A NANOPARTICLE FOR USE IN THE TREATMENT OF AN OCULAR DISEASE

Abstract

The present invention relates to a nanoparticle for use in the treatment of ocular diseases, in particular diseases of the retina (“retinopathies”) or of optic neuropathies, in particular glaucoma.

Claims

1-15. (canceled)

16. A method of effectively preventing or treating, in a patient, one or several of an inflammatory component, an immune-response component, an angiogenic component and a neurodegenerative component of a retinal disease or of an optic neuropathy; wherein said method comprises administering a nanoparticle to a patient in need thereof; wherein said nanoparticle comprises: a core comprising a drug that has one or several of the following activities: anti-inflammatory activity, immune-suppressive activity, anti-angiogenic activity, neuroprotective activity, gene therapeutic activity and regulatory activity on gene expression; an amphiphilic shell surrounding said core, said amphiphilic shell comprising at least one phospholipid and, optionally, at least one surfactant; a targeting ligand binding to a receptor expressed on the surface of retinal pigment epithelial (RPE) cells and/or endothelial cells and/or optic nerve cells; said targeting ligand being covalently coupled to said amphiphilic shell.

17. The method according to claim 16, wherein effectively preventing or treating said one or several of an inflammatory component, an immune-response component, an angiogenic component, and a neurodegenerative component of said retinal disease or of said optic neuropathy manifests itself in one or several of: an increase in intracellular availability of said drug in retinal pigment epithelial (RPE) cells and/or in optic nerve cells and/or in ocular endothelial cells; an extended residence time of said drug in retinal pigment epithelial (RPE) cells and/or in optic nerve cells and/or in ocular endothelial cells; an interference with the VEGF-signalling pathway in the eye; a suppression or reduction of retinal neovascularization a suppression or reduction of inflammation in the eye; a suppression or reduction of an immune-response in the eye; and a suppression or reduction of neurodegeneration and of neuronal cell death in the eye.

18. The method according to claim i6, wherein said retinal disease is selected from retinal dystrophy and neovascular retinal diseases; and wherein said optic neuropathy is glaucoma.

19. The method according to claim 17, wherein said increase in intracellular availability of said drug in retinal pigment epithelial (RPE) cells and/or in optic nerve cells and/or in ocular endothelial cells is an increase in intracellular availability of said drug in comparison to an intracellular availability of said drug in retinal pigment epithelial (RPE) cells and/or in optic nerve cells and/or in ocular endothelial cells, observed when said drug is administered as a free drug that is not comprised within a nanoparticle; and/or said extended residence time of said drug in retinal pigment epithelial (RPE) cells and/or in optic nerve cells and/or in ocular endothelial cells, is a residence time of said drug in retinal pigment epithelial (RPE) cells and/or in optic nerve cells and/or in ocular endothelial cells, in the range of from at least 1 day-at least 5 days; and/or said interference with the VEGF-signalling pathway in the eye is an inhibition of the expression or activity of the VEGF-receptor, or is an inhibition of the expression or activity of VEGF; and/or said reduction of retinal neovascularization is a reduction of retinal neovascularization down to 50% or less of retinal neovascularization observed in an untreated retina affected by said retinal disease; and/or said reduction of inflammation in the eye is a reduction of inflammation down to 50% or less of inflammation observed in an untreated eye affected by said retinal disease; and/or said reduction of immune-response in the eye is a reduction of immune-response down to 50% or less of immune-response observed in an untreated eye affected by said retinal disease; and/or said reduction of neurodegeneration and of neuronal cell death in the eye is a reduction of neuronal cell death down to 80% or more of the level of neuronal cell death observed in an untreated eye affected by said optic neuropathy.

20. The method according to claim 16, wherein said receptor expressed on the surface of retinal pigment epithelial (RPE) cells and/or endothelial cells and/or optic nerve cells, is selected from a G-protein coupled receptor, an integrin, and a scavenger receptor.

21. The method according to claim 16, wherein said drug that has one or several of anti-inflammatory activity, immune-suppressive activity, anti-angiogenic activity, neuroprotective activity, gene therapeutic activity and regulatory activity on gene expression is selected from anti-inflammatory drugs, immunesuppressive drugs, anti-angiogenic drugs, neuroprotective drugs, and nucleic acids.

