VIRUS-MIMETIC NANOPARTICLES
20220409743 · 2022-12-29
Inventors
- SARA MALSANKA-FIGUEROA (NÜRNBERG, DE)
- DANIEL FLEISCHMANN (REGENSBURG, DE)
- ACHIM GÖPFERICH (SINZING, DE)
Cpc classification
A61K31/197
HUMAN NECESSITIES
A61K47/6937
HUMAN NECESSITIES
A61K47/593
HUMAN NECESSITIES
A61K47/60
HUMAN NECESSITIES
A61K47/62
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61K47/69
HUMAN NECESSITIES
A61K31/197
HUMAN NECESSITIES
A61K31/4418
HUMAN NECESSITIES
A61K47/59
HUMAN NECESSITIES
A61K47/60
HUMAN NECESSITIES
A61K47/62
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
The present invention relates to a nanoparticle comprising a nanomaterial and at least a first ligand and a second ligand tethered to the nanoparticle. The present invention further relates to a nanoparticle for use as a medicament or diagnostic agent. The present invention also relates to a nanoparticle for use in a method of preventing or treating a disease selected from diabetic nephropathy, glomerulonephritis, glomerular VEGF A dysregulation, endothelial VEGF A dysregulation, diabetic retinopathy, rheumatoid arthritis, age-related macular degeneration, and cancer such as breast cancer. Furthermore, the present invention relates to a method of preparing a nanoparticle.
Claims
1-17. (canceled)
18. A nanoparticle, comprising a nanomaterial and at least a first ligand and a second ligand, wherein said first ligand is capable of mediating an attachment of said nanoparticle to a target cell, and wherein said second ligand is capable of mediating an internalization of said nanoparticle into said target cell.
19. The nanoparticle according to claim 18, wherein said first ligand is a non-agonistic agent binding to a GPCR, and/or an agent binding to glycoprotein and/or glycolipid on a target cell surface.
20. The nanoparticle according to claim 18, wherein said second ligand is any of i) an agent binding to an integrin, ii) an agonistic agent binding to a GPCR, iii) an agent binding to an ectoenzyme, and/or iv) an agent binding to a transferrin-receptor.
21. The nanoparticle according to claim 18, further comprising a therapeutic agent.
22. The nanoparticle according to claim 18, wherein said first ligand and said second ligand are each coupled to said nanomaterial.
23. The nanoparticle according to claim 18, wherein said nanomaterial comprises more than one block-copolymer chain, and wherein said first ligand is coupled to a first block-copolymer chain of said nanomaterial and said second ligand is coupled to a second block-copolymer chain of said nanomaterial, and wherein said first block-copolymer chain is longer than said second block-copolymer chain.
24. The nanoparticle according to claim 23, wherein said first block-copolymer chain comprises PEG in a range of from 1 k to 20 k, and/or comprises PLA in a range of from 5 k to 40 k.
25. The nanoparticle according to claim 18, wherein said second ligand is enzymatically activated prior to said internalization of said nanoparticle into said target cell.
26. The nanoparticle according to claim 18, wherein said target cell is selected from a mesangial cell, an endothelial cell, a B cell, a T cell, a macrophage, a dendritic cell, and a tumor cell.
27. The nanoparticle according to claim 18, wherein said nanoparticle has a size of from 5 nm to 1000 nm.
28. The nanoparticle according to claim 18, wherein a ratio of said first ligand to said second ligand is in the range of from 2:1 to 1:2.
29. The nanoparticle according to claim 18, wherein said nanoparticle has a particle avidity for a targeted receptor of from 1 pM to 100 nM.
30. The nanoparticle according to claim 18, wherein said nanomaterial comprises PEG and wherein said nanoparticle has a ligand density of ligand/PEG of at least 5%.
31. A medicament or a diagnostic agent, comprising a nanoparticle as defined in claim 18.
32. A method of preventing or treating a disease selected from diabetic nephropathy, glomerulonephritis, glomerular VEGF A dysregulation, endothelial VEGF A dysregulation, diabetic retinopathy, rheumatoid arthritis, age-related macular degeneration, and cancer, wherein said method comprises administering an effective amount of a nanoparticle, as defined in claim 18, to a patient in need thereof.
33. A method of preparing a nanoparticle, as defined in claim 18, comprising the steps: a) providing, in any order, one or several nanomaterial(s) and, optionally, a therapeutic agent; b) optionally, preparing a block-copolymer from any of said one or several nanomaterial(s); c) coupling, in one or more steps, a first ligand and a second ligand thereto; d) providing a therapeutic agent, if not already provided in step a); e) preparing and obtaining a nanoparticle using the ligands coupled to said nanomaterial and said therapeutic agent.
34. The method according to claim 34, wherein said obtaining in step e) comprises obtaining nanoparticles having a polydispersity index of from 0.01 to 0.5.
35. The nanoparticle according to claim 18, wherein said nanomaterial comprises any of polyethylene glycol (PEG), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), an oxazoline-derived polymer, a poly(aminoacid), a polysaccharide, a phospholipid, a sphingolipid, cholesterol, a PEG-lipid, a block-copolymer, gold, or a qdot material.
36. The nanoparticle according to claim 18, wherein said first ligand is angiotensin II receptor type 1 (AT1r), human neuropeptide Y1-receptor, C-X-C chemokine receptor type 4, heparan sulfate, a sialoglycoprotein, a ganglioside, or a mannose receptor.
37. The nanoparticle according to claim 18, wherein said second ligand is αVβ3 integrin, αVβ5 integrin, AT1r, legumain, a membrane-type matrix metalloproteinase, angiotensin converting enzyme (ACE), or an agent binding to a transferrin-receptor.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0071] The present invention is now further described by reference to the following figures.
[0072] All methods mentioned in the figure descriptions below were carried out as described in detail in the examples.
[0073]
[0074] (A) NPs carrying EXP3174 and Ang-I on their corona (NPEXPAng-I) attach to the cell membrane through EXP3174-mediated AT1R-binding. Specific recognition is triggered through enzymatic Ang-I processing and Ang-Il-mediated internalization.
[0075] (B) Flow chart exemplifying the triple target cell recognition of decision-making NPs.
[0076]
[0077] (A) Assembly of ligand-decorated NPs.
[0078] (B) Molar ligand content of different NP species normalized to the PEG content,
[0079] (C) size and polydispersity index (PDI) and
[0080] (D) c-potential of the resulting NP formulations.
[0081] Results are presented as mean±SD of at least n=3 measurements.
[0082]
[0083] Interaction of ligand-decorated NPs with their target ATTR (A-D) and ACE (E-F) determined by intracellular calcium measurements. (A) Ligand affinity and (B) particle avidity for the AT1R. (C) IC50 values for the free and particle-bound ligands. (D) Kinetic measurement of the AT1R inhibition by ligand-decorated particles. (E) Michaelis-Menten kinetics of NPEXPAng-I and NAng-I. (F) Specificity constant (Kcat/km) for the free and particle-bound Ang-I calculated based on ligand and NP concentration. Results are presented as mean±SD of at least n=3 measurements. Levels of statistical significance are indicated as **p≤0.01, ***p≤0.001, ****p≤0.0001 and #p≤0.0001 and x≤0.001 comparing the AT1R inhibition of NPEXP and NPEXPAng-I at different time points. n.s.: non-significant.
