Senescence-associated nanoparticle; formulations, methods, and uses thereof

Abstract

The present disclosure relates to a senescence-associated beta-galactosidase (SA-β-Gal) degradable polymeric nanoparticle, formulations, methods of producing said nanoparticle and uses in senotherapy. Said nanoparticle is capable of precisely and efficiently deliver and release a senotherapeutic active ingredient, namely a senolytic active ingredient, in organ-specific senescent cells, particularly the senescent liver cells, increasing the senotherapeutic index of the senolytic active ingredient and inducing apoptosis in senescent cells overexpressing SA-β-Gal.

Description

D E S C R I P T I O NSENESCENCE-ASSOCIATED NANOPARTICLE; FORMULATIONS, METHODS, AND USES THEREOFTECHNICAL FIELD

[0001] The present disclosure relates to a senescence-associated beta-galactosidase degradable polymeric nanoparticle for the delivery and release of a senotherapeutic active ingredient.BACKGROUND

[0002] Cell senescence is defined as a state of proliferative arrest associated with structural and functional alterations including the production of a senescence associated secretory phenotype (SASP)1. The accumulation of these cells in living systems has been shown to be detrimental for tissue / organ function2-3. Over the past decade numerous strategies have been developed to target and eliminate senescent cells2. Clear evidence exists that the removal of senescent cells can effectively stave off tissue dysfunction.4-5

[0003] Among the pharmacological interventions investigated in the last 10 years, the removal of senescent cells with senotherapeutic active ingredients, namely, senolytic active ingredients has become one of the most explored interventions, with many currently in human clinical trials, including idiopathic pulmonary fibrosis, chronic kidney disease, Alzheimer's disease, osteoarthritis, diabetic macular oedema, SARS-CoV-2, among others6.

[0004] To prevent potential side effects of the senolytic active ingredients that act systemically (e.g. thrombocytopenia7), researchers have encapsulated the drug in nanoparticles (NPs) or modify the drug with a protective element to make it inactive unless part of the molecule is cleaved (prodrug), allowing selective activation within senescent cells811. These NPs can be designed to be internalized by both proliferative and senescent cells, but release the therapeutic agent exclusively within senescent cells12-13, thereby minimizing off-target effects. For example, mesoporous silica NPs were loaded with a senolytic active ingredient and coated with an oligosaccharide by senescence-associated p-galactosidase (SA-P-Gal), which is overexpressed in senescent cells, triggering drug release12-13. Others have explored the use of molecular imprinted NPs14or NPs conjugated with antibodies15 17to interact more specifically with epitopes in the membrane of senescent cells. However, many of these approaches face significant limitations, namely because in general de NPs are inorganic, which remain in the body and might have deleterious effects in cells12-13, or they have limited targeting effect in organ-specific senescent cells15 17. Thus, the development of NPs that can be easily eliminated from living systems and deliver more specifically the senotherapeutic drug in organ-specific senescent cells are still required. Another important aspect that remains elusive is the importance of NPs release kinetics of the senolytic activeingredient to maximize its senotherapeutic effect. It is recognized that NPs should be (i) internalized in enough concentration within the senescent cells, (ii) get entrapped in lysosomes, (iii) be cleaved by the enzyme SA-P-Gal that hydrolyses lactose to glucose and galactose, and (iv) the senolytic active ingredient able to cross the lysosome membrane to reach the cytoplasm to act in its molecular target(s). Previous studies have shown that the NPs that respond to SA-P-Gal are able to eliminate, at variable level, senescent cells but it is unclear how the release profile of the senolytic active ingredient from the NP affects the senotherapeutic activity of the senolytic active ingredient-loaded NPs.

[0005] In most of the interventions, the anti-senescence effects of the NPs were demonstrated in tumour xenografts13-18, atherosclerotic plaques15, organs such as lungs13and kidney16. Despite these promising results, there is a significant gap in evaluating the efficacy of these NPs in the liver, a critical organ that takes up the majority of the NPs after intravenous administration19-20. It remains to be demonstrated the senotherapeutic activity of these NPs in the liver, particularly in aged animals, which often exhibit an accumulation of senescent cells in this organ. Specifically, which liver cells are the most effective in the internalization of the NPs and which cellular compartment benefits the most from senotherapeutic NPs. It is recognized that aging is a major risk factor for the development of acute and chronic liver diseases21. During physiological aging, it has been reported that both rodents and humans showed an increase in hepatic and sinusoidal dysfunction, with elevated hepatic vascular resistance and increased portal pressure, and an increase in senescent liver cell accumulation22. It has been also shown that liver cell senescence mediates hepatic steatosis23. The effect of senotherapeutic drugs, but not senotherapeutic NPs and NPs, has been demonstrated in different pre-clinical (not yet in clinical) contexts of liver diseases such as hepatic steatosis23, partial hepatectomy24and biliary atresia and biliary cirrhosis25. Thus, demonstrating effectiveness in the liver, particularly in aged models with high senescent cell burdens, is a key step in expanding the therapeutic potential of these NPs and addressing a critical unmet need in senescence-targeted therapies. The elimination of senescent cells by senotherapeutic interventions is being evaluated in several clinical trials for the treatment of age-related diseases. However, these approaches present several limitations, including a low senotherapeutic index (i.e., the safety margin between the dose of the drug that produces a desired effect and the dose that produces unwanted side effects is relatively low) and, in some cases, concerns regarding their elimination. One of the fundamental technical challenges is the development of a therapeutic agent or delivery system that selectively targets senescent cells within aging tissues, while sparing proliferative healthy cells. Current approaches lack sufficient specificity, often resulting in collateral damage to non-senescent cells, which leads to undesirable side effects such as tissue degeneration or immune responses. The problem is further compounded by the heterogeneous nature of senescent cell populations, which exhibit variable markers and signalling pathways across different tissue types and disease states. As a result, conventional treatments not only suffer from low efficacy but also struggle with systemic toxicity, reducing their overall clinical viability. Therefore, there is anurgent need for a more precise targeting mechanism that can effectively differentiate senescent cells from healthy counterparts, potentially leveraging unique molecular markers or cellular pathways exclusive to senescence.

[0006] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.GENERAL DESCRIPTION

[0007] The present disclosure relates to senescence-associated beta-galactosidase (SA-P-Gal) degradable polymeric nanoparticle for the delivery and release of a senotherapeutic active ingredient, namely a senolytic active ingredient. The NP of the present disclosure is endocytosed by both proliferative and senescent cells, but only in the latter is it degraded by SA-P-Gal at a sufficient level in the endolysosome to release the encapsulated senolytic active ingredient, which can then cross a damaged endolysosomal membrane, reaching the cell cytoplasm and inducing apoptosis in senescent cells overexpressing SA-P-Gal.

[0008] The present disclosure is capable of precisely target organ-specific senescent cells and be easily eliminated from living systems after delivering the senolytic active ingredient. Surprisingly, the senotherapeutic index of the senolytic active ingredient-loaded NP of the present disclosure is at least 44 times higher than that observed for the soluble senolytic active ingredient due to the efficient delivery to senescent cells. Particularly, the present disclosure targets liver senescent cells and accumulates in these cells releasing the senolytic active ingredient.

[0009] The present disclosure is biocompatible and biodegradable and represents a significant advancement over conventional inorganic particles, offering a safer, more efficient system for the delivery and release of senolytic active ingredients, having an enhanced targeting capability. Such improvements enable selective treatment of senescent cells in specific tissues or organs while minimizing exposure to proliferative cells and reducing systemic side effects.

[0010] An aspect of the present disclosure relates to a polymeric nanoparticle for delivering and releasing an active ingredient, wherein the nanoparticle may comprise:a cyclodextrin-based polymer;an adamantane-modified oligosaccharide cleavable by senescence-associated p-galactosidase; a senotherapeutic active ingredient;wherein the cyclodextrin-based polymer bound to the adamantane-modified oligosaccharide; wherein the senolytic active ingredient may be entrapped in the polymeric nanoparticle.

[0011] In an embodiment for better results, the degradation rate of the nanoparticle is more than 1.5% per minute, under physiological conditions at 37°C. The nanoparticle degradation rate constant (k = 0.03± 0.015 min1) was determined by fitting the data obtained by DLS of NP count decrease (%) ([NP] = 100 ug / mL in PBS) at different time points (0, 1, 2, 4 and 24 hours) under the presence of p-galactosidase, to an exponential decay equation. This characteristic enables the nanoparticle to be efficiently degraded by senescence-associated p-galactosidase within the endolysosomal compartment, facilitating the release of its content into the cell cytoplasm due to the high permeability of endolysosomes in senescent cells.

[0012] In an embodiment for better results, the cyclodextrin-based polymer may be formed by acrylated p-cyclodextrin monomers and amine monomers (Table 1); preferably linked via Michael-type addition, enabling the NPs internalization by senescent cells and the efficient release of the senotherapeutic active ingredient.Table 1. Information of chemical name, CAS number and vendor of the monomers used to synthesize the library of p-cyclodextrin-based polymers.

[0013] In an embodiment for better results, the cyclodextrin-based branched polymer molecular weight may range from 5 kDa - 150 kDa; preferably 10 kDa - 100 kDa; more preferably 30 kDa - 70 kDa; even more preferably 45 kDa - 55 kDa.

[0014] In an embodiment for better results, the cyclodextrin-based polymer may be bound to the adamantane-modified oligosaccharide via non-covalent interactions; preferably host-guest interactions where the cyclodextrin-based polymer molecule (host) provides a cavity or binding site that accommodates the adamantane-modified oligosaccharide (guest).

[0015] In an embodiment for better results, the macrocycle of the cyclodextrin-based polymer (host) to form host-guest interactions may be replaced by other macrocyclic molecules, such as: alfa- or gammacyclodextrins, cucurbiturils, calixarenes, pillararenes, porphyrins, among others.

[0016] In an embodiment for better results, the adamantane of the modified oligosaccharide polymer (guest) may be replaced with other molecules to form host-guest interactions, such as: naphthalene,anthracene, pyrene, ferrocene, quaternary ammonium derivatives, alkylammonium derivatives, tertbutyl derivatives, dye molecules (rhodamine, fluorescein, among others), among others.

[0017] In an embodiment for better results, the adamantane-modified oligosaccharide may be galactan-adamantane.

[0018] In an embodiment for better results, the galactan-adamantane molecular weight may range from 10 kDa - 150 kDa; preferably 60 kDa - 110 kDa, more preferably 85 kDa - 95 kDa.

[0019] In an embodiment for better results, the degree of substitution of adamantane per galactose monomer, may range from 1% - 30%; preferably 5% - 25%; more preferably 8% - 16%; even more preferably 10%. The degree of substitution of adamantane per galactose monomer may be determined by proton nuclear magnetic resonance, more specifically between the proton integral of peaks of galactan (5 = 3.5-4.6 ppm) and adamantane (5 = 1.5-2.7 ppm).

[0020] In an embodiment for better results, the oligosaccharide cleavable by senescence-associated -galactosidase may be replaced by other oligosaccharides / polysaccharides, such as: polylactose, agarose, guar gum, among others.

[0021] In an embodiment for better results, the cyclodextrin-based polymer / adamantane-modified oligosaccharide ratio may be 30% (w / w) - 70% (w / w); preferably 35% (w / w) - 65% (w / w), more preferably 50% (w / w) - 55 % (w / w).

[0022] In an embodiment for better results, the senotherapeutic active ingredient may be a senolytic active ingredient, or a senomorphic active ingredient, or a senescence-inhibitor active ingredient, or mixtures thereof.

