HK1208344B - Cell-specific targeting by nanostructured carrier systems - Google Patents

Description

The present invention relates to nanostructured carrier systems according to the claims for use in the treatment of diseases of the liver and/or kidney, comprising one or more polymers and/or lipids as well as one or more polymethine dyes as targeting units for the targeted transport of the nanostructured carrier system into a target tissue. The invention also relates to the cell-specific transport of one or more pharmaceutical active ingredients into a specific target tissue (cell-specific targeting), and to the use of the nanostructured carrier systems according to the invention for the prevention and/or treatment of diseases of the liver and/or kidney.

In the current state of the art, the use of nanoparticles with coupled dyes in clinical diagnostics is known, for example, for the detection of organ functions or protein expressions in the diagnosis of pathological conditions, or for proteome analyses. In this context, the coupled dyes, usually fluorescent dyes such as cyanines, are used as markers, and their fluorescence and absorption properties are measured.

WO20121013247A1 describes the use of polymethine fluorescent dyes for determining an organ function, particularly the function of the liver or kidney. In this method, the dye is used as a marker in a tissue or body fluid, such as blood or urine, and is radiatively excited. The fluorescence emission of the dye is then detected, the data are recorded and evaluated to determine the organ function under investigation.

In WO2010/116209A1, it is described how a clinical condition based on abnormal Selectin secretion can be detected using fluorescent dyes and fluorescence spectroscopy.

Rungta, P. et al., "Selective Imaging and Killing of Cancer Cells with Protein-Activated Near-Infrared Fluorescing Nanoparticles," Macromol Biosci, 2011, 11: pp. 927-37 describes nanoparticles containing indocyanine green for the visualization and treatment of liver cell carcinoma.

WO2013/051732A1 describes liposomes comprising near-infrared (NIR) dyes for the treatment of tumors.

In the current state of the art, the use of nanoparticles for targeted delivery of active ingredients to specific tissues is still known (Sheridan, C., Proof of concept for next-generation nanoparticle drugs in humans. Nat Biotechnol, 2012. 30(6): p. 471-3; Gratton, S.E. et al., The effect of particle design on cellular internalization pathways. Proc Natl Acad Sci U S A, 2008. 105(33): p. 11613-8; Jiang, N. et al., Targeted Gene Silencing of TLR4 using liposomal nanoparticles for preventing liver ischemia reperfusion injury. American Journal of Transplantation, 2011. 11: p. 1835-44).

Targeted or cell-specific transport of a drug, also known as "drug targeting" or "targeted drug delivery," refers to the directed and selective accumulation and release of an active ingredient at a desired site of action, aiming to increase the effectiveness of the drug while reducing systemic side effects on surrounding tissues. Transported drugs are often antibodies, peptides, or small molecules such as oligonucleotides or nucleic acids.

The active substance-carrying nanoparticles known from the prior art are used in tumor therapy and function according to the following mechanisms: Either the nanoparticle is provided with a coating layer or an antibody. If the nanoparticle is coated with an aqueous layer, it becomes invisible to the immune system. When this nanoparticle is injected and not attacked by the immune system, it diffuses through the "leaky" blood vessels in the tumor, which have significantly larger openings (fenestrations) compared to normal blood vessels, and is taken up by surrounding cells via endocytosis, as these cells also exhibit increased permeability compared to healthy cells. A disadvantage is that not only the desired cells take up the nanoparticle, but also other (healthy) cells, to which the nanoparticle is transported non-specifically via the blood vessels. This can cause severe side effects. Another disadvantage is that this transport is limited to tumor tissue, meaning that transport into other tissues, such as the liver or kidney, is not possible. This transport occurs passively, and the uptake is non-specific or not selective. In the second method, after its production, the nanoparticle is equipped with antibodies on its surface. The target of these constructs are cells with antigens to which these antibodies bind. This transport mechanism is also passive and non-selective.

The processes described above of passive accumulation of nanoparticles, liposomes, or macromolecules are referred to as the EPR effect ("enhanced permeability and retention"; enhanced permeability and retention) and represent a passive "drug targeting." As mentioned, the disadvantages are that these transport processes are not active and not selective.

Nowhere in the prior art is an active and selective transport or carrier system described, wherein active transport occurs selectively into a specific target tissue via special targeting units, and at the same time (pharmaceutical) active substances can be transported into the target tissue ("drug targeting"; cell-specific targeting), and the accumulation of the carrier system and, if applicable, of the (pharmaceutical) active substance in the target tissue not only is achieved but also can be tracked and verified via the targeting unit.

Therefore, there is a need to provide an improved transport or carrier system that enables an active and selective transport of carriers and active ingredients into a target tissue. There is also a continued need to use such a transport or carrier system for the delivery of pharmaceutical active ingredients in the treatment of diseases.

With the present invention, such a transport system is provided. The present invention relates to a unique, multifunctionally combinable, theranostic system for actively and selectively transporting various pharmaceutical active ingredients (for example, hydrophilic, lipophilic, hydrophobic, amphiphilic, anionic, and cationic substances) into a target tissue (targeted or cell-specific drug transport, or "drug targeting").

The present invention relates, in its first aspect, to a nanostructured carrier system according to the claims for use in the treatment of diseases of the liver and/or kidney, comprising at least one polymer and/or at least one lipid and at least one polymethine dye, wherein the at least one polymethine dye acts as a targeting unit to enable the targeted transport of the nanostructured carrier system into a target tissue.

If the inventive nanostructured carrier system comprises polymers, it is referred to herein as "nanoparticle"; if it comprises lipids, it is referred to herein as "liposome." If the inventive nanostructured carrier system comprises both polymers and lipids, it is referred to herein as either "nanoparticle" or "liposome." Accordingly, according to the invention, the terms "nanoparticle" and "liposome" are used synonymously and also refer to a nanostructured carrier system that comprises both polymers and lipids.

Nanoparticles are structures smaller than 1 µm and can be composed of multiple molecules. They generally exhibit a higher surface-to-volume ratio, which provides increased chemical reactivity. These nanoparticles can consist of polymers, which are characterized by the repetition of certain units (monomers). The polymers are covalently linked together through the chemical reaction of these monomers (polymerization). If these polymers have partially hydrophobic properties, they can form nanostructures in aqueous environments (e.g., nanoparticles, micelles, vesicles). Due to their hydrophobic properties, lipids can also be used to form nanoparticles (micelles, liposomes).

A preferred embodiment of the present invention relates to a nanostructured carrier system, wherein the at least one polymer is selected from the group consisting of polyesters, poly(meth)acrylates, polystyrene derivatives, polyamides, polyurethanes, polyacrylonitriles, polytetrafluoroethylenes, silicones, polyethylene glycols, polyethylene oxides, and polyoxazolines and their copolymers, preferably in various compositions, such as statistical, gradient, alternating, block, graft or star copolymers, or the at least one lipid is selected from the group consisting of saturated and unsaturated fatty acids, preferably cholesterol, palmitic acid, phospholipids, sphingolipids and glycolipids. Preferably, the polymer or lipid according to the invention is a biocompatible polymer or lipid.

The polymer according to the invention is particularly preferably a hydrophobic, hydrophilic, amphiphilic, anionic and/or cationic polymer. In particular, the polymer is selected from the group consisting of PLGA, PLA, PCL, PGA, PDMAEMA, PMMA, PMAA, PEI, PEtOx, and PEG.

As "targeting unit" in the sense of the invention, substances are meant which actively and selectively cause the transport of the nanostructured carrier system according to the invention into a specific target tissue. According to the invention, targeting units are polymethine dyes. The terms "targeting unit" and "polymethine dye" are used synonymously according to the invention.

