transplantation .
  • the disclosure also provides an innovative method for producing liposomes containing at least one active ingredient as well as a method for producing HA-coated liposomes having a molecular weight greater than 17000 Da .
  • the disclosure further relates to a method for producing HA bound to a phospholipid in the form of a conjugate (HA-f) .
  • FIG. 1 Cell viability results after incubation of primary myofibroblast lines (LFs) derived from patients with chronic lung rejection (CLAD;A) , fibrosis- associated collagen disease (ILD-CTD;B) or immortalised human lung carcinoma cell line (A549;C) with imatinib- containing liposomes (LIPIm) , HA-coated liposomes containing imatinib (LIP-HAIm) and imatibin (Im) alone, at the same imatinib concentration (30 pM) after 24, 48 and 72 h. Data are represented as mean ⁇ standard deviation of the percentage compared to the control set at 100%. Statistical analysis: **, p ⁇ 0.01 vs. CTR; *, p ⁇ 0.05 vs . CTR .
  • FIG. 1 Results of western blot protein quantification performed on the cell lysate of CLAD- and ILD-CTD-derived LFs after treatment for 24 h with LIPIm, LIP-HAIm and imatinib (Im) .
  • CTR Quantification of phosphorylated cAbl normalized to control
  • CTR quantification of type I collagen normalized to control
  • FIG. 3 Representative images acquired with the confocal microscope of peripheral blood-derived monocytes incubated for 1 h with fluorescent liposomes (LIP) and HA-coated liposomes (LIP-HA) .
  • the cell nuclei were labelled with a specific fluorophore (DAPI) .
  • White circles indicate the presence of LIP or LIP-HA within the monocytes.
  • FIG. 1 Results of survival (A) and body weight gain (B) of mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES (HEPES) , bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib (IM IP) intraperitoneally, bleomycin and liposomes with imatinib and hyaluronic acid (LIPO-IM- HA) .
  • Sham saline
  • CTR bleomycin and saline
  • HEPES bleomycin and HEPES
  • IM IT bleomycin and imatinib intratracheally
  • IM IP bleomycin and imatinib
  • LIPO-IM- HA bleomycin and liposomes with imatinib and hyaluronic acid
  • Hemocytometer cell count results in bronchoalveolar lavage fluid in the presence of trypan blue dye (A) .
  • Hemocytometer count results of neutrophils (B) , macrophages (C) and lymphocytes (D) in the presence of Wright-Giemsa dye in bronchoalveolar lavage fluid in mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES (HEPES) , bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib intraperitoneally (IM IP) , bleomycin and liposomes with imatinib and hyaluronic acid (LIPO-IM-HA) .
  • FIG. 6 Representative images acquired with the light microscope of lung tissue sections stained with hematoxylin and eosin from mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES (HEPES) , bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib intraperitoneally (IM IP) , bleomycin and liposome with imatinib and hyaluronic acid intratracheally (LIPO-IM-HA) , Aschroft score (A) indicates histological damage.
  • Sham saline
  • CTR bleomycin and saline
  • HEPES bleomycin and HEPES
  • IM IT bleomycin and imatinib intratracheally
  • IM IP bleomycin and imatinib intraperitoneally
  • LIPO-IM-HA Aschroft score
  • Figure 7 Representative images acquired with the light microscope of Masson's trichrome-stained lung tissue sections of mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES (HEPES) , bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib intraperitoneally (IM IP) , bleomycin and liposomes with imatinib and hyaluronic acid (LIPO-IM-HA) , collagen quantitation (A) .
  • Sham saline
  • CTR bleomycin and saline
  • HEPES bleomycin and HEPES
  • IM IT bleomycin and imatinib intratracheally
  • IM IP bleomycin and imatinib intraperitoneally
  • LIPO-IM-HA bleomycin and liposomes with imatinib and hyaluronic acid
  • FIG 8. Representative images acquired with the light microscope of the lung tissue sections on which an immunohistochemical analysis was performed with the TGF- lp antibody of the mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES (HEPES) , bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib intraperitoneally (IM IP) , bleomycin and liposomes with imatinib and hyaluronic acid (LIPO-IM-HA) , quantification of TGF-lp expression (A) .
  • Sham saline
  • CTR bleomycin and saline
  • HEPES bleomycin and HEPES
  • IM IT bleomycin and imatinib intratracheally
  • IM IP bleomycin and imatinib intraperitoneally
  • LIPO-IM-HA quantification of TGF-lp expression
  • Figure 9 Representative images acquired with the light microscope of the lung tissue sections on which immunohistochemical analysis was performed with the CD4 antibody from mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib intraperitoneally (IM IP) , bleomycin and liposomes with imatinib and hyaluronic acid (LIPO-IM-HA) , quantification of CD4 expression (A) .
  • Sham saline
  • CTR bleomycin and saline
  • IM IT bleomycin and HEPES bleomycin and imatinib intratracheally
  • IM IP bleomycin and imatinib intraperitoneally
  • LIPO-IM-HA bleomycin and liposomes with imatinib and hyaluronic acid
  • Figure 10 Representative images acquired with the light microscope of lung tissue sections on which immunohistochemical analysis was performed with the CD8 antibody of mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES (HEPES) , bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib intraperitoneally (IM IP) , bleomycin and liposomes with imatinib and hyaluronic acid (LIPO-IM- HA) , quantification of CD8 expression (A) .
  • Sham saline
  • CTR bleomycin and saline
  • HEPES bleomycin and HEPES
  • IM IT bleomycin and imatinib intratracheally
  • IM IP bleomycin and imatinib intraperitoneally
  • LIPO-IM- HA quantification of CD8 expression
  • This disclosure relates to a liposomal nanovehicle functionali zed on the surface with hyaluronic acid (HA) in order to facilitate the selective delivery of an active ingredient towards diseased cells and alveolar macrophages present in chronic and severe respiratory diseases .
  • HA hyaluronic acid
  • Liposomes are phospholipid vesicles that have a membrane , the liposomal membrane , comprising a lipid bilayer, general ly a phospholipid bilayer . Liposomes have nanometric dimensions , enclose an aqueous core and may be used in the clinic for the delivery of active ingredients . They can encapsulate both hydrophilic and lipophilic molecules , allowing the gradual release of the active ingredient , thus modi fying its pharmacokinetic profile .
  • the liposomes obj ect of the present disclosure are HA-coated liposomes having a molecular weight higher than 17000 Daltons , preferably between 20000 and 60000 Daltons .
  • HA is directly bound to the head of a phospholipid of the lipid bilayer of the liposomal membrane .
  • the HA is directly bound to the head of a membrane phospholipid comprising an amine group .
  • the HA is directly bound to an amine group of a membrane phospholipid .
  • the weight ratio of hyaluronic acid to membrane phospholipids is between 1 and 5 .
  • the phospholipid bilayer of the liposomal membrane may also comprise cholesterol .
  • the phospholipid of the phospholipid bilayer of the liposomal membrane may be selected from 1 , 2-dipalmitoyl- sn-glycero-3-phosphoethanolamine ( DPPE ) , phosphatidylglycerol ( PG) , phosphatidylcholine ( PC ) and combinations thereof .
  • DPPE 2-dipalmitoyl- sn-glycero-3-phosphoethanolamine
  • PG phosphatidylglycerol
  • PC phosphatidylcholine
  • PC phosphatidylcholine
  • the coated liposomes obj ect of the present disclosure may have an average diameter ranging from 100 nm to 300 nm, preferably equal to 250 nm .
  • the mean diameter value was analyzed by the quasi-elastic light scattering ( QELS ) technique at 25 ° C .
  • the coated liposomes have a Z potential value ranging from - 10 mV to -50 mV, preferably equal to -33 mV .
  • the liposomes obj ect of the present disclosure may contain at least one active ingredient . From the electron microscope analysis it was also observed that the liposomes have a spherical shape and the presence of the precipitated active ingredient inside them was confirmed .
  • the liposomes retain approximately 95% of the initial active ingredient content , in particular imatinib, after storage at 4 ° C for 3 weeks . During this period, no appreciable changes in diameter and Z-potential , nor precipitation or aggregation phenomena are observed .
  • the encapsulation method carried out by the Inventors and described below provides a series of advantages , among which it allows to obtain stable formulations : the Inventors have in fact observed that 20% of imatinib, as an active ingredient , is released in 24 hours in HEPES buf fer and 30% in 72 hours ; in serum the release is about 50% in 24 hours and 80% in 72 hours .
  • the liposomes obj ect of the present disclosure as well as the compositions containing them may be used to deliver the active ingredients contained therein to speci fic targets and may also be administered by inhalation .
  • the diseases which may be ef fectively treated with the liposomes obj ect of the present disclosure are respiratory diseases , preferably pulmonary fibrotic diseases .
  • treatable diseases may be selected from idiopathic pulmonary fibros is , pulmonary fibrosis associated with collagen diseases and bronchiolitis obliterans .
  • Bronchiolitis obliterans may result from lung transplantation or chronic mani festation after allogeneic bone marrow stem cell transplantation .
  • the liposomes obj ect of the present disclosure coated with HA having a speci fic molecular weight represent an absolute innovation and an important step forward in the treatment of these serious diseases , with a considerable economic and social impact .
  • Bronchiolitis obliterans which represents the failure of a lung transplant or the expression of chronic pulmonary graft , is associated with high costs to the healthcare system, requiring frequent evaluations and hospitali zations , costly therapies and complex support in the phase of disease progression, while pulmonary fibrosis is often the cause of severe disability and death in patients .
  • pulmonary fibrosis is often the cause of severe disability and death in patients .
  • idiopathic form it is not associated with other systemic diseases
  • systemic sclerosis or autoimmunebased inflammatory myopathy it is often unresponsive to the immunosuppressive therapy adopted for these pictures .
  • mesenchymal cell growth factors such as PDGF, TGF-beta and VEGF
  • the action of these factors is mediated by several protein kinases that mediate apoptosis resistance , inhibition of LFs proli feration and extracellular matrix production .
  • TK tyrosine kinase
  • imatinib which not only inhibits BCR-ABL, but is equally potent against other receptors with tyrosine kinase properties , such as PDGFRa and c-KIT .
  • nintedanib Another compound, already widely used in the treatment of pulmonary fibrosis , both idiopathic and associated with collagen diseases , and administered orally, is nintedanib, which is able to inhibit PDGF a and B , FGF 1-3 and EGFR 1-3 receptor-associated TK .
  • Liposomal formulations containing anti fungal or anticancer drugs are commercially available for intravenous administration .
  • liposomal amikacin which is indicated in the treatment of mycobacterial pulmonary diseases refractory to first- line antibiotic therapy
  • liposomal cyclosporine under registration for bronchiolitis obliterans
  • Commercially available formulations are not functionali zed and therefore do not have a speci fic cellular target . Therefore , to date , there is no liposomal formulation with active targeting capability in advanced development that may also be administered by inhalation .
  • the local administration of active ingredients via the liposomes described here is extremely ef fective in that it allows the active ingredients to speci fically reach the cells most involved in the pathogenic process , i . e . cells expressing the CD44 receptor ( LFs , inflammatory cells , activated endothelial cells ) .
  • LFs primary fibroblastic cells isolated from the lung of patients with collagen disease- associated fibrosis ( interstitial lung disease in collagen tissue disease , ILD-CTD) or from patients with chronic lung rej ection ( chronic lung allograft dys function, CLAD) express CD44 in a high percentage ( >90% )
  • the Inventors of the present application used these primary cell lines to test the internali zation and function of HA-coated liposomes with speci fic molecular weight and containing imatinib as active ingredient .