[0023] In an embodiment for better results, the senolytic active ingredient may be selected from a list consisting of: navitoclax (ABT-263) (CAS No.: 923564-51-6); venetoclax (Cas No.: 1257044-40-8), quercetin (CAS No.: 117-39-5), dasatinib (CAS No.: 302962-49-8), fisetin (CAS No.: 528-48-3), 17-DMAG (CAS No.: 467214-21-7), SSK1 (CAS No.: 2629250-69-5), A1331852 (CAS No.: 1430844-80-6), A1155463 (CAS No.: 1235034-55-5), procyanidin Cl (CAS No.: 37064-30-5), luteolin (CAS No.: 491-70-3), enzastaurin (CAS No.: 170364-57-5), piperlongumine (CAS No.: 20069-09-4), geldanamycin (CAS No.: 30562-34-6), tanespimycin (CAS No.: 75747-14-7), ansamycin (CAS No.: 72559-06-9), resorcinol (CAS No.: 102-29-4), purine- and pyrimidine-like N-terminal inhibitors, Sting inhibitors (e.g. H-151 (CAS No.: 941987-60-6)), curcumin (CAS No.: 458-37-7) and analogues, cardiac glycosides (such as ouabain (CAS No.: 11018-89-6), proscil laridin A (CAS No.: 466-06-8), digoxin (CAS No.: 20830-75-5), ouabagenin (CAS No.: 508-52-1), bufalin (CAS No.: 465-21-4), K-strophanthin (CAS No.: 560-53-2), strophanthidin (CAS No.: 66-28-4)), aspirin (CAS No.: 50-78-2), PZ15227 (CAS No.: 2143464-25-7), ARV825 (CAS No.: 1818885-28-7), fenofibrate (CAS No.: 49562-28-9), azithromycin (CAS No.: 83905-01-5), roxithromycin (CAS No.: 80214-83-1), tamatinib (R406) (CAS No.: 841290-80-0), mitoTam (CAS No.: 1634624-73-9),panobinostat (CAS No.: 404950-80-7), AT-406 (CAS No.: 1071992-99-8), ganetespib (CAS No.: 888216-25-9), Foxo4-DRI (CAS No.: 2460055-10-9), UBX0101, RG7112 (CAS No.: 939981-39-2), P5091 (CAS No.: 882257-11-6), P22077 (CAS No.: 1247819-59-5), and mixtures thereof.

[0024] In another embodiment, the senolytic active ingredient may be replaced by a senomorphic active ingredient selected from a list consisting of: rapamycin (CAS No.: 53123-88-9), metformin (CAS No.: 657-24-9), resveratrol (CAS No.: 501-36-0), SR12343 (CAS No.: 2055101-86-3), SB203580 (CAS No.: 152121-47-6), UR13756, BIRB796 (CAS No.: 285983-48-4), MK2.III (CAS No.: 1186648-22-5), PF-3644022 (CAS No.: 1276121-88-0), ruxolitinib (CAS No.: 941678-49-5), KU-55933 (CAS No.: 587871-26-9), KU-60019 (CAS No.: 925701-46-8), atorvastatin (CAS No.: 134523-00-5), pravastatin (CAS No.: 81131-70-6), pitavastatin (CAS No.: 147526-32-7), simvastatin (CAS No.: 79902-63-9), apigenin (CAS No.: 520-36-5), kaempferol (CAS No.: 520-18-3), quercetin (CAS No.: 117-39-5), epigallocatechin gallate (CAS No.: 989-51-5), genistein (CAS No.: 446-72-0), oleuropein aglycone (CAS No.: 31773-95-2), hydroxytyrosol (CAS No.: 10597-60-1), and mixtures thereof.

[0025] In an embodiment for better results, the senotherapeutic active ingredient content released within 4 hours may be 60% - 90%, preferably 80% - 100%, more preferably 90%.

[0026] In an embodiment for better results, the mass of senotherapeutic active ingredient per mg of nanoparticle may range from 10 pg - 400 pg; preferably 13 pg - 340 pg; wherein the loading efficiency is 5% - 50%, preferably 10% - 60%, more preferably 7% - 11%.

[0027] In an embodiment for better results, the nanoparticle size measured by ranges from 40 nm -400 nm; preferably 100 nm - 300 nm, more preferably 20 nm - 260 nm. The nanoparticle size may be determined by dynamic light scattering (DLS).

[0028] In an embodiment for better results, the nanoparticle may be coated or bound to antibodies, peptides, nanobodies or other recognition molecules that interact more specifically with proteins or receptors expressed in senescent cells such as uPAR, dipeptidyl peptidase 4 (or CD26), B2M, TPP1, LYAG, VCAM1, FBLN1, GALNS, CATC, IBP7, LRC15, CREL1, EZRI, CAVN2, PCYOX, SDCB1, MAZB1, HEXB, PCP, PTGIS, CSPG4, SYNPO, CD248, FKB11, GOT1B, among others that one skilled in the art might recognize.

[0029] In an embodiment for better results, the disassembly of the formulation may be obtained by other overexpressed enzymes in the lysosomes of senescent cells, such as other hydrolases (a-fucosidase, arylsulfatase A, a-mannosidase, N-acetyl-p-hexosaminidase, among others), including proteases (cathepsin, gelsolin, fibronectin, metalloproteinase, among others).

[0030] In an aspect related to the present disclosure, the nanoparticle may be used in medicine or veterinary. Preferably, the nanoparticle of the present disclosure may be used in the prevention or treatment of age-related diseases or disorders; preferably in the prevention or treatment of age-related liver diseases or disorders.

[0031] In an embodiment for better results, the nanoparticle of the present disclosure may be used as an improver of cell death in senescent cells; preferably liver senescent cells. The results of the present disclosure are unexpectedly due the accumulation of the nanoparticle of the present disclosure in the liver.

[0032] In an aspect related to the present disclosure, the nanoparticle may be used to coat a medical device to render them with anti-senescence properties; in particular a stent, a catheter, an implantable prothesis, among others.

[0033] Another aspect of the present disclosure relates to a formulation comprising the nanoparticle described in the present disclosure in a therapeutical amount and a suitable pharmaceutical carrier or vehicle.

[0034] In an embodiment for better results, the formulation comprising the nanoparticle described in the present disclosure may further comprise a second active ingredient; preferably wherein the second active ingredient is select from: anti-inflammatory active ingredients, chemotherapeutic active ingredients, antifibrotic active ingredients, immunomodulatory active ingredients, stress modulator ingredients, or growth factor inhibitor or enhancer ingredient, or mixtures thereof.

[0035] In an embodiment for better results, the formulation comprising the nanoparticle described in the present disclosure may be an aqueous solution.

[0036] In an embodiment for better results, the formulation comprising the nanoparticle described in the present disclosure, wherein the polydispersity index may be 0.02 - 0.5, preferably 0.1 - 0.4, more preferably 0.1-0.2. The polydispersity index may be determined by DLS.

[0037] In an embodiment for better results, the formulation comprising the nanoparticle described in the present disclosure, wherein the zeta potential may be -5 mV - -20 mV; - preferably 10 mV - -15 mV.

[0038] In an embodiment for better results, the formulation comprising the nanoparticle described in the present disclosure may consist of: powders, tablets, capsules, syrups, creams, ointments, gels, lotions, suspensions, emulsions, patches, inhalers, nebulizers, suppositories, enemas, eye drops, ophthalmic ointments, nasal sprays, or nasal drops.

[0039] In an embodiment for better results, the formulation comprising the nanoparticle described in the present disclosure in oral, intravenous, intramuscular, subcutaneous, transdermal, inhalation, rectal, vaginal, buccal, sublingual, topical, intrathecal, nasal, or ocular administration forms.

[0040] Another aspect of the present disclosure relates to a method for producing the nanoparticle described in the present disclosure, that may comprise the steps:providing a cyclodextrin-based polymer, an aqueous solution of an adamantane-modified oligosaccharide, and a senotherapeutic active ingredient;mixing the cyclodextrin-based polymer and the senotherapeutic active ingredient in an organic solvent;adding the adamantane-modified oligosaccharide to the mixture of the cyclodextrin-based polymer and the senotherapeutic active ingredient; preferably at 250 rpm, room temperature for 2 hours; to bind the cyclodextrin-based polymer with the adamantane-modified oligosaccharide and entrap the senotherapeutic active ingredient; as result the cyclodextrin-based polymer (host) provides a cavity or binding site that accommodates adamantane-modified oligosaccharide (guest) entrapping the senotherapeutic active ingredient;precipitating, forming, and collecting the nanoparticle formulation;optionally filtering or centrifuging the nanoparticle formulation.

[0041] In an embodiment for better results, the method may comprise the precipitating, forming and collecting step using a microfluidics apparatus.

[0042] In an embodiment for better results, the encapsulation efficiency of the nanoparticle described in the present disclosure may be 35% - 100%, preferably 30% -50%, more preferably 35 - 40%.BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0044] Figure 1: Schematic representation of the strategy of the present disclosure to identify a degradable NP with high senotherapeutic index. Initially, a library of 42 NPs with diverse physicochemical and degradation properties was synthesized. These NPs were formed by supramolecular hostguest interactions of cyclodextrin-based polymers with adamantane-modified galactan, an oligosaccharide of galactose. The NPs are both internalized by proliferative and senescent cells but only in the second ones are degraded at sufficient level to release the senotherapeutic to cell cytoplasm which then induces cell apoptosis.

[0045] Figure 2: Illustration of the results of an embodiment of the present disclosure related to the synthesis of p-cyclodextrin polymers. (A) Reaction scheme for the synthesis p-cyclodextrin polymers (Pl-P42). (B) Monomers used for the synthesis of the library: acrylated p-cyclodextrin (P-CD-AC) and amines (1-42).

[0046] Figure 3: Schematic representation of the optimization of NP formation and disassembly. (A) Schematic representation of the NP assembly through host-guest interactions between p-cyclodextrin polymers (Pl to P42) and adamantane-modified galactan (Gai-Ada) to encapsulate the senolytic drug Navitoclax. (B) Photograph of different solutions showing the successful formation of NPs: 1) aqueous solution of Gai-Ada, 2) mixture of p-cyclodextrin polymer randomly selected (P10) with Gai-Ada (host-guest ratio of 2:1) and 3) aqueous solution of polymer PIO. (C) Responsiveness of the assembled NPs with different synthesized Gai-Ada to the enzyme P-Galactosidase. The NPs (1 mg / mL) were formed with the selected polymer and incubated with P-Galactosidase (Aspergillus oryzae, 20 units) for 4 h at 37 °C. NPs count decrease was determined by DLS by the ratio between the number of NPs before addition of the enzyme and the number of NPs determined 4 h after enzyme addition. Results are expressed as Means ± SEM (n = 3).

[0047] Figure 4: Illustration of results of an embodiment of the present disclosure concerning the senotherapeutic index of soluble Navitoclax and Navitoclax-encapsulated NPs. (A) High-throughput screening of the NP library encapsulating Navitoclax. The black bars show the fold increase of cell viability in the best two formulations regarding soluble Navitoclax. The selection of the concentration of Navitoclax (100 nM) was based on the average NP library loading efficiency. The cells were incubated with the NPs for 4 h, washed and cell survival was evaluated after 48 h. The results are expressed as Means ± SEM (n = 3-6). (B - E) Quantification of cell survival (%) and half maximal inhibitory concentration (IC50) of Nav (B) and the best nanoformulation hits from the screening, NP4 (C) and NP17 (D), as well as a non-hit nano formulation (E) on proliferative HUAECs (HUAECs) and gamma irradiation-induced senescent HUAECs (sHUAECs). The senolytic index for soluble Nav or Nav-loaded on NP formulations is shown in the tables below. The results are expressed as Means ± SEM (n = 3).