The polymethine dye according to the invention is a symmetric or asymmetric polymethine of general structure I, II, III, or IV: where a. n represents the numerical values 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, b. R1 to R17 are the same or different and can be hydrogen, one or more alkyl, tertiary alkyl, cycloalkyl (the groups "alkyl" and "cycloalkyl" also include olefinic structures), aryl, carboxyaryl, dicarboxyaryl, heteroaryl, or heterocyclic aliphatic groups, alkoxy, alkylthio, aryloxy, arylthio, heteroaryloxy, heteroarylthio, a hydroxyl, nitro or cyano group, an alkyl-substituted or cyclic amine function, and/or two ortho-standing groups, for example R3 and R4, R13 and R14 and/or R1 and R2 and R11 and R12 and/or R7 and R9 form another aromatic,heteroaromatic, aliphatic or heteroaliphatic rings can be formed; c. at least one of the substituents R1-R17 carries a solubilizing or ionizable or ionized substituent, such as SO3−, (-SO3H), PO3^2−, COOH, OH or NR3+, cyclodextrins or sugars, which determine the hydrophilic properties of these polymethine dyes, wherein this substituent can also be bound to the polymethine dye via a spacer group; and d. at least one of the substituents R1-R17 carries a reactive group (linker), such as isocyanates, isothiocyanates, hydrazines, amines, mono- and dichloro- or mono- and dibromo-triazines, aziridines, epoxides, sulfonyl halides, acid halides, carboxylic anhydrides, N-hydroxysuccinimide esters, imido-esters, carboxylic acids, glyoxal, aldehydes, maleimides or iodacetamides and phosphoramidite derivatives or azides, alkynes or olefins, wherein this substituent can also be attached to the polymethine dye via a spacer group;e. the aromatic, heteroaromatic, aliphatic or heteroaliphatic spacer group consisting of structural elements such as -[(CH2)a-Y-(CH2)b]c- or -[(C6H4)a-Y-(C6H4)b]-, where Y is the same or different and includes CR2-, O-, S-, SO2, SO2NH-, NR-, COO- or CONR functions, which are attached to one of the substituents R1-R17, and a and b are the same or different with numerical values from 0-18, and with numerical values for c from 0-18; f. the substituents R8 and R9, in corresponding cases with n = 2, 3, 4 or 5, can also be present 2-, 3-, 4- or 5-fold, and they can be the same or different.

According to the invention, the terms "targeting unit" and "polymethine dye" are used synonymously.

The targeting units (polymethine dyes) are conjugated to the polymer via a linker.

General structure: Polymer - Linker - Targeting unit:

The general structure of an inventive linker is described as follows: at least one structural unit (polymer and/or targeting unit) carries a reactive group (linker), such as isocyanates, isothiocyanates, hydrazines, amines, mono- and dichloro- or mono- and dibromo-triazines, aziridines, epoxides, sulfonyl halides, acid halides, carboxylic anhydrides, N-hydroxysuccinimide esters, imido esters, carboxylic acids, glyoxal, aldehydes, maleimides or iodacetamides and phosphoramidite derivatives or azides, alkynes or olefins, wherein this substituent can also be attached via a spacer group to the polymethine compound and/or the polymer. Through this reactive group, the targeting unit is linked to the polymer (or vice versa) via a covalent bond.

The chemical bonds between the polymer or targeting unit and the linker can be chosen as either biostable or biodegradable. One or more different targeting units can be bound to a polymer. Also, polymers equipped with different targeting units can be mixed within a nanoparticle. In this case, both the polymer, the targeting unit, or both can differ. Instead of a polymer, under the same conditions as described above, a lipid can be used instead of a polymer, and correspondingly, a liposome can be used instead of a nanoparticle.

In a preferred embodiment of the present invention, the at least one polymethine dye of the nanostructured carrier system is selected from the group consisting of DY635, DY-680, DY-780, DY-880, DY-735, DY-835, DY-830, DY-730, DY-750, DY-850, DY-778, DY-878, DY-704, DY-804, DY-754, DY-854, DY-700, DY-800, ICG, and DY-IRDYE 800CW. Furthermore preferred are the polymethine dyes DY-630, DY-631, DY-632, DY-633, DY-634, DY-636, DY-647, DY-648, DY-649, DY-650, DY-651, DY-652, DY-590, DY-548, DY-495, and DY-405. These are polymethine dyes used as targeting units that enable selective transport into hepatocytes or renal parenchymal cells. The general structures of a hepatocyte-targeting unit according to the invention and a parenchymal cell-targeting unit according to the invention, as well as corresponding examples, are shown in Table 2 in Figure 8.These targeting units have a selectivity for a specific cell type (hepatocytes or renal parenchymal cells) and can transfer this cell selectivity to a nanoparticle or liposome when they are chemically bound to it. The selectivity of the targeting unit arises from its interaction with influx transporters that are expressed by the target cells. Furthermore, the targeting units exhibit fluorescent properties in the red to infrared range. These fluorescent properties can also be transferred to the nanostructured carrier system, more specifically to the nanoparticle or liposome, thereby making not only the accumulation of the dye but also (when connected to the nanoparticle or liposome) the accumulation of the nanoparticle or liposome in the blood and tissue detectable.

The polymethine dyes according to the invention act as targeting units that selectively transport the nanostructured carrier system into the target tissue. The selectivity is crucial for successful transport into the "correct" tissue and exclusively into this tissue, representing a significant advantage over the prior art. The polymethine dyes serve as transporter ligands for tissue-specific transporters. The following properties are important for a polymethine dye to function as such a transporter ligand: (1) hydrophobicity and (2) the combination with a specific structure. These properties are decisive for being recognized by a tissue-specific transporter as a ligand (selectivity of the dye). When the polymethine dye is bound to a polymer or lipid, so that it becomes exposed on the outside after the production of the nanoparticle or liposome,He transfers his selectivity to the nanoparticle or liposome. Following systemic or local application, the following processes occur, which are crucial for the selectivity of the nanoparticle or liposome: The nanoparticle or liposome passes by different tissues with at least one exposed polymethine dye. The polymethine dye is recognized and interacts with tissue-specific basolateral or apical influx transporters due to its hydrophobicity and structure at the cell surface. However, the interaction of the polymethine dye with the influx transporter does not lead to a direct transport of the entire nanostructured carrier system, the nanoparticle, or the liposome through this transporter, as it has too high a molecular weight and size when covalently and stably bound to the nanoparticle or liposome.The interaction of the polymethine dye with the Influx transporter rather leads to accumulation and immobilization of the nanoparticle or liposome on the cell surface. The accumulation and immobilization of the nanoparticle or liposome on the cell surface enhances the interaction between the cell membrane and the nanoparticle or liposome, resulting in cellular uptake (endocytosis) of the nanoparticle or liposome.

Cell selectivity arises from the specific interaction of the polymethine dye, which is coupled to the nanoparticle or liposome, and which is recognized by the corresponding tissue-specific influx transporter. Influx transporters for the polymethine dyes according to the invention have been defined for hepatocytes and renal parenchymal cells.

Inventive polymethine dyes that are specifically taken up by influx transporters of the basolateral membrane of hepatocytes make the nanoparticle specific for hepatocytes. According to current scientific knowledge and FDA, the following are classified as influx transporters in hepatocytes:

Name	Gen
SLCO1B1
SLCO1B3
SLCO2B1
SLCO1A2
SLC13A3
SLC10A1
SLC22A1
SLC22A3
SLC22A7
SLC22A6
SLC22A8
SLCO2A1

The ligands of these transporters are particularly all polymethine dyes that have a structure as shown in Table 2 (Fig. 8a-e), left column.