  • LIP-HAIm speci fic molecular weight
  • the presence of HA exerts a cryoprotective ef fect , capable of favoring the lyophili zation of compositions comprising the liposomes .
  • the lyophili zation in turn determines an increase in the stability of the compositions and makes it possible to obtain compositions in the form of powders which may also be administered as aerosols .
  • the composition comprising the liposomes may also be in liquid or solid form .
  • the Inventors of the present application have also developed a method for producing liposomes , optionally coated with HA, comprising an active ingredient which has proved to be simple and particularly ef fective in terms of the quantity of active ingredient loaded into the liposomal core .
  • the active ingredient is inserted into the aqueous core of the liposomes using the pH gradient technique , preferably using citrate buf fer .
  • This technique allows to obtain the precipitation of the active ingredient in the aqueous core o f the vesicles and a high encapsulation ef ficiency, equal to 85% for imatinib, therefore higher than that obtained using, for example , an ammonium sulphate solution ( 70% ) , already described above .
  • the method for producing liposomes containing at least one active ingredient obj ect of the present disclosure comprises the steps of :
  • a buf fer solution comprising at least one salt , preferably selected from sodium salt , potassium salt , ammonium salt and obtaining a second solution,
  • the phospholipid usable in the disclosed method may be selected from DSPC ( 1 , 2-distearoyl-sn-glycerol-3- phosphocholine ) , phosphatidylglycerol ( PG) , phosphatidylcholine ( PC ) , combinations thereof .
  • PC usable in the disclosed method may be either of natural origin, for example from soy and/or egg, or synthetic .
  • synthetic PC may comprise fully hydrogenated soy PC and/or PC with the two lipid chains having a number of carbon atoms ranging from 12 to 18 ( C12-C18 ) .
  • a polyethylene glycol modi fied phospholipid, 1 , 2 -disteroyl-sn- 3-phosphoethanolamine-N- [methoxy (polyethylene glycol ) ] may also be used .
  • at least one lipid, for example belonging to the class o f sterols , for example cholesterol may also be added to the solvent .
  • the solvent usable in the phospholipid solubili zation step may be selected from the group consisting of chloroform, dichloromethane ( DCM or methylene chloride ) , methanol , ethanol and mixtures thereof .
  • the solvent evaporation step from the first solution may be carried out under reduced pressure and then under vacuum .
  • the solvent may be removed by evaporation, for example using a rotary vacuum evaporator (Rotavapor®) .
  • the evaporation of the solvent is promoted by the reduction of the pressure which lowers the boiling temperature of the solvent .
  • the salt contained in the buf fer solution of lipid film hydration step may be selected from sodium citrate , sodium phosphate , sodium sulphate , sodium acetate , ammonium citrate , ammonium phosphate , ammonium sulphate and ammonium acetate .
  • the pH of the buf fer solution comprising a salt preferably sodium citrate , may be between 4 and 5 .
  • the Inventors have observed that optimal results in terms of encapsulation yield of the active ingredient are obtained when the salt contained in the buf fer solution is sodium citrate .
  • the pH of the buf fer solution comprising sodium citrate is 4 . 38 .
  • the method does not involve the use of ammonium sulphate .
  • the solution obtained in the lipid film hydration step may be subj ected to sonication, for example in a sonication bath, and to stirring, preferably in a vortex, until the lipid film is completely detached .
  • the solution may then be subj ected to extrusion by using an extruder, preferably at a temperature between 20°C and 60°C, more preferably equal to 40°C to obtain the suspension comprising liposomes.
  • the extrusion may be carried out under nitrogen pressure, preferably between 100 psi and 800 psi using filters, preferably made of polycarbonate, in which the pore diameter may be between 800 nm and 100 nm.
  • the suspension comprising liposomes may be subjected to a pH change step in order to achieve a suspension pH preferably between 7 and 7.6.
  • the pH change step may be carried out by subjecting the suspension to gel filtration on a packed column, for example with Sepharose CL-4B, eluting with buffer, preferably HEPES, pH 7.4.
  • buffer preferably HEPES, pH 7.4.
  • the active ingredient may be added to the suspension comprising the liposomes dissolved in a buffer solution in a concentration between 0.1 mg/100 pl and 1 mg/100 pl, preferably equal to 0.5 mg/100 pl.
  • the buffer solution may comprise for example HEPES and/or phosphate buffer .
  • the active ingredient may be a protein kinase enzyme inhibitor, preferably a tyrosine kinase enzyme inhibitor, more preferably selected in the group consisting of imatinib, nintedanib and other derivatives belonging to the tyrosine kinase inhibitor class.
  • a protein kinase enzyme inhibitor preferably a tyrosine kinase enzyme inhibitor, more preferably selected in the group consisting of imatinib, nintedanib and other derivatives belonging to the tyrosine kinase inhibitor class.
  • the liposomal suspension added with the active ingredient may be kept for a period of time comprised between 20 minutes and 60 minutes, preferably equal to 30 minutes, at a temperature comprised between 30°C and 40°C, preferably equal to 37°C.
  • the liposomal suspension may also be subjected to a purification step in order to remove the nonencapsulated active ingredient.
  • the purification step may be carried out by gel filtration or molecular exclusion chromatography.
  • the method may allow to produce HA-coated liposomes.
  • HA may be added to the buffer solution, preferably HA having a molecular weight higher than 17000 Daltons.
  • HA is added in the form of conjugate with at least one phospholipid (HA-f) .
  • conjugate refers to a product obtained from the chemical reaction between two components that leads to the formation of a covalent bond.
  • Such a HA-f conjugate may be obtained by a reductive amination reaction between HA and a phospholipid containing an amine group, for example phosphatidylethanolamine (PE) .
  • PE phosphatidylethanolamine
  • this conjugate only one molecule of PE is bound to HA, and the synthetic process is particularly complex as the molecular weight of HA increases, which in previous works was between 4000 and 17000 Daltons.
  • HA having a molecular weight greater than 17000 Daltons, preferably between 20000 and 60000 Daltons, may be solubilized in at least one halogenated solvent to obtain a solution of solubilized HA.
  • the solvent may comprise DCM as halogenated solvent.
  • the solvent may comprise DCM and dimethyl sulfoxide (DMSO) , preferably in a 1:1 weight ratio.
  • the HA solubilization step may be carried out by sonication, preferably in a sonicator bath, more preferably for a period comprised between 30 minutes and 120 minutes, preferably equal to 90 minutes.
  • HA in particular having a molecular weight above 17000 Daltons may be added to the solvent in a concentration between 0.02 mmol and 0.1 mmol.
  • the solution comprising solubilized HA may be heated to a temperature ranging from 40°C to 70°C, preferably equal to 60°C.
  • the phospholipid comprising an amine group preferably DPPE, is added to obtain a mixture comprising HA and phospholipid comprising an amine group.
  • the phospholipid comprising an amine group is added solubilized in the solvent, preferably comprising chloroform and methanol.
  • the mixture may comprise chloroform and methanol in a 1:1 weight ratio.
  • the phospholipid may be added to the solvent in a concentration between 0.02 mmol and 0.1 mmol.
  • the mixture comprising HA and phospholipid including an amino group is brought to a temperature between 18°C and 24°C, preferably equal to 20°C (room temperature) .
  • the mixture may be subjected to a pH change in order to bring the pH to a value of 4.5.
  • the pH adjustment may be carried out by adding an acid, for example acetic acid .
  • the mixture may be brought to a temperature ranging from 40°C to 70°C, preferably equal to 60°C for a period of time ranging from 1 hour to 3 hours, more preferably equal to 2 hours .
  • the mixture comprising HA and phospholipid including an amine group is added with at least one reducing agent to obtain a solution comprising the HA-f conjugate.
  • the reducing agent may be selected from borohydrides , sodium cyanoborohydride and sodium triacet-oxy-borohydride (STAB) .
  • the reducing agent is a borohydride, more preferably STAB.
  • the solution comprising the HA-f conjugate may be kept at a temperature ranging from 50°C to 70°C, preferably 60°C for a period of time ranging from 80 hours to 120 hours, preferably for a period of 96 hours.
  • the solution may be subjected to purification and optionally washed.
  • the purification may be carried out by dialysis.
  • the washing may be carried out for example with DCM.
  • the solution may then be subjected to a lyophilization step to obtain the lyophilized HA-f conjugate.
  • the lyophilized conjugate may be solubilized and added to the lipid film in the hydration step, in particular in the buffer solution comprising a salt, so as to obtain HA-coated liposomes containing an active ingredient.
  • the weight ratio of hyaluronic acid to membrane phospholipids is between 1 and 5.
  • At least one fluorescenceemitting compound for example fluorescein, may be added to the buffer solution in the lipid film hydration step.
  • the liposomes obtained with the described method may have an average diameter between 100 nm and 300 nm, when determined using the quasi-elastic light scattering (QELS) technique.
  • QELS quasi-elastic light scattering
  • the diameter is preferably about 200 nm; in the case of HA-coated liposomes, the diameter is preferably about 250 nm.
  • the presence of HA on the surface of the liposomes therefore leads to an increase in the value of the average particle diameter.
  • the Z-potential value may be around -30 mV for uncoated liposomes and slightly more negative, around -33 mV, for HA-coated liposomes, due to the presence of the negatively charged carboxyl groups of HA.
  • a composition comprising 1 , 2-distearoyl-sn- glycerol-3-phosphocholine (DSPC) , cholesterol (COL) and phosphatidylglycerol (PG) in a molar ratio of 70:30:3 was used to prepare the liposomes.
  • DSPC 2-distearoyl-sn- glycerol-3-phosphocholine
  • COL cholesterol
  • PG phosphatidylglycerol
  • the components were solubilized in chloroform and then the lipid film was prepared by evaporating the solvent under reduced pressure in the Rotavapor® and then under vacuum.
  • the lipid film was hydrated with citrate buffer (solution of citric acid and sodium citrate, pH 4.38) , sonicated and vortexed.
  • the liposomes were extruded under nitrogen pressure through polycarbonate filters (pore diameter 400 and 200 nm) using an extruder (Extruder; Lipex, Vancouver, Canada) at a temperature of 40°C. They were then subjected to gel filtration on a column packed with Sepharose CL-4B eluting with HEPES buffer pH 7.4 to change the pH of the liposomal suspension.
  • a solution of imatinib mesylate in HEPES buffer (0.5 mg/100 pl) was then added to the liposomal suspension and incubated for 30 minutes at 37°C; the liposomal preparation was then purified on a packed column with Sepharose CL-4B eluting with HEPES buffer pH 7.4, removing the unencapsulated active ingredient by gel filtration.
  • the same procedure was used to obtain the HA-coated liposomes by replacing the PG with the HA-f conjugate.
  • the HA-f conjugate was obtained by conjugating HA of molecular weight 44000 Da with the phospholipid 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) .
  • DPPE dipalmitoyl-sn-glycero-3-phosphoethanolamine
  • the HA-f conjugate was obtained by a reductive amination reaction between HA and a phospholipid containing an amino group (PE) .
  • PE phospholipid containing an amino group
  • HA with a molecular weight of 44000 Da was solubilized in a mixture of DCM and DMSO (1:1) in a sonicator bath for 90 minutes.
  • the solution was then brought to room temperature, acidified to pH 4.5 with acetic acid and then left for 2 hours at 60°C.
  • solubilized STAB was added to the same DPPE mixture and the reaction proceeded for 96 hours at 60°C.
  • the mixture was dialyzed against water and subsequently washed several times with DCM and finally lyophilized.
  • the conjugate was solubilized in citrate buffer and added to the lipid film during the hydration step.
  • Fluorescent liposomes were prepared by adding a fluorescein solution in HEPES buffer during the lipid film hydration step and then subjected to extrusion and purification by gel filtration.