[0048] Figure 5: Illustration of results of an embodiment of the present disclosure related to the NP17 degradation and Nav release studies in the presence of p -galactosidase. (A) Scheme representation of the effect of the enzyme P-Galactosidase on Nav-NPs and performed assays. (B) Chromatogram obtained from the HPLC analysis of the disassembled NPs in DMSO to determine the host / guest polymer ratio of NP17. The retention time of Gai-Ada is 4.3 min. (C) Representative image of NP17 obtained by TEM. Bar corresponds to 200 nm. (D) Distribution of NP diameters as evaluated by TEM and DLS analysis. (D) Navitoclax release profile from NP17 with or without enzyme P-Galactosidase (P-Gal and no P-Gal, respectively) as well as denatured P-Galactosidase (P-Gal d.). NP17 (0.1 mg / mL, [Nav]loaded = 7.6 ± 0.51 pM (7.8 ± 0.52 pg / mL)) was suspended in 1 mL PBS (pH = 4.5), stirred at 309C and at different time points a certain sample volume was removed, centrifuged and the amount of Navitoclax released was determined by HPLC relative to a calibration curve. (E) NPs count decrease evaluated by DLS upon addition of enzyme P-Galactosidase (1 unit) to NP17 (50 pg / mL). (D) Percentage of galactan hydrolysed from NP17 upon addition P-Galactosidase, assessed by galactose determination assay (P-galactose dehydrogenase (Gal-DH)). Results are expressed as Means ± SEM (n = 3).

[0049] Figure 6: Illustration of results of an embodiment of the present disclosure related to the NP17 biodistribution in old rats (16 months-old) and uptake of NP17-Cy5 by liver cells. (A) Ex vivo images of the tissues at 24 h and 72 h after injection with NP17-Cy5. (B) Quantification of NP biodistribution in rat tissues collected at 24 h or 72 h post-injection (n = 1, each time point), as measured by Cy5fluorescence. (C-E) Wistar rats (n=2) were injected with NP17-Cy5 (corresponding to 0.2 mg / Kg / day of Navitoclax) and sacrificed after 4 h post-injection. The liver was perfused, enzymatically digested and mechanically disaggregated to yield a multicellular suspension. Hepatocytes (Hep), LSECs, Kupffer cells (KCs) and hepatic stellate cells (HSCs) were isolated from the cell suspension initially by a density gradient centrifugation and then by differential adherence time to coated substrates (see Materials and Methods). (C) Representative images of the different cell types isolated from the liver, 4 h upon IV injection. Magnification is 40x and scale is 50 pm. (D) Quantification of the total area occupied by the NP17-Cy5, normalised by the number of nuclei per field. Each dot represents different fields (n > 5) of Wistar rats with 20 months (n = 2). (E) NP average size in each cell. Each dot represents a different field (n = 2).

[0050] Figure 7: Illustration of results of an embodiment of the present disclosure related to the effect of NP17 in liver function of old rats (20 months-old). (A) Schematic representation of the animal experiments set up. (B) Changes in portal pressure of rats treated with vehicle or NP17. (C) Scanning Electron Micrograph of the internal surface of the fenestrated liver sinusoids demonstrating the low porosity of the endothelium from rats injected with vehicle or NP17. Scale bar corresponds to 1 pm. (D) Quantification of fenestration frequency. (E) Representative images of fibrosis as evaluated by Sirius red staining. Scale bar corresponds to 500 pm. (F) Quantification of tissue area positive for Sirius red staining. In B, D and F, values are presented as box and whiskers plots with min to max values. P-values above data refer to the comparison of young versus old.

[0051] Figure 8.1-8.2: Illustration of results of an embodiment of the present disclosure related to the effect of NP17 in the expression of senescence markers in the liver of old rats (20 months-old). (A, D, G, J) Representative images for the expression of p21 (A), pl6 (D), HMGB1 (G) and vWF (J) in endothelial and non-endothelial cells of old rat livers treated with vehicle or NP17. Magnification is 50x and scale bar is 50 pm. (B, C, E, F, H, I, K) Quantification of the expression of p21 (B and C; n > 7 / group; a total of 50, 42 and 59 images were analyzed from Veh, Nav and NP17, respectively, each image with >100 nuclei), pl6 (E and F; n > 6 / group; a total of 43, 43 and 49 images were analyzed from Veh, Nav and NP17, respectively, each image with >100 nuclei), HMGB1 (H and I; n > 8 / group; a total of 44, 41 and 52 images were analyzed from Veh, Nav and NP17, respectively, each image with >20 nuclei) and vWF (J and K; n > 6 / group; 5-6 region of interest (area = 0.2 mm2) were analysed per microscopic image) collected from endothelial and non-endothelial cells of old rat livers treated with vehicle or NP17. For HMGB1 quantification at least 5 large blood vessels and 500 sinusoidal ECs were included in counting per animal. Each dot in the graph corresponds to the analysis of an independent animal. Values are presented as box and whiskers plots with min to max values. P-values above data refer to the comparison to the vehicle treatment.DETAILED DESCRIPTION

[0052] The present disclosure relates to senescence-associated beta-galactosidase (SA-p-Gal) degradable nanoparticle formulation for the delivery and release of a senolytic active ingredient. A novel library of nanoparticles that are degradable by SA-p-Gal overexpressed in senescent cells was designed. The novelty of this library relies in the fact that the NPs are prepared by host-guest interactions between SA- -Gal-cleavable oligosaccharides derivatized with adamantane and cyclodextrin-based branched polymers with variable composition, being all the components organic and degradable (relevant to avoid cell transformation effects observed in previous inorganic formulations tested for the release of senolytic active ingredients), the NP of the present disclosure respond to SA-p-Gal with variable degradation kinetics based in their chemical composition, releasing the senolytic active ingredient at different rates, and are able to release most of the encapsulated senolytic active ingredient than some of the formulations tested before (90% of the initial loading of the formulation is released versus 30% in other formulations). Navitoclax, also known as ABT-263, was selected as a proof-of-concept senolytic active ingredient. It has been reported to target Bcl-2 family members such as Bel— 2, Bel— xl, and Bcl-w, inducing apoptosis in senescent cells, which, akin to cancer cells, rely on anti-apoptotic defences. However, Navitoclax is also a chemotherapeutic agent, with reported toxic side effects including transient thrombocytopenia and neutropenia. The NPs of the present disclosure were characterized regarding NP efficiency formation, size, charge, encapsulation and loading efficiency. Next, the in vitro senotherapeutic potential of the formulation was evaluated against senescent endothelial cells versus proliferative ones, and the half-maximal inhibitory concentration (IC5o) values calculated for the hit formulations. Finally, the in vivo senotherapeutic potential of one of the hit formulations was attested in an aged rat model. The results indicate that the release kinetics of the senolytic active ingredient affects the efficacy of the formulation, furthermore that NPs accumulate higher in liver sinusoidal endothelial cells than in other liver cells and they are able to decrease the number of senescent cells and ameliorate liver function.

[0053] The following sections provide a detailed description of the materials and methods used in the embodiments of the present disclosure.Synthesis of acrylated p-cyclodextrin

[0054] The acrylated p-cyclodextrin monomer (P-CD-AC) was synthesized and purified according to a previously reported procedure by Cao et al.26. Briefly, under ice-bath conditions, p-cyclodextrin (P-CD) (0.5 g, 0.44 mmol, Alfa Aesar) was dissolved in 15 mL of / V, / V'-dimethylformamide (DMF, VWR Chemicals) and then triethylamine (EtaN) (0.51 mL, 3.68 mmol, Sigma-Aldrich) was added. The resulting mixture was then flushed with nitrogen to remove oxygen from the media and acryloyl chloride (AC) (0.28 mL, 3.45 mmol, Sigma-Aldrich) dissolved in 5 mL of DMF was added dropwise using a constant pressure dropping funnel. Then reaction was then carried at room temperature for 24 h in a sealed container. Aftercompletion of the reaction, to remove any undissolved solid impurities (triethylamine hydrochloride salt), the product was centrifuged at low speed and the filtrate evaporated to dryness using a rotary distillation. Acetone (VWR Chemicals) was then added to precipitate a solid product, filtered and dried under vacuum for 24 h. The derivatization of P-CD with acrylate groups was confirmed via proton nuclear magnetic resonance (1H-NMR) analyses, dissolving the resulting white powder in deuterium oxide (D2O).Synthesis of p-cyclodextrin-based polymersThe library of polymers was synthesized via Michael-type addition, were the -CD-AC (100 pL, 200 mM, DMSO) was mixed with the 42 different amine monomers (190 pL, 200 mM, DMSO) (Table 1). The reaction was carried out in a 2 mL Eppendorf under stirring at 250 rpm (IKA KS4000), at 60 °C for 5 days. At the end of the 5 days, the unreacted vinyl groups were capped with the respective amines 1-42 (20 mL, 200 mM, DMSO) for 2 h (60 °C, 250 rpm). The end product was stored at -20 °C until further use. Synthesis of Galactan-Adamantane (Gai-Ada) polymer

[0055] For the synthesis of the Gai-Ada polymer, to an aqueous solution of galactan (Gal) (166 pmol; P(1.4)-galacto-oligosaccharide, from potato; low molecular weight, Megazyme; monosaccharide (%): galactose: arabinose: rhamnose: galacturonic acid = 87:3:4:6) was added l,l'-carbonyldiimidazole (CDI) (38.3 pmol, Sigma-Aldrich) and the solution was left under stirring for 10 min up to 1 h at RT and 250 rpm. Then 1-adamantemethylamine (Ada) (33.3 pmol, Sigma-Aldrich) in dimethylformamide (DMF, VWR Chemicals) was added and the reaction was carried for additional 24 h, under stirring (250 rpm) at RT. To remove unreacted Ada, the product was dialyzed for 24 h against water (Spectr / Por, MWCO 12-14 kDa). The dialyzed solution was centrifuged at 10.000 g for 10 min and the resultant supernatant lyophilized for at least 48 h. NMR spectroscopy was performed to determine the degree of substitution (DS) of Ada per galactose monomer, by dissolving the Gai-Ada in deuterium oxide (D2O).Characterization of polymers by NMR spectrometry

[0056] 1H NMR spectra were recorded on a Bruker Avance III spectrometer at 400 MHz. The spectra were recorded in deuterated solvents using a pulse of 90°, relaxation delay of 4.0 s. Tetramethylsilane (TMS) was used as the internal standard. Diffusion-ordered spectroscopy (DOSY) experiments were performed on a Bruker NEO 750 with a probe of type PA TXI 750S5 H&F-C / N-D-05 Z to determine the molecular weight of Gai-Ada (dissolved in D2O, 4 mg / mL) and the polymer P17 (dissolved in DMSO-d6, 8 mg / mL). The number of linear gradient steps was set to be 16. The standard Bruker pulse program, "Iedbpgp2s", was used. NMR spectra were analysed using the software Mnova from Mestrelab Research (v. 10.0.1).Synthesis of NP library

