Composition and method for making lipid nano-carriers and the corresponding lipid nano-carriers for biomedical use

Abstract

The invention concerns a composition for making lipid nano-carriers, which have an aqueous phase and a lipid phase, as well as a method for making the lipid nano- carriers in which the composition is used. The lipid nano-carriers thus obtained are also provided.

Description

[0001] “COMPOSITION AND METHOD FOR MAKING LIPID NANO-CARRIERS AND THE CORRESPONDING LIPID NANO-CARRIERS FOR BIOMEDICAL USE”

[0002] FIELD OF THE INVENTION

[0003] The present invention concerns a composition for making lipid nano-carriers, a method for making lipid nano-carriers and the lipid nano-carriers thus obtained. In particular, the lipid nano-carriers are to be made using microfluidics.

[0004] BACKGROUND OF THE INVENTION

[0005] The impressive advances in technology of recent decades has made it possible to identify new genetic alterations and cellular transduction mechanisms underlying oncological and hematological diseases, not only expanding our knowledge on the pathogenic mechanisms of neoplastic diseases, but also defining new molecular targets that can potentially be targeted pharmacologically.

[0006] Designing a new drug is not always sufficient, because very often the molecular target is not easily accessible, such as for proteins within the cell cytoplasm or plasma membrane for example, or in order to modify the gene sequence within the nucleus. Also, very often the drugs are rapidly eliminated from the bloodstream or degraded, significantly reducing their clinical activity and residence time in the human body.

[0007] For these reasons, it is not only necessary to develop new specific molecular target drugs so that they are selectively directed toward tumor cells or other target cells, and reduce toxicity to healthy cells, but it is also essential to improve the way the drug is administered and delivered to the target cell so as to increase what is known as its half-life, that is, the residence time of the drug in circulation, and bioavailability, that is, the biologically active part of the drug.

[0008] In fact, a new drug needs to be protected from rapid degradation and elimination, and transported to the right place (target tissue) in order to work. In recent years, numerous new approaches to drug administration have been developed and others are being tested; many of these approaches provide the delivery of the drug to the target site using a “carrier” that works like a taxi, carrying the single drug to the place of interest where it is released, rather than as a bus that can carry different molecules and to different places. Some recent drug delivery technologies provide to encapsulate, that is, introduce into nanometric carriers, different active substance of interest, allowing to overcome the problems typically associated with their free delivery, such as low bioavailability, short half-life, onset of side effects and lack of a specific target. In fact, in this way, the molecules of interest travel in the bloodstream to the target cells, protected inside droplets (either lipid or of a different nature) that act as a shield against degradation and reduce the elimination of the drug in free form.

[0009] Among the methodologies for the production of lipid nano-carriers (LNCs), one of the most widespread is the microfluidic methodology. The microfluidic system consists in mixing two phases, typically an organic one and an aqueous one which, through a fast process, leads to the formation of an emulsion of very small lipid droplets with the aqueous component, that is, the LNCs, trapped inside. Microfluidic technology is not part of the present invention, since it has already been extensively studied and discussed in scientific and patent literature for the production of different nanoparticles, such as for example in the article “Translating the fabrication of protein-loaded poly(lactic-co-glycolic acid) nanoparticles from bench to scale-independent production using microfluidics” published by Roces Carla B et al. in DRUG DELIVERY AND TRANSLATIONAL RESEARCH, SPRINGER, vol. 10, no. 3, p. 582-593 (XP037127775). This technology is in fact particularly effective, and the LNCs obtained at the end of the process appear homogeneous as a granulometric distribution with a low polydispersity index, often consisting of single layer unilamellar vesicles (SUVs), and have a precise surface charge useful for their stability and delivery. In addition, lipid-based nanometric delivery systems are often well tolerated after in vivo administration, showing good biocompatibility.

