Cell-specific targeting using nanostructured delivery systems

Abstract

The invention relates to a nanostructured delivery system comprising at least one polymer and/or at least one lipid and at least one polymethine dye, wherein the at least one polymethine dye acting as a targeting unit brings about the targeted transport of the nanostructured delivery system into a target issue. The invention also relates to a pharmaceutical composition and the uses of the nanostructured delivery system for transporting said system and, optionally, a pharmaceutical active ingredient into the target tissue, as well for treating liver and/or kidney diseases.

Claims

1. A method for targeted transport into a target tissue, comprising contacting a target tissue with a nanostructured delivery system comprising at least one polymer and/or at least one lipid and at least one polymethine dye, wherein the at least polymethine dye triggers 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, or III: ##STR00002## where a. n stands for the numerical values 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; b. R.sup.1-R.sup.17 may be the same or different and may be hydrogen, one or more alkyl, tert-alkyl, cycloalkyl, olefinic structures, aryl, carboxyaryl, dicarboxyaryl, heteroaryl or heterocycloaliphatic radicals, alkyloxy, alkylmercapto, arlyoxy, arylmercapto, heteroaryloxy, heteroarylmercapto groups, hydroxyl, nitro or cyano group, an alkyl-substituted or cyclic amine function and/or two ortho-position radicals, together may form an additional aromatic, heteroaromatic, aliphatic or heteroaliphatic ring; wherein alkyl or cycloalkyl radicals comprising fused ring systems between R.sup.3 and R.sup.4 or R.sup.13 and R.sup.14 form only one ring fusion; c. at least one of the R.sup.1-R.sup.17 substituents has a solubilizing and/or ionizable or ionized substituent, which determines the hydrophilic properties of these polymethine dyes, wherein this substituent may also be bound to the polymethine dye by a spacer group; d. at least one of the R.sup.1-R.sup.17 substituents has a reactive group (linker), wherein this substituent may also be bound to the polymethine dye by a spacer group; and e. an aromatic, heteroaromatic, aliphatic or heteroaliphatic spacer group is bound to one of the R.sup.1-R.sup.17 substituents; f. the R.sup.8 and R.sup.9 substituents with corresponding n=2, 3, 4 or 5, may also be present 2×, 3×, 4× or 5×, and these may be the same or different.

2. The method according to claim 1, wherein the at least one polymethine dye triggers the uptake of the nanostructured delivery system into the cells of the target tissue.

3. The method according to claim 1, wherein the nanostructured delivery system additionally comprises at least one active pharmaceutical ingredient.

4. The method according to claim 1, wherein the at least one polymethine dye 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.

5. The method according to claim 1, wherein the at least one polymer is selected from the group consisting of polyesters, poly(meth)acrylates, polystyrene derivatives, polyamides, polyurethanes, polyacrylonitriles, olytetrafluoroethylenes, silicones, polyethylene glycols, polyethylene oxides and polyoxazolines and their copolymers or the at least one lipid is selected from the group consisting of saturated and unsaturated fatty acids.

6. The method according to claim 2, 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, OATP4A1, and OATP4C1.

7. The method according to claim 3, wherein the at least one active pharmaceutical ingredient is selected from the group consisting of inductors, contrast media, nucleic acids, proteins.

8. The method of claim 1, wherein accumulation of the nanostructured delivery system and/or its components in the target tissue is detectable by means of the fluorescence properties of the at least one polymethine dye.

9. The method according to claim 3, wherein the target tissue is liver and/or kidneys of a subject with a disease of the liver and/or kidneys, and the method serves to treat the disease of the liver and/or kidneys.

10. The method according to claim 9, wherein the disease is selected from the group consisting of infectious diseases involving damage to the liver and/or kidneys, liver failure, cirrhosis of the liver, metabolic diseases of the liver, excretory dysfunctions of the liver, liver tumors, primary liver tumors, kidney tumors, primary kidney tumors, nephritis conditions, chronic and acute renal failure and diseases that cause subsequent damage to the liver and/or kidneys.

11. The method according to claim 1, wherein the solubilizing and/or ionizable or ionized substituent is selected from the group consisting of SO.sub.3.sup.−, (—SO.sub.3H), PO.sub.3.sup.2, COOH, OH or NR.sub.3.sup.+, cyclodextrins, and sugar.