Inventive polymethine dyes that are specifically taken up by influx transporters of the basolateral membrane of renal parenchymal cells (especially proximal tubular cells) make the nanoparticle specific for these cell types. According to current scientific knowledge and FDA guidelines, the following influx transporters of renal parenchymal cells (especially proximal tubular cells) are known:

Name	Gen
OCT2	SLCO1B1
OAT1	SLCO1 B3
OAT3	SLC22A8
OATP4A1	SLCO4A1
OATP4C1	SLCO4C1
OCT1	SLC22A1
OCT3	SLC22A3
PGT	SLCO2A1

The ligands of these transporters are particularly all polymethine dyes that have a structure as shown in Table 2 (Fig. 8a-e), right column.

Another preferred embodiment of the invention relates to a nanostructured carrier system, wherein at least one polymethine dye causes the uptake of the nanostructured carrier system into the cells of the target tissue via at least one tissue-specific transporter. Particularly preferably, the tissue-specific transporter is selected from the group consisting of OATP1B1, OATP-C, OATP2, LST-1, OATP1B3, OATP8, OATP2B1, OATP1A2, NaDC3, SDCT2, NTCP, OCT1, OCT3, OAT2, OAT1, OAT3, PGT, OCT2, OAT1, OATP4A1, and OATP4C1.

The terms "tissue-specific transporter," "transporter," and "influx transporter" are used synonymously according to the invention.

The terms "nanoscale carrier system," "nanoparticle," and "liposome" are used synonymously in the invention in connection with transport to and uptake into the target tissue via a tissue-specific transporter.

After the nanostructured carrier system, or the nanoparticle or liposome, has been taken up by the target tissue, there occurs the release of the polymethine dye and a pharmaceutical active ingredient according to the invention.

Release of a nanoparticle as part of the nanostructured carrier system: 1. Acidification of the endosome → destabilization of the nanoparticle, degradation of the polymer by spontaneous or enzymatic cleavage; 2. Release of active substances (which can penetrate the endosome); 3. Release of the active ingredient, desorption of the dye from the polymer; 4. Polymer components are directed into various metabolic pathways, the dye is excreted.

Release of a liposome as part of the nanostructured carrier system: 1. Uptake by endosomes → acidification → fusion of the liposome with the endosomal membrane after endocytosis or direct fusion of the liposome with the cell membrane; 2. In both cases, the active ingredient is directly released into the cytoplasm; 3. If the targeting unit (polymethine dye) is connected to the lipid via a labile bond, this bond can be cleaved and the dye is released. With the use of a biostable bond, the polymethine dye remains attached to the lipid. If the lipid is subsequently degraded, the polymethine dye can be secreted together with a small lipid residue. It is likely that part of the lipid can be incorporated into the cell membranes along with the polymethine dye.

The nanostructured carrier system according to the invention further comprises at least one pharmaceutical active ingredient. Preferably, the at least one pharmaceutical active ingredient is selected from the group consisting of low-molecular substances, in particular inhibitors, inducers or contrast agents, as well as high-molecular substances, in particular potentially therapeutically usable nucleic acids (e.g., short interfering RNA, short hairpin RNA, micro RNA, plasmid DNA) and proteins (e.g., antibodies, interferons, cytokines). The following table exemplarily describes active ingredients, whose specific administration by the nanostructured carrier system of the present invention enables new therapeutic options:

Glucokortikoide	Decortin	Organtransplantation	Leber, Niere
Zytostatika z.B. Alkylantien	Cyclophosphoamid	Organ transplantation , Tumore	Leber, Niere
Antimetabolite	Methotrexat	Organtransplantation, Tumore	Leber, Niere
Interkalantien	Mitoxantron	Organtransplantation, Tumore	Leber, Niere
Antikörper	Rituximab (Anti-CD20), Daclizumab (Anti-CD25)	Organtransplantation, Tumore	Leber, Niere
Interferone	IFN-β, IFN-γ	Orqantransplantation	Leber, Niere
Phospho-Inositol-3 Kinase Inhibitoren	D-116883, AS605240, IPI-145	Tumore, Sepsis	Leber, Niere
Coxibe	Celecoxib, Etoricoxib	Akutes Nierenversagen	Niere
JNK-Inhibitoren	CC-401, Celgene	Malaria	Leber
Röntgenkontrastmittel	Peritrast	Diagnose z.B. von Tumore	Leber, Niere
Paramagnetische Röntgenkontrastmittel	Gadopentetat-Dimeglumin (Magnevist)	Diagnose z.B. von Tumore	Leber, Niere

The pharmaceutical active ingredient is particularly preferred to be a lipophilic, hydrophobic, hydrophilic, amphiphilic, anionic and/or cationic pharmaceutical active ingredient.

Under the term "pharmaceutical active ingredient," the invention relates to any inorganic or organic molecule, substance, or compound that exhibits a pharmacological effect. The term "pharmaceutical active ingredient" is used herein synonymously with the terms "medicinal product" and "medication."

The nanostructured carrier system according to the present invention represents a previously unique, multifunctional theranostic system for actively and selectively transporting various substances, particularly pharmaceutical active ingredients (e.g., hydrophilic or lipophilic small molecules as well as nucleic acids), into a specific target tissue. The transport of the pharmaceutical active ingredient is facilitated by targeting units, polymethine dyes, as part of the nanostructured carrier system, which interact with tissue-specific transporters on the target cell. By choosing the polymethine dye(s) (DY), the pharmaceutical active ingredient(s), and the polymer(s) or lipid(s), as well as varying their parameters, it is possible to produce nanoparticles or liposomes that are specifically tailored for the respective application, especially for the pharmaceutical active ingredient to be transported and/or the target tissue.In this way, it is possible to efficiently transport one or more pharmaceutical active ingredients as part of the nanostructured carrier system into a specific tissue or cell type (target tissue) and release them there. The pharmaceutical active ingredients can be those that, without being enclosed in a nanoparticle or liposome, have only low or no bioavailability, or exhibit low or no stability in vivo, or are intended to act only in specific organs or cells (target tissue). The specificity and accumulation of the nanostructured carrier system (nanoparticle or liposome) and/or its components, such as polymers, lipids, or pharmaceutical active substances, in the target tissue can be checked and monitored via the fluorescence properties in the red to infrared range of the non-toxic polymethine dye(s).also detected.

"Target tissue" refers to all tissues, organs, or cells into which the transport of a nanostructured carrier system and/or its components, particularly a pharmaceutical active ingredient, is possible and meaningful. Target tissues are especially all tissues, organs, or cells into which the transport of one or more pharmaceutical active ingredients is possible and meaningful, for example for the treatment or diagnosis of a disease. In the context of the invention, "target tissue" includes the liver, kidney, and tumors originating from these tissues, such as hepatocellular carcinomas or hypernephromas. The terms "target tissue," "target cell," "cells of a target tissue," and "organ" are used synonymously herein.

By conjugating the inventive polymethine dyes (hereinafter referred to as DY) to polymers or lipids, functionalized polymers (e.g., DY-PLGA, DY-PLA, DY-PCL) and functionalized lipids are produced. These are then used for the production of nanoparticles or liposomes, preferably using simple or double emulsification techniques or precipitation techniques. It is possible to individually adjust the nanoparticles or liposomes to a specific question. The diverse possibilities are exemplified in Table 1 (Figure 1). The polymethine dyes can be conjugated with a wide variety of different polymers or lipids, so that highly selective nanostructured carrier systems can be provided by the special combination of a polymethine dye with a lipid or polymer. The synthesis of functionalized polymers is schematically shown in Figure 2 and explained in detail in Example 1. The preparation of the inventive functionalized polymers or lipids, nanoparticles and liposomes, as well as the inclusion of pharmaceutical active ingredients, can be carried out according to conventional methods known from the prior art. Preferred manufacturing processes are disclosed in the examples and figures of the present invention.