[0057] NPs were prepared by dissolving the guest-polymer (Gai-Ada ([Ada] = 0.5 mM)) in 1.0 mL water, and then a pre-mix of p-CD-based polymer ([ -CD] = 1.0 mM in DMSO) with Navitoclax (100 pM, Octagon Chemicals) was added. For the synthesis of empty NPs, the Navitoclax volume was replaced with DMSO. The mixture was stirred in shaker at 250 rpm, room temperature for 2 h. The NPs were then centrifuged 3 times at 10000 g for 10 min to remove free Navitoclax. NP formation efficiency (%) was defined as the ratio of total weight of obtained NP and theoretical weight of the initial polymers (Equation 1). The concentration of Navitoclax was determined by a calibration curve (y = 0.01118x + 0.004478) and reading the supernatants in a microplate reader at X = 355 nm (Synergy Hl, BioTek, USA). Encapsulation efficiency (EE) was defined as the ratio of actual and original amount of Navitoclax encapsulation in the NPs (Equation 2). Drug loading (DL) was defined as the ratio of actual drug loaded and the mass of NPs (Equations 3).100(1)

[0058] %FE - Percentage of nanoparticle formation efficiency. M(NP) is defined as the weight of the NP obtained after purification and freeze-drying and M (Polymers) is the theoretical polymers weight used for NP synthesis.(Total Drua — Free Drag)F = - - -Total (2)

[0059] %EE - Percentage of Encapsulation Efficiency. Total drug refers to total amount added in weight. Free drug refers to total amount of drug that was not incorporated in the NP.Free Drsta)— * 100%Total of NP (3)

[0060] %DL - Percentage of Drug Loading. Total of NP refers to total concentration of NP in the solution.NP characterization: size and zeta potential analyses

[0061] The diameter and zeta potential of NPs was measured by photon correlation spectroscopy (PCS) using quasi-elastic light scattering equipment (ZetaPALS analyser, Brookhaven Instruments Corp.,Holtsville, NY) and ZetaPlus™ Particle Sizing Software (version 4.03). The scattered light was collected at fixed angle of 90°. To measure NPs size, a suspension of NP in water (molecular biology grade, Fisher Scientific) was added to a cuvette (50 pg / mL, 1.5 mL), allowed to stabilize for 10 min and then analysed at room temperature (3 times). The zeta potential of NPs was determined in a 1.0 mM KCI solution at 25 °C (50 pg / mL, 1.5 mL). All data were recorded as the mean of 5 measurements runs. NP stability in water and cell culture medium (EGM-2) with fetal bovine serum (FBS, 10%) was measured by DLS with a NP concentration of 50 pg / mL.NP characterization: TEM analyses

[0062] A suspension of NP17 (500 pg / mL) was prepared in molecular grade water. A droplet of the suspension was added to the surface of an ultrathin carbon coated 400 mesh copper grid and left air-dry for 5 h at room temperature in a closed petri dish. NPs were viewed with a JEOL JEM-2010-HT microscope. Digital images were acquired with a fast-readout "OneView" 4k x 4k CCD camera that operates at 25 fps (300 fps with 512 x 512 pixel) and features drift correction. The diameter of NPs was analysed with the Particle Analysis tool from ImageJ.NP characterization: disassembly studies by DLS analyses

[0063] NP disassembly in the presence of P-Galactosidase from Aspergillus oryzae (G5160, > 8units / mg, Sigma-Aldrich / or the denatured enzyme (10 min at 70 °C in a water bath) was measured by DLS to assess NP count decreased over time. Nav-loaded NPs (50 pg / mL) were resuspended in 1.5 mL of 0.1M acetate buffer (pH = 4.5) and P-Galactosidase (1 unit) was added and stirred at 250 rpm and 30 °C. At different time points, the average count rate was determined as for the sample without the addition of the enzyme.NP characterization: degradation of galactan in the NPs

[0064] High purity galactose dehydrogenase from E. Coli (GDH) (Megazyme) was used for the determination of D-galactose upon hydrolysis of galactan (3.5 mg / mL), adamantane-modified galactan (3.5 mg / mL) or NPs (1 mg / mL) in the presence of p-galactosidase. The amount of D-galactose was measured in the reserve reaction (NAD+to NADH) at 25 °C in a reaction mixture containing 0.1 M Tris. HCI buffer (pH = 8.7), 2.0 mM NAD+and a suitable amount of enzyme (4 units for Gal and Gai-Ada and 20 units for NPs) to obtain a measurable increase in the absorbance at 340 nm using a Synergy Hl microplate reader. The concentration of D-galactose hydrolysed from Gai-Ada polymers or NPs was determined by the linear regression equation, y = 1.911x - 0.04756 (R2= 0.9894).NP characterization: Navitoclax release studies

[0065] Nav-loaded NPs (0.1 mg / mL or 1 mg / mL) was suspended in 1 mL of 0.1M acetate buffer (pH = 4.5) and p-galactosidase (2 or 20 units) was added and stirred at 250 rpm and 30 °C. At different time points, aliquots were taken, centrifuged and the amount of Navitoclax released was determined by HPLC and calculated by the linear regression equation, y = 641107x (R2= 0.9916) (absorbance at 254 nm,integration of the peak at a retention time of 14.4 min), obtained with the soluble Navitoclax. As a control, the same procedure was performed without adding the enzyme to the NPs suspension (or adding the denatured enzyme), as a control.

[0066] Analytical RP-HPLC was performed on a Shimazo Prominence-1 LC-2030 C 3D, using a XBridge C183.5 pm 4.6 x 250 mm column (Waters). Analytes were separated at a flow rate of ImL / min with a 2 min linear gradient of 5% solvent A (acetonitrile (Fisher Scientific) + 0.08% of trifluoracetic acid (TFA) (Sigma-Aldrich)) - 95% solvent B (water + 0.08% of TFA) followed by an 8 min linear gradient to 95% solvent A-5% solvent B, which was held for an additional 5 min and then returned to initial conditions over 5 min.NP characterization: ratio of host-guest polymers

[0067] Lyophilized Nav-loaded NPs (50 pg) were resuspended in DMSO to disassembly the NPs and the amount of Gai-Ada polymer was determined by HPLC and calculated by the linear regression equation, y = 4069217x (R2= 0.9987) (absorbance at 215 min, integration of the peak at a retention time of 4.33 min), obtained with the soluble Gai-Ada polymer.Labelling of NPs with Cy5-NHS

[0068] Navitoclax-loaded NPs were labelled with Cyanine5 (Cy5) NHS ester (Lumiprobe, UK) for some in vitro and in vivo studies. In brief, NPs (1 mg / mL) were diluted in 2% of DMSO and in 20 mM of NaHCOs buffer (pH 8.5), and then conjugated with the fluorophore at a weight ratio (w / w) of 1:100 (Cy5:NP) for in vitro studies and 1:75 (Cy5:NPs) for in vivo studies. This conjugation process took place during 2 h at room temperature under stirring at 250 rpm. The resulting NPs were centrifuged twice at 10.000 g for 10 min to remove unbounded Navitoclax and Cy5. The pellet was then resuspended in milligrade water. The supernatant, containing free Cy5, was collected and quantified by using Synergy Hl microplate reader at X = 650 nm (BioTek). The NPs were aliquoted, snap-frozen with liquid nitrogen and stored at -80 °C.Cell culture

[0069] Human Umbilical Artery Endothelial Cells (HUAECs) line (Cell Applications, USA) was used and cultured in Endothelial Cell Growth 2 (EGM-2 Promocell) with the following supplements: endothelial cell growth medium supplement mix (PromoCell), penicillin-streptomycin (PS, 50 U / mL: 50 mg / mL, Invitrogen, USA) and 2% fetal bovine serum (FBS, Life Technologies). Cells were cultured in 0.1% gelatine-coated well plates in passages between 5 and 7. Primary LSECs isolated from human liver (Innoprot, Spain) were purchased and cultured in Endothelial Cell Medium (ECM, Innoprot) with the following supplements: Endothelial Cell Growth Supplement (Innoprot), penicillin / streptomycin solution (Innoprot), and FBS (Innoprot, Ref: P60104). Cells were cultured in a fibronectin (2 pg / cm2) coated flask in passages between 6 and 8. A549 cells (CCL-185TM, ATCC) are a lung cancer cell line and were usedand cultured at a density of 26,700 cells / cm2in Dulbecco's Modified Eagle Medium (DMEM, Corning) with 10% FBS (Gibco) and pen-strep (Gibco 50 U / mL:50 mg / mL). Once confluence was achieved, A549 cells were harvested, by incubation with 0.25% Trypsin / PBS for 10 min, centrifuged at 1100 rpm for 5 min, resuspended in DMEM and then plated for subsequent steps. All cells were incubated in 20% O2 and 5% CO2 at 37°C, with media changed every 2-3 days. Cells were routinely tested for mycoplasma using the universal Mycoplasma Detection Kit (ATCC).Induction of cell senescence

[0070] To induce senescence, HUAECs, LSECs or A549 cells were irradiated in a Petri dish or 6-well plate, with 10 GY Gamma Irradiation (RS 2000 irradiator, RadSource Technologies) after reaching a confluency of 60-80%. First, cells were washed once with lx DPBS (GibcoTM, Thermofisher Scientific), then irradiated and washed again with lx DPBS. Then, cells were incubated for 3 to 4 days in culture media, then plated at a density of 62,500 cell / cm2to a 96 well plate and incubated again in culture media. The screening assay took place on the 7thday after irradiation for HUAECs or on the 10thday for LSECs and A549 cells, when cells showed a relevant senescence phenotype. Irradiated and proliferative (non-irradiated) cells were submitted to the same procedures. Proliferative cells were plated to the 96 well plate with a cell density of 15,625 cells / cm2. To induce cell senescence with doxorubicin, LSECs were plated at a density of 12xl03cells / cm2in T25 flasks and when 70-80% confluency was reached, cells were exposed for 24 h to doxorubicin hydrochloride (200 nM, TargetMol). Cells were then washed thrice with lx PBS and left for one day before replating in 96-multiwells. At day 6 post-Dox, cells were characterized for different senescent markers.Senescent cell characterization: SA- -galactosidase (SA-P-Gal) activity

[0071] The senescence level of irradiated HUAECs was assessed with a fluorescent substrate for p-galactosidase, C12FDG (5-dodecanoylaminofluorescein di-p-D-galactopyranoside, ThermoFisher Scientific). Briefly, cells were incubated with fresh medium for 1 h in a CC -free incubator, C12FDG was added (10 pM) and incubated for 2 h. To evaluate SA-P-Gal in LSECs, a colorimetric assay (ab65351, Abeam) was used according to manufacturer's instructions. Cells were counterstained with 4', 6'-diamino-2-fenil-indol (DAPI, 2 ng / pL) solution for nuclei detection. Images were acquired in IN Cell Analyser 2200 (GE Healthcare Life Sciences) and data were expressed as percent of total DAPI-positive cells.Senescent cell characterization: quantitative real time polymerase chain reaction (qRT-PCR) analyses

[0072] RNA from cells was isolated with RNeasy Mini Kit (ref. 74104, Qiagen) and its concentration measured with a Nanodrop Spectrophotometer (Thermo Fisher Scientific) at 260 nm of absorbance. Reverse transcription to cDNA was performed with qScript® cDNA SuperMix (#95161-025, Quantabio), according to manufacturer's specifications, in the CFX Connect™ Real-Time System (Bio-Rad). qRT-PCR was run by using NZYSpeedy qPCR Green Master Mix (#MB22403, NZYtech) in a CFX96™ Real-Time PCRDetection System (Bio-Rad) for 40 cycles. Samples were measured in triplicates and the mean cycle threshold (Ct) values were quantified. Quantification of target genes was normalized to the housekeeping gene to each cell, according to the 2-AACTmethod. The list of primers for SYBR Green technology can be found in Table 2.Table 2. List of pre-designed mouse primer pairs from Sigma-Aldrich.Evaluation of the senotherapeutic potential of NPs or Nav against senescent cells

