Virus-mimetic nanoparticles

Abstract

To provide a nanoparticle comprising a nanomaterial and at least a first ligand and a second ligand tethered to the nanoparticle.SOLUTION: The present invention further 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 of preventing or treating a disease selected from 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.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to nanoparticles comprising a nanomaterial and at least a first ligand and a second ligand.The present invention further relates to nanoparticles for use as a medicine or diagnostic agent.The present invention also relates to nanoparticles for use in a method for preventing or treating a disease selected from 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 for preparing nanoparticles. [Background technology]

[0002] 2. Background of the Invention A myriad of nanomaterials have been developed over the last few years as carriers for drug treatments or diagnostic methods. To endow them with the ability to identify target cells in vivo with sufficient specificity, ligands that bind to cell receptors have been tethered to their surface. However, simply following this old paradigm, although it increases the avidity of nanomaterials, has proven insufficient for unambiguous cell identification. At present, even nanomaterials presenting several different ligands for hetero-multivalent binding are unable to discriminate between different cell types.

[0003] In contrast, viruses are nanoparticles (NPs) with ultimate target cell specificity. In contrast to synthetic biomedical nanomaterials, viruses use a sequential, multi-step recognition process for cell identification. Since many current nanoparticle-based approaches lack sufficient specificity, exploiting a viral targeting strategy may be a viable option to overcome this limitation. Thus, by mimicking the sequential recognition strategy of viruses, such as influenza A virus, with nanomaterials, it may be possible to specifically target cells. In particular, viral target cell recognition is likely to be advantageous in vivo if the particles undergo surface modifications due to protein adsorption.

[0004] In particular, the initial step of virus attachment to cell membranes, which does not result in particle uptake but increases the virus particle density on the cell surface, is missing in current nanoparticle design strategies. This initial attachment to glycolipids and glycoproteins or specific receptors has been found to be essential for virus infectivity. Furthermore, if the particles are subject to clearance after reaching the target tissue, they become unsuitable for drug delivery purposes. In the case of the mesangium, early reports have shown that small particles with diameters of 70 ± 25 nm or less penetrated glomerular endothelial fenestrations with diameters of about 80-100 nm. However, these particles have undergone mesangial clearance. This problem of known nanoparticles, which are often rapidly cleared from tissues, may be overcome if the nanoparticles are internalized by the target cells in the target tissue.

[0005] Maslanka Figueroa et al. [1] relates to polymer nanoparticles containing a ligand which is angiotensin-I.

[0006] Sah et al. [2] relates to nanoparticles containing block copolymers and drugs. The present invention aims to provide nanomaterials with virus-mimetic cell identification mechanisms to address cells in vitro and in vivo. Furthermore, the present invention aims to provide an efficient accumulation of said nanoparticles in target tissues in vivo. It is a further object of the present invention to provide a drug delivery system which allows for the delivery of a drug or diagnostic agent to a target tissue. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Maslanka Figueroa et al., Proc. Natl. Acad. Sci. (2019)201902563 [Non-Patent Document 2] Sah E. et al., Journal of Nanomaterials, Volume 2015, Article ID 794601 Summary of the Invention [Means for solving the problem]

[0008] Summary of the Invention In the following, the elements of the present invention are described. Although these elements are listed in specific embodiments, it should be understood that these elements can be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be interpreted as limiting the present invention to only the specifically described embodiments. This description should be understood to support and encompass embodiments that combine two or more of the specifically described embodiments, or combine one or more of the specifically described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all described elements in this application should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0009] In a first aspect, the present invention provides a nanoparticle comprising a nanomaterial and at least a first ligand and a second ligand, - said first ligand is capable of mediating attachment of said nanoparticle to a target cell; - the second ligand is capable of mediating internalization of the nanoparticle into the target cell; - preferably, said nanomaterial comprises any of 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-poly-caprolactone, inorganic materials such as gold or qdot materials, and combinations thereof. Concerning nanoparticles.

[0010] In one embodiment, the first ligand is a non-agonistic agent that binds to a GPCR, 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 a target cell, such as heparan sulfate, sialoglycoprotein, ganglioside and mannose receptors, preferably EXP3174 or telmisartan.

[0011] In one embodiment, the second ligand is either i) an agent that binds to an integrin, such as αVβ3 integrin or αVβ5 integrin, preferably selected from RGD, a cyclic RGD-peptide having the sequence of SEQ ID NO:1, and derivatives thereof; ii) an agonistic agent that binds to a GPCR, such as AT1r, preferably activated angiotensin-II; iii) an agent that binds to an ectoenzyme, such as legumain, membrane-type matrix metalloproteinase and angiotensin-converting enzyme (ACE), preferably angiotensin-I; and / or iv) an agent that binds to the transferrin receptor.

[0012] In one embodiment, the nanoparticles further comprise a therapeutic agent, preferably either pirfenidone or cinaciguat.

[0013] 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.

[0014] In one embodiment, the nanomaterial comprises more than one block copolymer chain, the first ligand is coupled to a first block copolymer chain of the nanomaterial, the second ligand is coupled to a second block copolymer chain of the nanomaterial, and the first block copolymer chain is longer than the second block copolymer chain, preferably at least 1.5 times as long as the second block copolymer chain, and more preferably at least 3 times as long as the second block copolymer chain.

[0015] In one embodiment, the first block copolymer chain comprises PEG in the range of 1k-20k, preferably 1k-10k, and / or PLA in the range of 5k-40k, preferably 10k-20k, and optionally the first block copolymer chain comprises PEG in the range of 5k-40k, preferably 10k-20k. 5k -PLA 10k and the second block copolymer chain is PEG 2k -PLA 10k It is.

[0016] In one embodiment, the second ligand is enzymatically activated prior to the internalization of the nanoparticle into the target cell.

[0017] In one embodiment, the target cell is selected from a mesangial cell, an endothelial cell, such as a retinal endothelial cell, a B cell, a T cell, a macrophage, a dendritic cell, and a tumor cell.

[0018] In one embodiment, the particles have a size of 5 nm to 1000 nm, preferably 10 nm to 150 nm, more preferably 20 nm to 100 nm.

[0019] In one embodiment, the ratio of said first ligand to said second ligand ranges from 2:1 to 1:2, preferably 1:1.

[0020] In one embodiment, the particles have a particle avidity for the targeted receptor of between 1 pM and 100 nM, preferably between 50 pM and 1 nM.

[0021] 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.

[0022] In a further aspect, the present invention relates to a nanoparticle as defined in any of the above embodiments for use as a pharmaceutical or diagnostic agent.

[0023] In a further aspect, the present invention relates to a nanoparticle as defined in any of the above embodiments for use in a method for preventing or treating a disease selected from 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.

[0024] In a further aspect, the present invention provides a method for preparing nanoparticles comprising the steps of: a) providing, in any order, one or more nanomaterials, preferably including 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, e.g., PEG-PLA or PEG-poly-caprolactone, inorganic materials, e.g., gold or qdot materials, and any combination thereof, and optionally a therapeutic agent; b) optionally preparing a block copolymer from either said one or several nanomaterials; c) coupling thereto, in one or more steps, a first ligand and a second ligand, preferably by DCC / NHS coupling or EDC / NHS coupling; d) providing a therapeutic agent, preferably a lipophilic therapeutic agent, if not already provided in step a); e) Preparing and obtaining nanoparticles using the ligands coupled to the nanomaterial and the therapeutic agent, preferably by nanoprecipitation. The present invention relates to a method comprising the steps of:

[0025] In one embodiment, said obtaining step in step e) comprises obtaining nanoparticles having a polydispersity index between 0.01 and 0.5, preferably between 0.01 and 0.3, more preferably between 0.01 and 0.1.

[0026] In this embodiment, the nanoparticle, the nanomaterial, the therapeutic agent, the block copolymer, the first ligand and the second ligand are as defined above.

[0027] In a further aspect, the present invention relates to a pharmaceutical composition comprising the nanoparticles as defined above and a pharma- ceutically acceptable excipient.

[0028] In a further aspect, the present invention relates to a method for preventing or treating a disease comprising administering an effective amount of the nanoparticles and / or the pharmaceutical composition to a patient in need thereof.

[0029] In this aspect, the disease, the nanoparticles, and the pharmaceutical composition are as defined above.

[0030] In a further aspect, the invention relates to the use of nanoparticles in the manufacture of a medicament for the treatment of a disease.

[0031] In this embodiment, the nanoparticles and the disease are as defined above.

[0032] In a further aspect, the present invention relates to the use of nanoparticles in the manufacture of a diagnostic agent for the diagnosis and / or prognosis of a disease.

[0033] In this embodiment, the nanoparticles and the disease are as defined above.

[0034] Detailed Description Insufficient availability in the tissue of interest due to adverse physicochemical properties is a common cause of drug failure. Viruses overcome this constraint by embedding their nucleic acid in nanoscale particles and directing with high specificity to their target cells. Nanotechnology has provided a wealth of nanocarriers for drug delivery, but their ability to unambiguously identify the cells of interest has remained mediocre. We show herein that particles with a virus-like ability to identify cells by three consecutive checks for cell identity have a superior ability to identify mesangial cells in vivo compared to conventional nanoparticles. In mice, this resulted in a 15-fold higher accumulation in the kidney mesangium and subsequent massive cellular uptake. The present invention provides a surprisingly effective tool for transporting drugs into target tissues, which is suitable for use in the treatment of various diseases, such as diabetic nephropathy, for which no drug therapy currently exists.

[0035] We investigated the mechanism by which angiotensin-II is expressed in the NP corona to mediate receptor attachment. For example, we designed particles bearing EXP3174, a type 1 receptor (AT1R) ligand (Figure 1A). As a G protein-coupled receptor (GPCR) antagonist, it has the paramount advantage that binding cannot induce cellular NP uptake, but only membrane binding, thus preventing particle uptake by off-target cells that only bear the AT1R.

[0036] As a second recognition criterion, we endowed the particles with the ability to probe the cell surface for the presence of targets, e.g., angiotensin-converting enzyme (ACE), which recognizes the proligand angiotensin-I (Ang-I) in the particle corona and converts it to the active ligand angiotensin-II (Ang-II).

[0037] As a third recognition step, a ligand such as Ang-II binds to a target such as AT1R and, as an agonist, triggers cellular uptake of the particle upon receptor binding. The entire process of target cell recognition can be best illustrated with a flow chart (Figure 1B). The particles were tested in vitro for their target receptor avidity and target cell specificity. Furthermore, we evaluated how the simultaneous presentation of two ligands directed to the same receptor, an antagonist that promotes cell membrane binding and an agonist that supports cellular internalization, affects the ability of NPs to mediate cellular uptake. Finally, we showed that particles with such a virus-mimetic triple recognition strategy outperformed conventional NPs in reaching mesangial cells in vivo.

[0038] Since NPs are typically distributed in organisms by passive transport mechanisms, their appearance in a particular tissue is a matter of their physicochemical properties. However, the percentage of particles that accumulate in the tissue of interest can be increased if they can actively interact with the cells of interest. To confirm the identity of the cells, it is not enough for the NPs to have ligands that bind to the respective receptors. The particles of the present invention clearly demonstrate that a strategy of stepwise identification of cells is more advantageous, in particular a strategy that includes a first ligand to mediate attachment to the target cell and a second ligand to mediate internalization of the nanoparticle into the target cell.

[0039] Using viruses as templates, we have designed NPs that can carry out a sequence of "if-then-else" decisions one after the other. In each single step, the NP searches the cell with the help of a ligand or substrate for the presence of a receptor or ectoenzyme, respectively. If it is successful, the next identification step follows, otherwise (else), the particle "decides" that the cell cannot be the target cell. As with viruses, this helps to avoid NP uptake by the "wrong" cell type.

[0040] As an example, a receptor belonging to the family of GPCR, such as AT1R, was used for the decision making particle. If the cell identity is detected using a ligand, such as a GPCR-antagonist, such as EXP3174 or telmisartan, then the positive outcome of this interaction is that the particle binds to the cell surface and remains there. If the next interaction cannot be positive, then it is clear that there is a risk that the particle will remain on the off-target cell. However, in this case, as can be expected due to the thermodynamic equilibrium between free and bound particles, the decrease in the concentration of free particles in vivo over time shifts the equilibrium so that the particle dissociates from the "wrong" target over time. In contrast, if the cell identity is detected using an agonist for the same GPCR, such as Ang-II, then the positive answer of the cell is particle internalization. Thus, by carefully selecting the target and type of ligand for interaction, the particles can be endowed with logic that may allow for even more covert target cell identification than the exemplary targets investigated in this study. An example is local ocular application in retinal tissue, where the particles can distinguish between more than 60 cell types present, for example by specifically targeting endothelial cells.

[0041] The terms "nanoparticle" and "NP" as used herein refer to a nanomaterial structure comprising a first ligand and a second ligand. In particular, a nanoparticle is a nano-object in which all three external dimensions are at the nanoscale, e.g., liposomes, polymeric nanoparticles, micelles, lipid nanocapsules, liposomes, inorganic nanoparticles, e.g., gold nanoparticles or Qdots. In one embodiment, the nanoparticles provide excellent biocompatibility and highly tunable composition. Nanoparticles can be produced from a wide variety of materials, e.g., polymers, biomolecules, and metals. In one embodiment, the nanoparticles of the present invention are virus-mimetic nanoparticles capable of transporting drugs into target tissues, e.g., in the mesangium of the kidney. In one embodiment, the nanoparticles comprise pirfenidone and / or cinaciguat and are for use in 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, the nanoparticles are prepared using nanoprecipitation by injecting the polymer mixture dropwise into an aqueous phase. In one embodiment, the nanoparticles of the present invention accumulate in the target tissue, such as kidney tissue, within a very short time after administration of the nanoparticles, i.e., <1 hour.

[0042] In one embodiment, the nanoparticles of the present invention are very small, i.e., <80 nm, and due to their small size, they can rapidly exit the bloodstream through fenestrated endothelium and accumulate in target tissues such as mesangial tissue. In one embodiment, the nanoparticles further comprise a therapeutic agent, preferably one of pirfenidone and cinaciguat. In one embodiment, the particles have a size between 5 nm and 1000 nm, preferably between 10 nm and 150 nm, more preferably between 20 nm and 100 nm. In one embodiment, the particle size is measured using dynamic light scattering, particle scattering diffusometry, nanoparticle tracking analysis, atomic force microscopy or transmission electron microscopy, preferably dynamic light scattering or transmission electron microscopy. The size of a particle is determined by its diameter for any line segment that passes through the center of a spherical particle and whose end point lies on the spherical particle. In the case of non-spherical particles, the diameter is for the longest line segment that passes through the center of the non-spherical particle and whose end point lies on the particle. In one embodiment, the average diameter is for the average of the diameters of the nanoparticles contained in a batch of nanoparticles. In one embodiment, the particles of the 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 measurements, 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 invention are for use in medicine. In one embodiment, the nanoparticles have a zeta potential that is positive, negative or neutral, for example, between -20 mV and 0 mV, or between -15 mV and -5 mV. In one embodiment, the nanoparticles comprise at least one core comprising a nanomaterial and optionally a polymer and / or linker on its surface, and said first ligand and said second ligand are coupled to said polymer and / or linker. In one embodiment, the particles have a polymer core.

[0043] The term "viral mimetic" as used herein refers to an approach that mimics the cell targeting approach of viruses. The viral mimetic particles of the present invention are internalized by target cells through a recognition process that has at least two sequential steps. In such a two-step process, the first step is that a first ligand on the particle, such as EXP3174 or telmisartan, binds to a target expressed on the target cell, such as angiotensin receptor on mesangial cells. Subsequently, the particle is internalized into the target cell, which is mediated by a second ligand, such as activated angiotensin II or a cyclic amino acid sequence (cycloArg-Gly-Asp-D-Phe-Lys; SEQ ID NO: 1), that binds to a target on the target cell, thereby initiating the endocytosis of the particle. In one embodiment, the sequential presentation of the first and second ligand is achieved by i) stereocontrol mediated by using a longer linker, e.g. a longer PEG-PLA chain, for the first ligand compared to a shorter linker, e.g. a shorter PEG-PLA chain, used for the second ligand, and / or ii) an activation step where the second ligand needs to be activated so that it is able to mediate internalization, e.g. conversion of angiotensin I to angiotensin II. In one embodiment, the sequential presentation of the ligands allows a 15-fold higher accumulation of the nanoparticles in target cells, such as mesangial cells, compared to conventional particles without ligand modification. In one embodiment, the particles have a particle avidity for the targeted receptor of 1 pM to 100 nM, preferably 50 pM to 1 nM. In one embodiment, the nanoparticles of the invention are for use as pharmaceutical or diagnostic agents. In one embodiment, the nanoparticles of the invention are for use in a method of preventing or treating a disease selected from 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. In one embodiment, the terms "nanoparticle", "viral mimetic particle" and "decision-making nanoparticle" are used interchangeably.