[0010] Despite this, once in circulation, in some cases the LNCs are recognized as elements foreign to the body by the immune system’s cells, which are activated to eliminate them, mainly through phagocytosis. LNC phagocytosis can be controlled and / or prevented through the functionalization of the vector surfaces through the insertion of molecules and proteins extracted from the immune system’s cells. In this way, the LNCs have greater bioavailability, staying in circulation longer, or greater targeting efficiency.

[0011] In fact, the presence of self -molecules on the surface of the vectors, as binding molecules (for example, membrane proteins), allows to target the LNCs onto the cells whose membranes present the corresponding receptors. The interaction between these molecules makes the administration of the LNC site-specific, the LNC being able, once bound to the target cell, to release its contents inside the cell, avoiding possible side effects on cells of no interest.

[0012] To date, several formulations have been developed for the production of nanocarriers functionalized with proteins extracted from human cells, such as red blood cells, platelets and white blood cells, through multiple manufacturing technologies; these nanosystems show good tolerability from in vitro and in vivo studies on animal models, while there are still few applications in the clinical field, aimed at a small group of diseases. Furthermore, the main objective of current formulations is to deliver drugs to the target cells or, in the case of mRNA vaccines for example, to deliver small portions of viral or tumor mRNA to induce an immunological response against infecting agents or tumor cells. In particular, in the latter case, the mRNA delivery is aimed only at the production of portions of immunogenic but non-infectious viral or tumor proteins, without altering gene expression in any way. This allows to reprogram a cell deficient for a specific protein or for several proteins, or to induce the expression of proteins ad hoc which allow to recognize tumor cells. In this way, the cells would be reprogrammed while preserving their gene heritage, reducing all the risks related to the modification of the human genome with current technologies that exploit viral vectors, transposomes, or enzymes of bacterial origin. In addition, the in situ delivery of proteins would allow the cells to be engineered in vivo, without resorting to in vitro expansion and subsequent re-infusion, and would allow costs to be reduced and the methodology to be expanded to a broader cohort of patients.

[0013] In fact, one of the main problems of cell therapies that recognize tumor cells and kill them is patient-specificity. For example, known cell therapies with engineered lymphocytes provide to modify the lymphocyte genome that begins to express a receptor capable of recognizing the tumor cell and then killing it. This process provides harvesting the patient’s lymphocytes, engineering with traditional methods (viral, transposomes, or Crispr), in vitro expansion of the engineered cells, and re-infusion into the patient.

[0014] This process can fail in several points due to lack of engineering and cell expansion, but an important limitation is that this process, which resembles a hematopoietic stem cell transplant, has to necessarily provide the infusion of lymphocytes that are HLA-compatible with the recipient, to prevent the engineered lymphocytes from recognizing not only the tumor but also the peripheral tissues as foreign, causing damage (for example, see those processes that go under the name of graft versus host disease (GvHD)), or transplant disease toward the host. For this reason, it is the patient’s lymphocytes that are directly engineered, to eliminate the HLA (human leukocyte antigens) incompatibility variable. There is therefore the need to perfect a formulation and a system for the release of substances that can overcome at least one of the disadvantages of the state of the art.

[0015] For this reason, it is necessary to solve the technical problem of providing LNCs that can release proteins and / or gene material toward healthy or tumor cells, and that at the same time can be used with any patient. In fact, patents US-A1- 2022 / 378700 and AU-A4-2020103700 disclose already known solutions that, however, only partly solve the aforementioned problems, such as formulations capable of carrying mRNA molecules, encoding specific chimeric proteins or synthetic exosomes consisting of biopolymers and lipids, respectively.

[0016] One purpose of the present invention is to make a composition for the microfluidic production of LNCs that allow to release human and / or engineered proteins toward healthy and tumor cells.

[0017] Another purpose of the present invention is to make a composition for the microfluidic production of patient non-specific LNCs without immunogenic proteins.

[0018] The Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.

[0019] SUMMARY OF THE INVENTION

[0020] The present invention is set forth and characterized in the independent claims. The dependent claims describe other characteristics of the present invention or variants to the main inventive idea.