Another disclosure relates to a pharmaceutical composition containing a nanostructured carrier system according to the invention as well as suitable excipients and additives.

Under "auxiliary and adjuvant substances," any pharmacologically compatible and therapeutically meaningful substance is understood to be a substance that is not a pharmaceutical active ingredient, but can be formulated together with the pharmaceutical active ingredient in the pharmaceutical formulation to influence the qualitative properties of the pharmaceutical preparation, particularly to improve them. Preferably, the auxiliary and/or adjuvant substances do not exert or, with regard to the intended treatment, do not have any significant or at least any unwanted pharmacological effect. Suitable auxiliary and adjuvant substances include, for example, pharmaceutically acceptable inorganic or organic acids, bases, salts and/or buffer substances. Examples of inorganic acids are hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid and phosphoric acid, with hydrochloric acid and sulfuric acid being particularly preferred. Examples of suitable organic acids are malic acid,Tartaric acid, maleic acid, succinic acid, acetic acid, formic acid, and propionic acid, and in particular preferably ascorbic acid, fumaric acid and citric acid. Examples of pharmaceutically acceptable bases are alkali hydroxides, alkali carbonates and alkali ions, preferably sodium. Mixtures of these substances can be used in particular for adjusting and buffering the pH value. Preferred buffer substances according to the invention are further PBS, HEPES, TRIS, MOPS and other physiologically compatible buffer substances. Further suitable excipients and additives are solvents or diluents, stabilizers, suspending agents, preservatives, fillers and/or binders as well as other conventional excipients and additives known in the art. The selection of the excipients and the amounts to be used depend on the pharmaceutical active ingredient and the type of administration.Pharmaceutical compositions of the present invention are preferably administered parenterally, particularly intravenously. For all parenteral applications, preparations in the form of suspensions and solutions as well as easily reconstitutable dry preparations are suitable.

The production of a pharmaceutical composition can be carried out by any method known in the art.

The dosage of the components of a pharmaceutical composition according to the invention depends on various factors, such as the type of active pharmaceutical ingredient, the disease, the condition of the patient (mammal, preferably human) to whom the pharmaceutical composition according to the invention is administered, and the route of administration, for example, parenteral, intravenous, or other methods. Such parameters are known to those skilled in the art, and therefore the determination of dosages falls within their general technical knowledge.

Another disclosure relates to the use of a nanostructured carrier system or a pharmaceutical composition according to the disclosure for the active and selective transport of the nanostructured carrier system or the pharmaceutical composition into a target tissue, wherein the transport is achieved by means of at least one polymethine dye as a targeting unit. Particularly preferably, the at least one polymethine dye facilitates the uptake of the nanostructured carrier system or the pharmaceutical composition into the cells of the target tissue via at least one tissue-specific transporter. In particular, the accumulation of the nanostructured carrier system and/or its components in a target tissue is detectable by means of the fluorescent properties of the at least one polymethine dye. As components of the nanostructured carrier system (nanoparticle or liposome), in addition to the at least one polymethine dye, at least one polymer, at least one lipid, and/or at least one pharmaceutical active ingredient are to be understood.

Another disclosure relates to a nanostructured carrier system or a pharmaceutical composition according to the disclosure for use as a medicinal product.

The nanostructured carrier system according to the invention or the pharmaceutical composition according to the disclosure is suitable for use in the treatment of diseases of the liver and/or kidney, preferably infectious diseases causing damage to the liver and/or kidney, such as malaria and hepatitis C, liver failure, for example drug-induced liver failure and fulminant liver failure, liver cirrhosis, for example alcohol-induced liver cirrhosis, metabolic disorders of the liver, such as Wilson's disease and Dubin-Johnson syndrome, excretory dysfunctions of the liver, liver tumors, primary liver tumors, for example hepatocellular carcinoma, angiosarcoma and hepatoblastoma, kidney tumors, primary kidney tumors, for example clear cell carcinoma, papillary carcinoma and chromophobic carcinoma, nephritis, chronic and acute renal failure, as well as diseases that cause consecutive damage to the liver and/or kidney, for example sepsis.

The nanostructured carrier systems and targeting units according to the invention, particularly polymethine dyes, offer a unique opportunity to combine diagnosis with therapy within a single molecule. Thus, predictions about the effectiveness of the therapy can be made based on the uptake of the free targeting structure, but also the therapy using the same targeting unit bound to the nanoparticle or liposome can be monitored and controlled. Due to the high flexibility of the targeting structure in the linker region, the targeting units can be chemically attached to various lipids and polymers. Furthermore, due to the chemical structure of the targeting unit, it remains very stable, in contrast to biological targeting units (e.g., antibodies or peptides), and is accessible for chemical purification and analysis. This enables high reproducibility and controllability in the synthesis.Due to the property of the targeting unit as a ligand for tissue-specific transporters, it can be excreted in vivo after desorption from the polymer, thereby avoiding intracellular accumulation and toxicity. With current developments in imaging, particularly in the field of multispectral optoacoustic tomography, the targeting unit can be directly detected. Furthermore, contrast agents for computer-assisted X-ray tomography or magnetic resonance tomography can also be incorporated into the nanoparticles or liposomes according to the invention, allowing them to be localized as well. Such a diverse and cell-specific system, which connects diagnosis and therapy via a fluorescent dye in the red to infrared range as a targeting unit, which in turn is efficiently excreted by the liver and kidneys due to its selectivity toward biomarkers, is unique so far.