[0044] The term "nanomaterial" as used herein relates to a material having any external dimension on the nanoscale or having a nanoscale internal or surface structure. In one embodiment, the nanomaterial preferably comprises any of 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 stearic PEG, block copolymers, such as PEG-PLA or PEG-poly-caprolactone, inorganic materials, such as gold or qdot 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, the first ligand is coupled to a first block copolymer chain of the nanomaterial, the second ligand is coupled to a second block copolymer chain of the nanomaterial, the first block copolymer chain being 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-20k, preferably 1k-10k, and / or PLA in the range of 5k-40k, preferably 10k-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, one ligand is coupled to one PEG molecule. The term "k" as used herein refers to a polymer chain, e.g., a block copolymer chain, PEG and / or PLA, in kilodaltons (kDa). For example, PEG in the range of 1k-20k refers to PEG in the range of 1kDa-20kDa, e.g., PEG 5kDa In one embodiment, PEG 5k -PLA10k and PEG 2k -PLA 10k are respectively 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 is PEG (PEG 総 ), the particles comprising at least 5%, preferably at least 15%, more preferably at least 25% ligand / PEG. 総 and the term "ligand" includes both the first and second ligands. In one embodiment, the ligand density of the ligand / PEG is ≦50%.

[0045] The term "first ligand" as used herein refers to a ligand capable of mediating the attachment of nanoparticles to target cells. In one embodiment, the first ligand only initiates the binding of the particle to the target cell, but does not initiate the internalization of the particle into the target cell, and subsequent binding of a second ligand to the target cell is required 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 induces the binding of the nanoparticle to the target cell, such as an antibody or an antigen-binding fragment thereof, a peptide, an aptamer, a DNA nanostructure, a receptor ligand and a receptor. In one embodiment, the first ligand is a non-agonistic agent that binds to a GPCR, 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 a target cell, such as heparan sulfate, sialoglycoprotein, ganglioside, and mannose receptors, preferably EXP3174 or telmisartan. In one embodiment, the non-agonistic agent that binds to a GPCR induces binding of the particle to a target cell, but does not induce internalization of the particle into the target cell, such as endocytosis. In one embodiment, the first ligand and / or the second ligand binds to a target that is not ubiquitously expressed, but is predominantly expressed on target cells in a target tissue. In one embodiment, the first ligand and / or the second ligand binds to a target that is not ubiquitously expressed, but is predominantly expressed on target cells in a target tissue. In one embodiment, the first ligand and / or the second ligand binds to a target that is not ubiquitously expressed, such as K DIt binds to a target structure on a target cell with an affinity of <100 nM. In one embodiment, the first ligand and / or the second ligand have a molecular weight of <1500 Da. In one embodiment, the terms "target" and "target structure" are used interchangeably. In one embodiment, the term "target structure" refers to a protein, peptide, nucleic acid, saccharide, glycolipid and / or glycoprotein that is presented on the surface of a target cell. In one embodiment, the first ligand is any AT1R antagonist that has 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, biologically active derivatives thereof. In one embodiment, the "biologically active derivative" has the same biological function, such as the same binding function and / or therapeutic function, as EXP3174 or telmisartan, respectively.

[0046] The term "non-agonistic agent", as used herein, relates to an agent that binds to a target structure without having an agonistic effect on said target structure, preferably without having an effect on said target structure that mediates internalization into a target cell.

[0047] The term "secondary ligand" as used herein relates to a ligand capable of mediating the internalization of the nanoparticle into a target cell. In one embodiment, the binding of the second ligand to a target structure, e.g., a receptor on a target cell, initiates the internalization of the nanoparticle into the target cell. In one embodiment, the target structure on the target cell is any structure that is typically expressed on the surface of the target cell, e.g., a surface molecule, a receptor and / or a biomarker. In one embodiment, the target structure on the target cell is a molecule that is typically 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, e.g., an antibody or antigen-binding fragment thereof, a peptide, an aptamer, a DNA nanostructure, a receptor ligand and a receptor. In one embodiment, the second ligand is either i) an agent that binds to an integrin, for example, αVβ3 integrin, preferably selected from RGD, cyclic RGD-peptide having the sequence of SEQ ID NO: 1, and derivatives thereof, ii) an agonistic agent that binds to a GPCR, for example, AT1r, preferably activated angiotensin-II, iii) an agent that binds to an ectoenzyme, for example, legumain, membrane-type matrix metalloproteinase and angiotensin-converting enzyme (ACE), preferably angiotensin-I, and / or iv) an agent that binds to a transferrin receptor. In one embodiment, the second ligand is enzymatically activated before the internalization of the nanoparticle into the target cell, for example, angiotensin-I is activated to angiotensin-II by ACE before the binding of the second ligand to AT1r and the internalization of the particle into the target cell. In one embodiment, enzymatic activation of the second ligand is performed by an ectoenzyme on the surface of the target cell, preferably said enzymatic activation comprises enzymatic cleavage of said second ligand, thereby providing an activated second ligand.In an alternative 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 ranges from 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 specificity for the target cell.

[0048] In one embodiment, when the second ligand targets AT1r, the second ligand is angiotensin-I, which is preferred over the second ligand is angiotensin-II, because the intermediate step of the enzymatic activation of angiotensin-I to angiotensin-II allows the nanoparticle to have increased specificity. In one embodiment, when the first ligand is EXP3174 and the second ligand is angiotensin-II, or angiotensin-I 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, which is EXP3174, binds to AT1R as an antagonist, and the second ligand, which is angiotensin-II, optionally after enzymatic activation, binds to AT1R as an agonist. After the binding of the first ligand, for example, EXP3174, there is still an available AT1R binding site for the second ligand. In one embodiment, the binding site of the target bound by the first ligand and the binding site of the target bound by the second ligand are different binding sites or are the same binding site but bound sequentially.

[0049] In one embodiment, the second ligand is shielded from binding to said target cell by i) steric hindrance, e.g., a block copolymer chain or linker of the second ligand that is shorter than the block copolymer chain or linker of the first ligand, and / or ii) the requirement for enzymatic activation of the second ligand before it can mediate internalization.

[0050] The term "capable of mediating attachment" as used herein relates to the binding ability of the first ligand to a target cell. The first ligand is capable of mediating attachment, i.e. the first ligand induces the binding of the nanoparticle to the target cell, preferably by targeting a target structure on the target cell. In one embodiment, said binding of the first ligand to the target cell does not induce the internalization of the nanoparticle into the target cell. In one embodiment, for the internalization of the nanoparticle into the target cell, a further interaction is required, i.e. an interaction of the second ligand with the target cell, preferably comprising a second ligand that targets a target structure on the target cell that is the same or different than 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 of the target structure, e.g., an agonist binding site and an antagonist binding site.

[0051] The term "capable of mediating internalization" as used herein relates to the ability of the second ligand to induce internalization of the nanoparticle into a target cell. In one embodiment, the second ligand binds to a target structure on the target cell, thereby initiating internalization of the nanoparticle into the target cell. In one embodiment, said ability to induce internalization may include the ability to be activated, for example by enzymatic activation, before inducing internalization. In one embodiment, said internalization involves either receptor-mediated endocytosis, clathrin-coated pits and / or caveolae.

[0052] The term "target cells" as used herein relates to cells involved in a pathological condition, i.e. a disease. In one embodiment, the treatment of a disease comprises targeting said disease-involved cells with the nanoparticles of the invention. In one embodiment, the nanoparticles of the invention function as a drug delivery system to transport a drug to said target cells. In one embodiment, said target cells are selected from mesangial cells, endothelial cells, e.g. retinal endothelial cells, and tumor cells.

[0053] The term "therapeutic agent" as used herein relates to any substance intended for medical treatment. In one embodiment, the therapeutic agent is contained in the particle body, i.e., core, of the nanoparticle, e.g., inside the nanoparticle and / or throughout the particle nanomaterial, and / or is coupled to the nanoparticle using a linker. In one embodiment, the therapeutic agent is a lipophilic therapeutic agent and is encapsulated and / or contained by the particle body. In one embodiment, the therapeutic agent is non-covalently or covalently coupled to the particle, e.g., by a cleavable linker. 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 anti-fibrotic agent or a chemotherapeutic agent. In one embodiment, the therapeutic agent is either pirfenidone or cinaciguat. The term "particle body" as used herein relates to the main supporting structure of the particle. For example, the particle body may relate to the lipid bilayer of a liposome or to the core structure of a polymer and / or solid lipid particle. In one embodiment, the nanoparticles of the present invention are loaded with a therapeutic agent for specifically treating target cells, such as mesangial cells, with the therapeutic agent. In one embodiment, the therapeutic agent loaded into the particle, such as pirfenidone or cinaciguat, is dissolved in a polymer phase, incorporated into said particle during the preparation of the particle polymer core, and / or dissolved in an aqueous phase contained by the particle, such as an aqueous phase contained by liposomes, and / or dissolved in a lipid phase contained by the particle, such as an aqueous phase contained by liposomes. Pirfenidone is a TGF-beta antagonist and has been proposed as a candidate for treating mesangium-related pathological fibrosis. Cinaciguat (BAY 58-2667) is a soluble guanylate cyclase (sGC) activator and has been proposed for treating diabetic nephropathy. In one embodiment, the therapeutic agent pirfenidone or cinaciguat is efficiently incorporated into the polymer core of the nanoparticle without significantly changing the properties of the particle, which is possible due to the lipophilic properties of the therapeutic agent.In one embodiment, the nanoparticles are for use in the treatment of diabetic nephropathy and the therapeutic agent is an anti-fibrotic agent, hi one embodiment, the nanoparticles are for use in the treatment of cancer and the therapeutic agent is a chemotherapeutic agent.

[0054] The present invention further relates to a composition comprising the nanoparticles of the present invention and a pharma- ceutically acceptable excipient. The nanoparticles of the present invention can be mixed with suitable auxiliary substances and / or additives to obtain a pharma- ceutically acceptable composition. Such substances include pharmacologically acceptable substances that increase the stability, solubility, biocompatibility or biological half-life of the nanoparticles, or substances that need to be included for certain routes of application, such as intravenous solutions, sprays, band-aids or pills. The present invention also relates to a composition comprising the nanoparticles of the present invention for use in medicine, such as for use in a method for preventing or treating a disease selected from 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.

[0055] The nanoparticles of the present invention use a novel virus-mimetic recognition principle for target cells, such as mesangial cells, which results in highly 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 the nanoparticles and / or linked to the nanoparticles, allowing targeted treatment of target cells in target tissues, such as mesangial cells in the mesangium. In one embodiment, the nanoparticles containing a therapeutic agent are used to prevent or treat diabetic nephropathy. In one embodiment, the nanoparticles of the present invention allow target cells to be targeted using a recognition process having at least two steps. In one embodiment, the nanoparticles of the present invention comprise a selective AT1 antagonist, such as EXP3174, as a first ligand, and further comprise a therapeutic agent, preferably pirfenidone and / or cinaciguat. In one embodiment, the nanoparticles of the present invention are EXPcRGD nanoparticles, i.e. nanoparticles comprising EXP3174 as a first ligand and cRGD, in particular cRGDfK, as a second ligand, or the nanoparticles are EXPAng-I nanoparticles, i.e. nanoparticles comprising 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 a therapeutic agent, preferably pirfenidone and / or cinaciguat. In one embodiment, the nanoparticles are nanoparticles comprising a selective AT1 antagonist, such as EXP3174, as a first ligand, preferably EXPcRGD nanoparticles, and further comprise a therapeutic agent, which is cinaciguat. In one embodiment, the nanoparticles comprise nanomaterials comprising or consisting of PLGA and / or PEG-PLA.

[0056] The term "administering" as used herein relates to the administration of an agent, such as the nanoparticles of the invention and / or the pharmaceutical composition of the invention, which may be achieved by any method that allows the agent to reach the target cells. These methods include, for example, injection, oral ingestion, inhalation, intranasal delivery, topical administration, deposition, implantation, suppository, or any other method of administration that gives the nanoparticles access to the target cells. Injection may refer to intravenous, intradermal, subcutaneous, intramuscular or intraperitoneal injection. Implantation may include inserting an implantable drug delivery system comprising the nanoparticles of the invention and / or a hydrogel comprising the nanoparticles, in particular in It may be related to hydrogels injected subcutaneously and / or intraperitoneally that gel in situ and release nanoparticles in a delayed manner. Suppositories include glycerin suppositories. Inhalation includes administering nanoparticles using aerosols in an inhaler, either alone or attached to a carrier that can be absorbed. Nanoparticles can be suspended in liquid, for example, in colloidal form.

[0057] An "effective amount" is the amount of nanoparticles or pharmaceutical composition that alleviates the symptoms found for a disease and / or condition.

[0058] The term "patient" as used herein relates to a human or animal, preferably a mammal. Treating a patient is meant to include, for example, preventing, treating, reducing symptoms of a disease or condition, such as cancer or diabetic nephropathy, or curing such a disease or condition.

[0059] The term "block copolymer" as used herein refers to a polymer that includes two or more homo- or copolymer subunits linked by covalent bonds. An intermediate non-repeating subunit that is a junction block may be included. A block copolymer is composed of blocks of different polymerized monomers. The term "block copolymer chain" refers to a chain of block copolymers.

[0060] The terms "DCC / NHS coupling" and "EDC / NHS coupling" as used herein refer to coupling reactions. A common method for synthesizing NHS-activated molecules is to mix NHS with, for example, the desired carboxylic acid and a small amount of an organic base in an anhydrous solvent. Then, a coupling reagent, for example, dicyclohexylcarbodiimide (DCC) or ethyl(dimethylaminopropyl)carbodiimide (EDC), is added to form a highly reactive activated intermediate. In one embodiment, the first and second ligands 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.

[0061] We herein successfully show that virus-mimetic NPs that double / triple check cell identity enable enhanced NP accumulation in targeted MCs in vivo. By combining antagonistic ligands that mimic the initial cell attachment of the virus with an enzyme-mediated target cell recognition process, the particles had exceptionally high in vitro target avidity along with exceptional target cell specificity. We also demonstrate that simultaneous hetero-multivalent binding of particle-tethered agonists and antagonists to the same GPCR surprisingly results in particle uptake. Overall, non-specific size-mediated passive targeting is not sufficient to achieve satisfactory particle accumulation in MCs. Even traditional particle functionalization with a single ligand appears to be an insufficient approach. However, by mimicking the complex multi-step virus-target cell binding and recognition process, we obtained particles that can identify and accumulate in MCs. This opens new options for the delivery of drugs for the treatment of various diseases, including renal diseases.

[0062] The inventors further fabricated adenovirus-mimetic block copolymer nanoparticles that effectively targeted glomerular mesangial cells due to sterically controlled sequential ligand-receptor interactions. The hetero-multivalent NPs thereby not only exhibited precisely tunable physicochemical characteristics, but also excellent avidity for both targeting motifs, resulting in substantial AT1r binding in the picomolar concentration range, and significantly increased mesangial cell uptake compared to non-functionalized NPs. Benefiting from these features, the virus-mimetic NPs were able to selectively target mesangial cells even in the surrounding environment of off-target cells. Moreover, the hetero-multivalent NPs showed the necessary in vivo robustness, resulting in efficient in vivo accumulation in the mesangial region with only minor off-target deposition in the kidney. Strikingly, the hetero-multivalent EXPcRGD NPs thereby exhibited much better mesangial targeting compared to homo-functionalized cRGD or EXP NPs. Because we were able to target the same distinct cell types in vivo using two different virus-inspired concepts, we conclude that mimicking viral infection patterns allows for a highly effective targeting concept. Furthermore, successful mesangial cell targeting will allow for sophisticated treatment of mesangial-associated kidney pathologies, as this dramatically increases drug delivery compared to all other approaches currently available.

[0063] The invention will now be further described with reference to the following drawings.

[0064] All methods referred to in the following figure descriptions were performed as described in detail in the Examples. [Brief description of the drawings]

[0065] [Figure 1]Figure 1 shows virus-mimetic attachment and target cell recognition. (A) NPs carrying EXP3174 and Ang-I on their corona (NPEXPAng-I) attach to the cell membrane via EXP3174-mediated AT1R binding. Specific recognition is triggered via enzymatic Ang-I processing and Ang-II-mediated internalization. (B) Flowchart illustrating the triple target cell recognition of decision-making NPs.

[0066] [Diagram 2] Figure 2 shows nanoparticle characterization. (A) Assembly of ligand-modified NPs. (B) Molar ligand content of different NP species normalized to PEG content, (C) size and polydispersity index (PDI) and (D) zeta potential of the resulting NP formulations. Results are presented as mean ± SD of at least n = 3 measurements.

