Virus mimicking nanoparticles

Abstract

The present invention relates to a nanoparticle comprising a nanomaterial and at least a first ligand and a second ligand tethered to the nanoparticle. The present invention also relates to a nanoparticle for use as a medicament or diagnostic agent. The present invention also relates to a nanoparticle for use in a method for preventing or treating a disease selected from the group consisting of diabetic nephropathy, glomerulonephritis, glomerular VEGF A dysregulation, endothelial VEGF A dysregulation, diabetic retinopathy, rheumatoid arthritis, age-related macular degeneration and cancer, such as breast cancer. Furthermore, the present invention relates to a method of preparing a nanoparticle.

Description

Technical Field

[0001] This invention relates to nanoparticles comprising nanomaterials and at least a first ligand and a second ligand. The invention also relates to nanoparticles for use as pharmaceuticals or diagnostic agents. Furthermore, the invention relates to nanoparticles used in methods for preventing or treating diseases selected from diabetic nephropathy, glomerulonephritis, glomerular VEGF A dysregulation, endothelial VEGF A dysregulation, diabetic retinopathy, rheumatoid arthritis, age-related macular degeneration, and cancers (e.g., breast cancer). Additionally, the invention relates to a method for preparing nanoparticles. Background Technology

[0002] In recent years, numerous nanomaterials have been developed as carriers for drug therapy or diagnostics. To enable them to recognize target cells with sufficient specificity in vivo, ligands that bind to cell receptors have been bound to their surfaces. However, simply following this old paradigm increases the affinity of nanomaterials but proves insufficient for definitive cell recognition. Currently, even nanomaterials with multiple different ligands for heterovalent binding cannot distinguish between different cell types.

[0003] In contrast, viruses are nanoparticles (NPs) with ultimate target cell specificity. Compared to synthetic biomedical nanomaterials, viruses utilize a sequential, multi-step recognition process for cell recognition. Since many current nanoparticle-based methods lack sufficient specificity, utilizing virus-targeting strategies may be a viable option to overcome this limitation. Therefore, mimicking the sequential recognition strategies of viruses (e.g., influenza A virus) with nanomaterials could allow for specific cell targeting. In particular, viral target cell recognition may be advantageous in vivo, where particles are surface-modified due to protein adsorption.

[0004] In particular, the initial step of viral attachment to the cell membrane (which does not lead to particle uptake but increases the density of viral particles on the cell surface) is missing in current nanoparticle design strategies. This initial adhesion to glycolipids and glycoproteins or specific receptors has been found to be crucial for viral infectivity. More importantly, if particles are cleared once they reach the target tissue, they are insufficient for drug delivery purposes. In the mesangial case, early reports showed that small particles with a diameter of 70 ± 25 nm or smaller were penetrating the glomerular endothelial fenestrations with a diameter of approximately 80–100 nm. However, these particles were cleared by the mesangium. If nanoparticles are internalized by target cells in the target tissue, the problem of the known rapid clearance of nanoparticles from tissues can be overcome.

[0005] Maslanka Figueroa et al. [1] have discussed polymer nanoparticles containing ligands of angiotensin-I.

[0006] Sah et al. [2] have worked on nanoparticles containing block copolymers and drugs. Summary of the Invention

[0007] This invention aims to provide nanomaterials equipped with a virus-mimicking cell recognition mechanism for addressing cells in vitro and in vivo. Furthermore, an object of this invention is to provide nanoparticles that allow for efficient accumulation of said nanoparticles in target tissues in vivo. Another object of this invention is to provide a drug delivery system that allows for the delivery of drugs or diagnostic agents to target tissues.

[0008] The elements of the invention will now be described. These elements are listed with particular embodiments; however, it should be understood that they can be combined in any manner and in any number to produce other embodiments. The various examples and preferred embodiments described should not be construed as limiting the invention to the explicitly described embodiments. This description should be understood to support and cover embodiments that combine two or more explicitly described embodiments or combine one or more explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context otherwise requires, any permutation and combination of all elements described in this application should be considered as disclosed in the description of this application.

[0009] In a first aspect, the present invention relates to a nanoparticle comprising a nanomaterial and at least a first ligand and a second ligand.

[0010] - wherein the first ligand mediates the attachment of the nanoparticles to the target cells, and

[0011] - wherein the second ligand is capable of mediating the internalization of the nanoparticles into the target cells, and

[0012] - Preferably, the nanomaterial comprises any of the following: polyethylene glycol (PEG), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), oxazoline-derived polymers, poly(amino acids), polysaccharides, phospholipids, sphingolipids, cholesterol, PEG-lipids, block copolymers such as PEG-PLA or PEG-polycaprolactone, inorganic substances such as gold or quantum dot materials, and combinations thereof.

[0013] In one embodiment, the first ligand is a non-agonist that binds to GPCRs (e.g., angiotensin II receptor type 1 (AT1r), human neuropeptide Y1 receptor, and CXC chemokine receptor type 4), and / or a reagent that binds to glycoproteins and / or glycolipids on the surface of target cells, such as heparan sulfate, sialic acid glycoprotein, gangliosides, and mannose receptors, preferably EXP3174 or telmisartan.

[0014] In one embodiment, the second ligand is any of the following: i) a reagent that binds to integrins such as αVβ3 integrin or αVβ5 integrin, preferably selected from RGD, cyclic RGD peptides having the sequence of SEQ ID NO.1 and their derivatives; ii) an agonist that binds to GPCRs such as AT1r, preferably activated angiotensin-II; iii) a reagent that binds to extracellular enzymes such as legumain, membrane matrix metalloproteinases and angiotensin-converting enzyme (ACE), preferably angiotensin-I; and / or iv) a reagent that binds to transferrin receptors.

[0015] In one embodiment, the nanoparticles further comprise a therapeutic agent, preferably any one of pirfenidone and cinaciguat.

[0016] In one embodiment, the first ligand and the second ligand are each coupled to the nanomaterial, preferably each coupled to a block copolymer chain of the nanomaterial.

[0017] In one embodiment, the nanomaterial comprises more than one block copolymer chain, wherein the first ligand is coupled to the first block copolymer chain of the nanomaterial and the second ligand is coupled to the second block copolymer chain of the nanomaterial, wherein the first block copolymer chain is longer than the second block copolymer chain, preferably at least 1.5 times longer than the second block copolymer chain, more preferably at least 3 times longer than the second block copolymer chain.

[0018] In one embodiment, the first block copolymer chain comprises PEG in the range of 1k to 20k, preferably 1k to 10k, and / or PLA in the range of 5k to 40k, preferably 10k to 20k, wherein the first block copolymer chain is PEG. 5k -PLA 10k And the second block copolymer chain is PEG. 2k -PLA 10k .

[0019] In one embodiment, the second ligand is enzymatically activated prior to the internalization of the nanoparticles into the target cells.

[0020] In one embodiment, the target cells are selected from mesangial cells, endothelial cells such as retinal endothelial cells, B cells, T cells, macrophages, dendritic cells, and tumor cells.

[0021] In one embodiment, the nanoparticles have a size of 5 nm to 1000 nm, preferably 10 nm to 150 nm, and more preferably 20 nm to 100 nm.

[0022] In one embodiment, the ratio of the first ligand to the second ligand is in the range of 2:1 to 1:2, preferably 1:1.

[0023] In one embodiment, the nanoparticles have a particle affinity for the target receptor of 1 pM to 100 nM, preferably 50 pM to 1 nM.

[0024] In one embodiment, the nanomaterial comprises PEG, and the particles have a ligand density of at least 5%, preferably at least 15%, more preferably at least 25% ligand / PEG.

[0025] In another aspect, the present invention relates to nanoparticles as defined in any of the above embodiments, which are used as pharmaceuticals or diagnostic agents.

[0026] In another aspect, the present invention relates to nanoparticles as defined in any of the above embodiments, methods for preventing or treating diseases selected from diabetic nephropathy, glomerulonephritis, glomerular VEGF A dysregulation, endothelial VEGF A dysregulation, diabetic retinopathy, rheumatoid arthritis, age-related macular degeneration, and cancers such as breast cancer.

[0027] In another aspect, the present invention relates to a method for preparing nanoparticles, comprising the following steps:

[0028] a) Provide one or more nanomaterials in any order, preferably comprising any of the following: polyethylene glycol (PEG), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), oxazoline-derived polymers, poly(amino acids), polysaccharides, phospholipids, sphingolipids, cholesterol, PEG-lipids, block copolymers such as PEG-PLA or PEG-polycaprolactone, inorganic substances such as gold or quantum dot materials, combinations thereof, and optionally therapeutic agents;

[0029] b) Optionally, block copolymers are prepared from any of the one or more nanomaterials;

[0030] c) Couple the first and second ligands to it in one or more steps, preferably by DCC / NHS- or EDC / NHS- coupling;

[0031] d) Provide a therapeutic agent, preferably a lipophilic therapeutic agent, if not provided in step a);

[0032] e) Nanoparticles are prepared and obtained using ligands coupled to the nanomaterial and the therapeutic agent, preferably by nanoprecipitation.

[0033] In one embodiment, the obtaining in step e) includes obtaining nanoparticles with a polydispersity index of 0.01 to 0.5, preferably 0.01 to 0.3, more preferably 0.01 to 0.1.

[0034] In this regard, the nanoparticles, the nanomaterials, the therapeutic agent, the block copolymer, the first ligand, and the second ligand are as defined above.

[0035] In another respect, the present invention relates to pharmaceutical compositions comprising nanoparticles as defined above and pharmaceutically acceptable excipients.

[0036] In another aspect, the present invention relates to methods for preventing or treating diseases, wherein the methods include administering an effective amount of nanoparticles and / or pharmaceutical compositions to a patient in need.

[0037] In this regard, the disease, the nanoparticles, and the pharmaceutical composition are as defined above.

[0038] In another aspect, the present invention relates to the use of nanoparticles in the preparation of medicaments for treating diseases.

[0039] In this regard, the nanoparticles and the disease are as defined above.

[0040] In another aspect, the present invention relates to the use of nanoparticles in the preparation of diagnostic agents for disease diagnosis and / or prognosis.

[0041] In this regard, the nanoparticles and the disease are as defined above.

[0042] Detailed description

[0043] Poor utilization in target tissues due to unfavorable physicochemical properties is a common cause of drug failure. Viruses overcome this limitation by embedding their nucleic acids into nanoscale particles and making them highly specific to their target cells. While nanotechnology has provided a large number of nanocarriers for drug delivery, their ability to definitively identify target cells remains moderate. The inventors demonstrate herein that particles with a virus-like ability to identify cells by three consecutive checks of cell identity exhibit superior ability to recognize mesangial cells in vivo compared to conventional nanoparticles. In mice, this resulted in a 15-fold increase in accumulation in the renal mesangium, followed by significant cellular uptake. This invention provides a surprisingly effective tool for delivering drugs to target tissues, and this tool is applicable to the treatment of a variety of diseases, such as diabetic nephropathy, for which there is currently no drug therapy.

[0044] The inventors designed particles that carry, for example, EXP3174 (angiotensin-II type 1 receptor (AT1R) ligand) in the NP corona to mediate receptor attachment. Figure 1 A). As a G protein-coupled receptor (GPCR) antagonist, its greatest advantage is that binding does not trigger cellular NP uptake, but only membrane binding, thereby preventing off-target cells carrying only AT1R from taking up particles.

[0045] For the second recognition criterion, the inventors have equipped the particles with the ability to detect the presence of cell surface targets such as angiotensin-converting enzyme (ACE), which recognizes the pro-ligand angiotensin-I (Ang-I) in the particle crown and converts it into the active ligand angiotensin-II (Ang-II).

[0046] As a third recognition step, the ligand (e.g., Ang-II) binds to the target (e.g., AT1R) and, acting as an agonist, triggers cellular uptake of the granule upon receptor binding. The entire process of target cell recognition can best be illustrated by a flowchart (…). Figure 1 B) To illustrate. The target receptor affinity and target cell specificity of the particles were examined in vitro. Furthermore, the effect of the simultaneous presentation of two ligands targeting the same receptor (an antagonist that promotes cell membrane binding and an agonist that supports cell internalization) on the ability of NPs to mediate cellular uptake was evaluated. Finally, the inventors demonstrated that particles with this virus-mimicking triple recognition strategy outperform conventional NPs in reaching mesangial cells in vivo.

[0047] Since nanoparticles (NPs) are typically distributed throughout organisms via passive transport mechanisms, their presence in specific tissues depends on their physicochemical properties. However, if they can actively interact with target cells, the proportion of particles accumulating in the target tissue can be increased. Equipping NPs with ligands that bind to their respective receptors to identify cells is insufficient. The particles of this invention clearly demonstrate that a strategy of stepwise cell identification, particularly one comprising a first ligand for mediating attachment to target cells and a second ligand for mediating the internalization of nanoparticles into target cells, is more advantageous.

[0048] Using a virus as a template, the inventors designed an NP capable of performing a series of "if-then-else" decisions, made one after another. In each individual step, the NP, with the help of a ligand or substrate, probes the cell for the presence of a receptor or extracellular enzyme, respectively. If successful, the next recognition step is performed; if unsuccessful (otherwise), the particle "decides" that the cell cannot be a target cell. Like a virus, this helps prevent "wrong" cell types from taking up the NP.

[0049] For example, receptors belonging to the GPCR family, such as AT1R, are used to determine particle identity. In the case of detecting cell identity with ligands (e.g., GPCR antagonists, such as EXP3174 or telmisartan), a positive result of this interaction is that the particle binds to the cell surface and remains there. If the next interaction is not positive (otherwise), it is clear that the particle is at risk of becoming stranded on off-target cells. However, in this case, due to the thermodynamic equilibrium between free and bound particles, as can be expected, the concentration of free particles in vivo decreases over time, altering this equilibrium so that the particle dissociates from the “wrong” target over time. Conversely, if cell identity is detected using an agonist of the same GPCR, such as Ang-II, the positive answer for the cell is that the particle is internalized. Therefore, by carefully selecting the type of interacting target and ligand, particles can be equipped with logic that allows the identification of more hidden target cells than the exemplary targets studied in this study. One example is a local ocular application in retinal tissue, where particles are able to distinguish more than 60 cell types present, for example, by specifically targeting endothelial cells.

[0050] As used herein, the terms "nanoparticle" and "NP" refer to nanomaterial structures comprising a first ligand and a second ligand. Specifically, nanoparticles are nanoobjects having all three external dimensions at the nanoscale, such as liposomes, polymer nanoparticles, micelles, lipid nanocapsules, inorganic nanoparticles such as gold nanoparticles, or quantum dots (Qdots). In one embodiment, the nanoparticles provide excellent biocompatibility and highly tunable compositions. Nanoparticles can be made from a variety of materials, such as polymers, biomolecules, and metals. In one embodiment, the nanoparticles of the present invention are virus-mimicking nanoparticles capable of delivering drugs to target tissues, such as the renal mesangium. In one embodiment, the nanoparticles comprise pirfenidone and / or cinasciguat and are intended for the treatment of diabetic nephropathy. In one embodiment, the nanoparticles of the present invention comprise a biodegradable block copolymer comprising PEG and PLA. In one embodiment, the first and second ligands are covalently coupled to the nanoparticles via DCC / NHS or EDC / NHS. In one embodiment, the block copolymer is dissolved in acetonitrile and mixed with PLGA (70 / 30, m / m) to obtain a polymer mixture. In one embodiment, nanoparticles are prepared by nanoprecipitation by dropwise injection of the polymer mixture into an aqueous phase. In one embodiment, the nanoparticles of the present invention accumulate in target tissues, such as kidney tissue, within a very short time (i.e., <1 hour) after application of the nanoparticles.

[0051] In one embodiment, the nanoparticles of the present invention are very small, i.e., <80 nm, and due to their small size, are able to rapidly exit the bloodstream through the porous endothelium and accumulate in target tissues such as mesangial tissue. In one embodiment, the nanoparticles further comprise a therapeutic agent, preferably any one of pirfenidone and cinasciguat. In one embodiment, the particle size is from 5 nm to 1000 nm, preferably from 10 nm to 150 nm, more preferably from 20 nm to 100 nm. In one embodiment, particle size is measured using dynamic light scattering, particle scattering diffusion, nanoparticle tracking analysis, atomic force microscopy, or transmission electron microscopy, preferably dynamic light scattering or transmission electron microscopy. The particle size is determined by its diameter, which relates to any straight line segment passing through the center of a spherical particle and whose endpoint lies on the spherical particle. In the case of non-spherical particles, the diameter relates to the longest line segment passing through the center of the non-spherical particle and whose endpoint lies on the particle. In one embodiment, the average diameter relates to the average diameter of the nanoparticles contained in a batch of nanoparticles. In one embodiment, the particles of the present invention have a polydispersity index of 0.01 to 0.5, preferably 0.01 to 0.3, more preferably 0.01 to 0.1. In one embodiment, the polydispersity index is measured using dynamic light scattering, particle scattering-diffusion method, nanoparticle tracking analysis, atomic force microscopy, or transmission electron microscopy, preferably dynamic light scattering or transmission electron microscopy. In one embodiment, the terms "nanoparticle" and "particle" are used interchangeably. In one embodiment, the nanoparticles of the present invention are used in medicine. In one embodiment, the nanoparticles have a positive, negative, or neutral polarity. - Potential, for example -20mV to 0mV, or -15mV to -5mV. In one embodiment, the nanoparticles comprise at least one core containing nanomaterials and optionally a polymer and / or connector on its surface, wherein the first and second ligands are coupled to the polymer and / or connector. In one embodiment, the particle has a polymer core.