The invention is further illustrated by means of figures: Figure 1 shows an overview of the possible variations of an inventive nanoparticle and their influence on the physicochemical properties of the nanoparticles themselves and on the biological consequences (Table 1). Figure 2 schematically represents the functionalization of the inventive polymers. A: Synthesis of the functionalized PLGA polymer via EDC coupling of the polymethine dye DY635 to the carboxylic acid end group of PLGA to obtain DY635-PLGA-NP (also referred to as DY635-PLGA; both terms are used synonymously in the present invention). B: SEC elution profile of the functionalized PLGA polymers with UV and IR detectors. The synthesis and functionalization are further described in detail in Example 1. Figure 3 shows the structure and preparation of the inventive nanoparticles.The individual ultrasound steps are marked by gray needles (arrows). A detailed description is also provided in Example 2. A: Structure of the nanoparticles and their preparation using a simple emulsification technique. The hydrophobic polymer is shown in dark gray, with the hydrophobic active ingredient represented in medium gray, and the surfactant (surfactant) in light gray within water. B: Structure of the nanoparticles and their preparation using a double emulsification technique. The hydrophobic polymer is shown in dark gray, with the hydrophilic active ingredient represented in white. The upper light gray layer is again water with surfactant. C: Overview (cross-sections) of possible variations of an inventive nanoparticle and their influence on the physicochemical properties of the nanoparticles themselves and on the biological consequences (Table 1).The hydrophobic polymer or lipid is shown in black, a possible pharmaceutical active ingredient in gray, galactose and DY635 as transporters for cell-specific uptake into hepatocytes, with only the nanoparticles according to the invention retaining cell specificity. Figure 4 shows the results of the characterization of a selection of nanoparticles according to the invention. The box plots include the 0.25 to 0.75 quantile. The median is indicated as a horizontal bar, and the mean as a square. The whiskers show the maximum and minimum values, respectively. A: The size of the PLGA nanoparticles does not differ from the siRNA/PEI-loaded PLGA nanoparticles (approximately 180 nm). In contrast, the DY635-PLGA nanoparticles are significantly larger (approximately 260 nm).B: The z-potential of PLGA nanoparticles is slightly negative. By using DY635-PLGA nanoparticles, the potential shifts to a weakly positive z-potential (no significance). Upon loading with siRNA/PEI polyplexes (siRNA/PEI + PLGA nanoparticles), the z-potential changes significantly and becomes strongly positive (+76 mV). C: To determine the endotoxin content, nanoparticle (NP) solutions at a concentration of 25 mg NP/ml, which is also used in vivo, were examined. The endotoxin contamination varied between samples from 0.4 to 0.6 ng/ml. However, the value was always below the FDA limit (2.5 ng/ml). D: NP solutions at 25 mg/ml are also used for the hemolysis and aggregation assay. For the assay, DY635-PLGA nanoparticles were used.as they are also used in the in vivo experiments. Further description can be found in Example 3. Figure 5 shows the uptake kinetics and characterization of RNAi in Hepa1-6 cells in vitro. A: Diagram (Heatmap) describing the time- and concentration-dependent RNAi. The axes show time in hours (h) versus siRNA concentration (ng/100,000 cells). The change in HMGCR expression compared to untreated controls is displayed in grayscale percentages (scale above the image). For the points in the heatmap, siRNA concentrations of 1, 5, 10, 25, 50, 100, 200, 400 ng / 100,000 cells are used and measured after 12, 16, 24, 32,40 or 48 hours were examined. For each time point, three independent replicates were generated. The results were then normalized against the HMGCR gene expression level of untreated Hepa1-6 cells and further normalized using HPRT gene expression. B: Uptake of nanoparticles by Hepa1-6 cells after 0 and 30 minutes (min). DY635 is visualized in the Cy5 channel on the LSM. Cell nuclei are stained with DAPI after washing and fixing the cells. Further description can be found in Example 4. Figure 6 shows the organ specificity and kinetics of a nanoparticle according to the invention in the liver, kidney, spleen, and heart. A: Comparison of the decay kinetics of DY635 versus DY635-PLGA-NP. Mean values from 3 ROIs in the liver.Error bars represent the SEM. B+C: Overlay of images from the Cy5 (DY635)-channel (B: light gray to white, C: light gray) and DAPI (background) channel on the IVM at different time points. D-G: 5 µm organ sections 10 minutes after injection of DY635-PLGA-NPs. On the images, DY635-PLGA-NPs or DY635 (Cy5-channel, green in the image) and cell nuclei (DAPI-stained, red in the image) are overlaid. F,G: Here, the staining is additionally overlaid with the liver structure visualized in phase contrast (blue in the image). Further description can be found in Example 5. Figure 7 shows the secretion pathway of a nanoparticle according to the invention. Secretion pathway of DY635-PLGA-NPs. A: For calculating the plasma disappearance rate, a standard curve is created in untreated plasma with DY635-PLGA nanoparticles.A standard series with DY635 in bile was used for bile secretion. B: shows the percentage of DY635 "recovery" (recovery) in the bile. The data points were calculated based on the data from A. The curve was approximated using OriginPro 8.5, QuickFit: Exponential Decay with Offset. Figure 8 shows the inventive targeting units, namely polymethine dyes. Figure 8 shows the general structure of a hepatocyte-targeting unit and the general structure of a renal parenchymal cell-targeting unit, including the linker to the polymer or lipid, as well as examples of such hepatocyte-targeting units and parenchymal cell-targeting units (Table 2). The targeting units have selectivity for a specific cell type (hepatocytes or renal parenchymal cells) and can transfer this cell selectivity to a nanoparticle or liposome.when they are connected via a chemical bond. The selectivity of the targeting unit arises from its interaction with influx transporters, which are expressed by the target cells. Furthermore, the targeting units have fluorescent properties in the red to infrared range. These fluorescent properties can also be transferred to the nanostructured carrier system, more precisely to the nanoparticle or liposome, thereby making not only the accumulation of the dye but also (if connected to the nanoparticle or liposome) the accumulation of the nanoparticle or liposome in the blood and tissue detectable. Figure 9 shows the effectiveness of the inventive nanostructured carrier systems in transporting a pharmaceutical active ingredient. A: Plasma cholesterol levels after two injections of the nanostructured carrier systems,which transported an siRNA against HMGCR or received two injections of a control substance. The figure shows a median bar plot; error bars represent the standard error of the mean, the numbers in the bars indicate the number of animals in each group, and significance was determined by a two-sided U-test, ** significance level 0.01. Figure 9A: The results show that the described approach makes it possible to significantly reduce plasma cholesterol concentration. The organ-specific nanostructured delivery system showed the strongest effect. As can be seen from Figure 9B, the organ-specific nanostructured delivery system achieved a strong and organ-specific effect in hepatocytes. In contrast, the non-specific nanostructured delivery system showed no specific and weaker down-regulation of HMGCR.Figure 10 shows a novel interaction of a novel polymethine dye, DY-635, used as a targeting unit, with a human basolateral transporter of hepatocytes. A and B show bar plots of the mean values, with error bars representing the standard error of the mean; all experiments were performed six times. The significance was determined by a two-tailed U-test, **p < 0.01, *p < 0.01.

The invention will be demonstrated below with examples, without being limited to them.

Examples Example 1: Synthesis of Functionalized Polymers

The synthesized nanoparticles are based on the hydrophobic polymer poly(lactic-co-glycolic acid) (PLGA), which is biocompatible and biodegradable. This polymer can be covalently linked to an amine-functionalized dye via coupling reagents such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) due to its active carboxylic acid group ("acid terminated"). In this case, the polymethine dye DY635 was used (see Figure 2). Each 100th polymer chain was functionalized. The polymers were then separated from free DY635 dye by dialysis and purified by precipitation. Characterization was performed using size-exclusion chromatography (SEC), where a UV/Vis detector and a refractive index (RI) detector were combined. The graphical representation of the synthesis and a SEC elution profile are shown in Figure 2.

Example 2: Production of nanoparticles

After the functionalization of the polymers (Example 1), nanoparticles were produced using simple (A) and double (B) emulsion methods. High-frequency ultrasound was used, which, with the help of surface-active substances (surfactants), here polyvinyl alcohol (PVA), promotes the formation of nano-scale particles. The hydrophobic polymers were dissolved in ethyl acetate, a solvent immiscible with water (25 mg/ml). As surfactant (tenside), 0.3% PVA (polyvinyl alcohol) in purified water was used, while the total polymer concentration was 2.5 mg/ml. The polymer suspension in ethyl acetate was added to water containing surfactant and nanoparticle formation occurred via ultrasound (A). If hydrophilic substances are to be encapsulated, the hydrophilic substance is first dissolved in water and then added to the polymer in ethyl acetate, followed by ultrasound treatment. After that, water containing surfactant is added again, and nanoparticle formation occurs via ultrasound. The results of the emulsion technique are shown in Figure 3.

The nanoparticles produced in this way, with a diameter of approximately 200 nm, were then stirred under an air stream until all the organic solvent (ethyl acetate) had evaporated, thus stabilizing the particles in water. To remove the excess surfactant, the nanoparticles were thoroughly washed at least twice with ultrapure water. This can be supported by vortexing and incubation in an ultrasonic bath. Finally, the particles were lyophilized, and their mass was determined.