[0067] [Diagram 3] FIG. 3 shows in vitro interactions with AT1R and ACE. Interaction of ligand-modified NPs with their targets AT1R (A-D) and ACE (E-F) determined by intracellular calcium measurements. (A) Ligand affinity and (B) particle avidity for AT1R. (C) IC50 values ​​for free and particle-bound ligand. (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) for free and particle-bound Ang-I calculated based on ligand and NP concentrations. Results are shown as mean ± SD of at least n=3 determinations. The levels of statistical significance are indicated 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.

[0068] [Figure 4]Figure 4 shows the cellular internalization of NPEXPAng-I (red) in target rat mesangial cell (rMC) cells (white) transfected with YFP-tagged AT1R (green) at different incubation times (pAT1R-rMC). Scale bar 20 μm.

[0069] [Diagram 5] Figure 5 shows the uptake specificity of the virus mimetic NPEXPAng-I. (A) Ligand-mediated internalization of NPEXPAng-I, NPAng-I and NPEXP in rMCs inhibited by free EXP3174 and captopril (see also Figures 11 and 12). (B) Uptake of NPEXPAng-I in AT1R and ACE positive rMCs and HK-2 cells and AT1R and ACE negative HeLa cells. Specificity of particle uptake in co-culture of target rMCs with off-target (C) NCI-H295-R or (D) HeLa cells analyzed via flow cytometry. (E) CLMS images of particle uptake (red) in green stained (CTG) rMCs (green) in co-culture 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 the mean ± SD of at least n = 3 determinations. Statistical significance levels are indicated 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.

[0070] [Figure 6] Figure 6 shows NP distribution in mouse kidney. (A) NPEXPAng-I fluorescence located in kidney glomeruli (white arrows). (B) Control non-targeted NPMeO does not accumulate in kidney glomeruli and lacks particle-associated fluorescence. Blue: DAPI staining of cell nuclei; Green: tissue autofluorescence; Red: NP-associated fluorescence. From left to right, boxed regions are shown as enlarged views.

[0071] [Figure 7] FIG. 7 shows the evaluation of NP-associated fluorescence detected in kidney glomeruli analyzed via fluorescence microscopy. (A) Images of kidney glomeruli (dotted circles) of mice treated with different particle formulations. Scale bar 40 μm. See also FIG. 14B. (B) Quantitative analysis of complete glomerular NP-fluorescence. (C) Comparison of particle-associated fluorescence in the outer and inner cortical glomeruli. (D) Glomerular localization of NPEXPAng-I determined via CLSM of MC and integrin-α8 staining (scale bar 20 μm). Results in (C) and (D) are shown as mean ± SD of at least n=120 fluorescence measurements of n=6 mice per NP sample. The level of statistical significance is indicated as ****p≦0.0001. ns: not significant.

[0072] [Figure 8] FIG. 8 shows ligand coupling to PEG-PLA block copolymer. (A) Lys-Ang-I and (B) EXP3174 were coupled to carboxylic acid-terminated or amine-terminated PEG5k-PLA10k using EDC / NHS or DCC / NHS chemistry, respectively. (C) Complete polymer functionalization demonstrated by quantification of molar ligand and PEG content. (D) Absence of unreacted NH2 polymer end groups on EXP3174 modified polymers was determined using flurescamine. Student's t-test was performed using GraphPad Prism 6.0 to assess statistical significance. The level of statistical significance is presented as ****p≦0.0001 comparing MeO- and EXP3174-fluorescence with NH2-terminated PEG5k-PLA10k. For detailed methodology, see Example 1.

[0073] [Figure 9]Figure 9 shows the maximum calcium signal and inhibition by NPEXP. rMCs were stimulated simultaneously with Ang-II and NPEXP, and the resulting intracellular calcium response was immediately measured for 1 min. At the NPEXP concentration used, EXP3174 ligand did not inhibit agonist-induced calcium signals for the duration of 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. For detailed methodology, see Example 1.

[0074] [Figure 10] FIG. 10 shows the uptake of different particle formulations over time in AT1R-positive pAT1R-rMCs analyzed via CLSM. (A) NPEXP is not internalized in the cell line and is mostly located at the cell membrane and in the filipodia between cells that form large clusters over time. Receptor binding is shown by the colocalization of NP-associated and receptor-associated fluorescence. (B) NPAng-I is internalized by cells as shown by their cytoplasmic localization. (C) NPMeO is not associated with cells due to the lack of tethered ligands that allow specific targeting. Cells: white; AT1R-YFP: green; NP formulations: red. Scale bar 20 μm. For detailed methods, see Example 1.

[0075] [Figure 11]FIG. 11 shows that EXP3174 counteracts the reduced uptake due to steric hindrance of the Ang-I ligand by the long polymer chains on NPEXPAng-I. NPAng-I (gray) with 20% Ang-I density was prepared with COOH-PEG5k-PLA10k of varying polymer densities and analyzed for their cellular uptake using flow cytometry. Concomitantly, NPEXPAng-I (yellow) was prepared with EXP3174-PEG5k-PLA10k of varying densities to compare the effect of a second ligand on the steric hindrance of Ang-I. Functionalization of the long polymer chains on NPEXPAng-I with EXP3174 counteracted the reduced uptake due to steric hindrance of the Ang-I ligand when non-functionalized long polymers were added, and significantly increased particle internalization. Results are shown as the mean ± SD of at least n=3 measurements. Two-way ANOVA with Sidak's multiple comparison test was performed using GraphPad Prism 6.0 to evaluate statistical significance. The level of statistical significance is indicated as ****p≦0.0001. For detailed method, see Example 1.

[0076] [Figure 12] Figure 12 shows the specificity of NP uptake analyzed via CLSM. Cells were pre-incubated with free EXP3174 for 30 min before the addition of different NP formulations (NPEXPAng-I, NPAng-I and NPEXP). Inhibition of the target receptor resulted in suppression of particle-associated fluorescence. Scale bar 20 μm. For detailed methodology, see Example 1.

[0077] [Figure 13]FIG. 13 shows the uptake of (A) NPEXP and (B) NPAng-I in co-cultures of target and off-target cells. NPEXP shows accumulation in rMC and NCI-H205R cells, because both of these cells have AT1R. In contrast, co-cultures of rMC and HeLa cells show preferential accumulation of NPEXP in rMC, because HeLa cells express only a small amount of the receptor on the cell membrane (see [3]). NPAng-I shows higher specificity, because they preferentially accumulate in target rMC over off-target cells (HeLa or NCI-H295R cells), which lack ACE, and which have the necessary machinery (ACE and AT1R) for their internalization. Cell nuclei: blue; off-target cells (HeLa or NCI-H295R): white; target cells (rMC): green; NP: red. Scale bar 20 μm. See Example 1 for detailed methods.

[0078] [Figure 14]FIG. 14 shows the in vivo distribution of different NP formulations. (A) Renal distribution of NPAng-I and NPEXP in mouse kidney. NPAng-I shows small NP-associated fluorescence in the majority of glomeruli, in contrast to NPEXP, which did not accumulate in this region. Glomeruli are marked with white arrows for better visualization. From left to right, boxed areas are shown as enlargements. DAPI staining of cell nuclei: blue; tissue autofluorescence: green; NP-associated fluorescence: red. (B) Renal distribution of free dye used to label NPs. CF647 was injected into mice as a control to evaluate its distribution in the kidney. Strong fluorescence could be detected in the tubule region, without fluorescence in the glomeruli (marked with white circles). This demonstrates that the fluorescence seen for the particle-samples comes from the particles themselves and not from leaked dye that is freely filtered due to its low molecular weight. (C) Plasma retention of different NP formulations after 1 hour of circulation in NRMI mice. NP fluorescence in plasma 1 h after injection was measured and normalized to the fluorescence measured 5 min after injection (initial particle blood fluorescence). Non-targeted NPs (NPMeO) show the highest blood circulation time due to the stealth effect conferred by their PEG-shell. Even though 40% of all polymers on the NPEXPAng-I surface are coupled to ligands, which reduces the particle stealth effect, they can be consistent with the blood retention of non-targeted particles. They show significantly higher fluorescence in plasma after 1 h compared to particles functionalized by only one ligand (NPAng-I and NPEXP). NPAng-I, which has a specific two-step virus-mimetic recognition mechanism, also shows significantly better blood retention than NPEXP, which is indicative of commonly targeted NPs. As a control, the free dye (CF647) used to label the particles was further injected into the mice, which subsequently disappears rapidly from the blood circulation to its free filtration (6% of initial fluorescence after 1 h). Results in (C) are shown as the mean ± SD of at least n=6 samples. Student's t-test was performed using GraphPad Prism 6.0 to assess statistical significance.The levels of statistical significance are indicated as *p≦0.05, ***p≦0.001 and ****p≦0.0001. ns: not significant. For detailed methods, see Example 1.

[0079] [Figure 15] Figure 15 shows an exemplary in vivo use of the decision-making nanoparticle of the present invention, which comprises a first ligand for attachment to a target cell and a second ligand for recognition of the nanoparticle / its internalization into said target cell. The nanoparticle of the present invention can be used to target various target tissues, such as the mesangium. The nanoparticle can be customized to target a specific target tissue by selecting the first and second ligands that target target receptors and / or molecules on the surface of the target cell.

[0080] [Figure 16] Figure 16 shows transverse kidney sections from particle-treated animals. A) Viral mimetic particles show high accumulation in the renal corpuscle (circle labels). B) Unmodified nanoparticles without primary and secondary ligands show no significant uptake into glomerular tissue.

[0081] [Figure 17] FIG. 17 shows that adenoviral mimetic NPs enter glomerular mesangial cells via a synergistic combination of passive and active targeting strategies. (a) Upon administration, NPs reach the glomerular region of the renal filtration system via the afferent arteriole, which then branches into the glomerular capillary system. (b) Within the glomerular capillaries, NPs cannot pass through the renal filter due to their reticular structure, but can extravasate through endothelial fenestrations, thereby reaching the mesangium located in the gap. (c) Viral mimetic NPs can then effectively infiltrate mesangial cells via initial binding to the angiotensin II receptor type 1 (AT1r) and subsequent αVβ3 integrin-mediated endocytosis. (AA: afferent arteriole; EA: efferent arteriole; MC: mesangial cell; PO: podocyte; FP: podocyte; ET: endothelium; BM: basement membrane).

[0082] [Figure 18] FIG. 18 shows the characterization of adenovirus-mimetic NPs. (a) Particle design of hetero-multivalent EXPcRGD NPs as well as homo-functionalized or non-functionalized NP species. (b) DLS analysis. All particle types were produced below the size threshold of 60 nm (PDI: polydispersity index) without significant aggregation. (c) Zeta potential measurements. Addition of COOH-PEG2k-PLA10k to the polymer mix results in a negative surface charge. (d) / (e) Quantification of cRGDfK and EXP3174 ligand surface density, respectively, after NP production. The final surface content was directly proportional to the amount of ligand functionalized polymer pre-added for both cRGDfK (R2=0.9957) and EXP3174 (R2=0.9871). Results show the mean ± SD (n=3).

[0083] [Figure 19] Figure 19 shows the AT1r interaction of adenovirus mimetic NPs. (a) Intracellular calcium levels after AT1r stimulation of rMCs treated with free EXP3174 or particle-bound EXP3174. Both EXPcRGD NPs (IC50=276±31pM) and EXP NPs (IC50=552±73pM) effectively bound to AT1r, thereby inhibiting AT1r and resulting in low Ca2+ influx upon AT1r stimulation by angiotensin II (ATII). This effect was even stronger than that for free EXP3174 (IC50=2.66±0.9nM) due to increased avidity from multivalency. (b) AT1r activity for rMCs treated with NPs without AT1r ligand. Neither non-functionalized control NPs nor cRGD NPs were able to significantly bind to AT1r and produced maximal calcium levels upon ATII stimulation, confirming the specificity of the assay (M = molar concentration of either NPs or EXP3174). Results show the mean ± SD (n = 3).

[0084] [Figure 20]Figure 20 shows in vitro internalization of NPs by target mesangial cells. (a) CLSM analysis of rMC stained with CTDR after incubation with 0.05 mg / mL AlexaFluor™ 568-labeled EXPcRGD nanoparticles. NP-associated fluorescence (purple) was detectable in vesicular structures (grey) within the rMC cytosol. With increasing incubation time, endocytic vesicles both grew in size, number and intensity, indicating fusion of vesicles into larger endosomes (scale bar 20 μm). (b) Flow cytometry analysis of NP uptake into rMC. Hetero-multivalent EXPcRGD NPs showed substantially increased cellular uptake compared to control NPs as well as homo-functional NPs. (c) Flow cytometry results after 60 min of NP incubation. EXP cRGD NPs showed the greatest cell association compared to all other NP types, whereas addition of excess free cRGDfK (c = 500 μM) sharply reduced the fluorescent signal to the level of EXP NPs, indicating the αVβ3 dependence of NP uptake. Results show the mean ± SD (n = 3). ◇ P < 0.0001, ○ P < 0.001, **** P < 0.0001 (AFU, arbitrary fluorescence units).

[0085] [Figure 21]FIG. 21 shows TEM analysis of NP interaction with mesangial cells. (a) EXPcRGD NPs accumulated in numerous vesicular structures (black arrows) within rMC. Vesicles of different sizes were present in both the outer and inner parts of the cell cytosol, indicating intracellular processing and fusion into larger endosomes. Furthermore, a significant number of NPs were still located at the cell membrane, suggesting that the particles underwent a stepwise process of initial cell binding and subsequent endocytosis (the image on the right shows a zoomed-in view of the black box on the left). (b) In contrast, EXP NPs accumulated only at the cell border, where they bound to distinct surface structures of rMC cells, indicating possible interaction with membrane-bound AT1r (the image in the top left corner shows a zoomed-in view of the black box). (c) Control NPs showed only negligible interaction with rMC, and gold-enhanced NPs were barely visible.

[0086] [Figure 22]Figure 22 shows the mesangial cell selectivity of EXPcRGD NPs in an in vitro co-culture assay. (a) CLSM analysis of CTG-stained rMCs (green) co-cultured with CTDR-labeled HeLa or NCI-H295R cells (gray) that acted as off-target cells. Cell nuclei were stained with Hoechst 33258 (blue). For rMC / HeLa co-cultures (top row), NP-derived fluorescence (purple) could only be detected within the region of rMCs, indicating selective EXPcRGD NP uptake into target cells. Co-culture of rMCs with AT1r-expressing NCI-H295R cells results in a diverse NP distribution (bottom row). EXPcRGD NPs thereby also bound to the surface of NCI-H295R cells, resulting in a diffuse pattern around the cell borders. However, efficient particle uptake into round endocytic vesicles could only be seen in mesangial cells (scale bar 20 μm). Flow cytometric analysis of rMCs with both HeLa (b) and NCI-H295R (c) supported the CLSM results, since EXPcRGD NPs showed significantly higher cellular association with rMCs in both cases. Interestingly, the addition of excess amounts of free EXP3174 (c=1 mM) in (c) sharply reduced NP interaction with NCI-H295R cells, indicating that EXPcRGD NPs could no longer bind to NCI-H295R cells via AT1r. Results show the mean ± SD (n=3). ****P<0.0001 (ns: not significant. AFU, arbitrary fluorescence units).

[0087] [Diagram 23] Figure 23 shows that EXPcRGD NPs exhibit strong intraglomerular accumulation in vivo. Transverse kidney cryosections were imaged using a fluorescent microscope. To facilitate histological evaluation, cell nuclei were stained with DAPI (blue) and tissue autofluorescence was recorded (green). EXPcRGD NPs (red) were found to accumulate almost exclusively in the glomerular region of the cortex (white circles), while fluorescence in the tubular region was negligible (from upper left to lower right, images show zoomed-in views of the white squares).

[0088] [Figure 24] FIG. 24 shows that EXPcRGD NPs exhibit significantly enhanced accumulation in mesangial cells. (a) Fluorescence microscopy analysis revealed that high fluorescence levels within the glomeruli (white circles) could be detected primarily for EXPcRGD NPs. EXP NPs showed moderate accumulation in the glomeruli, while control NPs and cRGD NPs did not produce any signal to any significant extent (scale bar 20 μm. Calibration bar: values ​​from 0 to 65535 gray). (b) Accurate quantification of intraglomerular fluorescence intensity was achieved by assessing the integrated density per area of ​​the glomerulus for a sufficient number of glomeruli. Thereby, EXPcRGD NPs showed much higher fluorescence intensity per glomerulus compared to all other particle types. Results show the mean ± SD (n=60). ****P<0.0001 (AFU, arbitrary fluorescence unit). (b) Antibody staining for the mesangial surface marker integrin α-8 showed that EXPcRGD NP-associated fluorescence (red) colocalized with areas covered by mesangial cells (yellow), indicating that EXPcRGD NPs were able to selectively infiltrate into mesangial cells via endocytosis. Scale bar 20 μm.