[0052] As used herein, the term "viral mimicry" refers to a method that mimics a cell-targeting approach to a virus. The viral mimicry particles of the present invention are internalized into target cells via a recognition process having at least two consecutive steps. In this two-step process, the first step involves a first ligand on the particle (e.g., EXP3174 or telmisartan) binding to a target expressed on the target cell (e.g., angiotensin receptor on mesangial cells). Subsequently, the particle is internalized into the target cell, mediated by a second ligand binding to a target on the target cell, such as activated angiotensin II or a cyclic amino acid sequence (cyclic Arg-Gly-Asp-D-Phe-Lys; SEQ ID No. 1), thereby initiating endocytosis of the particle. In one embodiment, the sequential presentation of the first and second ligands is achieved by: i) spatial control mediated by a longer linker for the first ligand, such as a longer PEG-PLA chain, compared to a shorter linker for the second ligand, such as a shorter PEG-PLA chain, and / or by ii) an activation step, wherein the second ligand must be activated to mediate internalization, such as the conversion of angiotensin I to angiotensin II. In one embodiment, the sequential presentation of the ligands allows for up to 15-fold higher accumulation of nanoparticles in target cells, such as mesangial cells, compared to conventional particles without ligand modification. In one embodiment, the particle affinity of the particles for the target receptor is 1 pM to 100 nM, preferably 50 pM to 1 nM. In one embodiment, the nanoparticles of the present invention are used as a pharmaceutical or diagnostic agent. In one embodiment, the nanoparticles of the present invention are used in a method for the prevention or treatment of diseases selected from diabetic nephropathy, glomerulonephritis, glomerular VEGFA dysregulation, endothelial VEGFA dysregulation, diabetic retinopathy, rheumatoid arthritis, age-related macular degeneration, and cancers (e.g., breast cancer). In one implementation, the terms "nanoparticles," "virus-mimicking particles," and "decision nanoparticles" are used interchangeably.

[0053] As used herein, the term "nanomaterial" refers to a material having any external size at the nanoscale or having an internal or surface structure at the nanoscale. In one embodiment, the nanomaterial preferably comprises polyethylene glycol (PEG), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), oxazoline-derived polymers, poly(amino acids), polysaccharides, phospholipids, sphingolipids, cholesterol, PEG-lipids such as DPSE-PEG and acid stearate PEG, block copolymers such as PEG-PLA or PEG-polycaprolactone, inorganic materials such as gold or quantum dot materials, and combinations thereof. In one embodiment, the first ligand and the second ligand are each coupled to the nanomaterial, preferably each coupled to a block copolymer chain of the nanomaterial. In one embodiment, the nanomaterial comprises more than one block copolymer chain, and the first ligand is coupled to a first block copolymer chain of the nanomaterial and the second ligand is coupled to a second block copolymer chain of the nanomaterial, wherein the first block copolymer chain is longer than the second block copolymer chain, preferably at least 1.5 times longer than the second block copolymer chain, more preferably at least 3 times longer than the second block copolymer chain. In one embodiment, the first block copolymer chain comprises PEG in the range of 1k to 20k, preferably 1k to 10k, and / or PLA in the range of 5k to 40k, preferably 10k to 20k. In one embodiment, the PEG chain of the first block copolymer chain is longer than the PEG chain of the second block copolymer chain. In one embodiment, a ligand is coupled to a PEG molecule. As used herein in the context of polymer chains (e.g., block copolymer chains, PEG, and / or PLA), the term "k" refers to kilodaltons (kDa). For example, PEG in the range of 1k to 20k involves PEG in the range of 1kDa to 20kDa, such as PEG 5kDa In one implementation scheme, PEG 5k -PLA 10k and PEG 2k -PLA 10k Each involves PEG 5kDa -PLA 10kDa and PEG 2kDa -PLA 10kDa In one embodiment, the first block copolymer chain is PEG. 5k -PLA 10k And the second block copolymer chain is PEG. 2k -PLA 10k In one embodiment, the nanomaterial contains a total amount of PEG (PEG 总 The particles contain at least 5%, preferably at least 15%, more preferably at least 25% ligand / PEG. 总The ligand density, where the term "ligand" includes both the first ligand and the second ligand. In one embodiment, the ligand density of the ligand / PEG is ≤50%.

[0054] As used herein, the term "first ligand" refers to a ligand capable of mediating nanoparticle attachment to target cells. In one embodiment, the first ligand only initiates the binding of the particle to the target cell without initiating the internalization of the particle into the target cell, and a second ligand is required to subsequently bind to the target cell to initiate the internalization of the particle into the target cell. In one embodiment, the first ligand is covalently or non-covalently coupled to the nanoparticle. In one embodiment, the first ligand is any biomolecule that triggers the binding of the nanoparticle to the target cell, such as an antibody or its antigen-binding fragment, peptide, aptamer, DNA nanostructure, receptor ligand, and receptor. In one embodiment, the first ligand is a non-agonist that binds to GPCRs such as angiotensin II receptor type 1 (AT1r), human neuropeptide Y1 receptor, and CXC chemokine receptor type 4, and / or an agent that binds to glycoproteins and / or glycolipids on the surface of the target cell, such as heparan sulfate, sialic acid glycoprotein, gangliosides, and mannose receptors, preferably EXP3174 or telmisartan. In one embodiment, a non-agonist binding to the GPCR triggers particle binding to target cells but does not trigger particle internalization (e.g., endocytosis) into the target cells. In one embodiment, the first ligand and / or the second ligand bind to a target that is not universally expressed but is primarily expressed on target cells in the target tissue. In one embodiment, the first ligand and / or the second ligand bind with an affinity K D <100 nM binds to target structures on target cells. In one embodiment, the first ligand and / or the second ligand has a molecular weight ≤1500 Da. In one embodiment, the terms "target" and "target structure" are used interchangeably. In one embodiment, the term "target structure" refers to proteins, peptides, nucleic acids, carbohydrates, glycolipids, and / or glycoproteins present on the surface of target cells. In one embodiment, the first ligand is any AT1R antagonist having a free carboxylic acid residue for functionalization. In one embodiment, the first ligand is a selective AT1 antagonist, such as EXP3174 (losartan carboxylic acid) or telmisartan, which are potent and selective AT1 antagonists, or optionally their bioactive derivatives. In one embodiment, the "bioactive derivative" has the same biological function as EXP3174 or telmisartan, such as the same binding and / or therapeutic function.

[0055] As used herein, the term "non-agonist" refers to an agent that binds to a target structure and does not have an agonistic effect on said target structure, preferably one that does not have an effect on the internalization of said target structure into the target cell.

[0056] As used herein, the term "second ligand" refers to a ligand capable of mediating the internalization of nanoparticles into target cells. In one embodiment, binding of the second ligand to a target structure (e.g., a receptor) on the target cell triggers the internalization of the nanoparticle into the target cell. In one embodiment, the target structure on the target cell is any structure normally expressed on the surface of the target cell, such as surface molecules, receptors, and / or biomarkers. In one embodiment, the target structure on the target cell is a molecule that is normally overexpressed in cells in a pathological state compared to healthy cells. In one embodiment, the first ligand and the second ligand target the same target structure on the target cell or target different target structures on the target cell. In one embodiment, the second ligand is covalently or non-covalently coupled to the nanoparticle. In one embodiment, the second ligand is any biomolecule that triggers the internalization of the nanoparticle into the target cell, such as an antibody or its antigen-binding fragment, a peptide, an aptamer, a DNA nanostructure, a receptor ligand, and a receptor. In one embodiment, the second ligand is any of the following: i) an agent that binds to integrins such as αVβ3 integrin, preferably selected from RGD, cyclic RGD-peptides having the sequence of SEQ ID NO. 1, and their derivatives; ii) an agonist that binds to GPCRs such as AT1r, preferably activated angiotensin-II; iii) an agent that binds to extracellular enzymes such as aspartate endopeptidase, membrane-type matrix metalloproteinases, and angiotensin-converting enzyme (ACE), preferably angiotensin-I; and / or iv) an agent that binds to transferrin receptors. In one embodiment, the second ligand is enzymatically activated prior to the internalization of the nanoparticle into the target cell, for example, angiotensin-I is activated by ACE to angiotensin-II prior to the binding of the second ligand to AT1r and the internalization of the particle into the target cell. In one embodiment, the enzymatic activation of the second ligand is carried out by an extracellular enzyme on the surface of the target cell, wherein preferably, the enzymatic activation includes enzymatic cleavage of the second ligand to provide the activated second ligand. In another embodiment, the second ligand is not enzymatically activated prior to mediating internalization. In one embodiment, the ratio of the first ligand to the second ligand is in the range of 2:1 to 1:2, preferably 1:1. In one embodiment, the second ligand binds to the target cell after the first ligand binds to the target cell. In one embodiment, the sequential binding of the first and second ligands to the target cell increases the specificity for the target cell.

[0057] In one embodiment, if the second ligand targets AT1r, angiotensin-I is preferred over angiotensin-II because the intermediate step of enzymatically activating angiotensin-I to angiotensin-II allows for increased nanoparticle specificity. In one embodiment, if the first ligand is EXP3174 and the second ligand is angiotensin-II or angiotensin-I to be activated to angiotensin-II, both the first and second ligands bind to the same target structure on the target cell, namely AT1R. In this embodiment, the first ligand, EXP3174, binds to AT1R as an antagonist, and the second ligand, for angiotensin-II (optionally after enzymatic activation), binds to AT1R as an agonist. After the first ligand (e.g., EXP3174) binds, there are still available AT1R binding sites for the second ligand. In one embodiment, the binding sites of the target bound by the first ligand and the target bound by the second ligand are different binding sites or the same binding sites but bound sequentially.

[0058] In one embodiment, the second ligand is shielded from binding to the target cell by: i) steric hindrance, for example, by a block copolymer chain or linker of the second ligand being shorter than that of the first ligand, and / or ii) by the second ligand being enzymatically activated before it can mediate internalization.

[0059] As used herein, the term "capable of mediating attachment" refers to the ability of a first ligand to bind to a target cell. Capable of mediating attachment means that the first ligand triggers the binding of the nanoparticle to the target cell, preferably through targeting a target structure on the target cell.

[0060] In one embodiment, the binding of the first ligand to the target cell does not trigger the internalization of the nanoparticle into the target cell. In another embodiment, further interaction is required for the internalization of the nanoparticle into the target cell, namely, the interaction of the second ligand with the target cell, preferably including a second ligand targeting a target structure on the target cell (which may be the same as or different from the target structure of the first ligand). In one embodiment, the first and second ligands may bind to the same target structure, but to different sites on the target structure, such as an agonist binding site and an antagonist binding site.

[0061] As used herein, the term "capable of mediating internalization" refers to the ability of a second ligand to trigger the internalization of nanoparticles into target cells. In one embodiment, the second ligand binds to a target structure on the target cell, thereby initiating the internalization of nanoparticles into the target cell. In one embodiment, the ability to trigger internalization may include the ability to be activated prior to triggering internalization, for example, through enzymatic activation. In one embodiment, the internalization involves any of receptor-mediated endocytosis, clathrin-coated pits, and / or caveolae.

[0062] As used herein, the term "target cell" refers to cells involved in a pathological condition (i.e., a disease). In one embodiment, treatment of the disease includes targeting the disease-involved cells with the nanoparticles of the present invention. In one embodiment, the nanoparticles of the present invention are used as a drug delivery system to deliver a drug to the target cells. In one embodiment, the target cells are selected from mesangial cells, endothelial cells such as retinal endothelial cells, and tumor cells.

[0063] As used herein, the term "therapeutic agent" refers to any substance intended for use in medical treatment. In one embodiment, the therapeutic agent is contained within the particle body (i.e., the core) of a nanoparticle, for example, within the nanoparticle and / or throughout the particle nanomaterial, and / or coupled to the nanoparticle using a connector. In one embodiment, the therapeutic agent is a lipophilic therapeutic agent and is encapsulated and / or contained within the particle body. In one embodiment, the therapeutic agent is non-covalently or covalently coupled to the particle, for example, through a cleavable connector. In one embodiment, the therapeutic agent may be covalently or non-covalently coupled to any component of the nanoparticle. In one embodiment, the therapeutic agent is an antifibrotic agent or a chemotherapeutic agent. In one embodiment, the therapeutic agent is any of pirfenidone and cinasciguat. As used herein, the term "particle body" refers to the main supporting structure of a particle. For example, the particle body may refer to a lipid bilayer or polymer of a liposome and / or the core structure of a solid lipid particle. In one embodiment, the nanoparticles of the present invention are loaded with a therapeutic agent for the specific treatment of target cells, such as mesangial cells, with the therapeutic agent. In one embodiment, a therapeutic agent (e.g., pirfenidone or cinasciguat) to be loaded into the particles is dissolved in a polymer phase and incorporated into the particles during the preparation of the polymer core of the particles, and / or dissolved in an aqueous phase contained in the particles, such as an aqueous phase contained in liposomes, and / or dissolved in a lipid phase contained in the particles, such as a lipid phase contained in liposomes. Pirfenidone is a TGF-β antagonist and has been proposed as a candidate drug for the treatment of mesangial-associated pathological fibrosis. Cinasciguat (BAY 58-2667) is a soluble guanylate cyclase (sGC) activator and has been proposed for the treatment of diabetic nephropathy. In one embodiment, the therapeutic agent pirfenidone or cinasciguat is efficiently incorporated into the polymer core of the nanoparticles without significantly altering the properties of the particles, which is possible due to the lipophilic nature of the therapeutic agent. In one embodiment, the nanoparticles are used to treat diabetic nephropathy and the therapeutic agent is an antifibrotic agent. In one embodiment, the nanoparticles are used to treat cancer and the therapeutic agent is a chemotherapeutic agent.

[0064] This invention also relates to compositions comprising the nanoparticles of the present invention and pharmaceutically acceptable excipients. The nanoparticles of the present invention can be mixed with suitable excipients and / or additives to obtain pharmaceutically acceptable compositions. Such substances include pharmacologically acceptable substances that increase the stability, solubility, biocompatibility, or biological half-life of the nanoparticles, or substances that are necessary for certain routes of administration (e.g., intravenous solutions, sprays, Band-Aids, or pills). This invention also relates to compositions comprising the nanoparticles of the present invention for medical use, such as methods for the prevention or treatment of diseases selected from diabetic nephropathy, glomerulonephritis, glomerular VEGF A dysregulation, endothelial VEGF A dysregulation, diabetic retinopathy, rheumatoid arthritis, age-related macular degeneration, and cancers such as breast cancer.

[0065] The nanoparticles of the present invention utilize a novel virus-mimicking recognition principle for target cells such as mesangial cells, resulting in efficient accumulation of the nanoparticles in target tissues such as the mesangium. In one embodiment, the nanoparticles are combined with a suitable therapeutic agent, i.e., the therapeutic agent is incorporated into and / or linked to the nanoparticles, which allows for targeted treatment of target cells in target tissues, such as mesangial cells in the mesangium. In one embodiment, the nanoparticles containing the therapeutic agent are used for the prevention or treatment of diabetic nephropathy. In one embodiment, the nanoparticles of the present invention allow for the targeting of target cells using a recognition process having at least two steps. In one embodiment, the nanoparticles of the present invention contain a selective AT1 antagonist (e.g., EXP3174) as a first ligand and also contain a therapeutic agent, preferably pirfenidone and / or cinasciguat. In one embodiment, the nanoparticles of the present invention are EXPcRGD nanoparticles, i.e., nanoparticles containing EXP3174 as a first ligand and cRGD (especially cRGDfK) as a second ligand, or the nanoparticles are EXPAng-I nanoparticles, i.e., nanoparticles containing EXP3174 as a first ligand and Ang-I as a second ligand. In one embodiment, the EXPcRGD nanoparticles and / or EXPAng-I nanoparticles further comprise one or more therapeutic agents, preferably pirfenidone and / or cinastegua. In one embodiment, the nanoparticles are nanoparticles comprising a selective AT1 antagonist (e.g., EXP3174) as a first ligand, preferably EXPcRGD nanoparticles, and further comprising a therapeutic agent, which is cinastegua. In one embodiment, the nanoparticles comprise nanomaterials comprising or composed of PLGA and / or PEG-PLA.

[0066] As used herein, the term "application" refers to the administration of a pharmaceutical agent, such as the nanoparticles of the present invention and / or the pharmaceutical compositions of the present invention, which can be achieved by any method that allows the pharmaceutical agent to reach target cells. These methods include, for example, injection, oral administration, inhalation, nasal delivery, topical application, deposition, implantation, suppositories, or any other method of administration via nanoparticles to access target cells. Injection may involve intravenous, intradermal, subcutaneous, intramuscular, or intraperitoneal injection. Implantation may include insertion into an implantable drug delivery system comprising the nanoparticles of the present invention and / or may involve hydrogels comprising nanoparticles, particularly hydrogels for subcutaneous and / or intraperitoneal injection that are gelled in situ and have a delayed release of the nanoparticles. Suppositories include glycerin suppositories. Inhalation includes the administration of nanoparticles as an aerosol in an inhaler, alone or attached to an absorbable carrier. Nanoparticles may be suspended in a liquid, for example, in colloidal form.

[0067] "Effective amount" is the amount of nanoparticles or pharmaceutical composition that alleviates the symptoms of the identified disease and / or condition.

[0068] As used herein, the term "patient" refers to a person or animal, preferably a mammal. Treatment of a patient is intended to include, for example, prevention, treatment, relief of symptoms of a disease or condition, or cure of a disease or condition, such as cancer or diabetic nephropathy.

[0069] As used herein, the term "block copolymer" refers to a polymer comprising two or more homopolymer or copolymer subunits linked by covalent bonds. It may contain intermediate non-repeating subunits, which are linking blocks. Block copolymers consist of blocks of different polymeric monomers. The term "block copolymer chain" refers to the chain of a block copolymer.

[0070] As used herein, the terms “DCC / NHS-coupling” and “EDC / NHS-coupling” refer to coupling reactions. A common method for synthesizing NHS-activated molecules involves mixing NHS with, for example, a desired carboxylic acid and a small amount of organic base in an anhydrous solvent. A coupling agent such as dicyclohexylcarbodiimide (DCC) or ethyl(dimethylaminopropyl)carbodiimide (EDC) is then added to form a highly active activation intermediate. In one embodiment, a first ligand and a second ligand are coupled to the nanomaterial in at least two steps. In one embodiment, the first and second ligands are coupled to the nanomaterial via a linker, via a fusion protein, and / or via PEG.