[0021] In accordance with the above purposes and to resolve the technical problem described above in a new and original way, also achieving considerable advantages compared to the state of the prior art, a composition according to the present invention for the microfluidic production of lipid nano-carriers LNCs comprises a lipid phase and an aqueous phase.

[0022] In accordance with one aspect of the present invention, the lipid phase comprises at least one lipid chosen from phosphatidylcholine-based lipids, cationic and / or ionizable lipids, PEGylated lipids and mixtures thereof. Preferably, the aqueous phase comprises at least one protein and / or at least one peptide. The presence of nucleic acid can also be provided.

[0023] PEGylated lipids are understood to be lipids functionalized with a polyethylene glycol PEG. More precisely, they are modified lipids obtained by functionalizing the PEG polymer, in particular on the hydrocarbon terminal of the phospholipid.

[0024] In accordance with some embodiments, the aqueous phase comprises, in addition to the at least one protein and / or at least one peptide, at least one lipid.

[0025] Preferably, the at least one lipid of the lipid phase is chosen from DPPC, DSPC, DOPC, DOPE, DOTAP and their derivatives and mixtures of two or more of them, or from ALC-0315, or ALC-0159 and their derivatives and mixtures of two or more of them, or lipids PEGylated with PEG2000 and their derivatives.

[0026] In the context of the present patent application, derivatives are understood to be compounds that have the same chemical structure to which a functional group is added. This achieves the advantage of producing LNCs of liposomal and / or micellar nature capable of carrying proteins and / or peptides, and / or possibly nucleic acids in a predefined tissue of the human body. Phosphatidylcholine-based and / or ionizable and / or cationic lipids allow to carry proteins and / or peptides, while cationic lipids allow to carry nucleic acids.

[0027] According to some embodiments, the lipid phase comprises, in addition to the above mentioned at least one lipid, at least a second lipid chosen from PEG2000, DOTMA, PC, SOY PC, PG, ALC0159, lipid C12-200, CKK-E12, ALC-0315, NBD-PS and their derivatives, or lipids added with PEG-2000.

[0028] In accordance with some embodiments, the composition is free of HLA-class I molecules, so as to be patient non-specific.

[0029] Advantageously, the aqueous phase comprises a mix of proteins and / or peptides and / or amino acids and a mix of lipids. Preferably, the one or more proteins of the aqueous phase are of the type extracted from a membrane, in particular from a cell membrane, or of a recombinant nature. According to some embodiments, the proteins: lipids weight ratio in the aqueous phase is comprised between 1:0.1 and 1:200, preferably between 1 :1 and 1:100. Advantageously, the N / P ratio between the nucleic acid: lip id charges is comprised between 1 :0.1 and 1 :20, more preferably between 1:1 and 1:10. In the case of the N / P ratio (i.e., nitrogen / phosphate), it is instead the ratio between the positive charges (ammonium ion functional group -NH4+) and the negative charges (phosphate functional group -PHO4 ) of the lipid molecules and nucleic acids, respectively.

[0030] Advantageously, the molar ratio between two lipids in the lipid phase is comprised between 1:0.1 and 1 :5, preferably between 1:0.5 and 1:2.

[0031] In accordance with some embodiments, the lipid phase also comprises the polyethylene glycol PEG. The molar ratio between PEG and the at least one lipid is preferably comprised between 1:0.1 and 1:50, more preferably between 1:0.5 and 1:33, even more preferably between 1 :0.5 and 1 :20.

[0032] According to some embodiments, the lipid phase also comprises cholesterol. The molar ratio between cholesterol and the at least one lipid is comprised between 1:0.1 and 1 :5, preferably between 1 :0.5 and 1:3.

[0033] In accordance with another aspect of the present invention, lipid nano-carriers are provided with a composition as disclosed above, formulated as vesicles in which the aqueous phase is encapsulated in the lipid phase.

[0034] According to some embodiments, the lipid nano-carriers are free of HLA-class I molecules, so as to eliminate their patient-specificity.