Example 3: Characterization of Nanoparticles

Nanoparticles conjugated with DY635 (DY635-PLGA-NP) were produced and reproduced with constant parameters. The assays used for this purpose are listed below: Size: Measurement of the size of various nanostructured carrier systems dissolved in deionized water using dynamic light scattering (e.g., Zetasizer (Malvern Instruments GmbH)) or electron microscopic images. Shape: Determination of shape by electron microscopic images. Charge: Measurement of the charge of various nanostructured carrier systems dissolved in deionized water using the Zetasizer (Malvern Instruments GmbH) by determining the electrophoretic signal (ζ-potential, surface charge). Endotoxins: Endotoxin measurement using the LAL chromogenic assay according to Guilfoyle, D.E. et al., Evaluation of a chromogenic procedure for use with the Limulus lysate assay of bacterial endotoxins in drug products. J Parenter Sci Technol, 1985. 39(6): p. 233-6. Hemolysis: Measurement of hemoglobin concentration of erythrocytes incubated with the particles in physiological buffer for 1 hour. When the erythrocyte membrane is damaged, the measurable hemoglobin concentration in the supernatant increases. Aggregation: Measurement of the absorbance of erythrocytes incubated with the polymers in physiological buffer. Samples with cell aggregates show lower absorbance than homogeneously distributed, non-aggregated cells.

The results are shown in Figure 4. A: Size, B: Charge, C: Endotoxin contamination. For size determination, dynamic light scattering was used, and the charge was determined by zeta potential. D: Furthermore, it was shown that DY635-PLGA-NP do not have lytic properties in blood and do not cause aggregation of erythrocytes. E and F: In electron microscopic images (SEM), the particles show a round or spherical shape, both unloaded and without targeting (E) as well as with targeting and loaded (F).

Example 4: Induction of "Drug-Associated Effects" by RNAi and uptake in vitro ("Proof of Concept")

Execution for Figure 6A: Hepa1-6 cells were cultured under standard conditions (37°C, 5% CO2, DMEM with 4.5g/l glucose, 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin) in 6-well plates (100,000 cells per 9.6 cm²). After 24 hours, different concentrations of the nanostructured carrier system, which was prepared as described in Example 7B, were added to the wells and incubated for various periods (concentrations and incubation times are indicated in Figure 5A). After the incubation period, the cells were washed with Hank's Balanced Salt Solution (HBSS) and lysed with RLT buffer (Qiagen GmbH) supplemented with 1% β-mercaptoethanol. The mRNA was isolated from the lysate and analyzed by RT-qPCR. The values were then normalized to the expression level of hypoxanthine-guanine phosphoribosyltransferase, and the HMGCR expression level (HMGCR: 3-hydroxy-3-methylglutaryl-coenzyme A reductase or HMG-CoA) was compared to non-transfected Hepa1-6 cells.

Execution for Figure 6B: Hepa1-6 cells were cultured on Chamber Slides (Nunc, Thermo Scientific GmbH) under standard culture conditions (5,000 cells/1.5 cm²). After 24 hours, the cells were incubated with 100 µg/ml (final concentration) of DY-635-modified nanostructured delivery system (prepared as described in Example 7B). After 30 minutes of incubation under standard culture conditions with the nanostructured delivery system, the cells were washed with HBSS and fixed for 15 minutes with 5% formalin (pH 7). Subsequently, the slides were washed and the cell nuclei were stained with DAPI. For evaluation by laser scanning microscopy, the cells were mounted with VectaShield (Vector Labs, Inc.) and covered with a coverslip. The nanostructured delivery system was detected by modification with DY-635 at 633 nm (excitation), while the cell nuclei were visualized at 460 nm (excitation).

The results are shown in Figure 5. A: By siRNA transfection in Hepa1-6 cells, the HMGCR gene expression could be downregulated by up to 70%. HMGCR (3-hydroxy-3-methylglutaryl-Coenzyme-A reductase or HMG-CoA) represents the key enzyme of a central metabolic pathway - cholesterol biosynthesis. The downregulation of this metabolic gene demonstrated in this experiment shows the effectiveness of the active substance transport according to the invention through the nanostructured carrier systems. Furthermore, an increased plasma cholesterol level plays a central role in the development of atherosclerosis. This treatment method using the nanostructured carrier systems of the invention represents an interesting alternative to conventional treatment with statins, and also marks the first step towards gene transport for humans with hereditary elevated cholesterol levels (familial hypercholesterolemia). B: shows that DY635-PLGA-NPs are taken up by Hepa1-6 cells (mouse hepatoma cell line) within 30 minutes. Such a rapid and intense uptake of a nanoparticle has not been described in the prior art so far.

Example 5: In vivo targeting: Organ specificity and description of the secretion pathway

The preparation of the nanostructured carrier system for this experiment was carried out as described in Example 2 (B). For injection, the freeze-dried nanostructured carrier system was dissolved in a sterile 5% glucose solution (Glucosteril G5, Fresenius SE & Co KGaG) using an orbital mixer and an ultrasonic bath.

Execution (Figure 6A): Mice or rats were venously catheterized (jugular vein). Subsequently, the liver was prepared ex situ on an intravital microscope. Then, DY-635 (13 pmol/g body weight (BW)) or the nanostructured carrier system carrying a DY-635 modification (6.5 µg/g BW) was administered intravenously. The specific fluorescence of DY-635 at 633 nm in the liver was measured over time, and various regions of interest (ROIs) in the acquired images were quantified over time. Representative images of the measurement of DY-635 (Figure 6B) or the DY-635-modified nanostructured carrier system (Figure 6C) are shown in Figure 6. DY-635 or the DY-635-modified nanostructured carrier system was visualized by the fluorescence of DY-635 at 633 nm.The liver structure was visualized using the autofluorescence of NADH/NADH+ at 450 nm. To demonstrate organ specificity, male mice received 6.5 µg of the DY-635-modified carrier system per gram of body weight via a central venous catheter. Ten minutes after injection, the animal was humanely euthanized, and the organs were removed and cryo-prepared for histological processing. Subsequently, 5 µm thick sections of the organs were cut on a cryostat and counterstained with DAPI. All organs were then analyzed under identical settings regarding DAPI-stained cell nuclei (at 430 nm) and nanoparticles (at 633 nm).

The results are shown in Figure 6. C: The nanoparticles DY635-PLGA-NP are already taken up by hepatocytes after 1 minute (paving stone-like signal-rich areas, shown after 1 and 10 minutes (1 min, 10 min)). After approximately 50 minutes (50 min), almost all of the dye DY635 has been excreted from the liver. Similar results were observed with DY635. B: Overall, a similar picture is seen as in previous studies with the pure dye DY635. A: shows the decay rate of DY635 in the liver. The changed decay rate of DY635 intensity in the liver of DY635 and DY635-PLGA-NP shows that the dye DY635 remains bound to the polymer PLGA even intracellularly and is only released and excreted after hydrolysis of the PLGA. D-G: show organ-specificity, which was checked using different organ sections. In the liver (D), a strong accumulation is visible after injection of DY635-PLGA-NP (green). In the spleen (E), heart (F) and kidneys (G), however, hardly any (spleen, heart) or no (kidney) nanoparticles are visible.

Example 6: Secretion pathway of the nanoparticle DY635-PLGA

Through this experiment, the plasma disappearance rate and bile secretion of DY635-PLGA nanoparticles or the polymer dye DY635 were investigated. For this purpose, male rats (strain: RccHan:WIST) were instrumented (catheters placed in the V. jugularis, A. carotis, and Ductus choledochus). Subsequently, the substance to be tested was injected via the venous catheter. Then, blood was sampled at short time intervals from the arterial catheter and bile from the catheter in the Ductus choledochus. The blood was then further processed into plasma. The amount of dye DY635 was subsequently measured by fluorimetry using a calibration curve. DY635-PLGA-NPs were found to be maximum in the arterial blood after 4 minutes and were almost completely taken up by organs within 15 minutes (min) after DY635-PLGA-NP injection. Slightly delayed, as described earlier, since DY635 must first be released from the nanoparticles, DY635 is secreted into the bile (Figure 6, Figure A). The (calculated) recovery of DY635 in the bile of 95% also demonstrates the high specificity of DY635-PLGA-NPs for hepatocytes (Figure 6, Figure B).