[0071] The inventors have successfully demonstrated herein that viral mimicry of NPs with dual / triple cell identity checks allows for enhanced NP accumulation in target cells (MCs) in vivo. By combining antagonistic ligands mimicking the initial cell attachment of the virus with enzymes mediating the target cell recognition process, these particles exhibit very high in vitro target affinity and excellent target cell specificity. The inventors have also demonstrated that simultaneous heterovalent binding of agonists and antagonists to the same GPCRs of the particles unexpectedly leads to particle uptake. Overall, nonspecific size-mediated passive targeting is insufficient to achieve satisfactory particle accumulation in MCs. Even conventional particle functionalization using a single ligand appears to be an inadequate approach. However, by mimicking the complex multi-step viral target cell binding and recognition process, the inventors have obtained particles capable of recognition and accumulation in MCs. This opens new options for drug delivery for the treatment of a variety of diseases, including kidney disease.

[0072] The inventors further fabricated adenovirus-mimicking block copolymer nanoparticles that efficiently target glomerular mesangial cells due to spatially controlled sequential ligand-receptor interactions. Therefore, the heteropolyvalent NP not only exhibits precisely tunable physicochemical properties but also demonstrates excellent affinity for both target motifs, resulting in substantial AT1r binding and significantly increased mesangial cell uptake in the picomolar range compared to unfunctionalized NPs. Utilizing these characteristics, the virus-mimicking NP can selectively target mesangial cells, even in the context of off-target cells. Furthermore, the heteropolyvalent NP exhibits the necessary in vivo robustness, leading to efficient accumulation in the mesangial region with only small off-target deposition within the kidney. Clearly, the heteropolyvalent EXPcRGD NP thus demonstrates significantly better mesangial targeting compared to the homofunctional cRGD or EXP NPs. Since the inventors were able to target the same unique cell types in vivo using two different virus-inspired concepts, they conclude that the mimicry of viral infection patterns allows for highly efficient targeting of concepts. Furthermore, successful mesangial cell targeting allows for precise treatment of mesangial-related kidney diseases because it significantly increases drug delivery compared to all other currently available methods. Attached Figure Description

[0073] The invention will now be further described with reference to the following figures.

[0074] All methods mentioned in the accompanying drawings are performed as described in detail in the embodiments.

[0075] Figure 1 The simulation shows viral attachment and target cell recognition.

[0076] (A) NPs carrying EXP3174 and Ang-I on their crown (NPEXPAng-I) attach to the cell membrane via EXP3174-mediated AT1R binding. Specific recognition is triggered by enzymatic Ang-I processing and Ang-II-mediated internalization.

[0077] (B) An example illustrates the flowchart of the triple target cell recognition of decision NP.

[0078] Figure 2 The characterization of the nanoparticles is shown.

[0079] (A) Assembly of ligand-modified NPs.

[0080] (B) Molar ligand content of different NP substances normalized relative to PEG content.

[0081] (C) Size and polydispersity index (PDI) and

[0082] (D) The obtained NP preparation -potential.

[0083] The results are shown as the average of at least n = 3 measurements ± SD.

[0084] Figure 3 In vitro interactions with AT1R and ACE were demonstrated.

[0085] The interaction between ligand-modified NPs and their targets AT1R (AD) and ACE (EF) was determined by intracellular calcium measurements. (A) Ligand affinity and (B) particle affinity for AT1R. (C) IC50 values ​​of free and particle-bound ligands. (D) Kinetic measurements of AT1R inhibition by ligand-modified particles. (E) Michaelis-Menten kinetics of NPEXPAng-I and NAng-I. (F) Specificity constants (Kcat / km) of free and particle-bound Ang-I calculated based on ligand and NP concentrations. Results are shown as mean ± SD of at least n = 3 measurements. Statistical significance levels are expressed as **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, and #p ≤ 0.0001 and x ≤ 0.001, comparing AT1R inhibition of NPEXP and NPEXPAng-I at different time points. ns: not significant.

[0086] Figure 4 This image shows the internalization of NPEXPAng-I (red) in target rat mesangial cells (rMC) transfected with YFP-labeled AT1R (green) at different incubation times (pAT1R-rMC). Scale bar: 20 μm.

[0087] Figure 5 This demonstrates the uptake specificity of the virus mimicking NPEXPAng-I.

[0088] (A) Ligand-mediated internalization of NPESPAng-I, NPESPAng-I, and NPEXP in rMCs is inhibited by free EXP3174 and captopril (see also) Figure 11 and 12 (B) Uptake of NPESPAng-I in AT1R and ACE-positive rMCs and HK-2 cells, and AT1R and ACE-negative HeLa cells. Specificity of particle uptake in target rMCs co-cultured with off-target (C) NCI-H295-R cells or (D) HeLa cells, analyzed by flow cytometry. (E) CLMS image of particle uptake (red) in green-stained (CTG) rMCs (green) co-cultured with deep red-stained (CTDR) off-target HeLa or NCI-H295R cells (white). Scale bar 20 μm. (See also...) Figure 13 Results are presented as mean ± SD of at least n = 3 measurements. Statistical significance levels are expressed as **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, and #p ≤ 0.0001, comparing NP uptake in cells with and without captopril or EXP3174 inhibition. ns: not significant.

[0089] Figure 6 The distribution of NPs in mouse kidneys is shown. (A) NPEXPAng-I fluorescence is located in the glomeruli (white arrows). (B) Control untargeted NPMeO did not accumulate in the glomeruli, and the glomeruli lacked granule-related fluorescence. Blue: DAPI staining of cell nuclei; green: tissue autofluorescence; red: NP-related fluorescence. Square areas from left to right are shown as magnification.

[0090] Figure 7 The evaluation of NP-related fluorescence detected in the glomeruli is shown by fluorescence microscopy analysis.

[0091] (A) Images of mouse glomeruli (dashed circles) treated with different particle formulations. Scale bar 40 μm. See also Figure 14 (B) Quantitative analysis of NP-fluorescence in intact glomeruli. (C) Comparison of granule-related fluorescence in glomeruli of the epithelial and endothelial layers. (D) Glomerular localization of NPEXPAng-I as determined by CLSM and integrin-α8 staining of MCs (scale bar 20 μm). Results in (C) and (D) are expressed as mean ± SD of at least n = 120 fluorescence measurements for each NP sample (n = 6 mice). Statistical significance is expressed as p ≤ 0.0001. ns: not significant.

[0092] Figure 8 The coupling of the ligand with the PEG-PLA block copolymer is shown.

[0093] (A) Lys-Ang-I and (B) EXP3174 were respectively treated with carboxylic acid or amine-terminated PEG using EDC / NHS or DCC / NHS chemistry. 5k -PLA 10k (C) Quantification of molar ligand and PEG content revealed complete polymer functionalization. (D) Fluoroamine was used to confirm the absence of unreacted NH2 polymer end groups in the EXP3174-modified polymer. Student's t-tests were performed using GraphPad Prism 6.0 to assess statistical significance. Statistical significance levels are expressed as p ≤ 0.0001, comparing NH2-terminated PEGs. 5k -PLA 10k The fluorescence of MeO- and EXP3174- was observed. See Example 1 for detailed methods.

[0094] Figure 9 The maximum calcium signaling and inhibition of NPEXP are shown.

[0095] rMCs were simultaneously stimulated with Ang-II and NPEXP, and the resulting intracellular calcium response was measured immediately for 1 minute. At the NPEXP concentration used, the EXP3174 ligand did not inhibit agonist-triggered calcium signaling during the assay. Therefore, the effect of EXP3174 on Ang-II measurements was considered negligible. Results are shown as the mean ± SD of at least n = 3 measurements. Detailed methodology is described in Example 1.

[0096] Figure 10 The results show the intake of different particulate formulations over time in AT1R-positive pAT1R-rMCs analyzed by CLSM.

[0097] (A) NPEXP is not internalized in cell lines and is primarily located on the cell membrane and on filipodia between large clusters of cells over time. Receptor binding is shown by co-localization of NP and receptor-associated fluorescence. (B) NPAng-I is internalized by cells, as indicated by its cytoplasmic localization. (C) NPMeO does not bind to cells because they lack tethered ligands capable of achieving specific targeting. Cells: white; AT1R-YFP: green; NP-formulation: red. Scale bar 20 μm. See Example 1 for detailed methods.

[0098] Figure 11 The results showed that EXP3174 offset the reduced uptake caused by the steric hindrance of the Ang-I ligand due to the long polymer chain on NPEXPAng-I.

[0099] Preparation of COOH-PEG with different polymer densities 5k NPAng-I (gray) with a 20% Ang-I density of PLA10k was prepared, and their cellular uptake was analyzed by flow cytometry. Simultaneously, EXP3174-PEG with different densities was prepared. 5k -PLA10k NPESPAng-I (yellow) was used to compare the effect of the second ligand on the steric hindrance of Ang-I. Functionalization of long polymer chains with EXP3174 on NPESPAng-I offset the reduced uptake due to steric hindrance of the Ang-I ligand when adding unfunctionalized long polymers and significantly increased particle internalization. Results are shown as mean ± SD of at least n = 3 measurements. Two-way ANOVA and Sidak multiple comparison tests were performed using GraphPad Prism 6.0 to assess statistical significance. Statistical significance is expressed as p ≤ 0.0001. Detailed methodologies are described in Example 1.

[0100] Figure 12 The specificity of NP uptake analyzed by CLSM was demonstrated.

[0101] Cells were pre-incubated with free EXP3174 for 30 minutes before adding different NP formulations (NPEXPAng-I, NPAng-I, and NPESP). Inhibition of the target receptor resulted in inhibition of particle-associated fluorescence. Scale bar 20 μm. See Example 1 for detailed methods.

[0102] Figure 13 The uptake of (A) NPEXP and (B) NPAng-I in co-culture of target cells and off-target cells is shown.

[0103] NPEXP showed accumulation in rMCs and NCI-H205R cells because both carry AT1R. Conversely, co-culture of rMCs and HeLa cells showed preferential accumulation of NPEXP in rMCs because HeLa cells express only a small number of receptors on their cell membranes (see [3]). NPAng-I showed higher specificity because they preferentially accumulated in target rMCs carrying the equipment (ACE and AT1R) required for their internalization, relative to off-target cells lacking ACE (HeLa or NCI-H295R cells). Nuclei: blue; Off-target cells (HeLa or NCI-H295R): white; Target cells (rMCs): green; NPs: red. Scale bar 20 μm. See Example 1 for specific methods.

[0104] Figure 14 The in vivo distribution of different NP formulations is shown.

[0105] (A) Kidney distribution of NPAng-I and NPEXP in mouse kidneys. NPAng-I showed small NP-related fluorescence in most glomeruli, while NPEXP did not accumulate in this region. Glomeruli are marked with white arrows for visualization. Square areas from left to right are shown at magnification. DAPI staining of cell nuclei: blue; tissue autofluorescence: green; NP-related fluorescence: red. (B) Kidney distribution of free dyes used to label NPs. CF647 was injected into mice as a control to assess its distribution in the kidneys. Strong fluorescence was detected in the tubular region, while no fluorescence was observed in the glomeruli (marked with white circles). This indicates that the fluorescence seen in the particulate samples comes from the particles themselves, rather than from leaked dye that is freely filtered out due to its low molecular weight. (C) Plasma retention of different NP formulations one hour after circulation in NRMI mice. NP fluorescence in plasma was measured 1 hour after injection and normalized relative to fluorescence measured 5 minutes after injection (initial particulate blood fluorescence). Untargeted NPs (NPMeO) exhibited the highest blood circulation time, attributed to the cloaking effect conferred by their PEG shell. Although 40% of all polymers on the NPESPAng-I surface were ligand-coupled, which reduced the cloaking effect of the particles, they were able to match the blood retention of untargeted particles. They depicted significantly higher fluorescence in plasma after 1 hour compared to particles functionalized with only one ligand (NPAng-I and NPESP). NPAng-I, carrying a specific two-step virus-mimicking recognition mechanism, also showed significantly superior blood retention compared to NPESP, which represents the typically targeted NP. As a control, the free dye (CF647) used to label the particles was additionally injected into mice and disappeared rapidly from blood circulation after free filtration (6% of the initial fluorescence after 1 hour). Results in (C) are presented as mean ± SD of at least n = 6 samples. Student's t-test was performed using GraphPad Prism 6.0 to assess statistical significance. Statistical significance levels are expressed as *p≤0.05, ***p≤0.001, and ****p≤0.0001. ns: not significant. See Example 1 for detailed methods.

[0106] Figure 15 Exemplary in vivo use of the decision nanoparticles of the present invention is shown, comprising a first ligand for attachment to target cells and a second ligand for recognition / internalization of the nanoparticles into said target cells. The nanoparticles of the present invention can be used to target a variety of target tissues, such as mesangiums. By selecting the first and second ligands that target target receptors and / or molecules on the surface of target cells, nanoparticles for targeting specific target tissues can be tailored.

[0107] Figure 16 A transverse kidney slice of an animal treated with granules is shown.

[0108] A) Virus mimic particles showed high accumulation in the renal corpuscles (circular markers).

[0109] B) Unmodified nanoparticles without carrying the first and second ligands did not show significant absorption into glomerular tissue.

[0110] Figure 17 This indicates that adenovirus-mimicking NPs enter glomerular mesangial cells through a synergistic combination of passive and active targeting strategies. (a) After administration, NPs reach the glomerular region of the renal filtration system via afferent arterioles, which then shunt into the glomerular capillary system. (b) Within the glomerular capillaries, due to their reticular structure, NPs cannot pass through the renal filters but can leak through endothelial fenestrations to reach the interstitial mesangium. (c) Therefore, viral-mimicking NPs can effectively infiltrate mesangial cells via initial binding to angiotensin II receptor type 1 (AT1r) and subsequent αVβ3 integrin-mediated endocytosis. (AA: afferent arteriole; EA: efferent arteriole; MC: mesangial cell; PO: podocyte; FP: foot process; ET: endothelium; BM: basement membrane.)

[0111] Figure 18 Characterization of adenovirus-mimicking NPs is shown. (a) Particle design of heteropolyvalent EXPcRGD NPs and isofunctionalized or nonfunctionalized NPs. (b) DLS analysis. All particle types were fabricated below the 60 nm size threshold without significant aggregation. (PDI: Polydispersity Index). (c) Zeta potential measurement. COOH-PEG 2k -PLA 10k Adding to the polymer mixture results in a negative surface charge. (d) / (e) are the quantifications of the surface densities of cRGDfK and EXP3174 ligands after NP fabrication, respectively. The final surface content is compared with the previously added cRGDfK(R 2 =0.9957) and EXP3174(R 2 The amount of ligand-functionalized polymers is proportional to the amount of polymers with a ligand ratio of 0.9871. The results represent the mean ± SD (n = 3).

[0112] Figure 19 The AT1r interaction of adenovirus-mimicking NPs is shown. (a) Intracellular calcium levels in rMCs treated with free or particle-bound EXP3174 after AT1r stimulation. EXPcRGD NP(IC 50 =276±31pM) and EXP NP(IC 50 =552±73pM) effectively binds to and inhibits AT1r, resulting in low Ca2+ influx into AT1r upon stimulation with angiotensin II (ATII). Due to the affinity gain from multivalent derivation, free EXP3174 (IC) effectively binds to and inhibits AT1r, leading to low Ca2+ influx into AT1r upon stimulation with angiotensin II (ATII). 50This effect was even stronger with (M = 2.66 ± 0.9 nM). (b) AT1r activity of rMCs treated with NPs without AT1r ligands. Neither the unfunctionalized control NP nor the cRGD NP significantly bound AT1r, resulting in the highest calcium levels upon ATII stimulation and confirming the specificity of the assay. (M = molar concentration of NP or EXP3174). Results represent mean ± SD (n = 3).

[0113] Figure 20 The internalization of NP by target mesangial cells in vitro was demonstrated. (a) with 0.05 mg mL -1 Alexa Fluor TM CLSM analysis of CTDR-stained rMCs after incubation with 568-labeled EXPcRGD nanoparticles. NP-related fluorescence (purple) can be detected in vesicle structures within the rMC cytosol (gray). The size, number, and intensity of endocytic vesicles increased with increasing incubation time, indicating vesicle fusion into larger endosomes. (Scale bar 20 μm.) (b) Flow cytometry analysis of NP uptake in rMCs. Heterovalent EXPcRGD NPs showed significantly increased cellular uptake compared to control NPs and isofunctional NPs. (c) Flow cytometry results after 60 min of NP incubation. Although EXPcRGD NPs showed the greatest cell binding compared to all other NP types, the fluorescence signal decreased sharply to EXP NP levels upon addition of excess free cRGDfK (c = 500 μM), indicating an αVβ3-dependent NP uptake. Results represent mean ± SD (n = 3). ◇P<0.0001, οP<0.001, ****P<0.0001. (AFU, any fluorescence unit).

[0114] Figure 21 TEM analysis of NP interactions with mesangial cells is shown. (a) EXPcRGD NPs accumulate in numerous vesicular structures (black arrows) within rMCs. Vesicles of varying sizes are present both outside and inside the cytosol, indicating intracellular processing and fusion into larger endosomes. Furthermore, a large number of NPs remain on the cell membrane, suggesting a stepwise process of initial cell binding and subsequent endocytosis. (The image on the right shows a magnified view of the black box on the left). (b) In contrast, EXP NPs accumulate only at the cell boundary, where they bind to different surface structures of rMC cells, suggesting possible interaction with membrane-bound AT1r. (The image in the upper left shows a magnified view of the black box). (c) Control NPs show only negligible interactions with rMCs, with almost no gold-enhanced NPs visible.