[0035] In accordance with another aspect of the present invention, there is provided a method for making lipid nano-carriers comprising the steps of making available a composition as disclosed above and of mixing the lipid phase and the aqueous phase, preferably by means of a microfluidic circuit, in particular to achieve a controlled nanoprecipitation in laminar flow regime in order to create vesicles in which the aqueous phase is mixed in the lipid phase. A process of nanoprecipitation is thus obtained that is subsequently controlled by the principles of thermodynamics which allows the formation of LNCs.

[0036] According to some embodiments, the mixing step is carried out by means of a microfluidic system. Preferably, the microfluidic system comprises at least one pump per phase and a microchip equipped with a circuit with channels smaller than 1mm in diameter, preferably comprised between 300pm and 800pm in diameter. Advantageously, the circuit is Y-shaped, that is, it comprises an inlet channel for the lipid phase and an inlet channel for the aqueous phase that join in a laminar flow regime and with an appropriate flow rate and flow ratio in a single outlet channel for the vesicles. More preferably, the circuit has a total length of at least 5 cm, even more preferably of up to 10 cm. The total length of the circuit is understood as the total length of the channels that compose it.

[0037] In accordance with some embodiments, the volume ratio between the lipid phase and the aqueous phase is comprised between 1 : 1 and 1 :20, preferably between 1 :2 and 1 :10.

[0038] In accordance with some embodiments, the lipid phase and the aqueous phase are fed at a flow rate of at least 2 mL / min, for example 4 mL / min, 5 mL / min, 6 mL / min, 7 mL / min, 8 mL / min, 9 mL / min or 10 mL / min.

[0039] According to some embodiments, the method comprises a step of removing the HLA-class I molecules. This removal step, which occurs before the mixing step, can be performed by means of immunoprecipitation, for example using a dedicated kit.

[0040] DESCRIPTION OF THE DRAWINGS

[0041] These and other aspects, characteristics and advantages of the present invention will become apparent from the following description of some embodiments, given as a non-restrictive example with reference to the attached drawings wherein:

[0042] - figs. 1A, IB and 1 C represent the characterization of lipid nano-carriers according to the present invention, using DLS, Nanosight and transmission electron microscopy, respectively;

[0043] - fig. 2 A is a confocal microscopy image showing the uptake of lipid nano-carriers by peripheral blood mononuclear cells with co-localization of the proteins transported by the nano-carriers;

[0044] - fig. 2B shows different graphs representative for the expression of CD3 on cells treated (w) or not treated (w / o) with the lipid nano-carriers according to the invention, at different times;

[0045] - fig. 3 shows graphs obtained using flow cytometry which represent the expression of proteins on the lipid nano-carriers according to the invention using Flaer-FITC and HLA-DR-SN780; - fig. 4A shows the granulometry of the nano-carriers loaded with RNA and with DOTMA; and

[0046] - fig. 4B shows the cytotoxicity of the lipid nano-carriers according to the invention at different concentrations, using MTT assay on CHO (Chinese Hamster Ovary) cells;

[0047] - figs, from 5A to 5D show the potential of LNCs without the HLA complex, the main immunogenic antigens.

[0048] We must clarify that the phraseology and terminology used in the present description, as well as the figures in the attached drawings also in relation as to how described, have the sole function of better illustrating and explaining the present invention, their purpose being to provide a non-limiting example of the invention itself, since the scope of protection is defined by the claims.

[0049] To facilitate comprehension, the same reference numbers have been used, where possible, to identify identical common elements in the drawings. It is understood that elements and characteristics of one embodiment can be conveniently combined or incorporated into other embodiments without further clarifications. DESCRIPTION OF SOME EMBODIMENTS OF THE PRESENT INVENTION

[0050] Unless otherwise defined, all the technical and scientific terms used here and hereafter have the same meaning as commonly understood by a person with ordinary experience in the field of the art to which the present invention belongs. Even if methods and materials similar or equivalent to those described here can be used in practice and in the trials of the present invention, the methods and materials are described hereafter as an example. In the event of conflict, the present application shall prevail, including its definitions. The materials, methods and examples have a purely illustrative purpose and shall not be understood restrictively.