  • the liposomal preparations were stored at a temperature of 4 °C.
  • the average diameter and poly-dispersion index of the liposomes were determined at 25°C by means of the quasi-elastic light scattering (QELS) technique, and the surface charge was assessed by Z-potential determination by performing triplicate measurements.
  • the morphology of the liposomes was determined by cryo-TEM (cryogenic- Transmission Electron Microscopy) analysis.
  • the poly- dispersion index was less than 0.2 for the uncoated liposomes and the HA-coated liposomes object of this disclosure .
  • the amount of imatinib encapsulated in the liposomes was determined by RP-HPLC.
  • liposomal fractions were diluted with acetonitrile (by a different factor depending on the presumed imatinib concentration) , the mixture was sonicated, vortexed, centrifuged and the supernatant was filtered (0.45 pm PTFE) and analyzed by HPLC (the analytical conditions used are described in the scientific article Imatinib assay by HPLC with photodiode-array UV detection in plasma from patients with chronic myeloid leukaemia: comparison with LC-MS/MS Clinica Chimica Acta (2010) 411:140-146) .
  • the amount of imatinib encapsulated in liposomes was between 0.35 mg/100 pl and 0.40 mg/100 pl.
  • the lipid concentration was determined by phosphorus analysis, as described in Phosphorus assay in column chromatography J Biol Chem. (1959) 234:466-468. An average lipid concentration of 3.8 mM was determined.
  • the release kinetics of imatinib were assessed by incubating liposomal formulations with and without HA at 37°C in HEPES buffer or serum. At set times, an aliquot of liposomal formulation was taken, purified by gel filtration, and the amount of encapsulated active ingredient was determined by HPLC and compared with the initial value. The physical stability of the liposomes was assessed under storage conditions (4°C) by monitoring the average diameter, Z-potential and amount of encapsulated drug at set time intervals.
  • Tests were conducted on an immortalized human lung carcinoma line (A549) and primary myofibroblast (LFs) lines isolated from bronchoalveolar lavage (BAL) of patients with CLAD or ILD-CTD. All these lines show a high rate of CD44 expression.
  • A549 immortalized human lung carcinoma line
  • LFs primary myofibroblast lines isolated from bronchoalveolar lavage
  • BALs from patients with CLAD and ILD-CTD are first filtered with sterile gauze and subsequently centrifuged at 1800 rpm for 10 minutes. The supernatant is collected and stored at -20°C, while the cell pellet is resuspended with saline and centrifuged again at 1800 rpm for 10 minutes. The pellet obtained is counted to grow 6 x 10 6 cells in DMEM culture medium with 10% fetal bovine serum (BBS) and 1% penicillin/ streptomycin and L- glutamine added. After approximately 10-15 days, foci of LFs are formed, which are isolated and cultured for experiments.
  • BBS fetal bovine serum
  • the cells used express high levels of the CD44 protein, as described in the scientific article Antibody-engineered nanoparticles selectively inhibit mesenchymal cells isolated from patients with chronic lung allograft dysfunction. Nanomedicine (Lond) . (2015) 10: 9-23.
  • HA-modified liposomes in CD44-expressing cells, 0.2 x 10 6 A549 or LFs cells were plated and after 24 h of culture to allow cell adhesion, they were incubated with uncoated or HA-coated fluorescent liposomes (LIP and LIP-HA) for 4 and 24 h at 37°C.
  • LIP and LIP-HA uncoated or HA-coated fluorescent liposomes
  • the cells were analyzed by cytof luorimetry and confocal microscopy, showing that LIP-HA enters both A549 and LFs of CLAD and ILD-CTD more efficiently than LIP.
  • the Inventors evaluated the effect of liposomes containing imatinib as active ingredient on cell viability using both A549 and LFs derived from CLAD and ILD-CTD.
  • the viability assay was carried out by MTT assay, which gives the possibility to quantify cell viability by means of mitochondrial activity.
  • 3 x 10 3 A549 cells or LFs were plated and incubated with LIP- HAIm, LIPIm, imatinib by incubating at the same concentration of imatinib (30 uM) for up to 72 h.
  • the method of hydration of the lipid film followed by extrusion is used, for both encapsulation of the native active ingredient ( as described in Ef ficacy of targeted liposomes and nano- cochleates containing imatinib plus dex-ketoprof en against fibrosarcoma Drug Dev Res . ( 2019 ) 80 : 556-565 ; Antiproli ferative activity and VEGF expression reduction in MCF7 and PC-3 cancer cells by paclitaxel and imatinib co-encapsulation in f olate-targeted liposomes AAPS Pharm Sci Tech .
  • the solubility of imatinib in citrate buffer and ammonium sulphate solution was determined by preparing a suspension of the active ingredient in each of the two media, incubating it at 25°C for 24 hours and analyzing the filtrate via HPLC .
  • the peculiar characteristic of the liposomes disclosed here is that they act preferentially on fibroblasts and inflammatory cells (in particular macrophages and lymphocytes expressing CD44) , which are the main effectors (key players) of chronic progressive fibrosing diseases. On myofibroblasts, in particular, they act by inhibiting their proliferation, migration and extracellular matrix deposition. In vivo efficacy test
  • mice Male GDI mice (25-30 g, Envigo, Milan, Italy) were housed in a controlled location. They received food and water ad libitum. The study was approved by the Animal Care Review Board (OPBA) of the University of Messina. All in vivo experiments followed the new US, European, Italian and ARRIVE guidelines.
  • OPBA Animal Care Review Board
  • bleomycin sulphate (1 mg/kg body weight) was administered via a single intratracheal injection. A volume of 100 pL was injected, immediately followed by 300 pL of air to ensure delivery to the distal airways. Treatments were administered every 5 days starting on day 10 after bleomycin administration. Twenty-eight days after bleomycin injection, animals were sacrificed and tissues were harvested for further analysis.
  • mice received bleomycin administration and were treated daily with the vehicle (saline solution (50 pL) ) ;
  • mice received bleomycin administration and were treated intratracheally with hepes (50 pL) ;
  • mice received bleomycin administration and were treated intratracheally with Imatinib (50 pL of 150 pg/ml) ;
  • mice received bleomycin administration and were treated daily with imatinib (10 mg/ml) intraperitoneally (IP) ;
  • LIPO-IM-HA mice received bleomycin administration and were treated daily with liposome with hyaluronic acid-functionalized imatinib (LIPO-IM-HA) (50 pL of 150 pg/ml) ;
  • mice were euthanized 28 days after bleomycin instillation and tissues were collected for lesion analysis .
  • Survival rate and body weight gain mortality and body weight were assessed daily up to 28 days.
  • Bronchoalveolar lavage (BAL) : twenty-eight days after bleomycin instillation, the mice were sacrificed and the tracheas were cannulated to perform the lavage. A total of 0.5 ml of phosphate-buff ered saline (PBS) was used. The recovered BAL fluid was centrifuged, the pelleted cells were harvested and the supernatants were stored at -20°C. In the presence of trypan blue dye, total BAL cells were counted using a hemocytometer. For a differential leucocyte count, the bronchoalveolar lavage (BAL) wash was stained with Wright-Giemsa. After staining, the differential count was performed using the standard morphological protocol under the light microscope .
  • PBS phosphate-buff ered saline
  • lung tissue samples were collected 28 days after bleomycin injection. After fixing the tissues in buffered formaldehyde solution (10% in PBS) , histological sections were stained with hematoxylin and eosin and evaluated using a Laica DM6 microscope (Milan, Italy) . The severity of pulmonary fibrosis was graded on a scale from 0 to 8. Lung sections were stained with Masson's trichrome for fibrosis.
  • MPO activity was defined as the amount of enzyme that degrades 1 pmol peroxide per minute at 37 °C and was expressed in units per gram of wet tissue weight.
  • the pellets were resuspended in a second buffer containing 150 mM sodium chloride (NaCl) , 1% Triton X- 100, 1 mM ethylene glycol tetraacetic acid (EGTA) , 10 mM tris-chloric acid (HC1) pH 7.4, 0.2 mM PMSF, 1 mM ethylenediaminetetraacetic acid (EDTA) , 0.2 mM sodium orthovanadate and 20 pm leupeptin. After centrifugation at 4°C and 15000 g for 30 min, the nuclear proteins containing the supernatants were stored at -80°C for further analysis.
  • NaCl sodium chloride
  • HC1 tris-chloric acid
  • PMSF 1 mM ethylenediaminetetraacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • anti-IkB- alpha (1:1000, Santa Cruz Biotechnology) or anti-NF-kB p65 (1:1000; Santa Cruz Biotechnology) or anti-p-p38 (1:1000, Santa Cruz Biotechnology) or anti-p-JNK (1:1000, Santa Cruz Biotechnology) or anti-MMP2 (1:1000, Santa Cruz Biotechnology) or anti-MMP9 (1:1000, Santa Cruz Biotechnology) or anti-TRAF-6 (1:1000, Santa Cruz Biotechnology) in IxPBS, 5% w/v skimmed milk powder and 0.1% Tween-20 and incubated at 4 °C, overnight.
  • the membranes were incubated with peroxidase-conj ugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson Immuno Research) for 1 hour at room temperature. To verify that the membranes were loaded with equal amounts of protein, they were also incubated with antibody against laminin (1:1000; Santa Cruz Biotechnology) and beta-actin (1:1000; Santa Cruz Biotechnology) . The signals were detected with the enhanced chemiluminescence detection system reagent according to the manufacturer's instructions (SuperSignal West Pico Chemiluminescent Substrate, Pierce) .
  • Immunohistochemical analysis the antibodies that were incubated O/N on the brain sections were anti-TGF- beta (Millipore, 1:500 in PBS, v/v, AB152, Burlington, MA, USA) , anti-CD4 (Santa Cruz Biotechnology, 1:300 in PBS, v/v, 65G10 sc-32258, Dallas, TX, USA) and anti-CD8 (Santa Cruz Biotechnology, 1:50 in PBS, v/v, LB509 sc- 58480, Dallas, TX, USA) .
  • TGF- beta Millipore, 1:500 in PBS, v/v, AB152, Burlington, MA, USA
  • anti-CD4 Santa Cruz Biotechnology, 1:300 in PBS, v/v, 65G10 sc-32258, Dallas, TX, USA
  • anti-CD8 Santa Cruz Biotechnology, 1:50 in PBS, v/v, LB509 sc- 58
  • the Image J IHC profiler plug-in was used for densitometric analysis. When this is selected, it automatically draws a histogram profile of the deconstructed DAB image and a corresponding score log is shown.
  • the histogram profile corresponds to the positive pixel intensity value obtained by the computer program.
  • mice Twenty-eight days after bleomycin instillation, the HEPES-treated mice showed recruitment of inflammatory cells in the bronchoalveolar lavage fluid, compared to the sham group (Figure 5A) .
  • neutrophils Figure 5B
  • macrophages Figure 5C
  • lymphocytes Figure 5D
  • LIPO-IM-HA significantly reduced the infiltration of inflammatory cells into the bronchoalveolar lavage fluid.
  • Collagen deposition lung damage produced by bleomycin is characterized by increased collagen deposition and severe fibrosis. Masson's trichrome staining showed an increased blue area in the tissues of vehicle-treated mice, compared to sham tissue. LIPO-IM- HA significantly reduced fibrosis and collagen deposition, as shown by the Masson' s trichrome soluble collagen assay ( Figure 7) .
  • One or more embodiments of this disclosure may therefore involve liposomes coated with hyaluronic acid (HA) , wherein the hyaluronic acid has a molecular weight above 17000 Daltons , preferably between 20000 Daltons and 60000 Daltons .