[0084]
[0085]
[0086] (A) Ligand-mediated internalization of NPEXPAng-I, NPAng-I and NPEXP in rMCs inhibited by free EXP3174 and captopril (see also
[0087]
[0088]
[0089] (A) Images of kidney glomeruli (dotted circles) of mice treated with the different particle formulations. Scale bar 40 μm. see also
[0090]
[0091] (A) Lys-Ang-I and (B) EXP3174 were linked to carboxylic acid- or amine-ended PEG.sub.5k-PLA.sub.10k using EDC/NHS or DCC/NHS chemistry, respectively. (C) Complete polymer functionalization shown by the quantification of the molar ligand and PEG content. (D) Absence of unreacted NH.sub.2 polymer end groups on EXP3174-modified-polymer was determined using flurescamine. A Student's t-test was performed using GraphPad Prism 6.0 to assess statistical significance. Levels of statistical significance ae indicated as ****p≤0.0001 comparing the fluorescence of MeO—and EXP3174—with NH.sub.2-terminated PEG.sub.5k-PLA.sub.10k. For detailed methods see Example 1.
[0092]
[0093] rMCs were stimulated simultaneously with Ang-II and NPEXP and the resulting intracellular calcium response measured immediately for 1 minute. At the used NPEXP concentrations, the EXP3174 ligand did not inhibit the agonist-triggered calcium signal during the assay duration. Therefore, the influence of EXP3174 on the Ang-II-measurement was considered negligible. Results are shown as mean±SD of at least n=3 measurements. For detailed methods see Example 1.
[0094]
[0095] (A) NPEXP are not internalized in the cell line and mostly locate on the cellular membrane and filipodia between cells forming big clusters over time. Receptor binding is shown by the colocalization of NP- and receptor-associated fluorescence. (B) NPAng-I are internalized by the cells as depicted by their cytoplasmic localization. (C) NPMeO do not associate with cells due to their lack of a tethered ligands enabling a specific targeting. Cells: white; AT1R-YFP: green; NP-formulations: red. Scale bar 20 μm. For detailed methods see Example 1.
[0096]
[0097] NPAng-I (grey) with 20% Ang-I density were prepared with varying polymer densities of COOH-PEG.sub.5k-PLA10k and analysed for their cellular uptake using flow cytometry. Concomitantly, NPEXPAng-I (yellow) were prepared with varying densities of EXP3174-PEG.sub.5k-PLA10k to compare the effect of the second ligand on the stearic hindrance of Ang-I. Functionalization of long polymer chains with EXP3174 on NPEXPAng-I counterbalanced the decreased uptake due to stearic hindrance of the Ang-I ligand when adding non-functionalized long polymers, and significantly increased the particle internalization. Results are presented as mean±SD of at least n=3 measurements. A 2-way ANOVA with Sidak's multiple comparisons test was performed using GraphPad Prism 6.0 to assess statistical significance. Levels of statistical significance ae indicated as ****p≤0.0001. For detailed methods see Example 1.
[0098]
[0099] Cells were preincubated for 30 minutes with free EXP3174 prior to the addition of the different NP formulations (NPEXPAng-I, NPAng-I and NPEXP). Inhibition of the target receptor resulted in the suppression of the particle-associated fluorescence. Scale bar 20 μm. For detailed methods see Example 1.
[0100]
[0101] NPEXP show accumulation in rMCs and NCI-H205R cells, as they both carry the AT1R. Contrary, the co-culture of rMCs and HeLa cells shows preferential accumulation of NPEXP in rMCs, as HeLa cells express only minor amounts of the receptor on the cell membrane (cf. [3]). NPAng-I show a higher specificity as they preferentially accumulate in target rMCs, which carry the necessary equipment for their internalization (the ACE and the ATTR), over off-target cells lacking ACE (HeLa or NCI-H295R cells). Cell nuclei: blue; Off-target cells (HeLa or NCI-H295R): white; Target cells (rMCs): green; NPs: red. Scale bar 20 μM. For detailed method see Example 1.
[0102]
[0103] (A) Kidney distribution of NPAng-I and NPEXP in mice kidneys. NPAng-I show a small NP-associated fluorescence in the majority of glomeruli contrary to NPEXP which did not accumulate in this area. The glomeruli are marked with white arrows for better visualization. From left to right squared out regions are shown as magnifications. DAPI staining of cell nuclei: blue; Tissue autofluorescence: green; NP-associated fluorescence: red. (B) Kidney distribution of the free dye used to label the NPs. CF647 was injected into mice as a control to assess its distribution in the kidney. Strong fluorescence could be detected in the tubular area, with no fluorescence in the glomeruli (marked with a white circle). This demonstrates that the fluorescence seen for the particle-samples comes from the particles themselves and not from leaked dye, which is freely filtrated due to its low molecular weight. (C) Plasma residence of different NP-formulations after one-hour circulation in NRMI mice. NP fluorescence in plasma 1 hour after injection was measured and normalized to the fluorescence measured 5 minutes after injection (initial particle blood fluorescence). Non-targeted NPs (NPMeO) show the highest blood circulation time, which is due to the stealth effect conferred by their PEG-shell. Even though 40% of all the polymers on the NPEXPAng-I surface are ligand-coupled, which decrease the particle stealth effect, they are able to match the blood residence of non-targeted particles. They depict a significant higher fluorescence in plasma after 1 h compared to particles functionalized with only one ligand (NPAng-I and NPEXP). NPAng-I, which carry a specific two-step virus-mimetic recognition mechanism also show a significant superior blood residence than NPEXP, which represent commonly targeted NPs. As a control, the free dye used to label the particles (CF647) was additionally injected into mice and it rapidly disappears from the blood circulation after its to its free filtration (6% of the initial fluorescence after 1 h). Results in (C) are presented as mean±SD of at least n=6 samples. A Student t test was performed using GraphPad Prism 6.0 to assess statistical significance. Levels of statistical significance are indicated as *p≤0.05, ***p≤0.001 and ****p≤0.0001. n.s.: non-significant. For detailed methods see Example 1.
[0104]
[0105]
[0106] A) virus mimetic particles show high accumulation in the renal corpuscles (round labelling).
[0107] B) unmodified nanoparticles, which do not carry the first ligand and the second ligand, do not show significant uptake into glomerular tissue.
[0108]
[0109]
[0110]
[0111]
[0112]
[0113]
[0114]
[0115]
[0116]
[0117]
[0118]
[0119]
[0120] A) Relative blood plasma fluorescence after NP injection. Control NPs exhibited maximal blood circulation values with almost 50% residual blood plasma fluorescence after 60 min of injection. In contrast to EXP NPs as well as EXPcRGD NPs, that both showed tolerable residual concentrations, cRGD NPs were rapidly cleared from the blood. Results represent mean±SD (n=.sub.3). ****P<0.0001, ***P<0.001, **P<0.01. (n.s.: not significant.)
[0121] B) Fluorescence imaging of kidney cryosections after injection of free CF™ 6.sub.47 fluorescent dye. To visualize cell nuclei, sections were DAPI-stained (blue). After injection of a comparable molarity of free dye, strong fluorescence signals (red to white) could be detected in tubular areas of the kidney, indicating free renal filtration of the low-molecular dye. As expected, no intraglomerular accumulation could be detected (white circles). (Calibration bar: 0-65535 Gray Value.)
[0122]
[0123]
[0124]
[0125]
[0126] In the following, reference is made to the examples, which are given to illustrate, not to limit the present invention.
