Specific tissue-targeted lipid nanoparticles, compositions and applications thereof

Abstract

The present invention relates to nanoparticles capable of crossing biological barriers. In particular, it refers to lipid nanoparticles configured to cross the blood-brain barrier, target skeletal tissues and effectively penetrate the skin barrier. The invention also encompasses compositions and applications thereof.

Description

[0001] SPECIFIC TISSUE-TARGETED LIPID NANOPARTICLES, COMPOSITIONS AND APPLICATIONS THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to nanoparticles capable of crossing biological barriers. In particular, it refers to lipid nanoparticles configured to cross the blood-brain barrier, target skeletal tissues and effectively penetrate the skin barrier. The invention also encompasses compositions and applications thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] The development of more efficient drugs is currently experiencing significant growth. Among the various approaches being explored, advancements in drug delivery systems are one of the most heavily researched and exploited fields.

[0006] Nanoparticles have become a significant tool in drug delivery systems, particularly for their ability to encapsulate therapeutic agents and deliver them to specific tissues or cells within the mammalian organism. In particular, lipid nanoparticles (LNPs), mainly represented by liposomes and extracellular vesicles, have gained considerable attention due to their biocompatibility, biodegradability, and capability to deliver a wide range of molecules, including small drugs, peptides, and nucleic acids, and its ability to reach less available tissues and organs.

[0007] However, despite advancements in nanoparticle technology, challenges remain in effectively targeting certain tissues and biological barriers. One major obstacle in drug delivery is the bloodbrain barrier (BBB), a highly selective membrane that protects the central nervous system (CNS) by preventing potentially harmful substances from entering the brain. Many therapeutic agents struggle to cross the BBB, thus limiting treatment options for neurological conditions such as brain cancer, neurodegenerative diseases, and other disorders affecting the CNS.

[0008] Numerous studies have consistently indicated a possible connection between schizophrenia and BBB dysfunction. A physical alteration of the BBB has been observed in humans with symptoms of the disease (Mahmud, S.Z.L., et al (2022). Investigation of Blood-Brain Barrier Disruption in Schizophrenia using Magnetization Transfer-ASL at 7T. Annual meeting ISMRM-ESMRMB). This could be due to the presence of inflammatory cytokines, such as tumor necrosis factor a (TNF- a), IL-6 and other chemokines, which could damage endothelial microvascularity (Yarlagadda, A., et al (2010). The blood brain barrier and the role of ratiometric molecular analysis in schizophrenia. Psychiatry (Edgmont) 7, 20-23; Pan, HZ, et al (2003). Interactions of cytokines with the blood-brain barrier: implications for feeding. Curr Pharm Des 9, 827-831. 10.2174 / 1381612033455332; Schwarz, M.J., et al (1998). Blood-cerebrospinal fluid barrier impairment as indicator for an immune process in schizophrenia. Neurosci Lett 253, 201-203. 10.1016 / s0304-3940(98)00655-7). The complex dysfunction of the blood-brain barrier (BBB) in psychosis, particularly in schizophrenia, is emphasized, suggesting possible consequences such as alteration of neuronal and synaptic function, an increase in the permeability to inflammatory molecules and a reduction in the efficacy of drugs in antipsychotic treatments (Pollak, T.A. et al (2018). The blood-brain barrier in psychosis. Lancet Psychiatry 5, 79-92. 10.1016 / S2215- 0366(17)30293-6).

[0009] Extracellular vesicles (EVs) are nanometer-sized particles characterized by the presence of a lipid bilayer structure that encapsulates proteins, nucleic acids and other metabolites (Van Niel, G. et al (2018). Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol 19, 213-228; Witwer, K.W., et al. (2021). Updating MISEV: Evolving the minimal requirements for studies of extracellular vesicles. J Extracell Vesicles 10, e12182). These play various roles in physiology and pathology, such as autocrine and paracrine communication (Raposo, G., et al (2019). Extracellular vesicles: a new communication paradigm? Nat Rev Mol Cell Biol 20, 509- 510; Thery, C. et al. (2018). Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 7, 1535750). Furthermore, they have a high potential to be adapted as new generation nanocarriers. It is widely known that EVs can cross the blood-brain barrier (BBB). The BBB is composed of closely organized endothelial cells that line the cerebral blood vessels and is accompanied by other cell types such as astrocytes and pericytes. The main function of the BBB is to regulate the passage of nutrients, oxygen and other molecules, such as drugs or pathogens, between the blood and brain tissue. This selective permeability helps maintain the brain microenvironment and protects it from fluctuations in the bloodstream that could upset its delicate balance (Zhou, W., et al (2023). Bidirectional Communication Between the Brain and Other Organs: The Role of Extracellular Vesicles. Cell Mol Neurobiol, 1-22; Daneman, R. et al (2015). The blood-brain barrier. Cold Spring Harb Perspect Biol 7, a020412; Sweeney, M.D., et al (2019). Blood-Brain Barrier: From Physiology to Disease and Back. Physiol Rev 99, 21-78. 10.1152 / physrev.00050.2017). The literature has reported that EVs are crucial in the interaction of the brain with other tissues such as heart, liver, pancreas or lungs (Wang, D. et al. (2023). Extracellular vesicles: Critical bilateral communicators in periphery-brain crosstalk in central nervous system disorders. Biomed Pharmacother 160, 114354). Furthermore, EVs have been implicated in the pathology of neurodegenerative diseases (Andras, I.E., and Toborek, M. (2016). Extracellular vesicles of the blood-brain barrier. Tissue Barriers 4, e1131804). They are known to contribute to intercellular transmission of diseases such as Alzheimer's (Goetzl, E.J., et al (2016). Decreased synaptic proteins in neuronal exosomes of frontotemporal dementia and Alzheimer's disease. Faseb J 30, 4141-4148; Mullins, R.J. et al (2017). Exosomal Biomarkers of Brain Insulin Resistance Associated With Regional Atrophy in Alzheimer's Disease. Hum Brain Mapp 38, 1933-1940); Parkinson's (Valencia, J., et al. (2022). The Potential Roles of Extracellular Vesicles as Biomarkers for Parkinson's Disease: A Systematic Review. International Journal of Molecular Sciences 23. ARTN 11508) or amyotrophic lateral sclerosis (ALS) (Gagliardi, D., et al (2021). Extracellular vesicles and amyotrophic lateral sclerosis: from misfolded protein vehicles to promising clinical biomarkers. Cell Mol Life Sci 78, 561-572. 10.1007 / S00018-020-03619-3; Barbo, M., et al (2023). Extracellular Vesicles as Potential Biomarkers in Amyotrophic Lateral Sclerosis. Genes-Basel 14. ARTN 325

[0010] 10.3390 / genes14020325), although they could also have neuroprotective roles (Takeuchi, T. (2021). Pathogenic and protective roles of extracellular vesicles in neurodegenerative diseases. J Biochem 169, 181-186. 10.1093 / jb / mvaa131). In addition, they are emerging as potential biomarkers in psychotic disorders. For example, microRNAs (miRNAs) related to schizophrenia have been found encapsulated in circulating EVs (Wei, H. et al. (2015). Detection of circulating miRNA levels in schizophrenia. Am J Psychiatry 172, 1141-1147.

[0011] 10.1176 / appi.ajp.2015.14030273).

[0012] Liposomes, a type of extracellular vesicle, have shown significant potential as drug delivery systems due to their ability to encapsulate a wide range of compounds, including hydrophobic and hydrophilic cargoes (Foad, Rommasi et al. (2021). Liposomal Nanomedicine: Applications for Drug Delivery in Cancer Therapy.. Nanoscale Research Letters, doi: 10.1186 / S11671-021- 03553-8; Nikita, R., et al (2020). Liposomes: A Novel Drug Delivery System: An Overview. Asian Journal of Pharmaceutical Research, 10(1):23-28. doi: 10.5958 / 2231-5691.2020.00005.2). These lipid-based nanocarriers offer several advantages, such as a improved bioavailability of drugs, reduced systemic side effects and improved penetration of the cargo into target tissues. Furthermore, the integration of imaging and therapeutic capabilities into liposome-based drug carriers has been proposed, leading to the development of multifunctional nanoplatforms (Yibo, Liu., et al (2021). Targeted liposomal drug delivery: a nanoscience and biophysical perspective.. 6(2):78-94. doi: 10.1039 / D0NH00605J).

[0013] The mechanisms behind how EVs cross the BBB are not yet fully understood. Most hypothetical pathways are known to facilitate communication between peripheral tissues and the nervous system (Matsumoto, J., et al (2017). The Transport Mechanism of Extracellular Vesicles at the Blood-Brain Barrier. Curr Pharm Des 23, 6206-6214). It has been suggested that EVs could be potential biomarkers to evaluate the state of the BBB and as mediators to aid immune neuroinvasion (Ramirez, S.H., et al (2018). Extracellular vesicles: mediators and biomarkers of pathology along CNS barriers. Fluids Barriers CNS 15, 19). Furthermore, several components of the BBB, such as endothelial cells or pericytes, can influence the biogenesis and functionality of these EVs depending on their state and microenvironment (Saint-Pol, J., et al (2020). Targeting and Crossing the Blood-Brain Barrier with Extracellular Vesicles. Cells 9). The specific mechanisms of EV uptake are mainly regulated by their lipid composition, glycoproteins and proteins (Mulcahy, L.A., et al (2014). Routes and mechanisms of extracellular vesicle uptake. J Extracell Vesicles 3. 10.3402 / jev.v3.24641).

[0014] In addition to the BBB, delivering therapeutic agents specifically to skeletal tissues also poses significant difficulties. The targeted delivery to bones and muscles is critical for the treatment of conditions such as osteoporosis, arthritis, and muscular dystrophy. Current delivery systems often exhibit poor specificity for these tissues, leading to suboptimal therapeutic outcomes and systemic side effects.

[0015] Thus, the clinical significance of improving drug targeting to the skeleton for conditions like bone fragility and osteoporosis is underscored by the need for treatments that can specifically address the deteriorating bone architecture and enhance bone strength while minimizing side effects (Luo, G., et al (2024). Enhancing osteoporosis treatment using a targeted, sustained-release drug delivery system based on macrocyclic amphiphile. International Journal of Pharmaceutics, 661, 124457; Pop, P. A., Lazar, L., & Marcu, F. M. (2013). Significance of Kinetotherapy in Rehabilitation Treatment of Osteoporosis, american society of mechanical engineers). Current therapeutic strategies are often hampered by non-specific drug targeting, leading to undesirable skeletal side effects and limited efficacy {Luo et al., 2024). Novel drug delivery systems, such as hydroxyapatite-responsive carriers and nanotechnology-based approaches, have been developed to target bone tissue more effectively, ensuring controlled release and enhanced drug stability {Chen, J., et al (2020). Nanocomposites drug delivery systems for the healing of bone fractures. International Journal of Pharmaceutics, 585, 119477; Luo et al., 2024; Chen, J., et al (2020). Nanocomposites drug delivery systems for the healing of bone fractures. International Journal of Pharmaceutics, 585, 119477).

[0016] Interestingly, while hydroxyapatite serves as a general targeting moiety for the skeletal system, the use of bisphosphonates, polymeric oligopeptides, and nanoparticles has been explored to improve targeting methods for osteoporosis and other skeletal diseases {Sawamoto, K., et al (2020). Bone-Specific Drug Delivery for Osteoporosis and Rare Skeletal Disorders. Current Osteoporosis Reports, 18(5), 515-525). These advancements in bone-targeting strategies are crucial given the complexity of bone architecture and the challenge of delivering drugs to specific bone regions, such as avascular cartilage lesions {Chen et al., 2020; Sawamoto et al., 2020).

[0017] Moreover, transdermal drug delivery, which involves administering drugs through the skin, faces limitations due to the skin's natural barrier properties. The outermost layer of the skin, the stratum corneum, provides a highly impermeable barrier, preventing many drugs from penetrating effectively into deeper tissues, particularly hydrophilic and large molecules {Badilli, U., et al (2018). Chapter 9 - Lipid-based nanoparticles for dermal drug delivery. In Organic Materials as Smart Nanocarriers for Drug Delivery (pp. 369-413). elsevier; Medi, B. M. et al (2017). Electroporation for Dermal and Transdermal Drug Delivery (pp. 105-122). While some advancements have been made in enhancing transdermal delivery, many formulations still face poor absorption rates, limited drug bioavailability, and the need for frequent dosing. Overcoming this barrier is essential to fully exploit the potential of transdermal and topical therapies.