[0073] 7 days (HUAECs) or 10 days (LSECs and A549) after irradiation, the cells were exposed for 4 h to different concentrations of Navitoclax, NP library or empty NPs. Subsequently, the cells underwent three washes with supplemented culture media and were left for an additional 48 h for further analysis, with no changes made to the media during this period. At day 6 post-Dox, LSECs were exposed for 4 h to NP17 at different concentrations (1 or 10 pg / mL). Then, cells were washed thrice with supplemented culture media and left for 48 h for further analysis. As a control, proliferative cells were also incubated with NPs. Cell viability was evaluated using Hoechst H33342 (Sigma-Aldrich, 0.25 pg / mL) and Propidium Iodide (PI, Sigma-Aldrich, 0.25 pg / mL) staining. Images were acquired on a high-content microscope (In Cell Analyzer 2200 / 2000, GE Healthcare) and at least 10 random fields per well were imaged (20x objective). Results were analysed using IN Cell Developer Toolbox software (GE Healthcare). The number of cells positive to propidium iodide were normalized to the total nuclei count. Results were normalized to the non-treated condition, i.e., cells only exposed to cell media.Lysosome colocalization studies

[0074] For lysosome colocalization studies, Nav-loaded NPs-Cy5 (1 pg / mL) were incubated with HUAECs (proliferative and irradiated) for 1 h and 4 h. Cells were washed 3 times in lx PBS and incubated with Hoechst H33342 (Sigma-Aldrich, 0.2 pg / mL) and 100 nM LysoTracker™ Red DND-99 (Invitrogen) for 30 min. Cells were washed once in PBS and incubated in medium and Trypan Blue 1:100 (Gibco™ Trypan Blue Solution, 0.4). Then, cells were imaged on a high-content microscope (In Cell 2200, GE Healthcare) with a 20x objective (12 random fields per well). The obtained images were then analysed with ImageJ co-localization macro Just Another Colocalization Plugin (JACoP). Mander's coefficient was used as the colocalization readout.0-galactosidase knockdown studies

[0075] For the transient knockdown of beta-galactosidase, A549 were seeded in a T75 flask (26,700 cells / cm2) in Dulbecco's Modified Eagle Medium (DMEM, Corning) with 10% foetal bovine serum (FBS, Gibco)) and pen-strep (Gibco 50 U / mL:50 mg / mL) and irradiated at 10 Gy once confluent. Three days post-irradiation, A549 cells were plated in a 96-well plate (30000 cells / well). At day 6 post-irradiation, medium was changed to DMEM without P / S and cells were transfected with ON-TARGETplus Human GLB1 (2720) siRNA - SMARTpool (siGLBl, Bioportugal) or with miRIDIAN microRNA mimic Negative Control (Scramble miRNA, Dharmacon™) at a final quantity of 5 pmol. Transfections were carried on using Lipofectamine’ RNAiMAX Reagent, according to manufacturer's instructions. Forty-eight hours after transfection, cells were incubated with NPs (1 pg / mL). After 4 h, cells were washed 3 times in lx PBS and stained with Hoechst H33342 and PI. Forty-eight hours after incubation with NPs, 12 random fields per well were imaged on a high-content microscope (In Cell 2200, GE Healthcare) with a 20x objective. DAPI channel was used for the Hoechst stain and Texas Red / Cy3 for the PI stain. The obtained images were then analysed using the IN Cell Investigator software (GE Healthcare Life Science), using a protocol that counts total nucleus per well stained with Hoechst and total apoptotic nucleus stained positive for PI, giving a total cell count per well and cell count of both Hoechst and PI positive cells.In vivo study: biodistribution of the NPs

[0076] Biodistribution was assessed by intravenous injection of Cy5-NP17 (equivalent to 0.2 mg / kg / day of Navitoclax). After the designated time points (24 or 72 h), animals were euthanized via a pentobarbital overdose (200 mg / kg, Nembutal) and perfused with physiological saline solution containing 1% heparin to facilitate exsanguination. Subsequently, selected organs were extracted, and fluorescence was analysed using the IVIS LUMINA SERIES III (PerkinElmer). Average radiant efficiency was calculated using Living Image 4.7.3 Software (PerkinElmer).In vivo study: efficacy study of the NPs

[0077] Aged male Wistar rats (Janvier Laboratories, France; 20 months old) were administered with Navitoclax (50 mg / kg / day; n=8) by gavage or injected through the penile vein with NP17 (correspondingto 0.2 mg / kg / day of navitoclax; n=9) or vehicle (PBS; n=8). For the preparation of the formulations, Navitoclax was dissolved in 10% DMSO + 90% PEG-400 and NPs were diluted in PBS. The treatments were administered for 2 weeks (4 doses per week), with a 1-week of interval in between. For the injection of the NPs or the vehicle, rats were anesthetized with isoflurane. Animals were allowed to recover for an additional 4 weeks without any manipulation. On the day of the sacrifice, liver hemodynamic was performed prior to organs collection. Animals were caged in pairs on a 12:12 lightdark cycle in environmentally controlled animal facilities at the Institut d'lnvestigacions Biomediques August Pi i Sunyer (IDIBAPS). All experiments were approved by the Laboratory Animal Care and Use Committee of the University of Barcelona and were conducted in accordance with European Community guidelines for the protection of animals used for experimental or other scientific purposes (EEC Directive 86 / 609).In vivo study: liver hemodynamics

[0078] Previous to the study, animals were food-deprived for 8-12 h to avoid alterations in splanchnic hemodynamics. The animals were anesthetized with inhaled isoflurane (Isovet) during the whole study. Mean arterial pressure (MAP) and heart rate (HR) were measured by cannulating the femoral artery, and portal pressure (PP) was measured by cannulating the ileocolic vein. The measures were performed with heparinized p50 catheter (Portex) connected to a pressure probe. Portal blood flow (PBF) was measured after the splanchnic vein bifurcation, next to the entrance to the liver, with specific nonconstrictive perivascular ultrasonic transit-time flow probe (Transonic Systems Inc.). Pressure and flow probes were connected to a Powerlab (4SP) and data was displayed into LabChart v5.5.6 software file. Hemodynamic parameters were acquired following a 20 min stabilization period27. After completion of the hemodynamic study, blood samples were collected via the cava vein. Finally, animals were euthanized through pentobarbital overdose (200 mg / kg, Nembutal) and tissue samples were collected for molecular, ultrastructural, and histological determinations.In vivo study: organ collection and blood processing

[0079] During sacrifice, organs were weighed, washed in PBS and immediately snap-frozen (for gene expression analysis), formalin-fixed (for immunohistochemistry) or placed in OCT for subsequent analysis. For paraffin embedded organs, they were fixed for 20-24 h at 4 °C using 10% formalin, washed twice with PBS, placed in 70% ethanol, and again washed in PBS before embedding in paraffin. To reduce the intrinsic variability of the organs, the same section of the organs was always collected depending on the experiment to be performed. Blood was collected in EDTA K3E tubes and centrifuged at 4000 g for 20 min at 4 °C to isolate plasma, or in Serum gel Z / l.l tubes and centrifuged at 10.000 g for 5 min at 4 °C.In vivo study: toxicity analyses

[0080] Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and albumin were determined through standard methods by Echevarne Laboratories (Barcelona, Spain).In vivo study: scanning electron microscopy (SEM) analyses

[0081] Liver sinusoidal ultrastructure was characterized using electron microscopy as previously described22. Briefly, 5 animals of each group were perfused through portal vein with a solution containing 2.5% glutaraldehyde and 2% paraformaldehyde (PFA) and fixed overnight at 4 °C. Samples were washed 3 times with 0.1 M cacodylate buffer. Liver sections were fixed with 1% osmium in cacodylate buffer, dehydrated in ethanol, and dried with hexamethyldisilazane. Six randomly selected blocks from each animal were mounted onto stubs, and sputter coated with gold. 10 images per animal were acquired at a resolution of 15,000x using a Jeol 6380 Scanning Electron Microscope (JEOL Ltd). Liver sinusoidal fenestrations were quantified using Biodock, Al Software Platform (Biodock 2023).In vivo study: plasma proteomics

[0082] For cytokine and chemokine measurements, plasma-EDTA samples from vehicle and NPs-treated rats were analysed at Eve Technologies (Canada) for the quantification of a large array of SASP factors using the Rat Cytokine Array / Chemokine Array 27 Plex, RD27.In vivo study: immunofluorescence (IHC-IF) and immunocytochemistry (ICC) analyses

[0083] Liver from rats was collected, formalin-fixed paraffin-embedded to obtain 4 pm sections. Upon deparaffinization and rehydration, heat-mediated antigen retrieval with 10 mM TRIS-EDTA buffer (pH 9) was performed and, whenever necessary, slides were permeabilized with 0.1-1% Triton X-100. Samples were blocked with antibody diluent with background reducing agent (Dako) or 1:60 normal goat serum diluted in 0.1% bovine serum albumin for 30 min. Then, slides were incubated overnight at 4°C with primary antibodies, followed by 30 min incubation at room temperature with respective secondary antibodies (Supplemental Table 7). To detect Galectin 9 accumulation in the endolysosomes, cells were fixed with PFA (4%) for 10 min and permeabilized in PBS with 0.1% Triton for 10 min. After 3 washes with PBS (5 min each), cells were blocked with 1% BSA and 0.3 M Glycine in PBS for 45 min at RT followed by incubation with the primary antibody for Galectin 9 at 4°C in PBS with BSA (1 %). On the next day, after 3 washes in PBS, cells were incubated with Alexa fluor-488 donkey anti-mouse for 1 h at RT. Cell nuclei was stained with DAPI (2 pg / mL) and slides were mounted with Vectashield mounting media (Vector Laboratories). At least 5 different images per animal were obtained in LSM 710 confocal microscope with 40x magnification. Quantifications were performed using ImageJ or QuPath software's.In vivo study: Sirius red staining

[0084] For fibrosis evaluation, 4 pm sections were stained with 0.1% Sirius red in picric acid aqueous solution (Sigma). Ten images from each slide at 40x magnification were analysed using a microscope BX51 (Olympus) equipped with a digital camera. Sirius red-stained area was quantified using ImageJsoftware (NIH), and values are represented by mean of fibrosis percentage per total area. Sinusoidal fibre was evaluated by the elimination of fibre associated with large vessels.In vivo study: RNA In Situ Hybridization (RNA-ISH)

[0085] RNA In Situ Hybridization (RNA-ISH) was performed with the RNAScope’ Multiplex Fluorescent V2 Assay from Advanced Cell Diagnostics (ACD), with minor modifications to the manufacturer's recommended protocol. Briefly, paraffin sections were baked in ACD HybEZ™ II Hybridization System (Cat. No. 240200ACD-2, SERIAL 2017 33072) for 60 min at 60 °C and immediately submerged twice in xylene, followed by 100% ethanol. Sections were incubated with RNAscope Hydrogen Peroxidase solution (Ref. 322335) for 10 min and washed with distilled water. Antigen retrieval was performed for 45 min in a water bath at 100 °C with RNAscope IX Target Retrieval Reagent (Ref. 322001). Slides were rinsed in distilled water, dry in 100% ethanol for 2 min, and a hydrophobic barrier (Ref.310018) was designed around each section. Slides were then incubated with RNAscope Protease Plus (Ref. 322331) for 30 min at 40 °C, washed with distilled water, incubated with target probe for p21 (Rn-Cdknla, Ref.42851) or pl6 (Rn-Cdkn2a, Ref. 423861) for 2 h at 40 °C, and washed with RNAscope IX Wash Buffer (WB; Ref. 320058). RNAscope Multiplex Fluorescent Detection Reagents (Ref. 323110) were used for detection of mRNA targets by using (i) AMP1, 30 min at 40 °C, (ii) AMP2, 30 min at 40 °C, (iii) AMP3, 15 min at 409C, (iv) HRP-C1 probe, 15 min at 409C, (v) Opal Dye 570 (Ref. FP1488001KT), diluted 1:1000 in RNAscope Multiplex TSA buffer (Ref. 322809), 30 min at 40 °C, and (vi) FL v2 HRP blocker, 15 min at 40 °C. Sections were washed twice with WB between each step. Sections were counterstained with DAPI (2 pg / mL) and mounted with Vectashield mounting media. At least 5 z-stack images (z = 0.3 pm) per slide were acquired using the confocal microscope LSM 710 (Zeiss) with a 40x objective. Quantification was performed in QuPath (0.4.3) and the percentage of positive cells was assessed based on the number of cells with >1 dot / cell.In vivo study; isolation of LSEC, KC, HSC and Hepatocytes cells for Cy5-NP17 detection