EXAMPLES
Example 1: Materials and Methods
[0127] Cell culture
[0128] The cell lines used in this study were cultured at 37° C. and 5% CO.sub.2. rMCs, NCI-H295R and HeLa cells were cultured in RPMI1640 medium (Sigma Aldrich) supplemented with 10% FBS and insulin-transferrin-selenium and 100 nM hydrocortisone. HK-2 cells were maintained in DMEM-F12 (1:1) medium (Sigma Aldrich) supplemented with 10% FBS. pAT1R-rMCs were obtained by transfecting rMCs with a plasmid encoding the AT1R with a YFP-tag (CXN2-HA-AT1R-YFP) (cf. [4]) using the commercially available transfection reagent Lipofectamine 2000 following the manufacturer's instructions. pAT1R-rMCs were cultured in RPMI1640medium supplemented with 10% FBS and 600 μg/ml geneticin (G418). The cell lines were characterized for their target AT1R and ACE expression as shown previously [1, 3].
[0129] Mice
[0130] The experimental procedures on animals were carried out according to the national and institutional guidelines and were approved by the local authority (Regierung von Unterfranken, reference number: 55.2-2532-2-329). The mice indicated in the Key Resources Table have been used in this study at the age of 10 weeks. Only female mice were used in all experiments. They were kept under Specific pathogen Free (SPF) housing facilities, under standard conditions (50±5% relative humidity, temperature of 21±1° C., air exchange >8 AC/h and light period of 12h:12h (L:D)).
[0131] Polymer Preparation: Block Copolymer Synthesis
[0132] PEG-PLA block-copolymers (COOH-PEG.sub.2k-PLA.sub.10k, COOH-PEG.sub.5k-PLA.sub.10k, NH.sub.2-PEG.sub.5k-PLA.sub.10k, and MeO-PEG.sub.5k-PLA.sub.10k) were synthesized through ring opening polymerization of cyclic 3,6-dimethyl-1,4-dioxane-2,5-dione (lactide). In short, the lactide was recrystallized prior to use from anhydrous ethyl acetate and dried under vacuum at 40° C. for 12 hours at room temperature (r.t.). COOH-PEG.sub.5k-OH, COOH-PEG.sub.2k-OH, Boc-NH-PEG.sub.5k-OH or MeO-PEG.sub.5k-OH were used as macroinitiators for the ring opening polymerization. They were solved (0.3 mmol) in anhydrous DCM and mixed with the purified lactide (18 mmol). 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.9 mmol) was added as a catalyst. The polymerization was quenched after 1 hour with benzoic acid (4.6 mmol). The resulting polymers were precipitated in diethyl ether and dried under vacuum at 40° C. (for COOH-PEG.sub.2k-PLA.sub.10k, COOH-PEG.sub.5k-PLA.sub.10k, and MeO-PEG.sub.5k-PLA.sub.10k) or 35° C. (for Boc-NH-PEG.sub.5k-PLA.sub.10k) for 12 hours. For the cleavage of the protective Boc group, Boc-NH-PEG-PLA was dissolved in 50% (v/v) TFA/DCM and stirred at r.t. for 30 minutes. Afterwards, it was diluted with the triple volume of DCM and washed with saturated sodium bicarbonate solution three times. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting NH.sub.2-PEG.sub.5k-PLA.sub.10k was purified through precipitation in diethyl ether and subsequently dried under vacuum at 35° C. for 12 hours.
[0133] Polymer Preparation: Ligand Coupling
[0134] For the preparation of Ang-I-modified polymers (see also
[0135] Polymer Preparation: Fluorescence Labelling of PLGA
[0136] For in vitro and in vivo particle detection, fluorescently labelled PLGA was used in the particle core. To that end, TAMRA-amine (for CLSM) and CF6467-amine (for flow cytometry and in vivo experiments) were covalently coupled to carboxylic acid-terminated 13.4 kDa PLGA. Briefly, 5 μmol acid-terminated PLGA were dissolved in anhydrous DMF and activated for 2 h at r.t. with 129 μmol DMTMM (25-fold excess). Afterwards, 1 μmol fluorescent dye was dissolved in DMF, added dropwise to the PLGA and reacted for 72 h at r.t. in the dark. The reaction product was diluted (DMF<10%) and dialyzed against ultrapure water, using a 3.5 kDa molecular weight cut-off regenerated cellulose dialysis membrane (Spectrum Laboratories) over 34 hours (with medium change after 30 minutes, 2, and 6 hours) under light exclusion. Fluorescently labelled PLGA was then lyophilized over 3 days.
[0137] NP Preparation and Characterization: Particle Preparation
[0138] For NP preparation, PEG-PLA block-copolymers and 13.4 kDa PLGA were mixed at a 70:30 mass ratio to a final concentration of 10 mg/mL in ACN. For ligand-modified particles COOH-PEG.sub.2k-PLA.sub.10k and ligand-modified polymers were mixed accordingly so that 20% of the polymers making up the NP-structure were modified with Ang-I (NPAng-I) or/and EXP3174 (NPEXP and NPEXPAng-I, respectively). NPs were prepared via bulk nanoprecipitation of polymer mixtures in vigorously stirring 10% DPBS (v/v) (pH 7.4) to a final concentration of 1 mg/ml. Particles were stirred for 2 hours to ensure the evaporation of the organic solvent, and concentrated by ultracentrifugation using a 30-kDa molecular weight cutoff Microsep advance centrifugal device (Pall Life Sciences) for 20 minutes at 756 g.
[0139] NP Preparation and Characterization: Dynamic Light Scattering and ζ-Potential
[0140] Size and ζ-potential of the resulting particles were determined in 10% PBS at a constant temperature of 25° C. using 1 mg/mL or 3.5 mg/ml concentrations, respectively, with a ZetaSizer Nano ZS (Malvern Instruments) equipped with a 633 He—Ne laser at a 173° backscatter angle and the Malvern Zetasizer software version 7.11. The cuvette position was set at 4.65 mm and the attenuator optimized automatically by the device. Disposable microcuvettes (Brand) and a folded capillary cell (Malvern Instruments) were used for size and 4-potential measurements, respectively.
[0141] NP Preparation and Characterization: Particle Quantification
[0142] Quantification of particle PEG concentration was performed using a colorimetric iodine complexing assay and correlated with the gravimetrical NP content determined via lyophilization. In short, the particle samples were diluted in ultrapure water to a PEG concentration in the 5-30 μg/mL range. Dilutions in ultrapure water of MeO-PEG-OH (0-40 μg/mL) were used as standards for the calibration curve. 140 μL of samples or standards were mixed with 60 μL of a 2:1 (v/v) mixture of 5% (m/v) barium chloride solution in 1 N HCl and a 0.1 N aqueous iodine solution. The samples and standards were transferred into a 96-well plate and their absorbance at 535 nm measured using a FUOstar microplate reader (BMG Labtech). The correlation of the particle PEG content with the exact polymer concentration was determined gravimetrically after sample lyophilization. The molar particle concentration was calculated from the particle mass determined through the colorimetric iodine complexing assay, the particle density (1.25 g/cm.sup.3) and the hydrodynamic diameter of the NPs obtained through DLS measurements assuming a spherical particle shape. Ligand concentration on the particle corona was quantified using a BCA assay, and fluorometrically for Ang-I and EXP3174, respectively, as described above.