[0018] Traditional chemical enhancers can increase skin permeability but often cause irritation and show limited effectiveness with larger molecules. In contrast, natural compounds and advanced delivery systems, such as lipid-based nanoparticles, offer safer and more effective alternatives for enhancing drug absorption through the skin {Raghav, R. S. eta! (2024). A Comprehensive Review on Potential Chemical and Herbal Permeation Enhancers Used in Transdermal Drug Delivery Systems. Recent Advances in Drug Delivery and Formulation, 18(1), 21-34; Badilli et al., 2018). Nanoparticles, such as solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), and polymeric nanocarriers, improve drug stability, enable controlled release, and facilitate penetration through the skin barrier (Khan, S. U., et al (2024). Nanotherapeutic approaches for transdermal drug delivery systems and their biomedical applications. European Polymer Journal, 207, 112819.).

[0019] Despite their potential, nanoparticles may still struggle to fully overcome the stratum corneum. For example, raloxifene nanoparticles required the addition of permeation enhancers like menthol to effectively penetrate the skin, indicating that nanoparticles alone may not always suffice (Otake, H., & Nagai, N. (2024). Development of Transdermal Formulation Based on Nanotechnology and Elucidation of Its Drug Delivery Pathways. Yakugaku Zasshi : Journal of the Pharmaceutical Society of Japan, 144(5), 505-510.).

[0020] In summary, while nanoparticles significantly enhance transdermal drug delivery, the effectiveness of these systems often depends on additional strategies to overcome the skin’s natural barriers. Further innovation in both nanoparticle technology and permeation-enhancing methods remains crucial for optimal therapeutic outcomes (Chen, Y., & Feng, X. (2022). Gold nanoparticles for skin drug delivery. International Journal of Pharmaceutics, 625, 122122.; Khan et al., 2024).

[0021] The authors of the present invention has carried out a huge experimental effort to reach those molecules, present in the composition of EVs, responsible for the aforementioned specific tissue targeting capabilities.

[0022] As a result of that experimental work, they have identified a shared set of 12 lipids as the main drivers of EVs that confer the capacity of crossing the BBB and reaching brain tissues, as well as specifically targeting skeletal tissues and trespassing the skin barrier.

[0023] Thanks to these important findings, similar structures and other nanocarriers can be advantageously edited in order to increase the efficiency of their arrival in these tissues. The present invention thus addresses the limitations and needs of the state of the art by providing lipid nanoparticles configured to cross the blood-brain barrier, target skeletal tissues, and effectively penetrate the skin barrier. The nanoparticles feature a unique lipid composition that surprisingly enhances their ability to traverse these biological barriers, making them highly suitable for the targeted delivery of therapeutic agents. In addition to their improved targeting capabilities, these nanoparticles offer potential applications in a variety of therapeutic contexts, ranging from neurological diseases to musculoskeletal disorders and transdermal drug delivery.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Sorting of EVs from mice brain tissues. (A) Control (brain cn 8 and brain cn 15) and DiD control (brain cd 12) dot plots of EV events. (B) Brain (B5 brain, B7 brain and B9 brain) dot plots of EV events. (C) Bar graph representation of the correspondent percentage of fluorescence positive particles for all conditions, previously lablled and in vivo administered. ***; p<0.001.

[0026] Figure 2: Pathway enrichment of EV unique lipids.

[0027] Figure 3: Sorting of EVs from mice skeletal tissues. (A) Dot plots of EV events detected in skeletal tissues of mice previously adiministered with EVs containing the nanoparticles composition of the invention. (B) Percentage of events of administered EVs of the invention that reach skeletal tissues in vivo after ex vivo evaluation.

[0028] Figure 4. Representative analysis of fluorescently labeled Minudik® vesicle penetration into skin. A. Confocal micrographs of the application site at 0 h and 1 h. Columns: left, DAPI (nuclei); center, red lipid-dye signal from Minudik® vesicles; right, merged channels with tissue context. Scale bar = 100 pm. B. Normalized distribution of the fluorescent signal across tissue layers (total skin, epidermis, dermis) at 0 h and 1 h, illustrating the progressive accumulation of vesicles within the epidermis and upper dermis over time.

[0029] Figure 5. Comparative in-vivo exposure of Minudik® vesicles after oral vs intravenous dosing. A. Grouped bar chart of fold-change vs baseline at 0, 2, 4, and 24 h for oral and intravenous (i.v.) administration. Both routes rise similarly from baseline at each time point (means ± error bars), indicating no route-specific advantage within the measured window. B. Exposure summary (AUC0h-2h-4h-24h) for oral and i.v. dosing. The integrated signals are comparable between routes, supporting no clear difference in overall exposure under the tested conditions. DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0030] It is noted that throughout the present disclosure the following lipids shall be refer as follows: a. Brassylic acid shall be refer to as lipid A or A. b. PC(38:4) shall be refer to as lipid B or B. c. Cer(d35:0) shall be refer to as lipid C or C. d. Myristic acid shall be refer to as lipid D or D. e. CE(22:5) shall be refer to as lipid E or E. f. DG36 shall be refer to as lipid F of F. g. DG34 shall be refer to as lipid G or G. h. PC(P-38:5) shall be refer to as lipid H or H, and i. DG44:2 shall be refer to as lipid I or I.

[0031] As used herein, lipid names follow LIPID MAPS conventions. For Table 1 , “Mass” refers to the observed m / z of the base adduct measured under the LC-HRMS conditions described for the methods; “RT” is the chromatographic retention time in minutes. Typical adducts are as follows: triacylglycerols, diacylglycerols, and cholesteryl esters as [M+NH4]+; phosphatidylcholines (diacyl and ether) as [M+H]+with a diagnostic m / z 184 phosphocholine fragment in MS / MS; ceramides as [M+H]+generating characteristic long-chain-base fragments; fatty acids as [M-H]“; and fatty amides / ethanolamides as [M+H]+. Unless otherwise noted, identifications in Table 1 are supported by accurate mass within <5 ppm error, class-consistent retention behavior, and classspecific MS / MS fragments. Where two names are shown for a single feature (e.g., palmitoylethanolamide / oleamide), the entry indicates isobaric or co-eluting species that are distinguished by MS / MS when resolved; if not fully resolved, both designations are reported. For clarity around specific entries in Table 1 (please note that the content of table 1 should prevail in case of any inconsistencies):

[0032] PC (38:4) — Phosphatidylcholine (total acyl carbons: 38; double bonds: 4)

[0033] Cer (d35:0) — Ceramide (with a dihydroxy sphingoid base; total carbons: 35; double bonds: 0) CE (22:5) — Cholesteryl ester of docosapentaenoic acid (cholesteryl docosapentaenoate) DG 36 — Diacylglycerol (total acyl carbons: 36) DG 34 — Diacylglycerol (total acyl carbons: 34)

[0034] PC (P-38:5) / PC (0-38:6) — Plasmenyl-phosphatidylcholine (PC-P, a plasmalogen, 38:5) / Plasmanyl-phosphatidylcholine (PC-O, an ether PC, 38:6)

[0035] DG 44:2 — Diacylglycerol (total acyl carbons: 44; double bonds: 2) palmitoylethanolamide — N-palmitoylethanolamine (PEA); IIIPAC: N-(2- hydroxyethyl)hexadecanamide

[0036] TG (47:0) — Triacylglycerol (total acyl carbons: 47; double bonds: 0) TG (50:3) — Triacylglycerol (total acyl carbons: 50; double bonds: 3) The present disclosure pertains to tissue-targeting lipid nanoparticles engineered to cross the blood-brain barrier (BBB) and / or to target skeletal tissues. As illustrated in the examples of the present specification, fluorescently labeled extracellular vesicles (EVs) capable of homing to brain and bone were isolated from the corresponding tissues by flow cytometry, with a mean of 1.23X 1011vesicle particles quantified per condition. Lipidomics by liquid chromatography-tandem mass spectrometry (LC-MS / MS) revealed a reproducible signature in which lipid A (Brassylic acid) is predominant at about fifty-four percent of the normalized lipid content, lipids B-E (respectively PC(38:4), Cer(d35:0), Myristic acid and CE(22:5) contribute intermediate amounts between five and fifteen percent each, and lipids F-l (respectively DG36, DG34, PC(P-38:5) and DG44:2) shall are minor constituents each below five percent on average; minimum and maximum values across biological replicates reflect natural variability while preserving the same predominance of A and the same relative ordering of secondary and minor contributors. Lipid nanoparticles configured to reproduce this signature, whether derived from synthetic sources, from natural sources, or from combinations thereof, display the ability to vehiculize payloads across the bloodbrain barrier and / or to localize to skeletal tissues, thereby enabling treatment of central nervous system (CNS) disorders and skeletal diseases. As used herein, the lipid nanoparticles can be derived from synthetic sources; from natural sources; or from hybrids of synthetic and natural sources whose final lipid distribution is compositional-adjusted to fall within the ranges defined below.

[0037] In one embodiment corresponding to an independent particle aspect, there is provided a lipid nanoparticle suitable to cross the blood-brain barrier and / or target skeletal tissues, comprising a lipid composition comprising at least two, three, four, five, six, seven, eight or nine of the following lipids: A, B, C, D, E, F, G, H and I, and optionally J (palmitoylethanolamide), K (Triacylglycerol (TG) (47:0) and L (TG (50:3); for use in the treatment of diseases related to dysfunctions of the central nervous system or of the skeletal system.

[0038] According to the present invention, the term “lipidic nanoparticle” refers to extracellular vesicles (EVs), liposomes, solid lipid nanoparticles (SLNs), and nanostructured lipid carriers (NLCs), which are notable for their ability to enhance drug solubility, bioavailability, and provide controlled and sustained release. The lipid matrix of these nanoparticles is composed of biocompatible and biodegradable lipids, which can be solid or a combination of solid and liquid lipids, as in the case of NLCs, providing high entrapment efficiency for active constituents. In particular, EVs are lipid- bilayer-bound particles that can contain a variety of bioactive molecules such as lipids and proteins. The lipid composition of EVs is reflective of their cellular origin and contributes to their membrane curvature, which is a key feature in their identification and targeting. The shape of lipidic nanoparticles can be spherical or more complex. The diameter of the lipid nanoparticle ranges from 30 to 950 nm. More preferably, the lipid nanoparticle has a diameter smaller than 200 nm. As indicated later on in the specificaiton, the lipid nanoparticle of the invention can be obtained from a) biological or natural sources, which may include eukaryotic cells such as immune cells, neurons, epithelial cells, and stem cells, as well as prokaryotic cells like bacteria. In mammals, these can be derived from bodily fluids such as blood, urine, saliva, tears, and cerebrospinal fluid, which demonstrates their presence in all physiological fluids. Moreover, these can be harvested from cell cultures where they are secreted by cultured cells under controlled laboratory conditions. Additionally, the lipid nanoparticles of the invention can be obtained from by-products of the fermented food industry, including those generated during the production of beer, wine, kombucha, and yogurt. Specifically, brewery spent yeast, a by-product of beer production, is a rich biological source for that end. Similarly, by-products from wine fermentation, such as yeast and other cellular debris, can also serve as valuable sources. In kombucha production, the symbiotic culture of bacteria and yeast (SCOBY) offers another potential source of lipid nanoparticles. Likewise, the fermentation process involved in yogurt production yields bacterial cultures and cellular materials that can be harvested and used as biological source.

[0039] However, it is important to note that not all vesicles derived from these biological sources will necessarily possess the specific lipid profile of the invention. In cases where the obtained vesicles do not exhibit the desired profile, they will be subjected to additional editing or modification to ensure they incorporate the required lipids. Alternatively, lipid nanoparticles of the invention can be obtained through b) either chemical or biological synthesis in the form of liposomes, through methods used in the state of the art. For example, using the high aspect ratio microfluidic vertical flow method. This method of nanoscale liposome synthesis employs microfluidic devices with a significant disparity between the height and width of the channels, which allows for precise control of fluid flow and the formation of liposomes with uniform size and low polydispersity. This technique leverages the vertical orientation of flow focusing to enhance the throughput of liposome production, thereby overcoming the limitations of traditional microfluidic methods that typically have low production rates (Han, J. Y. et al. (2023). Scalable Liposome Synthesis by High Aspect Ratio Microfluidic Flow Focusing. Methods in Molecular Biology (Clifton, N.J.), 2622, 87- 93. https: / / doi.org / 10.1007 / 978-1-0716-2954-3_7; Hood, R. R., & Devoe, D. L. (2015). High- Throughput Continuous Flow Production of Nanoscale Liposomes by Microfluidic Vertical Flow Focusing. Small, 11 (43), 5790-5799). Another widely used method for synthesizing lipid nanoparticles that has undergone recent optimization is ultrafast acoustofluidic micromixing. This method of nanoscale liposome synthesis involves the use of acoustic waves to rapidly mix fluids in microfluidic channels, facilitating the formation of liposomes with controlled size and distribution. This technique leverages the rapid convective and diffusive mixing induced by acoustic streaming to synthesize liposomes, which are spherical vesicles composed of lipid bilayers (Agha, A. et al (2024). Integration of acoustic micromixing with cyclic olefin copolymer microfluidics for enhanced lab-on-a-chip applications in nanoscale liposome synthesis. Biofabrication, 16(4), 045004; Zhao, S., et al. (2021). Fabrication of tunable, high-molecular- weight polymeric nanoparticles via ultrafast acoustofluidic micromixing. Lab on a Chip, 21 (12), 2453-2463). The addition of glycerol to the solvent in the acoustofluidic process significantly reduce liposome size (Pourabdollah Vardin, A., & Yesiloz, G. (2023). Nanoscale Liposome Synthesis for Drug Delivery Applications via Ultrafast Acoustofluidic Micromixing. Hittite Journal of Science and Engineering, 10(3), 237-241). The methods above are adapted to obtain lipid nanoparticles with the required lipids in their membrane or lipid bilayer. Subsequente editions of the nanoparticles thus obtained can be made to link the desired proteins or other molecules.