[0051] All measurements are carried out at 25°C (ambient temperature) and at atmospheric pressure, unless otherwise indicated, with the exception of cell cultures that require specific conditions in the incubator. All temperatures are in degrees Celsius, unless otherwise indicated.

[0052] All percentages and ratios indicated shall be understood to refer to the weight of the total composition (w / w), unless otherwise indicated.

[0053] All percentage ranges indicated here are given with the provision that the sum with respect the overall composition is 100%, unless otherwise indicated.

[0054] All the ranges reported here shall be understood to include the extremes, including those that report a range “between” two values, unless otherwise indicated.

[0055] The present description also includes the ranges that derive from uniting or overlapping two or more ranges described, unless otherwise indicated.

[0056] The present description also includes the ranges that can derive from the combination of two or more punctual values described, unless otherwise indicated.

[0057] Several compositions for the microfluidic production of LNCs for delivery of human and / or engineered proteins and gene material to healthy human cells and human tumor cells have been optimized. The use of LNCs in which the HLA-class I molecules have been removed through immunoprecipitation also allows to eliminate the patient-specificity of the vesicles, thus allowing them to be produced on a large scale and be used in a large cohort of patients, eliminating the need for HLA compatibility to reduce the risk of GvHD and adverse reactions to a minimum.

[0058] The applicability of this system has been verified both in the oncological field and in the hematological field, as well as in the field of protein delivery, to instruct some specific target cells, and / or in the field of nucleic acid delivery. In a more strictly hematological field, the system has been tested so as to engineer cells so that they express a whole set of deficient proteins without modifying the gene pool.

[0059] Paroxysmal nocturnal hemoglobinuria (PNH), a benign disease that falls into the broader group of marrow insufficiencies which also includes acquired aplastic anemia (AA), myelodysplastic syndromes (MDS) and large granular lymphocyte leukemia, was chosen as the pathology to verify the applicability of the proteindelivery of this formulation.

[0060] In particular, in PNH, somatic mutations of the X-linked gene PIGA (phosphatidylinositol glycan class A), involved in the biosynthesis of the lipid anchor glycosylphosphatidylinositol (GPI), cause the deficiency of the lipid anchor in the membrane. Normally, the GPI anchor allows, in fact, the anchoring in the membrane of a series of proteins that do not carry the sequence to attach to the membrane. The absence of the GPI tail prevents the correct positioning of these proteins, called GPI-anchored, which, therefore, are absent in patients with PNH. Various mutations result in varying degrees of GPI deficiency, up to the most severe cases where the cells completely lack the entire set of the GPI-anchored proteins, including the complement regulatory proteins CD55 and CD59. Therefore, the GPI-deficient cells are more sensitive to complement-mediated cell lysis, which is the main pathogenic event of PNH-correlated symptoms (anemia, hemoglobinuria and thrombosis). However, to date, the only pharmacologically active therapy targeted for the treatment of patients with PNH is complement inhibition, reducing its activation rate. Therefore, current pharmacological therapies tend to modulate the end effect and not modify the triggering event.

[0061] Another area in which the feasibility and applicability of the system has been verified is the delivery of membrane proteins to target cells in order to induce the expression of previously absent proteins, including specific proteins to recognize tumor antigens. These scenarios see the clinical use of LNCs loaded with HLA- class I deprived human white blood cell proteins to make them patient-specific, so that they can be used on a large scale. The presence of selected proteins on the surface of the nanovectors allows them to be targeted directly on the cells of interest, such as for example lymphocytes, tumor cells, and / or healthy cells with protein deficiency, in order to restore normal protein expression (as in the case of GPI-anchored proteins in PNH), or to induce the expression of chimeric receptors for tumor cell recognition, or to induce direct membrane exposure of the proteins of interest.