22. The method according to claim 21, wherein said anti-inflammatory drugs are selected from glucocorticoids; COX-inhibitors; non-steroidal anti-inflammatory drugs (NSAIDs); anti-inflammatory prodrugs,; and activators of soluble guanylate cyclase (sGC); said immune-suppressive drugs are selected from TNF-alpha inhibitors; Cyclosporins; mTOR-inhibitors; calcineurin inhibitors; inosinemonophosphate-dehydrogenasae inhibitors; folic acid antagonists; nitroimidazole-based immunesuppressants; and dihydro-orotate-dehydrogenase inhibitors; said anti-angiogenic drugs are selected from inhibitors of VEGF-receptor (VEGFR) or of VEGF; antifungal drugs; folic acid antagonists; tyrosine kinase inhibitors; anti-diabetics; tricycle anti-depressants; statins; sartans; coumarine and coumarine derivatives; and IGF-1 receptor inhibitors; said neuroprotective drugs are selected from immunosuppressant, anti-inflammatory and anti-oxidative drugs; and said nucleic acids are selected from DNA, RNA, LNA, PNA, oligonucleotides of any of the foregoing.

23. The method according to claim 16, wherein said nanoparticle is a lipid nanoparticle, and said shell comprises a phospholipid selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, phosphoinositides, phosphatidylinositol monophosphate, phosphatidylinositol bisphosphate, phosphatidylinositol triphosphate, ceramide phosphorylcholine, ceramide phosphorylethanolamine, ceramide phosphoryllipid and mixtures of any of the foregoing, and wherein said shell further comprises a surfactant.

24. The method according to claim 16, wherein said core is a) an oily core, and said drug is a lipohilic drug; or b) an aqueous core, and said drug is a hydrophilic drug.

25. The method according to claim i6, wherein said core comprises an oily or aqueous phase and said drug, said drug being dispersed in said oily or aqueous phase, said drug being dispersed in said oily or aqueous phase in the form of particles.

26. The method according to claim 16, wherein said core comprises a solvent and said drug, said drug being dissolved or dispersed in said solvent, wherein is or comprises lipids, in particular mono-, di- or trigylcerides.

27. The method according to claim 16, wherein said nanoparticle, in particular said lipid nanoparticle, has a size in the range of from 5 nm to 100 nm.

28. The method according to claim 16, wherein said nanoparticle, when administered to a patient as a sample of a plurality of nanoparticles, shows an enrichment in at least one of blood, spleen and eyes of said patient, by a factor of >3, in comparison to nanoparticles without a targeting ligand binding to an integrin.

29. The method according to claim 28, wherein said nanoparticle, when administered to a patient as a sample of a plurality of nanoparticles, shows an enrichment in the eyes of said patient, wherein said enrichment occurs in the retinae of said eyes or in or at the optic nerve.

30. The method according to claim 16, wherein, in said method of effectively preventing or treating one or several of an inflammatory component, an immune-response component, an angiogenic component, and a neurodegenerative component of said retinal disease or of said optic neuropathy, said nanoparticle is administered to a patient as a sample of a plurality of such nanoparticles, wherein such administration is performed as a) a systemic administration selected from an intravenous administration, a subcutaneous administration, an intramuscular administration, a nasal administration, a pulmonal administration, more preferably an intravenous administration, or b) a local administration selected from an intraocular administration, a subretinal administration, and an administration to the cornea, more preferably an intravitreal administration, even more preferably in the vicinity of the retina of the respective eye of said patient.

31. The method according to claim 18, wherein the retina disease is selected from hereditary retinal dystrophy, and the glaucoma is open-angle glaucoma or angle-closure glaucoma.

32. The method according to claim 20, wherein said integrin is selected from ανβ3-integrin and ανβ5-integrin, and wherein said targeting ligand is selected from a peptide having an amino acid sequence RGD, a cyclic peptide having an amino acid sequence of cyclo(-Arg-Gly-Asp-D-Phe-Cys) and derivatives thereof, or wherein said targeting ligand is a phospholipid.