  • HA hyaluronic acid
  • the l iposomes may have an average liposome diameter si ze between 100 nm and 300 nm, preferably 250 nm . In one or more embodiments , the liposomes may exhibit a Z potential value between - 10 mV and -50 mV, preferably -33 mV .
  • Liposomes may have HA covalently bound to the head of a phospholipid of the lipid bilayer of the liposomal membrane .
  • the HA is bound to an amine group of the head of a membrane phospholipid .
  • liposomes may comprise at least one active ingredient , preferably a protein kinase enzyme inhibitor, most preferably selected from the group consisting of imatinib, nintedanib and other derivatives belonging to the tyrosine kinase inhibitor class .
  • active ingredient preferably a protein kinase enzyme inhibitor, most preferably selected from the group consisting of imatinib, nintedanib and other derivatives belonging to the tyrosine kinase inhibitor class .
  • LIPO- IM-HA when admini stered intratracheally is able to exert a potent anti fibrotic ef fect that is superior to the free drug either administered intratracheally or systemically ( intraperitoneally) .
  • ef fect is mediated by an important anti-inflammatory function that is revealed by numerous analyses of broncho-alveolar lavage and the expression of pro-inflammatory and pro- fibrotic factors in the tissue of treated animals .
  • the disclosure provides a composition comprising the disclosed liposomes .
  • Such composition may further comprise at least one pharmaceutically acceptable vehicle .
  • the composition may further comprise diluents and/or excipients , also for inhalation administration .
  • the liposomes and/or the composition comprising them may be intended for use in medicine , pre ferably for inhalation administration, more preferably for use in the treatment of at least one respiratory disease , even more preferably at least one pulmonary fibrotic disease , for example selected from idiopathic pulmonary fibrosis , pulmonary fibrosis associated with collagen diseases , bronchiolitis obliterans .
  • the disclosure includes a procedure for the production of liposomes to carry at least one active ingredient comprising the steps of : solubili zing at least one phospholipid in a solvent , preferably a halogenated solvent , and obtaining a first solution,
  • - hydrating the lipid film in a solution containing at least one salt preferably selected from sodium salt , potassium salt , ammonium salt and obtain a second solution, subj ecting the second solution to extrusion and obtaining a suspension comprising liposomes .
  • the solvent may be selected from the group consisting of chloroform, methanol , ethanol , DCM, and mixtures thereof .
  • extrusion may be conducted using an extruder, preferably at a temperature between 20 ° C and 60 ° C, more preferably 40 ° C .
  • the method for producing liposomes may further comprise the step of adding at least one active ingredient to the suspension comprising liposomes to obtain liposomes comprising an active ingredient .
  • the active ingredient may be added to the suspension comprising the liposomes in a concentration between 0 . 1 mg/ 100 pl and 1 mg/ 100 pl preferably 0 . 5 mg/ 100 pl .
  • the method for producing liposomes may provide that HA, preferably having a molecular weight greater than 17000 Da, bound to at least one phospholipid is added in the step of hydrating the lipid film .
  • HA bound to at least one phospholipid may be obtained by the steps of :
  • solubili zing HA preferably having a molecular weight above 17000 Da, in a solvent including at least one halogenated solvent to obtain a solution of solubili zed HA,
  • the disclosure provides a method for obtaining HA, preferably having a molecular weight greater than 17000 Da, bound to at least one phospholipid, comprising the steps of : - solubili zing HA in a solvent comprising at least one halogenated solvent to obtain a solution of solubili zed HA, - adding to the solution of solubili zed HA at least one phospholipid, preferably comprising an amine group, preferably DPPE , and obtaining a mixture comprising HA and at least one phospholipid, adding to said mixture comprising HA and at least one phospholipid at least one reducing agent to obtain a solution comprising HA bound to the at least one phospholipid .

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Abstract

Liposomes coated with hyaluronic acid having a molecular weight greater than 17000 Daltons, preferably between 20000 and 60000 Daltons. The liposomes may contain an active ingredient, such as, for example, a protein kinase enzyme inhibitor, preferably selected from imatinib, nintedanib and other derivatives belonging to the tyrosine kinase inhibitor class. The liposomes may be effectively used in the treatment, preferably by inhalation, of respiratory diseases.

Description

"Liposomes , compositions comprising the same , uses thereof"
★ ★ ★
Field of the invention
This disclosure relates to liposomes , their use as vehicles for medical applications , and methods for producing them .
Technological background
The application of nanotechnologies in the therapeutic field is extremely innovative and associated with numerous advantages , including : 1 ) increase in the bioavailability of several drugs ; 2 ) possibility of exploiting alternative routes of administration ( such as inhalation delivery which allows high doses of the drug to be reached at the level of the target organ cells ) ; 3 ) possibility of directing the treatment , through functionali zation, towards a speci fic cell population ( targeting) . These innovative aspects are particularly relevant in the context of chronic respiratory diseases . Many drugs cannot be administered by inhalation, as they are either insoluble in water or toxic to the epithelial cells of the respiratory system . Therefore , the use of functionalised nanovectors could allow to reach adequate concentrations of the active ingredient in the target cells , sparing the respiratory epithelium and signi ficantly limiting the systemic toxic ef fects . This approach, often proposed, has not yet found an application, as the vectors developed so far have been found to be poorly biocompatible or of too complex synthesis for large-scale clinical application .
Summary of the invention
The aim of this disclosure is to provide biocompatible carriers of active ingredients , which are ef fective in the treatment of diseases for which there is an urgent need for a well-tolerated resolutive therapy, which do not present toxicity, and which are able to optimi ze the quantity of active ingredient at the target organ .
According to the invention, the above-mentioned purpose is achieved by means of the solution speci fically referred to in the appended claims , which form an integral part of this disclosure .
This disclosure relates to liposomes coated with hyaluronic acid (HA) having a molecular weight greater than 17000 Daltons , preferably between 20000 Daltons and 60000 Daltons .
The liposomes may contain an active ingredient in the core , in particular a protein kinase enzyme inhibitor, preferably a tyrosine kinase enzyme inhibitor, more preferably selected from the group consisting of imatinib, nintedanib and other derivatives belonging to the tyrosine kinase inhibitor class .
This disclosure also provides compositions comprising the liposomes and their uses .
Speci fically, the disclosed liposomes and the compositions comprising them may be used in medicine , to deliver active ingredients to speci fic target cells . In one or more embodiments , the disclosed liposomes and the compositions comprising them may be ef fectively used in the treatment of respiratory diseases , pre ferably pulmonary fibrotic diseases . Respiratory diseases may be selected from idiopathic or inflammation-based pulmonary fibrosis , particularly diseases associated with collagen diseases , and idiopathic bronchiolitis obliterans or secondary to bone marrow or lung transplantation .
The disclosure also provides an innovative method for producing liposomes containing at least one active ingredient as well as a method for producing HA-coated liposomes having a molecular weight greater than 17000 Da . The disclosure further relates to a method for producing HA bound to a phospholipid in the form of a conjugate (HA-f) .
Brief description of the figures
One or more embodiments will be described, purely by way of non-limiting example with reference to the attached figures, listed below.
Figure 1. Cell viability results after incubation of primary myofibroblast lines (LFs) derived from patients with chronic lung rejection (CLAD;A) , fibrosis- associated collagen disease (ILD-CTD;B) or immortalised human lung carcinoma cell line (A549;C) with imatinib- containing liposomes (LIPIm) , HA-coated liposomes containing imatinib (LIP-HAIm) and imatibin (Im) alone, at the same imatinib concentration (30 pM) after 24, 48 and 72 h. Data are represented as mean ± standard deviation of the percentage compared to the control set at 100%. Statistical analysis: **, p <0.01 vs. CTR; *, p <0.05 vs . CTR .
Figure 2. Results of western blot protein quantification performed on the cell lysate of CLAD- and ILD-CTD-derived LFs after treatment for 24 h with LIPIm, LIP-HAIm and imatinib (Im) . (A) Quantification of phosphorylated cAbl normalized to control (CTR = 1) and (B) quantification of type I collagen normalized to control (CTR = 1) .
Figure 3. Representative images acquired with the confocal microscope of peripheral blood-derived monocytes incubated for 1 h with fluorescent liposomes (LIP) and HA-coated liposomes (LIP-HA) . The cell nuclei were labelled with a specific fluorophore (DAPI) . White circles indicate the presence of LIP or LIP-HA within the monocytes.
Figure 4. Results of survival (A) and body weight gain (B) of mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES (HEPES) , bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib (IM IP) intraperitoneally, bleomycin and liposomes with imatinib and hyaluronic acid (LIPO-IM- HA) .
Figure 5. Hemocytometer cell count results in bronchoalveolar lavage fluid in the presence of trypan blue dye (A) . Hemocytometer count results of neutrophils (B) , macrophages (C) and lymphocytes (D) in the presence of Wright-Giemsa dye in bronchoalveolar lavage fluid in mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES (HEPES) , bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib intraperitoneally (IM IP) , bleomycin and liposomes with imatinib and hyaluronic acid (LIPO-IM-HA) .
Figure 6. Representative images acquired with the light microscope of lung tissue sections stained with hematoxylin and eosin from mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES (HEPES) , bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib intraperitoneally (IM IP) , bleomycin and liposome with imatinib and hyaluronic acid intratracheally (LIPO-IM-HA) , Aschroft score (A) indicates histological damage. Evaluation of myeloperoxidase activity (B) in lung tissue samples from mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES (HEPES) , bleomycin and imatinib intratracheally (IM IT) , bleomycin and intraperitoneal imatinib (IM IP) , bleomycin and liposomes with imatinib and hyaluronic acid (LIPO-IM- HA) .
Figure 7. Representative images acquired with the light microscope of Masson's trichrome-stained lung tissue sections of mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES (HEPES) , bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib intraperitoneally (IM IP) , bleomycin and liposomes with imatinib and hyaluronic acid (LIPO-IM-HA) , collagen quantitation (A) .
Figure 8. Representative images acquired with the light microscope of the lung tissue sections on which an immunohistochemical analysis was performed with the TGF- lp antibody of the mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES (HEPES) , bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib intraperitoneally (IM IP) , bleomycin and liposomes with imatinib and hyaluronic acid (LIPO-IM-HA) , quantification of TGF-lp expression (A) .
Figure 9. Representative images acquired with the light microscope of the lung tissue sections on which immunohistochemical analysis was performed with the CD4 antibody from mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib intraperitoneally (IM IP) , bleomycin and liposomes with imatinib and hyaluronic acid (LIPO-IM-HA) , quantification of CD4 expression (A) .
Figure 10. Representative images acquired with the light microscope of lung tissue sections on which immunohistochemical analysis was performed with the CD8 antibody of mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES (HEPES) , bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib intraperitoneally (IM IP) , bleomycin and liposomes with imatinib and hyaluronic acid (LIPO-IM- HA) , quantification of CD8 expression (A) .
Detailed description of the invention
In the following specification, a number of specific details are provided to enable a thorough understanding of the embodiments. The embodiments can be practiced without one or more of the speci fic details , or with other methods , components , materials , etc . In other cases , well-known structures , material s or operations are not shown or described in detail to avoid obscuring aspects of the embodiments .
Reference throughout this disclosure to "one embodiment only" or "an embodiment" indicates that a particular aspect , structure or feature described in connection with the embodiment is included in at least one embodiment . Therefore , the appearance of the expressions " in one embodiment" or " in an embodiment" at various places throughout the present disclosure do not necessarily all refer to the same embodiment . Furthermore , the particular aspects , structures or features can be combined in any suitable way in one or more embodiments .
The headings provided here are for convenience only and do not interpret the scope or purpose of the embodiments .