[0086] Wistar rats were injected with Cy5-NP17 (corresponding to 0.2 mg / Kg / day of Navitoclax) for 4 h (n = 2), as reported above. In vivo cell internalization of Cy5-NP17 were assessed by isolation of primary hepatocytes and non-parenchymal cells using the '4 in 1' protocol as previously reported28. Briefly, liver was perfused, digested with 0.015 % collagenase A (103586, Roche, Sant Cugat del Valles, Spain), and mechanically disaggregated, obtaining a multicellular suspension. Hepatocytes were purified by low-speed centrifugation, and non-parenchymal cells were separated using a three-phase iodixanol (Optiprep™, Sigma-Aldrich, Saint Louis, MO, USA) density gradient centrifugation. Subsequently, the upper interphase, which contained the HSCs, was directly seeded, whereas the lower one, enriched in LSECs and KCs, was further purified by differential adherence time to non-coated substrates. Highly pure (>95%) and viable (80-95%) cells were seeded at high density on collagen treated p-Dish 35 mm high (Ibidi GmbH, Germany). Isolated cells were stained with NucBlue Live ReadyProbes (Invitrogen) for 20minutes. Cells were visualized with TCS SPE Confocal System with DM I 4000 B (Leica), with a 40x objective. NPs were quantified using Analysis Particle tool in Image J.Statistical analysis

[0087] Statistical testing was performed using GraphPad® Prism 9.0 Software. Outliers were excluded by ROUT analysis (Q= 1 %). Kolmogorov-Smirnov normality or Shapiro-Wilk tests were used to evaluate normal distribution of data. Normally distributed data were tested with independent sample Student's T test and one-way ANOVA (Tukey's post hoc test) for two or three groups, respectively. Results are presented as Means ± SEM or as Box & Whisker Plot with min to max values. Differences between groups were considered significant when p < 0.05.

[0088] The following sections provide a detailed description of the results of the embodiments of the present disclosure.Preparation and characterization of NP library

[0089] In an embodiment for better results, the NPs library was prepared through host-guest interactions of cyclodextrin-based polymers (host) with adamantane-modified oligosaccharides (guest), in the presence of Navitoclax (Fig. 1). The binding affinity of adamantane to p-cyclodextrin is ~105M1, and is reported in the literature that a Ka>104M1is required for a cyclodextrin complex with a guest molecule to exist as an inclusion complex in in vivo. In the context of the present disclosure galactan (Gal), a P(l,4)-galacto-oligosaccharide, with high percentage of galactose that is cleaved by SA- -Gal was selected. To prepare a library of NPs with variable SA-p-Gal degradation profile and Navitoclax release kinetics acryloylated-cyclodextrin reacted with diamines (in dimethylsulfoxide, for 5 days, 60 °C), using a Michael-type addition reaction, to obtain branch polymers (Fig. 2). This strategy allowed to obtain polymers in a high-throughput way, avoiding long purification steps. The acryloylated P-CD monomer was synthesized by reaction of P-CD with acryloyl chloride in the presence of triethylamine. The degree of substitution (DS) of 3.8 ± 0.2, defined as the number of vinyl groups per P-CD molecule (each P-CD has 7 glucose units; thus, 3.8 out of the 7 units were modified with a vinyl group), was obtained by1H-NMR. Monomers of diamines were selected based on their chemical properties (hydrophobicity / hydrophilicity, composition, structure) or by the fact that they have been used with success in previous NP libraries. At the end of the 5 days reaction, the unreacted vinyl groups were capped with the respective amines. To validate the overall synthetic strategy, one of the polymers was characterized. The efficiency of the capping procedure was high, as no measurable vinyl protons signals (6.0-6.5 ppm) was observed in the1H-NMR of P10.

[0090] In an embodiment for better results, the guest polymer was prepared by the reaction of Gal with different molar ratios of 1-adamantemethylamine (Ada) in the presence of 1,1'-carbonyldiimidazole (CDI) in H2O / DMF (50 / 50%, v / v) at room temperature for 24 h. The1H-NMR analysis, proton peaks of galactan (5 = 3.5-4.6 ppm) and adamantane (5 = 1.5-2.7 ppm), showed anaverage DS ranging from 5.21 ± 0.6% to 25.2 ± 3.1%. In order to verify if the adamantane-modified galactan (Gai-Ada) guest polymer was hydrolysed by SA-P-Gal, the enzyme p-galactosidase (Aspergillus oryzae) was added, and the galactose, the end product of galactan hydrolysis was analysed by p-galactose dehydrogenase (Gal-DH) assay. In this assay, the conversion of NAD+in NADH was followed by reading the absorbance change at 340 nm. The results of Gai-Ada hydrolysis with different DS in the presence of p-galactosidase, room temperature for 1 h, showed a decrease from 60% with unmodified galactan to 40% hydrolysis with Gai-Ada with a DS of about 25 %.

[0091] Prior to the NP library synthesis, the DS of adamantane in galactan was optimized to be suitable for highest NP degradation upon incubation with the enzyme p-galactosidase. To form effective enzyme responsive NPs, a compromise between NP formation efficiency (lower amount of adamantane impacts in NP yield formation) and NP count decrease (higher amount of adamantane impacts on the hydrolysis of galactan) should be taking in consideration.

[0092] In an embodiment for better results, the selected NP was obtained through nanoprecipitation of a mixture of p-CD-polymer in DMSO ([P-CD] = 1.0 mM) and Navitoclax (100 pM) to an aqueous solution of Gai-Ada ([Ada] = 0.5 mM) (Fig. 3A). The mixture was incubated in an orbital shaker shaking at 250 rpm at room temperature for 2 h. Formation of NPs was validated visually, since separated host and guest polymers do not show any turbidity when dispersed in an aqueous solution, while the mixture exhibits a clear Tyndal effect, indicating the existence of abundant NPs (Fig. 3B).

[0093] In an embodiment for better results, the NP efficiency formation for NP10 with Gai-Ada with low DS (DS = 5%) was 15.5 ± 0.6% (i.e. percentage of mass recovered from the initial polymers used for NPs synthesis) while for Gai-Ada with high DS (DS = 25%) was 34.5 ± 6.2%. On the other hand, in the presence of the p-galactosidase, degradation of NP10 with Gai-Ada with low DS was ~6 fold higher than Gai-Ada with high DS, as measured by NP count decrease using dynamic light scattering (DLS) measurements. In this way, the intermediate value of DS (10%) with average NP formation efficiency and high NP count decrease was adopted to synthesize the library with 42 possible NPs.

[0094] In an embodiment for better results, the synthesized NPs library was characterized to obtain the NPs formation efficiency and their physico-chemical properties, size, zeta potential, encapsulation and loading efficiency. The NP formation efficiency was determined taking into account the mass of polymers used for the synthesis of the NPs and the resulting NP mass after freeze-drying. The mean efficiency of the NP library formation was 28.0% ± 4.1. The NPs were then characterized by DLS to obtain NPs size, polydispersity and zeta potential. The NPs had an average size of 250.6 nm ± 16.3 and an average polydispersity of 0.22, while the majority of the NPs showed negative zeta potential, - 10.6 ± I.87 mV. Then, we evaluated the encapsulation efficiency and loading efficiency of the NP library. The mean encapsulation and loading efficiency of Navitoclax were also calculated to be 36.6% ± 3.6 and II.3% ± 1.5, respectively, indicating that such supramolecular nanoparticles are a good hydrophobic-drug-loading system. NPs composed by the more hydrophobic monomers 10, 7 , 19 showed the highest loading efficiency.

[0095] In an embodiment for better results, the stability of some formulations in cell culture medium was evaluated. The results showed that the selected NPs did not show a major increase in size with time (< 20% increase), while the NPs count also remained constant, indicating that the NPs were stable during 24 h.

[0096] The results showed that NPs with diverse size and charge were produced. Depending in the type of formulation, between 13.7 pg and 339.7 pg of Navitoclax was encapsulated per mg of NP. Finally, it was demonstrated that the NPs were stable in cell culture medium.In vitro senotherapeutic potential of the formulations

[0097] In an embodiment for better results, the senotherapeutic potential of Nav-loaded NPs was evaluated in senescent endothelial cells (human umbilical artery endothelial cells, sHUAECs), using proliferative cells as controls. The senescence phenotype was induced by gamma irradiation (10 Gy) and characterized after 7 days. Cells showed high activity of SA-P-Gal (60% of the cells were labelled for C12FDG, a substrate that is cleaved by intracellular SA-P-Gal) and high expression of mRNA encoding cell cycle arrest genes such as p21 and pl6, and low expression of mRNA encoding lamin Bl, all markers of cell senescence.

[0098] In an embodiment for better results, to evaluate the cytotoxicity of the NPs (in this case without Navitoclax; referred as "empty NPs"), senescent and proliferative endothelial cells were exposed to the NPs (NP1, NP4 and NP6) for 4 h, washed to remove non-internalized NPs and finally evaluated for cell survival at 48 h. The formulations had no measurable impact in cell viability for the concentrations tested. Then, to assess the suitable concentration of Nav-loaded NPs to use in the high-throughput screening of the NP library, several concentrations of NP1, NP4 and NP6 NPs were tested, incubated for 4h, washed, and the cell survival of sHUAECs and HUAECs was assessed at 48 h. Cell survival (evaluated by propidium iodide (PI) staining) was obtained by high-content microscopy analysis. The NP1, NP4 and NP6 formulations showed significant induction of cell death in senescent cells at concentrations of 0.5 or 1 pg / mL, while no measurable cell death was observed in proliferative cells for the same concentrations. Increasing the concentration of the Nav-loaded NPs (10, 25, 50 pg / mL) increased significantly cell death in senescent cells but also in proliferative cells. Based on these results, the senotherapeutic activity of the entire NP library was done at a concentration of 1 pg / mL of Nav-loaded NPs. The library of 37 Nav-loaded NPs was incubated for 4 h, washed to remove non-internalized NPs and cell survival assessed at 48 h. As a control, soluble Nav was used (0.05 or 0.1 pM, equivalent to the average loaded Nav in NPs). The results showed an average mortality rate on senescent cells of 65% at 48 h. In contrast, proliferativecells did not show significant cell death with the NP library, except for NP13, NP35 and NP36 formulations.