12. The method according to claim 1, wherein the reactive group is selected from the group consisting of isocyanates, isothiocyanates, hydrazines, amines, mono- and dichloro- or mono- and dibromotriazines, aziridines, epoxies, sulfonyl halides, acid halides, carboxylic anhydrides, N-hydroxysuccinimide esters, imido esters, carboxylic acids, glyoxal, aldehyde, maleimide or iodacetamide and phosphoramidite derivatives, azides, alkynes or olefins.

13. The method according to claim 1, wherein the aromatic, heteroaromatic, aliphatic, or heteroaliphatic spacer group consists of structural elements of formula [(CH.sub.2).sub.a—Y—(CH.sub.2).sub.b].sub.c or [(C.sub.6H.sub.4).sub.a—Y—(C.sub.6H.sub.4).sub.b], where Y may be the same or different and comprises CR.sub.2—, O—, S—, SO.sub.2, SO.sub.2NH—, NR—, COO— or CONR functions; a and b may be the same or different and have numerical values of 0-18, and wherein c has a numerical value of 0-18.

14. The method according to claim 5, wherein the polymer comprises random, gradient, alternating, block, graft or star copolymers.

15. The method according to claim 5, wherein the saturated and unsaturated fatty acids are selected from the group consisting of cholesterol, palm ethyl acid, phospholipids, sphingolipids, and glycolipids.

Description

(1) The invention will also be illustrated as an example on the basis of the figures:

(2) FIG. 1 shows an overview of the possible variations of a nanoparticle according to the invention and its influence on the physicochemical properties of nanoparticles themselves and on the biological consequences (Table 1).

(3) FIG. 2 shows schematically the functionalization of the polymers according to the invention. A: Synthesis of the functionalized PLGA polymer by EDC coupling of the polymethine dye DY635 to the carboxylic acid terminal group of the PLGA to form DY635-PlGA-NP (or also referred to as DY635-PLGA; the two terms are used as synonyms in the present invention). B: SEC elugram of the functionalized PLGA polymers with UV and IR detectors. Synthesis and functionalization are also described in detail in Example 1.

(4) FIG. 3 shows the design and production of nanoparticles according to the invention. The individual ultrasonic steps are characterized by gray needles (arrows). A detailed description can also be found in Example 2. A: Structure of the nanoparticles and their production by means of single emulsion technique. The hydrophobic polymer is shown in the dark gray with the hydrophobic active ingredient shown in the medium gray and the surfactant in water shown in the light gray. B: Structure of the nanoparticle and its production by means of the double emulsion technique. This shows the hydrophobic polymer in the dark gray and the hydrophobic active ingredient shown in white. The upper light gray layer is then again water with surfactant. C: Overview (cross sections) of the possible variations on a nanoparticle according to the invention and the influence thereof on the physicochemical properties of the nanoparticle itself and on the biological consequences (Table 1). The hydrophobic polymer or lipid is shown in black, and a possible active pharmaceutical ingredient, namely galactose and DY635 as the transporter for cell-specific uptake into hepatocytes, is/are shown in gray, where only the nanoparticles according to the invention retain their cell specificity.

(5) FIG. 4 shows the results of characterization of a selection of nanoparticles according to the invention. The box plots comprise the 0.25 to 0.75 quantile. The median is plotted as a horizontal bar, the mean as a cube. The whiskers each represent a highest and lowest value, respectively. A: The size of the PLGA nanoparticles does not differ from that of the siRNA/PEI-loaded PLGA nanoparticles (approximately 180 nm). However, the DY635-PLGA nanoparticles are significantly larger (approximately 260 nm). B: The zeta potential of PLGA nanoparticles is slightly negative. Due to the use of DY635-PLGA nanoparticles, the potential fluctuates into the weakly positive zeta potential (not significant). Due to the loading with the siRNA/PEI polyplexes (siRNA/PEI+PLGA nanoparticle), the zeta potential changes significantly and becomes strongly positive (+76 mV). C: For determining the amount of endotoxin, nanoparticle (NP) solutions in a concentration of 25 mg NP/mL, such as those also used in vivo, were investigated. The endotoxin load fluctuated between 0.4 mg/mL and 0.6 mg/mL in the samples. However, the value was always below the FDA limit value (2.5 ng/mL). D: NP solutions of 25 mg/mL are also used for the hemolysis and aggregation assays. Nanoparticles were used for the DY635-PLGA assay, such as those also used in the in vivo experiments. E and F: electron micrographs (SEM), E: unloaded, without targeting. F: loaded, with targeting. A further description of this can be found in Example 3.