[0115] Figure 22Mesangial cell selectivity of EXPcRGD NPs in in vitro co-culture assays is shown. (a) CLSM analysis of CTG-stained rMCs (green) co-cultured with CTDR-labeled HeLa or NCI-H295R cells (grey) acting as off-target cells. For rMC / HeLa co-culture (top row), NP-derived fluorescence (purple) was detected only within the rMC region, indicating selective EXPcRGD NP uptake into the target cells. Co-culture of rMCs with AT1r-expressing NCI-H295R cells resulted in a divergent NP distribution (bottom row). Thus, EXPcRGD NPs also bind to the surface of NCI-H295R cells, resulting in a diffusion pattern around the cell boundary. However, efficient granule uptake into round endocytic vesicles was only observed in mesangial cells. (Scale bar 20 μm). Flow cytometry analysis of rMCs with both HeLa (b) and NCI-H295R (c) supported the CLSM results, as EXPcRGD NPs showed significantly higher cell binding to rMCs in both cases. Interestingly, the addition of excess free EXP3174 (c = 1 mM) to (c) drastically reduced the interaction of NPs with NCI-H295R cells, indicating that EXPcRGD NPs could no longer bind to NCI-H295R cells via AT1r. Results represent mean ± SD (n = 3). ****P < 0.0001. (ns: not significant. AFU, arbitrary fluorescence units).

[0116] Figure 23 This indicates that EXPcRGD NPs exhibit strong intraglomerular accumulation in vivo. Transverse frozen sections of the kidney were imaged using fluorescence microscopy. For ease of histological evaluation, cell nuclei were stained with DAPI (blue) and tissue autofluorescence (green) was recorded. EXPcRGD NPs (red) were found to accumulate almost exclusively in the cortical glomerular region (white circles), while fluorescence in the tubular region was negligible. (The image shows a magnified view within the white boxes from top left to bottom right).

[0117] Figure 24The results show that EXPcRGD NP exhibits significantly enhanced accumulation in mesangial cells. (a) Fluorescence microscopy analysis revealed high fluorescence levels primarily detected within the glomeruli (white circles) for EXPcRGD NP. While EXP NP showed moderate accumulation in the glomeruli, control NP and cRGD NP did not produce any comparable signal. (Scale bar 20 μm. Calibration bar: 0–65535 gray values). (b) Precise quantification of intraglomerular fluorescence intensity was achieved by assessing the integrated density of each glomerular region in a sufficient number of glomeruli. Therefore, EXPcRGD NP showed significantly higher fluorescence intensity per glomerulus compared to all other particle types. Results represent mean ± SD (n = 60). ****P < 0.0001. (AFU, arbitrary fluorescence units). (b) Antibody staining of the mesangial surface marker integrin α-8 showed that EXPcRGD NP-related fluorescence (red) colocalized with the mesangial cell-covered area (yellow), indicating that EXPcRGD NP can selectively infiltrate mesangial cells via endocytosis. Scale bar 20 μm.

[0118] Figure 25 The synthetic concept of ligand-functionalized PEG-PLA block copolymers is shown. (a) Iso-bifunctionalized PEG polymers (①) with different chain lengths (2kDa / 5kDa) are mixed with 3,6-dimethyl-1,4-dioxane-2,5-dione (②) to generate NH2-PEG by ring-opening polymerization. 5k -PLA10k and COOH-PEG 2k -PLA 10k (③). (b) Subsequently, NH2-PEG 5k -PLA 10k EXP3174-PEG was obtained by covalent coupling of DCC / NHS chemistry with the carboxyl group of EXP3174(④). 5k -PLA 10k (⑤). (c) In addition, COOH-PEG 2k -PLA 10k The cRGDfK-PEG is chemically linked to the lysine residue of cRGDfK(⑥) via EDC / NHS, resulting in a shorter cRGDfK-PEG. 2k -PLA 10k (⑦). (d) The synthesized EXP3174-PEG was determined by comparing the molar concentrations of PEG and EXP3174. 5k -PLA 10k The coupling efficiency was high. Therefore, the molar concentration did not change significantly, indicating successful functionalization. (e)cRGDfK-PEG 2k -PLA 10kThe coupling efficiency was also within a reasonable range, with only negligible differences in the molar concentrations of PEG and cRGDfK. The results represent the average ± SD (n = 3).

[0119] Figure 26 Gold-labeled NPs are shown for TEM visualization. NPs were gold-labeled by covalently attaching ultrasmall gold nanoparticles (diameter: 2.2 nm) to the carboxyl groups of PLGA and then using them to fabricate NPs. After incubation of rMC with the labeled NPs, the particle cores were gold-enhanced by depositing more gold particles on the NP cores, thereby increasing the electron density of the sample and enabling visualization under a TEM microscope, where the NPs appear as dark dots.

[0120] Figure 27 The results show the quantification of αVβ3 expression in different cell types as investigated by (a) flow cytometry and (b) CLSM. For cell counting analysis, nonspecific binding sites were blocked with DPBS solution containing 2% BSA, and 10⁵ cells were counted. Anti-CD51 / 61 antibody was diluted 1:20 in 0.1% BSA-dPBS solution. Mouse IgG1,κ isotype control (FC) was used as a nonspecific control and incubated for 1 hour. Cells then underwent several steps of washing with DPBS and centrifugation. Finally, the samples were resuspended in DPBS and analyzed by flow cytometry as previously described (FACSCalibur, excitation: 633 nm, emission: 661 / 16 nm bandpass filter). Therefore, rMCs showed the highest αVβ3-derived fluorescence level, while HeLa and NCI-H295R cells both showed negligible signals, significantly lower than rMCs. Results represent mean ± SD (n = 3). ****P < 0.0001, **P < 0.01, *P < 0.05 (AFU, arbitrary fluorescence units). To confirm the flow cytometry results, rMCs were seeded into 8-well Ibidi slides (15,000 cell wells). -1 Cells were stained with αVβ3 integrins as described above. Cells were then washed with DPBS, fixed in DPBS solution with 4% PFA, and analyzed on a Zeiss LSM 710. CLSM images showed strong integrin signals colocalized with rMC cell bodies, indicating abundant αVβ3 expression in mesangial cells. Scale bar 20 μm.

[0121] Figure 28 The results of fluorescence imaging are shown.

[0122] A) Relative plasma fluorescence after NP injection. Control NP showed the highest circulating value, with nearly 50% residual plasma fluorescence 60 minutes after injection. cRGD NP was rapidly cleared from the blood compared to EXP NP and EXPcRGD NP, which showed tolerable residual concentrations. Results represent mean ± SD (n = 3). ****P < 0.0001, ***P < 0.001, **P < 0.01. (ns: not significant).

[0123] B) Injecting free CF TM Fluorescence imaging of frozen kidney sections after 647 fluorescent dyeing. Sections were stained with DAPI (blue) for visualization of cell nuclei. Strong fluorescence signals (red to white) were detected in the renal tubular regions after injection of comparable molar concentrations of free dye, indicating free renal filtration of low-molecular-weight dye. As expected, no accumulation was detected in the glomeruli (white circles). (Calibration bar: 0-65535 grayscale values).

[0124] Figure 29 This demonstrates the concept of NP-assisted cinacigua delivery. Heterovalent EXPcRGD NPs enter target renal mesangial cells via a previously described sequence recognition sequence. Following successful endocytosis, the NP undergoes lysosomal degradation, leading to the release of cinacigua (small dot). CCG then activates and stabilizes mesangial GC, resulting in enhanced, NO-mediated cGMP production. Multiple profibrotic pathways are inhibited via cGMP-mediated activation of protein-regulated kinase (PGK1-α), leading to an overall reduction in mesangial cell profibrotic remodeling.

[0125] Figure 30 An exemplary experimental setup is shown. For all experiments, free cinnarizine (c = 2 μM) was compared to CCG carrying a concentration of 0.2 μM or EXPcRGD NP without the drug. The amount of free cinnarizine was selected based on previous studies that demonstrated potent effects within this concentration range.

[0126] Figure 31Western blot analysis showing the effects of cinasciguat on drug targets. a) sGC stimulation and stabilization. Both free CCG and CCG-loaded EXPcRGD NPs significantly increased sGC levels during 24 hours of incubation, while the CCG-free control NP had no significant effect. b) PGK1-α activation. To assess PGK1-α activity, phosphorylation of its substrate, vasodilatory stimulating phosphoprotein (VASP), was evaluated. Therefore, both free CCG and CCG-loaded EXPcRGD NPs showed a significant increase in the P-VASP / VASP ratio, while the control NP did not cause a detectable change. (n = 3; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05).

[0127] Figure 32 The study demonstrated the antifibrotic and antiproliferative effects of NP loaded with cinaciguat. Mesangial cells were pre-incubated with free CCG or NP for 4 hours, then loaded with 10 ng / mL... -1 TGF-β incubation for 48 hours induced fibrosis and hyperproliferative changes. a) MTT assay showed an antiproliferative effect. Although mesangial cells showed significant hyperproliferation upon TGF-β stimulation, pre-incubation with free CCG or CCG-loaded NPs significantly reduced this effect. b) Western blot analysis of α-SMA and c) collagen I levels showed that both fibrosis markers were significantly reduced in CCG or CCG-loaded NPs compared to the TGF-β control. (n=3; ****P<0.0001; ***P<0.001; ns not significant). d) LSM analysis further supported the results of b), showing that mesangial cells showed significantly reduced α-SMA production after pre-incubation with CCG or CCG-loaded NPs. Scale bar = 2 μm.

[0128] The invention will be described below with reference to embodiments, which are given for illustrative purposes and not for limiting the invention. Detailed Implementation

[0129] Example

[0130] Example 1: Materials and Methods

[0131] Cell culture

[0132] The cell lines used in this study were cultured at 37°C and 5% CO2. rMC, NCI-H295R, and HeLa cells were cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% FBS, insulin-transferrin-selenium, and 100 nM hydrocortisone. HK-2 cells were maintained in DMEM-F12 (1:1) medium (Sigma-Aldrich) supplemented with 10% FBS. pAT1R-rMC was obtained by transfecting rMC with a plasmid (CXN2-HA-AT1R-YFP) encoding an AT1R with a YFP tag (see [4]) using the commercially available transfection reagent Lipofectamine 2000 according to the manufacturer's instructions. pAT1R-rMC was cultured in RPMI 1640 medium supplemented with 10% FBS and 600 μg / ml genimycin (G418). The cell lines were characterized by their target AT1R and ACE expression, as previously described [1,3].

[0133] mice

[0134] Animal experimental procedures were conducted in accordance with national and institutional guidelines and approved by the local authorities (Regierung von Unterfranken, Reference No.: 55.2-2532-2-329). Mice listed in the Key Resources Table were used in this study at 10 weeks of age. Only female mice were used in all experiments. They were kept in specific pathogen-free (SPF) facilities under standard conditions (50 ± 5% relative humidity, 21 ± 1°C temperature, air exchange >8 AC / h, and a 12h:12h (L:D) photocycle).

[0135] Polymer preparation: Block copolymer synthesis

[0136] PEG-PLA block copolymer (COOH-PEG) 2k -PLA 10k COOH-PEG 5k -PLA 10k NH2-PEG 5k -PLA 10k and MeO-PEG 5k -PLA 10k The product was synthesized via ring-opening polymerization of cyclic 3,6-dimethyl-1,4-dioxane-2,5-dione (lactide). Briefly, the lactide was recrystallized from anhydrous ethyl acetate before use and dried under vacuum at 40°C for 12 hours at room temperature (rt). COOH-PEG 5k -OH, COOH-PEG 2k -OH, Boc-NH-PEG 5k-OH or MeO-PEG 5k -OH groups were used as macromolecular initiators for ring-opening polymerization. They were dissolved (0.3 mmol) in anhydrous DCM and mixed with purified lactide (18 mmol). 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.9 mmol) was added as a catalyst. The polymerization was quenched with benzoic acid (4.6 mmol) after 1 hour. The resulting polymer was precipitated in diethyl ether and heated at 40 °C (for COOH-PEG). 2k -PLA 10k COOH-PEG 5k -PLA 10k and MeO-PEG 5k -PLA 10k ) or 35℃ (for Boc-NH-PEG) 5k -PLA 10k The mixture was dried under vacuum for 12 hours. To cleave the protective Boc group, Boc-NH-PEG-PLA was dissolved in 50% (v / v) TFA / DCM and stirred at room temperature for 30 minutes. It was then diluted with three times its volume of DCM and washed three times with a saturated sodium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting NH2-PEG... 5k -PLA 10k Purification was achieved by precipitation in diethyl ether, followed by vacuum drying at 35°C for 12 hours.

[0137] Polymer preparation: ligand coupling

[0138] To prepare Ang-I modified polymers (see also...) Figure 8 ), 14 μmol of COOH-PEG 5k -PLA 10k The polymer was activated for 2 hours with an excess of 25 mol of EDC and NHS in DMF solution. Then, the reaction was quenched for 20 minutes with 863 μmol of 2-mercaptoethanol, followed by dropwise addition of a DMF solution of 66 μmol of DIPEA and 17 μmol of Lys-Ang-I. After 48 hours, the resulting polymer was diluted in ultrapure water (Millipore) to a DMF concentration below 10% and dialyzed for 24 hours using a regenerated cellulose dialysis membrane with a molecular weight cutoff of 6-8 kDa (medium changed after 30 minutes, 2 hours, and 6 hours) to remove unreacted ligands and reagents. For the preparation of EXP3174-modified polymers (see also...), see... Figure 896.4 μmol of EXP3174 was activated for 2 hours with an equimolar amount of DCC and NHS in DMF solution. The resulting urea byproduct was then removed by centrifugation (5 min, 12,000 x g) followed by filtration through a 0.2 μm Rotilabo PTFE injection filter. 27.6 μmol of NH2-PEG was then added. 5k -PLA 10k A DMF solution and 17.5 mol excess DIPEA were added to the activated ligand and reacted for 20 h. The ligand-modified polymer was purified as follows: precipitation was performed in ice-cold 1:5 (v / v) diethyl ether: methanol, followed by dialyzing with 10% ethanol in 10 mM borate buffer (pH 8.5) for 24 h to remove excess free ligands (medium changed after 30 min and 6 h) using a regenerated cellulose dialysis membrane with a molecular weight cutoff of 6-8 kDa (Spectrum Laboratories), and then dialyzing with ultrapure water for 24 h to remove buffer salts (medium changed after 30 min, 2 h, and 6 h). The ligand-modified block copolymer was lyophilized for 72 h, followed by ligand coupling confirmation. For this purpose, the polymer was dissolved in ACN at a concentration of 40 mg / ml and precipitated in stirred ultrapure water to produce polymer micelles (final concentration 1 mg / ml). PEG content was quantified using a colorimetric iodine complexation assay, and conjugated Ang-I was quantified using a Pierce BCA assay kit following the manufacturer's instructions using a FLUOstar Omega microplate reader. ELISA reader was used to measure PEG content at λ... ex =250nm and λ em Quantitative analysis of EXP3174 was performed at 370 nm. NH2-PEG was identified using fluorescent amines. 5k -PLA 10k There are no unreacted NH2 end groups (see also) Figure 8 ).

[0139] Polymer preparation: Fluorescent labeling of PLGA

[0140] For in vitro and in vivo particle detection, fluorescently labeled PLGA was used in the particle core. For this purpose, TAMRA-amine (for CLSM) and CF6467-amine (for flow cytometry and in vivo experiments) were covalently coupled to carboxylic acid-terminated 13.4 kDa PLGA. Briefly, 5 μmol of acid-terminated PLGA was dissolved in anhydrous DMF and activated for 2 h at room temperature with 29 μmol DMTMM (25-fold excess). Then, 1 μmol of fluorescent dye was dissolved in DMF and added dropwise to the PLGA, and reacted in the dark at room temperature for 72 h. The reaction product was diluted (DMF < 10%) and dialyzed with ultrapure water for 34 h in the dark using a 3.5 kDa regenerated cellulose dialysis membrane (Spectrum Laboratories) (changing the medium after 30 min, 2, and 6 h). The fluorescently labeled PLGA was then lyophilized for 3 days.

[0141] NP preparation and characterization: Particle preparation

[0142] For NP preparation, PEG-PLA block copolymer and 13.4 kDa PLGA were mixed in ACN at a mass ratio of 70:30 to a final concentration of 10 mg / mL. For ligand-modified particles, COOH-PEG... 2k -PLA 10k The ligand-modified polymers were mixed accordingly, such that 20% of the polymers constituting the NP structure were modified with Ang-I (NPAng-I) and / or EXP3174 (NPEXP and NPESPAng-I, respectively). NPs were prepared by bulk nanoprecipitation of the polymer mixture in vigorously stirred 10% DPBS (v / v) (pH 7.4) to a final concentration of 1 mg / ml. The particles were stirred for 2 hours to ensure the organic solvent evaporated, and then concentrated by ultracentrifugation at 756 g for 20 minutes using a Microsep Advance centrifuge with a molecular weight cutoff of 30 kDa (Pall Life Sciences).

[0143] NP preparation and characterization: dynamic light scattering and zeta potential

[0144] Using a 633He-Ne laser equipped with a 173° backscattering angle and a ZetaSizer Nano ZS (Malvern Instruments) with Malvern Zetasizer software version 7.11, the size and zeta potential of the obtained particles were determined at a concentration of 1 mg / mL or 3.5 mg / mL in 10% PBS at a constant temperature of 25°C. The cuvette position was set to 4.65 mm, and the attenuator was automatically optimized by the device. Disposable microcuvettes (Brand) and folded capillary cells (Malvern Instruments) were used for size and zeta potential measurements, respectively.

[0145] NP preparation and characterization: particle quantization

[0146] The particle PEG concentration was quantified using a colorimetric iodine complexation assay and correlated with the gravimetric NP content determined by lyophilization. In short, particle samples were diluted in ultrapure water to a PEG concentration ranging from 5–30 μg / mL. A dilution of MeO-PEG-OH (0–40 μg / mL) in ultrapure water was used as a standard for the calibration curve. 140 μL of sample or standard was mixed with 60 μL of a 2:1 (v / v) mixture of 5% (m / v) barium chloride in 1N HCl solution and 0.1N iodine aqueous solution. Samples and standards were transferred to 96-well plates, and their absorbance at 535 nm was measured using a FUOstar microplate reader (BMG Labtech). The correlation between particle PEG content and accurate polymer concentration was determined by gravimetric analysis after sample lyophilization. Molar particle concentration was determined by particle mass and particle density (1.25 g / cm³) as determined by the colorimetric iodine complexation assay. 3 The hydrodynamic diameter of the NP, obtained by DLS measurement, was calculated, assuming a spherical particle shape. As described above, the concentration of ligands on the particle crown was quantified using the BCA assay, and Ang-I and EXP3174 were quantified by fluorescence, respectively.