[0099] In an embodiment for better results, to identify the top formulations from the NPs screening the fold increase of cell survival in proliferative versus senescent endothelial cells was analysed (Fig. 4A). The results showed that the best formulations to deliver Navitoclax in endothelial cells were NP4 and NP17, when compared with soluble Navitoclax. Next, a half maximal inhibitory concentration ( IC5o) study was carried out in endothelial cells to evaluate the apoptotic effect of soluble Navitoclax, the best hits NP4 and NP17, as well as a non-hit formulation (Fig. 4B to 4E). For the curve fitting, the three-parameter logistic regression technique was applied, hence allowing to obtain the IC5o values for each bioassay. The concentration of Navitoclax required to induce death in 50% of the proliferative cells increased from 4.04 pM for the soluble drug to 4.68 pM and 9.82 pM for NP4 and NP17, respectively. This indicates that Navitoclax released by the NPs, particularly from NP17, is less effective in inducing cell death of proliferative cells. On the other hand, the concentration of Navitoclax required to induce death in 50% of the senescent cells decreased from 0.354 pM for the soluble drug to 9 nM or 21 nM when the drug is encapsulated in the formulations NP4 or NP17, respectively. The results from the non-hit formulation did not show any improvement in the senolytic index corroborating the results from the screening assay.

[0100] In an embodiment for better results, taking in consideration the senotherapeutic activity results, NP17 was selected for subsequent studies. To further confirm the senotherapeutic activity of NP17 formulation, a dose-response assay was performed in liver sinusoidal endothelial cells (LSECs) in which the senescence phenotype was induced by gamma irradiation. As shown before for senescent HUAECs, senescent LSECs showed high activity of SA-P-Gal (80% of the cells were labelled for C12FDG) and high expression of mRNA encoding cell cycle arrest genes such as p21 and pl6, as well as IL-6 a SASP marker. The dose-response assay results showed that this NP had an improved senolytic index over soluble Navitoclax (~475x) and indicates that this effect is mainly mediated not only by a higher degree of protection on proliferative cells from cytotoxic activity, but also an efficient delivery to senescent cells (Fig. 4F and 4G). To further confirm that NP17 can eliminate senescent cells originated from a different stressor, their senotherapeutic activity against doxorubicin (Dox)-induced cell senescence was evaluated. LSECs cultured in the presence of Dox showed increased levels of SA-P-Gal (> 80%), higher nuclei area, lower level of proliferation (Ki67) and higher percentage of p21+cells as compared to control cells (not treated with Dox). Importantly, NP17 induced higher death of senescent cells as compared to proliferative cells.

[0101] In an embodiment for better results, the characterization of NP17 showed that the NPs had: (i) an average size of 255 ± 23 nm as demonstrated by DLS and TEM analysis, (ii) a host / guest polymer ratio in the NP of 52 ± 3%, (iii) Gai-Ada and P17 polymers with a molecular weight of 92 ± 5 kDa and 53 ± 7kDa, as determined by DOSY experiments, (iii) a loading efficiency of Navitoclax of 8 ± 1%, (iv) a capacity to release more than 90% of the loaded cargo as determined by HPLC and (v) a decrease of 60% of the NP count rate (DLS) when the enzyme P-Gal was added to the solution (Fig. 5).

[0102] In an embodiment for better results, the results showed that part of the NPs (NP4 and NP17) induced significant cell death in senescent but not in proliferative endothelial cells at relatively low concentrations (1 pg / mL). Importantly, the senolytic index of NP17 formulation was more than 44-fold (sHUAECs) and 475-fold (sLSECs) higher than soluble Navitoclax.Mechanism behind the senotherapeutic potential of the NPs: the relevance of drug release kinetics

[0103] In an embodiment for better results, to demonstrate that the senolytic effect of Nav-loaded NPs is dependent in the degradation of the formulations by lysosomal p-galactosidase, a lung carcinoma cell line (A549 cells) was used. This cell line has been reported to demonstrate that the levels of lysosomal p-galactosidase activity were determinant for the activation of a prodrug.8Initially, A549 cells were exposed to gamma irradiation to induce a senescence phenotype, including an enhanced expression of lysosomal p-galactosidase. Then, cells were transfected with siRNA to knockdown Glbl expression or with scramble siRNA as a control. When these cells were exposed to NP17 without Navitoclax (referred as empty NPs) the viability of both cells was not affected. However, when both cells were exposed to NP17 with Navitoclax, the knockdown in the lysosomal p-galactosidase expression increased cell survival as expected. This indicates that the selective disintegration of NPs is, to some extent, influenced by the enhanced SA-P-Gal expression in senescent cells.

[0104] In an embodiment for better results, to investigate the relevance of drug release kinetics in the senotherapeutic activity of the formulations, initially, NPs that in the presence of the enzyme p-galactosidase (without cells) had variable degradation profiles were identified. From these analyses, three formulations were selected, having a rapid (NP4 and NP17) and a slow (NP16) degradation profile. From these results, it seems that the formulations that degrade faster have higher senotherapeutic activity (and thus senolytic index) than the ones that degrade slower (Figure 4). Importantly, the differences in the senotherapeutic activity were not due to differential uptake of the formulations by senescent cells or proliferative cells in the case of NP17 and NP16 formulations since they showed similar levels of NP internalization.

[0105] In an embodiment for better results, to determine whether Nav-loaded NPs induced endolysosomal compartment disruption, galectin-9 was monitored by immunofluorescence, which is a very sensitive sensor of membrane damage and has been used to demonstrate endosomal escape of therapeutic molecules, including chloroquine. The results showed that senescent LSECs had higher galectin 9 foci than proliferative cells which indicates higher leakage of the endolysosomalT1compartment. NP17 induced endolysosomal membrane damage in proliferative as in senescent cells and thus facilitating the escape of Navitoclax to the cell cytoplasm.

[0106] The results showed that (i) the senolytic effect of Navitoclax-loaded NPs was dependent in the degradation of the NPs, (ii) Navitoclax release kinetics from the formulation influenced its senotherapeutic activity and (iii) endolysosomal membrane leakage in senescent cells is increased after Navitoclax-loaded NPs uptake.In vivo senotherapeutic potential of one of the hit NPs

[0107] In an embodiment for better results, the full potential of the NP17 formulation in aged rats was evaluated. Previous studies indicated that the liver of aged rats (20 months old) showed signs of cell senescence confirmed by the higher levels of pl6 and lower levels of telomere lengths and SIRT1 protein than young livers (3 months old).22In the current study, the characterization of senescence phenotype in aged rat livers (20 months old) was further extended. In this way, the expression of senescence markers such as pl6, p21, HMGB1, vWF by immunofluorescence and RNAish in young (3 months) and aged rats (20 months) was analysed. The results showed that LSECs expressed higher levels of senescence markers in aged than in young rats. This senescence phenotype was also observed in non-endothelial (parenchymal) liver cells as demonstrated by an increase in p21 expression, although not statistically significant, and pl6 expression.

[0108] In an embodiment for better results, to evaluate the biodistribution of NP17 in aged rats, the formulation was initially labelled with cyanine5 dye and then administered by intravenous injection at a concentration of 2.5 mg / kg / day. The results showed a higher accumulation in the liver, when compared with the other organs (Fig. 6). Moreover, the isolation of liver cells 4 h upon injection of N17 indicate a higher accumulation in LSECs and hepatic stellate cells (HSCs), quantified by the NP area normalized by the number of nuclei. Interestingly, the area of NPs is reduced in hepatocytes, suggesting the degradation of the NP by the sinusoidal cells (Fig. 6C-E).

[0109] In an embodiment for better results, the effect of NP17 formulation in liver function was evaluated. Aged rats were treated for 2 weeks (4 injections each week, with one week of interval) with vehicle (i.v. PBS), soluble Navitoclax (oral gavage, 50 mg / kg / day) or NP17 (i.v., corresponding to 0.2 mg / kg / day of loaded navitoclax). Liver function was assessed 4 weeks after the treatment (Fig. 7 Table 3). Systemic hemodynamic parameters were not altered in rats after treatment with NP17 or Navitoclax (Table 3). Importantly, rats treated with NP17 showed a decrease in portal pressure (8.67 ± 0.20 mmHg) as compared to rats treated with vehicle (9.33 ± 0.25 mmHg) (Fig. 7B), which was the consequence of a reduction in the hepatic vascular resistance leading to a better perfusion, as evidenced by increased portal blood flow (Table 3). In addition, rats treated with NP17 showed a liver sinusoid with no alterations in fenestrae porosity but a significant increase in the number of fenestrae as compared torats receiving vehicle (Fig. 7C and 7D). Moreover, analysis of extracellular matrix deposition in rats treated with NP17 showed a decrease in hepatic fibrosis, as demonstrated by reduction of Sirius Red staining (Fig. 7E and 7F), and a decrease of sinusoidal fibres. Intrahepatic neovascularization analysis of young vs old rats shows a decrease in the vasculature, which after the treatment of old rats with NP17 showed a tendency to increase when compared with the vehicle. Additionally, NP17 treatment decreased significantly the production of selected components of the SASP, namely, EGF, Fractalkine, IP-10 and Leptin when compared with the treatment with the vehicle.Table 3. Morphometry data and hemodynamic assessment 4 weeks after treatment. Values are Mean ± SEM (n=8). Data followed normality and statistical significance was evaluated by ANOVA test with Tukey's post hoc correction. *Veh vs. Nav, +Veh vs. NP17, ¥Nav vs. NP17. No differences were found between Nav and NP17 rats. BW, body weight; SMABF, superior mesenteric arterial blood flow; ALT, alanine transaminase; AST, aspartate transaminase; Portal blood flow index and Intrahepatic resistance index are normalized by liver weight (g).

[0110] In an embodiment for better results, the impact of NP17, Nav and vehicle in the senescent phenotype of LSECs was evaluated. The results indicate that rats treated with NP17 showed a significant reduction in liver senescence relative to vehicle as demonstrated by a significant decrease in: (i) percentage of either endothelial or non-endothelial cells expressing p21 (Fig. 8A, 8B and 8C), (ii) pl6 (Fig. 8D, 8E and 8F), (ii) percentage of either sinusoidal endothelial cells or large blood vessels expressing HMGBl (Fig. 8G, 8H and 81) and (iv) sinusoidal expression of the endothelial marker vWF (Fig.8J, 8K).

[0111] In an embodiment for better results, the results obtained in aged rats indicate: (i) a clear senescence phenotype in liver sinusoidal endothelial cells and in other liver cells (parenchymal cells); (ii) NP17 formulation administered intravenously accumulated mainly in LSECs, followed by HSCs; (iii) NP17 formulation improved key liver function parameters and (iv) NP17 formulation decreased cell senescence in the liver both in the endothelial and non-endothelial compartments.

[0112] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0113] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The abovedescribed embodiments are combinable.