(6) FIG. 5 shows the uptake kinetics and characterization of the RNAi into Hepa 1-6 cells in vitro. A: Diagram (heat map) describing the time- and concentration-dependent RNAi. The time is plotted in hours (h) on one axis against the siRNA concentration (ng/100,000 cells) on the other axis. The change in HMGCR expression in comparison with untreated controls is imaged in gray scale in percent (scale shown above the graph). For the points in the heat map, siRNA concentrations of 1, 5, 10, 25, 50, 100, 200 and 400 ng/100,000 cells were used and were tested after 12, 16, 24, 32, 40 or 48 hours. Three independent replicates were generated for each point in time. The result was then relativized against the HMGCR gene expression level of untreated Hepa 1-6 cells and normalized with the help of the HPRT gene expression. B: Uptake of the nanoparticles into Hepa 1-6 cells after 0 and 30 minutes (min). DY635 is visualized on LSM in the Cy5 channel. The cell nuclei were stained with DAPI after washing and fixing the cells. A further description of this process can be found in Example 4.

(7) FIG. 6 shows the organ specificity and kinetics of a nanoparticle according to the invention in the liver, kidneys, spleen and heart. A: Comparison of the decay kinetics of DY635 versus DY635-PLGA-NP. Averages from 3 ROIs in the liver. Error bars show the SEM. B+C: Superimposing the images of Cy5 (DY635) channel (B light gray to white, C light gray) and DAPI (background) channel on the IVM at different points in time. D-G: 5 μm organ sections 10 minutes after DY635-PLGA-NP injection. DY635-PLGA-NP and/or DY635 (Cy5 channel, shown in green in the image) and the cell nuclei (DAPI-stained, shown in red in the image) are superimposed in the images. F, G: The stainings here are additionally superimposed on the liver structure (shown in blue in the image) visualized in phase contrast. A further description can be found in Example 5.

(8) FIG. 7 shows the secretion route of a nanoparticle according to the invention. The secretion route of DY635-PLGA-NP: A: For calculating the plasma shrinkage rate, a standard curve is prepared in untreated plasma with DY635-PLGA nanoparticles. A standard series with DY635 in bile was used for the bile secretion. B: This shows the percentage DY635 “recovery” in the bile. The measurement points were computer-generated based on data from A. The curve was approximated by using OriginPro 8.5, QuickFit: exponential decay with offset approximated.

(9) FIG. 8 shows targeting units according to the invention, namely polymethine dyes. FIG. 8 shows the general structure for a hepatocyte targeting unit and the general structure for a renal parenchyma cell targeting unit, specifying the linker to the polymer and/or lipid as well as examples of such hepatocyte targeting units and parenchyma cell targeting units (Table 2). The targeting units have a selectivity for one cell type (hepatocytes or renal parenchyma cells) and can transfer this cell selectivity to a nanoparticle or a liposome when they are bound to it by a chemical bond. The selectivity of the targeting unit occurs due to the interaction with influx transporters which are expressed by the target cells. The targeting units also have fluorescence properties in the red to infrared range. These fluorescence properties can be transferred to the nanostructured delivery system, more specifically to the nanoparticle or the liposome, so that not only can an accumulation of the dye be detected in the blood and in the tissue but also (if it is bonded to the nanoparticle and/or the liposome) accumulation of the nanoparticle and/or of the liposome can also be detected.

(10) FIG. 9 shows the efficacy of the nanostructured delivery systems according to the invention in the transport of an active pharmaceutical ingredient. A: Plasma cholesterol levels after two injections of the nanostructured delivery systems which transport a siRNA toward the HMGCR and/or after two injections of a controlled substance. This figure shows a median bar plot, error bars describe the mean error of the standard deviation, the numbers in the bars describe the number of animals in each group, the significances were determined by a two-tailed U-test, ** significance level 0.01.