[0040] In another embodiment consistent with the foregoing, said lipid composition comprises at least elements A to I so that each of A, B, C, D, E, F, G, H and I is present. In a preferred embodiment aligned with the lipidomics data, the lipid nanoparticle comprises a lipid fraction characterized in that the lipid fraction comprises, preferably by percentages obtained from LC-MS / MS data and preferably normalized within each sample to total one hundred percent, each of elements A-l, and the lipid fraction simultaneously satisfies the following percentages by weight (wt%): A in a wt% between forty-five and sixty percent, B in a wt% between 8 and eighteen percent, C in a wt% between 2.4 and twelve percent, D in a wt% between 3.5 and twelve percent, E in a wt% between 2.4 and twelve percent, F in a wt% between zero-point-five and five percent, G in a wt% between zero-point-five and five percent, H in a wt% between zero-point-five and five percent, and I in a wt% between zero-point-five and five percent, wherein preferably the sum of elements A to I above is ninety-eight to one hundred percent on the same normalized basis. It is noted that the balance to 100 wt% can consist of trace components selected from impurities, residual solvents, water, salts, or unassigned lipids.

[0041] It is noted that as indicated in the present invention, a non limiting example for deriving the percentages obtained from LC-MS / MS data is presented in the examples. It is preferably noted that percentages obtained from LC-MS / MS data and preferably normalized within each sample to total one hundred percent, means intra-sample scaling of raw ion intensities preferably so that the total signal for identified lipids in a given sample equals one hundred percent (please refer to the examples). It is further noted that while other analytical techniques may be used to estimate composition (including internal-standard-corrected LC-MS / MS reported as mass percent or mole percent, shotgun lipidomics with isotope dilution reported as mole percent, high-performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD) or with charged aerosol detection (CAD), phosphorus-31 nuclear magnetic resonance (31P-NMR) headgroup analysis combined with mass-spectrometry subclass assignment, phosphate assay with mass- spectrometry subclassing, or thin-layer chromatography (TLC) densitometry with verified linear response), the much preferred method for calculating the weight percent (wt%) values recited herein is LC-MS / MS. In a further explicit embodiment, lipids other than elements A to I can be optionally present, preferably in a combined amount of zero to two percent on the same normalized basis, such as elements J to L.

[0042] In a preferred embodiment consistent with the predominance of A observed in tissue-targeting vesicles, element A is present in a wt% between fifty and fifty-eight percent. In another embodiment, element B is present in a wt% between ten and 18 percent In another preferred embodiment, element C is present in a wt% between 2.4 and ten percent. In another preferred embodiment, each of lipids D-E is present in a wt% between 2.4 and ten percent, preferably between five and 10 percent. In an additional preferred embodiment, each of lipids F-l is present in a wt% between one and four percent, preferably one-point-five to three-point-five or four percent. In grouped-fraction embodiments, the group sum B plus C plus D plus E is present in a wt% between 25 and forty-five percent, and the group sum F plus G plus H plus I is present in a wt% between eight and 13 percent.

[0043] In a preferred embodiment, the lipid nanoparticle is characterized by comprising a lipid fraction represented by reference normalized weight percentages A 53.81 , B 13.70, C 7.91 , D 7.02, E 6.68, F 3.33, G 3.24, H 2.20 and I 2.10, each entry being deemed met when the measured value lies within a selectable tolerance of plus / minus thirty percent, preferably plus / minus twenty percent, preferably plus / minus ten percent or preferably plus / minus five percent of the listed value; that is, for variations of ±30%, ±20%, ±10% or ±5% or less. In some embodiments, the lipid nanoparticle is thus characterized by a lipid fraction having component amounts that correspond to the values reported above within a tolerance selected from ±30%, ±20%, ±15%, ±10%, ±5%, or ±1 % of any of these values. As used herein, ‘±Y% of the value’ denotes a relative deviation around the value. For example, a value of 25 wt% with a tolerance of ±10% permits 22.5-27.5 wt%. Unless indicated otherwise, the component wt% values are expressed relative to the lipid fraction and the sum of the lipid components equals 100 wt% (±1 wt% due to rounding). Where a listed component is omitted or is below the limit of quantification, its amount is 0 wt% and the remaining lipid components constitute the balance to 100 wt%.

[0044] In an alternative embodiment, the lipid nanoparticle comprises a lipid fraction characterized by a ratio signature computed from percentages obtained from LC-MS / MS data and preferably normalized within each sample to total one hundred percent, wherein each of elements A-l is present, and the signature simultaneously satisfies all of the following: the ratio A to (B+C+D+E) is between 1.2:1 and 2.0:1 ; the ratio A to (F+G+H+l) is between 3.0:1 and 7.0:1 ; the ratio (B+C) to (D+E) is between 1.2:1 and 2.0:1 ; the ratio (B+F) to (D+E+G) is between 1.0:1 and 2.5:1 ; the ratio A to B is between 3.0:1 and 5.0:1 ; the ratio A to C is between 5.0:1 and 9.0:1 ; the ratio B to D is between 1.3:1 and 2.5:1 ; the ratio C to E is between 0.8:1 and 1.5:1 ; the ratio (B+E) to (C+D) is between 1.1 :1 and 1.8:1 ; the ratio (B+C+D+E) to (F+G+H+l) is between 2.0:1 and 5.0:1 ; the ratio F to G is between 0.7:1 and 1.4:1 ; and the ratio H to I is between 0.7:1 and 1.5:1. In a preferred embodiment, when B, F, D, E and G are present, they are present at a ratio [B+F] to [D+E+G] between 1.0:1 and 2.5:1 , thereby restating and supporting the (B+F):(D+E+G) relationship.

[0045] In a further embodiment, the lipid nanoparticle that satisfies any one of the quantitative embodiments described above also satisfies the ratio signature simultaneously, thereby providing an explicit basis that the particle meets both composition windows and ratio criteria in the same sample. In a further embodiment, the diameter of the nanoparticle is lower than 950 nm, preferably lower than two hundred nanometers, for example a number-average diameter less than two hundred nanometers as measured by nanoparticle tracking analysis (NTA) or dynamic light scattering (DLS).

[0046] In a further embodiment, the lipid nanoparticle further comprises at least one peptide of sequence selected from the list consisting of Sequence Identification Numbers (SEQ ID NO) 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. In a more specific embodiment, the peptide of sequence is SEQ ID NO 11 , and the peptide can be associated with different zones of the nanoparticle including surface- displayed, bilayer-inserted as a lipopeptide, luminally located within the aqueous interior, or interfacially anchored within the membrane.

[0047] In further embodiments applicable to all of the foregoing, the lipid nanoparticles can encapsulate, intercalate or otherwise carry nucleic acids including messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA) mimics, antisense oligonucleotides (ASO) and DNA plasmids; protein and enzyme cargos; peptides; small molecules; and diagnostic agents including fluorescent dyes and contrast agents for positron emission tomography (PET), magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT) and computed tomography (CT). The lipid nanoparticles of the present invention can be thus loaded with a wide range of molecules, including, but being not limited to, bioactive molecules, active compounds, excipients, etc. (those molecules include metabolites, peptides, proteins, lipids, RNAs, DNAs, organic and inorganic compounds, etc.) and their membrane can also be edited by the addition or deletion of different molecules (metabolites, peptides, proteins, lipids, RNAs, DNAs, organic and inorganic compounds, etc) and the incorporation of signaling-oriented proteinaceous, through multifaceted processes widely used in the state of the art, to enhance the delivery and targeting of therapeutic agents.

[0048] In a further independent aspect corresponding to a composition, there is provided a composition comprising a population of lipid nanoparticles according to any of the embodiments described above and optionally a pharmaceutically acceptable carrier, for use in the treatment of diseases related to dysfunctions of the central nervous system or of the skeletal system. In a further embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of nanoparticles in the population comply with the characterization required by any one of the particle embodiments, including the composition windows and / or the ratio signature and, where applicable, size criteria. In a further embodiment, the population comprises 1 xio10to 5*1012particles per dose, preferably about 1.23x1011particles.

[0049] In a further embodiment, the composition is provided in a dosage form selected from solution, suspension, lyophilized cake, or buffered infusion, optionally comprising cryoprotectants and / or stabilizers such as trehalose, sucrose, mannitol, antioxidants or chelators, with the formulation chosen such that the measured lipid fractions remain within the specified composition ranges or ratio signature upon storage and reconstitution. In a further embodiment, the nanoparticles in the composition have an average diameter of between thirty and nine hundred and fifty nanometers preferably with a diameter lower than two hundred nanometers as recited above.

[0050] In a further independent aspect corresponding to medical use, the lipid nanoparticle as defined in any of the particle embodiments or the composition as defined in any of the composition embodiments is for use in a method of treatment as a nanocarrier of molecules to the central nervous system, explicitly providing basis for CNS delivery. In a parallel medical-use embodiment, the lipid nanoparticle as defined in any of the particle embodiments or the composition as defined in any of the composition embodiments is for use in a method of treatment as a nanocarrier of molecules to the skeletal system, explicitly providing basis for skeletal delivery. In further embodiments, the lipid nanoparticle or composition is for use in a method of treatment of a disease selected from the group consisting of Parkinson’s disease, multiple sclerosis and glioblastoma, and, in a further embodiments, for use in a method of treatment of a disease selected from the group consisting of osteoporosis, osteoarthritis, bone cancer and fractures. These use embodiments are supported by the demonstrated ability of the particles to cross the blood-brain barrier and / or to target skeletal tissues and by the flow-cytometric recovery of fluorescently labeled EVs from brain and skeleton followed by LC-MS / MS analysis that established the characteristic lipid signature. Without being bound by theory, an A-dominant membrane accompanied by secondary contributions from B-E and minor contributions from F-l modulates bilayer curvature, rigidity and surface protein partitioning to enhance receptor- mediated or adsorptive-mediated transcytosis across brain endothelium and to promote affinity for bone microenvironments including hydroxyapatite and osteoblast or osteoclast membranes, thereby enabling the nanoparticles to vehiculize payloads to these sites.