[0089] [Diagram 25]Figure 25 shows the synthesis concept for ligand-functionalized PEG-PLA block copolymers. (a) Hetero-bifunctional PEG polymers ((1)) of varying chain length (2 kDa / 5 kDa) were mixed with 3,6-dimethyl-1,4-dioxane-2,5-dione ((2)) to create NH2-PEG5k-PLA10k as well as COOH-PEG2k-PLA10k ((3)) via ring-opening polymerization. (b) NH2-PEG5k-PLA10k was subsequently covalently coupled to the carboxyl group of EXP3174 ((4)) via DCC / NHS chemistry to give EXP3174-PEG5k-PLA10k ((5)). (c) Furthermore, COOH-PEG2k-PLA10k was coupled to the lysine residue of cRGDfK ((6)) via EDC / NHS chemistry to yield the shorter cRGDfK-PEG2k-PLA10k ((7)). (d) The coupling efficiency for the synthesized EXP3174-PEG5k-PLA10k was determined by comparing the molar concentrations of both PEG and EXP3174. Thereby, the molar concentrations did not vary significantly, indicating successful functionalization. (e) The coupling efficiency for cRGDfK-PEG2k-PLA10k was also within a reasonable range, with only negligible differences in the molar concentrations of PEG and cRGDfK. Results show the mean ± SD (n=3).

[0090] [Figure 26] Figure 26 shows gold-tagged NPs that allow for enhanced TEM visualization. NPs were labeled with gold by covalently attaching very small gold nanoparticles (diameter: 2.2 nm) to the carboxyl groups of PLGA that was then used to manufacture NPs. After incubation of rMC with the labeled NPs, the particle cores were enriched with gold by depositing additional gold particles on the NP cores, thereby increasing the electron density of the sample and allowing visualization in a TEM microscope, where the NPs appeared as dark black spots.

[0091] [Figure 27]FIG. 27 shows (a) flow cytometry results and (b) quantification of αVβ3 expression by different cell types investigated by CLSM. For cytometry analysis, non-specific binding sites were blocked with 2% BSA in DPBS and 105 cells were incubated for 1 hour with AlexaFluor® anti-CD51 / 61 antibody (AlexaFluor® mouse IgG1, kappa isotype control (FC) serving as a non-specific control) at a dilution of 1:20 in 0.1% BSA in DPBS. Cells then underwent multiple steps of DPBS washing and centrifugation. Finally, samples were resuspended in DPBS and analyzed using flow cytometry as previously described (FACS Calibur, excitation: 633 nm, emission: 661 / 16 nm bandpass filter). Fluorescence levels from αVβ3 were thereby greatest for rMC, while both HeLa and NCI-H295R cells showed negligible signals, both of which were significantly lower than for rMC. Results show the 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 (15,000 cells / well) in 8-well Ibidi slides and stained for αVβ3 integrin as described above. Cells were then washed with DPBS, fixed with 4% PFA in DPBS, and analyzed on a Zeiss LSM 710. CLSM images showed strong integrin signal colocalized with rMC cell bodies, indicating substantial αVβ3 expression by mesangial cells. Scale bar 20 μm.

[0092] [Figure 28]FIG. 28 shows the results of fluorescence imaging. A) Relative plasma fluorescence after NP injection. Control NPs showed maximum blood circulation values ​​60 min after injection, with nearly 50% residual plasma fluorescence. In contrast to EXP NPs as well as EXPcRGD NPs, both of which showed acceptable residual concentrations, cRGD NPs were rapidly cleared from the blood. Results show mean ± SD (n=3). ****P<0.0001, ***P<0.001, **P<0.01. (ns: not significant). B) Fluorescence imaging of kidney cryosections after injection of free CF™647 fluorescent dye. Sections were stained with DAPI (blue) to visualize cell nuclei. After injection of a comparable molar concentration of free dye, strong fluorescent signals (red to white) could be detected in the tubular regions of the kidney, indicating free renal filtration of the small molecule dye. As expected, no intraglomerular accumulation was detectable (white circles) (calibration bar: values ​​from 0 to 65535 Gray).

[0093] [Figure 29] Figure 29 illustrates the concept of NP-assisted cinaciguat delivery. Hetero-multivalent EXPcRGD NPs enter target renal mesangial cells via the previously described sequential recognition sequence. After successful endocytosis, NPs undergo endolysosomal degradation, resulting in the release of cinaciguat (small dots). CCG then activates and stabilizes mesangial sGC, leading to enhanced NO-mediated cGMP production. Through cGMP-mediated activation of protein-regulated kinase (PGK1-α), several profibrotic pathways are inhibited, resulting in an overall reduction in profibrotic remodeling of mesangial cells.

[0094] [Diagram 30] Figure 30 shows an exemplary experimental setup. For all experiments, free cinaciguat (c=2 μM) was compared to EXPcRGD NPs with CCG at a concentration of 0.2 μM or without drug. The amount of free cinaciguat was chosen according to previous studies that showed a strong effect in this concentration range.

[0095] [Diagram 31] FIG. 31 shows a Western blot analysis of the effect of cinaciguat on drug targets. a) sGC stimulation and stabilization. Both free CCG and EXPcRGD NPs loaded with CCG lead to a substantial increase in sGC levels during 24 hours of incubation, while control NPs lacking CCG showed no significant effect. b) PGK1-α activation. To assess PGK1-α activity, the phosphorylation of its substrate, vasodilator-stimulated phosphoprotein (VASP), was evaluated. Both free CCG and CCG-bearing EXPcRGD NPs thereby showed a significant increase in the P-VASP / VASP ratio, while control NPs did not produce a detectable change (n=3; ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05).

[0096] [Figure 32-1] FIG. 32 shows the antifibrotic and antiproliferative effects of NPs loaded with cinaciguat. Mesangial cells were preincubated with free CCG or NPs for 4 hours before incubation with 10 ng / mL TGF-β for 48 hours to induce fibrotic and hyperproliferative changes. a) MTT assay shows antiproliferative effect. Mesangial cells showed significant hyperproliferation upon TGF-β stimulation, whereas preincubation with free CCG or CCG-loaded NPs considerably reduced this effect. b) Western blot analysis of both α-SMA and c) collagen I levels showed a dramatic reduction of both fibrosis markers for CCG or CCG-loaded NPs compared to TGF-β control (n=3; ****P<0.0001; ***P<0.001; ns not significant). d) LSM analysis further supported the results from b), showing that mesangial cells showed significantly reduced production of α-SMA upon pre-incubation with CCG or CCG-bearing NPs. Scale bar=2 μm. [Figure 32-2] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0097] Reference is made below to examples, given to illustrate but not to limit the invention. EXAMPLES

[0098] Example 1 material and method cell culture 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) supplemented with 10% FBS and insulin-transferrin-selenium and 100 nM hydrocortisone. HK-2 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 600 μg / ml geneticin (G418). 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 encoding AT1R with a YFP tag (CXN2-HA-AT1R-YFP) (see [4]) using the commercial 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 geneticin (G418). Cell lines were characterized for their target AT1R and ACE expression as previously shown [1, 3].

[0099] mouse Experimental procedures on animals were performed according to national and institutional guidelines and approved by local authorities (Regierung von Unterfranken, reference number: 55.2-2532-2-329). Mice shown in the Key Resources Table were used in this study at 10 weeks of age. Only female mice were used in all experiments. The mice were kept in specific pathogen-free (SPF) housing under standard conditions (relative humidity of 50±5%, temperature of 21±1° C., air exchange >8 AC / h, and a 12 h:12 h (L:D) light period).

[0100] Polymer Preparation: Block Copolymer Synthesis 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 ) was synthesized via ring-opening polymerization of cyclic 3,6-dimethyl-1,4-dioxane-2,5-dione (lactide). Briefly, lactide was recrystallized from anhydrous ethyl acetate and dried under vacuum at room temperature (rt) for 12 h at 40 °C before use. 5k -OH, COOH-PEG 2k -OH, Boc-NH-PEG 5k -OH or MeO-PEG 5k -OH was used as a macroinitiator for ring-opening polymerization. They were dissolved in anhydrous DCM (0.3 mmol) 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 h. The resulting polymer was precipitated in diethyl ether and incubated at 40 °C (COOH-PEG) for 12 h. 2k -PLA 10k , COOH-PEG 5k -PLA 10k and MeO-PEG 5k -PLA 10k ) or 35 °C (Boc-NH-PEG 5k -PLA 10k For cleavage of the Boc protecting group, Boc-NH-PEG-PLA was dissolved in 50% (v / v) TFA / DCM and stirred at rt for 30 min. It was then diluted with 3 volumes of DCM and washed 3 times with 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 10kwas purified via precipitation in diethyl ether followed by drying under vacuum at 35° C. for 12 hours.

[0101] Polymer Preparation: Ligand Coupling For the preparation of the Ang-I modified polymer (see also FIG. 8), 14 μmol of COOH-PEG 5k -PLA 10k EXP3174 was activated with 25 molar excess of EDC and NHS in DMF for 2 h. The reaction was then quenched by adding 863 μmol of 2-mercapthoethanol for 20 min, followed by dropwise addition of 66 μmol of DIPEA and 17 μmol of Lys-Ang-I in DMF. After 48 h, the resulting polymer was diluted in ultrapure water (Millipore) to a DMF concentration below 10% and dialyzed using a 6-8 kDa molecular weight cutoff regenerated cellulose dialysis membrane for 24 h (with medium changes after 30 min, 2 h, and 6 h) to remove unreacted ligand and reagents. For the preparation of EXP3174-modified polymer (see also Figure 8), 96.4 μmol of EXP3174 was activated with equimolar amounts of DCC and NHS in DMF for 2 h. The resulting urea by-product was then removed by centrifugation (5 min, 12,000×g) and subsequent filtration using a 0.2 μm Rotilabo PTFE syringe filter. 5k -PLA 10kand 17.5 molar excess of DIPEA were added to the activated ligand and allowed to react for 20 h. The ligand-modified polymer was purified by precipitation in ice-cold 1:5 (v / v) diethyl ether:methanol and subsequent dialysis against 10% ethanol in 10 mM borate buffer (pH 8.5) for 24 h to remove excess free ligand (with medium changes after 30 min and 6 h), and then dialyzed against ultrapure water to remove buffer salts using 6-8 kDa molecular weight cut-off regenerated cellulose dialysis membranes (Spectrum Laboratories) for 24 h (with medium changes after 30 min, 2 h, and 6 h). The ligand-modified block copolymer was lyophilized for 72 h before ligand coupling confirmation. To that end, the polymer was solubilized in ACN at a concentration of 40 mg / ml and precipitated in stirred ultrapure water to create polymer micelles (final concentration 1 mg / ml). PEG content was quantified using a colorimetric iodine complexation assay and coupled Ang-I was quantified using a Pierce BCA assay kit according to the manufacturer's instructions using a FLUOstar Omega microplate reader. EXP3174 was quantified using a λ 2000 s fluorometric plate reader using an LS-5S fluorescent plate reader. ex = 250 nm and λ em Quantified fluorescently at 370 nm. 5k -PLA 10k The absence of unreacted NH2 end groups on the amine was determined using fluorescamine (see also FIG. 8).

[0102] Polymer preparation: fluorescent labeling of PLGA For in vitro and in vivo particle detection, fluorescently labeled PLGA was used in the particle core. For that purpose, TAMRA-amine (for CLSM) and CF6467-amine (for flow cytometry and in vivo experiments) were covalently coupled to 13.4 kDa carboxylic acid-terminated PLGA. Briefly, 5 μmol of acid-terminated PLGA was dissolved in anhydrous DMF and activated with 129 μmol of DMTMM (25-fold excess) for 2 h at rt. Then, 1 μmol of fluorescent dye was dissolved in DMF, added dropwise to PLGA, and reacted in the dark for 72 h at rt. The reaction product was diluted (DMF<10%) and dialyzed against ultrapure water using a 3.5 kDa molecular weight cutoff regenerated cellulose dialysis membrane (Spectrum Laboratories) for 34 h (with medium changes after 30 min, 2 h, and 6 h) in the dark. The fluorescently labeled PLGA was then freeze-dried for 3 days.

[0103] Preparation and characterization of NPs: Particle preparation For NP preparation, PEG-PLA block copolymer and 13.4 kDa PLGA were mixed in a mass ratio of 70:30 to a final concentration of 10 mg / mL in ACN. For ligand-modified particles, COOH-PEG 2k -PLA 10k and ligand modified polymers were mixed accordingly such that 20% of the polymers making up the NP structure were modified with Ang-I (NPAng-I) or / and EXP3174 (NPEXP and NPEXPAng-I, respectively). NPs were prepared to a final concentration of 1 mg / ml via bulk nanoprecipitation of the polymer mixture in 10% DPBS (v / v) (pH 7.4) with vigorous stirring. The particles were stirred for 2 h to ensure evaporation of the organic solvent and concentrated by ultracentrifugation at 756 g for 20 min using a 30 kDa molecular weight cut-off Microsep advance centrifugation device (Pall Life Sciences).

[0104] Preparation and characterization of NPs: Dynamic light scattering and zeta potential The size and ζ-potential of the resulting particles were determined in 10% PBS at a constant temperature of 25°C using a ZetaSizer Nano ZS (Malvern Instruments) equipped with a 633 He-Ne laser at a backscattering angle of 173° and Malvern Zetasizer software version 7.11, using a concentration of 1 mg / mL or 3.5 mg / ml, respectively. The cuvette position was set at 4.65 mm and the attenuator was automatically optimized by the device. Disposable microcuvette (Brand) and folding capillary cells (Malvern Instruments) were used for size and ζ-potential measurements, respectively.

[0105] Preparation and characterization of NPs: Particle quantification Quantification of particle PEG concentration was performed using a colorimetric iodine complexation assay and correlated with gravimetric NP content determined via lyophilization. Briefly, particle samples were diluted in ultrapure water to PEG concentrations ranging from 5 to 30 μg / mL. Dilutions of MeO-PEG-OH in ultrapure water (0 to 40 μg / mL) were used as standards for the calibration curve. 140 μL of sample or standard was mixed with 60 μL of a 2:1 (v / v) mixture of a 5% (m / v) solution of barium chloride in 1 N HCl and a 0.1 N aqueous iodine solution. Samples and standards were transferred into a 96-well plate and their absorbance at 535 nm was measured using a FUOstar microplate reader (BMG Labtech). Correlation of particle PEG content with exact polymer concentration was determined gravimetrically after sample lyophilization. Particle concentration in molar terms was determined via a colorimetric iodine complexation assay. Particle mass, particle density (1.25 g / cm 3 ), and the hydrodynamic diameter of the NPs obtained via DLS measurements assuming a spherical particle shape. The ligand concentrations on the particle corona were quantified fluorimetrically for Ang-I and EXP3174, respectively, using the BCA assay, as described above.

[0106] Intracellular calcium measurement To assess the AT1R interaction of different NP formulations, ratiometric Fura-2 Ca 2+ The chelator method was used as previously described [1, 3] using AT1R-positive rMCs. For that purpose, rMCs were seeded in T-150 flasks (Corning) and incubated until confluent. 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. The cells were incubated for 1 h under gentle agitation (50 rpm) and light-protected. Afterwards, the cell suspension was washed with DPBS by centrifugation (2×, 200g, 5 min, RT) and resuspended in Leibovitz medium supplemented with 2.5 mM probenecid at a count of 2 million cells / mL. To determine particle avidity and ligand affinity for AT1R (Figure 3A-C), 45 μl of rMCs (90,000 cells / well) loaded with Fura-2 in suspension were incubated with 10 μL of different samples (NPs, or free ligands ranging from 1 nM to 300 μM (ligand concentration)) in a 96-well half-area microplate for 30 min at rt. Afterwards, cells were stimulated with 45 μL of 300 nM Lys-Ang-II aqueous solution and the resulting calcium signal was recorded instantly for 1 min / well using a FLUOstar Omega microplate reader (BMG Labtech) with excitation of 340 / 20 nm and 380 / 20 nm and emission bandpass filters of 510 / 20 nm. To determine the kinetics of AT1R interaction (Figure 3D), the same procedure was used, but samples were incubated with cells for different periods of time (5 to 320 min). Maximum and minimum signal ratios were determined by stimulating cells with 0.1% Triton-X 100 or 0.1% Triton-X 100 combined with 45 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), respectively. Intracellular calcium concentration was determined using a K dValues ​​were calculated following Grynkiewicz's postulate. Statistical significance was assessed via Student's t-test (Figure 3C) and two-way ANOVA with Sidak's multiple comparisons (Figure 3D) using GraphPad Prism 6.0.