(11) FIG. 9 A: These results show that it is possible through the approach described here to significantly lower the plasma cholesterol concentration. The organ-specific nanostructured delivery system shows the greatest effect. It is clear from FIG. 9 B that through the organ-specific nanostructured delivery system, an organ-specific and strong effect is achieved in hepatocytes. However, the nonspecific nanostructured delivery system does not show any specific or weaker down-regulation of the HMGCR.

(12) FIG. 10 shows the interaction according to the invention of a polymethine dye according to the invention as the targeting unit, DY-635, with a human basolateral hepatocyte transporter. FIGS. 10 A, B show bar plots of the mean value, with error bars describing the mean error of the standard deviation. All experiments here were performed six times each. The significances were determined by a bilateral U-test, **significance level 0.01, *significance level 0.01.

(13) The invention is demonstrated below on the basis of examples, although it is not limited to them.

EXAMPLES

Example 1: Synthesis of Functionalized Polymers

(14) The synthesized nanoparticles are based on the hydrophobic polymer poly(lactic-co-glycolic acid) (PLGA), which is biocompatible and biodegradable. This polymer can be bound covalently to an amine-functionalized dye on the basis of its active carboxylic acid group (“acid terminated”) by means of coupling reagents such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide). The polymethine dye DY-635 was used here (see FIG. 2). Every 100th polymer chain was functionalized. The polymers were then separated from the free dye DY-635 by dialysis and purified by precipitation. The characterization was based on size exclusion chromatography (SEC), in which a UV/Vis detector and an RI (refractive index) detector were combined. Figure shows the graphic plot of the synthesis and an SEC elugram.

Example 2: Production of Nanoparticles

(15) After functionalization of the polymers (Example 1), nanoparticles were produced by a single emulsion (A) and by a double emulsion (B) using high-frequency ultrasound, which promotes the formation of nanoscale particles with the help of surface-active substances (surfactants), i.e., polyvinyl alcohol (PVA) here. The hydrophobic polymers were therefore dissolved in ethyl acetate, a solvent that is not miscible with water (25 mg/mL). The surfactant used was 0.3% PVA (polyvinyl alcohol) in ultrapure water, where the total polymer concentration was 2.5 mg/mL. The polymer suspension in ethyl acetate was added to water with surfactant and nanoparticles were formed by using ultrasound (A). If hydrophilic substances were enclosed, then the hydrophilic substance was first dissolved in water and then added to the polymer in ethyl acetate and treated ultrasonically. Next, water with surfactant was added and nanoparticles were again formed by using ultrasound. FIG. 3 shows the results obtained by this emulsion technique.

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

Example 3: Characterization of the Nanoparticles

(17) Nanoparticles of DY-635-conjugated PLGA (DY635-PLGA-NP) were produced with constant parameters and reproduced. The assays used for this are explained below: Size: measurement of the size of the various nanostructured delivery systems dissolved in deionized water by dynamic light scatter (for example, Zetasizer (Malvern Instruments GmbH)) or by electron micrographs. Shape: determination of shape by electron micrographs. Charge: measurement of the various nanostructured delivery systems dissolved in deionized water using a Zetasizer (Malvern Instruments GmbH) by determining the electrophoretic signal (zeta potential, surface charge). Endotoxins: endotoxin measurement by LAL chromogenic assay according to D. E. Guilfoyle, et al., Evaluation of a chromogenic procedure for use with the Limulus lysate assay of bacterial endotoxins drug products. J Parenter Sci Technol, 1985, 39(6): pp. 233-6. Hemolysis: measurement of the hemoglobin concentration of erythrocytes which were incubated with the particles in physiological buffer for one hour. The measurable hemoglobin concentration in the supernatant increases when there is damage to the erythrocyte membrane. Aggregation: Measurement of the absorption of erythrocytes incubated with the polymers in physiological buffer. Samples with cell aggregates show a lower absorption than homogeneously distributed unaggregated cells.