This disclosure relates to a liposomal nanovehicle functionali zed on the surface with hyaluronic acid (HA) in order to facilitate the selective delivery of an active ingredient towards diseased cells and alveolar macrophages present in chronic and severe respiratory diseases .
Liposomes are phospholipid vesicles that have a membrane , the liposomal membrane , comprising a lipid bilayer, general ly a phospholipid bilayer . Liposomes have nanometric dimensions , enclose an aqueous core and may be used in the clinic for the delivery of active ingredients . They can encapsulate both hydrophilic and lipophilic molecules , allowing the gradual release of the active ingredient , thus modi fying its pharmacokinetic profile .
The liposomes obj ect of the present disclosure are HA-coated liposomes having a molecular weight higher than 17000 Daltons , preferably between 20000 and 60000 Daltons . HA is directly bound to the head of a phospholipid of the lipid bilayer of the liposomal membrane . Speci fically, the HA is directly bound to the head of a membrane phospholipid comprising an amine group . More preferably, the HA is directly bound to an amine group of a membrane phospholipid . In one or more embodiments , the weight ratio of hyaluronic acid to membrane phospholipids is between 1 and 5 .
The phospholipid bilayer of the liposomal membrane may also comprise cholesterol .
The phospholipid of the phospholipid bilayer of the liposomal membrane may be selected from 1 , 2-dipalmitoyl- sn-glycero-3-phosphoethanolamine ( DPPE ) , phosphatidylglycerol ( PG) , phosphatidylcholine ( PC ) and combinations thereof . In one or more embodiments , PC of both natural ( soy and/or egg) and synthetic origin may be used . In one or more embodiments , synthetic PC may comprise fully hydrogenated soy PC and/or PC having the two lipid chains with a number of carbon atoms between 12 and 18 ( C12-C18 ) .
The coated liposomes obj ect of the present disclosure may have an average diameter ranging from 100 nm to 300 nm, preferably equal to 250 nm . The mean diameter value was analyzed by the quasi-elastic light scattering ( QELS ) technique at 25 ° C . In one or more embodiments , the coated liposomes have a Z potential value ranging from - 10 mV to -50 mV, preferably equal to -33 mV .
In one or more embodiments , the liposomes obj ect of the present disclosure may contain at least one active ingredient . From the electron microscope analysis it was also observed that the liposomes have a spherical shape and the presence of the precipitated active ingredient inside them was confirmed .
The Inventors observed that the liposomes retain approximately 95% of the initial active ingredient content , in particular imatinib, after storage at 4 ° C for 3 weeks . During this period, no appreciable changes in diameter and Z-potential , nor precipitation or aggregation phenomena are observed .
The encapsulation method carried out by the Inventors and described below provides a series of advantages , among which it allows to obtain stable formulations : the Inventors have in fact observed that 20% of imatinib, as an active ingredient , is released in 24 hours in HEPES buf fer and 30% in 72 hours ; in serum the release is about 50% in 24 hours and 80% in 72 hours .
The liposomes obj ect of the present disclosure as well as the compositions containing them may be used to deliver the active ingredients contained therein to speci fic targets and may also be administered by inhalation .
Speci fically, the diseases which may be ef fectively treated with the liposomes obj ect of the present disclosure are respiratory diseases , preferably pulmonary fibrotic diseases . In one or more embodiments , treatable diseases may be selected from idiopathic pulmonary fibros is , pulmonary fibrosis associated with collagen diseases and bronchiolitis obliterans . Bronchiolitis obliterans may result from lung transplantation or chronic mani festation after allogeneic bone marrow stem cell transplantation .
These clinical conditions with very di f ferent etiology share some fundamental characteristics : 1 ) they are rare diseases for which there is an urgent need for well-tolerated resolutive therapy; 2 ) are characteri zed by an uncontrolled proli feration of myofibroblasts ( LFs ) , triggered and sustained by a chronic inf lammatory/autoimmune inj ury .
Characteri zation studies of LFs , obtained from bronchoalveolar lavage samples of patients suf fering from these diseases , have shown, at the level of the cell membrane , an overexpression o f CD44 , a glycoprotein that is also the speci fic receptor for HA.
The liposomes obj ect of the present disclosure coated with HA having a speci fic molecular weight (higher than 17000 Daltons , preferably between 20000 Daltons and 60000 Daltons ) represent an absolute innovation and an important step forward in the treatment of these serious diseases , with a considerable economic and social impact .
Bronchiolitis obliterans , which represents the failure of a lung transplant or the expression of chronic pulmonary graft , is associated with high costs to the healthcare system, requiring frequent evaluations and hospitali zations , costly therapies and complex support in the phase of disease progression, while pulmonary fibrosis is often the cause of severe disability and death in patients . In the idiopathic form, it is not associated with other systemic diseases , while in the form associated with systemic sclerosis or autoimmunebased inflammatory myopathy, it is often unresponsive to the immunosuppressive therapy adopted for these pictures .
In recent years , numerous studies have aroused great interest regarding the role of several mesenchymal cell growth factors , such as PDGF, TGF-beta and VEGF, in the pathogenesis of di f fuse lung fibrosis . The action of these factors is mediated by several protein kinases that mediate apoptosis resistance , inhibition of LFs proli feration and extracellular matrix production . Among the numerous tyrosine kinase ( TK) inhibitors developed in recent years , the first and most widely used is imatinib, which not only inhibits BCR-ABL, but is equally potent against other receptors with tyrosine kinase properties , such as PDGFRa and c-KIT .
Another compound, already widely used in the treatment of pulmonary fibrosis , both idiopathic and associated with collagen diseases , and administered orally, is nintedanib, which is able to inhibit PDGF a and B , FGF 1-3 and EGFR 1-3 receptor-associated TK .
Systemic administration of these inhibitors results in signi ficant toxicity, which in turn limits their use and dosage , thereby reducing the amount of active ingredient at the target organ level .
Liposomal formulations containing anti fungal or anticancer drugs are commercially available for intravenous administration . Recently, two liposomal formulations for inhalation use have been produced : liposomal amikacin (which is indicated in the treatment of mycobacterial pulmonary diseases refractory to first- line antibiotic therapy) and liposomal cyclosporine (under registration for bronchiolitis obliterans ) . Commercially available formulations , however, are not functionali zed and therefore do not have a speci fic cellular target . Therefore , to date , there is no liposomal formulation with active targeting capability in advanced development that may also be administered by inhalation .
As demonstrated below, the local administration of active ingredients via the liposomes described here is extremely ef fective in that it allows the active ingredients to speci fically reach the cells most involved in the pathogenic process , i . e . cells expressing the CD44 receptor ( LFs , inflammatory cells , activated endothelial cells ) .
After finding that primary fibroblastic cells ( LFs ) isolated from the lung of patients with collagen disease- associated fibrosis ( interstitial lung disease in collagen tissue disease , ILD-CTD) or from patients with chronic lung rej ection ( chronic lung allograft dys function, CLAD) express CD44 in a high percentage ( >90% ) , the Inventors of the present application used these primary cell lines to test the internali zation and function of HA-coated liposomes with speci fic molecular weight and containing imatinib as active ingredient . The results described below show that the surface coating with HA having a speci fic molecular weight ( LIP-HAIm) allows to obtain liposomes with active targeting ability and high uptake by cells expressing the CD44 receptor .
Speci fically, the advantage of using HA having a molecular weight greater than 17000 Daltons , preferably between 20000 and 60000 Daltons , consists in a higher af finity towards the CD44 receptor, compared to nonfunctionali zed liposomes . In addition, the presence of HA exerts a cryoprotective ef fect , capable of favoring the lyophili zation of compositions comprising the liposomes . The lyophili zation in turn determines an increase in the stability of the compositions and makes it possible to obtain compositions in the form of powders which may also be administered as aerosols . In one or more embodiments , the composition comprising the liposomes may also be in liquid or solid form .
The Inventors of the present application have also developed a method for producing liposomes , optionally coated with HA, comprising an active ingredient which has proved to be simple and particularly ef fective in terms of the quantity of active ingredient loaded into the liposomal core .
In the invention disclosed herein, the active ingredient is inserted into the aqueous core of the liposomes using the pH gradient technique , preferably using citrate buf fer . This technique allows to obtain the precipitation of the active ingredient in the aqueous core o f the vesicles and a high encapsulation ef ficiency, equal to 85% for imatinib, therefore higher than that obtained using, for example , an ammonium sulphate solution ( 70% ) , already described above .
Speci fically, the method for producing liposomes containing at least one active ingredient obj ect of the present disclosure comprises the steps of :
- solubili zing at least one phospholipid in at least one halogenated solvent and obtaining a first solution,
- evaporating the solvent from said first solution and obtaining a lipid film,
- hydrating the lipid film in a buf fer solution comprising at least one salt , preferably selected from sodium salt , potassium salt , ammonium salt and obtaining a second solution,
- subj ecting the second solution to extrusion and obtaining a suspension comprising liposomes ,
- adding to said suspension comprising liposomes at least one active ingredient and obtaining liposomes containing said active ingredient .
The phospholipid usable in the disclosed method may be selected from DSPC ( 1 , 2-distearoyl-sn-glycerol-3- phosphocholine ) , phosphatidylglycerol ( PG) , phosphatidylcholine ( PC ) , combinations thereof . PC usable in the disclosed method may be either of natural origin, for example from soy and/or egg, or synthetic . In one or more embodiments , synthetic PC may comprise fully hydrogenated soy PC and/or PC with the two lipid chains having a number of carbon atoms ranging from 12 to 18 ( C12-C18 ) . A polyethylene glycol modi fied phospholipid, 1 , 2 -disteroyl-sn- 3-phosphoethanolamine-N- [methoxy (polyethylene glycol ) ] (mPEG-DSPE ) may also be used . In one or more embodiments , at least one lipid, for example belonging to the class o f sterols , for example cholesterol , may also be added to the solvent .
The solvent usable in the phospholipid solubili zation step may be selected from the group consisting of chloroform, dichloromethane ( DCM or methylene chloride ) , methanol , ethanol and mixtures thereof .
In one or more embodiments , the solvent evaporation step from the first solution may be carried out under reduced pressure and then under vacuum . The solvent may be removed by evaporation, for example using a rotary vacuum evaporator (Rotavapor®) . The evaporation of the solvent is promoted by the reduction of the pressure which lowers the boiling temperature of the solvent .
The salt contained in the buf fer solution of lipid film hydration step may be selected from sodium citrate , sodium phosphate , sodium sulphate , sodium acetate , ammonium citrate , ammonium phosphate , ammonium sulphate and ammonium acetate . The pH of the buf fer solution comprising a salt , preferably sodium citrate , may be between 4 and 5 . The Inventors have observed that optimal results in terms of encapsulation yield of the active ingredient are obtained when the salt contained in the buf fer solution is sodium citrate . Preferably, the pH of the buf fer solution comprising sodium citrate is 4 . 38 . In one or more embodiments , particularly when the active ingredient is imatinib, the method does not involve the use of ammonium sulphate .
The solution obtained in the lipid film hydration step may be subj ected to sonication, for example in a sonication bath, and to stirring, preferably in a vortex, until the lipid film is completely detached .
The solution may then be subj ected to extrusion by using an extruder, preferably at a temperature between 20°C and 60°C, more preferably equal to 40°C to obtain the suspension comprising liposomes.
The extrusion may be carried out under nitrogen pressure, preferably between 100 psi and 800 psi using filters, preferably made of polycarbonate, in which the pore diameter may be between 800 nm and 100 nm.
The suspension comprising liposomes may be subjected to a pH change step in order to achieve a suspension pH preferably between 7 and 7.6.