[0114] The following dependent claims further set out particular embodiments of the disclosure. References1 Varela-Eirin, M. & Demaria, M. Cellular senescence. Curr Biol 32, R448-R452 (2022).https: / / doi.org:10.1016 / j. cub.2022.04.0032 Gasek, N. S., Kuchel, G. A., Kirkland, J. L. & Xu, M. Strategies for Targeting Senescent Cells in Human Disease. Nat Aging 1, 870-879 (2021). https: / / doi.org:10.1038 / s43587-021-00121-8Gorgoulis, V. et al. Cellular Senescence: Defining a Path Forward. Cell 179, 813-827 (2019). https: / / doi.org:10.1016 / j.cell.2019.10.005Xu, M. et al. Senolytics improve physical function and increase lifespan in old age. Nat Med 24, 1246-1256 (2018). https: / / doi.org:10.1038 / s41591-018-0092-9Baker, D. J. et al. Clearance of pl6lnk4a-positive senescent cells delays ageing-associated disorders. Nature 479, 232-236 (2011). https: / / doi.org:10.1038 / naturel0600Chaib, S., Tchkonia, T. & Kirkland, J. L. Cellular senescence and senolytics: the path to the clinic. Nature Medicine 28, 1556-1568 (2022). https: / / doi.org:10.1038 / s41591-022-01923-yKile, B. T. The role of apoptosis in megakaryocytes and platelets. Br J Haematol 165, 217-226 (2014). https: / / doi.org:10.1111 / bjh.12757Gonzalez-Gualda, E. et al. Galacto-conjugation of Navitoclax as an efficient strategy to increase senolytic specificity and reduce platelet toxicity. Aging Cell 19, el3142 (2020). https: / / doi.org:10.1111 / acel.13142Cai, Y. et al. Elimination of senescent cells by beta-galactosidase-targeted prodrug attenuates inflammation and restores physical function in aged mice. Cell Res 30, 574-589 (2020). https: / / doi.org:10.1038 / s41422-020-0314-9Ni, S. et al. Pro-senescence neddylation inhibitor combined with a senescence activated betagalactosidase prodrug to selectively target cancer cells. Signal Transduct Target Ther 7, 313 (2022). https: / / doi.org:10.1038 / s41392-022-01128-2Guerrero, A. et al. Galactose-modified duocarmycin prodrugs as senolytics. Aging Cell 19, el3133 (2020). https: / / doi.org:10.1111 / acel.13133Agostini, A. et al. Targeted cargo delivery in senescent cells using capped mesoporous silica nanoparticles. Angew Chem Int Ed Engl 51, 10556-10560 (2012). https: / / doi.org:10.1002 / anie.201204663Munoz-Espin, D. et al. A versatile drug delivery system targeting senescent cells. EMBO Mol Med 10 (2018). https: / / doi.org:10.15252 / emmm.201809355Akang E. Ekpenyong-Akiba et al. Detecting and targeting senescent cells using molecularly imprinted nanoparticles. Nanoscale Horizons 4, 11 (2019).Pham, L. M. et al. Targeting and clearance of senescent foamy macrophages and senescent endothelial cells by antibody-functionalized mesoporous silica nanoparticles for alleviating aorta atherosclerosis. Biomaterials 269, 120677 (2021). https: / / doi.org:10.1016 / j. biomaterials.2021.120677Qu, A. et al. An NIR-Responsive DNA-Mediated Nanotetrahedron Enhances the Clearance of Senescent Cells. Adv Mater 32, e2000184 (2020). https: / / doi.org:10.1002 / adma.202000184Li, S. et al. Chiral Cu(x) Co(y) S Nanoparticles under Magnetic Field and NIR Light to Eliminate Senescent Cells. Angew Chem Int Ed Engl 59, 13915-13922 (2020). https: / / doi.org:10.1002 / anie.202004575Galiana, I. et al. Preclinical antitumor efficacy of senescence-inducing chemotherapy combined with a nanoSenolytic. J Control Release 323, 624-634 (2020). https: / / doi.org:10.1016 / j.jconrel.2020.04.045Kumar, R. et al. In vivo biodistribution and clearance studies using multimodal organically modified silica nanoparticles. ACS Nano 4, 699-708 (2010). https: / / doi.org:10.1021 / nn901146y Zhang, Y. N., Poon, W., Tavares, A. J., McGilvray, I. D. & Chan, W. C. W. Nanoparticle-liver interactions: Cellular uptake and hepatobiliary elimination. J Control Release 240, 332-348 (2016). https: / / doi.org:10.1016 / j.jconrel.2016.01.020Sanfeliu-Redondo, D., Gibert-Ramos, A. & Gracia-Sancho, J. Cell senescence in liver diseases: pathological mechanism and theranostic opportunity. Nat Rev Gastroenterol Hepatol (2024). https: / / doi.org:10.1038 / s41575-024-00913-4Maeso-Diaz, R. et al. Effects of aging on liver microcirculatory function and sinusoidal phenotype. Aging Cell 17, el2829 (2018). https: / / doi.org:10.1111 / acel.12829Ogrodnik, M. et al. Cellular senescence drives age-dependent hepatic steatosis. Nat Common 8, 15691 (2017). https: / / doi.org:10.1038 / ncommsl5691Ritschka, B. et al. The senotherapeutic drug ABT-737 disrupts aberrant p21 expression to restore liver regeneration in adult mice. Genes Dev 34, 489-494 (2020). https: / / doi.org:10.1101 / gad.332643.119Jannone, G. et al. Senescence and senotherapies in biliary atresia and biliary cirrhosis. 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Claims

C L A I M S1. Polymeric nanoparticle for delivering and releasing an active ingredient, wherein the nanoparticle comprises:a cyclodextrin-based polymer;an adamantane-modified oligosaccharide cleavable by senescence-associated p-galactosidase; a senotherapeutic active ingredient;wherein the cyclodextrin-based polymer is bound to the adamantane-modified oligosaccharide; wherein the senolytic active ingredient is entrapped in the polymeric nanoparticle.

2. Nanoparticle according to the previous claim, wherein the cyclodextrin-based polymer is formed by acrylated p-cyclodextrin monomers and amine monomers; preferably linked via Michael-type addition.

3. Nanoparticle according to any of the previous claims, wherein the cyclodextrin-based branched polymer molecular weight may range from 5 kDa - 150 kDa; preferably 10 kDa - 100 kDa; more preferably 30 kDa - 70 kDa; even more preferably 45 kDa - 55 kDa.

4. Nanoparticle according to any of the previous claims, wherein the degradation rate of the nanoparticle is superior to 1.5% per minute under physiological conditions at 37°C.

5. Nanoparticle according to any of the previous claim, wherein the cyclodextrin-based polymer is bound to the adamantane-modified oligosaccharide via non-covalent interactions; preferably hostguest interactions.

6. Nanoparticle according to any of the previous claims, wherein the adamantane-modified oligosaccharide is galactan-adamantane.

7. Nanoparticle according to the previous claim, wherein the galactan-adamantane molecular weight ranges from 10 kDa - 150 kDa; preferably 60 kDa - 110 kDa, more preferably 85 kDa - 90 kDa.

8. Nanoparticle according to any of the previous claims 3-4, wherein the degree of substitution of adamantane per galactose monomer is 1% - 30%; preferably 5% - 25%; more preferably 8% - 16%; even more preferably 10%.

9. Nanoparticle according to any of the previous claims, wherein the cyclodextrin-based polymer / adamantane-modified oligosaccharide ratio is 30% (w / w) - 70% (w / w); preferably 35% (w / w) - 65% (w / w), more preferably 50% (w / w) - 55 % (w / w).

10. Nanoparticle according to any of the previous claims, wherein the senotherapeutic active ingredient is a senolytic active ingredient, or a senomorphic active ingredient, or a senescence-inhibitor active ingredient, or mixtures thereof.

11. Nanoparticle according to any of the previous claims, wherein the senolytic active ingredient is selected from a list consisting of: navitoclax, venetoclax, quercetin, dasatinib, fisetin, 17-DMAG, SSK1, A1331852, A1155463, procyanidin Cl, luteolin, enzastaurin, piperlongumine, geldanamycin, tanespimycin, alvespimycin (17-DMAG), ansamycin, resorcinol, purine- and pyrimidine-like N- terminal inhibitors, Sting inhibitors (e.g. H-151), curcumin and analogues, cardiac glycosides (such as ouabain, proscillaridin A, digoxin, ouabagenin, bufalin, K-stropanthin, strophanthidin), aspirin, PZ15227, ARV825, fenofibrate, azithromycin, roxithromycin, tamatinib (R406), mitoTam, panobinostat, AT-406, ganetespib, Foxo4-DRI, UBX0101, RG7112, P5091, P22077, and mixtures thereof.

12. Nanoparticle according to any of the previous claims, wherein the senomorphic active ingredient is selected from a list consisting of: rapamycin, metformin, resveratrol, SR12343, SB203580, UR13756, BIRB796, MK2.III, PF-3644022, ruxolitinib, KU-55933, KU-60019, atorvastatin, pravastatin, pitavastatin, simvastatin, apigenin, kaempferol, quercetin, epigallocatechin gallate, genistein, oleuropein aglycone, hydroxytyrosol, and mixtures thereof.

13. Nanoparticle according to any of the previous claims, wherein the senotherapeutic active ingredient content released within 4 hours is 60% - 100%, preferably 80% - 90%, more preferably 90%.

14. Nanoparticle according to any of the previous claims, wherein the mass of senotherapeutic active ingredient per mg of nanoparticle ranges from 10 pg - 400 pg; preferably 13 pg - 340 pg.

15. Nanoparticle formulation according to any of the previous claims, wherein the nanoparticle size measured by dynamic light scattering ranges from 40 nm - 400 nm; preferably 100 nm - 300 nm, more preferably 240 nm - 260 nm.

16. Nanoparticle according to any of the previous claims, for use in medicine or veterinary.

17. Nanoparticle according to the previous claim, for use in the prevention or treatment of age-related diseases or disorders; preferably in the prevention or treatment of age-related liver diseases or disorders.

18. Nanoparticle according to any of the previous claims 15-16, as an improver of cell death in senescent cells; preferably liver senescent cells.

19. Formulation comprising the nanoparticle according to any of the previous claims in a therapeutical amount and a suitable pharmaceutical carrier or vehicle.

20. Formulation further comprising a second active ingredient; preferably wherein the second active ingredient is select from: anti-inflammatory active ingredients, chemotherapeutic active ingredients, antifibrotic active ingredients, immunomodulatory active ingredients, stress modulator ingredients, or growth factor inhibitor or enhancer ingredient, or mixtures thereof.

21. Formulation according to the previous claim, wherein the formulation is an aqueous solution.

22. Formulation according to any of the previous claims 19-21, wherein the polydispersity index is 0.02 - 0.5, preferably 0.1 - 0.4, more preferably 0.1-0.2.

23. Formulation according to any of the previous claims 19-22, wherein the zeta potential is -5 mV - -20 mV; - preferably 10 mV - -15 mV.

24. Formulation according to any of the previous claims 19-23, consisting of: powders, tablets, capsules, syrups, creams, ointments, gels, lotions, suspensions, emulsions, patches, inhalers, nebulizers, suppositories, enemas, eye drops, ophthalmic ointments, nasal sprays, or nasal drops.

25. Formulation according to any of the previous claims 19-24, in oral, intravenous, intramuscular, subcutaneous, transdermal, inhalation, rectal, vaginal, buccal, sublingual, topical, intrathecal, nasal, or ocular administration forms.

26. Medical device comprising the nanoparticle formulation according to any of the previous claims 19- 25, wherein the medical device is coated by a nanoparticle formulation.

27. Method for producing the nanoparticle formulation according to any of the previous claims, comprising the steps:providing a cyclodextrin-based polymer, an aqueous solution of an adamantane-modified oligosaccharide, and a senotherapeutic active ingredient;mixing the cyclodextrin-based polymer and the senotherapeutic active ingredient in an organic solvent; preferably under constant stirring;adding the adamantane-modified oligosaccharide to the mixture of the cyclodextrin-based polymer and the senotherapeutic active ingredient to bind the cyclodextrin-based polymer with the adamantane-modified oligosaccharide and entrap the senotherapeutic active ingredient; precipitating, forming, and collecting the nanoparticle formulation;optionally filtering or centrifuging the nanoparticle formulation.

28. Method for producing the nanoparticle formulation according to the previous claim comprising the precipitating, forming and collecting step using a microfluidics apparatus.

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