(18) The results are shown in FIG. 4. A: size, B: charge, C: endotoxin load. For the size measurement, the dynamic light scatter was used and the charge was determined by means of the zeta potential. D: in addition it was shown that DY635-PLGA-NP does not have any lysine properties in blood and these do not lead to aggregation of erythrocytes. E and F: the particles have a round or spherical shape in the electron micrographs (SEM), both unloaded and without targeting (E) as well as loaded and with targeting (F).

Example 4: Triggering Drug-Associated Effects by RNAi and Uptake In Vitro (“Proof of Concept”)

(19) Procedure for FIG. 6 A: Hepa 1-6 cells were cultured under standard culture conditions (37° C., 5% CO.sub.2, DMEM 4.5 g/L glucose, 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin) in 6-well plates (100,000 cells per 9.6 cm.sup.2). After 24 hours, various concentrations of the nanostructured delivery system which was prepared as described in Example 7 B were added to the wells and incubated for various periods (concentrations and incubation times can be seen in FIG. 5 A). After the incubation time, the cells were washed with Hank's Balanced Salt Solution (HBSS) and lysed with RLT buffer (Qiagen GmbH), to which 1% (3-mercaptoethanol was added. The mRNA was isolated from the lysate and analyzed in RT-qPCR. The values were then normalized on the hypoxanthin-guanine-phosphoribosyl transferase expression level and the HMGCR expression level (HMGCR: 3-hydroxy-3-methylglutaryl-coenzyme-A-reductase coenzyme or HMG-CoA) was compared with nontransfected Hepa 1-6 cells.

(20) Procedure for FIG. 6 B: Hepa 1-6 cells were cultured under standard culture conditions on chamber slides (Nunc, Thermo Scientific GmbH) (5,000 cells/1.5 cm.sup.2). After 24 hours, the cells were mixed with 100 μg/mL (final concentration) DY-635-modified nanostructured delivery system (produced as described in Example 7 B). After incubating for 30 minutes under standard culture conditions with the nanostructured delivery system, the cells were washed with HBSS and fixed with 5% formalin (pH 7) for 15 minutes. Then the microscope slides were washed with the cells and the cell nuclei were stained with DAPI. For analysis by laser scanning microscopy, the cells were moistened with VectaShield (Vector Labs, Inc.) and sealed with a cover glass. The nanostructured delivery system was detected by the modification with DY-635 at 633 nm (excitation) and the cell nuclei were visualized at 460 nm (excitation).

(21) The results are shown in FIG. 5. A: The HMGCR gene expression could be downregulated by up to 70% by siRNA transfection in Hepa 1-6 cells. HMGCR (3-hydroxy-3-methylglutaryl-coenzyme-A-reductase or HMG-CoA) embodies the key enzyme of a central metabolism—the cholesterol biosynthesis. The downregulation of this metabolic gene demonstrated in this experiment shows the efficacy of the transport of the active ingredient according to the invention by the nanostructured delivery system. Furthermore, an elevated plasma cholesterol level is of central importance in the development of arteriosclerosis. The treatment method presented here using the nanostructured delivery systems according to the invention is an interesting alternative to traditional treatment with statins and in particular this is the first in the direction of gene transport for humans with a congenitally elevated cholesterol level (familial hypercholesterolemia). B: This shows that DY635-PLGA-NP is taken up by Hepa 1-6 cells (murine hepatocyte cell line) within 30 minutes. Such a rapid and intense uptake of a nanoparticle has not been described before in the prior art.

Example 5: In Vivo Targeting: Organ Specificity and Description of the Secretion Route

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

(23) Procedure (FIG. 6 A): Mice or rats were catheterized through a vein (jugular vein). Next the liver was prepared ex situ using an intravital microscope. Next DY-635 (13 pmol/g body weight (BW)) or the nanostructured delivery system carrying a DY-635 modification (6.5 μg/g BW) was injected venously and the specific fluorescence of DY-635 was measured at 633 nm in the liver over time and various Regions of Interest (ROIs) were quantified in the images recorded over time. FIG. 6 shows representative images of the measurement of DY-635 (FIG. 6 B) or DY-635-modified nanostructured delivery systems (FIG. 6 C). DY-635 or the DY-635-modified nanostructured delivery system was represented by the fluorescence of DY-635 at 633 nm. The liver structure was represented by the autofluorescence of NADH/NADH+ at 450 nm. To represent the organ specificity, male mice were injected with 6.5 μg of the DY-635-modified delivery system per g BW through a central venous catheter. The animal was euthanized painlessly 10 minutes after the injection and the organs were removed and cryoprepared for the histological workup. Next, 5-μm-thick sections of the organs were prepared with the cryotome and these were counterstained with DAPI. Then all the organs were examined at the same settings with regard to the DAPI-stained cell nuclei (at 430 nm) and with regard to the nanoparticle (at 633 nm).