Example 7: Incorporation of pharmaceutical active ingredients into nanoparticles

After functionalizing the polymers or lipids with the targeting unit (Example 1), nanoparticle are prepared by simple (A) and double (B) emulsification.

(A) Nanoparticles from a simple emulsion

If hydrophilic substances are to be encapsulated, the single-emulsion technique was used. Here, the active ingredient is enclosed in a hydrophobic polymer core by hydrophobic interactions. The active ingredient was dissolved together with the polymer in a suitable organic solvent. A suitable organic solvent is one that is neutral towards both the polymer and the active ingredient, i.e., it does not chemically alter them and has no effect on their stability. In this case, ethyl acetate was used. The mixture was coated with a hydrophilic solution. To stabilize the nanoparticles and increase the yield, a surfactant can be added to the hydrophilic solution, as is done with double-emulsion nanoparticles (see Double-Emulsion Nanoparticles). The two phases were mixed using high-energy ultrasound, which is emitted coaxially to an electrode vertically immersed into the sample. As a result, nanoparticles were formed.

(B) Double Emulsion Nanoparticles

For production, the hydrophobic polymers were dissolved in a suitable solvent at high concentration. A suitable solvent is an organic one that is neutral towards both the polymer and the active ingredient, i.e., it does not chemically alter them and has no effect on their stability. In this case, ethyl acetate was used. The concentration of the polymers depends on the size, hydrophilicity, solubility, and stability of the polymer. Suitable concentrations range between 2 and 50 mg/mL. The active ingredient was dissolved in purified water at a suitable concentration. A suitable concentration of the active ingredient depends on the chemical properties of the active ingredient and the capacity of the nanoparticles. Subsequently, the shell polymer dissolved in the organic solvent was coated onto the active ingredient dissolved in aqueous solution.The polymer and organic solvents had to be present in the sample in at least a 10-fold excess. By irradiating high-energy ultrasound coaxially with an electrode immersed perpendicularly into the sample, hydrophobic nanoparticles were formed outwardly. The active ingredient was thereby enclosed within a hydrophilic core by interacting with hydrophilic groups of the nanoparticle. In the second step, a suitable surfactant was dissolved in pure water at an appropriate concentration. A surfactant concentration is adequate if it is sufficient to produce nanoparticles. The concentration depends on the environmental conditions and must be determined experimentally. It is usually between 0.01 and 5% (w/v). Then, an appropriate amount of surfactant was added to the sample,that the concentration of polymer was only at least 1/10 of the original amount. Again, two phases formed, which were mixed by high-frequency ultrasound emitted coaxially to a vertically immersed electrode. By adding surface-active substances (surfactants), for example, surfactants such as polyvinyl alcohol, the formation of water-soluble nano-sized particles was ensured.

For illustration, an approach is described in which hydrophilic, polyethylenimine (PEI)-complexed small interfering RNA (siRNA) were encapsulated into PLGA nanoparticles. The PLGA was previously modified with DY-635, so that every 200th chain carries a dye molecule: (1) 2.4 µL PEI (1 mg/ml) were mixed with 2 µL siRNA (1 µg/µL) and then diluted with 45.6 µL of purified water. The resulting mixture is referred to as polyplexes, since the anionic siRNA interacts with the cationic PEI, and one PEI molecule binds and stabilizes the siRNA in a dense network. (2) 325 mg of DY-635-conjugated PLGA were dissolved in a total of 12.35 ml ethyl acetate. (3) 90 µL of the polymer solution from (2) were mixed with 50 µL of polyplexes from (1) using high-frequency ultrasound (as described above). (4) 1 mL of 0.3 wt% PVA in purified water was added to the mixture and ultrasound was applied. (5) The resulting nanoparticles were purified and freeze-dried.

Purification (for (A) and (B))

The nanoparticles produced in this way had a diameter depending on the shape and material of the vessels, the intensity of the ultrasound, and the substance concentration, and ranged in size from 120 to 220 nm. After production, the solvent was removed under sterile conditions. To remove excess surfactant, the nanoparticles were washed multiple times (at least twice) by centrifugation, decanting the supernatant, and resuspending the nanoparticles in sterile purified water. Finally, the particles were lyophilized and their mass was determined.

Example 8: Incorporation of pharmaceutical active ingredients into liposomes

After functionalizing the polymers or lipids with the targeting unit (Example 1), liposomes were prepared as follows. 1. Preparation of a 50 mM lipid solution, for example 1:1 DOPC:DSPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine:1,2-Distearoyl-sn-glycero-3-phosphocholine) + 30% cholesterol + 5% N-dodecanoyl-DOPE in chloroform/methanol (2:1 vol/vol). The DOPC can be modified with a polymethine dye prior to use. 2. Evaporation of the chloroform/methanol solvent (approx. 30 min, 90 rpm) in a rotary evaporator. 3. The lipids were then dissolved in 1 ml of a 7:3 vol/vol mixture of DMSO:EtOH. 4. Subsequently, the hydrophilic dextran was dissolved as an active ingredient in a suitable buffer, namely PBS (Phosphate Buffered Solution), at a concentration of 1 mg/ml. 5. Then, 0.3 ml of the lipid solution was added dropwise to the dextran solution. The dextran solution was kept in motion at 750 rpm on a magnetic stirrer during the dropwise addition. 6. The liposomes were then separated using a mini-extruder. 7. Subsequently, the liposome solution was aliquoted into 1 ml vials and subjected to 10 cycles of freezing in liquid nitrogen and thawing in warm water. 8. Afterwards, the liposomes were again extruded 10 times. 9. Then, the liposomes were dialyzed against PBS for 16 hours using a pre-prepared dialysis cassette (MWCO = 20 kDa). 10. Finally, the liposomes can be freeze-dried, stored, or used.

Example 9: Influence of cholesterol biosynthesis by the organ-specific transport of a siRNA targeting HMG-CoA reductase (HMGCR) in DY-635 modified nanostructured carrier systems

Male FVB/NRj mice (10 weeks old) were treated twice with the DY-635 modified nanostructured carrier system via intravenous injection, with a 24-hour interval between treatments. A dose of 6.5 µg of the nanostructured carrier system per gram of body weight was injected. The preparation of the carrier system was carried out as described in Example 7 (B), where 3 µg of siRNA against HMGCR or 3 µg of scrambled siRNA (siRNA without effect) were encapsulated together with 108 µg PEI in 3 mg DY-635-modified PLGA. Sixteen hours after the second injection, the animals were humanely euthanized and blood as well as organs were collected for analysis. Blood was collected in lithium-heparin monovettes and processed into plasma. For evaluation of the therapeutic efficacy, total cholesterol in plasma was determined, as well as changes in gene expression in various organs by qPCR for specificity. These values were compared to the cholesterol levels and HMGCR expression levels of healthy FVB/NRj mice (10 weeks old) and control groups. The control groups consisted of the following: Treatment with a DY-635-modified, thus hepatocyte-specific nanostructured carrier system and an ineffective scrambled siRNA; treatment with a nanostructured carrier system that did not contain any DY-635 modification and otherwise did not differ from the therapeutic construct; and animals that received only a 5% glucose solution.