33. The method according to claim 23, wherein said surfactant is glycerol polyethylene glycol ricinoleate.

34. The method according to claim 24, wherein said lipophic drug is selected from cyclosporine A; activators of soluble guanylate cyclase (sGC); glucocorticoids; statins; tacrolimus; Coenzyme Q10 (CoQ10); Vitamin E; citicoline; palmitoylethanolamide; melatonin; and SC79; and said hydrophilic drug is selected from anti-VEGF peptides and anti-VEGFR peptides; tricyclic anti-depressants; and growth factors.

35. The method according to claim 26, wherein the fatty acid component(s) of said mono-, di- or tri-glycerides has(have) a chain length of fatty acids in the range of from 6-18 carbon atoms.

Description

[0116] The invention is now further described by reference to the figures wherein

[0117] FIG. 1 shows the size and size distribution of LNCs and RGD-LNCs. Size (bars) and size distribution (polydispersity, black squares) of LNCs and RGD-LNCs were measured in 10% DPBS at 25° C. using dynamic light scattering.

[0118] FIG. 2 shows the biodistribution of non-modified LNCs and RGD-LNCs after systemic administration and 1 h circulation time. Nanoparticle concentration in tissues was analysed by determining the fluorescence. Date is expressed as mean ±SEM (n=6). Levels of statistical significance are indicated as **(P<0.01), ***(P<0.001) and ****(P<0.0001); Results are indicated by percentage initial dose (ID) per gram organ. Percentage initial dose per gram organ after systemic administration of 100 μl of either 20 mg/ml LNCs or RGD-LNCs after 1 h circulation time.

[0119] FIG. 3 shows the bioavailability of RGD-LNCs compared to LNCs after systemic administration and 1 h circulation time. Nanoparticle concentration in tissues was analysed by determining the fluorescence. Date is expressed as mean ±SEM (n=6). Levels of statistical significance are indicated as **(P<0.01), ***(P<0.001) and ****(P<0.0001); Results are indicated by percentage initial dose per eye. More specifically, FIG. 3 shows the Percentage initial dose (ID) per eye after systemic administration of 100 μl of either 20 mg/ml LNCs or RGD-LNCs after 1 h circulation time.

[0120] FIG. 4 shows the Microscopic analysis of flat-mounted retinas. Fluorescence images show capillary-associated nanoparticle accumulation for RGD-LNCs compared to LNCs. Z-stack pictures of RGD-LNCs treated retinas show capillary-associated fluorescence through all three retinal vascular layers (lower plexus, intermediate plexus and upper plexus). More specifically, FIG. 4 shows a Microscopic analysis of flat-mounted retinas.

[0121] TOP: Fluorescence images of whole retina show a marked increase in nanoparticle accumulation for RGD-LNCs, with a clear capillary association. Scale bar: 500 μm.

[0122] BOTTOM: In a z-stack picture of the marked area, RGD-LNC fluorescence can be clearly seen through all three retinal vascular layers. Scale bar: 50 μm.

[0123] FIG. 5 shows fluorescence images of cryosection of the posterior mouse eye after the application of RGD-LNCs and LNCs (white) and 1 h circulation time, nuclei stained with DAPI (blue) and F-actin stained with Phalloidin-TRITC (orange). Revealing excessive RGD-LNC accumulation in retinal and choroidal vessels and particularly in RPE cells. More specifically, FIG. 5 shows Fluorescence images of cryosection of the posterior mouse eye after the application of RGD-LNCs and LNCs (white) and 1 h circulation time, nuclei stained with DAPI (blue) and F-actin stained with Phalloidin-TRITC (orange) for a better orientation in the tissue. Revealing RGD-LNC accumulation in retinal and choroidal vessels and particularly in RPE cells.

[0124] FIG. 6 shows fluorescence images of cryosections of the posterior mouse eye after the application of CsA loaded RGD-LNCs and in untreated mice. The top panel shows healthy mice (Normoxia), and the bottom panel shows mice with retinopathy (ROP). Both groups were treated at P12 with CsA RGD-LNCs or were not treated. Staining was performed using DAPI or an anti-VEGF-R2-antibody. As can be seen in the case of healthy mice, no differences between the treatment groups were observed. For mice with ROP, the treatment using CsA RGD-LNCs reduced the VEGF-R2-expression to value comparable to healthy animals.