(24) The results are shown in FIG. 6. C: The DY635-PLGA-NP nanoparticles were taken up in the hepatocytes after just 1 minute (cobblestone-type signal-rich areas), imaging after 1 and 10 minutes (1 min, 10 min). After about 50 minutes (50 min), almost all the DY635 dye has been eliminated from the liver. Similar results were found with DY635. B: On the whole, the results are similar to those obtained in the earlier studies with the pure dye DY635. A: This shows the decay rate of DY635 in the liver. The altered decay rate of the DY635 intensity in the liver from DY635 and DY635-PLGA-NP shows that the DY635 dye is also still bound intracellularly to the PLGA polymer and is released and eliminated only after hydrolysis of the PLGA. D-G: This shows the organ specificities which were verified by means of various organ sections. After injection of DY635-PLGA-NP (green), the liver shows a marked accumulation. However, hardly any nanoparticles (spleen, heart) or no nanoparticles at all (kidneys) are visible in the spleen (E), the heart (F) and the kidneys (G).

Example 6: Secretion Route of the Nanoparticle DY635-PLGA

(25) On the basis of this experiment, the plasma decay rate and the bile secretion of the DY635-PLGA nanoparticle and/or of the polymethine dye DY635 was investigated. This was done using male rats (strain: RccHan:WIST) with instrumentation (catheter in the jugular vein, carotid artery, common bile duct). Next, the substance to be tested is injected through the venous catheter. Then blood is taken from the arterial catheter at short intervals, and bile is taken from the catheter in the common bile duct. The blood is then processed further to plasma. The amount of DY635 was then measured by fluorimetry based on a calibration curve. DY635-PLGA-NP could be detected in the arterial blood at most after 4 minutes and was taken up almost completely into the organs up to 20 minutes after injection of DY635-PLGA-NP, i.e., within 15 minutes (min). There was a slight delay because as already described, DY635 must first be released from the nanoparticles and then DY635 is secreted into the bile (FIG. 6, illustration A). The (calculated) 95% recovery of DY635 in the bile also shows the high specificity of DY635-PLGA-NP for hepatocytes (FIG. 6, illustration B).

Example 7: Inclusion of Active Pharmaceutical Ingredients in Nanoparticles

(26) After functionalization of the polymers or lipids with the targeting unit (Example 1), nanoparticles were produced by single emulsion (A) and double emulsion (B).

(27) (A) Nanoparticles from a Single Emulsion

(28) If hydrophilic substances were to be included, the single emulsion technique was used. In this case the active ingredient is enclosed in a hydrophobic polymer core by hydrophobic interactions. The active ingredient was then dissolved together with the polymer in a suitable organic solvent. An organic solvent is suitable when it is neutral with respect to both the polymer and the active ingredient, i.e., it does not trigger any chemical changes therein and has no influence on their stability. Ethyl acetate was used in the present case. The mixture was overlayered with the hydrophilic solution. For stabilization of the nanoparticles and to increase the yield, a surfactant may be added to the hydrophilic solution in the case of double emulsion nanoparticles (cf. double emulsion nanoparticles). The two phases were combined by high-energy ultrasound emitted coaxially with an electrode immersed perpendicularly into the sample. This resulted in nanoparticles.