[0051] In addition, it is further noted that as indicated in the examples of the present invention, the data presented herein collectively supports the hypothesis that Minudik® EVs, when applied topically, are capable of penetrating the stratum corneum and distributing within the viable epidermis and superficial dermis within one hour, while systemic absorption appears to be absent. In this sense, lipid nanoparticles or nanoparticle compositions of the present invention are further useful as effective drug delivery strategies that can penetrate the skin barrier, crucial for the treatment of skin diseases, certain cancers, and in advancing cosmetic formulations. The skin, being the body's largest organ, serves as a formidable barrier, making it challenging for therapeutic agents to reach deeper layers where they are most needed. In cancer treatment, particularly for skin cancers like melanoma, delivering chemotherapeutic agents directly through the skin can improve drug concentration at the tumor site while minimizing systemic side effects. For chronic skin diseases such as psoriasis and eczema, transdermal drug delivery offers a way to deliver treatments more effectively, reducing inflammation and promoting healing directly at the affected areas. In cosmetics, advanced transdermal delivery systems can enhance the penetration of active ingredients, leading to more effective anti-aging, moisturizing, and skin-repairing products. By overcoming the skin barrier, these drug delivery strategies offer more targeted, efficient, and less invasive treatment options, improving outcomes in both medical and cosmetic applications. Therefore, in a further embodiment of the invention corresponding to medical use, the lipid nanoparticle as defined in any of the particle embodiments or the composition as defined in any of the composition embodiments is for use in a method of treatment as a nanocarrier of molecules to the skin barrier, dermis and / or epidermis, explicitly providing basis for skin delivery. In particular, for the treatment or prevention of skin diseases, certain cancers, and in advancing cosmetic formulations. In cancer treatment, particularly for skin cancers like melanoma. For chronic skin diseases such as psoriasis and eczema. the lipid nanoparticle as defined in any of the particle embodiments or the composition as defined in any of the composition embodiments is for use in a method of treatment as a nanocarrier of molecules to the central nervous system, explicitly providing basis for CNS delivery. In a parallel medical-use embodiment, the lipid nanoparticle as defined in any of the particle embodiments or the composition as defined in any of the composition embodiments is for use in a method of treatment as a nanocarrier of molecules to the skeletal system, explicitly providing basis for skeletal delivery. In further embodiments, the lipid nanoparticle or composition is for use in a method of treatment of a disease selected from the group consisting of Parkinson’s disease, multiple sclerosis and glioblastoma, and, in a further embodiments, for use in a method of treatment of a disease selected from the group consisting of osteoporosis, osteoarthritis, bone cancer and fractures. These use embodiments are supported by the demonstrated ability of the particles to cross the blood-brain barrier and / or to target skeletal tissues and by the flowcytometric recovery of fluorescently labeled EVs from brain and skeleton followed by LC-MS / MS analysis that established the characteristic lipid signature. Without being bound by theory, an A- dominant membrane accompanied by secondary contributions from B-E and minor contributions from F-l modulates bi layer curvature, rigidity and surface protein partitioning to enhance receptor- mediated or adsorptive-mediated transcytosis across brain endothelium and to promote affinity for bone microenvironments including hydroxyapatite and osteoblast or osteoclast membranes, thereby enabling the nanoparticles to vehiculize payloads to these sites. In addition, it is further noted that as indicated in the examples of the present invention, the data presented herein collectively supports the hypothesis that Minudik® EVs, when applied topically, are capable of penetrating the stratum corneum and distributing within the viable epidermis and superficial dermis within one hour, while systemic absorption appears to be absent. In this sense, lipid nanoparticles or nanoparticle compositions of the present invention are further useful as effective drug delivery strategies that can penetrate the skin barrier, crucial for the treatment of skin diseases, certain cancers, and in advancing cosmetic formulations. The skin, being the body's largest organ, serves as a formidable barrier, making it challenging for therapeutic agents to reach deeper layers where they are most needed. In cancer treatment, particularly for skin cancers like melanoma, delivering chemotherapeutic agents directly through the skin can improve drug concentration at the tumor site while minimizing systemic side effects. For chronic skin diseases such as psoriasis and eczema, transdermal drug delivery offers a way to deliver treatments more effectively, reducing inflammation and promoting healing directly at the affected areas. In cosmetics, advanced transdermal delivery systems can enhance the penetration of active ingredients, leading to more effective anti-aging, moisturizing, and skin-repairing products. By overcoming the skin barrier, these drug delivery strategies offer more targeted, efficient, and less invasive treatment options, improving outcomes in both medical and cosmetic applications. Therefore, in a further embodiment of the invention corresponding to medical use, the lipid nanoparticle as defined in any of the particle embodiments or the composition as defined in any of the composition embodiments is for use in a method of treatment as a nanocarrier of molecules to the skin barrier, dermis and / or epidermis, explicitly providing basis for skin delivery. In particular, for the treatment or prevention of skin diseases, certain cancers, and in advancing cosmetic formulations. In cancer treatment, particularly for skin cancers like melanoma. For chronic skin diseases such as psoriasis and eczema.

[0052] In a further aspect, the lipid nanoparticle (LNP) as defined in any particle embodiment, or the composition as defined in any composition embodiment, is configured for administration to a subject via a route selected from oral, intravenous (i.v.), intramuscular, subcutaneous, intradermal, intrathecal, intranasal, inhalation, transdermal, buccal, sublingual, rectal, intraarticular, and topical administration. In a preferred embodiment, the LNP or composition is formulated for oral administration. In certain examples, the LNP comprises an enteric or pH- responsive coating and / or a mucus-penetrating surface (e.g., PEGylated or zwitterionic corona) to enhance gastrointestinal transit, protect cargo from gastric and intestinal enzymes, and promote trans-epithelial uptake.

[0053] In another embodiment, the LNP or composition is formulated for intravenous (i.v.) administration. In certain examples, the LNP includes a long-circulating surface (e.g., PEG), an optional targeting ligand (e.g., peptide or small molecule) and / or membrane-active helper lipids to promote endosomal escape after tissue uptake.

[0054] In further embodiments, the route of administration is combined with nanocarrier action to deliver a therapeutic, prophylactic, or diagnostic molecule selected from small molecules, peptides, proteins, nucleic acids (e.g., mRNA, siRNA, saRNA, ASO, plasmid DNA), CRISPR components, imaging agents, or combinations thereof. In some embodiments, the LNP provides (i) cargo protection from enzymatic degradation, (ii) enhanced epithelial or endothelial translocation, (iii) cell-specific uptake, and / or (iv) controlled or sustained release at the target site.

[0055] In an embodiment, the LNP as defined in any particle embodiment, or the composition as defined in any composition embodiment, is for use in a method of treatment as a nanocarrier of molecules to the central nervous system (CNS), thereby providing basis for CNS delivery. In certain embodiments, the LNP is administered via the oral or i.v route. In further embodiments, the LNP or composition is for use in a method of treatment of a CNS disease selected from Parkinson’s disease, multiple sclerosis, and glioblastoma.

[0056] In an embodiment, the LNP as defined in any particle embodiment, or the composition as defined in any composition embodiment, is for use in a method of treatment as a nanocarrier of molecules to the skeletal system, thereby providing basis for skeletal delivery. In certain embodiments, the LNP is administered via the oral or i.v route. In further embodiments, the LNP or composition is for use in a method of treatment of a skeletal disease selected from osteoporosis, osteoarthritis, bone cancer, and fractures.

[0057] In an embodiment, the LNP as defined in any particle embodiment, or the composition as defined in any composition embodiment, is for use in a method of treatment as a nanocarrier of molecules to the skin barrier, thereby providing basis for skin delivery. In certain embodiments, the LNP is administered via the oral or i.v route. It is further noted that production of the lipid nanoparticles is not limited by any specific process and can employ any suitable method known in the art for assembling lipid vesicles or extracellular- vesicle-mimetic particles; the invention is expressly not limited to any particular manufacturing apparatus, order of addition, or parameter set, provided that the resulting particles meet the composition ranges and / or ratio signature and size specifications recited above. Across all embodiments in which percentages are recited, LC-MS / MS with intra-sample scaling to a total of one hundred percent per sample is the much preferred method for calculating wt% values, with alternative analytical techniques considered acceptable when they provide corresponding values.

[0058] DETAILED DESCRIPTION OF THE INVENTION

[0059] There is an unmet medical need for an effective drug delivery system, which can cross biological barriers and transport active components to brain, skeletal and skin tissues.

[0060] It is thus an object of the present invention to address those needs and to provide nanoparticles suitable as carrier systems capable not only of reaching the BBB, but also targeting skeletal tissues, and tresspassing the skin barrier for the treatment of diseases related to dysfunctions of the central nervous system, skeletal system and skin diseases.

[0061] In the present invention, this object has been solved by providing a set of twelve lipids as the main drivers of nanoparticles to reach the BBB, targeting skeletal tissues, and tresspassing the skin barrier.

[0062] Therefore, in a first aspect, the present invention refers to a specific tissue-targeted lipid nanoparticle, configured to cross the blood-brain barrier, target skeletal tissues, and penetrate the skin barrier, comprising at least two of the following lipids in its composition:

[0063] - brassylic acid,

[0064] - myristic acid,

[0065] - palmitoylethanolamide (PEA),

[0066] - Diacylglycerol (DG) (36:0),

[0067] - DG (34:0),

[0068] - DG (44:2), - Triacylglycerol (TG) (47:0),

[0069] - TG (50:3),

[0070] - Phosphatyldylcholine (PC) (P-38:5)) / PC (0-38:6),

[0071] - PC (38:4)

[0072] - Ceramide (Cer) (d35:0) and

[0073] - Cholesterol ester (CE) (22:5).

[0074] Some of these lipids have shown relationships with EVs release and activity, such as myristic acid in hepatic cell lines, especially these have been related to EVs at the lipid family level (Speziali, G. et al. (2018). Myristic acid induces proteomic and secretomic changes associated with steatosis, cytoskeleton remodeling, endoplasmic reticulum stress, protein turnover and exosome release in HepG2 cells. J Proteomics 181, 118-130). However, their association at the isoform level is identified for the first time in this study. Unsaturated lipids in EVs have also an influence in membrane fluidity and signalisation, for example in endothelial cells (Sudha, Srivastava., et al (1983). Fluidity, Permeability And Antioxidant Behavior Of Model Membranes Incorporated With Alpha-Tocopherol And Vita mi n-E Acetate) or some highly invasive tumor cells, due to their membrane conductivity (Giusy Di Conza et al (2021). Tumor-induced reshuffling of lipid composition on the endoplasmic reticulum membrane sustains macrophage survival and pro- tumorigenic activity. Nature Immunology, doi: 10.1038 / S41590-021-01047-4).

[0075] The specific lipid profile of the nanoparticles of the present invention contributes to their excellent improved biocompatibility and bioavailability properties.

[0076] In a particular embodiment, the lipid nanoparticle comprises at least the combination of two lipids selected from brassylic acid and myristic acid, or DG (36:0) and DG (34:0) or TG (47:0) and CE (22:5).

[0077] In another particular embodiment, the lipid nanoparticle comprises at least the combination of three lipids selected from DG (34:0), TG (50:3) and PC (P-38:5) / PC (0-38:6), or DG (44:2), PC (38:4) and Cer (d35:0) or TG (47:0), TG (50:3) and CE (22:5). In a preferred embodiment, the lipid nanoparticle comprises the combination of the twelve lipids listed above (brassylic acid, myristic acid, PEA, DG (36:0), DG (34:0), DG (44:2), TG (47:0), TG (50:3), (PC) (P-38:5)) / PC (0-38:6), PC (38:4), Cer (d35:0) and CE (22:5)).

[0078] According to the present invention, the term “I ipidic nanoparticle” refers to extracellular vesicles (EVs), liposomes, solid lipid nanoparticles (SLNs), and nanostructured lipid carriers (NLCs), which are notable for their ability to enhance drug solubility, bioavailability, and provide controlled and sustained release. The lipid matrix of these nanoparticles is composed of biocompatible and biodegradable lipids, which can be solid or a combination of solid and liquid lipids, as in the case of NLCs, providing high entrapment efficiency for active constituents. In particular, EVs are lipid- bilayer-bound particles that can contain a variety of bioactive molecules such as lipids and proteins. The lipid composition of EVs is reflective of their cellular origin and contributes to their membrane curvature, which is a key feature in their identification and targeting. The shape of lipidic nanoparticles can be spherical or more complex. The diameter of the lipid nanoparticle ranges from 30 to 950 nm. More preferably, the lipid nanoparticle has a diameter smaller than 200 nm.

[0079] The lipid nanoparticle of the invention can be obtained from a) biological or natural sources, which may include eukaryotic cells such as immune cells, neurons, epithelial cells, and stem cells, as well as prokaryotic cells like bacteria. In mammals, these can be derived from bodily fluids such as blood, urine, saliva, tears, and cerebrospinal fluid, which demonstrates their presence in all physiological fluids. Moreover, these can be harvested from cell cultures where they are secreted by cultured cells under controlled laboratory conditions. Additionally, the lipid nanoparticles of the invention can be obtained from by-products of the fermented food industry, including those generated during the production of beer, wine, kombucha, and yogurt. Specifically, brewery spent yeast, a by-product of beer production, is a rich biological source for that end. Similarly, byproducts from wine fermentation, such as yeast and other cellular debris, can also serve as valuable sources. In kombucha production, the symbiotic culture of bacteria and yeast (SCOBY) offers another potential source of lipid nanoparticles. Likewise, the fermentation process involved in yogurt production yields bacterial cultures and cellular materials that can be harvested and used as biological source. However, it is important to note that not all vesicles derived from these biological sources will necessarily possess the specific lipid profile of the invention. In cases where the obtained vesicles do not exhibit the desired profile, they will be subjected to additional editing or modification to ensure they incorporate the required lipids.

[0080] Alternatively, lipid nanoparticles of the invention can be obtained through b) either chemical or biological synthesis in the form of liposomes, through methods used in the state of the art. For example, using the high aspect ratio microfluidic vertical flow method. This method of nanoscale liposome synthesis employs microfluidic devices with a significant disparity between the height and width of the channels, which allows for precise control of fluid flow and the formation of liposomes with uniform size and low polydispersity. This technique leverages the vertical orientation of flow focusing to enhance the throughput of liposome production, thereby overcoming the limitations of traditional microfluidic methods that typically have low production rates (Han, J. Y. et al. (2023). Scalable Liposome Synthesis by High Aspect Ratio Microfluidic Flow Focusing. Methods in Molecular Biology (Clifton, N.J.), 2622, 87-93. https: / / doi.org / 10.1007 / 978-1-0716-2954-3_7; Hood, R. R., & Devoe, D. L. (2015). High- Throughput Continuous Flow Production of Nanoscale Liposomes by Microfluidic Vertical Flow Focusing. Small, 11(43), 5790-5799).