[0125] FIG. 7 shows Fluorescence images of retina whole-mounts at P17 of healthy mice (Normoxia) or mice with retinopathy (ROP), treated at P12 with either RGD-LNCs, CsA RGD-LNCs or free CsA. Revealing in the case of healthy mice, no differences between the treatment groups. For mice with ROP, the tremendous positive effect of CsA RGD-LNCs on the neovascularization can be visualized, whereas there a hardly any differences in the retinae of RGD-LNC treated and CsA treated mice compared to control. More specifically, FIG. 7 shows Fluorescence images of retina whole-mounts at P17 of healthy mice (Normoxia) or mice with retinopathy (ROP), treated at P12 with either RGD-LNCs, CsA RGD-LNCs or free CsA. Revealing in the case of healthy mice, no differences between the treatment groups. For mice with ROP, the tremendous positive effect of CsA RGD-LNCs on the neovascularization can be visualized, whereas there a hardy any differences in the retinae of RGD-LNC treated and CsA treated mice compared to control. Vessels stained with FITC-dextran. Scale bar: 500 μm.

[0126] FIG. 8 shows the quantification of percentage neovascularization. Analyzation of retina whole-mounts at P17 of healthy mice (Normoxia) or mice with retinopathy (ROP), treated at P12 with either RGD-LNCs, CsA RGD-LNCs or free CsA. Revealing in the case of healthy mice, no differences between the treatment groups. For mice with ROP, the tremendous positive effect of CsA RGD-LNCs on the neovascularization can be visualized, whereas there is no effect of free CsA and a slight effect of RGD-LNCs. More specifically, FIG. 8 shows Quantification of percentage neovascularization. Analyzation of retina whole-mounts at P17 of healthy mice (normoxia) or mice with retinopathy (ROP), treated at P12 with either RGD-LNCs, CsA RGD-LNCs or free CsA. Revealing in the case of healthy mice, no differences between the treatment groups. For mice with ROP, the tremendous positive effect of CsA RGD-LNCs on the neovascularization can be visualized, whereas there is no effect of free CsA and a slight effect of RGD-LNCs.

[0127] FIG. 9 shows the quantification of the total CsA amount in ng. Analyzation of eyes via UHPLC-MS at P17 of mice with retinopathy (ROP), treated at P12 with either CsA RGD-LNCs or free CsA. Revealing the presence of considerable amounts of CsA only in the eyes of mice treated with CsA RGD-LNCs, indicating the increased availability of CsA due RGD-LNC transport. More specifically, FIG. 9 shows a Quantification of the total CsA amount in the eye in ng. Analyzation of eyes via UHPLC-MS at P17 of mice with retinopathy (ROP), treated at P12 with either CsA RGD-LNCs or free CsA. Revealing the presence of considerable amounts of CsA only in the eyes of mice treated with CsA RGD-LNCs, indicating the increased availability of CsA due RGD-LNC transport.

[0128] FIG. 10 shows fluorescence images of cryosection of the posterior eye of an untreated mouse or after the application of RGD-LNCs (white) and 1 h circulation time; nuclei stained with DAPI (blue). Revealing RGD-LNC accumulation in the optic nerve, especially the optic nerve head and the area of the RPE that is directly adjacent to the optic nerve.

[0129] Moreover, reference is made to the examples which are given to illustrate, not to limit the present invention.

EXAMPLES

Example 1

Production of Lipid Nanoparticles

[0130] Lipid nanoparticles were chosen as a delivery system, because of their biocompatible nature, simplicity of preparation without using organic solvents and capacity to encapsulate a broad range of drugs with various solubility characteristics. Furthermore, RPE cells depend on the supply with lipids such as cholesterol, triglycerides, fatty acids and phospholipids. This requires that LDL and VLDL nanoparticles, which are classical lipid transporters, can penetrate the endothelial cell layer of the choroid and travel across the Bruch Membrane to transport lipids from the blood to RPE cells. The present inventors hypothesised that LNCs that consist of an oily core, made of medium-chain triglycerides (MCT), surrounded by a mixture of lecithin (Lipoid® S75-3) and a pegylated surfactant (Kolliphor® HS 15) would be ideal to mimic HDL and LDL. They have a thick outer layer of phospholipids similar to their biological counterparts. Their preparation was as follows:

[0131] 887.5 mg Kolliphor® HS15, 30 mg Lipoid® S75-3, 415 mg MCT, 12 mg NaCl and 655.8 mg water were subjected to three cycles of progressive heating and cooling between 90 and 60° C. To quantify particles after modification and purification, fluorescent dyes (DiI, DiO or DiD 1.5% (w/w)) were added to the initial mixture. During the last cycle, an irreversible shock was induced by dilution with 5 ml water at the phase inversion temperature, leading to the formation of stable LNCs. Afterwards, additional magnetic stirring was applied for 5 min at room temperature. The final dispersion was filtered through a 0.22 μm regenerated cellulose (RC) membrane for sterilization and stored at room temperature in the dark.

[0132] To prepare drug-loaded LNCs, 35.3 mg CsA were dissolved in MCT and particles were prepared as described above.

[0133] The preparation is based on the phase-inversion temperature phenomenon of an emulsion leading to lipid nanoparticle formation with good mono-dispersity and leading to the formation of nanoparticles with a diameter of approx. 50 nm [Heurtault B. et al., A Novel Phase Inversion-Based Process for the Preparation of Lipid Nanocarriers., Pharm. Res., 19, 2002]. As a next step, the LNCs were grafted with a targeting ligand. First, cyclo(-Arg-Gly-Asp-D-Phe-Cys) (RGD) peptides were coupled to the amphiphilic DSPE-PEG2000-maleimide using conjugation chemistry between the thiol group present on the cyclic structure of the peptide and the maleimide. Next, the conjugate was inserted in the shell of the LNCs by post-insertion method, by simply heating up the mixture of DSPE-conjugate and LNCs to favour the transfer of DSPE-PEG molecules from micelles to LNCs. Modified LNCs were dialyzed against DPBS overnight using Spectra/Por® Float-A-Lyzer® G2 MWCO 300 kDa (Sigma-Aldrich, Germany) and subsequently centrifuged twice (15 min, 4000 g) using an Amicon® Ultra-4 MWCO 100 kDa centrifugal filter (Merck, Germany) for further purification.

[0134] To that end, firstly ligand molecules were coupled to the amphiphilic DSPE-PEG2000-maleimide using the conjugation chemistry between the thiol-group present on the cyclic structure of the peptide and the maleimide. Next, the conjugate or DSPE-mPEG2000 was inserted in the shell of the LNCs by post-insertion method [Perrier T., et al., Post-insertion into Lipid Nanoparticles (LNCs): From experimental aspects to mechanisms., Int. J. Pharm., 396, 2010].

[0135] As a ligand, a small, cyclic peptide was used, cyclo(-Arg-Gly-Asp-D-Phe-Cys) (RGD), that is highly potent and αvβ3 integrin specific.

Example 2

Targeted Administration of Lipid Nanoparticles

[0136] The targeting concept chosen by the present inventors was proved in vivo as follows: 100 μl of either 20 mg/ml LNCs or RGD-LNCs were injected systemically into healthy mice and were allowed to circulate for 1 h. Afterwards, mice were sacrificed, and the content blood, organs and eyes were collected. Blood, organs and eyes were homogenized in lysis buffer and afterwards all samples were centrifugated and nanoparticle concentration in the supernatant was measured via fluorescence. Actual Nanoparticle content was quantified using a calibration curve, made individually for each organ by spiking homogenates with defined nanoparticle amounts.

[0137] The inventors found that RGD-LNCs are able to sufficiently circulate in the blood and additionally they showed extended blood circulation in contrast to non-modified LNCs (FIG. 2). Moreover, it reveals that RGD-modified LNCs are able to accumulate efficiently in the eye, in contrast to non-modified LNCs (FIG. 3), with an enhancement of bioavailability by factor 10.

[0138] For a deeper insight in the nanoparticle localization in the eye, fluorescent microscope pictures of retina flat mounts were taken (FIG. 4). To that end, 100 μl of either 20 mg/ml LNCs or RGD-LNCs were injected systemically into healthy mice and were allowed to circulate for 1 h. Afterwards, mice were sacrificed, eyes collected and placed in 4% PFA for 1 h. Then retina was isolated and retina flatmounts were prepared. Afterwards, fluorescently labelled LNCs were detected using fluorescence microscopy. RGD-LNCs binding to the retinal microvasculature was clearly evident, whereas non-modified LNCs could hardly be found in the retina. By having a closer look into the retinal vasculature, RGD-LNCs are not only able to accumulate within all different plexus of the retina, indicated by fluorescence through the whole z-stack of the retina, but bind to smaller as well as to larger vessels (FIG. 4).