[0143] Intracellular Calcium Measurements
[0144] In order to assess the AT1R interaction of the different NP formulations a ratiometric Fura-2 Ca.sup.2+ chelator method was used as previously described [1, 3] using AT1R positive rMCs. For that purpose, rMCs were seeded in T-150 flasks (Corning) and incubated until confluent. Subsequently, they were trypsinized, centrifuged (200g, 5 min) and resuspended in Leibovitz medium supplemented with 5 μM Fura-2, AM (Thermo Fisher), 0.05% Pluronic F-127, and 2.5 mM Probenecid. Cells were incubated for 1 hour, light protected, with gentle agitation (50 rpm). Afterwards, the cell suspension was washed with DPBS by centrifugation (2×, 200 g, 5 min, RT), and resuspended in Leibovitz medium supplemented with 2.5 mM Probenecid at a count of 2 million cells/mL. To determine the particle avidity and ligand affinity for the AT1R (
[0145] Enzyme Kinetic Measurements
[0146] The Michaelis-Menten kinetics for NPAng-I and NPEXPAng-I were determined as previously described [1] using rabbit lung ACE (Sigma Aldrich) as a soluble surrogate for the cell membrane-bound enzyme. In short, different concentrations of NPs (corresponding to 10-120 μM Ang-I) were incubated with 18 μM of enzyme for different time periods (5, 15, 30, 60, 90 and 120 min) to convert the Ang-I on the particle corona to the AT1R-active ligand Ang-II. The resulting Ang-II was quantified by direct intracellular calcium measurements. To that end, rMCs were loaded with Fura-2 dye as described above. Then, 10 μL samples were pipetted onto a 96-well half area plate and 90 μL of a Fura-2-loaded rMC-suspension (90,000 cells/well) were injected on top of them. The resulting calcium signal was recorded immediately over 1 minute using a FLUOstar Omega microplate reader (BMG, Labtech), as described above. In order to rule out the interference of the EXP3174 ligand on NPEXPAng-I under the experimental conditions used, NPEXP were used as a control (see
[0147] Cellular Distribution of NPs: Confocal Microscopy
[0148] In order to determine the cellular distribution of the different particle formulations (
[0149] Cellular Distribution of NPs: Flow Cytometry
[0150] Analysis of particle uptake through flow cytometry (
[0151] NP Target Cell Specificity: Flow Cytometry
[0152] To assess the NP uptake in different cells lines (
[0153] The particle specificity in co-culture of target and off-target cells was investigated through flow cytometry (
[0154] NP Target Cell Specificity: Confocal Microscopy
[0155] To confirm the flow cytometry experiments, CLSM analysis of the particle specificity in co-culture (
[0156] NP Kidney Distribution In Vivo
[0157] To assess the kidney distribution of the different NP formulations (NPEXPAng-I, NPAng-I, NPEXP and NPMeO), 100 μL of a 120 nM NP solution (equivalent to approximatively 10 mg/ml NPs) were injected via the vena jugularis in 10-week old female NMRI mice (Charles River) that were anesthetized with isoflurane inhalation and buprenorphine (0.1 mg/kg body weight) (n=6 for each particle sample). Additionally, as a control 100 μL of the free dye used to fluorescently label the particles (CF647) was injected in the same concentration contained in a particle sample (approximatively 50 μM). After 5 min a blood sample was collected via i.v. punction while mice were still under anesthesia. After 1 h of particle circulation mice were anaesthetized with ketamine/xylazine, a final blood sample was collected and they were killed through perfusion fixation with 4% PFA. The kidneys were harvested and cut transversally. They were cryoprotected by placing them in phosphate buffer (0.1 M pH 7.4) supplemented with 18% sucrose and 1% PFA overnight. Afterwards, they were frozen in liquid nitrogen-cooled 2-propanol (−40° C.) and embedded in Tissue Tek® O.C.T.™ Compound for cryosections. Kidneys were cut into 5 μm sections using a CryoStar NX70 cryostat (Thermo Fisher Scientific) and transferred onto Superfrost™ plus glass slides. For better visualization cell nuclei were stained with DAPI (12.5 μg/ml in DPBS) prior to section imaging using an Axiovert 200M (Zeiss) fluorescence microscope and Zen software (Zeiss). Images of the whole kidney were acquired using a lox objective (
[0158] Immunohistochemistry
[0159] To assess the glomerular localization of NPs, freshly cut 5 μm kidney cryosections were washed for 5 min with DPBS, 5 min with DPBS supplemented with 0.1% sodium dodecyl sulfate (SDS) and 5 min with DPBS prior to 10 min-blockage with 5% BSA in DPBS supplemented with 0.04% Triton-X (m/v). Sections were washed again with DPBS (5 min) and incubated overnight in a 1:200 solution of the primary polyclonal goat anti-Integrin-α8 antibody in DPBS supplemented with 0.5% BSA and 0.004 Triton-X (m/v) at 4° C. Then, they were washed for 5 minutes in DPBS and incubated for 1 h with the Cyt-anti-goat secondary antibody (1:400) and DAPI (12.5 μg/ml) in DPBS supplemented with 0.5% BSA and 0.04% Triton-X at r.t. in the dark. Cryosections were washed with DPBS and ultrapure water before they were mounted using Dako Faramount Mounting Medium and analyzed using a Zeiss LSM 700 microscope and Fiji software, as described above.
[0160] Quantification and statistical analysis
[0161] Statistical analysis was performed using GraphPad Prism Software 6.0. Student t tests or two-way ANOVA with a Sidak's or Turkey's multiple comparison test were employed to evaluate statistical significance as indicated in the method details. Levels of statistical significance and “n” numbers for each experiment are indicated in the text and figure legends.
Example 2: Block Copolymers Allow for a Virus-Mimetic Particle Design
[0162] All materials and methods mentioned in this example were as described in the previous example.
[0163] For the development of virus-mimetic NPs, the present inventors coupled the ligands EXP3174 and Ang-I to poly(ethylene glycol)5k-poly(lactic acid)10k (PEG-PLA) block copolymers (
Example 3: NPs Recognize Target Receptors In Vitro
[0164] All materials and methods mentioned in this example were as described in the previous examples.
[0165] To confirm the particles' ability to triple check a cell's identity was initially assessed in vitro. Particle avidity for the target receptor, which mediates primary attachment and subsequent internalization, was investigated using calcium mobilization assays, since the stimulation or silencing of the Gq-coupled AT1R with an agonist or antagonist results in a cytosolic Ca.sup.2+ influx or its suppression, respectively. To that end, ATTR-positive rat mesangial cells (rMCs) were incubated for a 30 minute period with varying concentrations of either free ligands or NP-formulations prior to stimulation with Ang-II and recording the resulting calcium signal. As depicted in
[0166] The coupling to linkers leads to an affinity loss that is compensated by the high avidity multivalent binding of several receptors simultaneously (
[0167] To assess kinetics of cell/particle interactions, intracellular calcium measurements were performed over a 5.5-hour period by incubating NPs with rMCs at a concentration corresponding to 10 μM ligand. The extent to which they could silence calcium signaling triggered by the present free agonist served as a measure for the completeness to which the respective particles had bound via their ligands to the AT1R in the cell surface at different time points (
[0168] A prerequisite for particle internalization is the ability of ACE to activate Ang-I to Ang-II. Therefore, the present inventors investigated the enzyme kinetics for NPEXPAng-I, to determine whether the presence of the antagonist on the particle surface would hinder the enzymatic reaction. A soluble form of ACE was incubated for varying time periods with different particle concentrations and the resulting Ang-II on the NP corona was quantified running calcium mobilization assays. The interference of the EXP3174 ligand in the assay was assessed by measuring the signal inhibition exhibited by NPEXP (
Example 4: Decision-Making NPs are Target-Cell Specific
[0169] All materials and methods mentioned in this example were as described in the previous examples.