The pH change step may be carried out by subjecting the suspension to gel filtration on a packed column, for example with Sepharose CL-4B, eluting with buffer, preferably HEPES, pH 7.4.
The active ingredient may be added to the suspension comprising the liposomes dissolved in a buffer solution in a concentration between 0.1 mg/100 pl and 1 mg/100 pl, preferably equal to 0.5 mg/100 pl. The buffer solution may comprise for example HEPES and/or phosphate buffer .
The active ingredient may be a protein kinase enzyme inhibitor, preferably a tyrosine kinase enzyme inhibitor, more preferably selected in the group consisting of imatinib, nintedanib and other derivatives belonging to the tyrosine kinase inhibitor class.
The liposomal suspension added with the active ingredient may be kept for a period of time comprised between 20 minutes and 60 minutes, preferably equal to 30 minutes, at a temperature comprised between 30°C and 40°C, preferably equal to 37°C.
The liposomal suspension may also be subjected to a purification step in order to remove the nonencapsulated active ingredient.
The purification step may be carried out by gel filtration or molecular exclusion chromatography.
In one or more embodiments, the method may allow to produce HA-coated liposomes. For this purpose, in the lipid film hydration step, HA may be added to the buffer solution, preferably HA having a molecular weight higher than 17000 Daltons.
Specifically, HA is added in the form of conjugate with at least one phospholipid (HA-f) .
The term conjugate refers to a product obtained from the chemical reaction between two components that leads to the formation of a covalent bond.
Such a HA-f conjugate may be obtained by a reductive amination reaction between HA and a phospholipid containing an amine group, for example phosphatidylethanolamine (PE) . In this conjugate only one molecule of PE is bound to HA, and the synthetic process is particularly complex as the molecular weight of HA increases, which in previous works was between 4000 and 17000 Daltons.
To produce HA-f, HA having a molecular weight greater than 17000 Daltons, preferably between 20000 and 60000 Daltons, may be solubilized in at least one halogenated solvent to obtain a solution of solubilized HA. Preferably, the solvent may comprise DCM as halogenated solvent.
In one or more embodiments, the solvent may comprise DCM and dimethyl sulfoxide (DMSO) , preferably in a 1:1 weight ratio. The HA solubilization step may be carried out by sonication, preferably in a sonicator bath, more preferably for a period comprised between 30 minutes and 120 minutes, preferably equal to 90 minutes.
HA, in particular having a molecular weight above 17000 Daltons may be added to the solvent in a concentration between 0.02 mmol and 0.1 mmol.
The solution comprising solubilized HA may be heated to a temperature ranging from 40°C to 70°C, preferably equal to 60°C. To the solution comprising solubilized HA, the phospholipid comprising an amine group, preferably DPPE, is added to obtain a mixture comprising HA and phospholipid comprising an amine group.
In one or more embodiments, the phospholipid comprising an amine group is added solubilized in the solvent, preferably comprising chloroform and methanol. Preferably, the mixture may comprise chloroform and methanol in a 1:1 weight ratio. The phospholipid may be added to the solvent in a concentration between 0.02 mmol and 0.1 mmol.
The mixture comprising HA and phospholipid including an amino group is brought to a temperature between 18°C and 24°C, preferably equal to 20°C (room temperature) .
The mixture may be subjected to a pH change in order to bring the pH to a value of 4.5. The pH adjustment may be carried out by adding an acid, for example acetic acid .
The mixture may be brought to a temperature ranging from 40°C to 70°C, preferably equal to 60°C for a period of time ranging from 1 hour to 3 hours, more preferably equal to 2 hours .
The mixture comprising HA and phospholipid including an amine group is added with at least one reducing agent to obtain a solution comprising the HA-f conjugate. The reducing agent may be selected from borohydrides , sodium cyanoborohydride and sodium triacet-oxy-borohydride (STAB) . Preferably, the reducing agent is a borohydride, more preferably STAB.
The solution comprising the HA-f conjugate may be kept at a temperature ranging from 50°C to 70°C, preferably 60°C for a period of time ranging from 80 hours to 120 hours, preferably for a period of 96 hours.
The solution may be subjected to purification and optionally washed.
The purification may be carried out by dialysis. The washing may be carried out for example with DCM. The solution may then be subjected to a lyophilization step to obtain the lyophilized HA-f conjugate.
The lyophilized conjugate may be solubilized and added to the lipid film in the hydration step, in particular in the buffer solution comprising a salt, so as to obtain HA-coated liposomes containing an active ingredient. In one or more embodiments, the weight ratio of hyaluronic acid to membrane phospholipids is between 1 and 5.
In one or more embodiments, in order to obtain fluorescent liposomes, at least one fluorescenceemitting compound, for example fluorescein, may be added to the buffer solution in the lipid film hydration step.
The liposomes obtained with the described method may have an average diameter between 100 nm and 300 nm, when determined using the quasi-elastic light scattering (QELS) technique.
In the case of uncoated liposomes, the diameter is preferably about 200 nm; in the case of HA-coated liposomes, the diameter is preferably about 250 nm. The presence of HA on the surface of the liposomes therefore leads to an increase in the value of the average particle diameter. In addition, the Z-potential value may be around -30 mV for uncoated liposomes and slightly more negative, around -33 mV, for HA-coated liposomes, due to the presence of the negatively charged carboxyl groups of HA.
Example of preparation and characterization of liposomes containing imatinib
A composition comprising 1 , 2-distearoyl-sn- glycerol-3-phosphocholine (DSPC) , cholesterol (COL) and phosphatidylglycerol (PG) in a molar ratio of 70:30:3 was used to prepare the liposomes.
The components were solubilized in chloroform and then the lipid film was prepared by evaporating the solvent under reduced pressure in the Rotavapor® and then under vacuum.
Subsequently, the lipid film was hydrated with citrate buffer (solution of citric acid and sodium citrate, pH 4.38) , sonicated and vortexed. The liposomes were extruded under nitrogen pressure through polycarbonate filters (pore diameter 400 and 200 nm) using an extruder (Extruder; Lipex, Vancouver, Canada) at a temperature of 40°C. They were then subjected to gel filtration on a column packed with Sepharose CL-4B eluting with HEPES buffer pH 7.4 to change the pH of the liposomal suspension. A solution of imatinib mesylate in HEPES buffer (0.5 mg/100 pl) was then added to the liposomal suspension and incubated for 30 minutes at 37°C; the liposomal preparation was then purified on a packed column with Sepharose CL-4B eluting with HEPES buffer pH 7.4, removing the unencapsulated active ingredient by gel filtration.
The same procedure was used to obtain the HA-coated liposomes by replacing the PG with the HA-f conjugate. The HA-f conjugate was obtained by conjugating HA of molecular weight 44000 Da with the phospholipid 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) .
The reaction was carried out following the synthesis reported in the scientific publication Hyaluronic acid-coated liposomes for active targeting of gemcitabine. European Journal of Pharmaceutics and Biopharmaceutics (2013) 85: 373-380 with some modifications in order to carry out the conjugation reaction using polymer with a significantly higher molecular weight, as described below.
The HA-f conjugate was obtained by a reductive amination reaction between HA and a phospholipid containing an amino group (PE) . In this conjugate only one molecule of PE is bound to HA and the synthetic process is particularly complex with the increase in the molecular weight of HA which in previous works was between 4000 Da and 17000 Da.
HA with a molecular weight of 44000 Da was solubilized in a mixture of DCM and DMSO (1:1) in a sonicator bath for 90 minutes.
Next, the solution was heated to 60°C and an equimolar amount of DPPE solubilized in a 1:1 mixture of chloroform and methanol was added.
The solution was then brought to room temperature, acidified to pH 4.5 with acetic acid and then left for 2 hours at 60°C.
Finally, solubilized STAB was added to the same DPPE mixture and the reaction proceeded for 96 hours at 60°C.
At the end of the reaction, the mixture was dialyzed against water and subsequently washed several times with DCM and finally lyophilized. The conjugate was solubilized in citrate buffer and added to the lipid film during the hydration step.
Fluorescent liposomes were prepared by adding a fluorescein solution in HEPES buffer during the lipid film hydration step and then subjected to extrusion and purification by gel filtration.
The liposomal preparations were stored at a temperature of 4 °C.
The average diameter and poly-dispersion index of the liposomes were determined at 25°C by means of the quasi-elastic light scattering (QELS) technique, and the surface charge was assessed by Z-potential determination by performing triplicate measurements. The morphology of the liposomes was determined by cryo-TEM (cryogenic- Transmission Electron Microscopy) analysis. The poly- dispersion index was less than 0.2 for the uncoated liposomes and the HA-coated liposomes object of this disclosure .
The amount of imatinib encapsulated in the liposomes was determined by RP-HPLC. For this purpose liposomal fractions were diluted with acetonitrile (by a different factor depending on the presumed imatinib concentration) , the mixture was sonicated, vortexed, centrifuged and the supernatant was filtered (0.45 pm PTFE) and analyzed by HPLC (the analytical conditions used are described in the scientific article Imatinib assay by HPLC with photodiode-array UV detection in plasma from patients with chronic myeloid leukaemia: comparison with LC-MS/MS Clinica Chimica Acta (2010) 411:140-146) . The amount of imatinib encapsulated in liposomes was between 0.35 mg/100 pl and 0.40 mg/100 pl.
The lipid concentration was determined by phosphorus analysis, as described in Phosphorus assay in column chromatography J Biol Chem. (1959) 234:466-468. An average lipid concentration of 3.8 mM was determined.
The release kinetics of imatinib were assessed by incubating liposomal formulations with and without HA at 37°C in HEPES buffer or serum. At set times, an aliquot of liposomal formulation was taken, purified by gel filtration, and the amount of encapsulated active ingredient was determined by HPLC and compared with the initial value. The physical stability of the liposomes was assessed under storage conditions (4°C) by monitoring the average diameter, Z-potential and amount of encapsulated drug at set time intervals.
In vitro efficacy test
Tests were conducted on an immortalized human lung carcinoma line (A549) and primary myofibroblast (LFs) lines isolated from bronchoalveolar lavage (BAL) of patients with CLAD or ILD-CTD. All these lines show a high rate of CD44 expression.
The isolation of LFs was carried out following the procedure described in the scientific article Antibody- engineered nanoparticles selectively inhibit mesenchymal cells isolated from patients with chronic lung allograft dysfunction. Nanomedicine (Lond) (2015) 10:9-23.
Briefly, BALs from patients with CLAD and ILD-CTD are first filtered with sterile gauze and subsequently centrifuged at 1800 rpm for 10 minutes. The supernatant is collected and stored at -20°C, while the cell pellet is resuspended with saline and centrifuged again at 1800 rpm for 10 minutes. The pellet obtained is counted to grow 6 x 106 cells in DMEM culture medium with 10% fetal bovine serum (BBS) and 1% penicillin/ streptomycin and L- glutamine added. After approximately 10-15 days, foci of LFs are formed, which are isolated and cultured for experiments. The cells used express high levels of the CD44 protein, as described in the scientific article Antibody-engineered nanoparticles selectively inhibit mesenchymal cells isolated from patients with chronic lung allograft dysfunction. Nanomedicine (Lond) . (2015) 10: 9-23.
In order to test the internalization efficacy of HA-modified liposomes in CD44-expressing cells, 0.2 x 106 A549 or LFs cells were plated and after 24 h of culture to allow cell adhesion, they were incubated with uncoated or HA-coated fluorescent liposomes (LIP and LIP-HA) for 4 and 24 h at 37°C.
After treatment, the cells were analyzed by cytof luorimetry and confocal microscopy, showing that LIP-HA enters both A549 and LFs of CLAD and ILD-CTD more efficiently than LIP.