[0107] Enzyme kinetic measurements Michaelis-Menten kinetics for NPAng-I and NPEXPAng-I were determined as previously described [1] using rabbit lung ACE (Sigma Aldrich) as a soluble surrogate for the cell membrane-bound enzyme. Briefly, different concentrations of NPs (corresponding to 10-120 μM Ang-I) were incubated with 18 μM enzyme for different periods of time (5, 15, 30, 60, 90 and 120 min) to convert Ang-I on the particle corona to Ang-II, the ligand for AT1R activity. The resulting Ang-II was quantified by direct intracellular calcium measurements. For that purpose, rMCs were loaded with Fura-2 dye as described above. Then, 10 μL of the samples were pipetted onto 96-well half-area plates and 90 μL of the Fura-2-loaded rMC suspension (90,000 cells / well) was injected on top of them. The resulting calcium signal was instantly recorded for 1 min using a FLUOstar Omega microplate reader (BMG, Labtech) as described above. NPEXP was used as a control to exclude interference of EXP3174 ligand on NPEXPAng-I under the experimental conditions used (see Figure 9). rMCs (90,000 cells / well) were stimulated simultaneously with different NPEXP concentrations and Lys-Ang-II (400 nM) and the resulting calcium signal was instantly recorded for 1 min. Measured calcium concentrations were converted to pmol of hydrolyzed product using known concentrations of free Lys-Ang-II ranging from 1 nM to 300 μM. The rate of reaction (pmol / min) over a 15 min incubation time was plotted against the substrate concentrations used in the assay using GraphPad Prism 6.0 to determine the Michaelis-Menten constants (Km) for particle-based and ligand-based concentrations (Figure 3E). To calculate the specificity constant (Kcat / Km) used to compare different substrates for the same enzyme, the catalytic constant (Kcat) was obtained using the same software (Figure 3F). Statistical significance (Figure 3F) was assessed via two-way ANOVA with Tukey's multiple comparison test using GraphPad Prism 6.0.

[0108] Cellular distribution of NPs: confocal microscopy To determine the cellular distribution of the different particle formulations (Figure 4), pAT1R-rMCs were seeded at a density of 10,000 cells / well in 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 pre-warmed NP solutions (0.2mg / ml) in Leibovitz medium (LM) supplemented with 0.1% bovine serum albumin (BSA). Afterwards, the NPs were discarded and the cells were washed extensively with DPBS, followed by cell staining with 1x CellMask™ Deep Red Plasma Membrane Stain for 5 minutes and fixing with 4% paraformaldehyde (PFA) in DPBS for 10 minutes at rt. Images were acquired using a Zeiss LSM 700 microscope with the focal plane set at 1.4 μm using Zen software (Carl Zeiss Microscopy). For inhibition of particle uptake and binding, cells were preincubated with 1 mM free EXP3174 or captopril prior to particle addition. Images were analyzed using Fiji software.

[0109] Cellular distribution of NPs: flow cytometry Analysis of particle uptake via flow cytometry (Figure 5A) was performed as previously described [1]. Briefly, rMCs were seeded at a density of 30,000 cells / well in 24-well plates and incubated (37°C) for 48 h. Pre-warmed NP solution (0.7 mg / ml in LM supplemented with 0.1% BSA) was pipetted on top of the cells, which were washed with DPBS and then incubated at 37°C for 45 min. To confirm uptake specificity, cells were incubated with 1 mM captopril and / or EXP3174 for 30 min prior to particle addition. Afterwards, the particle solution was discarded and cells were washed extensively with DPBS, trypsinized and centrifuged (2x, 200g for 5 min, 4°C). NP-associated cellular fluorescence was analyzed in DPBS using a FACS Calibur cytometer (Becton Dickinson). Fluorescence was recorded using excitation at 633 nm and a 661 / 16 nm bandpass filter. The population of viable cells was gated using Flowing software 2.5.1. (Turku Centre for Biotechnology) and the geometric mean of NP-associated fluorescence was analyzed. Statistical significance (Figure 5A) was assessed via Student's t-test using GraphPad Prism 6.0.

[0110] NP target cell specificity: flow cytometry To evaluate NP uptake in different cell lines (Figure 5B), rMC, HK-2 and HeLa cells were seeded at a density of 30,000, 50,000 or 100,000 cells / well, respectively, in 24-well plates and incubated (37 °C) for 48 h. Then, pre-warmed NP solution (0.7 mg / ml in LM supplemented with 0.1% BSA) was added on top of the cells and treated as described above. Statistical significance (Figure 5B) was assessed via two-way ANOVA with Sidak's multiple comparison test using GraphPad Prism 6.0.

[0111] Particle specificity in co-cultures of target and off-target cells was investigated via flow cytometry (Figure 5C-D) as previously described [1]. Briefly, rMCs stained with CTG (10 μM in serum-free medium, 30 min, 37 °C) were seeded in co-culture with unstained off-target NCI-H295R or HeLa cells at a density of 10,000 and 75,000 cells / well, respectively, in 24-well plates and incubated for 48 h. Warm NP solution at a concentration of 0.02 mg / ml in LM supplemented with 0.1% BSA was subsequently added on top of the cells and incubated for 45 min at 37 °C. Afterwards, the particles were discarded and the cells were processed for flow cytometry analysis, as described above. Statistical significance was assessed via Student's t-test using GraphPad Prism 6.0.

[0112] NP target cell specificity: confocal microscopy To confirm the flow cytometry experiments, CLSM analysis of particle specificity in co-cultures (Figure 5E and Figure 13) was performed as previously described [1]. Briefly, target CTG-stained rMCs (10 μM, 30 min, 37°C) were seeded in co-culture with off-target CTDR-stained HeLa or NCI-H295R cells (25 μM, 30 min, 37°C) at a density of 2,000 and 10,000 cells / well, respectively, and incubated for 24 h. Cell nuclei were then stained with Hoechst 33258 (5 μg / ml in DPBS) for 20 min, and a pre-warmed 0.02 mg / mL NP solution in LM supplemented with 0.1% BSA was pipetted on top of the cells and incubated for 45 min. The NP solution was then discarded, and the cells were washed extensively with DPBS and fixed with 4% PFA in DPBS for 10 min (rt). Images were acquired and analyzed using a Zeiss LSM 700 microscope and Fiji software, respectively, as described above.

[0113] NP renal distribution in vivo To evaluate the renal distribution of the different NP formulations (NPEXPAng-I, NPAng-I, NPEXP, and NPMeO), 100 μL of 120 nM NP solution (equivalent to approximately 10 mg / ml NP) was injected via the jugular vein in 10-week-old female NMRI mice (Charles River) anesthetized with isoflurane inhalation and buprenorphine (0.1 mg / kg body weight) (n=6 for each particle sample). In addition, as a control, 100 μL of free dye (CF647) used to fluorescently label the particles was injected at the same concentration (approximately 50 μM) included in the particle sample. After 5 minutes, blood samples were collected via iv puncture while the mice were still under anesthesia. After 1 hour of particle circulation, the mice were anesthetized with ketamine / xylazine, a final blood sample was collected, and sacrificed via perfusion fixation with 4% PFA. The kidneys were harvested and sectioned transversely. They were cryoprotected by placing them overnight in phosphate buffer (0.1M pH 7.4) supplemented with 18% sucrose and 1% PFA. They were then frozen (-40°C) in liquid nitrogen-cooled 2-propanol and embedded in Tissue Tek® OCT™ Compound for cryosectioning. Kidneys were cut into 5 μm sections using a CryoStar NX70 cryostat (Thermo Fisher Scientific) and transferred onto Superfrost™ Plus glass slides. For better visualization, cell nuclei were stained with DAPI (12.5 μg / ml in DPBS) before section imaging using an Axiovert 200M (Zeiss) fluorescent microscope and Zen software (Zeiss). Images of whole kidneys were acquired using a 10× objective (Figure 6). For glomerular fluorescence quantification, images were acquired using a 40x objective (average of 120 glomeruli per sample) and analyzed using Fiji software (Schneider et al., 2012). For better visualization, a look-up table "Red Hot" was applied to the particle-associated fluorescence. The area of ​​each glomerulus was quantified and the fluorescent area was gated. The integrated fluorescence density of each gated area was then quantified and correlated with the total glomerular area.Statistical significance was assessed via Student's t-test using GraphPad Prism 6.0. To compare particle-associated fluorescence of the inner and outer cortex, the cortex was divided into two equal sections and glomerular fluorescence was analyzed as described above. To assess statistical significance, a two-way ANOVA with Sidak's multiple comparison test was performed using GraphPad Prism 6.0.

[0114] NP-associated fluorescence in plasma was measured using a FLUOstar Omega microplate reader (BMG Labtech) with excitation and emission wavelengths of 640 nm and 680 nm, respectively. Fluorescence 1 h after injection was correlated with the initial fluorescence of a sample obtained 5 min after injection.

[0115] immunohistochemistry To assess glomerular localization of NPs, freshly cut 5 μm kidney cryosections were washed for 5 min in DPBS, washed for 5 min in DPBS supplemented with 0.1% sodium dodecyl sulfate (SDS), washed for 5 min in DPBS, and then blocked for 10 min in 5% BSA in DPBS supplemented with 0.04% Triton-X (m / v). Sections were washed again in DPBS (5 min) and incubated overnight at 4 °C in a 1:200 solution of primary polyclonal goat anti-integrin-α8 antibody in DPBS supplemented with 0.5% BSA and 0.004% Triton-X (m / v). They were then washed for 5 min in DPBS and incubated for 1 h at rt in the dark 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. Cryosections were washed with DPBS and ultrapure water, after which they were mounted using Dako Faramount mounting medium and analyzed using a Zeiss LSM 700 microscope and Fiji software as described above.

[0116] Quantification and statistical analysis Statistical analysis was performed using GraphPad Prism software 6.0. Student's t-test or two-way ANOVA with Sidak or Tukey's multiple comparison test was used to assess statistical significance as detailed in the methods. The level of statistical significance and the "n" number for each experiment are indicated in the text and figure legends.

[0117] Example 2 Block copolymers enable virus-mimetic particle design All materials and methods referred to in this example were as described in the previous examples.

[0118] For the development of virus-mimetic NPs, we coupled the ligands EXP3174 and Ang-I to poly(ethylene glycol) 5k-poly(lactic acid) 10k (PEG-PLA) block copolymers (Figure 8), which were blended with poly(lactic-co-glycolic acid) PLGA for NP fabrication via bulk nanoprecipitation, which gave the particles sufficient stability in vivo. The remaining non-functionalized polymer was PEG-PLA terminated with carboxylic acid (COOH-PEG2k-PLA10k) with shorter 2k PEG and 10k PLA blocks (Figure 1A). By modifying the polymer with ligands prior to NP preparation, precise control of the ligand density can be obtained. Particles were prepared such that 20% of their PEG chains were modified with Ang-I and another 20% with EXP3174 (NPEXPAng-I) (Figure 2B). The ligand density was maintained at a maximum of 40% to avoid steric hindrance and non-specific interactions between the ligands. As controls, ligand-free methoxy-PEG-terminated particles (NPMeO) and particles with 20% of either Ang-I or EXP (NPAng-I and NPEXP, respectively) were assembled (Figure 1A). By combining long ligand-bearing polymers with shorter unfunctionalized polymers for particle preparation, the size of the NPs could be maintained below 80 nm, providing the particles with the ability to pass through endothelial fenestrations of mesangial capillaries (Figure 2C). Carboxylic acid-terminated block copolymers were selected as fillers providing an overall negative particle charge ideal for avoiding nonspecific electrostatic adsorption to negative cell membranes (Figure 2D).

[0119] Example 3 NPs recognize target receptors in vitro All materials and methods referred to in this example were as described in the previous examples.

[0120] To confirm the ability of particles to triple check, cellular identity was first assessed in vitro. Particle avidity for target receptors mediating primary attachment and subsequent internalization was investigated using a calcium mobilization assay, which demonstrates that stimulation or silencing of Gq-coupled AT1R by agonists or antagonists, respectively, increases cytosolic Ca 2+ AT1R-positive rat mesangial cells (rMCs) were incubated with varying concentrations of either the free ligand or the NP formulations for a period of 30 min before stimulation with Ang-II, and the resulting calcium signals were recorded. As shown in Figure 3A, control experiments with free EXP3174 and Ang-II revealed high affinity of both compounds for AT1R in the nanomolar range (IC50 values ​​of 0.6 ± 0.4 and 1.5 ± 0.1 nM, respectively). Ang-I shows a lower affinity (IC50 0.9 ± 0.6 μM), since receptor binding and activation occurs only after enzymatic conversion to Ang-II by ACE present in the cell membrane.

[0121] Coupling to the linker results in an affinity loss that is compensated by simultaneous high avidity multivalent binding of several receptors (Figures 3B and 3C). Since their main interaction is with ACE, particles with Ang-I only (NPAng-I) show lower avidity for AT1R (IC50 of 9.4 ± 0.4 nM) than particles with EXP3174 (IC50 of 0.4 ± 0.1 nM). Nevertheless, particle binding of Ang-I results in a significant decrease in IC50 values ​​compared to the free ligand, due to the promotion of enzymatic cleavage at the NP interface and subsequent multivalent binding. NPs modified with EXP3174, in contrast, had an avidity of the same order of magnitude as for the free ligand. Surprisingly, particles NPEXPAng-I with both ligands showed a synergistic effect on receptor binding, since they had a significantly higher avidity (IC50 of 0.2±0.09 nM) for AT1R than either of the particles with only one type of ligand (Figure 3C). This proves that these ligands do not interfere with each other's interaction, since after enzymatic activation of Ang-I to Ang-II, both ligands target the same receptor in a simultaneous agonistic and antagonistic manner. Particles without any functionalization (NPMeO) did not evoke any response, confirming that the assay was ligand-specific (Figure 3B).

[0122] To evaluate the kinetics of cell / particle interaction, intracellular calcium measurements were performed over a 5.5-h period by incubating NPs with rMC at a concentration corresponding to 10 μM of ligand. The extent to which they could silence calcium signaling induced by the free agonist served as a measure for the completeness with which each particle bound to AT1R in the cell surface via their ligand at different time points (Figure 3D). Particles with only Ang-I on their surface showed slow receptor binding, since they needed to be first activated by cell membrane-bound ACE into Ang-II-bearing particles before they could interact with AT1R. Receptor binding reached a maximum at about 40% after 1 h of incubation and remained constant over the duration of the assay. This points to a rapid internalization of the particles when a certain number of proligands are activated, and perhaps not all Ang-I is converted to Ang-II. Once Ang-II on the particle surface binds to the receptor, the particles are rapidly internalized (as they have picomolar AT1R avidity [1]), which means that it may not be necessary for all proligands to be activated for NP internalization to occur. This phenomenon is avoided when EXP3174 is added as an attachment factor on the particle surface. Very fast and complete receptor blockade occurs after only 5 minutes of particle incubation (similar for NPEXPAng-I and NPEXP). AT1R inhibition is maintained for almost the entire measurement, dropping to about 80% at the last time point, likely due to receptor upregulation and recycling. Attachment to the cell membrane by EXP3174 slows down the recognition process, allowing for higher activation of Ang-I to Ang-II, which can bind AT1R more efficiently. Comparing NPEXPAng-I and NPEXP, there is a significantly higher initial AT1R inhibition of NPEXPAng-I that becomes constant after 45 minutes of particle incubation. This is likely due to the combinatorial effect of the two ligands resulting in higher avidity for AT1R (Figure 3C).

[0123] A prerequisite for particle internalization is the ability of ACE to activate Ang-I to Ang-II. Therefore, we investigated the enzyme kinetics for NPEXPAng-I to determine whether the presence of an antagonist on the particle surface interferes with the enzymatic reaction. A soluble form of ACE was incubated with different particle concentrations for varying periods of time, and the resulting Ang-II on the NP corona was quantified by performing a calcium mobilization assay. The interference of the EXP3174 ligand in the assay was evaluated by measuring the signal inhibition exhibited by NPEXP (Figure 9). The Michaelis-Menten constants (Km) determined for both NPAng-I and NPEXPAng-I (Figure 3E) yielded values ​​of the same order of magnitude for both particle formulations as for the free ligand (see [1]). Furthermore, we determined the catalytic constant (Kcat) to calculate the specificity constant (Kcat / Km), a useful index for comparing the affinity of different substrates for the same enzyme (Figure 3F). The enzymatic activation of Ang-I on NPEXPAng-I corona was not significantly different from that on NPAng-I, indicating that ACE is not sterically hindered by the additional ligand EXP3174. Moreover, the Kcat / Km values ​​calculated based on the ligand concentration were equal for free Ang-I and particle-bound Ang-I. Furthermore, when Kcat / Km is calculated based on the NP concentration, the bound ligand is a significantly better substrate for the enzyme, which is a result of the binding of several ligand molecules on the particle surface to several enzyme molecules (Figure 3F).