[0147] Intracellular calcium measurement

[0148] To evaluate the AT1R interactions of different NP formulations, as previously described [1, 3], the ratio of Fura-2 Ca was used. 2+The chelating agent method was used, employing AT1R-positive rMCs. For this purpose, rMCs were seeded in T-150 flasks (Corning) and incubated until confluence. Subsequently, they were trypsinized, centrifuged (200g, 5 min), and resuspended in Leibovitz medium supplemented with 5 μM Fura-2 AM (Thermo Fisher), 0.05% Pluronic F-127, and 2.5 mM probenecid. Cells were incubated for 1 hour with gentle stirring (50 rpm) in the dark. Afterward, the cell suspension was washed with DPBS by centrifugation (2x, 200g, 5 min, RT) and resuspended at a count of 2 million cells / mL in Leibovitz medium supplemented with 2.5 mM probenecid. To determine the particle affinity and ligand affinity of AT1R (…),… Figure 3 AC), 45 μl of Fura-2-loaded rMC suspension (90,000 cells / well) and 10 μl of different samples (1 nM to 300 μM (ligand concentration) of NP or free ligand) were incubated in 96-well half-area microplates at room temperature for 30 min. Afterwards, cells were stimulated with 45 μl of 300 nM Lys-Ang-II aqueous solution, and the resulting calcium signal was immediately recorded for 1 min / well using a FLUOstar Omega microplate reader (BMG Labtech) with 340 / 20 nm and 380 / 20 nm excitation and 510 / 20 nm emission bandpass filters. To determine the kinetics of AT1R interactions (…),… Figure 3 D), using the same procedure, but with different incubation periods (5 to 320 minutes) between samples and cells. Maximum and minimum signal ratios were determined by stimulating cells with either 0.1% Triton-X 100 or 0.1% Triton-X 100 and 45 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). Following Grynkiewicz's hypothesis K... d Intracellular calcium concentration was calculated after the value was set to 225 nM. The Student's t-test was performed using GraphPad Prism 6.0. Figure 3 C) and two-factor ANOVA using Sidak's multiple comparisons (C) Figure 3 D) Assess statistical significance.

[0149] Enzyme kinetics measurement

[0150] Using rabbit lung ACE (Sigma Aldrich) as a soluble substitute for the cell membrane-binding enzyme, the Michaelis-Menten kinetics of NPAng-I and NPEXPAng-I were determined as previously described [1]. Briefly, different concentrations of NP (corresponding to 10–120 μM Ang-I) were incubated with 18 μM enzyme for different time periods (5, 15, 30, 60, 90, and 120 min) to convert Ang-I on the granular crown to the AT1R active ligand Ang-II. The resulting Ang-II was quantified by direct intracellular calcium measurement. For this purpose, Fura-2 dye was loaded onto rMCs as described above. Then, 10 μL of sample was pipetted into a 96-well half-area plate and 90 μL of Fura-2-loaded rMC suspension (90,000 cells / well) was injected on top. The resulting calcium signal was recorded immediately for 1 min using a FLUOstar Omega microplate reader (BMG, Labtech) as described above. To eliminate interference from NPEXPAng-I by the EXP3174 ligand under the experimental conditions used, NPEXP was used as a control (see [link to experimental data]). Figure 9 rMCs (90,000 cells / well) were simultaneously stimulated with different NPESP concentrations and Lys-Ang-II (400 nM), and the resulting calcium signal was recorded immediately for 1 minute. Known concentrations of free Lys-Ang-II ranging from 1 nM to 300 μM were used to convert the measured calcium concentrations to pmol of the hydrolysis products. The reaction rate (pmol / min) over a 15-minute incubation time was plotted against the substrate concentration used in the assay using GraphPadPrism 6.0 to determine particle-based and ligand-based concentrations. Figure 3 The Michaelis-Menten constant (Km) for E) was obtained. The catalytic constant (Kcat) was obtained using the same software to calculate the specificity constant (Kcat / Km) for comparing different substrates of the same enzyme. Figure 3 F). Statistical significance was assessed using a two-way ANOVA with Tukey's multiple comparison test in GraphPad Prism 6.0. Figure 3 F).

[0151] Cellular distribution of NPs: confocal microscopy

[0152] To determine the cell distribution of different particulate formulations ( Figure 4pAT1R-rMC cells were seeded at a density of 10,000 cells / well into 8-well μ-slides (Ibidi, Graefelfing, Germany) and incubated for 24 hours (37°C). They were then incubated for 15, 45, or 90 minutes with NP solution (0.2 mg / ml) preheated in Leibovitz medium (LM) supplemented with 0.1% bovine serum albumin (BSA). Afterward, the NP solution was discarded, and the cells were thoroughly washed with DPBS and then rinsed with 1x CellMask. TM Cells were stained with DeepRed Plasma Membrane Stain for 5 minutes, then fixed with 4% paraformaldehyde (PFA) in DPBS solution for 10 minutes at room temperature. Images were acquired using a Zeiss LSM 700 microscope with a focal plane set to 1.4 μm, using Zen software (Carl Zeiss Microscopy). For inhibition of particle uptake and binding, cells were pre-incubated with 1 mM free EXP3174 or captopril before adding particles. Images were analyzed using Fiji software.

[0153] Cellular distribution of NPs: flow cytometry

[0154] As previously described [1], particle uptake was analyzed by flow cytometry ( Figure 5 A). Briefly, rMCs were seeded at a density of 30,000 cells / well in 24-well plates and incubated for 48 hours (37°C). Preheated NP solution (0.7 mg / ml in LM supplemented with 0.1% BSA) was pipetted onto the top of the cells, washed with DPBS, and incubated at 37°C for 45 minutes. To confirm uptake specificity, cells were incubated with 1 mM captopril and / or EXP3174 for 30 minutes before adding particles. Afterward, the particle solution was discarded, and the cells were thoroughly washed with DPBS, trypsinized, and centrifuged (2x, 200 g, 5 min, 4°C). NP-related cellular fluorescence was analyzed in DPBS using a FACSCalibur cytometer (Becton Dickinson). Fluorescence was excited at 633 nm and recorded using a 661 / 16 nm bandpass filter. Viable cell populations were gated using Flowing software 2.5.1 (Turku Centre for Biotechnology), and the geometric mean of NP-related fluorescence was analyzed. Statistical significance was assessed using the Student's t-test with GraphPadPrism 6.0. Figure 5 A).

[0155] NP target cell specificity: flow cytometry

[0156] To assess NP uptake in different cell lines ( Figure 5B) rMC, HK-2, and HeLa cells were seeded in 24-well plates at densities of 30,000, 50,000, or 100,000 cells / well, respectively, and incubated for 48 hours (37°C). Then, preheated NP solution (0.7 mg / ml in LM supplemented with 0.1% BSA) was added to the top of the cells and the treatment was performed as described above. Statistical significance was assessed using a two-way ANOVA with Sidak multiple comparison test in GraphPad Prism 6.0. Figure 5 B).

[0157] As previously described [1], particle specificity in co-culture of target cells and off-target cells was studied by flow cytometry. Figure 5 (CD). In short, CTG-stained rMCs (10 μM in serum-free medium, 30 min, 37°C) were seeded into 24-well plates at densities of 10,000 and 75,000 cells / well, respectively, in co-culture with unstained off-target NCI-H295R or HeLa cells, and incubated for 48 h. Then, warm NP solution at a concentration of 0.02 mg / ml in LM supplemented with 0.1% BSA was added to the top of the cells, and incubated at 37°C for 45 min. Afterward, the particles were discarded, and the cells were treated as described above for flow cytometry analysis. Statistical significance was assessed using Student's t-test with GraphPad Prism 6.0.

[0158] NP target cell specificity: confocal microscopy

[0159] To confirm the flow cytometry experiment, CLSM analysis was performed on particle specificity in co-culture as previously described [1]. Figure 5 E and Figure 13 In summary, target CTG-stained rMCs (10 μM, 30 min, 37 °C) were seeded at densities of 2,000 and 10,000 cells / well, respectively, in co-culture with off-target HeLa or NCI-H295R cells stained with CTDR (25 μM, 30 min, 37 °C) and incubated for 24 h. Then, the cell nuclei were stained with Hoechst 33258 (5 μg / ml in DPBS) for 20 min, and 0.02 mg / mL NP solution preheated in LM supplemented with 0.1% BSA was pipetted onto the top of the cells and incubated for 45 min. The NP solution was then discarded, and the cells were thoroughly washed with DPBS and fixed with 4% PFA in DPBS solution (rt) for 10 min. Images were acquired and analyzed using a Zeiss LSM 700 microscope and Fiji software, as described above.

[0160] Distribution of NP in the kidneys

[0161] To evaluate the renal distribution of different NP formulations (NPEXPAng-I, NPAng-I, NPEXP, and NPMeO), 100 μL of a 120 nM NP solution (equivalent to approximately 10 mg / ml NP) was injected via the jugular vein into 10-week-old female NMRI mice (Charles River) (n=6 per particle sample) anesthetized with isoflurane inhalation and buprenorphine (0.1 mg / kg body weight). Additionally, as a control, 100 μL of the free dye (CF647) for fluorescently labeled particles was injected at the same concentration (approximately 50 μM) as contained in the particle samples. Blood samples were collected via intravenous puncture 5 minutes later while the mice were still anesthetized. After anesthetizing the particle-circulating mice with ketamine / toluidine for 1 hour, final blood samples were collected and the mice were killed by perfusion fixation with 4% PFA. Kidneys were harvested and transversely cut. They were cryoprotected overnight in phosphate-buffered saline (0.1 M pH 7.4) supplemented with 18% sucrose and 1% PFA. They were then frozen in liquid nitrogen-cooled 2-propanol (-40°C) and embedded in tissues. OCT TM The compound was used for cryosectioning. Kidney sections were cut into 5 μm sections and transferred to Superfrost using a CryoStar NX70 cryostat (Thermo Fisher Scientific). TM Plus, on a glass slide. To better visualize the cell nuclei, the nuclei were stained with DAPI (12.5 μg / ml in DPBS) before section imaging using an Axiovert 200M (Zeiss) fluorescence microscope and Zen software (Zeiss). Whole kidney images were acquired using a 10x objective lens. Figure 6 For glomeruli, quantitative fluorescence images were captured using a 40x objective lens (average 120 glomeruli per sample) and analyzed using Fiji software (Schneider et al., 2012). For better visualization, a lookup table “Red Hot” was applied to particle-related fluorescence. Regions within each glomerulus were quantified, and fluorescent regions were gated. The integrated fluorescence density of each gated region was then quantified and correlated with the entire glomerular region. Statistical significance was assessed using a Student's t-test with GraphPad Prism 6.0. To compare particle-related fluorescence in the endothelial and epithelial layers, the cortex was divided into two equal parts, and glomerular fluorescence was analyzed as described above. For assessing statistical significance, a two-way ANOVA with a Sidak multiple comparison test was performed using GraphPad Prism 6.0.

[0162] NP-related fluorescence in plasma was measured using a FLUOstar Omega microplate reader (BMG Labtech) with excitation and emission wavelengths of 640 and 680 nm, respectively. Fluorescence at 1 hour post-injection was correlated with the initial fluorescence of samples obtained 5 minutes post-injection.

[0163] Immunohistochemistry

[0164] To assess glomerular localization of NPs, freshly cut 5 μm frozen kidney sections were washed for 5 min with DPBS, followed by 5 min with DPBS supplemented with 0.1% sodium dodecyl sulfate (SDS), then 5 min with DPBS, and blocked for 10 min with 5% BSA in DPBS supplemented with 0.04% Triton-X (m / v). The sections were washed again with DPBS (5 min) and incubated overnight at 4°C in a 1:200 solution of polyclonal goat anti-integrin-α8 primary antibody supplemented with 0.5% BSA and 0.004% Triton-X (m / v). They were then washed for 5 min with DPBS and incubated in the dark at room temperature for 1 h with Cy2-anti-goat secondary antibody (1:400) and DAPI (12.5 μg / ml) in DPBS supplemented with 0.5% BSA and 0.04% Triton-X. Frozen sections were washed with DPBS and ultrapure water, then fixed with Dako Faramount Mounting Medium, and analyzed using a Zeiss LSM 700 microscope and Fiji software, as described above.

[0165] Quantitative and statistical analysis

[0166] Statistical analysis was performed using GraphPad Prism software 6.0. Student's t-test or two-way ANOVA with Sidak or Turkey multiple comparison tests was used to assess statistical significance as shown in the methodological details. The statistical significance level and the number "n" for each experiment are indicated in the text and graph legends.

[0167] Example 2: Block copolymers allow for the design of virus-mimicking particles

[0168] All materials and methods mentioned in this embodiment are as described in the previous embodiments.

[0169] To develop virus-mimicking NPs, the inventors coupled ligand EXP3174 and Ang-I to a poly(ethylene glycol) 5k-poly(lactic acid) 10k(PEG-PLA) block copolymer (… Figure 8This was blended with poly(lactic acid-co-glycolic acid) PLGA for the fabrication of NPs via bulk nanoprecipitation, ensuring sufficient stability of the particles in vivo. The remaining nonfunctionalized polymer is carboxyl-terminated PEG-PLA (COOH-PEG2k-PLA10k) with short 2k PEG and 10k PLA blocks. Figure 1 A). By modifying the polymer with ligands prior to the preparation of NPs, precise control over the ligand density can be achieved. Particles were prepared such that 20% of the PEG chains were modified with Ang-I, and the remaining 20% ​​were modified with EXP3174 (NPEXPAng-I). Figure 2 B). Ligand density was maintained at a maximum of 40% to avoid steric hindrance and nonspecific interactions between ligands. As a control, ligand-free methoxy-PEG-terminated particles (NPMeO) and particles carrying 20% ​​Ang-I or EXP (NPAng-I and NPEXP, respectively) were assembled. Figure 1 A). By combining polymers carrying long ligands with shorter, nonfunctionalized polymers for particle preparation, the size of NPs can be maintained below 80 nm, thereby endowing the particles with the ability to pass through mesenteric capillary endothelial pores. Figure 2 C). The selection of carboxylic acid-terminated block copolymers as fillers provides an ideal overall negative particle charge, thus avoiding non-specific electrostatic adsorption to the negative cell membrane. Figure 2 D).

[0170] Example 3: NP recognizes target receptors in vitro

[0171] All materials and methods mentioned in this embodiment are as described in the previous embodiments.

[0172] To confirm the ability of particles to perform a triple check on cell identity, an initial in vitro assessment was conducted. Particle affinity for target receptors mediating initial attachment and subsequent internalization was investigated using a calcium mobilization assay, as stimulation or silencing of Gq-coupled AT1R with agonists or antagonists resulted in cytosolic calcium mobilization. 2+ Inflow or inhibition. For this purpose, AT1R-positive rat mesangial cells (rMCs) were incubated for 30 minutes with different concentrations of free ligands or NP formulations, then stimulated with Ang-II and the resulting calcium signals were recorded. Figure 3 As shown in Figure A, control experiments using free EXP3174 and Ang-II demonstrated that both compounds exhibited high affinity for AT1R in the nanomolar range (IC50 values ​​of 0.6 ± 0.4 and 1.5 ± 0.1 nM, respectively). Ang-I showed low affinity (IC50 0.9 ± 0.6 μM) because receptor binding and activation only occurred after conversion to Ang-II by ACE enzymes present in the cell membrane.

[0173] Coupling with the adapter leads to a loss of affinity, which is compensated for by the simultaneous high-affinity multivalent binding of multiple receptors. Figure 3 B and 3C). Particles carrying only Ang-I (NPAng-I) showed a lower affinity for AT1R (IC50 9.4 ± 0.4 nM) than particles carrying EXP3174 (IC50 0.4 ± 0.1 nM) because their primary interaction is with ACE. However, Ang-I particle binding resulted in a significantly lower IC50 value compared to the free ligand, due to the promotion of enzymatic cleavage at the NP interface and subsequent multivalent binding. In contrast, EXP3174-modified NPs exhibited the same order of magnitude affinity as the free ligand. Surprisingly, particles carrying both ligands, NPEXPAng-I, showed a synergistic effect in receptor binding, as their affinity for AT1R (IC50 0.2 ± 0.09 nM) was significantly higher than either of the particles carrying only one ligand. Figure 3 C). This demonstrates that the ligands do not hinder each other's interaction because, after Ang-I is enzymatically activated to Ang-II, both ligands simultaneously target the same receptor in an agonistic and antagonistic manner. The absence of any functionalized particles (NPMeO) confirms that the assay is ligand-specific, as they did not elicit any response. Figure 3 B).

[0174] To assess the kinetics of cell / particle interactions, NPs corresponding to 10 μM ligands were incubated with rMCs for intracellular calcium measurements over 5.5 hours. The degree to which they silenced calcium signals triggered by the presence of free agonists was used as a measure of the integrity of individual particles binding to AT1R on the cell surface via their ligands at different time points. Figure 3D). Particles carrying only Ang-I on their surface exhibited slow receptor binding because they initially required activation by cell membrane-bound ACE to become Ang-II-carrying particles in order to interact with AT1R. Receptor binding reached a maximum of about 40% after 1 hour of incubation and remained constant during the assay. This suggests that once a certain number of proligands are activated, the particles are rapidly internalized and that not all Ang-I may be converted to Ang-II. Once Ang-II on the particle surface binds to the receptor, the particles are rapidly internalized (because they have picomolar AT1R affinity[1]), which means that NP internalization may not require the activation of all proligands. This phenomenon can be avoided when EXP3174 is added to the particle surface as an attachment factor. Very rapid and complete receptor blockade occurs after only 5 minutes of particle incubation (similar for NPEXPAng-I and NPEXP). AT1R inhibition persisted for almost the entire measurement and decreased to about 80% at the last time point, likely due to receptor upregulation and recycling. EXP3174's attachment to the cell membrane slows down the recognition process, leading to higher Ang-I to Ang-II activation and thus more efficient binding to AT1R. Comparing NPEXPAng-I and NPEXP, NPEXPAng-I showed significantly higher initial AT1R inhibition, which leveled off after 45 minutes of particle incubation. This may be due to the combined effect of the two ligands resulting in a higher affinity for AT1R. Figure 3 C).