[0081] Another widely used method for synthesizing lipid nanoparticles that has undergone recent optimization is ultrafast acoustofluidic micromixing. This method of nanoscale liposome synthesis involves the use of acoustic waves to rapidly mix fluids in microfluidic channels, facilitating the formation of liposomes with controlled size and distribution. This technique leverages the rapid convective and diffusive mixing induced by acoustic streaming to synthesize liposomes, which are spherical vesicles composed of lipid bilayers (Agha, A. et al (2024). Integration of acoustic micromixing with cyclic olefin copolymer microfluidics for enhanced lab-on-a-chip applications in nanoscale liposome synthesis. Biofabrication, 16(4), 045004', Zhao, S., et al. (2021). Fabrication of tunable, high-molecular-weight polymeric nanoparticles via ultrafast acoustofluidic micromixing. Lab on a Chip, 21(12), 2453-2463). The addition of glycerol to the solvent in the acoustofluidic process significantly reduce liposome size (Pourabdollah Vardin, A., & Yesiloz, G. (2023). Nanoscale Liposome Synthesis for Drug Delivery Applications via Ultra fast Acoustofluidic Micromixing. Hittite Journal of Science and Engineering, 10(3), 237-241). The methods above are adapted to obtain lipid nanoparticles with the required lipids in their membrane or lipid bilayer. Subsequente editions of the nanoparticles thus obtained can be made to link the desired proteins or other molecules.

[0082] The lipid nanoparticles of the present invention can be loaded with a wide range of molecules, including, but being not limited to, bioactive molecules, active compounds, excipients, etc. (those molecules include metabolites, peptides, proteins, lipids, RNAs, DNAs, organic and inorganic compounds, etc.) and their membrane can also be edited by the addition or deletion of different molecules (metabolites, peptides, proteins, lipids, RNAs, DNAs, organic and inorganic compounds, etc) and the incorporation of signaling-oriented proteinaceous, through multifaceted processes widely used in the state of the art, to enhance the delivery and targeting of therapeutic agents.

[0083] Therefore, in a particular embodiment, the surface of the lipid nanoparticles of the invention can be functionalized with at least one peptide of sequence selected from SEQ ID NO 1 , SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5 (Malate dehydrogenase), SEQ ID NO 6, SEQ ID NO 7 (Dihydrolipoyldehydro genase), SEQ ID NO 8, SEQ ID NO 9 (EF-hand domain-containing protein), SEQ ID NO 10 (Serine / threonine-protein phosphatase), SEQ ID NO 11 (Enolase) and SEQ ID NO 12 (Peptidyl-prolyl cis-trans isomerase), enhancing targeting specificity of the lipid nanoparticles and delivery efficiency to target tissues.

[0084] Regarding the function of these peptides, Malate dehydrogenase is involved in glucose metabolism and the Krebs cycle (TCA) (Charles, S., et al (2024). 2. Malate dehydrogenase as a multi-purpose target for drug discovery. Essays in Biochemistry, doi: 10.1042 / ebc20230081). Dihydrolipoyl dehydrogenase is a mitochondrial enzyme involved in cellular respiration and implicated in oxidative stress processes (Stefan, Timm., et al (2024). 1. Mitochondrial Dihydrolipoamide Dehydrogenase (mtLPDI): Expression, Purification, Activity, and Redox Regulation, doi: 10. 1007 / 978-1 -0716-3802-6_5). EF-hand domain-containing protein is involved in the restructuring and regulation of cytoskeletal actin structure, providing stability to yeast 50. Serine / threonine phosphatases are broad mediators in cellular metabolism, growth, and cell cycle (Pieter, Vaneynde.et al (2022). The role of serine / threonine phosphatases in human development: Evidence from congenital disorders. Frontiers in Cell and Developmental Biology, doi: 10.3389 / fcell.2022. 1030119). Enolase is an enzyme involved in the reverse reaction of gluconeogenesis (Pei, Liang., et al (2021). Identification of an enolase gene and its physiological role in Spirometra mansoni.. Parasitology Research, doi: 10.1007 / S00436-021-07175-Y). Finally, peptidyl-prolyl cis-trans isomerase modifies the aminoacid folding (Rongmao, Qiu., et al (2023). Cis-trans isomerization of peptoid residues in the collagen triple-helix. Nature Communications, doi: 10. 1038 / S41467-023-43469-8).

[0085] The nanoparticles of the present invention display advantageous and compelling features as nanocarriers, nanovectors and / or nanocapsules for molecules delivery and / or encapsulation including low antigenicity, ability to trespass biological barriers, stable circulatory capacity and organ tropism. The nanoparticles can also be used by itself or after their molecular modification to enter endogenous routes of EVs in plant, animal and human cells (e.g. modulation of human cell autophagy mechanisms) in manifold applications within the novel bioeconomy and biomedical fields, e.g., food and / or food supplement industries for animal or human use, supplements or treatment for plants, biotech industries, pharmacy, cosmetic, etc.

[0086] Therefore, in another embodiment, the present invention refers to the use of the lipid nanoparticle of the invention as a nanocarrier, nanovector and / or nanocapsule for the release and / or encapsulation of molecules.

[0087] Further to the ability of crossing biological barriers, the nanoparticle of the present invention remain in brain tissues beyond the time-point of 24h, fact that reinforces the notion that these nanoparticles are further biocompatible with the central nervous system constituents.

[0088] The nanoparticles of the present invention are topically, intravenously and orally bioavailable, being the oral administration the most optimal administration via to achieve broader whole-body biodistribution of these nanoparticles, and lower accumulation in the liver, which is primarily linked to detoxification.

[0089] In another embodiment, the present invention refers to a composition comprising one or more lipidic nanoparticles of the invention. The nanoparticles of the invention, or the composition comprising said nanoparticles, can be used in the manufacture of a food and / or food supplement for animal or human use, a supplement for plants or a cosmetic product.

[0090] The invention also relates to compositions comprising one or more of these lipid nanoparticles for use in medicine. In particular, the nanoparticles, or compositions thereof, are useful in the treatment of diseases related to dysfunctions of the central nervous system such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and glioblastoma. These diseases present significant challenges in drug delivery due to the protective blood-brain barrier (BBB). This barrier, while essential for protecting the brain, also limits the passage of most therapeutic agents, reducing the effectiveness of traditional treatments. For neurodegenerative diseases like Alzheimer's and Parkinson's, where precise targeting of affected brain regions is crucial, existing drug delivery strategies often fail to deliver adequate concentrations of therapeutic agents to the desired sites. In the case of glioblastoma, a highly aggressive brain tumor, the BBB prevents many chemotherapeutic drugs from reaching the tumor in sufficient amounts, leading to poor outcomes. The use of the lipid nanoparticles of the invention allow to overcome these barriers, enhance drug penetration into the CNS, and improve therapeutic efficacy while minimizing side effects. These improvements could lead to better management and outcomes for patients suffering from these debilitating CNS disorders.

[0091] The lipid naniparticles, or lipid nanoparticle compositions, are also useful in the treatment of diseases of the skeletal system, such as osteoporosis, osteoarthritis, bone cancer, and fractures, which often require more effective drug delivery strategies to enhance treatment outcomes. In osteoporosis, current oral medications can lead to gastrointestinal side effects and poor patient adherence, while their systemic nature may result in insufficient drug concentration at the targeted bone sites. Osteoarthritis treatments also suffer from limited drug penetration into joint tissues, reducing the efficacy of pain relief and cartilage protection therapies. Bone cancers, like osteosarcoma, pose a significant challenge as chemotherapeutic agents often fail to achieve therapeutic levels at the tumor site due to poor vascularization of bone tissue, leading to inadequate treatment and increased risk of recurrence. Additionally, the management of fractures can benefit from targeted drug delivery to accelerate bone healing and reduce complications. The use of the nanoparticles of the invention will improve the precision, effectiveness, and safety of treatments for these skeletal disorders, ultimately leading to better patient outcomes. Finally, lipid nanoparticles or nanoparticle compositions are useful as effective drug delivery strategies that can penetrate the skin barrier, crucial for the treatment of skin diseases, certain cancers, and in advancing cosmetic formulations. The skin, being the body's largest organ, serves as a formidable barrier, making it challenging for therapeutic agents to reach deeper layers where they are most needed. In cancer treatment, particularly for skin cancers like melanoma, delivering chemotherapeutic agents directly through the skin can improve drug concentration at the tumor site while minimizing systemic side effects. For chronic skin diseases such as psoriasis and eczema, transdermal drug delivery offers a way to deliver treatments more effectively, reducing inflammation and promoting healing directly at the affected areas. In cosmetics, advanced transdermal delivery systems can enhance the penetration of active ingredients, leading to more effective anti-aging, moisturizing, and skin-repairing products. By overcoming the skin barrier, these drug delivery strategies offer more targeted, efficient, and less invasive treatment options, improving outcomes in both medical and cosmetic applications.

[0092] The following experimental section is provided purely by way of illustration and is not intended to limit the scope of the invention as defined in the appended claims. In the following experimental section, reference is made to the appended figures.

[0093] EXAMPLES

[0094] Characterization of the tissue-targeting abilities of lipid nanoparticles

[0095] Isolation of biological lipid nanoparticles

[0096] EVs were isolated following the published protocol developed by the inventors (Lorca, C., et al (2022). Industrial By-Products As a Novel Circular Source of Biocompatible Extracellular Vesicles. Adv Fund Mater 32). Beer products were selected, donated by Cerveses Ponent (Artesa de Segre, Lleida, Spain), as the source of EVs. Prior to EVs isolation, 15mL of beer by-products were centrifuged at 10000xg, 30 min at 4°C to obtain by-product 1 (BP1). Then, 3mL of BP1 were ultrafiltered in Vivaspin® PES 300kDa at 6000xg for 35 min at4°C. The concentrated supernatant was washed twice with 10mL of PBS using the same spinning, time and temperature conditions. Finally, concentrated EVs sample remaining in the upper side of the filter was collected and stored at -80°C for further downstream analysis. Fluorescent Labelling of EVs

[0097] EVs were prepared fresh prior to the in vivo study. EVs resuspended in PBS were labelled with 1 xio-6M of Vybrant DiD Cell-Labeling Solution (Invitrogen, Waltham, USA) at 37°C for 20 min. Subsequently, excess of dye was removed by filtration using a Vivaspin 10kDa MWCO filters following manufacturer’s instructions (5 min, 2000xg, 4°C). Passed-through liquid containing the unbound dye and fluorescent-labelled EVs were collected and stored at -80°C for further application.

[0098] Protein quantitation of EVs preparations

[0099] Bicinchoninic acid (BCA) assay BCA solution (B9643-1 L, Supelco, Merck, USA) and Copper (II) sulphate solution (C2284-25ML, Sigma Aldrich, Merck, USA) were used, according to the manufacturer’s instructions to estimate EV protein concentrations. 25pL of each sample was loaded in a 96-well plate, followed by 200pL of BCA / copper complex solution. The absorbance was measured at 562nm in a BioTek microplate reader.

[0100] In vivo administration of EVs preparations

[0101] In Vivo Administration of Fluorescent-Labeled EVs 10-weeks old C57BL / 6NT mice (n= 16) were housed in cages on a 12 h dark / light cycle at stable temperature (21 °C) with water provided ad libitum and fed with standard commercial chow for a minimum of 2 weeks (adaptation period) before starting the study. Mice were maintained in fasting conditions 16 h before the experimental procedure. Mice were treated with =3x109 particles suspended in 200 pL of PBS administered orally (oral) or by tail-intravenous injection (i.v.). Two different controls were also carried out; a control of the dye where the same amount of dye was mixed with PBS (CD) and a negative control where PBS were administered (CN). Mice were then euthanized at 25 h postadministration and brain and bone from ribs were excised.

[0102] Dissection of specific tissues and isolation of tissue lipid nanoparticles

[0103] Animals were humanely euthanized and brain, representative limb skeletal tissues and skin samples were dissected. The obtained tissues were subjected to isolation of EVs by PROSPR (Gallart-Palau, X. et al. (2016) Enrichment of extracellular vesicles from tissues of the central nervous system by PROSPR. Mol Neurodegeneration 11, 41, and the obtained preparations were subjected to analysis by dedicated flow citometry. Dedicated flow cytometry

[0104] Flow cytometry experiments were performed on a Beckman Coulter Cytoflex SRT cell sorter (with SSC detection in the violet laser). Red fluorescence from the EVs was detected using R 660 / 10 bandpass filter. Flow rate, fluorescence, and light scatter calibrations were performed on the day of the experiments. A buffer-only control (dPBS sample) was measured with the same flow cytometer and acquisition settings as all other samples passed at 2 evt / sec. Samples of brain and skeletal tissues usually passed at a 300-500 evt / sec for 5-10 minutes. We performed a trigger channel and threshold based on fluorescence. Fluorescence calibration was performed daily using Daily QC Calibration beads (C65719, Beckman Coulter, USA). Light scatter calibration was done with Megamix plus SSC Beads (Stago diagnostica) using SSC detection in both violet and green fluorescence (B 525 / 40 bandpass filter). EVs that were positive for green fluorescence were gated and eluted from the instrument for further analysis.