Confocal microscopy analysis confirms the greater internalization of LIP-HAs compared to LIPs, also showing that liposomes internalized by both A549s and LFs co-localize with cellular endosomes.
Having demonstrated the greater efficiency of LIP- HA compared to LIPs in being internalized by CD44- expressing cells, the Inventors evaluated the effect of liposomes containing imatinib as active ingredient on cell viability using both A549 and LFs derived from CLAD and ILD-CTD. The viability assay was carried out by MTT assay, which gives the possibility to quantify cell viability by means of mitochondrial activity. 3 x 103 A549 cells or LFs were plated and incubated with LIP- HAIm, LIPIm, imatinib by incubating at the same concentration of imatinib (30 uM) for up to 72 h.
These experiments showed that LIP-HAIm and LIPIm reduced the viability of LFs by 30%, while imatinib reduced their viability by 80%. In the case of A549, an increased reduction in cell viability by LIP-HAIm (30%) compared to LIPIm (0%) was observed after 72 h of treatment. Furthermore, in this cell line LIP-HAIm exert the same effect as imatinib alone (30%) (Figure 1) .
Knowing that the active ingredient imatinib specifically targets cAbl, it was decided to evaluate the efficacy of LIP-HAIm to inhibit cAbl activity only in CLAD and ILD-CTD LFs. To study cAbl activity, the phosphorylation level of the protein is assessed.
Western blot analysis showed, for LFs derived from patients with CLAD, a 50% reduction in cAbl activity with both LIPIm and LIP-HAIm, after 24 h of treatment. Analyzing LFs derived from ILD-CTD, a different activity of LIPIm and LIP-HAIm is observed, where LIPIm reduces cAbl activity by 60%, while LIP-HAIm reduces it by 70%. Interestingly, imatinib, for both CLAD- and ILD-CTD- derived cells, has no effect after 24 h on cAbl activity (Figure 2A) .
Finally, western blot analysis assessed the production of collagen type lai, a key factor in the extracellular matrix that makes up fibrotic lesions , after 24 h of treatment with LIP-HAIm, LIPIm and imatinib alone , again with the same amount of imatinib ( 30 uM) .
The Inventors showed that in the case of CLAD- derived LFs , imatinib delivered by LIP-HA reduced collagen production by 30% , while LIPIm reduced it by 20% . With regard to ILD-CTD-derived LFs , a 60% reduction in collagen expression was observed after treatment with LIP-HAIm and LIPIm . Imatinib alone showed activity in reducing collagen expression by 50% in CLAD LFs and 60% in ILD-CTD LFs ( Figure 2B ) .
These results demonstrate that encapsulation of the active ingredient within liposomes does not change its molecular properties , but maintains its speci fic activity .
Given the fact that macrophages are the main key players in the inflammatory process of the above- mentioned diseases , the ability of LIP and LI P-HA to interact with peripheral blood-derived monocytes was also evaluated . Confocal microscopic analyses showed that LIP-HA interacted more with monocytes than LIP ( Figure 3 ) .
In most cases , the method of hydration of the lipid film followed by extrusion is used, for both encapsulation of the native active ingredient ( as described in Ef ficacy of targeted liposomes and nano- cochleates containing imatinib plus dex-ketoprof en against fibrosarcoma Drug Dev Res . ( 2019 ) 80 : 556-565 ; Antiproli ferative activity and VEGF expression reduction in MCF7 and PC-3 cancer cells by paclitaxel and imatinib co-encapsulation in f olate-targeted liposomes AAPS Pharm Sci Tech . ( 2018 ) 19 : 201-211 ) and sodium deoxycholate conj ugate [ as described in Hyaluronan coated liposomes as the intravenous platform for delivery of imatinib mesylate in MDR colon cancer . International Journal of Biological Macromolecules (2015) 73:222-235] . In this procedure, the active ingredient is inserted into the aqueous core of the liposomes by the pH gradient technique using citrate buffer. This technique achieves precipitation of the active ingredient in the aqueous core of the liposomes with a high encapsulation efficiency of 85% for imatinib, which is higher than that obtained using an ammonium sulphate solution (70%) , previously described (Folate receptor targeted liposomes enhanced the antitumor potency of imatinib through the combination of active targeting and molecular targeting International Journal of Nanomedicine (2014) 9:2167-
2177) . The higher encapsulation yield obtained using citrate buffer rather than ammonium sulphate solution may result from the higher solubility of the active ingredient in citrate buffer (S= 330 mg/ml, pH=4.38) , compared to that in ammonium sulphate (S= 280 mg/ml, pH=5.75) . The solubility of imatinib in citrate buffer and ammonium sulphate solution was determined by preparing a suspension of the active ingredient in each of the two media, incubating it at 25°C for 24 hours and analyzing the filtrate via HPLC .
The peculiar characteristic of the liposomes disclosed here is that they act preferentially on fibroblasts and inflammatory cells (in particular macrophages and lymphocytes expressing CD44) , which are the main effectors (key players) of chronic progressive fibrosing diseases. On myofibroblasts, in particular, they act by inhibiting their proliferation, migration and extracellular matrix deposition. In vivo efficacy test
Materials and Methods
Animals: Male GDI mice (25-30 g, Envigo, Milan, Italy) were housed in a controlled location. They received food and water ad libitum. The study was approved by the Animal Care Review Board (OPBA) of the University of Messina. All in vivo experiments followed the new US, European, Italian and ARRIVE guidelines.
Induction of lung damage: bleomycin sulphate (1 mg/kg body weight) was administered via a single intratracheal injection. A volume of 100 pL was injected, immediately followed by 300 pL of air to ensure delivery to the distal airways. Treatments were administered every 5 days starting on day 10 after bleomycin administration. Twenty-eight days after bleomycin injection, animals were sacrificed and tissues were harvested for further analysis.
Experimental groups: the animals were divided into the following experimental groups (n=10) :
- Vehicle: mice received bleomycin administration and were treated daily with the vehicle (saline solution (50 pL) ) ;
- Hepes: mice received bleomycin administration and were treated intratracheally with hepes (50 pL) ;
- IM IT: mice received bleomycin administration and were treated intratracheally with Imatinib (50 pL of 150 pg/ml) ;
-IM IP: mice received bleomycin administration and were treated daily with imatinib (10 mg/ml) intraperitoneally (IP) ;
LIPO-IM-HA: mice received bleomycin administration and were treated daily with liposome with hyaluronic acid-functionalized imatinib (LIPO-IM-HA) (50 pL of 150 pg/ml) ;
- Sham: identical to the Vehicle group, but the animals received intratracheal instillation of saline (0.9% w/v) instead of bleomycin and were treated daily with the vehicle (saline) .
Mice were euthanized 28 days after bleomycin instillation and tissues were collected for lesion analysis .
Survival rate and body weight gain: mortality and body weight were assessed daily up to 28 days.
Bronchoalveolar lavage (BAL) : twenty-eight days after bleomycin instillation, the mice were sacrificed and the tracheas were cannulated to perform the lavage. A total of 0.5 ml of phosphate-buff ered saline (PBS) was used. The recovered BAL fluid was centrifuged, the pelleted cells were harvested and the supernatants were stored at -20°C. In the presence of trypan blue dye, total BAL cells were counted using a hemocytometer. For a differential leucocyte count, the bronchoalveolar lavage (BAL) wash was stained with Wright-Giemsa. After staining, the differential count was performed using the standard morphological protocol under the light microscope .
Histological examination: lung tissue samples were collected 28 days after bleomycin injection. After fixing the tissues in buffered formaldehyde solution (10% in PBS) , histological sections were stained with hematoxylin and eosin and evaluated using a Laica DM6 microscope (Milan, Italy) . The severity of pulmonary fibrosis was graded on a scale from 0 to 8. Lung sections were stained with Masson's trichrome for fibrosis.
Evaluation of myeloperoxidase activity (MPO) : MPO activity was defined as the amount of enzyme that degrades 1 pmol peroxide per minute at 37 °C and was expressed in units per gram of wet tissue weight.
Western blot analysis: briefly, the lung tissues from each mouse were homogenized in an extraction buffer containing 0.15 pM pepstatin A, 0.2 mM phenylmethylsulphonyl fluoride (PMSF) , 1 mM sodium orthovanadate and 20 pM leupeptin; homogenized at maximum setting for 2 min; and centrifuged at lOOOxg for 10 min at 4°C. The supernatants contain the cytosolic fractions, while the pellets represent the nuclear ones. The pellets were resuspended in a second buffer containing 150 mM sodium chloride (NaCl) , 1% Triton X- 100, 1 mM ethylene glycol tetraacetic acid (EGTA) , 10 mM tris-chloric acid (HC1) pH 7.4, 0.2 mM PMSF, 1 mM ethylenediaminetetraacetic acid (EDTA) , 0.2 mM sodium orthovanadate and 20 pm leupeptin. After centrifugation at 4°C and 15000 g for 30 min, the nuclear proteins containing the supernatants were stored at -80°C for further analysis. Specific primary antibody: anti-IkB- alpha (1:1000, Santa Cruz Biotechnology) or anti-NF-kB p65 (1:1000; Santa Cruz Biotechnology) or anti-p-p38 (1:1000, Santa Cruz Biotechnology) or anti-p-JNK (1:1000, Santa Cruz Biotechnology) or anti-MMP2 (1:1000, Santa Cruz Biotechnology) or anti-MMP9 (1:1000, Santa Cruz Biotechnology) or anti-TRAF-6 (1:1000, Santa Cruz Biotechnology) in IxPBS, 5% w/v skimmed milk powder and 0.1% Tween-20 and incubated at 4 °C, overnight. Subsequently, the membranes were incubated with peroxidase-conj ugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson Immuno Research) for 1 hour at room temperature. To verify that the membranes were loaded with equal amounts of protein, they were also incubated with antibody against laminin (1:1000; Santa Cruz Biotechnology) and beta-actin (1:1000; Santa Cruz Biotechnology) . The signals were detected with the enhanced chemiluminescence detection system reagent according to the manufacturer's instructions (SuperSignal West Pico Chemiluminescent Substrate, Pierce) . The relative expression of the protein bands was quantified by densitometry with the Bio-Rad ChemiDoc XRS software and standardized to beta-actin levels. Blot signal images (8 bit/600 dpi) were imported into the analysis software (Image Quant TL, v2003) . Immunohistochemical analysis: the antibodies that were incubated O/N on the brain sections were anti-TGF- beta (Millipore, 1:500 in PBS, v/v, AB152, Burlington, MA, USA) , anti-CD4 (Santa Cruz Biotechnology, 1:300 in PBS, v/v, 65G10 sc-32258, Dallas, TX, USA) and anti-CD8 (Santa Cruz Biotechnology, 1:50 in PBS, v/v, LB509 sc- 58480, Dallas, TX, USA) . To test the specificity of the antibodies, sections of 5 mice from each group were treated with a primary antibody or with a secondary antibody alone. Images were taken using a Laica DM6 microscope. The Image J IHC profiler plug-in was used for densitometric analysis. When this is selected, it automatically draws a histogram profile of the deconstructed DAB image and a corresponding score log is shown. The histogram profile corresponds to the positive pixel intensity value obtained by the computer program.
Statistical evaluation: all values in the figures and text are expressed as mean ± standard error of the mean (SEM) of N number of animals. Results were analyzed by one-way ANOVA followed by a Bonferroni post-hoc test for multiple comparisons. A p-value < 0.05 was considered significant. * p < 0.05 compared to sham, # p < 0.05 compared to vehicle, ** p < 0.01 compared to sham, ## p < 0.01 compared to vehicle, *** p < 0.001 compared to sham and ### p < 0.001 compared to vehicle.