[0175] A prerequisite for particle internalization is that ACE can activate Ang-I to Ang-II. Therefore, the inventors investigated the enzymatic kinetics of NPEXP Ang-I to determine whether the presence of antagonists on the particle surface would hinder the enzymatic reaction. Soluble ACE was incubated with different particle concentrations for different time periods, and the production of Ang-II on the NP crown was quantified by calcium mobilization assays. The interference of the EXP3174 ligand in the assay was assessed by measuring the signal inhibition exhibited by NPEXP. Figure 9 ). For NPAng-I and NPEXPAng-I ( Figure 3 E) The Michaelis Menten constant (Km) determined by both resulted in values ​​for both particulate formulations that were on the same order of magnitude as those for the free ligand (see [1]). Furthermore, the inventors determined the catalytic constant (Kcat) to calculate the specificity constant (Kcat / Km), a useful indicator for comparing the affinity of different substrates for the same enzyme. Figure 3F). Enzymatic activation of Ang-I on the NPEXPAng-I crown was not significantly different from that on NPAng-I, indicating that ACE is not sterically hindered by the additional ligand EXP3174. Furthermore, the Kcat / Km values ​​calculated based on ligand concentration were equal for both free and particle-bound Ang-I. More importantly, when Kcat / Km was calculated based on NP concentration, the bound ligand was a significantly better substrate for the enzyme, a result of the binding of multiple ligand molecules to multiple enzyme molecules on the particle surface. Figure 3 F).

[0176] Example 4: Decision NP is target cell specific.

[0177] All materials and methods mentioned in this embodiment are as described in the previous embodiments.

[0178] After successfully establishing the interaction between the particle and its individual target, the next step is to determine whether NPs carrying both antagonists and agonists on their crowns still trigger internalization in their target cells, and if so, whether the uptake is due to specific ligand-receptor interactions. Since the antagonist does not induce AT1R-mediated endocytosis while the agonist does, the inventors investigated the cellular localization of NPEXPAng-I in YFP-labeled AT1R-expressing rMCs (pAT1R-rMCs) using confocal laser scanning microscopy (CLSM). Figure 4 As shown, NPESPAng-I-related fluorescence was detected within the cells. It increased with increasing incubation time and strongly co-localized with AT1R fluorescence.

[0179] Therefore, specific particle uptake mediated by AT1R exists. However, particles carrying only the antagonist (NPEXP) are not internalized and are mainly located on the cell surface. Figure 10 A). Particle fluorescence also co-localizes with receptor fluorescence, indicating receptor-mediated attachment. Since NPAng-I is also internalized by cells ( Figure 10 B), therefore, the enzymatically produced Ang-II mediates the cellular uptake of NPEXPAng-I. Unexpectedly, with increasing incubation time, the receptors on the cell membrane underwent rearrangement ( Figure 4 The uptake progressed from a more dispersed and uniform distribution across the cell membrane (after 15 minutes) to more concentrated clusters (after 90 minutes), which strongly co-localized with NP fluorescence. This is further evidence that uptake is mediated by AT1R, as activation of receptors internalized in pits via clathrin (e.g., GPCRs, such as AT1R) promotes receptor clustering.

[0180] For NPEXPs, receptor rearrangement on the cell membrane also occurs, resulting from multivalent receptor binding facilitated by receptor movement on the cell surface. Once NPEXPs attach to receptors on the cell membrane, their lack of internalization leads to receptor-particle movement and further receptor binding on the cell membrane. Particles without ligands (NPMeO) are not taken up by the cell. Figure 10 C) confirmed that specific targeting mechanisms are crucial for mediating high cell internalization.

[0181] In summary, the inventors have demonstrated that the presence of antagonistic ligands that mediate attachment to the particle crown does not hinder subsequent particle internalization. More importantly, including additional ligands on the particle surface compensates for the targeting loss due to the steric hindrance of the Ang-I ligands by adding more long polymer chains. Figure 11 To further confirm particle specificity and ligand-mediated internalization, cells were pre-incubated with free EXP3174 or captopril (an ACE inhibitor) for 30 minutes before particle addition, thereby enabling flow cytometry analysis. Figure 5 A) and CLSM Figure 12 The suppression of particle-related fluorescence was analyzed.

[0182] Furthermore, the inventors examined granule internalization in different cell lines using flow cytometry. Figure 5 B). HeLa cells, which do not express ACE and express only low levels of AT1R, exhibited low particle uptake, which was nonspecific because it could not be inhibited by captopril or EXP3174. Conversely, rMC and HK-2 cells expressing both targets were able to take up these particles, as indicated by much higher particle-associated cellular fluorescence. Internalization was also mediated by activated proligands that bind to AT1R, as pre-incubation of cells with captopril or EXP3174 significantly suppressed cellular fluorescence. Thus, particles exhibit high specificity for their target cells. However, when NPs enter the body, they coexist with both target and off-target cells. Therefore, the inventors investigated whether NPESPAng-I could distinguish between them.

[0183] Target cells (rMCs) were seeded with excess off-target NCI-H295R or HeLa cells, both lacking ACE and expressing high and low AT1R levels, respectively. They were incubated with different NP formulations, and particle-associated fluorescence (PIF) was studied for each cell line by flow cytometry. Figure 5NPEXPAng-I exhibited excellent target cell specificity because they accumulated significantly more in target rMCs. Specificity was conferred by Ang-I, as NPAng-I also showed low accumulation in both off-target cell types. Conversely, NPEXP bound to the cell surface in rMCs to the same degree as in NCI-H295R cells expressing high AT1R levels, suggesting that a simple one-step recognition process is insufficient to confer particle selectivity. CLSM images confirmed the results of flow cytometry ( Figure 5 E and 13), where NPESPAng-I and NPAng-I fluorescence (red) were primarily associated with target rMCs (green) but not with off-target HeLa or NCI-H295R cells (white), while NPEXP fluorescence was found in both rMCs and NCI-H295R cells expressing AT1R. In summary, these results indicate that NPESPAng-I uptake is receptor-mediated and that initial cell attachment via the EXP3174 ligand does not reduce the particle specificity of target cells conferred by the virus-mimicking recognition process.

[0184] Example 5: NP-targeted in vivo MC

[0185] All materials and methods mentioned in this embodiment are as described in the previous embodiments.

[0186] Having demonstrated the complementary targeting ability and particle specificity of the two ligands on NPEXPAng-I in vitro, the next step is to determine whether the viral recognition mechanism leads to higher in vivo MC accumulation. To this end, targeting (NPEXPAng-I, NPAng-I, and NPEXP) will be performed. Figure 1 A) Non-targeted (NPMeO) particle formulations were injected into NRMI mice, and particle-related fluorescence was examined in frozen kidney sections. Figure 6 and 14 A). For example Figure 6 As shown in Figure A, NPEXPAng-I fluorescence was uniformly detected in all glomeruli of the kidney slices, while no fluorescence was observed in other kidney structures (e.g., tubules). Conversely, for the untargeted NPMeO, NP fluorescence was almost undetectable in the kidney slices. Figure 6 B). This indicates that simple size-dependent targeting is insufficient to achieve granule accumulation in MCs, as NPMeO may be cleared from the mesangium due to a lack of specific cell interactions. More importantly, NPEXP, as a target for NPs but unable to mediate cell internalization, also exhibited very low glomerular fluorescence (B). Figure 14 A), indicating that particle uptake is fundamental to achieving high MC accumulation. Furthermore, compared to NPAng-I, which lacks attachment factors, NPEXPAng-I achieved stronger and more uniform glomerular distribution (A). Figure 14A) indicates that the enhanced target cell recognition principle in vivo is highly advantageous.

[0187] To quantitatively assess NP-related fluorescence and better distinguish differences between different particulate formulations, images of the glomeruli were captured at higher magnification. Figure 7 A). Glomerular fluorescence quantitative analysis of virus-mimicking particles (NPEXPAng-I) with enhanced recognition mechanisms produced 15-fold higher fluorescence compared to non-targeted control particles (NPMeO) that showed only small fluorescent spots in some glomeruli.

[0188] Furthermore, the accumulation of NPEXPAng-I was significantly higher than that of single-ligand targeted particles (7 times and 5 times higher than NPEXP and NPAng-I, respectively). Figure 7 B). The detected fluorescence was particle-related, confirmed by the kidney distribution of the free dye (CF647) used for particle labeling, which showed strong tubular fluorescence but no glomerular fluorescence. Figure 14 B), because of its small size, it can filter freely. To assess the distribution of NP glomeruli, the fluorescence of epicortical and endocortical glomeruli was compared ( Figure 7 C). For all granule formulations, there were no significant differences between the two populations. This indicates that the granules are uniformly distributed throughout the glomeruli of the renal cortex, an essential prerequisite for treating glomerular-related diseases. Finally, since there are other cells in the glomeruli besides MCs that may internalize NPs, integrin-α8 was used as a marker for specific antibody staining of MCs to determine granule accumulation within them. Figure 7 As shown in Figure D, NPEXPAng-I fluorescence localization within antibody-stained MCs confirms that the particles can not only reach the glomerular mesangium but also be taken up by the MCs.

[0189] In summary, these results clearly demonstrate that size-mediated targeting is a necessary prerequisite for mesangial reach, but insufficient for particle accumulation in MCs. NP internalization appears to be essential to avoid mesangial clearance, explaining the lowest glomerular fluorescence in particles lacking this property (NPMeO and NPEXP). Implementation of the virus-mimicking recognition principle (NPAng-I) increases NP specificity and leads to particle uptake, resulting in higher MC accumulation. However, as in vitro studies have shown, promoting target cell recognition via initial virus-like cell attachment (NPEXPAng-I) significantly enhances the targeting potential of NPs, a result of the combined action of the two ligands.

[0190] Furthermore, the enhanced functionalization of NPESPAng-I did not lead to a reduction in particle blood retention. Typically, nanoparticles are coated with polymers such as PEG, which increases their circulation time and reduces plasma protein adsorption. A positive effect is usually offset by ligand functionalization, as off-target cells expressing the target receptor can bind to and interfere with NPs. However, quantification of plasma NP fluorescence one hour after injection showed that NPESPAng-I maintained circulation to the same extent as non-targeted NPMeO and significantly longer than other targeted formulations. Figure 14 (C) This may be due to higher particle specificity resulting from a more complex cell recognition process. Overall, the results indicate that NPs that target MCs and accumulate in large quantities within them can be developed by closely mimicking viral binding and internalization and combining it with optimal NP size.

[0191] Example 6: Materials and Methods

[0192] Material

[0193] Molecules of 2000 and 5000 g mol -1 Iso-bifunctional hydroxyl poly(ethylene glycol)carboxylic acid (COOH-PEG) 2k / 5k -OH) and a molecular weight of 2000 g mol -1 Hydroxy-poly(ethylene glycol) Boc-amine (Boc-NH-PEG) 2k -OH) was purchased from Jenkem Technology USA Inc. (Allen, Texas, USA), and the molecular weight is 5000 g mol. -1 Methoxylated poly(ethylene glycol) (MeO-PEG5k-OH) and Resomer RG 502 (PLGA) were purchased from Sigma-Aldrich (Taufkirchen, Germany). EXP3174 (also known as losartan carboxylic acid) was purchased from Santa Cruz (Heidelberg, Germany), while Cyclic RGDfK (cRGDfK) was purchased from Synpeptide Co. Ltd. (Shanghai, China). Alexa Fluor TM 568 hydrazide (Alexa568), CellTracker TM Green Dye (CTG) and CellTracker TM Deep Red Dye (CTDR) was purchased from Thermo Fisher Scientific (Schwert, Germany). Amine-functionalized spherical gold nanoparticles (Au) with an average diameter of 2.2 nm were used. 2.2 -NH2) is derived from Nanopartz Inc. (Lofland, Colorado, USA). GoldEnhance TMEM Plus kits were purchased from Nanoprobes (Yaphanck, NY, USA). Goat-derived integrin α-8 antibody was obtained from R&D Systems (Minneapolis, MINNESOTA, USA). Unless otherwise specified, all other chemicals were analytical grade from Sigma-Aldrich. Ultrapure water was obtained from Milli-Q water purification systems (Millipore, Billerica, MA, USA). NCI-H295R (CRL-2128) and HeLa (CCL-2) cells were purchased from ATCC (Manassas, VA, USA). All cell lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum, insulin-transferrin-selenium (ITS) (1x), and 100 nM hydrocortisone.

[0194] Polymer Synthesis

[0195] COOH-PEG 2k -PLA 10k Boc-NH-PEG 5k -PLA 10k and MeO-PEG 5k -PLA 10k The block copolymer was synthesized via ring-opening polymerization as described above. Briefly, a heterobifunctional PEG polymer (1 equivalent) was mixed with 3,6-dimethyl-1,4-dioxane-2,5-dione (70 equivalents) and 1,8-diazabicyclo[5.4.0]undec-7-ene (3 equivalents). The polymer mixture was stirred at room temperature (RT) for 1 hour (h) until polymerization was quenched with benzoic acid (14 equivalents). The resulting block copolymer was precipitated in diethyl ether, separated by filtration, and dried under vacuum. The molecular weight of the synthesized polymer was determined in deuterated chloroform at 295 K using a Bruker Avance 300 spectrometer (BrukerBioSpin GmbH, Rheinstein, Germany).

[0196] To prepare cRGDfK-PEG 2k -PLA polymer, as shown above, with the previously synthesized COOH-PEG 2k -PLA 10k Covalently coupled to the lysine residues of cRGDfK. In short, COOH-PEG... 2k -PLA 10kThe polymer was activated (1 equivalent) with 3-(ethyliminomethyleneamino)-N,N-dimethylpropyl-1-amine (EDC) / N-hydroxysuccinimide (NHS) (25 equivalents) at room temperature for 2 hours, followed by quenching with β-mercaptoethanol (BME) (30 equivalents). The activated polymer was reacted with cRGDfK (3 equivalents) and N,N-diisopropylethylamine (DIPEA) (10 equivalents) at room temperature for 24 hours. After precipitation of the resulting cRGDfK-coupled polymer in diethyl ether / methanol (15:1 v / v), free cRGDfK and excess reactants were removed by dialyzing with millipore water (mpH2O).

[0197] For EXP3174-PEG 5k -PLA 10k Initially, Boc-NH-PEG was cleaved. 5k -PLA 10k The Boc protecting group was removed. In short, the Boc-protected polymer was dissolved in dichloromethane (DCM) / trifluoroacetic acid (TFA) (1:1 V / V). After stirring for 30 minutes, excess TFA was neutralized with a saturated sodium bicarbonate solution. The organic phase was washed with mpH2O, and then polymer separation was performed as described above.

[0198] The resulting NH2-PEG 5k -PLA 10k EXP3174 was coupled to the carbonyl residues of the imidazole component. EXP3174 (3.5 equivalents) was activated at room temperature with N,N'-dicyclohexylcarbodiimide (DCC) / NHS (3.3 equivalents) for 2 hours. After removing the resulting dicyclohexylurea by centrifugation, NH2-PEG was added. 5k -PLA 10k (1 equivalent) and DIPEA (17.5 equivalent) were reacted at room temperature for 24 hours. The resulting EXP3174-PEG 5k -PLA 10k Precipitate in methanol / ether (1:5 V / V), then dialyze the product against ethanol / 100 mM borate buffer pH 8.5 / water (1 / 1 / 8 V / V) for 24 hours, followed by dialyze against mpH2O for 12 hours to remove unreacted EXP3174 and excess reactants.

[0199] PLGA labeled with fluorescent dyes

[0200] For particle visualization, the core-forming PLGA was covalently linked to a fluorescent dye prior to NP preparation. For this purpose, carboxylic acid-terminated PLGA was activated for 2 hours using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (DMTMM) as a catalyst. The activated PLGA was then reacted with Alexa Fluor. TM 568 hydrazide or CF TM 647amine was reacted at room temperature for 24 hours. The labeled PLGA was dialyzed against mpH2O for 24 hours to remove unreacted fluorescent dye.

[0201] PLGA labeled with nano gold

[0202] For electron microscopy analysis, PLGA was conjugated with gold nanoparticles. Initially, PLGA was activated in DCM with EDC and NHS for 2 hours. After removing the DCM under reduced pressure, the activated PLGA was dissolved in DMSO and combined with DIPEA and lyophilized monoamino gold nanoparticles (Au) with an average diameter of 2.2 nm. 2.2 The mixture was stirred at room temperature for 24 hours, and the gold-conjugated PLGA was precipitated in mpH2O, separated by centrifugation at 2500g for 10 minutes and lyophilized.

[0203] NP preparation

[0204] Block copolymer nanoparticles were fabricated using a common solvent evaporation technique. Appropriate amounts of PEG-PLA polymer and PLGA were mixed at a ratio of 70 / 30 (m / m) and diluted in acetonitrile (ACN) to a final concentration of 10 mg / mL. -1 To achieve the required ligand surface density for heterofunctional / homfunctional NP substances, according to Figure 18 The calibration depicted in d / e will be cRGDfK-PEG 2k -PLA and / or EXP3174-PEG 5k -PLA 10k With COOH-PEG 2k -PLA 10k Mix. Then the organic phase was added dropwise to 10% Dulbecco phosphate-buffered saline (DPBS) (7.5 mM, pH 7.4) under vigorous stirring and stirred at room temperature for 3 hours to remove the organic solvent.