[0105] Lipidomics sample preparation

[0106] To extract lipids of the EVs separated in the flow cytometer, 10pL of homogenized tissue were combined with 5pL of MiliQ water and 20pL of ice-cold methanol. After vigorous vortexing for 2 minutes, 250pL of methyl tert-butyl ether (MTBE), containing internal lipid were introduced. The samples underwent shaking in a water bath (ATU Ultrasonidos, Valencia, Spain) at 10°C for 30 minutes with an ultrasound frequency of 40 kHz and power of 100 W. Following this, 25pL of MiliQ water was added, and the mixture underwent centrifugation (1 ,400 g) at 10°C for 10 minutes to separate the organic phase (Pizarro, C., et al (2013). Plasma lipidomic profiling method based on ultrasound extraction and liquid chromatography mass spectrometry. Anal Chem 85, 12085- 12092). The lipid extracts in the upper phase were then analyzed using mass spectrometry. A quality control pool was created using all lipid extracts, while internal isotopically labeled lipid standards for each class were employed for signal normalization (Pradas, I., et al (2018). Lipidomics Reveals a Tissue-Specific Fingerprint. Front Physiol 9, 1165). Stock solutions of lipid standards were prepared by dissolving them in MTBE at a concentration of 1 mg / mL, and working solutions were diluted to 2.5pg / mL in MTBE.

[0107] LC-MS analysis for lipidomics.

[0108] EV lipid extracts underwent analysis using a method previously outlined in published literature (Castro-Perez, J.M., et al (2010). Comprehensive LC-MS E lipidomic analysis using a shotgun approach and its application to biomarker detection and identification in osteoarthritis patients. J Proteome Res 9, 2377-2389'). Lipid extracts were subjected to liquid chromatography-mass spectrometry using a LIPLC 1290 series coupled to ESIQ-TOF MS / MS 6545 (Agilent Technologies, Barcelona, Spainln the LIHPLC system, the sample compartment was maintained at a temperature of 4°C. Each sample was subjected to a 10pL application of lipid extract onto a 1.8pm particle 100 x 2.1mm id Waters Acquity HSS T3 column (Waters, Milford, MA, USA), which was heated to 55°C. The flow rate was set at 400pL / min, with solvent A consisting of 10mM ammonium acetate in a mixture of acetonitrile and water (40:60, v / v), while solvent B comprised 10mM ammonium acetate in a blend of acetonitrile and isopropanol (10:90, v / v). The gradient commenced at 40% mobile phase B, transitioned to 100% B over 10 minutes, and remained constant for 2 minutes. Finally, the system returned to 40% mobile phase B and underwent equilibration for 3 minutes. Duplicate runs of the samples were conducted to capture both positive and negative electrospray-ionized lipid species using a Time-of-Flight (TOF) mode. The TOF mode operated in full-scan mode, scanning from 100 to 3000 m / z with an extended dynamic range of 2 GHz. Nitrogen (N2) was utilized as the nebulizer gas at a flow rate of 5 L / min and a temperature of 350°C. The capillary voltage was set to 3500V, and the scan rate was adjusted to one scan per second. In-run calibration of the mass spectrometer was achieved through continuous infusion using a double spray with masses 121.050873 and 922.009798 for positive ion mode, and 119.036320 and 966.000725 for negative ion mode (Pradas, I., et al. (2019). Metformin induces lipid changes on sphingolipid species and oxidized lipids in polycystic ovary syndrome women. Sci Rep 9, 16033. 10.1038 / s41598-019-52263-w).

[0109] Lipidomic data pre-processing and annotation

[0110] MassHunter Qualitative Analysis Software (Agilent Technologies, Barcelona, Spain) was employed to acquire the molecular features of the samples. These features represent various comigrating ionic species of a particular molecular entity and were obtained using the Molecular Feature Extractor algorithm (Agilent Technologies, Barcelona, Spain). Additionally, MassHunter Mass Profiler Professional Software (Agilent Technologies, Barcelona, Spain) and Metabolanalyst Software (Chong, J., et al (2019). Using MetaboAnalyst 4.0 for Comprehensive and Integrative Metabolomics Data Analysis. Curr Protoc Bioinformatics 68, e86; Xia, J., et al (2016). Using MetaboAnalyst 3.0 for Comprehensive Metabolomics Data Analysis. Curr Protoc Bioinformatics 55, 14 10 11-14) were utilized for further analysis. Features with a minimum of 2 ions were exclusively chosen. Subsequently, the molecular attributes in the samples were aligned using a retention time window of 0.1 % ± 0.25 minutes and 30.0 ppm ± 2.0 millidaltons. Only features present in at least 70% of the QC samples were considered for correcting individual biases, with signal correction performed using a LOESS approach (Broadhurst, D. et al (2018). Guidelines and considerations for the use of system suitability and quality control samples in mass spectrometry assays applied in untargeted clinical metabolomic studies. Metabolomics 14, 72; Dunn, W.B. et al. (2011). Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry. Nat Protoc 6, 1060-1083). For annotation purposes, pertinent features, delineated by exact mass and retention time, were cross-referenced against the Human Metabolome Database (HMDB) (Wishart, D.S. etal. (2022). HMDB 5.0: the Human Metabolome Database for 2022. Nucleic Acids Res 50, D622-D631; Wishart, D.S. et al. (2018). HMDB 4.0: the human metabolome database for 2018. Nucleic Acids Res 46, D608-D617) (accuracy < 30ppm) and LIPID MAPS (Fahy, E. et al and Subramaniam, S. (2007). LIPID MAPS online tools for lipid research. Nucleic Acids Res 35, W606-612) databases (accuracy < 20ppm). The identities acquired were juxtaposed with the retention times of authentic standards. Subsequently, the identities were validated by comparing experimental MS / MS spectra against in silico libraries, utilizing both HMDB and LipidMatch, which is an R-based tool specifically designed for lipid identification (Koelmel, J.P., et al (2017). LipidMatch: an automated workflow for rule-based lipid identification using untargeted high- resolution tandem mass spectrometry data. BMC Bioinformatics 18, 331. 10.1186 / s12859-017- 1744-3). Furthermore, enrichment analysis were also performed using Metabolanalyst Software, as cited earlier.

[0111] Proteomics Characterization of Beer by-products derived EVs

[0112] Beer EVs underwent vesicle lysis and protein denaturation by dissolution in 16 M urea prepared in 100 x io-3M ammonium bicarbonate. Following a 20-minute incubation at room temperature, the samples were diluted with HPLC-grade water to achieve a final concentration of 8 M urea. Protein quantification in each sample was determined using the bicinchoninic acid (BCA) assay. The constituent proteins of the EVs were then prepared for shotgun proteomics according to previously established protocols (Gallart-Palau, X. et al (2020). Alzheimer's disease progression characterized by alterations in the molecular profiles and biogenesis of brain extracellular vesicles. Alzheimers Res Ther 12, 54) with slight modifications. In summary, solubilized EVs proteomes were reduced with 20 x 10'3M dithiothreitol at 30°C for 3 hours and alkylated with 40 x 10'3M iodoacetamide for 1 hour at room temperature, protected from light. Subsequently, trypsin digestion was carried out overnight at 37°C by adding 20 pg of sequencing-grade trypsin to the samples, followed by quenching with a final concentration of 0.5% formic acid (FA). The tryptic digested peptidomes were desalted using a 100 mg C18 Sep-pack cartridge (Waters, Milford, MA), with elution performed using 1 mL of 75% acetonitrile, 0.1 % FA. The eluates were then dried using a vacuum concentrator and stored at -20°C until further proteomics analysis.

[0113] Liquid Chromatography Tandem-Mass Spectrometry (LC-MS) of EV Proteomes

[0114] The desalted EVs peptidomes were reconstituted in 0.1 % formic acid (FA) before undergoing Liquid Chromatography Tandem-Mass Spectrometry (LC-MS / MS) analysis. The analysis was conducted using a nanoElute liquid chromatograph (Bruker Daltonics, MA, USA) at a flow rate of 300 nL / min, employing a 60-minute linear gradient of 3-45% acetonitrile (ACN). The nanoElute liquid chromatograph was online coupled to a state-of-the-art timsTOF Pro mass spectrometer (Bruker Daltonics, MA, USA), utilizing parallel accumulation-serial fragmentation (PASEF) data acquisition.

[0115] Proteomics Bioinformatics and Data Analysis

[0116] The proteomics raw data obtained from EVs was analysed using the specialized bioinformatics suite software PEAKS Studio X, with a precursor ion tolerance of 10 ppm and a fragment ion tolerance of 0.05 Da. In-house databases were constructed by amalgamating the existing proteome data available in the National Center for Biotechnology Information (NCBI) from all potential organisms present in each beer by product sample, based on existing literature. Carbamidomethyl of cysteine (Cys) was designated as a fixed modification during the proteomics bioinformatics database search. The PEAKS Studio software's PTM algorithm was employed for identifying protein post-translational modifications. A false discovery rate (FDR) of less than 1% was set for protein identification across all samples, with trypsin specified as the proteolytic enzyme. Subsequently, the data were exported to Microsoft Excel CSV files, and in-house generated macros were utilized for protein quantification analyses. Gene ontology (GO) analysis was conducted to compare EVs proteomes with human circulating EVs proteomes using PANTHER version 16.0. A list of proteins identified from human circulating EVs isolated by ultracentrifugation with a sucrose cushion was acquired from the authors' previously published work. RESULTS

[0117] EVs detection in brain tissues and isolation

[0118] After sacrifice, brain tissues from mice were isolated and processed for EV isolation. These samples were hypothesized to contain brain EVs from the same tissue, but also stained EVs that potentially cross the BBB. To test this, brain EV samples were submitted to flow cytometry (Figure 1). Measurements represented particles between 140 to 500 nm, as per set in the instrument and the beads used to equilibrate the measurements. As observed, control (brain cn 8, brain cn 15 and brain cn 12) and brain EV samples (B5 brain, B7 brain and B9 brain) presented a visually different dot plot distribution (Figure 1A, B). When observing the three brain samples (B5 brain, B7 brain and B9 brain), we have highlighted a red gated region, which corresponded to a green, fluorescent population (Figure 1 B). These were attributed to the presence of stained EVs in the tissue, which corresponded to and 1.53±0.67 % for unstained controls, 2.39±1.20 for DiD controls and 6.84±1.51 for EV samples detected at brain tissues (***; p<0.001) (Figure 1C). These stained EV were gated and sorted for downstream characterization.

[0119] EVs detection in skeletal tissues and isolation

[0120] Skeletal tissues from mice were dissected and processed for EV isolation as detailed in the examples and the above pragraph. These samples were hypothesized to contain skeletal EVs derived from the same tissue, along with stained EVs potentially crossing biological barriers. To examine this fact, skeletal EV samples underwent flow cytometry as above detailed. Figure 3 presents dedicated flow cytometry data demonstrating the distribution of extracellular vesicles (EVs) within bone tissue identified in our study. A clear population of stained vesicles, representing near 6% of the total nanoparticles detected, confirms the successful delivery of the administered EVs to the target tissue. These results indicate the efficacy of the delivery system in reaching the intended site. Additionally, a portion of the stained vesicles identified could be associated, in terms of diamater size, with specific compositions of bone cells (Figure 3). This suggests that the administered EVs not only reached the bone tissue but also retain their bioactivity, a fact further pursued experimentally.

[0121] The presence and distribution of these stained vesicles in this invention provide strong evidence of the EVs’ potential as tissue-targeted nanovectors, supporting their ability to localize within bone tissue at specific and measurable percentages, demonstrating their applicability and development in the invention detailed EV nanovector-based therapies.

[0122] Lipidomic characterisation of fluorescent by-products EVs that are detected in mice brain tissues Fluorescent EVs and brain EVs populations underwent a lipid extraction protocol to carry out a lipidomics study. These lipidomes were further compared with previously published data (Lorca et al, (2022)). Initially, 28 different components, defined by their mass, were identified after bioinformatics analysis, which corresponded to those unique in EVs that were found in brain tissues. When searching mass data into lipid databases to identify the components, several isoforms of fatty acids, glycerolipids such as Diradylglycerols (DG), Triradylglycerols (TG), phospholipids, such as phosphatidylcholine (PC), ceramides and cholesteryl ester (CE) were defined to be potentially found in our samples. To narrow down which isoforms were the predominant lipids in these samples, an MS / MS identification was performed, revealing 12 unique lipids for these by-products EVs, Brassylic acid, Myristic acid, Palmitoylethanolamide / Oleamide, DG(36:0), DG(34:0), DG(44:2), TG(47:0), TG(50:3), PC(P-38:5) / PC(O-38:6), PC(38:4), Cer(d35:0) and CE(22:5) (Table 1).