[0062] EXAMPLE 1 : LNCs to carry transmembrane proteins

[0063] Several formulations were developed and optimized for the production of protein- functionalized LNCs. The organic phase of the formulations was developed by trying to reproduce the amphipathic nature of the cell membrane; therefore, phosphatidylcholine-based lipids were mainly used, with or without net positive or negative charge or ionizable, conjugated with fluorescent molecules for in vitro / in vivo traceability.

[0064] The lipid phase comprises at least one lipid chosen from DPPC, DSPC, DOPC, DOPE, DOTAP and mixtures thereof. The lipid phase can also comprise, in addition, another lipid chosen from PEG2000, DOTMA, PC, SOY PC, PG, ALC0159, DPPC, lipid Cl 2-200, or CKK-E12. The system was also added with cholesterol and structured with or without the addition of poly-ethylene glycol (PEG).

[0065] An example of a lipid formulation of LNCs is: DPPC:DOPC:Cholesterol:Liss- Rhod-PE (1:1:1 :0.5), in which Liss-Rhod-PE is a derivative of DOPE. For the aqueous phase, several membrane-extracted protein: lipid ratios (range, 1-100) were evaluated.

[0066] Proteins were extracted from peripheral blood cells from healthy donors (authorization by the “Campania Sud” Ethics Committee, prot. / SCCE No. 249), isolated by means of Ficoll-Paque density gradient centrifugation; subsequently, the membrane proteins were isolated using the ProteoExtract Native Membrane Protein Extraction Kit and quantified using the Bradford test.

[0067] The mixing of the organic and aqueous phase was carried out using a microfluidic system, equipped with two pumps per phase that allows the solutions to be mixed using a microchip with a circuit with a total length of 8 cm, with a diameter of 600 pm (Y-shaped) under sterile conditions. In this case, the tested organic-aqueous phase ratio was 1 :2, while the total flow rate was kept constant at

[0068] 4 mL / min.

[0069] The formulations were examined at the morphological level using electron microscopy, and in terms of granulometry and surface charge using dynamic light scattering (DLS), showing a high homogeneity of the formulation and negatively charged surface that allows an optimal interaction with positively charged cell membranes, suggesting a high capacity for interaction and internalization of the particles formed (fig. 1).

[0070] Each production trial allowed to obtain a highly concentrated solution of LNCs, with on average a concentration of particles higher than one billion per mL. The morphology of the LNCs was analyzed using transmission electron microscopy (TEM) and the vesicles always proved to have a single unilamellar layer structure.

[0071] At a functional level, the LNCs were analyzed to verify protein and nucleic acid encapsulation efficiency. For protein quantification, no signal was detected from the assays carried out on the external dialysis water, suggesting the complete encapsulation of the proteins in the phospholipid layer, with an efficiency of 100% encapsulation.

[0072] In addition, confirmation of the successful in-membrane uptake was demonstrated using co-localization analysis in confocal microscopy and immunophenotyping by means of flow cytometry (fig. 2, in which the right panel shows the uptake and co-localization of the proteins carried by the LNCs (2 A); the left panel shows the expression of surface proteins by cells treated with LNCs (2B)).

[0073] Confocal microscopy showed the presence of the protein-loaded nano-carriers within the cellular cytoplasm and accumulation of specific proteins in the vesicle localization zone. In addition, the confirmation of the LNC uptake by the cells was also verified using immunophenotyping with flow cytometry.

[0074] To this end, the phospholipid composition of the LNCs was modified so as to obtain fluorescent particles conjugated with rhodamine, so that they could be traced. From the cytofluorimetric analysis, the peripheral blood mononuclear cells successfully incorporate the proteolipid vesicles, with the emergence of a positive population for rhodamine-labeled vesicles.