[0205] The NP dispersion was concentrated using a Pall Microsep filter (30 kDa molecular weight cutoff; Pall Corporation, New York, USA) by centrifugation at 1250 g for 25 minutes. To obtain the mass concentration of the manufactured NP, the PEG content was assessed using a colorimetric iodine complexation assay. The NP was then lyophilized and subjected to gravimetric analysis to obtain the ratio of PEG content to NP weight. This ratio was used in the following experiments to calculate the mass concentration based on the assessed PEG content of each NP substance.

[0206] NP characterization

[0207] NP size and zeta potential were evaluated using a Malvern Zetasizer Nano ZS (Malvern, Hellenberger, Germany). Samples were measured at an angle of 173° (25°C, RT) in 7.5 mM DPBS using a 633 nm He-Ne laser in a PMAA semi-micro cuvette (DLS; Brand, Wertheim, Germany) or a folded capillary cell (zeta potential; Malvern, Hellenberger, Germany).

[0208] cRGDfK Quantitative Analysis

[0209] The cRGDfK level on the NP surface was assessed based on arginine measurements. Briefly, 50 μL of NP sample (1 mg mL⁻¹) was mixed with 175 μL of a working solution consisting of 9,10-phenanthroquinone (150 μM ethanol solution) and 2N NaOH (6:1 V / V). After incubation at 60 °C for 3 hours, one equivalent of the sample was mixed with one equivalent of 1N HCl and incubated for another hour at room temperature. Finally, the levels were measured using Synergy thermometers at excitation wavelengths of 312 / 7 nm and emission wavelengths of 395 / 7 nm. TM Fluorescence was measured on a Neo2 multimode microplate reader (BioTek Instrument Inc., Winnowski, Vermont, USA). cRGDfK (0-40 μg mL) -1 The diluted solution was used as a calibrator. The molar concentration of cRGDfK and the ratio of molar cRGDfK content to molar PEG content were determined and plotted against theoretical values. Figure 18 d).

[0210] EXP3174 Quantization

[0211] To determine the surface level of EXP3174 on the manufactured particles, 1 equivalent of NP sample (1 mg / mL) was used. -1 Mix with 10 equivalents of 0.2M acetic acid. A diluted solution of EXP3174 in 0.2M acetic acid (0-30 μM) is used as a calibrator. (In Synergy)TM Fluorescence of samples and standards was measured using the Neo2 multimode microplate reader (see above) (excitation 250 / 10nm, emission 370 / 5nm). The molar concentration of EXP3174 and the ratio of EXP3174 molar content to PEG molar content were determined and plotted against theoretical values. Figure 18 e).

[0212] Calcium mobilization assay

[0213] To study the AT1r binding of NP, fura-2 was used as Ca. 2+ Chelating agents were used to measure intracellular calcium levels. Briefly, rMCs were incubated with 5 μM fura-2AM, 2.5 mM probenecid, and 0.05% Pluronics F-127 in Leibovitz L-15 medium at room temperature for 1 hour. Cells were then centrifuged (5 min, 200 g, RT) and resuspended in Leibovitz medium. 45 μL of different concentrations of NP or free EXP3174 were transferred into 96-well plates (Greiner Bio One, Frikenhausen, Germany), followed by 45 μL of rMC suspension (2 x 10⁻⁶). 6 mL -1 Next, the cells were incubated with the sample at room temperature for 45 minutes. After incubation, 10 μL of 30 nM AT II was added to each well to activate uninhibited AT1r, thereby inducing Ca2+. 2+ Injection was carried into the cytosol. Fluorescence signals were measured within the first 30 seconds after injection using a FluoStar Omega fluorescence microplate reader (BMG Labtech, Ortenberg, Germany), with excitation filters of 340 / 20 nm and 380 / 20 nm, and an emission filter of 510 / 20 nm. Ca was assessed by incubating the loaded cells with 0.1% Triton-X 100 and measuring fluorescence levels as described above. 2+ -Combined with Ca 2+ - Maximum unbound Fura-2 ratio. Similarly, minimum ratio was achieved by incubation with 0.1% Triton-X 100 and 45 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Intracellular calcium levels for each sample were calculated using the equation of Grynkiewicz et al. The half-maximum inhibitory concentration (IC50) was calculated using a GraphPad Prism (San Diego, CA, USA) and by applying an S-type dose-response equation (variable slope).

[0214] CLSM analysis

[0215] To analyze NP cell interactions in detail, rMCs were seeded at a density of 15,000 cells / well into 8-well slides (Ibidi, Grafelfin, Germany) and incubated at 37°C for 24 hours. For easier visualization of the cell cytosol, rMCs were stained with CTDR (25 μM, 45 min, 37°C) in serum-free RPMI 1640 medium before seeding. Alexa Fluor was used for further analysis. TM 568-labeled PLGA was used to manufacture NPs, which were then adjusted to 0.05 mg / mL in Leibovitz buffer supplemented with 0.1% BSA. -1 Mesangial cells were incubated with 250 μL of NP at 37 °C for 15, 45, and 90 min, washed with pre-warmed DPBS, and fixed with 4% paraformaldehyde (PFA) in DPBS for 10 min. Following the final washing step, the fixed samples were analyzed using a Zeiss LSM 710 (Carl Zeiss Microscopy GmbH, Jena, Germany).

[0216] Flow cytometry

[0217] To assess mesangial cell binding in NP samples, rMCs were seeded at a density of 40,000 cells / well in 24-well plates (Greiner Bio One, Frikenhausen, Germany) and incubated at 37°C for 48 hours. CF TM NPs were produced using PLGA labeled with 647 and adjusted to 0.05 mg NP / mL in Leibovitz buffer supplemented with 0.1% bovine serum albumin (BSA). -1 To confirm the entry of NP cells into α VFor β3-dependent uptake, 300 μL of free cRGDfK (c = 500 μM) was added to the relevant cell samples for 15 min prior to NP incubation. Cells were washed with DPBS and 300 μL of pre-warmed NP solution was added at 37 °C for 60 min. For the corresponding analysis of time-dependent uptake, cells were incubated for 120 min, and NP was removed after 0, 15, 30, 45, 60, 90, and 120 min. Cells were washed with DPBS, digested with trypsin, and centrifuged at 200 g and 4 °C for 5 min, followed by two more wash and centrifugation steps (DPBS, 200 g, 5 min, 4 °C). The final samples were resuspended in DPBS and analyzed using a FACS Calibur cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). NP-related fluorescence was excited at 633 nm, and the corresponding emission was recorded (661 / 16 bandpass filter). Flow cytometry data were analyzed using Flowing software 2.5.1 (Turku Center for Biotechnology, Turku, Finland). The geometric mean of cell-associated fluorescence was evaluated in live cell populations.

[0218] Transmission electron microscope

[0219] To assess the cellular localization of NPs, rMCs were seeded at a density of 12,000 cells / well in 24-well plates and incubated for 72 hours. The NP formulation containing gold nanoparticle-conjugated PLGA was diluted in Leibovitz buffer containing 0.1% BSA and administered at 0.05 mg / mL. -1 The concentration (V = 300 μL) was added for 45 minutes. After incubation, the samples were washed with DPBS and prepared for electron microscopy analysis. In short, cells were fixed with 2.5% PFA and 2.5% glutaraldehyde in 0.1 M sodium dimethylarsinate solution (Caco buffer) for 60 minutes at room temperature, washed with Caco buffer, and permeabilized with 0.1% Triton-X in DPBS for 10 minutes. After the washing step using mpH2O, GoldEnhance was used according to the manufacturer's instructions. TMSamples were gold-enhanced using the EM Plus kit (Nanoprobes Inc., Aphanke, New York, USA), followed by further washing and post-fixation in 2.5% sodium thiosulfate in mpH2O solution. Cells were stained with 0.5% osmium tetroxide and dehydrated in elevated concentrations of ethanol (50–99.5%). For counterstaining, uranyl acetate was used in 70% ethanol for 5 minutes. After embedding in Epon, ultrathin sections of 150 nm were imaged at magnifications of 6300x and 12500x using a 100kV Zeiss Libra 120 electron microscope (Carl Zeiss NTS GmbH, Haut-Cohen, Germany).

[0220] Co-culture experiment

[0221] To evaluate the cell selectivity of the manufactured NPs, the inventors used a co-culture design previously implemented by the inventors. For flow cytometry analysis, rMCs were seeded together with HeLa or NCI-H295R cells in 24-well plates at densities of 10,000 and 75,000 cells / well, respectively, and incubated at 37°C for 48 hours. To distinguish cell types, rMCs were stained with CTG (15 μM, 45 min, 37°C) in serum-free RPMI 1640 medium prior to seeding. The co-cultured cells were then treated with a solution of 0.05 mg / mL... -1 (V=300μL)CF TM NPs labeled with 647 were incubated together for 45 minutes. Sample preparation and flow cytometry analysis were performed as described above. Furthermore, rMC-related fluorescence was excited at 488 nm and recorded using a 530 / 30 bandpass filter. During data analysis, viable populations of stained rMC cells were further gated, and NP-related fluorescence was analyzed for cell specificity.

[0222] For CLSM analysis, rMC cells were CTG stained prior to seeding, as described above. To visualize all cell types, HeLa or NCI-H295R cells were also stained with CTDR (25 μM, 45 min, 37°C). (CellTracker) TM After incubation, rMCs and HeLa / NCI-H295R cells were seeded together in 8-well Ibidi slides at densities of 2,000 and 10,000 / 20,000 cells / well. After incubation at 37°C for 48 hours, cell nuclei were extracted using Hoechst 33258 (5 μg / mL in DPBS). -1 Stain for 20 minutes. Wash cells twice with pre-warmed DPBS and incubate at 37°C with 0.05 mg / mL. -1 Add Alexa Fluor at a concentration (V = 250 μL)TM NP labeled with 568 was incubated for 45 minutes. After NP incubation, the sample was processed as described above and analyzed using a Zeiss LSM 710 microscope.

[0223] In vivo cell targeting

[0224] Animal experiments were conducted in accordance with national and institutional guidelines and approved by the local authorities (Regierung von Unterfranken, reference number: 55.2-2532-2-329). Ten-week-old female NMRI mice (Charles River, Sulzfeld, Germany) were used as model animals. Buprenorphine (0.1 mg kg body weight) was administered. -1 After analgesia, mice were anesthetized with 2.5% isoflurane and injected with 100 μL of CF via the jugular vein. TM 647-labeled NP (c = 120 nM). Mice were kept anesthetized and initial blood samples were collected via intravenous puncture after 5 minutes. After 60 minutes, mice were anesthetized with ketamine / toluidine and final blood samples were collected. Animals were then euthanized by perfusion fixation. Both kidneys were removed and immediately transferred to a solution of 18% sucrose and 14% PFA in phosphate buffer (0.1 M pH 7.4). After 6 hours, the kidneys were washed with DPBS and cryoprotected at -80°C until further processing. For frozen sections, the organs were embedded in tissue. OCT TM Compounds (Sakura Finetek, Torrance, California, USA) were cut into 5 μm sections using a CryStar NX70 cryostat (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and fixed on Superfrost. TMPlus slides (Thermo Fisher Scientific, Schwert, Germany). To analyze NP kidney deposits and quantify glomerular fluorescence, sections were rinsed in DPBS and blocked for 10 min at room temperature with DPBS solution of 5% BSA supplemented with 0.04% Triton-X. After further rinsing in DPBS, samples were stained with cell nuclei using a 1:400 dilution of 4',6-diamidindole-2'-benzeneindole dihydrochloride (DAPI) in DPBS solution of 0.5% BSA and 0.04% Triton-X. After a final wash in DPBS and mpH2O, frozen sections were mounted with Mowiol mounting medium and analyzed on a Zeiss Axiovert 200M. For image analysis, Fiji software (Madison, Wisconsin, USA) was used. Glomerular fluorescence intensity was assessed by measuring the integrated density of regions exceeding a certain fluorescence threshold and dividing by the glomerular region. To assess the exact cellular location of NPs, kidney frozen sections were prepared as described above. After washing and blocking the sections, the samples were stained overnight at 4°C with goat-derived integrin α-8 antibody (diluted 1:200 in DPBS solution of 0.5% BSA / 0.04% Triton-X). The samples were then washed with DPBS and... Donkey anti-goat and DAPI were stained with a 1:400 dilution of 0.5% BSA / 0.04% Triton-X in DPBS solution for 1 hour at room temperature. After a final washing step, the samples were mounted and analyzed on a Zeiss LSM 710.

[0225] Example 7: Preparation of heteropolyvalent EXPcRGD NPs using a modular concept

[0226] All materials and methods mentioned in this embodiment are as described in Embodiment 6.

[0227] To generate NPs with desired adenovirus-mimicking properties, the inventors implemented a modular design based on the synergistic combination of different biocompatible polymer components into heteropolyvalent particulate matter. Figure 18 a) The overall polymer composition of the NP is intended to be similar to the inventors' previous influenza A-mimicking NP design in order to allow for a full comparison of the two targeting concepts. Therefore, the widely established poly(lactic acid-coglycolic acid) (PLGA) forms the hydrophobic NP core, ensuring not only enhanced structural integrity in aqueous media but also allowing for NP visualization via coupling with fluorescent dyes or gold nanoparticles. A poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) block copolymer, as a second component, provides the structural flexibility required to achieve the desired virus-mimicking NP design. In the first step, a long (PEG-PLA) block copolymer is synthesized via the previously described ring-opening polymerization. 5k-PLA 10k ) or shorter (PEG) 2k -PLA 10k PEG-PLA polymers with PEG chains ( Figure 25 a) Since EXP3174 was originally intended to bind to the AT1r mesoporous membrane as a freely moving ligand, it is associated with a longer and therefore more flexible PEG. 5k -PLA 10k Chain covalent coupling ( Figure 25 b). In contrast, the second ligand (cRGDfK) should not interact with surface-bound integrins unless first AT1r binding occurs and subsequent NP space proximity takes place. In this respect, it is linked to the shorter PEG. 2k -PLA 10k ( Figure 25 c). The surface densities of both cRGDfK and EXP3174 can be precisely tuned by mixing different amounts of ligand-functionalized or non-functionalized PEG-PLA polymers with PLGA prior to the fabrication of NPs via nanoprecipitation. Figure 18 d / e).

[0228] The inventors decided to prepare heterofunctional nanoparticles (EXPcRGD NPs) carrying 25% EXP3174 and 15% cRGDfK on their surface, thereby fully utilizing the receptor-binding capacity of the ligands while maintaining the structural integrity of the manufactured particles. The particles should be able to target cells by binding AT1r to the spatially flexible EXP3174, then reduce the spatial distance to the cell surface, subsequently activating αVβ3 integrin via the previously hidden cRGDfK, ultimately initiating NP endocytosis. Figure 17 c). Heterofunctional EXPcRGD NPs, as well as homofunctional (EXP NP / cRGD NPs) and nonfunctionalized methoxy-terminated particles (control NPs), were fabricated below the 60 nm size threshold and exhibited negative zeta potentials. Figure 18 b / C). These characteristics should not only facilitate successful extravasation through endothelial fenestrations ( Figure 17 (a / b) Furthermore, NP phagocytosis or prolonged serum protein adsorption should be prevented.

[0229] Example 8: Heterovalent EXPcRGD NPs exhibit excellent ligand affinity for target motifs.

[0230] All materials and methods mentioned in this embodiment are as described in Embodiments 6 and 7.

[0231] The inventors tested the binding of EXP3174-mediated NP to AT1r expressed in rat mesangial cells (rMCs). Because Gq-coupled AT1r and its major ligand angiotensin II (AT II) activation leads to calcium influx into the cytosol, intracellular calcium levels after ATII stimulation increased. 2+ The level can be used as a marker of AT1r activity after NP incubation. Therefore, low receptor activity indicates a high rate of EXP3174 binding, as the ligand itself acts as an effective antagonist. Figure 19 The intracellular Ca2+ in rMCs stimulated with AT II after pre-incubation with NP or free EXP3174 for 45 minutes was shown. 2+ Horizontal. EXPcRGD NP(IC) 50 =276±31pM) and EXPNP(IC 50 =552±73 pM) all showed excellent AT1r affinity, resulting in highly efficient inhibition of the receptor in the picomolar range and therefore the lowest intracellular Ca2+. 2+ level( Figure 19 a). Furthermore, the inhibitory potency of the NP type carrying EXP3174 is even higher than that of the free ligand (IC). 50 =2.66±0.9 nM). This strongly suggests that EXP3174 functionalized particles can interact with target receptors in a multivalent manner, thereby acquiring overall affinity. The discovery of IC50 values ​​for both EXPcRGD and EXP NP further supports this. 50 Within the same range of levels, the inventors concluded that the combination of EXP3174 and cRGDfK in one particle type does not significantly interfere with the binding capacity of EXP3174 itself. Furthermore, control NP and cRGD NP did not show any interaction with AT1r, thus failing to produce the maximum Ca2+ upon receptor stimulation. 2+ The signal was detected, and the specificity of the assay for AT1r was confirmed. Figure 19 b).

[0232] Having verified the AT1r binding ability of adenovirus-mimicking EXPcRGD NPs, the next step is to investigate the uptake of particles into rMCs via cRGDfK-αVβ3 interaction. Therefore, the inventors incubated mesangial cells with fluorescently labeled NPs and analyzed cell distribution using confocal laser scanning microscopy (CLSM). Cell Tracker was used to visualize the cell bodies. TM DeepRed (CTDR) preprocessing rMC. Figure 20 a shows Alexa Fluor TM568-labeled EXPcRGD NPs exhibited strong intracellular accumulation in globular structures representing endocytic vesicles. The number and intensity of accumulation increased over time. Furthermore, the vesicles appeared to enlarge with prolonged incubation. These findings support the ability of cRGDfK-functionalized NPs to bind to target cells and be taken up into intracellular vesicles via integrin-mediated endocytosis. Over time, these endocytic vesicles fuse with larger endosomes, thus increasing in size and intensity.