[0123] Table 1: Unique lipids which were identified in EVs that cross the BBB of treated mice The enrichment analysis of this lipids revealed that they were significantly involved in Neurotransmitter release cycle (p=0.000113), Transmission across Chemical Synapses and Neuronal System (p=0.000319). In a less significant meaning but higher enrichment ratio we have highlighted Defective SLC27A4 causes ichthyosis prematurity syndrome (IPS) (p= 0.0042) and Transport of fatty acids (p= 0.00467). Other relevant pathways involved in a less significative manner were involved in Fatty Acid metabolism, transport, G protein signalling, mitochondrial beta-oxidation o protein metabolism (Figure 2).

[0124] Proteomic fingerprint of the EVs that cross the BBB

[0125] In addition to the lipid composition study, an extraction and analysis of the protein composition of these vesicles isolated in mouse brains was conducted. This was aimed to draw a full fingerprint of the composition that enables these EVs to cross the mice BBB.

[0126] The following proteins related to different microorganisms that populate the EVs of the invention were obtained and confer these nanoparticles capacity to trespass biological barriers. To obtain a more detailed information of the functions and the protein composition of these vesicles, these were specifically subdivided into the different peptides found, which are uniquely present in these samples (Table 2).

[0127] Table 2. Unique proteins and peptides identified in EVs that cross the BBB of treated mice As such, 5 peptides were related to Malate dehydrogenase from Cyberlindnera fabianii, 2 peptides of Dihydrolipoyl dehydrogenase from Saccharomyces pastorianus, 2 peptides of EF- hand domain-containing protein from Hordeum vulgare subsp. vulgare, 1 peptide of Serine / threonine-protein phosphatase from Saccharomyces cerevisiae, 1 peptide of Enolase and Peptidyl-prolyl cis-trans isomerase D from Ashbya gossypii (Table 2).

[0128] The presence of the reported peptide sequence for each protein in the UniProt database was compared to determine its presence in other organisms, including mouse and human. The sequences associated with Malate dehydrogenase from Cyberlindnera fabianii are found between 70 and 90% in Cyberlindnera fabianii itself, as well as in other Cyberlindnera, Wickerhamomyces, Trichoderma, Hypocrea, Penicillium, Aspergillus, and more components of the Fungi kingdom. The sequences of Dihydrolipoyl dehydrogenase from Saccharomyces pastorianus are found in other Saccharomyces, other components of the Fungi kingdom, and Bacteria. The reported sequences for EF-hand domain-containing protein from Hordeum vulgare subsp. vulgare are found in other similar domains and in domains encoded in Calmodulin of multiple plants, yeasts, and fungi. The sequence associated with Serine / threonine-protein phosphatase is reported, matching 100%, with other fungi and yeasts, bacteria, and protists. The Enolase peptide is also found in entries related to phosphopyruvate hydratase, in both cases from plants, yeasts, fungi, and protists. Finally, the sequence of Peptidyl-prolyl cis-trans isomerase D is found in other fungi, yeasts, and protists.

[0129] Finally, a set of correlation was established between the list of lipids and peptides found in these extracellular vesicles that cross the BBB (Table 3). Positive relationships above 0.9 were found in Myristic acid, DG (36:0) and DG (34:0) with some of the peptides (Table 3).

[0130] Table 3: Correlation values between the lipidomic and proteomic fingerprint of EVs that cross the BBB.

[0131] The presence of correlation factors above 0.9 indicates that their coexistence in the composition of these vesicles enhances their bioavailability to the central nervous system and their crossing of the BBB.

[0132] Lipid signature of tissue-targeting Minudik vesicles

[0133] The fluorescently labeled EVs capable of tissue targeting (brain and skeleton) were isolated from targetted tissues by flow cytometry and analyzed by LC-MS / MS. A mean of 1.23 x 1011vesicle particles were quantified per condition. The lipid composition was determined, providing average, minimum and maximum raw intensities as well as normalized values expressed as relative percentage of the total lipid content per sample. Normalization was performed by intra-sample scaling. The analysis revealed a characteristic lipid profile in tissue-targeting Minudik vesicles. Brassylic acid was identified as the predominant lipid (-54% of normalized content), while PC(38:4), Cer(d35:0), Myristic acid and CE(22:5) showed intermediate contributions (5-15%). Minor fractions consisted of DG36, DG34, PC(P-38:5) and DG44:2 (<5% on average). The minimum and maximum values indicated the natural variability observed across biological replicates.

[0134] Table 4. Lipid composition of tissue-targeting (brain and skeleton) Minudik vesicles. The table shows the average, minimum and maximum intensities (relative abundance, raw MS signal) together with the normalized data expressed as percentage contribution to the total lipid content per sample. Minimum and maximum values represent the observed range across biological replicates. Normalization was performed by intra-sample scaling. A total of an average of 1.23 x 1011 vesicle particles were analyzed by LC-MS / MS.

[0135] This distribution defines a lipid signature that is characteristic of tissue-targeting Minudik vesicles, in which Brassylic acid predominates and is accompanied by secondary contributions from PC(38:4), Cer(d35:0), Myristic acid and CE(22:5), with minor contributions from DG36, DG34, PC(P-38:5) and DG44:2.

[0136] It is further noted that the ratio defined as [PC(38:4)+PC(P-38:5)] : [DG(36)+DG(34)+DG(44:2)] between 1.0:1 and 2.5:1.’ was verified using normalized lipid data from tissue-targeting EVs vesicles. Normalization was performed by intra-sample scaling, expressing each lipid as a relative proportion of the total lipid content per sample. Across the nine biological replicates analyzed, this ratio consistently fell within the range of 1.0:1 to 2.5:1 , as defined in the claim. The values ranged from 1.174 (minimum) to 2.224 (maximum), with a mean of 1.898 ± 0.354 (SD).

[0137] Synthesis of lipid nanoparticles with determined tissue-targeting abilities

[0138] Lipid nanoparticles with the tissue abilities detailed in this invention were sythesized as previously detailed by Zacheo et al (Lipid-Based Nanovesicles for Simultaneous Intracellular Delivery of Hydrophobic, Hydrophilic, and Amphiphilic Species. Front Bioeng Biotechnol. 2020 Jul 3;8:690. doi: 10.3389 / fbioe.2020.00690. PMID: 32719782; PMCID: PMC7350901). Briefly, a lipid mixture composed of GL, GP, SP, ST and FA in a ratio of 0.8:2:0.1 :0.1 :7 was used to synthesize the vesicles (final concentration: 5 mM in 5 mL of chloroform). The phospholipid solution was stirred for 1 hour with the cap closed to minimize solvent loss. Afterward, the cap was removed, and the solution was left to stir overnight at room temperature to allow solvent evaporation. PBS was then added, and the solution was sonicated for 4 minutes at 25 W power and 20 kHz frequency. To prevent overheating during sonication, the vial was placed in an ice bath.

[0139] The lipid profile of the nanoparticles obtained were characterized:

[0140] Tabla 5. Lipid profile(s) of functional endogenous and / or engineered nanoparticles Incorporation of signaling-oriented proteinaceous to tissue-targeted lipid nanoparticles

[0141] Using click chemistry, peptides and proteins were attached to the membrane of the isolated or synthesized lipid nanoparticles through a two-step reaction process as previously detailed by Jia et al 2018 (NRP-1 targeted and cargo-loaded exosomes facilitate simultaneous imaging and therapy of glioma in vitro and in vivo. Biomaterials. 2018 Sep; 178:302-316. doi: 10. 1016 / j. biomaterials.2018.06.029. Epub 2018 Jun 21. PMID: 29982104). First, the alkyne group was conjugated with a protein on the lipid nanoparticle membrane via a condensation reaction. Then, the peptide or protein, which contains an azido group, was attached to the alkyne group through triazole linkages. The reaction started by preparing a mixture containing 3.5 mg sulfo- N- hydroxysuccinimide and 29 mg 4-pentynoic acid in 1 mL of PBS, with the pH buffered to 7.4 using sodium bicarbonate. This solution was stirred on ice for 1 hour. Next, 46 mg of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) was added to the reaction mixture, which was then stirred on ice for another hour. Following this, the resultant reaction mixture was combined with the lipid nanoparticles and incubated at 20°C for 24 hours. The lipid nanoparticles was subsequently purified using ultrafiltration. In the second step, 160 pg of lipid nanoparticles (usually measured by protein content) in 300 pL PBS was combined with 14 pL of 0.32 M copper(ll) sulfate pentahydrate, 71 pL of 1.44 M l-ascorbic acid, 32.8 pL of 0.27 M bathophenanthroline disulfonic acid disodium salt trihydrate, and 0.2 mg of 8.5 mM azido-containing peptide / protein. These reagents resulted in a final reaction mixture with approximate concentrations of 10.5 mM copper(ll) sulfate, 240 mM l-ascorbic acid, 20.8 mM bathophenanthroline disulfonic acid, and 4.0 pM peptide / protein. This reaction mixture was stirred at room temperature under a nitrogen atmosphere for 4 hours. The modified lipid nanoparticles were then purified by ultrafiltration.

[0142] Following the application of click chemistry as detailed, the nanoparticle contained integrated protein sequences from proteins 1 , 3, 4, 5, and 6, demonstrating successful conjugation. Specifically, Malate dehydrogenase (Protein 1 , OS=Cyberlindnera fabianii) was incorporated, with key peptide sequences such as LFGVTTLDIVRA (SEQ ID NO 1) and AAGGIGQPLSLLLK (SEQ ID NO 2) attached to the nanoparticle’s surface. Additionally, sequences from the EF-hand domain-containing protein (Protein 3, OS=Hordeum vulgare subsp. vulgare) were included, exemplified by the peptides MKDTDSEEELKEAFRV (SEQ ID NO 8) and SLGQNPTEAELQDMINEVDADGNGTIDFPEFLNLMA (SEQ ID NO 9). From Serine / threonine-protein phosphatase (Protein 4, OS=Saccharomyces cerevisiae), the peptide YIFLGDYVDR (SEQ ID NO 10) was integrated into the nanoparticle. Additionally, Enolase (Protein 5, OS=Ashbya gossypii) contributed the peptide EALRMGSEVYHNLK (SEQ ID NO 11), ensuring functional diversity. Finally, Peptidyl-prolyl cis-trans isomerase D (Protein 6, OS=Ashbya gossypii) was incorporated with the sequence SIYGEKFEDENFARK (SEQ ID NO 12). These experiments were validated using mass spectrometry shotgun analysis to confirm the incorporation of protein sequences into the edited nanoparticle preparations. The shotgun approach confirms the successful conjugation of these proteins, verifying the nanoparticle’s composition and demonstrating the accuracy of the click chemistry process used in the experiments.

[0143] MINUDIK SKIN TOPICAL APPLICATION EXAMPLES

[0144] Nanovectors

[0145] Minudik® extracellular vesicles (EVs) were suspended in phosphate-buffered saline (PBS) at a concentration of 1 xio10particles / mL. The vesicle membranes were labeled with a lipophilic red fluorescent dye, which integrates into lipid bilayers. Unincorporated dye was removed through standard desalting and centrifugation procedures, resulting in a fluorescent EV preparation.

[0146] Animal and topical application

[0147] A juvenile domestic pig (Sus scrofa domesticus) was anesthetized following a standard veterinary protocol and remained under anesthesia for the duration of all procedures. The animal participated in a surgical training session at the CREBA Center (IRBLLEIDA), during which the topical dosing and tissue sampling described herein were conducted while the animal was anesthetized. Physiological parameters, including heart rate and respiration, were monitored, and ocular lubrication was administered. The dorsal area of the right auricle was gently cleansed with PBS and allowed to air-dry. A volume sufficient to wet the application site (~1 cm2) of the labeled EV suspension was topically applied and left uncovered.

[0148] Tissue collection

[0149] At approximately 0 hours (following application) and 1 hour post-dose, the treated area was excised. For each time point, a full-thickness skin block encompassing the application site was dissected. Histological procedures

[0150] Skin tissues were fixed in 4% paraformaldehyde (PFA) in PBS, cryoprotected in sucrose, embedded in OCT, sectioned using a Leica cryostat at a thickness of 16 pm, and mounted with Vectashield mounting medium containing DAPI.