(29) (B) Double Emulsion Nanoparticles

(30) For production, the hydrophobic polymers were dissolved in high concentration in a suitable solvent. An organic solvent is suitable when it is neutral with respect to both the polymer and the active ingredient, i.e., it does not alter them chemically and has no influence on their stability. Ethyl acetate was used in the present case. The concentration of the polymer depends on the size, hydrophilocity, solubility and stability of the polymer. Suitable concentrations here are between 2 and 50 mg/mL. The active ingredient was dissolved in ultrapure water in a suitable concentration. A suitable concentration of active ingredient depends on the chemical properties of the active ingredient and the capacitance of the nanoparticles. Following that, the shell polymer dissolved in the organic solvent was overlayered with the active ingredient dissolved in aqueous solution. The polymer and the organic solvent had to be present in the sample in an excess of at least tenfold. Particles that were hydrophobic on the outside were formed by bombarding with high-energy ultrasound coaxially with an electrode immerse in the sample. The active ingredient was thereby enclosed in a hydrophobic core due to interaction with hydrophilic groups of the nanoparticle in the interior. In the second step, a suitable surfactant was dissolved in ultrapure water in a suitable concentration. A surfactant concentration is adequate when it produces enough nanoparticles. The concentration depends on the ambient conditions and must be determined experimentally. It is usually between 0.01 and 5% (w/v). Then enough surfactant was added to the sample so that the concentration of polymer amount to only at least 1/10 of the starting amount. Again two phases were formed and were mixed by high-frequency ultrasound emitted coaxially to an electrode immersed perpendicularly into the sample. By mixing the surface-active substances (surfactants), i.e., polyvinyl alcohol in the present case, the formation of water-soluble nanoscale particles was ensured.

(31) For the sake of illustration, a batch is described, in which hydrophilic small interferin RNA (siRNA) complexed with polyethyleneimine (PEI) was enclosed in PLGA nanoparticles. The PLGA was first modified with DY-635, so that one out of every 200 chains would carry a dye molecule: (1) 2.4 μL PEI (1 mg/mL) was mixed with 2 μL siRNA (1 μg/μL) and mixed with 45.6 μL ultrapure water. The mixture is referred to below as a polyplex because the anionic siRNA and the cationic PEI interact with one another and a PEI binds and stabilizes the siRNA in a tight mesh network. (2) 325 mg DY-635-conjugated PLGA was dissolved in a total of 12.35 μL ethyl acetate. (3) 90 μL polymer solution from (2) was mixed with 50 μL polyplexes from (1) with high-frequency ultrasound (emitted as described above). (4) 1 mL PVA 0.3 wt % in ultrapure water was added to the mixture, which was then exposed to ultrasound. (5) The resulting nanoparticles were purified and freeze-dried.
Purification (for (A) and (B))

(32) The nanoparticles produced in this way had a diameter which was a function of the size and material of the vessels, the intensity of the ultrasound and the substance concentration and had a size of 120 to 220 nm. Under stable conditions, after producing the nanoparticles, the solvent was removed. To remove excess surfactant, the nanoparticles were washed several times (at least twice) by centrifuging, removing the supernatant and resuspending the nanoparticles in sterile ultrapure water. Then the particles were lyophilized and their mass was determined.

Example 8: Inclusion of Active Pharmaceutical Ingredients in Liposomes

(33) After functionalization of the polymers or lipids with the targeting unit (Example 1), liposomes were produced as follows: 1. Production of a 50 mM lipid solution from for example, 1:1 DOPC:DSPC (1,2-dioleolyl-sn-glycero-3-phosphocholine:1,2-distearol-sn-glycero-3-phospholine)+30% cholesterol+5% N-dod-DOPE in chloroform/methanol (2:1 vol/vol). Before being used, the DOPC can be modified with a polymethine dye. 2. Evaporation of the chloroform/methanol solvent (approximately 30 min, 90 rpm) in a rotary evaporator. 3. The lipids were then dissolved in 1 mL 7:3 vol/vol mixed DMSO:EtOH. 4. Next the hydrophilic dextran as the active ingredient was dissolved in a suitable buffer, namely PBS (phosphate buffered solution) to yield a concentration of 1 mg/mL. 5. 0.3 mL of the lipid solution was then added by drops to the dextran solution was then kept in motion at 750 rpm on a magnetic agitator while the dropwise addition was underway. 6. The liposomes were then separated in a miniextruder. 7. Next the liposome solution was aliquoted in 1-mL containers and alternately frozen in liquid nitrogen and then thawed in hot water 10 times. 8. Next the liposomes were separated 10 times in the extruder. 9. Then the liposomes were dialyzed in a prepared dialysis cassette (MWCO=20 kDa) against PBS for 16 hours. 10. Next the liposomes were freeze-dried, stored or used.