[0124] Example 4 Decision-making NPs are target cell specific All materials and methods referred to in this example were as described in the previous examples.

[0125] After particle interaction with their respective targets was successfully established, the next step was to determine whether NPs with antagonists as well as agonists on their corona still induce internalization by their target cells, and if so, whether uptake follows from specific ligand-receptor interactions. Since antagonists do not trigger AT1R-mediated endocytosis, whereas agonists do, we investigated the cellular localization of NPEXPAng-I in rMCs expressing YFP-tagged AT1R (pAT1R-rMCs) via confocal laser scanning microscopy (CLSM). As shown in Figure 4, NPEXPAng-I-associated fluorescence was found inside the cells. This increased with longer incubation times and was strongly colocalized with AT1R fluorescence.

[0126] Thus, there is specific particle uptake mediated by AT1R. However, particles with only antagonist (NPEXP) are not internalized by cells and are mostly located on the cell surface (Figure 10A). Particle fluorescence also colocalized with receptor fluorescence, demonstrating receptor-mediated attachment. Since NPAng-I was also internalized by cells (Figure 10B), enzymatically created Ang-II mediates cellular uptake of NPEXPAng-I. Unexpectedly, with increasing incubation time, there was a reorganization of receptors on the cell membrane from a more diffuse and uniform cell membrane distribution (after 15 min) to a more concentrated clustering (at 90 min) (Figure 4), which strongly colocalized with NP fluorescence. This is further evidence that uptake is mediated by AT1R, since activation of receptors that are internalized via clathrin-coated pits, e.g., GPCRs such as AT1R, promotes receptor clustering.

[0127] For NPEXP, receptor rearrangement on the cell membrane also occurs, which is the result of multivalent receptor binding promoted by receptor movement on the cell surface. When NPEXP attaches to receptors on the cell membrane, their lack of internalization can lead to receptor-particle mobility on the cell membrane and even receptor binding. Particles without ligand (NPMeO) are not taken up by cells (Figure 10C), confirming that a specific targeting mechanism is essential to mediate high cellular internalization.

[0128] Overall, we demonstrate that the presence of attachment-mediated antagonistic ligands linked to the particle corona does not interfere with subsequent particle internalization. Moreover, the inclusion of additional ligands on the particle surface compensated for the loss of targeting due to steric hindrance of the Ang-I ligand by the addition of more numerous and longer polymer chains (Figure 11). To further confirm particle specificity and ligand-mediated internalization, cells were preincubated with free EXP3174 or captopril, an ACE inhibitor that results in suppression of particle-associated fluorescence, for 30 min prior to particle addition and analyzed by flow cytometry (Figure 5A) and CLSM (Figure 12).

[0129] Furthermore, we tested particle internalization in different cell lines by flow cytometry (Figure 5B). HeLa cells, which do not express ACE but only trace levels of AT1R, showed low particle uptake, which was non-specific since it could not be suppressed by either captopril or EXP3174. In contrast, rMC and HK-2 cells, which express both targets, were able to take up particles, as shown by much higher particle-associated cellular fluorescence. Internalization was also mediated by activated proligand binding to AT1R, since pre-incubation of cells with captopril or EXP3174 significantly suppressed cellular fluorescence. Thus, particles show high specificity for their target cells. Nevertheless, when NPs enter the body, they are simultaneously presented with target and off-target cells. Therefore, we investigated whether NPEXPAng-I could distinguish between them.

[0130] Target cells (rMCs) were seeded with excess amounts of off-target NCI-H295R or HeLa cells, both of which lack ACE and express high and low AT1R levels, respectively. These were incubated with the different NP formulations, and each cell line was examined for particle-associated fluorescence via flow cytometry (Figure 5C-D). NPEXPAng-I accumulated significantly more in target rMCs, demonstrating exceptional target cell specificity. NPAng-I also showed low accumulation in both off-target cells, so specificity is conferred by Ang-I. In contrast, NPEXP bound to the cell surface in rMCs to the same extent as NCI-H295R cells, which express high AT1R levels, demonstrating that a simple one-step recognition process is not sufficient to confer particle selectivity. CLSM images confirmed the flow cytometry findings (Figs. 5E and 13), where NPEXPAng-I- and NPAng-I fluorescence (red) was mostly associated with target rMC (green) but not with off-target HeLa or NCI-H295R cells (white), whereas NPEXP fluorescence was found in both rMC and AT1R-expressing NCI-H295R cells. Taken together, these results demonstrate that NPEXPAng-I uptake is receptor-mediated and that initial cell attachment via the EXP3174 ligand does not reduce particle specificity for target cells conferred by the virus-mimetic recognition process.

[0131] Example 5 NPs target MCs in vivo All materials and methods referred to in this example were as described in the previous examples.

[0132] The complementary targeting ability and particle specificity of both ligands on NPEXPAng-I were confirmed in Having been demonstrated in vitro, the next step was to determine whether the virus recognition principle would result in higher in vivo MC accumulation. To that end, targeted particle formulations (NPEXPAng-I, NPAng-I and NPEXP) (Fig. 1A) as well as non-targeted particle formulations (NPMeO) were injected into NRMI mice and kidney cryosections were examined for particle-associated fluorescence (Figs. 6 and 14A). As shown in Fig. 6A, NPEXPAng-I fluorescence could be found uniformly throughout all glomeruli in kidney sections, with no fluorescence found in other kidney structures such as tubules. In contrast, for non-targeted NPMeO, NP fluorescence was barely detectable in kidney sections (Fig. 6B). This demonstrates that simple size-dependent targeting is not sufficient to achieve particle accumulation in MCs, as NPMeO is likely cleared from the mesangium due to lack of specific cellular interactions. Moreover, NPEXP, a targeted NP but unable to mediate cellular internalization, also showed negligible glomerular fluorescence (Figure S14A), demonstrating that particle uptake is critical for achieving high MC accumulation. Moreover, NPEXPAng-I achieved a much stronger and more uniform glomerular distribution than NPAng-I, which lacks attachment factors (Figure S14A), indicating that the principle of enhanced target cell recognition in vivo is highly advantageous.

[0133] To quantitatively assess NP-associated fluorescence and better discern differences between different particle formulations, images of glomeruli were acquired at higher magnification (Figure 7A). Quantitative analysis of glomerular fluorescence yielded 15-fold higher fluorescence for virus-mimetic particles with enhanced recognition mechanisms (NPEXPAng-I) compared to non-targeted control particles (NPMeO), which showed only small fluorescent spots in some glomeruli.

[0134] Moreover, NPEXPAng-I showed significantly higher accumulation than one-ligand targeted particles (7-fold and 5-fold higher than NPEXP and NPAng-I, respectively) (Figure 7B). That the detected fluorescence was particle-associated was confirmed by the kidney distribution of the free dye (CF647) used for particle labeling, which showed strong tubular but no glomerular fluorescence due to its small size that can be freely filtered (Figure 14B). To evaluate NP glomerular distribution, the fluorescence of glomeruli in the outer and inner cortex was compared (Figure 7C). For all particle formulations, there was no significant difference between the two populations. This indicates that the particles are distributed homogeneously in the glomeruli throughout the kidney cortex, which is an essential prerequisite for the treatment of glomerular-associated diseases. Finally, since apart from MCs, there were other cells in the glomerulus that could have internalized NPs, a specific antibody staining of MCs using integrin-α8 as a marker was performed to elucidate that the particles accumulated in MCs. As shown in Figure 7D, NPEXPAng-I fluorescence was localized inside antibody-stained MCs, confirming that the particles could not only reach the glomerular mesangium but also be taken up by MCs.

[0135] Taken together, the results clearly show that size-mediated targeting is a prerequisite to reach the mesangium but is insufficient to achieve particle accumulation in MCs. NP internalization appears essential to avoid mesangial clearance, which explains why particles lacking this attribute (NPMeO and NPEXP) result in the lowest glomerular fluorescence. Implementation of the virus-mimetic recognition principle (NPAng-I) increases NP specificity, resulting in particle uptake that then leads to higher MC accumulation. However, promoting target cell recognition via initial virus-like cell attachment (NPEXPAng-I) significantly enhances the targeting potential of NPs, which is the result of the combinatorial effect of the two ligands, as shown by in vitro studies.

[0136] Moreover, enhanced functionalization of NPEXPAng-I does not result in a decrease in particle blood retention. Generally, NPs are coated with polymers such as PEG, which increase their circulation time and reduce plasma protein adsorption. A positive effect that is usually countered by ligand functionalization, since off-target cells expressing targeted receptors can bind and interfere with NPs. Nevertheless, quantification of plasma NP fluorescence 1 h after injection showed that NPEXPAng-I remained in the circulation to the same extent as non-targeted NPMeO and significantly longer than other targeted formulations (Figure 14C). This is likely due to higher particle specificity resulting from a more complex cell recognition process. Overall, these results demonstrate that by closely mimicking viral binding and internalization and combining it with optimal NP size, it is possible to develop NPs that target and accumulate in large amounts in MCs.

[0137] Example 6 material and method material Heterobifunctional hydroxyl poly(ethylene glycol) carboxylates (COOH-PEG) with molecular masses of 2000 and 5000 g / mol were synthesized. 2k / 5k -OH) and hydroxyl poly(ethylene glycol) Boc-amine (Boc-NH-PEG) with a molecular mass of 2000 g / mol 2k Methoxypoly(ethylene glycol) (MeO-PEG) with a molecular mass of 5000 g / mol was purchased from Jenkem Technology USA Inc. (Allen, TX, USA). 5k-OH) and Resomer RG 502 (PLGA) were obtained from Sigma-Aldrich (Taufkirchen, Germany). EXP3174 (also known as Losartan Carboxylic Acid) was purchased from Santa Cruz (Heidelberg, Germany), whereas cyclic RGDfK (cRGDfK) was obtained from Synpeptide Co. Ltd. (Shanghai, China). AlexaFluor™ 568 Hydrazide (Alexa568), CellTracker™ Green Dye (CTG) and CellTracker™ Deep Red Dye (CTDR) were purchased from Thermo Fisher Scientific (Schwerte, Germany). Amine-functionalized spherical gold NPs (Au) with an average diameter of 2.2 nm were obtained from Synpeptide Co. Ltd. (Shanghai, China). 2.2 -NH2) was obtained from Nanopartz Inc. (Loveland, CO, USA). GoldEnhance™ EM Plus kit was purchased from Nanoprobes (Yaphank, NY, USA). Goat-derived integrin α-8 antibody was obtained from R&D Systems (Minneapolis, MN, USA). All other chemicals were purchased at analytical grade from Sigma-Aldrich unless stated differently. Ultrapure water was obtained from a Milli-Q water purification system (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 RPMI1640 medium containing 10% fetal bovine serum, insulin-transferrin-selenium (ITS) (1×) and 100 nM hydrocortisone.

[0138] Polymer synthesis COOH-PEG 2k -PLA 10k , Boc-NH-PEG 5k -PLA 10k and MeO-PEG 5k -PLA 10kBlock copolymers were synthesized via ring-opening polymerization as previously described. Briefly, heterobifunctional PEG polymers (1 equivalent = equiv) were mixed with 3,6-dimethyl-1,4-dioxane-2,5-dione (70 equivalents) and 1,8-diazabicylo[5.4.0] undec-7-ene (3 equivalents). The polymer mixture was stirred at room temperature (RT) for 1 h until the polymerization was quenched with benzoic acid (14 equivalents). The resulting block copolymers were precipitated in diethyl ether, isolated via filtration, and dried under vacuum. The molecular weights of the synthesized polymers were determined in deuterated chloroform at 295 K using a Bruker Avance 300 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany).

[0139] cRGDfK-PEG 2k For the preparation of the -PLA polymer, the previously synthesized COOH-PEG 2k -PLA 10k was covalently coupled to the lysine residues of cRGDfK as previously shown. 2k -PLA 10k (1 equiv.) was activated using 3-(ethyliminomethyleneamino)-N,N-dimethylpropan-1-amine (EDC) / N-hydroxysuccinimide (NHS) (25 equiv.) for 2 h at RT and then quenched with β-mercaptoethanol (BME) (30 equiv.). The activated polymer was reacted with cRGDfK (3 equiv.) and N,N-diisopropylethylamine (DIPEA) (10 equiv.) for 24 h at RT. After precipitation of the resulting cRGDfK-coupled polymer in diethyl ether / methanol (15:1 V / V), the free cRGDfK and excess reactants were removed using dialysis against millipore water (mpH2O).

[0140] EXP3174-PEG 5k -PLA 10kFor Boc-NH-PEG 5k -PLA 10k The Boc protecting group of was first cleaved. Briefly, the Boc-protected polymer was dissolved in dichloromethane (DCM) / trifluoroacetic acid (TFA) (1:1 V / V). After stirring for 30 min, the excess TFA was neutralized using saturated sodium bicarbonate solution. The organic phase was washed with mH2O, and then the polymer was isolated as described above.

[0141] The obtained NH2-PEG 5k -PLA 10k was coupled to EXP3174 via the carbonyl residue of the imidazole moiety. EXP3174 (3.5 equiv.) was activated with N,N'-dicyclohexylcarbodiimide (DCC) / NHS (3.3 equiv.) for 2 h at RT. After removal of the resulting dicyclohexylurea via centrifugation, NH 2- PEG 5k -PLA 10k (1 eq.) and DIPEA (17.5 eq.) were added and reacted at RT for 24 h. 5k -PLA 10k was precipitated in methanol / diethyl ether (1:5 V / V) and the product was dialyzed against ethanol / 100 mM borate buffer pH 8.5 / water (1 / 1 / 8 V / V) for 24 h, followed by dialysis against mHO for 12 h to remove unreacted EXP3174 and excess reactants.

[0142] PLGA labeling with fluorescent dyes For particle visualization, core-forming PLGA was covalently linked to a fluorescent dye before NP preparation. To that end, carboxylic acid-terminated PLGA was activated for 2 h using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as a catalytic agent. The activated PLGA was then reacted with either AlexaFluor™ 568 Hydrazide or CF™ 647 Amine at RT for 24 h. The labeled PLGA was dialyzed against mHO for 24 h to remove unreacted fluorescent dye.

[0143] PLGA labeling with nanogold For electron microscopy analysis, PLGA was conjugated to nanogold. PLGA was first activated with EDC and NHS in DCM for 2 h. After removal of DCM under reduced pressure, the activated PLGA was dissolved in DMSO and conjugated with DIPEA and freeze-dried monoamino gold nanoparticles (Au) with an average diameter of 2.2 nm. 2.2 After stirring at RT for 24 h, the gold-conjugated PLGA was precipitated in mHO, isolated via centrifugation at 2500 g for 10 min, and lyophilized.

[0144] NP preparation Block copolymer nanoparticles were fabricated using a common solvent evaporation technique. The corresponding amounts of PEG-PLA polymer and PLGA were mixed in a ratio of 70 / 30 (m / m) and diluted in acetonitrile (ACN) to a final concentration of 10 mg / mL. To reach the desired ligand surface density for hetero- / homo-functional NP species, cRGDfK-PEG 2k -PLA and / or EXP3174-PEG 5k -PLA 10k , according to the calibration shown in Figure 18d / e, 2k -PLA 10k The organic phase was then added dropwise to vigorously stirred 10% Dulbecco's Phosphate Buffered Saline (DPBS) (7.5 mM, pH 7.4) and stirred at RT for 3 h to remove the organic solvent.

[0145] The resulting NP dispersion was concentrated using Pall Microsep filters (molecular weight cut-off 30 kDa; Pall Corporation, NY, USA) via centrifugation at 1250 g for 25 min. To obtain the mass concentration of the produced NPs, the PEG content was assessed using a colorimetric iodine complexation assay. The NPs were then freeze-dried and gravimetrically analyzed to obtain the ratio of PEG content and NP weight. In the following experiments, this ratio was used to calculate the mass concentration from the estimated PEG content for each NP species.

[0146] NP characterization NP size and ζ-potential were assessed using a Malvern Zetasizer Nano ZS (Malvern, Herrenberg, Germany). Samples were measured (25 °C, RT) in 7.5 mM DPBS with a 633 nm He-Ne laser at an angle of 173° using either PMAA semi-microcuvette (DLS; Brand, Wertheim, Germany) or folded capillary cells (ζ-potential; Malvern, Herrenberg, Germany).

[0147] cRGDfK quantification The level of cRGDfK on the NP surface was evaluated based on the measurements of arginine. Briefly, 50 μL of NP sample (1 mg / mL) was mixed with 175 μL of working solution consisting of 9,10-phenanthrenequinone (150 μM in ethanol) and 2 N NaOH (6:1 V / V). After 3 h of incubation at 60 °C, 1 equivalent of sample was mixed with 1 equivalent of 1 N HCl and incubated for another hour at RT. Finally, fluorescence was measured with a Synergy™ Neo2 Multi-Mode Microplate Reader (BioTek Instrument Inc., Winooski, VT, USA) using an excitation wavelength of 312 / 7 nm and an emission wavelength of 395 / 7 nm. Dilutions of cRGDfK (0-40 μg / mL) served as calibrations. The molar cRGDfK concentration and the ratio of molar cRGDfK content to molar PEG content were determined and plotted against the theoretical values ​​(Figure 18d).