[0151] Confocal microscopy and micrographs analysis

[0152] Sections were imaged using a Zeiss laser-scanning confocal microscope equipped with a 10* objective lens. The red channel was employed to capture lipid-dye-labeled Minudik EVs, while the DAPI channel served as a nuclear counterstain, and transmitted light / autofluorescence provided tissue context. Imaging parameters, including laser power, gain, and pinhole settings, were maintained consistently across all conditions, and channels were acquired sequentially to prevent bleed-through. Micrographs were analyzed using ImageJ / Fiji software: images were imported via Bio-Formats, background subtraction was performed using the rolling-ball method, and regions of interest within the epidermis and dermis were manually segmented to quantify fluorescence intensity and / or area. Identical lookup tables, thresholds, and measurement settings were applied uniformly to all images.

[0153] MINUDIK SKIN TOPICAL APPLICATION RESULTS

[0154] Confocal micrographs demonstrated a dye-positive signal at the skin surface at 0 hours, with subsequent penetration observed by 1 hour, as indicated by fluorescence detected within the epidermis and upper dermis (Figure 4). Quantitative analysis of representative micrographs revealed an approximate distribution of the signal across layers at 1 hour, with approximately 42% of Minudik particles signal located in the epidermis and 5% in the dermis, relative to the total skin signal.

[0155] The data collectively support the hypothesis that Minudik® EVs, when applied topically, are capable of penetrating the stratum corneum and distributing within the viable epidermis and superficial dermis within one hour, while systemic absorption appears to be absent.

[0156] ORAL DATA MINUDIK

[0157] Throughout the evaluated time intervals (0, 2, 4, and 24 hours), Minudik® nanovectors exhibited no significant differences between intravenous and oral administration in their in vivo fluorescence measurements. As depicted in the grouped-bar plot (Figure 5), both administration routes demonstrate a similar increase from baseline at 2 hours, a comparable profile at 4 hours, and convergent accumulation by 24 hours. This overlap suggests the absence of a route-specific advantage in short-term tissue access under the tested conditions. This conclusion is further supported by the exposure summary (Figure 5), where the integrated signal over 24 hours is of similar magnitude for both oral and intravenous administration. Comparable area under the curve (AUC) values indicate that the overall tissue exposure achieved via the gastrointestinal route approximates that of direct systemic delivery. Collectively, these findings indicate that Minudik® EV-based nanovectors facilitate effective oral delivery, achieving tissue access comparable to intravenous administration. Consequently, the platform is suitable for the oral administration of therapeutic or diagnostic cargos intended to reach the target tissues described in the invention, while retaining the option of parenteral dosing when clinically indicated.

[0158] CLAUSES

[0159] 1. A lipid nanoparticle, comprising:

[0160] - a lipid composition comprising at least two of the following lipids,

[0161] - brassylic acid,

[0162] - myristic acid,

[0163] - palmitoylethanolamide,

[0164] - Diacylglycerol (DG) (36:0),

[0165] - DG (34:0),

[0166] - DG (44:2),

[0167] - Triacylglycerol (TG) (47:0),

[0168] - TG (50:3),

[0169] - Phosphatyldylcholine (PC) (P-38:5)) / PC (0-38:6),

[0170] - PC (38:4)

[0171] - Ceramide (Cer) (d35:0) and

[0172] - Cholesterol ester (CE) (22:5) wherein the lipid nanoparticle has a size between 30 and 950 nm and is configured to cross the blood-brain barrier, target skeletal tissues, and penetrate the skin barrier. 2. The lipid nanoparticle according to clause 1 wherein said nanoparticle is selected from the group comprising: a) lipid nanoparticles obtained from a natural source, and b) synthetic lipid nanoparticles obtained from chemical or biological synthesis methods.

[0173] 3. The lipid nanoparticle according to clause 2, wherein the nanoparticles a) are obtained from a by-product of the food industry.

[0174] 4. The lipid nanoparticle according to any of the previous clauses where the diameter of the nanoparticle is lower than 200 nm.

[0175] 5. The lipid nanoparticle according to any of the previous clauses further comprising at least one peptide of sequence selected from SEQ ID NO 1 , SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12.

[0176] 6. Use of a lipid nanoparticle, according to any one of clauses 1 to 5 , as a nanocarrier, nanovector and / or nanocapsule for the release and / or encapsulation of molecules.

[0177] 7. Composition comprising one or more lipid nanoparticles according to any one of clauses 1 to 5.

[0178] 8. The lipid nanoparticle according to any one of clauses 1 to 5 or the composition according to clause 7, for use in medicine.

[0179] 9. The lipid nanoparticle or the composition, according to clause 8, for use in the treatment of diseases related to dysfunctions of the central nervous system.

[0180] 10. The lipid nanoparticle or the composition for use, according to clause 9, wherein the disease is selected from Parkinson's disease, multiple sclerosis, and glioblastoma.

[0181] 11. The lipid nanoparticle or the composition, according to clause 8, for use in the treatment of diseases of the skeletal system.

[0182] 12. The lipid nanoparticle or the composition for use, according to clause 11 , wherein the disease is selected from osteoporosis, osteoarthritis, bone cancer, and fractures.

[0183] 13. The lipid nanoparticle or the composition, according to clause 8, for use in the treatment of skin diseases.

[0184] 14. The lipid nanoparticle or the composition for use, according to clause 13, wherein the disease is psoriasis or eczema.

Claims

44CLAIMS1. A lipid nanoparticle suitable to cross the blood-brain barrier and / or target skeletal tissues, comprising a lipid composition comprising at least two of the following lipids: a. Brassylic acid (element A); b. PC(38:4); c. Cer(d35:0); d. Myristic acid; e. CE(22:5); f. DG36; g. DG34; h. Phosphatyldylcholine (PC) (P-38:5)) / PC (0-38:6)); i. DG44:2; j. palmitoylethanolamide; k. Triacylglycerol (TG) (47:0); and l. TG (50:3); for use in the treatment of diseases related to dysfunctions of the central nervous system or of the skeletal system.

2. The lipid nanoparticle for use according to claim 1 , wherein said lipid composition comprises at least each of elements A to I of claim 1.

3. The lipid nanoparticle for use according to any one of claims 1 or 2, wherein the lipid nanoparticle comprises a lipid fraction, characterised in that the lipid fraction comprises each of elements A to I below in the indicated percentages by weight (wt%): a. Brassylic acid in a wt% between 45-60% (element A), b. PC(38:4) in a wt% between 8-18% (element B), c. Cer(d35:0) in a wt% between 2.4-12% (element C), d. Myristic acid in a wt% between 5-12% (element D), e. CE(22:5) in a wt% between 2.4-12% (element E), f. DG36 in a wt% between 0.5-5% (element F),45 g- DG34 in a wt% between 0.5-5% (element G), h. PC(P-38:5) in a wt% between 0.5-5% (element H), and DG44:2 in a wt% between 0.5-5% (element I), wherein the weight percentages are preferably obtained from LC-MS / MS data, wherein preferably the sum of the lipid components equals to between 98 to 100 wt% (±1 wt% due to rounding), more preferably wherein the sum of the lipid components equals 100 wt% (±1 wt% due to rounding).

4. The lipid nanoparticle for use according to claim 3, wherein lipids other than elements A to I are present in a combined amount of 0-2 wt%.

5. The lipid nanoparticle for use according to claim 3 or 4, wherein Brassylic acid is present in a wt% between 50-58%.

6. The lipid nanoparticle for use according to any one of claims 3-5, wherein lipid B is present in a wt% between 10-18%, each of lipids C and E are present in a wt% between 2.4-10% and element D is present in a wt% between 5-10%.

7. The lipid nanoparticle for use according to any one of claims 3-6, wherein each of lipids F-l is present in a wt% between 1-4%, preferably 1.5-3.5%.

8. The lipid nanoparticle for use according to any one of claims 3-7, wherein the group sum (B+C+D+E) is present in a wt% between 25-45%.

9. The lipid nanoparticle for use according to any one of claims 3-8, wherein the group sum (F+G+H+l) is present in a wt% between 8-13%.

10. The lipid nanoparticle for use according to any one of claims 3 to 9, wherein the lipid nanoparticle is characterized by comprising a lipid fraction comprising or consisting of ±30%, ±20%, ±15%, ±10%, ±5% or ±1% of each of the values indicated in the table below:46Brassylc acid 53,81PC(38:4) 13,70Cer(d35:0) 7.91Myristic acid 7,02CE(22:5) 6,68DG36 3,33DG34 3,24PC(P-38:5) 2,20DG44:2 2.10 wherein the above measured values are preferably obtained from LC-MS / MS data and wherein the sum of the lipid components equals 100 wt% (±1 wt% due to rounding).

11. A lipid nanoparticle for use according to any one of claims 1 or 2, wherein the lipid nanoparticle comprises a lipid fraction characterised by a ratio signature computed from percentages obtained from LC-MS / MS data and normalised within each sample to total 100%, wherein each of elements A-l of claim 3 is present, and the signature simultaneously satisfies: a. the ratio A : (B+C+D+E) is between 1.2:1 to 2.0: 1 ; b. the ratio A : (F+G+H+l) is between 3.0: 1 to 7.0: 1 ; c. the ratio (B+C) : (D+E) is between 1.2:1 to 2.0:1; d. the ratio (B+F) : (D+E+G) is between 1.0:1 to 2.5:1; e. the ratio A : B is between 3.0:1 to 5.0:1; f. the ratio A : C is between 5.0: 1 to 9.0: 1 ; g. the ratio B : D is between 1.3:1 to 2.5:1; h. the ratio C : E is between 0.8:1 to 1.5:1; i. the ratio (B+E) : (C+D) is between 1.1 :1 to 1.8:1; j. the ratio (B+C+D+E) : (F+G+H+l) is between 2.0:1 to 5.0:1; k. the ratio F : G is between 0.7:1 to 1.4:1 ; and l. the ratio H : I is between 0.7:1 to 1.5:1.

12. The lipid nanoparticle for use according to claim 11 , wherein B, F, D, E, G when present, are present at a ratio [B +F] : [D+E+G] between 1.0:1 and 2.5:

113. A lipid nanoparticle for use according to any one of claims 3 to 10, further characterised in that it satisfies simultaneously the ratio signature of any one of claims 11-12..

14. The lipid nanoparticle for use according to any preceding claim, wherein the diameter of the nanoparticle is between 30 nm and 950 nm, preferably lower than 200 nm.

15. The lipid nanoparticle for use according to any one of claims 1 to 14, wherein the lipid nanoparticle further comprises at least one peptide of sequence selected from the list consisting of SEQ ID NO 1 , SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ 5 ID NO 9, SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12.

16. The lipid nanoparticle for use according to the preceding claim, wherein the peptide of sequence is of SEQ ID NO 11.

17. A composition comprising a population of lipid nanoparticles according to any one of claims 1-16 and optionally a pharmaceutically acceptable carrier, for use in the treatment of diseases related to dysfunctions of the central nervous system or of the skeletal system.

18. The composition for use according to claim 17, wherein >80% of nanoparticles in the population comply with the characterisation required by any one of claims 1 to 16.

19. The composition for use according to claim 17 or 18, wherein the population comprises 1 xi01° to 5X 1012particles per dose, preferably about 1.23x1011particles.

20. The composition for use according to any one of claims 17 to 19, in a dosage form selected from solution, suspension, lyophilised cake, or buffered infusion, optionally comprising cryoprotectants and / or stabilisers.

21. The composition for use according to any one of claims 17 to 20, wherein the nanoparticles have a number-average diameter of between 30 and 950 nm,22. The lipid nanoparticle as defined in any of claims 1 to 16 or the composition as defined in any one of claims 17 to 21 , for use in a method of treatment as a nanocarrier of molecules to the central nervous system.

23. The lipid nanoparticle as defined in any of claims 1 to 16 or the composition as defined in any one of claims 17 to 21 , for use in a method of treatment as a nanocarrier of molecules to the skeletal system.

24. The lipid nanoparticle as defined in any of claims 1 to 16 or 22 or the composition as defined in any one of claims 17 to 22, for use in a method of treatment of a disease selected from the group consisting of Parkinson's disease, multiple sclerosis, and glioblastoma.

25. The lipid nanoparticle as defined in any of claims 1 to 16 or 23 or the composition as defined in any one of claims 17 to 21 or 23, for use in a method of treatment of a disease selected from the group consisting of osteoporosis, osteoarthritis, bone cancer, and fractures.

26. The lipid nanoparticle for use according to claims 1 to 16 or 22-25 or the composition for use according to claims 17 to 21 or 22-25, wherein the lipid nanoparticle or composition are administered via oral or via the intravenous route to a subject in need thereof.

27. The lipid nanoparticle for use according to claims 1 to 16 or 22-25 or the composition for use according to claims 17 to 21 or 22-25, wherein the lipid nanoparticle or composition are administered via oral to a subject in need thereof.

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