[0139] FIG. 5 shows a detailed image of the posterior mouse eye after the application of RGD-LNCs and LNCs (white) and 1 h circulation time, nuclei stained with DAPI (blue) and F-actin stained with Phalloidin-TRITC (orange) for a better orientation in the tissue. By having a closer look at the magnified section (rosa square), the accumulation of RGD-LNCs in retinal vessels as well as an additional accumulation in the RPE and the choroidal vessels can be seen. Especially compared to unmodified LNCs. These findings clearly confirm that RGD-modified LNCs are capable to target the posterior eye efficiently. Moreover, RGD-LNCs allow for double-play by targeting the site of pathologic manifestation as well as the central mainstay of pathomechanisms.

[0140] In order to take best advantage of that, an active compound that has various effects at the different locations was loaded into the nanoparticles. To that end, the drug Cyclosporin A (CsA) was chosen, as it is well known as an immunosuppressant and interferes at multiple intracellular sites with the VEGF signalling pathway [Freeman, D. J., Pharmacology and pharmacokinetics of cyclosporine., Clin. Biochemistry, 24, 1991]. CsA is able to suppress the intracellular VEGF signalling pathway and alleviates endothelial cell sprouting and proliferation. Additionally, CsA counteracts the TGFβ-related increase of VEGF production in RPE cells, the main source of VEGF in the retina [Rafiee, P., et al., Cyclosporin A differentially inhibits multiple steps in VEGF induced angiogenesis in human microvascular endothelial cells through altered intracellular signaling., Cell Comun. Sign., 2, 2004]. Furthermore, CsA possesses an anti-inflammatory potential and decreases interleukin-1β levels. In addition to that CsA restores damages of the blood-retina-barrier in an animal model of diabetes [A. Carmo, et al., Effect of cyclosporin-A on the blood-retinal barrier permeability in streptozotocin-induced diabetes, Mediators of inflammation, 9, 2000]. The fact that CsA has shown a significantly, but moderately alleviated progression of diabetic retinopathy after oral administration by transplantation patients, demonstrates the high therapeutic potential but suffer from an insufficient availability in the ocular vasculature [V. C. Chow, et al., Diabetic retinopathy after combined kidney-pancreas transplantation, Clin Transplant, 13, 1999].

[0141] Due to the high lipophilicity of CsA, it was directly dissolved in the oil phase of the initial emulsion and particles were prepared according to the standard protocol (see Example 1). High encapsulation efficiencies were achieved with values of 67% and a drug payload of 34.1 mg/g LNC.

Example 3

Mouse Model of Retinopathy of Prematurity

[0142] After confirming the targeting strategy in vitro and in vivo, the therapeutic concept for the treatment of neovascular ocular diseases was proven by using the mouse model of retinopathy of prematurity (ROP). This disease model is considered to be the standard model for retinopathy of prematurity and diabetic retinopathy, with the direct evaluation of neovascularization via retinal whole-mounts being the most meaningful outcome [Connor K. M., Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis, Nat. Protoc., 4, 2009]. Mice pups, 7 days old (postnatal day 7=P7), were exposed for 5 days to hyperoxic conditions (75±2% oxygen) in a sealed incubator. After 5 days, P12 (postnatal day 12=P12) mice were returned to room air. Maximal retinal neovascularization is known to occur 5 days after return to room air at P17 (postnatal day 17=P17). To that end, mice were treated at P12 with 20 μl of either DPBS (referred to as control), 20 mg/ml RGD-LNCs, 20 mg/ml CsA loaded RGD-LNCs or 0.68 mg/ml free CsA. After the single treatment at P12, mice were then at P17 anesthetized and perfused with 2 ml FITC dextran (green) for vessel staining. Afterwards, eyes were collected, and retina flat mounts were prepared. Those wholemounts were imaged as a whole using a fluorescence microscope. Additionally, whole retinal area and neovascular area can be measured and can be quantified as a percentage ratio.