Example 10: Demonstration of the Interaction of DY-635 with Hepatic Transporters

HEK-293T cells were transfected with human tissue-specific hepatocyte transporters. For Figure 10A, the uptake of the polymethine dye DY-635 as a targeting unit into these tissue-specific transporters was investigated. For this purpose, the cells were seeded onto 96-well plates, incubated for 24 hours under standard conditions, and then incubated for 5 minutes with DY-635 (final concentration in the well: 10 µmol/l) after medium exchange. Subsequently, the cells were lysed and the lysates were measured using fluorimetry. The amount of internalized DY-635 could be quantified using a DY-635 standard curve. As a control, the respective transporters were specifically inhibited (inhibitors and used final concentrations are described in Table 3 below). In this experiment, it was shown that DY-635 is a substrate for NTCP.The uptake via OCT1 can be considered negligible. In Figure 10B, it was investigated whether DY-635 binds as an inhibitor to the basolateral hepatocytic transporters. For this purpose, HEK-293T cells transfected with the tissue-specific transporters were seeded and incubated as described. After 24 hours, the cells were incubated for 5 minutes either with a radiolabeled, transporter-specific substrate, or with the radiolabeled specific substrate together with a specific inhibitor, or with DY-635 (final concentration of 10 µmol/l) (the substrates used and their concentrations are described in Table 3 below). The cells were then washed and lysed in well. For quantification of the uptake, the radioactive radiation of the substrates was measured.Here, it became evident that DY-635 is a strong inhibitor of OATP1B1 and OATP1B3. Additionally, OAT2 and OCT1 are also inhibited by DY-635. This demonstrates the strong interaction of DY-635 with tissue-specific hepatocytic transporters. It can be concluded that upon exposure of DY-635 at the surface of a nanostructured delivery system, it becomes immobilized on the cell surface of hepatocytes, leading to the subsequent endocytosis of the nanoparticle. Tabelle 3

OATP1B1	Rifampicin/ 5 µM
OATP1B3	Rifampicin/ 5 µM
OAT2	Indomethacine/100 µM
NTCP	Cyclosporin A/ 50 µM
NaDC3	Succinat/100 µM
OCT1	Decynium22/ 50 µM

Claims (8)

1. Nanostructurered delivery system, comprising at least one polymer, and/or at least one lipid and at least one polymethine dye for use in the treatment of diseases of the liver and/or the kidneys, wherein the at least polymethine dye, as a targeting unit, causes the transport of the nanostructured delivery system into the target tissue, and wherein the at least one polymethine dye is a symmetrical or asymmetrical polymethine of the general structure I, II, III or IV: wherein

   a. n stands for the numerical values 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

   b. R¹-R¹⁷ may be the same or different and maybe hydrogen, one or more alkyl, tert-alkyl, cycloalkyl or aryl-, carboxyaryl-, dicarboxyaryl-, heteroaryl- or heterocycloaliphatic radicals, alkyloxy-, alkylmercapto, heteroaryloxy-, heteroarylmercapto-, hydroxyl-, nitro- or cyano- group, an alkyl-substituted or cyclic amine function and/or two ortho-position radicals, e.g., R³ and R⁴, R¹³ and R¹⁴ and/or R¹ and R² and R¹¹ and R¹² and/or R⁷ and R⁹ together may form an additional aromatic, heteroaromatic, aliphatic or heteroaliphatic ring,

   c. at least one of the R¹ -R¹⁷ substituents has a solubilizing and/or ionizable or ionized substituent such as SO₃ ^- , (- SO₃H), PO₃ ^2-, COOH, OH or NR₃ ⁺, cyclodextrins or sugar, which determines the hydrophilic properties of these polymethine dyes, wherein this substituent may be bound to the polymethine dye also by a spacer group, and

   d. at least one of the R¹-R¹⁷ substituents has a reactive group (linker) such as isocyanates, isothiocyanates, hydrazines, amines, mono- and dichloro- or mono- and dibromotriazines, aziridines, epoxides, sulfonyl halides, acid halides, carboxylic anhydrides, N-hydroxysuccinimide esters, imido esters, carboxylic acids, glyoxal, aldehyde, maleimide or iodacetamide and phosphoramidite derivatives or azides, alkynes or olefins, wherein this substituent may be bound to the polymethine dye also by a spacer group,

   e. the aromatic, heteroaromatic, aliphatic or heteroaliphatic spacer group consists of structural elements such as [(CH₂)_a) - Y- (CH₂)_b]_c or [(C₆H₄)_a) - Y- (C₆H₄)_b], where Y may be the same or different and comprises CR₂-, O-, S-, SO₂, SO₂NH-, NR-, COO- or CONR functions, wherein it is bound to one of the R¹-R¹⁷ substituents, and a and b may be the same or different and have numerical values of 0-18 and numerical values for c of 0-18,

   f. the R⁸ and R⁹ substituents with corresponding n=2, 3, 4 or 5, may also be present 2x, 3x, 4x or 5x, and wherein these may be the same or different, and wherein

   the nanostructured delivery system comprises at least pharmaceutically active ingredient.
2. Nanostructured delivery system for the use according to claim 1, wherein the at least one polymethine dye causes the uptake of the nanostructured delivery system into the cells of the target tissue via at least one tissue-specific transporter.
3. The nanostructured carrier system for the use according to any one of the preceding claims, wherein the at least one polymethine dye is selected from the group consisting of DY-635, DY-680, DY-780, DY-880, DY-735, DY-835, DY-830, DY-730, DY-750, DY-850, DY-778, DY-878, DY-704, DY-804, DY-754, DY-854, DY-700, DY-800, ICG and DY-IRDYE 800CW.
4. The nanostructured delivery system for the use according to any one of the preceding claims, wherein the at least one polymer is selected from the group consisting of polyesters, poly(meth)acrylates, polystyrene derivatives, polyamides, polyurethanes, polyacrylonitriles, polytetrafluoroethylenes, silicones, polyethylene glycols, polyethylene oxides and polyoxazolines and copolymers thereof, preferably in various compositions, such as random, gradient, alternating, block, graft or stem copolymers, or wherein that at least one lipid is selected from the group consisting of saturated and unsaturated fatty acids, preferably cholesterol, palmitic acid, phospholipids, sphingolipids and glycolipids.
5. The nanostructured delivery system for the use according to any one of the preceding claims, wherein the at least one tissue-specific transporter is selected from the group consisting of OATP1B1, OATP-C, OATP2, LST-1, OATP1B3, OATP8, OATP2B1, OATP1A2, NaDC3, SDCT2, NTCP, OCT1, OCT3, OAT2, OAT1, OAT3, PGT, OCT2, OAT1, OATP4A1, OATP4C1.
6. Nanostructured delivery system for the use according to any one of the preceding claims, wherein the at least one pharmaceutically active ingredient is selected from the group consisting of low-molecular substances, in particular inducers or contrast agents, and higher molecular substances, in particular nucleic acids and proteins, preferably selected from the group consisting of glucocorticoids, cytostatics, antimetabolites, intercalants, antibodies, interferons, phosphoinositol 1-3 kinase inhibitors, coxibes, JNK inhibitors.
7. The nanostructured delivery system for the use according to any of the preceding claims, wherein the accumulation of the nanostructured delivery system and/or its components in a target tissue is detectable by means of the fluorescent properties of the at least one polymethine dye.
8. The nanostructured delivery system for the use according to any one of claims 1 to 7, wherein the disease is selected from the group consisting of infectious diseases with damage to the liver and/or kidney, liver failure, liver cirrhosis, metabolic diseases of the liver, excretory dysfunctions of the liver, liver tumors, primary liver tumors, kidney tumors, primary kidney tumors, nephritis, chronic and acute kidney failure and diseases, which cause a consecutive damage to the liver and/or kidney.