[0170] After the particle interaction with their individual targets had been successfully established, the next step was to determine if NPs carrying an antagonist as well as an agonist on their corona would still trigger internalization by their target cells, and if so, if the uptake ensued from a specific ligand-receptor interaction. As antagonists do not cause AT1R-mediated endocytosis and agonists do, the present inventors investigated via confocal laser scanning microscopy (CLSM) the cellular localization of NPEXPAng-I in rMCs expressing YFP-tagged AT1R (pAT1R-rMCs). As shown in
[0171] Accordingly, there is a specific particle uptake, mediated by the AT1R. However, particles carrying only the antagonist (NPEXP) were not internalized by the cells and located mostly on the cellular surface (
[0172] For NPEXP a receptor rearrangement on the cell membrane also occurred, which is a result of a multivalent receptor binding promoted by receptor movement on the cellular surface. Once NPEXP attach to a receptor on the cell membrane, their lack of internalization can lead to receptor-particle mobility on the cell membrane, and further receptor binding. Particles without ligands (NPMeO) were not taken up by the cells (
[0173] Overall, the present inventors demonstrate that the presence of an attachment-mediating antagonistic ligand linked to the particle corona does not hinder subsequent particle internalization. More so, the inclusion of an additional ligand on the particle surface compensated the targeting loss due to stearic hindrance of the Ang-I ligand by the addition of a higher number of long polymer chains (
[0174] Furthermore, the present inventors examined the particle internalization in different cell lines by flow cytometry (
[0175] Target cells (rMCs) were seeded together with an excess of off-target NCI-H295R or HeLa cells, which both lack the ACE and express high and low AT1R levels, respectively. They were incubated with the different NP formulations and each cell line was investigated for particle-associated fluorescence through flow cytometry (
Example 5: NPs Target MCs In Vivo
[0176] All materials and methods mentioned in this example were as described in the previous examples.
[0177] Since the complementary targeting ability of both ligands on NPEXPAng-I and the particle specificity was demonstrated in vitro the next step was to determine whether the viral recognition principle would lead to a higher in vivo MC accumulation. To that end, targeted (NPEXPAng-I, NPAng-I and NPEXP) (
[0178] In order to quantitatively assess the NP-associated fluorescence and better distinguish the differences among the different particle formulations, images of the glomeruli were taken at higher magnifications (
[0179] Additionally, NPEXPAng-I displayed significantly higher accumulation than one-ligand targeted particles (7- and 5-fold higher than NPEXP and NPAng-I, respectively) (
[0180] Taken together these results clearly show that size-mediated targeting is a necessary prerequisite to reach the mesangium, but insufficient to achieve particle accumulation in MCs. NP internalization seems to be imperative to avoid mesangial clearance, which explains that particles lacking this trait (NPMeO and NPEXP) lead to the lowest glomerular fluorescence. Implementing a virus-mimetic recognition principle (NPAng-I) increases NP specificity and results in particle uptake which in turn leads to a higher MC-accumulation. However, facilitating the target cell recognition via an initial virus-like cell attachment (NPEXPAng-I) significantly enhances the NP's targeting potential, a result of a combined effect of the two ligands, as shown by the in vitro studies.
[0181] Furthermore, the enhanced functionalization of NPEXPAng-I does not lead to a decrease in the particle blood residence. Generally, NPs are coated with polymers such as PEG, which increase their circulation time and decrease plasma protein adsorption. A positive effect, which is usually counteracted by ligand functionalization, as off-target cells expressing the targeted receptors can bind and interfere with the NPs. Nevertheless, quantification of the plasma NP fluorescence one hour after injection showed that NPEXPAng-I remained in circulation to the same extent as non-targeted NPMeO and significantly longer than the other targeted formulations (
Example 6: Materials and Methods
[0182] Materials
[0183] Heterobifunctional hydroxyl poly(ethylene glycol)carboxylic acid with a molecular mass of 2000 and 5000 g mol.sup.−1 (COOH-PEG.sub.2k/5k-OH) and hydroxyl poly(ethylene glycol)Boc-amine with a molecular mass of 2000 g mol.sup.−1 (Boc-NH-PEG.sub.2k-OH) were purchased from Jenkem Technology USA Inc. (Allen, Tex., USA) while methoxy poly(ethylene glycol)with a molecular mass of 5000 g mol.sup.−1 (MeO-PEG.sub.5k-OH) and Resomer RG 502 (PLGA) were obtained from Sigma-Aldrich (Taufkirchen, Germany). EXP3174 (also known as losartan carboxylic acid) was purchased from Santa Cruz (Heidelberg, Germany), while Cyclic RGDfK (cRGDfK) was obtained from Synpeptide Co. Ltd. (Shanghai. China). AlexaFluor™ 568 Hydrazide (Alexa568), CellTracker™ Green Dye (CTG) and CellTracker™ Deep Red Dye (CTDR) were purchased from Thermo Fisher Scientific (Schwerte, Germany). Amine-functionalized spherical gold NPs with an average diameter of 2.2 nm (Au.sub.2.2-NH.sub.2) were obtained from Nanopartz Inc. (Loveland, Colo., USA). GoldEnhance™ EM Plus kit was purchased from Nanoprobes (Yaphank, N.Y., USA). Goat-derived Integrin α-8 antibody was obtained from R&D Systems (Minneapolis, Minn., USA). All other chemicals were purchased from Sigma-Aldrich in analytical grade if not stated differently. Ultrapure water was obtained from a Milli-Q water purification system (Millipore, Billerica, Mass., USA). NCI-H295R (CRL-2128) and HeLa (CCL-2) cells were purchased from ATCC (Manassas, Va., USA). All cell lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum, Insulin-Transferrin-Selenium (ITS) (1×) and 100 nM hydrocortisone.
[0184] Polymer Synthesis
[0185] COOH-PEG.sub.2k-PLA.sub.10k, Boc-NH-PEG.sub.5k-PLA.sub.10k and MeO-PEG.sub.5k-PLA.sub.10k block copolymers were synthesized via a ring-opening polymerization as previously described. In brief, heterobifunctional PEG polymers (1 equivalent=equiv) were mixed with 3,6-dimethyl-1,4-dioxane-2,5-dione (70 equiv) and 1,8-diazabicylo [5.4.0] undec-7-ene (3 equiv). The polymer mixture was stirred for 1 hour (h) at room temperature (RT) until polymerization was quenched with benzoic acid (14 equiv). Resulting block-copolymer was precipitated in diethyl ether, isolated via filtration and dried under vacuum. Molecular weight of synthesized polymers was determined in deuterated chloroform at 295 K using a Bruker Avance 300 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany).