[0148] EXP3174 Quantification To determine the surface level of EXP3174 on the produced particles, 1 equivalent of NP sample (1 mg / mL) was mixed with 10 equivalents of 0.2 M acetic acid. Dilutions of EXP3174 (0-30 μM) in 0.2 M acetic acid served as calibration. Fluorescence of samples and standards was measured in a Synergy™ Neo2 Multi-Mode Microplate Reader (see above) (excitation 250 / 10 nm, emission 370 / 5 nm). EXP3174 molar concentration and the ratio of molar EXP3174 content to molar PEG content were determined and plotted against theoretical values ​​(Figure 18e).

[0149] Calcium mobilization assay To investigate the AT1r binding of NPs, we measured intracellular calcium levels by incubating fura-2 with Ca 2+The NPs were used as chelators for measurement. Briefly, rMCs were incubated with 5 μM fura-2AM, 2.5 mM probenecid and 0.05% Pluronics F-127 in Leibovitz's L-15 medium for 1 h at RT. Cells were then centrifuged (5 min, 200 g, RT) and resuspended in Leibovitz's medium. 45 μL of NPs or free EXP3174 at different concentrations were pipetted into a 96-well plate (Greiner Bio One, Frickenhausen, Germany) followed by 45 μL of rMC suspension (2 × 10 6 10 μL of 30 nM AT II was added to each well to activate uninhibited AT1r and induce Ca in the cell cytosol. 2+ Ca influx was induced as a result. Fluorescence signals during the first 30 seconds after injection were measured 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, respectively. 2+ Fura-2 bound to Ca 2+ The maximum ratio of Fura-2 to free Fura-2 was assessed by incubating the loaded cells with 0.1% Triton-X 100 and measuring the fluorescence levels as described above. Similarly, the minimum ratio was achieved by incubation with 0.1% Triton-X 100 combined with 45 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The intracellular calcium levels per sample were calculated using the equation of Grynkiewicz et al. The half maximal inhibitory concentration (IC 50 ) was calculated using GraphPad Prism (San Diego, CA, USA) applying a sigmoidal dose-response equation (variable slope).

[0150] CLSM analysis For detailed analysis of NP-cell interactions, rMCs were seeded in 8-well slides (Ibidi, Graefelfing, Germany) at a density of 15,000 cells / well and incubated at 37°C for 24 h. To facilitate visualization of cell cytosol, rMCs were stained with CTDR (25 μM, 45 min, 37°C) in serum-free RPMI1640 medium prior to seeding. NPs were fabricated using PLGA labeled with AlexaFluor™ 568 and adjusted to 0.05 mg / mL in Leibovitz's buffer supplemented with 0.1% BSA. Mesangial cells were incubated with 250 μL of NPs for 15, 45 and 90 min at 37°C, washed with pre-warmed DPBS and fixed with 4% paraformaldehyde (PFA) in DPBS for 10 min. After a final washing step, fixed samples were analyzed using a Zeiss LSM 710 (Carl Zeiss Microscopy GmbH, Jena, Germany).

[0151] Flow cytometry To evaluate mesangial cell association of NP samples, rMCs were seeded in 24-well plates (Greiner Bio One, Frickenhausen, Germany) at a density of 40,000 cells / well and incubated at 37° C. for 48 h. NPs were fabricated using PLGA labeled with CF™ 647 and adjusted to 0.05 mg NP / mL in Leibovitz's buffer supplemented with 0.1% bovine serum albumin (BSA). VTo confirm β3-dependence, 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 for 60 min at 37° C. For each analysis of time-dependent uptake, cells were incubated for a period of 120 min and NPs were removed after 0, 15, 30, 45, 60, 90 and 120 min. Cells were washed with DPBS, trypsinized and centrifuged at 200 g and 4° C. for 5 min, followed by two further washing and centrifugation steps (DPBS, 200 g, 5 min, 4° C.). Final samples were resuspended in DPBS and analyzed by FACS. Analysis was performed using a Calibur cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). NP-associated fluorescence was excited at 633 nm and the corresponding emission was recorded (661 / 16 bandpass filter). Flow cytometry data was analyzed using Flowing software 2.5.1 (Turku Centre The geometric mean of cell-associated fluorescence was assessed within a population of viable cells.

[0152] Transmission electron microscope To evaluate the cellular localization of NPs, rMCs were seeded in 24-well plates at a density of 12.000 cells / well and incubated for 72 h. NP formulations containing PLGA conjugated to nanogold were diluted in Leibovitz's buffer containing 0.1% BSA and added at a concentration of 0.05 mg / mL (V = 300 μL) for 45 min. After incubation, samples were washed with DPBS and prepared for electron microscopy analysis. Briefly, cells were fixed with 2.5% PFA and 2.5% glutaraldehyde in 0.1 M sodium cacodylate solution (Caco buffer) for 60 min at RT, washed with Caco buffer and permeabilized with 0.1% Triton-X in DPBS for 10 min. After a washing step with mpHO, the samples were enhanced with gold using the GoldEnhance™ EM Plus kit (Nanoprobes Inc., Yaphank, NY, USA) according to the manufacturer's specifications, then further washed and post-fixed in 2.5% sodium thiosulfate solution in mpHO. Cells were stained with 0.5% osmium tetroxide and dehydrated in rinse concentrations of ethanol (50-99.5%). For counterstaining, 2% uranyl acetate was applied for 5 min in 70% ethanol. After embedding in Epon, 150 nm ultrathin sections were imaged using a 100 kV Zeiss Libra 120 electron microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany) at magnifications of 6300× as well as 12500×.

[0153] Co-culture experiments To evaluate the cell selectivity of the fabricated NPs, we used a co-culture design that we have previously implemented. For flow cytometry analysis, rMCs were seeded together with HeLa or NCI-H295R cells at a density of 10,000 and 75,000 cells / well, respectively, in 24-well plates and incubated at 37°C for 48 hours. To distinguish between cell types, rMCs were stained with CTG (15 μM, 45 min, 37°C) in serum-free RPMI1640 medium prior to seeding. The co-cultured cells were then incubated for 45 min with NPs labeled with CF™ 647 at a concentration of 0.05 mg / mL (V=300 μL). Sample preparation and flow cytometry analysis were performed as described above. Furthermore, rMC-associated fluorescence was recorded using excitation at 488 nm and a 530 / 30 bandpass filter. During data analysis, the population of viable cells was further gated on stained rMC cells and NP-associated fluorescence was analyzed for cell specificity.

[0154] For CLSM analysis, rMC cells were stained with CTG before seeding as described above. To visualize all cell types, HeLa or NCI-H295R cells were also stained using CTDR (25 μM, 45 min, 37° C.). After CellTracker™ incubation, rMCs were seeded in 8-well Ibidi slides together with HeLa / NCI-H295R cells at densities of 2,000 and 10,000 / 20,000 cells / well. After 48 h of incubation at 37° C., cell nuclei were stained with Hoechst 33258 (5 μg / mL in DPBS) for 20 min. Cells were washed twice with pre-warmed DPBS and AlexaFluor™ 568-labeled NPs were added at a concentration of 0.05 mg / mL (V=250 μL) for 45 min at 37° C. After NP incubation, samples were treated as above and analyzed using a Zeiss LSM 710 microscope.

[0155] in vivo cell targeting Animal experiments were performed according to national and institutional guidelines and approved by local authorities (Regierung von Unterfranken, reference number: 55.2-2532-2-329). Female 10-week-old NMRI mice (Charles River, Sulzfeld, Germany) served as model animals. After analgesia with buprenorphine (0.1 mg / kg body weight), mice were anesthetized with 2.5% isoflurane and 100 μL of CF™647-labeled NPs (c=120 nM) were injected via the jugular vein. Mice were maintained under anesthesia and after 5 min, an initial blood sample was taken via iv puncture. After 60 min, mice were anesthetized with ketamine / xylazine, a final blood sample was taken, and animals were sacrificed via perfusion fixation. Both kidneys were removed and immediately transferred to a solution of 18% sucrose and 14% PFA in phosphate buffer (0.1M pH 7.4). After 6 hours, the kidneys were washed with DPBS and cryoprotected at -80°C until further processing. For cryosectioning, organs were embedded in Tissue Tek® OCT™ Compound (Sakura Finetek, Torrance, CA, USA), cut into 5 μm sections using a CryStar NX70 cryotome (Thermo Fisher Scientific, Waltham, MA, USA) and mounted on Superfrost™ Plus glass slides (Thermo Fisher Scientific, Schwerte, Germany). For analysis of NP renal deposition and glomerular fluorescence quantification, sections were rinsed in DPBS and blocked with 5% BSA supplemented with 0.04% Triton-X in DPBS for 10 min at RT. After further rinsing in DPBS, samples were stained for cell nuclei with 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI) at a dilution of 1:400 in 0.5% BSA and 0.04% Triton-X in DPBS. After a final washing step in DPBS and mpH2O, respectively, the frozen sections were mounted with Mowiol mounting medium and analyzed on a Zeiss Axiovert 200M. For image analysis, Fiji software (Madison, WI, USA) was used.Glomerular fluorescence intensity was assessed by measuring the integrated density of the area above a certain fluorescence threshold and dividing by the glomerular area. To assess the exact cellular location of NPs, kidney cryosections were prepared as described above. After washing and blocking of the sections, the samples were stained with goat-derived integrin α-8 antibody (1:200 dilution in 0.5% BSA / 0.04% Triton-X in DPBS) overnight at 4°C. The samples were then washed with DPBS and stained with Cy2® donkey anti-goat and DAPI at 1:400 dilution in 0.5% BSA / 0.04% Triton-X in DPBS for 1 h at RT. After a final washing step, the samples were mounted and analyzed on a Zeiss LSM 710.

[0156] Example 7 Preparation of hetero-multivalent EXPcRGD NPs using a modular concept All materials and methods referred to in this example were as described in Example 6.

[0157] To create NPs with the desired adenovirus-mimetic properties, we implemented a modular design based on the synergistic combination of different biocompatible polymer components into a hetero-multivalent particle species (Figure 18a). The overall polymer composition of the NPs was intended to be similar to our previous influenza A mimetic NP design so that both targeting concepts could be properly compared. Thereby, the widely established poly(lactic-coglycolic acid) (PLGA) forms the hydrophobic NP core, which not only ensures enhanced structural integrity in aqueous media but also allows NP visualization via the coupling of fluorescent dyes or nanogold. Poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) block copolymers as the second component provide the structural flexibility required to implement the pursued virus-mimetic NP design. In a first step, longer (PEG 5k -PLA 10k ) or shorter (PEG 2k -PLA 10k) PEG-PLA polymers bearing either PEG or PEG-PLA chains were synthesized via ring-opening polymerization as previously described (Figure 25a). Since EXP3174 was intended to initially bind to the mesangial AT1r as a freely mobile ligand, the longer and therefore more flexible PEG chains were used. 5k -PLA 10k The second ligand (cRGDfK) was covalently coupled to the shorter PEG chain (Figure 25b). In contrast, the second ligand (cRGDfK) should not be able to interact with the surface-bound integrin unless initial AT1r binding and subsequent spatial proximity of the NPs have occurred. In that regard, the second ligand (cRGDfK) was covalently coupled to the shorter PEG chain (Figure 25c). 2k -PLA 10k (Figure 25c). The surface density of both cRGDfK and EXP3174 could be precisely tuned by mixing well-defined amounts of either ligand-functionalized or non-functionalized PEG-PLA polymer with PLGA prior to NP fabrication via nanoprecipitation (Figure 18d / e).

[0158] We decided to prepare hetero-functionalized nanoparticles (EXPcRGD NPs) with 25% EXP3174 and 15% cRGDfK on their surface, thereby taking full advantage of the receptor binding capacity of the ligands, but preserving the structural integrity of the produced particles. The particles should be able to locate in the target cells by binding to AT1r via the sterically flexible EXP3174, then reduce the spatial distance to the cell surface, subsequently activate αVβ3 integrin via the previously hidden cRGDfK, and finally initiate NP endocytosis (Figure 17c). Hetero-functionalized EXPcRGD NPs below the size threshold of 60 nm as well as homo-functionalized (EXP NPs / cRGD NPs) and non-functionalized methoxy-terminated particles (control NPs) were produced, which showed negative zeta potential values ​​(Figures 18b / c). These features should not only promote successful extravasation through endothelial fenestrations (FIG. 17a / b), but also prevent NP phagocytosis or extensive serum protein adsorption.

[0159] Example 8 Hetero-multivalent EXPcRGD NPs exhibit superior ligand affinity to target motifs All materials and methods referred to in this example were as described in Examples 6 and 7.

[0160] We examined EXP3174-mediated NP binding to AT1r expressed by rat mesangial cells (rMC). q Activation of the coupled AT1r by its primary ligand, angiotensin II (AT II), results in calcium influx into the cell cytosol, and thus the intracellular Ca2+ concentration after AT II stimulation is 2+ The levels can be used as a marker of AT1r activity after NP incubation. Thereby, low receptor activity indicates a high ratio of bound EXP3174, since the ligand itself acts as a strong antagonist. Figure 19 shows the intracellular Ca of ATII-stimulated rMCs pre-incubated for 45 min with NP or free EXP3174. 2+ The level is shown. EXPcRGD NP(IC 50 = 276 ± 31 pM) and EXP NP (IC 50 = 552 ± 73 pM) both exhibited excellent AT1r avidity, which is consistent with highly effective inhibition of the receptor in the picomolar range and the resulting minimal cytosolic Ca 2+ Furthermore, the inhibitory potency of the EXP3174-bearing NP form was even higher (IC 50 = 2.66 ± 0.9 nM), strongly suggesting that the EXP3174 functionalized particles were able to interact with the target receptor in a multivalent fashion, resulting in increased overall avidity. 50Since the levels were found to be in the same range, we concluded that the combination of both EXP3174 and cRGDfK in one particle type did not significantly interfere with the binding capacity of EXP3174 itself. Furthermore, control NPs and cRGD NPs showed no interaction with the AT1r and showed no maximum Ca upon receptor stimulation. 2+ A signal was generated, confirming the specificity of the assay for AT1r (FIG. 19b).

[0161] After verifying the AT1r binding ability of adenovirus-mimetic EXPcRGD NPs, the next step was to investigate particle uptake into rMCs via cRGDfK-αVβ3 interaction. Therefore, we incubated mesangial cells with fluorescently labeled NPs and analyzed the cellular distribution via confocal laser scanning microscopy (CLSM). To visualize cell bodies, rMCs were pretreated with CellTracker™ Deep Red (CTDR). Figure 20a shows the strong intracellular accumulation of AlexaFluor™ 568-labeled EXPcRGD NPs in spherical structures indicative of endocytic vesicles. Over time, both the number and intensity of visible accumulations increased. Moreover, the vesicles appeared to increase in size with longer incubation times. These findings support that cRGDfK-functionalized NPs can bind to target cells and be incorporated into intracellular vesicles via integrin-mediated endocytosis. Over time, these endocytic vesicles fuse into larger endosomes, further increasing in size as well as strength.

[0162] Based on the CLSM results, we performed flow cytometry analysis of rMCs treated with NPs to determine cell-associated fluorescence over a 120-minute incubation period. As shown in FIG. 20b, the level of NP-derived fluorescence was greatest for EXPcRGD NPs compared to all other NP species over the entire incubation period. EXP NPs as well as control NPs showed only moderate fluorescence signals, whereas a significant level of cell association could be detected for cRGD NPs. Notably, the respective fluorescence levels reached a plateau after approximately 60 minutes, while the cell association of EXPcRGD NPs further increased. This strongly supports the hypothesis of sequential interactions between EXPcRGD NPs and their target cells, resulting in a prolonged increase in fluorescence levels compared to homo-functional cRGD NPs.

[0163] To investigate the effect of αVβ3 integrin on EXPcRGD NP cellular uptake, an excess of free cRGDfK (c=500 μM) was added, and then rMC was incubated with EXPcRGD NPs for 60 min. As a result, the level of cell-associated fluorescence was sharply reduced to a level comparable to that of EXP NPs or control NPs (FIG. 20c). Hetero-multivalent particles were apparently no longer able to target the required αVβ3 integrin and therefore were unable to substantially initiate endocytosis after AT1r binding.