[0143] FIG. 6 demonstrates the capacity of CsA-loaded RGD-LNCs to reduce the VEGF-R2 expression to a normal value, whereas in healthy mice, the VEGF-R2 levels are not altered. More specifically, FIG. 6 shows cryosections stained with DAPI and anti-VEGF-R2-antibody. The treatment using CsA RGD-LNCs resulted in a reduction of VEGF-R2 expression down to a healthy (“normal”) level. Indicating the interference with the VEGF-signalling pathway and the normalization rather the suppression of VEGF-R2 expression due to a endothelial and RPE cell specific anti-VEGF therapy.

[0144] Finally, the effect on retinal neovascularization was investigated. FIG. 7 depicts the visualized retinal neovascularization at P17, with detectable difference between CsA RGD-LNC treated mice and control mice, in the case of mice with retinopathy. If the mice were kept under normoxia and no retinal neovascularization can be detected, no negative impacts could be seen on retinal vessels after nanoparticle administration. Indicating that a prophylactic administration of CsA RGD-LNCs may cause no harm to the retinal vasculature. Additionally, no huge differences between control and RGD-LNC treated or CsA treated mice can be seen. The extend of neovascularization can be quantified as the percentage of neovascularization related to the whole retinal area and thereby subjective evaluation can be quantified. FIG. 8 reveals that there is no difference in the amount of neovascularization of healthy mice, independent of the treatment applied. In the case of mice with retinopathy, drug-free RGD-LNCs show a slight, but significant effect, while drug loaded RGD-LNCs seem to have a tremendous effect on the neovascularization after one-time treatment at P12 and free CsA seem to be totally ineffective. This reveals that the ligand-grafted delivery system according to the present invention is needed to achieve satisfactory results.

[0145] To prove the assumption, that a ligand-grafted delivery system is mandatory for the effectiveness of the drug, the amount of CsA at P17 of mice with retinopathy, treated on P12 was measured via UHPLC-MS. To that end, treated mice were anesthetized at P17, eyes were collected and front part of the eye, including the lens was discarded. Then posterior eye segment was placed into methanol, homogenized, centrifuged, filtered and analysed using UHPLC-MS. Samples were quantified with a calibration curve prepared from untreated mice eyes with spiked CsA. FIG. 9 reveals, that only when a delivery system is used, the there is still CsA present, indicating an extended residence time of the CsA in the eye and an increased availability of the drug.

[0146] In this model, that mimics ideally the pathomechanisms of retinopathy of prematurity, CsA loaded RGD-LNCs show tremendous effects on the overall pathogenesis, in particular on the neovascularization of the retina, as they are able to directly inhibit the neovascularization.

Example 4

Targeted Administration of Lipid Nanoparticles

[0147] Lipid nanoparticles, as prepared in example 2, were intravenously administered as described in example 2 to healthy mice and were allowed to circulate for 1 h. Afterwards, mice were sacrificed, the eyes were collected, fixated and cryoprotected. Afterwards, sagittal cryosections were prepared, and fluorescence microscopy images were taken upon staining with DAPI. The images are shown in FIG. 10 and demonstrate an accumulation of RGD-LNCs in optic nerve cells and the RPE area adjacent to the optic nerve thus demonstrating the possibility of effectively targeting optic nerve cells by such nanoparticles, thereby enabling an effective treatment (as opposed to a merely symptomatic treatment) of optic neuropathies, in particular of glaucoma. In accordance with embodiments of the present invention, the nanoparticles may be loaded with anti-inflammatory drugs, immune-suppressive drugs, and/or neuroprotective drugs, which may then become accumulated in the region of the optic nerve or the RPE in direct proximity of the optic nerve, due to the targeting of the nanoparticles.

[0148] The afore-mentioned examples show that the nanoparticles in accordance with the present invention make use of the novel combination of nanoparticles with a suitable ligand, such as an α.sub.νβ.sub.3-Integrin ligand (RGD), and a drug, such as Cyclosporin A. This combination enables a systemic, cell-specific therapy for all retinal diseases and optic neuropathies, in particular neovascular ocular diseases and glaucoma, which, in turn, means an improvement of the current therapeutic situation in all respects.