[0186] For preparation of cRGDfK-PEG.sub.2k-PLA polymers, previously synthesized COOH-PEG.sub.2k-PLA.sub.10k was covalently coupled to the lysine residue of cRGDfK as shown before. In short, COOH-PEG.sub.2k-PLA.sub.10k (1 equiv) was activated using 3-(ethyliminomethyleneamino)-N,N-dimethylpropan-1-amine (EDC)/N-hydroxysuccinimide (NHS) (25 equiv) for 2 h at RT, followed by quenching with β-mercaptoethanol (BME) (30 equiv). Activated polymer was reacted with cRGDfK (3 equiv) and N,N-diisopropylethylamine (DIPEA) (10 equiv) for 24 h at RT. After precipitation of resulting cRGDfK-coupled polymer in diethyl ether/methanol (15:1 V/V)), free cRGDfK and excess reactants were removed using dialysis against millipore water (mpH.sub.2O).
[0187] For EXP3174-PEG.sub.5k-PLA.sub.10k, the Boc-protecting group of Boc-NH-PEG.sub.5k-PLA.sub.10k was initially cleaved. In brief, Boc-protected polymer was dissolved in dichloromethane (DCM)/trifluoroacetic acid (TFA) (1:1 V/V). After stirring for 30 minutes (min), excess TFA was neutralized using a saturated sodium hydrogen carbonate solution. The organic phase was washed with mpH.sub.2O, followed by polymer isolation as described above. Resulting NH.sub.2-PEG.sub.5k-PLA.sub.10k was coupled to EXP3174 via the carbonyl residue of the imidazole component. EXP3174 (3.5 equiv) was activated with N,N′-dicyclohexylcarbodiimide (DCC)/NHS (3.3 equiv) for 2 h at RT. After removal of resulting dicyclohexylurea via centrifugation, NH.sub.2-PEG.sub.5k-PLA.sub.10k (1 equiv) and DIPEA (17.5 equiv) were added and reacted for 24 h at RT. Resulting EXP3174-PEG.sub.5k-PLA.sub.10k was precipitated in methanol/diethyl ether (1:5 V/V) and the product was dialyzed against ethanol/100 mM borate buffer pH 8..sub.5/water (1/1/8 V/V) for 24 hours followed by mpH.sub.2O for 12 h to remove unreacted EXP3174 and excess reactants.
[0188] PLGA Labeling with Fluorescent Dyes
[0189] For particle visualization, the core-forming PLGA was covalently linked to fluorescent dyes prior to NP preparation. To that end, carboxylic acid-terminated PLGA was activated for 2 h using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as a catalytical agent. Activated PLGA was then reacted either with AlexaFluor™568 Hydrazide or CFTM 647 amine for 24 h at RT. Labeled PLGA was dialyzed against mpH.sub.2O for 24 h to remove unreacted fluorescent dyes.
[0190] PLGA Labeling with Nanogold
[0191] For electron microscopy analysis, PLGA was conjugated to nanogold. PLGA was initially activated with EDC and NHS for 2 h in DCM. After DCM removal under reduced pressure, activated PLGA was dissolved in DMSO and mixed with DIPEA and lyophilized monoamino gold nanoparticles with an average diameter of 2.2 nm (Au.sub.2.2-NH.sub.2). After stirring at RT for 24 h, gold-conjugated PLGA was precipitated in mpH.sub.2O, isolated via centrifugation at 2500 g for 10 min and lyophilized.
[0192] NP Preparation
[0193] Block-copolymer nanoparticles were manufactured using a common solvent evaporation technique. Corresponding amounts of PEG-PLA polymers and PLGA were mixed at a ratio of 70/30 (m/m) and diluted in acetonitrile (ACN) to a final concentration of 10 mg mL.sup.−1. To reach the desired ligand surface density for hetero-/homo-functional NP species, cRGDfK-PEG.sub.2k-PLA and/or EXP3174-PEG.sub.5k-PLA.sub.10k were mixed with COOH-PEG.sub.2k-PLA.sub.10k according to the calibration depicted in
[0194] Resulting NP dispersions were concentrated via centrifugation at 1250 g for 25 min using Pall Microsep filters (molecular weight cut-off 30 kDa; Pall Corporation, N.Y., USA). To obtain the mass concentration of manufactured NPs, PEG content was assessed using a colorimetric iodine complexing assay. NPs were then lyophilized and gravimetrically analyzed to obtain the ratio of PEG content and NP weight. In the following experiments, this ratio was used to calculate mass concentration from the assessed PEG content for each NP species.
[0195] NP Characterization
[0196] NP size and ζ-Potential was evaluated using a Malvern Zetasizer Nano ZS (Malvern, Herrenberg, Germany). Samples were measured with a 633 nm He—Ne laser at an angle of 173° (25° C., RT) in 7.5 mM DPBS, using either PMAA semimicro cuvettes (DLS; Brand, Wertheim, Germany) or folded capillary cells (c-Potential; Malvern, Herrenberg, Germany).
[0197] cRGDfK Quantification
[0198] The level of cRGDfK on the NP surface was assessed based on the measurement of arginine. In brief, .sub.50 μL of NP samples (1 mg mL.sup.−1) were mixed with 175 μL of a working solution consisting of 9,10-phenanthrenequinone (150 μM in ethanol) and 2 N NaOH (6:1 V/V). After 3 h of incubation at 60° C., 1 equiv of sample was mixed with 1 equiv of 1 N HCl and incubated for another 1 h at RT. Finally, fluorescence was measured at a Synergy™ Neo2 Multi-Mode Microplate Reader (BioTek Instrument Inc., Winooski, Vt., USA) with an excitation wavelength of 312/7 nm and an emission wavelength of 395/7 nm. Dilutions of cRGDfK (0-40 μg mL.sup.−1) served as calibration. cRGDfK molarity as well as the ratio of molar cRGDfK content and molar PEG content were determined and plotted against the theoretical value (
[0199] EXP3174 Quantification
[0200] To determine the surface level of EXP3174 on manufactured particles, 1 equiv of NP samples (1 mg mL.sup.−1) was mixed with 10 equiv of 0.2 M acetic acid. Dilutions of EXP3174 in 0.2 M acetic acid (0-30 μM) served as calibration. Fluorescence of samples and standards was measured at a Synergy™ Neo2 Multi-Mode Microplate Reader (see above) (excitation 250/10 nm, emission 370/5 nm). EXP3174 molarity as well as the ratio of molar EXP3174 content and molar PEG content was determined and plotted against the theoretical value (
[0201] Calcium Mobilization Assay
[0202] In order to investigate AT1r binding of NPs, intracellular calcium levels were measured using fura-2 as a Ca.sup.2+ chelator. In brief, rMCs were incubated with 5 μM fura-2AM, 2.5 mM probenecid and 0.05% Pluronics F-127 in Leibovitz's L-15 medium for 1 h at RT. Cells were thereafter centrifuged (5 min, 200 g, RT) and resuspended in Leibovitz's medium. 45 μL of NPs or free EXP3174 at different concentrations were pipetted into 96-well plates (Greiner Bio One, Frickenhausen, Germany), followed by 45 μL of rMC suspension (2×10.sup.6 mL.sup.−1). In the following, cells were incubated with samples for 45 min at RT. After incubation, 10 μL of 30 nM AT II was added to each well to activate uninhibited AT1r and consequently induce Ca.sup.2+ influx into the cell cytosol. Fluorescence signal during the first 30 seconds after injection was measured using a FluoStar Omega fluorescence microplate reader (BMG Labtech, Ortenberg, Germany) with excitation filters at 340/20 nm and 380/20 nm and the emission filter at 510/20 nm, respectively. Maximal ratio of Ca.sup.2+-bound to Ca.sup.2+-unbound Fura-2 was evaluated by incubating loaded cells with 0.1% Triton-X 100 and measuring fluorescence levels as described above. Analogously, minimal ratio was achieved by incubation with 0.1% Triton-X 100 combined with 45 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). Levels of intracellular calcium per sample was calculated using the equation of Grynkiewicz et al. Half maximal inhibitory concentrations (IC.sub.50) were calculated using GraphPad Prism (San Diego, Calif, USA) and applying a sigmoidal dose-response equation (variable slope).