[0233] Based on the CLSM results, the inventors performed flow cytometry analysis on NP-treated rMCs and determined cell-related fluorescence during a 120-minute incubation period. Figure 20 As shown in b, EXPcRGD NP exhibited the highest NP-derived fluorescence level compared to all other NP substances throughout the incubation period. While EXP NP and control NP showed only moderate fluorescence signals, significant cell binding was detected in cRGD NP. Notably, the respective fluorescence levels plateaued after approximately 60 minutes, while cell binding of EXPcRGD NP further increased. This strongly supports the hypothesis of sequential interactions between EXPcRGD NP and its target cells, resulting in a prolonged increase in fluorescence level compared to the homofunctional cRGD NP.

[0234] To investigate the effect of αVβ3 integrin on EXPcRGD NP cell uptake, excess free cRGDfK (c = 500 μM) was added before incubating rMC with EXPcRGD NP for 60 minutes. As a result, cell-related fluorescence levels decreased sharply to levels comparable to those of EXP NP or control NP. Figure 20 c). The heteropolyvalent particles are clearly no longer able to address the necessary αVβ3 integrin, and therefore are essentially unable to initiate endocytosis after AT1r binding.

[0235] To further validate the concept of integrin-mediated NP endocytosis, the inventors decided to utilize transmission electron microscopy (TEM), which allows for the evaluation of NP-cell interactions at a much higher magnification level. To increase the electron density of the applied NPs and the corresponding TEM visibility, ultrasmall gold nanoparticles with an average diameter of 2.2 nm were covalently coupled to PLGA and then used for further NP fabrication. Figure 26 Mesangial cells incubated with these gold-labeled NPs can then be gold-enhanced to enhance and thus visualize the gold nuclei of the particles and assess their exact location. This retrospective gold enhancement offers the significant advantage that the physicochemical properties of the gold-labeled NPs are not significantly different from those of the unlabeled NPs, a situation not observed with commonly used gold NPs.

[0236] Figure 21Image a shows the cell bodies of two mesangial cells incubated with gold-labeled EXPcRGD NPs. Numerous round vesicles filled with gold-enhanced NPs were detected in the cytosol. The distribution pattern showed a significant similarity to the previously described CLSM results. Figure 20 a) This strongly supports ligand-mediated NP endocytosis. Furthermore, particle accumulation at the cell boundary was observed, indicating that these NPs remain bound to membrane-localized surface structures. These findings further suggest that administered EXPcRGD NPs interact with target cells in a stepwise process, first binding to AT1r and then integrin-mediated endocytosis. Based on this assessment, EXP NPs without surface-bound cRGDfK were only detectable on the rMC membrane, and no particle accumulation was observed in endocytic vesicles. Figure 21 b). Furthermore, cell-granule binding in the control NP was negligible ( Figure 21 c). The specificity of gold enhancement is manifested in the absence of gold accumulation in agranular cells.

[0237] Example 9: Ligand synergy leads to enhanced selectivity of mesangial cells in vitro

[0238] All materials and methods mentioned in this embodiment are as described in Embodiments 6 to 8.

[0239] It has been demonstrated that heteropolyvalent EXPcRGD NPs synergistically bind two key features of their surface ligands and present them in a spatially controlled manner. The inventors intend to demonstrate that this design can actually be used to increase mesangial cell selectivity. Therefore, the inventors conducted an in vitro-based assay in which target rMCs were co-cultured with a large number (5-10-fold) of off-target cells carrying either no or only one of the two target receptors. HeLa cells did not express AT1r or αVβ3-integrin to a significant extent; NCI-H295R cells were selected because they showed high AT1r but low αVβ3 expression. Figure 27 ).

[0240] To distinguish co-cultured cells in CLSM analysis, CellTracker was used. TM Green (CTG) staining was used to stain rMCs, while off-target cells were labeled with CTDR. Cellular distribution of NPs was assessed after 45 minutes of incubation with fluorescently labeled EXPcRGD NPs. In the rMC / HeLa co-culture model, granule-derived fluorescence was detected almost exclusively within the mesangial cell region. In contrast, HeLa cells showed only weak interaction with NPs, resulting in marginal fluorescence levels (…). Figure 22a). Based on this, the inventors concluded that EXPcRGD NPs can selectively target mesangial cells from HeLa cells due to differences in cell surface receptor expression. These findings are supported by flow cytometry analysis, which showed that the cell-associated fluorescence of EXPcRGD NPs in rMCs was significantly higher than that in off-target HeLa cells, while the cell-cell interaction of control NPs was negligible for both cell types. Figure 22 b). In contrast, co-culture of rMCs / NCI-H295Rs provided a different particle distribution. NP-related fluorescence was detected not only in rMCs but also in areas covered by NCI-H295R cells. However, the distribution pattern was significantly different. While fluorescence was found in rMCs within the previously observed round, vesicle-like structures, the fluorescence associated with NCI-H295Rs was more diffuse and enhanced primarily at the cell membrane. Figure 22 a). Therefore, the inventors concluded that although EXPcRGD NP accumulates in the endocytic vesicles of rMCs, it can only bind to AT1r present in the cell membrane of NCI-H295R cells, but cannot be taken up into the cytoplasm due to the lack of αVβ3 integrin. Furthermore, flow cytometry analysis showed that although NP-related fluorescence was higher in NCI-H295R cells than in HeLa cells, EXPcRGD NP still showed a significantly enhanced signal in mesangial cells. Figure 22 c). Notably, the addition of excessive free EXP3174 (c = 1 mM) prior to NP incubation resulted in a sharp decrease in fluorescence levels in NCI-H295R cells, while the cell-associated fluorescence of rMCs remained significantly high. This observation further supports the idea that EXPcRGD NPs can indeed target mesangial cells using both surface ligands, thereby benefiting from heterofunctional design.

[0241] In summary, the inventors’ co-culture model demonstrates that heteropolyvalent EXPcRGD NP has the ability to effectively recognize receptor-positive mesangial cells in the presence of off-target cells (which are not only numerically dominant but also express one of the two target receptors).

[0242] Example 10: In vivo accumulation of adenovirus-mimicking EXPcRGD NP in mesangial cells

[0243] All materials and methods mentioned in this embodiment are as described in Embodiments 6 to 9.

[0244] Both rMC binding and uptake studies successfully demonstrated that the inventors' viral mimicry concept of sequential ligand-receptor interactions enables heteropolyvalent EXPcRGD NPs to selectively target mesangial cells in vitro. However, transferring in vitro results to robust systems with sufficient in vivo efficiency has proven to be a major obstacle in nanoparticle design, as many strategies fail to provide the desired target specificity. Therefore, the inventors decided to evaluate the ability of NPs to actually reach the mesangial region in vivo, which requires not only the promotion of active cellular uptake but also sufficient passive accumulation in the target region. In this regard, fluorescently labeled NPs were injected into 10-week-old female NMRI mice. After 1 hour of NP circulation, the mice were sacrificed, and the kidneys were removed. Fluorescence analysis of the prepared frozen sections showed that EXPcRGD NPs accumulated efficiently in the glomerular region, while fluorescence in the tubular portion was negligible. Figure 23 In contrast, control NPs and homofunctional EXPs or cRGD NPs showed considerably low deposition in frozen kidney sections.

[0245] To quantify the observed differences, glomerular-related fluorescence levels were determined by assessing the glomerular fluorescence intensity in each region for all NP types. Figure 24 a / b). Therefore, compared with the control NP, the fluorescence intensity of EXPcRGD NP increased by more than 10-fold. Furthermore, the glomerular accumulation of heteropolyvalent NPs was significantly greater than that of the two isofunctional NP types. Notably, the fluorescence of cRGD NP was even lower than that of the nonfunctionalized control NP. The inventors hypothesized that cRGD NPs could not reach the glomerular region because shortly after injection, a large number of particles bound to endothelial cells expressing αVβ3 and thus left the bloodstream before reaching deeper areas of the kidney. This hypothesis was further supported by the following finding: among all particle types, the relative plasma level of cRGD NP was the lowest after 1 hour of incubation ( Figure 28 a). Conversely, in EXPcRGD NPs, the shorter cRGDfK-functionalized PEG-PLA chain prevents premature exposure to αVβ3 integrin by adding a longer EXP3174-functionalized PEG-PLA chain. Therefore, the heteropolyvalent particles avoid off-target deposition and thus successfully reach the glomerular region within the kidney. Antibody staining for the mesangial cell marker integrin-α8 further revealed that EXPcRGD NP-related fluorescence in the glomeruli can be detected almost entirely within mesangial cells. Figure 24 c), which proves the hypothesis of extravasation into the mesangial interstitium and subsequent endocytosis. Figure 17 (a / b). To verify that the fluorescence detected in the mesangial region originated from structurally intact NPs, the inventors injected an equivalent dose of free fluorescent dye into mice and analyzed the fluorescence deposition. Although the intraglomerular signal in these samples was negligible, the tubular cells exhibited very strong fluorescence levels ( Figure 28(b) This indicates that the low-molecular-weight dye is filtered by the kidneys compared to the injected NP substance. Therefore, the inventors conclude that intraglomerular fluorescence in all NP types originates from intact particles, since degradation would otherwise lead to increased tubular signaling.

[0246] Therefore, the in vivo studies discussed herein successfully demonstrate the potential of the novel adenovirus-mimicking NP design of this invention. Heterovalent EXPcRGD NPs efficiently accumulate in the mesangial region of the glomerulus, while isofunctional or unfunctionalized NPs do not. This strongly suggests that, in order to achieve adequate bioavailability levels, NPs must not only carry appropriate surface ligands but also be presented in a coordinated manner suitable for their respective targeting strategies. Furthermore, the accumulation of NPs in the mesangium also demonstrates the effectiveness of spatially controlled granule-cell interaction-mimicking adenovirus systems.

[0247] Example 11: The EXPcRGD NP loaded with Sina-Sina exhibits high efficiency

[0248] Nanoparticles (NPs) using the concept of target cell recognition mimicking influenza A or adenovirus were detected to accumulate efficiently within mesangial cells in the in vivo environment. In the next step, the experimental drug cinaciguat (BAY 58-2667) was encapsulated in an adenovirus-mimicking EXPcRGD NP. Cinaciguat (CCG) is a potent activator of soluble guanylate cyclase (sGC) and has been shown to significantly reduce mesangial fibrosis and glomerular damage in diabetic animal models. By encapsulating CCG in the inventors' promising NP material, the cell-selective delivery of CCG to pathological mesangial sites can be significantly increased, thereby improving therapeutic efficacy while minimizing off-target effects. Figure 29 ).

[0249] In the inventors' experimental setup, CCG was initially encapsulated in heteropolyvalent EXPcRGD NPs. Each resulting NP carried approximately 500-700 CCG molecules (data not shown). In all the following experiments, administration of 2 μM free cinnarizine was compared with approximately 0.5 nM of CCG-loaded EXPcRGD NPs (equivalent to 0.2 μM CCG) and drug-free EXPcRGD control NPs. Figure 30 The concentration of free cinacigua was selected based on previous publications that showed CCG had antifibrotic effects within this concentration range. However, NPs carrying CCG carried only 10% of their respective CCG amounts to test potential drug delivery effects.

[0250] To assess the effect of CCG-loaded EXPcRGD NP on target sGCs, mesangial cells were initially incubated for 24 hours, and protein levels were assessed using Western blotting analysis. Interestingly, the total amount of sGCs gradually increased after incubation with both the free drug and the CCG-loaded NP, indicating not only the previously shown activation but also the stabilizing effect of cinaciguat on sGCs. Figure 31 Conversely, the absence of Sina Sigwat's NP had no significant effect on sGC levels.

[0251] Finally, the antifibrotic and antiproliferative potential of NP-assisted CCG delivery was analyzed. In this regard, mesangial cells were initially incubated for 4 hours with free CCG, CCG-loaded EXPcRGD NP, or control NP without drug encapsulation. After 4 hours, 10 ng / mL of the drug was added. -1 Transforming growth factor β (TGF-β) was administered for 48 hours to induce fibrosis and hyperproliferative remodeling. While TGF-β administration led to a significant increase in mesangial cell proliferation, pre-incubation with free CCG and CCG-loaded NPs significantly reversed this effect. Figure 32 Furthermore, Western blot and fluorescence microscopy analyses of fibrosis markers α-SMA and collagen I revealed a similar effect of CCG (loaded with NP) in inhibiting mesangial cell-mediated fibrotic remodeling.

[0252] In summary, these results reveal two main findings:

[0253] 1. Both free and NP-encapsulated cinaciguat showed significant effects on its target enzyme sGC, leading to significant activation of the antifibrotic pathway. Figure 29 These results are consistent with previous findings on CCG and demonstrate the significant potential of the therapeutic agent in the treatment of fibrosis.

[0254] 2. In all experiments, even when the total amount of encapsulated CCG was only 10% of the free drug dose (0.2 μM vs. 2 μM), the effect of the EXPcRGD NP loaded with cinacigua was comparable to that of administration of free CCG. This suggests that the described NP has considerable potential for more efficient drug delivery to its intended intracellular targets.

[0255] References

[0256] [1]Maslanka Figueroa, S., Veser, A., Abstiens, K., Fleischmann, D., Beck, S., and Goepferich, A. (2019). Influenza A virus mimetic nanoparticles triggerselective cell uptake.Proc.Natl.Acad.Sci.201902563.

[0257] [2] Sah E. and Sah H. Journal of Nanomaterials, 2015, Article ID 794601.

[0258] [3]Hennig, R., Ohlmann, A., Staffel, J., Pollinger, K., Haunberger, A., Breunig, M., Schweda, F., Tamm, ER, and Goepferich, A. (2015). Multivalent nanoparticles bind the retinal and choroidal vasculature. J. Control. Release 220, 265–274.

[0259] [4]Inuzuka, T., Fujioka, Y., Tsuda, M., Fujioka, M., Satoh, AO, Horiuchi, K., Nishide, S., Nanbo, A., Tanaka, S., and Ohba, Y. (2016). Attenuation of ligand-induced activation of angiotensin II type 1 receptor signaling by the type 2 receptorvia protein kinase C.Sci.Rep.6,21613.

[0260] The features of the invention disclosed in the specification, claims and / or drawings can be used alone or in any combination thereof to form material for implementing the invention in various forms. sequence list <110> University of Regensburg <120> Virus-mimicking nanoparticles <130> FSP1V223205JW <150> EP19 219 424.9 <151> 2019-12-23 <160> 1 <170> BiSSAP 1.3.6 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Cyclic peptides <400> 1 Arg Gly Asp Phe Lys 1 5

Claims

1. A nanoparticle comprising a nanomaterial and at least a first ligand and a second ligand. -The first ligand mediates the attachment of the nanoparticles to the target cells, wherein the first ligand is EXP3174, and wherein the target cells are mesangial cells. -The second ligand is an integrin-binding agent, wherein the integrin-binding agent is the cyclic RGD peptide shown in SEQ ID NO. 1, and wherein the second ligand mediates the internalization of the nanoparticles into the target cells, and -The nanomaterials described herein consist of the following: Poly(lactic acid-co-glycolic acid) (PLGA) and block copolymer PEG-PLA, -The nanomaterial comprises more than one block copolymer chain, and the first ligand is associated with the first PEG-PLA block copolymer chain PEG. 5k -PLA 10k Coupling, and the second ligand is coupled with the second PEG-PLA block copolymer chain PEG 2k -PLA 10k Couplet.

2. The nanoparticles according to claim 1 further comprise a therapeutic agent.

3. The nanoparticles according to claim 2, wherein the therapeutic agent is any one of pirfenidone and cinasciguat.

4. The nanoparticle of claim 1, wherein the second ligand is enzymatically activated prior to internalization of the nanoparticle into the target cell.

5. The nanoparticles according to claim 1, wherein the nanoparticles have a size of 5 nm to 1000 nm.

6. The nanoparticles according to claim 1, wherein the nanoparticles have a size of 10 nm to 150 nm.

7. The nanoparticles according to claim 1, wherein the nanoparticles have a size of 20 nm to 100 nm.

8. The nanoparticles according to claim 1, wherein the ratio of the first ligand to the second ligand is in the range of 2:1 to 1:

2.

9. The nanoparticles according to claim 1, wherein the ratio of the first ligand to the second ligand is 1:

1.

10. The nanoparticles according to claim 1, wherein the nanoparticles have a particle affinity for the target receptor of 1 pM to 100 nM.

11. The nanoparticles according to claim 1, wherein the nanoparticles have a particle affinity for the target receptor of 50 pM to 1 nM.

12. The nanoparticles of claim 1, wherein the nanomaterial comprises PEG, and wherein the nanoparticles have a ligand density of at least 5% ligand / PEG.

13. The nanoparticles of claim 1, wherein the nanomaterial comprises PEG, and wherein the nanoparticles have a ligand density of at least 15% ligand / PEG.

14. The nanoparticles of claim 1, wherein the nanomaterial comprises PEG, and wherein the nanoparticles have a ligand density of at least 25% ligand / PEG.

15. Use of the nanoparticles according to any one of claims 1 to 14 in the preparation of a medicament for the prevention or treatment of diseases selected from diabetic nephropathy and glomerulonephritis.

16. A method for preparing nanoparticles as defined in any one of claims 1 to 14, comprising the following steps: a) Provide poly(lactic acid-co-glycolic acid) (PLGA) and block copolymer PEG-PLA in any order; b) Preparation of block copolymers from PEG-PLA; c) Couple the first and second ligands to it in one or more steps; d) Provide the treatment if it was not provided in step a); e) Nanoparticles are prepared and obtained using ligands coupled to the nanomaterials and the therapeutic agents.

17. The method of claim 16, further comprising providing a therapeutic agent in step a); and / or Step c) is performed via DCC / NHS- or EDC / NHS- coupling; and / or The therapeutic agent described therein is a lipophilic therapeutic agent; and / or Step e) is performed via nanoprecipitation.

18. The method of claim 16, wherein the obtaining in step e) comprises obtaining nanoparticles with a polydispersity index of 0.01 to 0.

5.

19. The method of claim 16, wherein the obtaining in step e) comprises obtaining nanoparticles with a polydispersity index of 0.01 to 0.

3.

20. The method of claim 16, wherein the obtaining in step e) comprises obtaining nanoparticles with a polydispersity index of 0.01 to 0.1.

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