Resul ts
Survival and body weight: LIPO-IM-HA administration significantly reduced mortality, as shown in Figure 4A, and body weight loss (Figure 4B) associated with pulmonary fibrosis.
Twenty-eight days after bleomycin instillation, the HEPES-treated mice showed recruitment of inflammatory cells in the bronchoalveolar lavage fluid, compared to the sham group (Figure 5A) . In particular, neutrophils (Figure 5B) , macrophages (Figure 5C) and lymphocytes (Figure 5D) were evaluated and a significant increase in the number of cells was observed. LIPO-IM-HA significantly reduced the infiltration of inflammatory cells into the bronchoalveolar lavage fluid.
Histological analysis: twenty-eight days after bleomycin administration, histological analysis of lung samples collected from vehicle and HEPES-treated mice showed tissue injury and extracellular matrix deposition, compared to sham animals. LIPO-IM-HA treatment significantly reduced lung damage, as shown by hematoxylin and eosin staining (Figure 6A) . In addition, LIPO-IM-HA was able to reduce neutrophil activity, as shown by the MPO assay (Figure 6B) .
Collagen deposition: lung damage produced by bleomycin is characterized by increased collagen deposition and severe fibrosis. Masson's trichrome staining showed an increased blue area in the tissues of vehicle-treated mice, compared to sham tissue. LIPO-IM- HA significantly reduced fibrosis and collagen deposition, as shown by the Masson' s trichrome soluble collagen assay (Figure 7) .
Fibrosis analysis: in order to assess fibrosis, immunohistochemical analysis for TGF-lp was also carried out. Vehicle-treated mice showed increased expression of TGF-lp, whereas LIPO-IM-HA significantly reduced its expression (Figure 8A) .
Immunohistochemical localization of CD4 : twentyeight days after bleomycin instillation, vehicle-treated animals showed increased positive CD4 expression, compared to sham animals. In animals treated with LIPO- IM-HA, CD4 expression was reduced (Figure 9A) .
Immunohistochemical localization of CD8 : twentyeight days after bleomycin instillation, vehicle-treated animals showed increased positive expression of CD8, compared to sham animals. In animals treated with LIPO- IM-HA, CD8 expression was reduced ( Figure 10A) .
One or more embodiments of this disclosure may therefore involve liposomes coated with hyaluronic acid (HA) , wherein the hyaluronic acid has a molecular weight above 17000 Daltons , preferably between 20000 Daltons and 60000 Daltons .
In one or more embodiments , the l iposomes may have an average liposome diameter si ze between 100 nm and 300 nm, preferably 250 nm . In one or more embodiments , the liposomes may exhibit a Z potential value between - 10 mV and -50 mV, preferably -33 mV .
Liposomes may have HA covalently bound to the head of a phospholipid of the lipid bilayer of the liposomal membrane . In one or more embodiments , the HA is bound to an amine group of the head of a membrane phospholipid .
In one or more embodiments , liposomes may comprise at least one active ingredient , preferably a protein kinase enzyme inhibitor, most preferably selected from the group consisting of imatinib, nintedanib and other derivatives belonging to the tyrosine kinase inhibitor class .
The results of in vi vo experiments therefore show that the administration of LIPO- IM-HA reduces the decrease in body weight and the increase in pro- inflammatory cells in the bronchoalveolar lavage fluid . Furthermore , treatment with LIPO- IM-HA reduces bleomycin-induced histological damage and collagen deposition . Immunohistochemical analyses demonstrate reduced expression of TGF-0 , CD-4 , CD- 8 in animals treated with LIPO- IM-HA compared to the CTR group . Western blot analyses show reduced activation of the Nf- kb pathway, MAPKs , MMPs and TRAF- 6 . In the bleomycin- induced fibrosis model , LIPO- IM-HA when admini stered intratracheally is able to exert a potent anti fibrotic ef fect that is superior to the free drug either administered intratracheally or systemically ( intraperitoneally) . Such ef fect is mediated by an important anti-inflammatory function that is revealed by numerous analyses of broncho-alveolar lavage and the expression of pro-inflammatory and pro- fibrotic factors in the tissue of treated animals .
In one or more embodiments , the disclosure provides a composition comprising the disclosed liposomes . Such composition may further comprise at least one pharmaceutically acceptable vehicle . In one or more embodiments , the composition may further comprise diluents and/or excipients , also for inhalation administration .
In one or more embodiments , the liposomes and/or the composition comprising them may be intended for use in medicine , pre ferably for inhalation administration, more preferably for use in the treatment of at least one respiratory disease , even more preferably at least one pulmonary fibrotic disease , for example selected from idiopathic pulmonary fibrosis , pulmonary fibrosis associated with collagen diseases , bronchiolitis obliterans .
In one or more embodiments , the disclosure includes a procedure for the production of liposomes to carry at least one active ingredient comprising the steps of : solubili zing at least one phospholipid in a solvent , preferably a halogenated solvent , and obtaining a first solution,
- evaporating the solvent from said first solution and obtaining a lipid film,
- hydrating the lipid film in a solution containing at least one salt , preferably selected from sodium salt , potassium salt , ammonium salt and obtain a second solution, subj ecting the second solution to extrusion and obtaining a suspension comprising liposomes .
In one or more embodiments , the solvent may be selected from the group consisting of chloroform, methanol , ethanol , DCM, and mixtures thereof .
In one or more embodiments , extrusion may be conducted using an extruder, preferably at a temperature between 20 ° C and 60 ° C, more preferably 40 ° C .
In one or more embodiments , the method for producing liposomes may further comprise the step of adding at least one active ingredient to the suspension comprising liposomes to obtain liposomes comprising an active ingredient . Preferably, the active ingredient may be added to the suspension comprising the liposomes in a concentration between 0 . 1 mg/ 100 pl and 1 mg/ 100 pl preferably 0 . 5 mg/ 100 pl .
In one or more embodiments , the method for producing liposomes may provide that HA, preferably having a molecular weight greater than 17000 Da, bound to at least one phospholipid is added in the step of hydrating the lipid film . Such HA bound to at least one phospholipid may be obtained by the steps of :
- solubili zing HA, preferably having a molecular weight above 17000 Da, in a solvent including at least one halogenated solvent to obtain a solution of solubili zed HA,
- adding to the solubili zed HA solution at least one phospholipid, preferably comprising an amine group, plus preferably DPPE , and obtaining a mixture comprising HA and at least one phospholipid, preferably comprising an amine group,
- adding to this mixture comprising HA and at least one phospholipid at least one reducing agent to obtain a solution comprising HA bound to at least one phospholipid .
In one or more embodiments , the disclosure provides a method for obtaining HA, preferably having a molecular weight greater than 17000 Da, bound to at least one phospholipid, comprising the steps of : - solubili zing HA in a solvent comprising at least one halogenated solvent to obtain a solution of solubili zed HA, - adding to the solution of solubili zed HA at least one phospholipid, preferably comprising an amine group, preferably DPPE , and obtaining a mixture comprising HA and at least one phospholipid, adding to said mixture comprising HA and at least one phospholipid at least one reducing agent to obtain a solution comprising HA bound to the at least one phospholipid .
Without prej udice to the basic principle , the implementation details and the embodiments may vary, even signi ficantly, with respect to what is illustrated here purely by way of non-limiting example , without thereby departing from the scope of protection .
This scope of protection is defined by the attached claims .

Claims

- 34 - CLAIMS
1 . Liposomes coated with hyaluronic acid (HA) , wherein HA has a molecular weight between 20000 Daltons and 60000 Daltons .
2 . Liposomes according to claim 1 , wherein the average diameter of the liposomes is between 100 nm and 300 nm, preferably is equal to 250 nm .
3. Liposomes according to claim 1 or claim 2 , wherein the Z potential value is comprised between - 10 mV and - 50 mV, preferably is -33 mV .
4 . Liposomes according to any one of the preceding claims , wherein HA is bound to the head of a phospholipid of the phospholipidic bilayer of the liposomal membrane .
5 . Liposomes according to any one of the preceding claims , wherein HA is linked to an amino group of the head of a membrane phospholipid .
6. Liposomes according to any one of the preceding claims , comprising at least one active principle .
7 . Liposomes according to claim 6 , wherein the active principle is an inhibitor of protein kinase enzymes , preferably an inhibitor of tyrosine kinase enzymes , more preferably selected in the group consisting of imatinib, nintedanib and other derivatives of the class of the tyrosine kinase enzyme inhibitor .
8 . Composition comprising liposomes according to any one of claims 1 to 7 .
9. Liposomes according to any one of claims 1 to 7 or composition according to claim 8 , for use in medicine .
10 . Liposomes or composition for use according to claim 9 , for a delivery through inhalatory administration .
11 . Liposomes according to any one of claims 1 to 7 or composition according to claim 8 , for use in the treatment of at least one respiratory disease , - 35 - preferably at least one pulmonary fibrotic disease, more preferably at least one pulmonary fibrotic disease selected from idiopathic pulmonary fibrosis and pulmonary fibrosis with an inflammatory basis, pulmonary fibrosis associated with collagen diseases, bronchiolitis obliterans.
12. Method for producing liposomes comprising the steps of:
- solubilizing at least one phospholipid in at least one halogenated solvent, and obtaining a first solution,
- evaporating the solvent from said first solution and obtaining a lipid film,
- hydrating the lipid film in a solution comprising at least one salt, preferably selected from sodium salt, potassium salt, ammonium salt and obtaining a second solution,
- subjecting the second solution to extrusion and obtaining a suspension comprising liposomes.
13. Method according to claim 12, wherein the solvent is selected in the group consisting of chloroform, methanol, ethanol, methylene chloride, mixtures thereof.
14. Method according to any one of claims 12 or 13, wherein the extrusion is carried out by using an extruder, preferably at a temperature between 20°C and 60°C, more preferably equal to 40°C.
15. Method according to any one of claims 12 to 14, wherein the method further comprises the step of adding to said suspension comprising liposomes at least one active principle and obtaining liposomes comprising at least one an active principle.
16. Method according to claim 15, wherein the active principle is added to the suspension comprising the liposomes in a concentration ranging from 0.1 mg/100 pl to 1 mg/100 pl, preferably equal to 0.5 mg/100 pl.
17. Method according to any one of claims 12 to 16, wherein HA is added in said solution compris ing at least one salt in said step of hydrating the lipid film .
18 . Method according to claim 17 , wherein HA added in said hydrating step has a molecular weight higher than 17000 Daltons and is bonded to at least one phospholipid in the form of conj ugate (HA- f ) .
19. Method according to any one o f claims 17 or 18 , wherein HA, preferably having a molecular weight higher than 17000 Da, bound to at least one phospholipid is obtained by the steps of : solubili zing HA, preferably having a molecular weight higher than 17000 Da, in a solvent comprising at least one halogenated solvent to obtain a solution of solubili zed HA,
- adding to the solution of solubili zed HA at least one phospholipid, preferably comprising an amino group, more preferably DPPE , and obtaining a mixture comprising HA and at least one phospholipid, preferably comprising an amino group,
- adding to said mixture comprising HA and at least one phospholipid at least one reducing agent to obtain a solution comprising HA bonded to at least one phospholipid .
20 . Method for obtaining HA, preferably with a molecular weight higher than 17000 Da, bonded to at least one phospholipid, which comprises the steps of :
- solubili zing HA in a solvent comprising at least one halogenated solvent to obtain a solubili zed HA solution,
- adding to the solubili zed HA solution at least one phospholipid, preferably comprising an amino group, preferably DPPE , and obtaining a mixture comprising HA and at least one phospholipid,
- adding to said mixture compri sing HA and at least one phospholipid at least one reducing agent to obtain a solution comprising HA bonded to the at least one phospholipid .
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Citations (1)

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