[0203] CLSM Analysis
[0204] For detailed analysis of NP-cell interaction, rMCs were seeded into 8-well slides (Ibidi, Gräfelfing, Germany) at a density of 15.000 cells well.sup.−1 and incubated for 24 hours at 37° C. In order to facilitate visualization of the cell cytosol, rMCs were stained with CTDR (25 μM, 45 min, 37° C.) in serum-free RPMI 1640 medium prior to seeding. NPs were manufactured using AlexaFluor™ 568-labeled PLGA and adjusted to 0.05 mg mL.sup.−1 in Leibovitz's buffer supplemented with 0.1% BSA. Mesangial cells were incubated with 250 μL of NPs for 15, 45 and .sub.90 min at 37° C., washed with prewarmed DPBS and fixed with 4% paraformaldehyde (PFA) in DPBS for 10 min. After a final washing step, fixed samples were analyzed using a Zeiss LSM 710 (Carl Zeiss Microscopy GmbH, Jena, Germany).
[0205] Flow Cytometry
[0206] To assess mesangial cell association of NP samples, rMCs were seeded into 24-well plates (Greiner Bio One, Frickenhausen, Germany) at a density of 40.000 cells well.sup.−1 and incubated for 48 h at 37° C. NPs were manufactured using CF™ 647-labeled PLGA and adjusted to 0.05 mg NP mL.sup.−1 in Leibovitz's buffer supplemented with 0.1% bovine serum albumine (BSA). To confirm α.sub.Vβ.sub.3-dependence of NP cell entry, 300 μL of free cRGDfK (c=500 μM) were added to the relevant cell samples for 15 minutes prior to NP incubation. Cells were washed with DPBS and 300 μL of prewarmed NP solutions were added for 60 minutes at 37° C. For respective analysis of time-dependent uptake, cells were incubated over a time period of 120 minutes with NPs being removed after 0, 15, 30, 45, 60, 90 and 120 minutes. Cells were washed with DPBS, trypsinized and centrifuged for 5 min at 200 g and 4° C., followed by two further washing and centrifugation steps (DPBS, 200 g, 5 min, 4° C.). Final samples were resuspended in DPBS and analyzed using a FACS Calibur cytometer (Becton Dickinson, Franklin Lakes, N.J., USA). NP-associated fluorescence was excited at 633 nm and corresponding emission was recorded (661/16 bandpass filter). Flow cytometry data was analyzed using Flowing software 2.5.1 (Turku Centre for Biotechnology, Turku, Finland). Within the population of viable cells, geometric mean of cell-associated fluorescence was evaluated.
[0207] Transmission Electron Microscopy
[0208] To evaluate cellular localization of NPs, rMCs were seeded into a 24-well plate at a density of 12.000 cells well.sup.−1 and incubated for 72 hours. NP formulations containing nanogold-conjugated PLGA were diluted in Leibovitz's buffer containing 0.1% BSA and added for 45 min at a concentration of 0.05 mg mL.sup.−1 (V=300 μL). After incubation, samples were washed with DPBS and prepared for electron microscopy analysis. In brief, cells were fixed with 2.5% PFA and 2.5% glutaraldehyde in a 0.1 M sodium cacodylate solution (Caco buffer) for 60 min at RT, washed with Caco buffer and permeabilized with 0.1% Triton-X in DPBS for 10 min. After a washing step with mpH.sub.2O, samples were gold enhanced using a GoldEnhance™EM Plus kit (Nanoprobes Inc., Yaphank, N.Y., USA) according to the manufacturer's specifications, followed by further washing and post-fixation in a 2.5% sodium thiosulfate solution in mpH.sub.2O. Cells were stained with 0.5% osmium tetroxide and dehydrated in rising concentrations of ethanol (50-99.5%) For counterstaining, 2% uranylacetate was applied for 5 min at 70% ethanol concentration. After embedding in Epon, ultrathin sections of 150 nm were imaged using a 100 kV Zeiss Libra 120 electron microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany) at a magnification of 6300× as well as 12500×.
[0209] Co-Culture Experiments
[0210] In order to assess cell selectivity of manufactured NPs, the present inventors used a co-culture design that had been previously implemented by the inventors. For flow cytometry analysis, rMCs were seeded together with HeLa or NCI-H295R cells in 24-well plates at a density of 10.000 and 75.000 cells well.sup.−1 respectively and incubated for 48 h at 37° C. To differentiate between cell types, rMCs were stained with CTG (15 μM, 45 min, 37° C.) in serum-free RPMI 1640 medium prior to seeding. Co-cultured cells were then incubated with CF™647-labeled NPs at a concentration of 0.05 mg mL.sup.−1 (V=300 μL) for 45 min. Preparation of samples and flow cytometry analysis was performed as described above. Additionally, rMC-associated fluorescence was excited at 488 nm and recorded using a 530/30 bandpass filter. During data analysis, the population of viable cells was further gated for stained rMC cells and NP-associated fluorescence analyzed concerning cell specificity. For CLSM analysis, rMC cells were CTG-stained prior to seeding as described above. To visualize all cell types, HeLa or NCI-H295R cells were also stained using CTDR (25 μM, 45 min, 37° C.). After CellTracker™ incubation, rMCs were seeded into 8-well Ibidi slides together with HeLa/NCI-H295R cells at a density of 2.000 and 10.000/20.000 cells well.sup.−1. After 48 h of incubation at 37° C., cell nuclei were stained with Hoechst 33258 (5 μg mL.sup.−1 in DPBS) for 20 minutes. Cells were washed twice with prewarmed DPBS and AlexaFluor™568-labeled NPs were added at a concentration of 0.05 mg mL.sup.−1 (V=250 μL) for 45 min at 37° C. After NP incubation, samples were treated as described above and analyzed using a Zeiss LSM 710 microscope.