[0075] In addition, the cells that incorporated the LNCs exhibit increased expression of numerous surface markers, such as CD 14, CD33, CD3, and HLA-DR, including the GPI-anchored proteins (identified with the GP1 tail marker, Flaer) (fig. 3), in a time-dependent manner. This internalization is efficient both in the mononuclear cells from peripheral blood of a healthy subject, and also from PNH patients. The treatment with proteolipid LNCs improved resistance to complement-mediated cell lysis, both in healthy subjects and also in PNH patients, pathologically deficient in the endogenous complement inhibitors CD55 and CD59.

[0076] EXAMPLE 2: Preparation of LNCs with HLA-Class free proteins

[0077] Several formulations were developed and optimized for the production of LNCs functionalized with proteins obtained from engineered cells.

[0078] The organic phase of the formulations was developed trying to reproduce the amphipathic nature of the cell membrane. Different formulations were developed and optimized using one or more of the following phospholipids: DOPE; DOTAP; DOTMA; PC; DSPC; DOPC; SOY PC; PG; ALC0159; DPPC; lipid C12-200; or CKK-E12 with or without the addition of polyethylene glycol (PEG).

[0079] For the aqueous phase, several proteins: lipids ratios (from 1:1 to 1 : 100) were evaluated, extracting membrane proteins in toto. In addition, the HLA-class I molecules, which determine the patient-specificity of the formulations, were removed by means of immunoprecipitation using the Dynabeads™ Protein G Immunoprecipitation kit.

[0080] The mixing of the organic and aqueous phase was carried out using a microfluidic system, equipped with two pumps per phase that allows the solutions to be mixed using a microchip with a circuit with a total length of 10 cm, with a diameter of 600 pm (Y-shaped) under sterile conditions. Different volume ratios between the organic-aqueous phases were tested (from 1:2 to 1:10), as well as the total flow rate (from 4 mL / min, 6 mL / min and 8 mL / min).

[0081] Granulometric investigations revealed that HLA-class I free LNCs had a granulometry similar to that of LNCs with HLA proteins, and the absence of the HLA-class I complex was then confirmed through cytofluorimetry investigations and then with confocal microscopy following cellular uptake of HLA-class I free LNCs, as shown in figs. 5A-5D.

[0082] EXAMPLE 3: Preparation of LNCs with cell-specific proteins

[0083] Several formulations were developed and optimized for the production of LNCs functionalized with specific proteins to make the uptake of LNCs selective for a cell type.

[0084] The organic phase of the formulations was developed trying to reproduce the amphipathic nature of the cell membrane. Different formulations were developed and optimized using one or more of the following phospholipids: DOPE; DOTAP; DOTMA; PC; DSPC; DOPC; SOY PC; PG; ALC0159; DPPC; lipid Cl 2-200; or CKK-E12 with or without the addition of polyethylene glycol (PEG).

[0085] For the aqueous phase, several proteins: lipids weight ratios were evaluated (from 1:1 to 1 :100), where the proteins of interest were extracted from cells.

[0086] For the encapsulation of the nucleic acids, the nucleic acids: lipids ratio was considered to be the N / P ratio between charges.

[0087] The mixing of the organic and aqueous phase was carried out using a microfluidic system, equipped with two pumps per phase that allows the solutions to be mixed using a microchip with a circuit with a total length of 10 cm, with a diameter of 600 pm (Y and S shaped) under sterile conditions. Different volume ratios between the organic-aqueous phases were tested (from 1:2 to 1:10), as well as the total flow rate (from 4 mL / min, 6 mL / min and 8 mL / min). The HLA-class I free LNCs showed similar granulometry to the LNCs loaded with generic membrane proteins; a uniform morphological structure always of single unilamellar vesicles (SUV) and surface charge.

[0088] The HLA-class I free LNCs then showed good cell uptake and an absence of toxicity up to 0.5 mg / mL.

[0089] EXAMPLE 4: Preparation of LNCs with cell-specific proteins and pharmacologically or biologically active molecules

[0090] Several formulations were developed and optimized for the production of LNCs functionalized with specific proteins to make the uptake of LNCs selective for a cell type and pharmacologically or biologically active molecules for intracellular direct release, in order to increase the selectivity and specificity of the molecule’s action and to reduce its degradation and elimination because protected by the lipid envelope.