Example 9: Influencing Cholesterol Biosynthesis by the Organ-Specific Transport of an siRNA Against HMG-CoA Reductase (HMGCR) in DY-635-Modified Nanostructured Delivery Systems

(34) Male FVB/NRj mice (10 weeks old) were treated twice at intervals of 24 hours with the DY-635-modified nanostructured delivery system by i.v. injection by injecting 6.5 μg of the nanostructured delivery system per kg body weight. The delivery system was produced as described in Example 7 (B), wherein PLGA-modified with 108 μg PEI was enclosed in 3 mg DY-635 for production of 3 μg siRNA against HMGCR or 3 μg scrambled siRNA (siRNA without effect). The animals were euthanized painlessly 16 hours after the second injection and both blood and organs were removed for analysis. The blood was removed in lithium heparin monovettes and processed to plasma. To determine the efficacy of the treatment, the total cholesterol was determined in the plasma, and for the specificity, the change in gene expression in various organs was determined in qPCR. These values were compared with the cholesterol and the HMGCR expression level of healthy FVB/NRj mice (10 weeks old) and control groups. The control groups had the following composition: treatment with a DY-635-modified and therefore hepatocyte-specific nanostructured delivery system and an ineffective scrambled siRNA; treatment with a nanostructured delivery system that did not contain any DY-635 modification but otherwise did not differ from the therapeutic construct; the animals received only the 5% glucose solution.

Example 10: Detecting the Interaction of DY-635 with Hepatocytic Transporters

(35) HEK-293T cells were transfected with human tissue-specific hepatocytic transporters. Then the uptake of the polymethine dye DY-635 as a targeting unit into these tissue-specific transporters was investigated for FIG. 10 A. To do so, the cells were sown onto 96 well plates, incubated for 24 hours under standard conditions, then incubated 5 minutes with DY-635 (final concentration in the well: 10 μmol/L) after changing the medium. Next the cells were lysed and the lysates were measured by fluorimetry. The quantity of DY-635 taken up was quantified by a DY-635 standard curve using the respective transporters which had been specifically inhibited for the controls (inhibitors and the final concentrations used are shown in the following Table 3). In this experiment, it was found that DY-635 is a substrate for NTCP. The uptake by OCT1 can be evaluated as negligible. In FIG. 10 B the question of whether DY-635 binds as an inhibitor to the basolateral hepatocytic transporters was investigated. For this purpose, the HEK-293T cells transfected with the tissue-specific transporters were sown and incubated as described above. After 24 hours the cells were incubated for 5 minutes with either a radioactively labeled transporter-specific substrate or with the radioactive-specific substrate with a specific inhibitor or with DY-635 (10 μmol/L final concentration) (the substrates and their concentrations are shown in Table 3 below). The cells were then washed and lysed in the well. To quantify the uptake, the radioactive radiation of the substrates was used. It was found here that DY-635 is a strong indicator for OATP1B1 and OATP1B3. OAT2 and OCT1 are also inhibited by DY-635. This illustrates the strong interaction of DY-635 with the tissue-specific hepatocytic transporters. It can be concluded that by exposure of DY-635 to the surface of a nanostructured transporter system, immobilization on the cell surface of the hepatocyte occurs, resulting in subsequent endocytosis of the nanoparticle.

(36) TABLE-US-00004 TABLE 3 Human Radioactively labeled hepatocytic transporter-specific Transporter-specific transporter substrate/concentration inhibitor/concentration OATP1B1 [.sup.3H]Estradiol/30 nM Rifampicin/5 μM OATP1B3 [.sup.3H]Sulfobromophthalein sp/50 nm Rifampicin/5 μM OAT2 [.sup.3H]cGMP/10 nM Indomethacine/100 μM NTCP [.sup.3H]Estradiol/30 nM Cyclosporin A/50 μM NaDC3 [.sup.13C]Succinate/10 μM Succinate/100 μM OCT1 [.sup.3H]1-Methyl-4- Decynium22/40 μM phenylpyridinium