[0164] To further validate the concept of integrin-mediated NP endocytosis, we decided to utilize transmission electron microscopy (TEM), which allowed the evaluation of NP-cell interactions at a much higher magnification level. To increase the electron density and the resulting TEM visibility of the applied NPs, ultrasmall gold nanoparticles with an average diameter of 2.2 nm were covalently coupled to PLGA, which was then used for further NP fabrication (Figure 26). Mesangial cells incubated with these gold-tagged NPs could then be enhanced with gold in order to sensitize the gold core of the particles and thus visualize it and evaluate their exact location. This retrospective gold enhancement offers the substantial advantage that the physicochemical characteristics of nanogold-labeled NPs are not significantly different from unlabeled NPs, which is not the case for the commonly utilized gold NPs.

[0165] Figure 21a shows the soma of two mesangial cells incubated with gold-tagged EXPcRGD NPs. Numerous round-shaped vesicles filled with gold-enhanced NPs could be detected within the cell cytosol. The distribution pattern showed remarkable similarity to the previously described CLSM results (Figure 20a), thereby strongly supporting ligand-mediated NP endocytosis. Moreover, particles were observed to accumulate at the cell border, indicating that these NPs were still bound to membrane-located surface structures. These findings further indicate that the applied EXPcRGD NPs interacted with the target cells in a stepwise process of prior binding to AT1r and subsequent integrin-mediated endocytosis. In accordance with this evaluation, EXP NPs without surface-bound cRGDfK could only be detected in the rMC membrane, and no particle accumulation was found in the endocytic vesicles (Figure 21b). Moreover, there was only a slight cell-particle association for the control NPs (Figure 21c). The specificity of the applied gold enhancement was demonstrated by the lack of gold accumulation in particle-free cells.

[0166] Example 9 Ligand synergy results in enhanced mesangial cell selectivity in vitro All materials and methods referred to in this example were as described in Examples 6-8.

[0167] Having demonstrated that hetero-multivalent EXPcRGD NPs synergistically combine both key features of their surface ligands and present them in a spatially controlled manner, we intended to demonstrate that this design could indeed be exploited to increase mesangial cell selectivity. Therefore, we implemented an in vitro-based assay, co-culturing target rMCs with a predominant number (5-10 fold) of off-target cells that either did not have the target receptor or only had one of the two target receptors. HeLa cells were chosen because they did not express either AT1r or αVβ3-integrin to any significant extent, whereas NCI-H295R cells showed high AT1r expression but low αVβ3 expression (Figure 27).

[0168] To distinguish between co-cultured cells in CLSM analysis, rMCs were stained using CellTracker™ Green (CTG), whereas off-target cells were marked with CTDR. After 45 min of incubation with fluorescently labeled EXPcRGD NPs, the cellular distribution of NPs was evaluated. In the rMC / HeLa co-culture model, particle-derived fluorescence could be detected almost exclusively in the area of ​​mesangial cells. HeLa cells, in contrast, only showed weak interactions with NPs, resulting in slight fluorescence levels (Figure 22a). Thus, we concluded that EXPcRGD NPs could selectively locate in mesangial cells among HeLa cells due to differences in receptor expression on the cell surface. These findings were supported by flow cytometry analysis showing that cell-associated fluorescence of EXPcRGD NPs was significantly higher in rMCs than in off-target HeLa cells, whereas cellular interactions for control NPs were only slight for both cell types (Figure 22b). In contrast, rMC / NCI-H295R co-cultures provided diverse particle distributions. NP-associated fluorescence could be found not only in rMCs but also within the area covered by NCI-H295R cells. However, the distribution patterns were significantly different. Fluorescence among rMCs was found in round vesicle-like structures, as seen previously, while NCI-H295R-associated fluorescence was more diffuse and was mainly enhanced in the cell membrane (Figure 22a). Therefore, we concluded that EXPcRGD NPs accumulated in endocytic vesicles of rMCs but could only bind to AT1r present in the cell membrane of NCI-H295R cells and could not be taken up into the cytosol due to the absence of αVβ3 integrin. Furthermore, flow cytometry analysis showed that although NP-associated fluorescence for NCI-H295R cells was higher compared to HeLa cells, EXPcRGD NPs still exhibited a significantly enhanced signal in mesangial cells (FIG. 22c).Strikingly, the addition of excess free EXP3174 (c=1 mM) prior to NP incubation resulted in a sharp decrease in the fluorescence levels for NCI-H295R cells, whereas cell-associated fluorescence for rMCs was still significantly higher. This observation further supports the view that EXPcRGD NPs can indeed utilize both surface ligands to target mesangial cells, thereby benefiting from a hetero-functional design.

[0169] Taken together, our co-culture model demonstrated that hetero-multivalent EXPcRGD NPs have the ability to effectively identify receptor-positive mesangial cells not only predominating in number, but also in the presence of off-target cells expressing one of the two targeted receptors.

[0170] Example 10 Accumulation of adenovirus-mimetic EXPcRGD NPs in mesangial cells in vivo All materials and methods referred to in this example were as described in Examples 6-9.

[0171] Both rMC binding and uptake studies successfully demonstrated that our virus-mimicking concept of sequential ligand-receptor interactions allows hetero-multivalent EXPcRGD NPs to selectively target mesangial cells in vitro. However, translating in vitro results into a robust system with sufficient in vivo efficiency has been shown to be a major obstacle in nanoparticle design, as many strategies have failed to deliver the desired target specificity. Therefore, we decided to evaluate the ability of NPs to actually reach the mesangial region in vivo, which not only requires active cellular uptake promotion, but also appropriate passive accumulation in the target region. For that, 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 extracted. Fluorescence analysis of the prepared frozen sections revealed that EXPcRGD NPs effectively accumulated in the glomerular region, while fluorescence in the tubular portion of the kidney was negligible (Figure 23). In contrast, control NPs as well as homo-functional EXP or cRGD NPs showed significantly lower deposition in kidney cryosections.

[0172] To quantify the observed differences, glomerular-associated fluorescence levels were determined by assessing glomerular fluorescence intensity per area for all NP types (Figure 24a / b). Thereby, EXPcRGD NPs showed more than a 10-fold increase in fluorescence intensity compared to control NPs. Furthermore, the glomerular accumulation of hetero-multivalent NPs was significantly greater than for both homo-functionalized NP types. Strikingly, cRGD NP fluorescence was even lower than for non-functionalized control NPs. We hypothesize that cRGD NPs could not reach the glomerular region because a predominant number of particles bind to αVβ3-expressing endothelial cells immediately after injection and, as a result, leave the bloodstream before reaching deeper regions of the kidney. This hypothesis was further supported by the finding that the relative plasma levels of cRGD NPs after 1 hour of incubation were the smallest among all particle types (Figure 28a). In contrast, in EXPcRGD NPs, the shorter cRGDfK-functionalized PEG-PLA chains were shielded from premature presentation to αVβ3 integrin by the addition of the longer EXP3174-functionalized PEG-PLA chains. As a result, the hetero-multivalent particles avoided off-target deposition and thus successfully reached the glomerular region in the kidney. Antibody staining for mesangial cell marker integrin-α8 further revealed that EXPcRGD NPs-associated fluorescence in the glomerulus could be found almost entirely within mesangial cells (FIG. 24c), proving the hypothesis of extravasation into the mesangial interstitium and subsequent endocytosis (FIG. 17a / b). To verify that the detected fluorescence in the mesangial region was derived from structurally intact NPs, we further injected a comparable dose of free fluorescent dye into mice and analyzed the fluorescent deposition. The intraglomerular signal for these samples was negligible, while tubular cells showed very strong fluorescence levels (Figure 28b). This indicated that the small molecule dye was renally filtered, in contrast to the injected NP species. Therefore, we concluded that the intraglomerular fluorescence for all NP types was derived from intact particles, since degradation would have otherwise led to an increase in the tubular signal.

[0173] Thus, the in vivo study described herein successfully demonstrated the potential of the new adenovirus-mimetic NP design of the present invention. Hetero-multivalent EXPcRGD NPs effectively accumulated in the mesangial region of the glomerulus, whereas homo-functionalized or non-functionalized NP species failed to do so. This strongly suggests that to reach a sufficient level of bioavailability, NPs must not only have appropriate surface ligands, but also present them in an organized format appropriate for the respective targeting strategy. Furthermore, NP accumulation in the mesangium also demonstrated that the adenovirus-mimetic system of sterically controlled particle-cell interaction is highly effective.

[0174] Example 11 EXPcRGD NPs loaded with cinaciguat show high efficiency Nanoparticles (NPs) using either influenza A mimetic or adenovirus mimetic target cell recognition concepts were detected to efficiently accumulate in mesangial cells in an in vivo situation. In the next step, the experimental drug cinaciguat (BAY 58-2667) was encapsulated in the adenovirus mimetic EXPcRGD NPs. Cinaciguat (CCG) is a potent activator of soluble guanylate cyclase (sGC) and has been shown to significantly reduce mesangial fibrosis and reduce glomerular damage in diabetic animal models. By encapsulating CCG in our promising NP species, the cell-selective delivery of CCG to pathological mesangial sites can be significantly increased, resulting in enhanced therapeutic efficacy with minimized off-target effects (Figure 29).

[0175] In our experimental setup, CCG was first encapsulated in hetero-multivalent EXPcRGD NPs. The resulting NPs had approximately 500-700 CCG molecules per NP (data not shown). In all following experiments, the administration of free cinaciguat at a concentration of 2 μM was compared to EXPcRGD NPs loaded with CCG at a concentration of approximately 0.5 nM (equivalent to 0.2 μM CCG) and to EXPcRGD control NPs without drug (Figure 30). The concentration of free cinaciguat was chosen according to previous publications that showed the antifibrotic effect of CCG in this concentration range. However, the CCG-bearing NPs had only 10% of the respective CCG amount to test possible drug delivery effects.

[0176] To evaluate the effect of EXPcRGD NPs loaded with CCG on target sGC, mesangial cells were first incubated for 24 hours, and protein amount was evaluated using Western blot analysis.Interestingly, the total amount of sGC was gradually increased after both incubation with free drug and NPs loaded with CCG, indicating not only the previously shown activating effect of cinaciguat on sGC, but also its stabilizing effect (Figure 31).In contrast, NPs lacking cinaciguat did not show any significant effect on sGC levels.

[0177] Finally, the antifibrotic and antiproliferative potential of NP-assisted CCG delivery was analyzed. For that, mesangial cells were treated with free CCG, CCG-loaded EXPcRGD The cells were first incubated for 4 h with either CCG NPs or control NPs without encapsulated drug. After 4 h, 10 ng / mL transforming growth factor beta (TGF-β) was added for 48 h to induce fibrotic and hyperproliferative remodeling. Administration of TGF-β resulted in a marked increase in mesangial cell proliferation, whereas pre-incubation with both free CCG and CCG-loaded NPs was able to significantly reverse this effect (Figure 32). Western blot and fluorescence microscopy analysis of fibrosis markers α-SMA and collagen I also revealed a similar effect of CCG (loaded NPs) on inhibiting profibrotic remodeling of mesangial cells.

[0178] Taken together, these results reveal two main findings: 1. Both free cinaciguat and NP-encapsulated cinaciguat show a significant effect on its target enzyme sGC, which leads to a significant activation of the described antifibrotic pathway (Figure 29). These results are in line with previous findings on CCG and indicate the outstanding potential of the therapeutic agent in antifibrotic treatment. 2. Throughout all experiments, EXPcRGD NPs loaded with cinaciguat showed comparable efficacy to the administration of free CCG, even though the overall amount of encapsulated CCG was only 10% of the free drug dose (0.2 μM vs. 2 μM), indicating the remarkable potential of the described NPs to more efficiently deliver pharmaceutical agents to their intended intracellular targets.

[0179] References [1] Maslanka Figueroa, S., Veser, A., Abstiens, K., Fleischmann, D., Beck, S., and Goepferich, A. (2019). Influenza A virus mimetic nanoparticles trigger selective cell uptake. Proc. Natl. Acad. Sci. 201902563. [2] Sah E. and Sah H. Journal of Nanomaterials, Volume 2015, Article ID 794601. [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. [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 receptor via protein kinase C. Sci. Rep. 6, 21613.

[0180] The features of the invention disclosed in this specification, the claims and / or the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in various forms thereof. The present invention provides, for example, the following items. (Item 1) A nanoparticle comprising a nanomaterial and at least a first ligand and a second ligand, - said first ligand is capable of mediating attachment of said nanoparticle to a target cell; - the second ligand is capable of mediating internalization of the nanoparticle into the target cell; - preferably, said nanomaterial comprises any of 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-poly-caprolactone, inorganic materials such as gold or qdot materials, and combinations thereof; Nanoparticles. (Item 2) 2. The nanoparticle according to item 1, wherein the first ligand is a non-agonistic drug that binds to a GPCR, such as angiotensin II receptor type 1 (AT1r), human neuropeptide Y1 receptor and CXC chemokine receptor type 4, and / or a drug that binds to glycoproteins and / or glycolipids on the surface of target cells, such as heparan sulfate, sialoglycoprotein, ganglioside and mannose receptors, preferably EXP3174 or telmisartan. (Item 3) 3. The nanoparticle according to item 1 or 2, wherein the second ligand is either i) an agent that binds to an integrin, such as αVβ3 integrin or αVβ5 integrin, preferably selected from RGD, a cyclic RGD-peptide having the sequence of SEQ ID NO: 1, and derivatives thereof, ii) an agonistic agent that binds to a GPCR, such as AT1r, preferably activated angiotensin-II, iii) an agent that binds to an ectoenzyme, such as legumain, membrane-type matrix metalloproteinase and angiotensin-converting enzyme (ACE), preferably angiotensin-I, and / or iv) an agent that binds to the transferrin receptor. (Item 4) The nanoparticles of any of the preceding items, further comprising a therapeutic agent, preferably either pirfenidone or cinaciguat. (Item 5) The nanoparticle of any of the preceding items, wherein the first ligand and the second ligand are each coupled to the nanomaterial, preferably each coupled to a block copolymer chain of the nanomaterial. (Item 6) 2. The nanoparticle of claim 1, wherein the nanomaterial comprises more than one block copolymer chain, the first ligand is coupled to a first block copolymer chain of the nanomaterial, the second ligand is coupled to a second block copolymer chain of the nanomaterial, and the first block copolymer chain is longer than the second block copolymer chain, preferably at least 1.5 times as long as the second block copolymer chain, and more preferably at least 3 times as long as the second block copolymer chain. (Item 7) 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, and optionally the first block copolymer chain comprises PEG in the range of 5k to 40k, preferably 10k to 20k. 5k -PLA 10k and the second block copolymer chain is PEG 2k -PLA 10k In item 6, The nanoparticles described herein. (Item 8) The nanoparticle of any of the preceding items, wherein the second ligand is enzymatically activated prior to the internalization of the nanoparticle into the target cell. (Item 9) The nanoparticles according to any of the preceding items, wherein 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. (Item 10) The nanoparticles according to any of the preceding items, having a size of 5 nm to 1000 nm, preferably 10 nm to 150 nm, more preferably 20 nm to 100 nm. (Item 11) The nanoparticles according to any of the preceding items, wherein the ratio of the first ligand to the second ligand is in the range of 2:1 to 1:2, preferably 1:1. (Item 12) The nanoparticles according to any of the preceding items, having a particle avidity for the targeted receptor of 1 pM to 100 nM, preferably 50 pM to 1 nM. (Item 13) The nanoparticles of any of the preceding items, wherein the nanomaterial comprises PEG and the nanoparticles have a ligand density of at least 5%, preferably at least 15%, more preferably at least 25% ligand / PEG. (Item 14) Nanoparticles according to any of the preceding items for use as a pharmaceutical or diagnostic agent. (Item 15) The nanoparticles according to any of the preceding items for use in a method for preventing or treating a disease selected from 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. (Item 16) 14. A method for preparing nanoparticles according to any one of items 1 to 13, comprising the steps of: a) providing, in any order, one or more nanomaterials, preferably including 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, e.g., PEG-PLA or PEG-poly-caprolactone, inorganic materials, e.g., gold or qdot materials, and any combination thereof, and optionally a therapeutic agent; b) optionally preparing a block copolymer from either said one or several nanomaterials; c) coupling a first ligand and a second ligand to said nanomaterial in one or more steps, preferably by DCC / NHS coupling or EDC / NHS coupling; d) providing a therapeutic agent, preferably a lipophilic therapeutic agent, if not already provided in step a); e) Preparing and obtaining nanoparticles using said ligand and said therapeutic agent coupled to said nanomaterial, preferably by nanoprecipitation. A method comprising: (Item 17) Item 17. The method according to item 16, wherein said obtaining step in step e) comprises obtaining nanoparticles having a polydispersity index of 0.01 to 0.5, preferably of 0.01 to 0.3, more preferably of 0.01 to 0.1.

Claims

[Claim 1] The invention described in the specification.

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