[0091] The organic phase of the formulations was developed trying to reproduce the amphipathic nature of the cell membrane. Different formulations were developed and optimized using one or more of the following phospholipids: DOPE; DOTAP; DOTMA; PC; DSPC; DOPC; SOY PC; PG; ALC0159; DPPC; lipid C 12-200; or CKK-E12 with or without the addition of polyethylene glycol (PEG).

[0092] For the aqueous phase, several proteinsdipids ratios were evaluated (from 1:1 to 1 :100), where the proteins of interest were extracted from cells.

[0093] Mixing of the organic and aqueous phase was performed using a microfluidic system under sterile conditions. Different volume ratios between the organic- aqueous phases were tested (from 1 :2 to 1:10), as well as the total flow rate (from 4 mL / min, 6 mL / min and 8 mL / min).

[0094] It is clear that modifications and / or additions of parts may be made to the composition, to the method and to the lipid nano-carriers as described heretofore, without departing from the field and scope of the present invention, as defined by the claims.

[0095] It is also clear that, although the present invention has been described with reference to some specific examples, a person of skill in the art will be able to achieve other equivalent forms of composition and method for making lipid nanocarriers and the corresponding lipid nano-carriers, having the characteristics as set forth in the claims and hence all coming within the field of protection defined thereby.

[0096] In the following claims, the sole purpose of the references in brackets is to facilitate their reading and they must not be considered as restrictive factors with regard to the field of protection defined by the claims.

Claims

CLAIMS1. Composition for making lipid nano-carriers, comprising a lipid phase and an aqueous phase, characterized in that said lipid phase comprises at least one lipid chosen from phosphatidylcholine-based lipids, cationic and / or ionizable lipids, PEGylated lipids and mixtures thereof, and in that said aqueous phase comprises at least one protein and / or at least one peptide.

2. Composition as in claim 1, characterized in that said liquid phase also contains at least one lipid.

3. Composition as in claim 1 or 2, characterized in that said at least one lipid of said lipid phase is chosen from DPPC, DSPC, DOPC, DOPE, DOTAP, ALC-0315, ALC-0159, PEG-2000, their derivatives and mixtures of two or more thereof.

4. Composition as in claim 3, characterized in that said lipid phase comprises, in addition to said at least one lipid, at least a second lipid chosen from PEG2000, DOTMA, PC, SOY PC, PG, ALC0159, lipid Cl 2-200, or CKK-E12.

5. Composition as in any of the previous claims, characterized in that said at least one protein is of the type extracted from a cell membrane, or from engineered cells.

6. Lipid Nano-carrier, characterized in that it has a composition as in one or more of the previous claims, and it is formulated as a vesicle in which said aqueous phase is encapsulated in said lipid phase.

7. Lipid Nano-carrier as in claim 6, characterized in that it is free of HLA-class I molecule.

8. Lipid Nano-carrier as in claim 6, characterized in that it transports human surface proteins.

9. Method for making Lipid Nano-carriers, characterized in that it provides to make available a composition as in any of the claims 1 to 5, and in that it provides to mix said lipid phase and said aqueous phase in order to form vesicles in which said aqueous phase is encapsulated in said lipid phase.

10. Method as in claim 9, characterized in that said mixing step is carried out by means of a microfluidic system.

11. Method as in claim 9, characterized in that said microfluidic system comprises at least one pump for each of said lipid phase and said aqueous phase, and a microchip equipped with a circuit with channels smaller than 1mm in diameter, preferably comprised between 300pm and 800pm in diameter.

12. Method as in any of the claims 9 to 11, characterized in that it provides, before the mixing step, a step of removal of HLA-class I molecules, preferably by immunoprecipitation.

13. Method as in any of the claims 10 to 12, characterized in that it provides a nanoprecipitation in laminar flow regime to create vesicles in which said aqueous phase is mixed in said lipid phase.

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