[0211] In Vivo Cell Targeting
[0212] Animal experiments were performed according to the national and institutional guidelines and were approved by the local authority (Regierung von Unterfranken, reference number: 55.2-2532-2-329). Female, 10-week-old NMRI mice (Charles River, Sulzfeld, Germany) acted as model animals. After analgesia with buprenorphin (0.1 mg kg body weight.sup.−1), mice were anaesthetized with 2.5% isoflurane and 100 μL of CF™ 647-labeled NPs (c=120 nM) were injected via the vena jugularis. Mice were kept in anesthesia and after 5 min, an initial blood sample was taken via i.v. punction. After 60 minutes, mice were anaesthetized with ketamine/xylazine, a final blood sample was taken, and animals were killed via perfusional fixation. Both kidneys were removed and immediately transferred to a 18% sucrose and 14% PFA solution in phosphate buffer (0.1 M pH 7.4). After 6 h, kidneys were washed with DPBS and cryoprotected at −80° C. until further processing. For cryosections, the organs were embedded in Tissue Tek® O.C.T.™ Compound (Sakura Finetek, Torrance, Calif., USA), cut into 5 μm sections using a CryStar NX70 cryotome (Thermo Fisher Scientific, Waltham, Mass., USA) and fixed on Superfrost198 plus glass slides (Thermo Fisher Scientific, Schwerte, Germany). For analysis of NP kidney deposition and glomerular fluorescence quantification, sections were rinsed in DPBS and blocked with 5% BSA supplemented with 0.04% Triton-X in DPBS for 10 min at RT. After further rinsing in DPBS, samples were stained for cell nuclei with a 1:400 dilution of 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) in 0.5% BSA and 0.04% Triton-X in DPBS. After a final washing step in DPBS and mpH.sub.2O respectively, cryosections were mounted with Mowiol mounting medium and analyzed at a Zeiss Axiovert 200M. For image analysis, Fiji software (Madison, Wis., USA) was used. Glomerular fluorescence intensities were evaluated by measuring the integrated density of areas over a certain fluorescence threshold and division by the glomerular area. In order to assess the exact cellular location of NPs, kidney cryosections were prepared as described above. After washing and blocking of sections, samples were stained overnight at 4° C. with a goat-derived Integrin α-8 antibody (1:200 dilution in 0.5% BSA/0.04% Triton-X in DPBS). Samples were thereafter washed with DPBS and stained with a 1:400 dilution of Cyt® donkey anti-goat and DAPI in 0.5% BSA/0.04% Triton-X in DPBS for ih at RT. After a final washing step, samples were mounted and analyzed at a Zeiss LSM 710.
Example 7: Preparation of Hetero-Multivalent EXPcRGD NPs Using a Modular Concept
[0213] All materials and methods mentioned in this example were as described in example 6.
[0214] In order to create NPs with the desired adenovirus-mimetic properties, the present inventors implemented a modular design that is based on the synergistic combination of different biocompatible polymer components into a hetero-multivalent particle species (
[0215] The present inventors decided to prepare hetero-functional nanoparticles that carry 25% EXP3174 and 15% cRGDfK on their surface (EXPcRGD NPs), thereby sufficiently exploiting the ligands' receptor binding capacities but preserving structural integrity of manufactured particles. Particles should be able to locate the target cell by binding to the AT1r via sterically flexible EXP3174, then lower the spatial distance to the cell surface and subsequently activate αVβ3 integrins via previously concealed cRGDfK, which eventually initiates NP endocytosis (
Example 8: Hetero-Multivalent EXPcRGD NPs Display Excellent Ligand Affinity for Target Motifs
[0216] All materials and methods mentioned in this example were as described in example 6 and 7.
[0217] The present inventors tested EXP3174-mediated NP binding to the AT1r expressed by rat mesangial cells (rMCs). As activation of G.sub.q-coupled AT1r with its primary ligand angiotensin II (AT II) results in a calcium influx into the cell cytosol, intracellular Ca.sup.2+ levels after AT II stimulation can be used as a marker for AT1r activity after NP incubation. Thereby, low receptor activity indicates a high ratio of bound EXP3174, as the ligand itself acts as a potent antagonist.
[0218] Having verified the AT1r binding capacity of adenovirus-mimetic EXPcRGD NPs, the next step was to investigate particle uptake into rMCs via cRGDfK-αVβ3 interaction. The present inventors therefore incubated mesangial cells with fluorescently labeled NPs and analyzed the cellular distribution via confocal laser scanning microscopy (CLSM). In order to visualize the cell body, rMCs were pre-treated with CellTracker™ Deep Red (CTDR).
[0219] Building on CLSM results, the present inventors performed flow cytometry analysis of NP-treated rMCs and determined the cell-associated fluorescence over an incubation period of 120 minutes. As shown in
[0220] To investigate the impact of αVβ3 integrin on EXPcRGD NP cell uptake, an excess of free cRGDfK (c=500 μM) was added prior to incubating rMCs with EXPcRGD NPs for 60 min. As a result, levels of cell-associated fluorescence sharply decreased to levels comparable with those of EXP NPs or Control NPs (
[0221] To further verify the concept of integrin-mediated NP endocytosis, the present inventors decided to utilize transmission electron microscopy (TEM), which enabled the assessment of NP-cell interactions at a much higher magnification level. In order to increase electron density and consequential TEM visibility of applied NPs, ultrasmall gold nanoparticles with an average diameter of 2.2 nm were covalently coupled to PLGA that was then used for further NP manufacture (
[0222]
Example 9: Ligand Synergism Leads to an Enhanced Mesangial Cell Selectivity In Vitro
[0223] All materials and methods mentioned in this example were as described in examples 6 to 8.
[0224] Having demonstrated that hetero-multivalent EXPcRGD NPs synergistically combine both key features of its surface ligands and present them in a sterically controlled manner, the present inventors intended to demonstrate that this design can actually be utilized to increase mesangial cell selectivity. The present inventors therefore implemented an in vitro based assay, in which target rMCs were co-cultured with a superior number (5-10 fold) of off-target cells carrying none or merely one of the two target receptors. While HeLa cells expressed neither AT1r nor αVβ3-integrin to a significant degree, NCI-H295R cells were chosen as they showed a high AT1r but low αVβ3 expression (
[0225] To differentiate between co-cultured cells in CLSM analysis, CellTracker™ Green (CTG) was used to stain rMCs while off-target cells were marked with CTDR. After 45 minutes of incubation with fluorescently labeled EXPcRGD NPs, cellular distribution of NPs was assessed. In the rMC/HeLa co-culture model, particle-derived fluorescence could almost exclusively be detected within the areas of mesangial cells. HeLa cells, in contrast, showed merely weak interaction with NPs, resulting in marginal fluorescence levels (
[0226] In summary, the present inventors' co-culture model demonstrated that hetero-multivalent EXPcRGD NPs have the capability to effectively identify receptor-positive mesangial cells in the presence of off-target cells that are not only prevailing in number but even express one of the two target receptors.
Example 10: Accumulation of Adenovirus-Mimetic EXPcRGD NPs in Mesangial Cells In Vivo
[0227] All materials and methods mentioned in this example were as described in examples 6 to 9.
[0228] Both rMC binding and uptake studies successfully showed that the present inventors' virus-mimetic concept of sequential ligand-receptor interaction enables hetero-multivalent EXPcRGD NPs to selectively target mesangial cells in vitro. However, transferring in vitro results into a robust system with sufficient in vivo efficiency has been shown to be the major obstacle in nanoparticle design as many strategies fail to deliver desired target-specificity. The present inventors therefore decided to assess the NPs' capability to actually reach mesangial areas in vivo which does not only require active cell uptake facilitation but also adequate passive accumulation in the target region. To that regard, fluorescently labeled NPs were injected into io-week-old female NMRI mice. After 1 h of NP circulation, mice were sacrificed, and kidneys were extracted. Fluorescence analysis of prepared cryosections revealed that EXPcRGD NPs effectively accumulated in glomerular areas while fluorescence in tubular parts of the kidney was neglectable (
[0229] To quantify observed differences, glomerulus-associated fluorescence levels were determined by assessing the glomerular fluorescence intensity per area for all NP types (
[0230] Thus, the herein discussed in vivo studies successfully demonstrated the potential of the new, adenovirus-mimetic NP design of the present invention. Hetero-multivalent EXPcRGD NPs effectively accumulated in mesangial areas of the glomerulus while homo-functional or unfunctionalized NP species failed to do so. This strongly suggests that in order to reach sufficient levels of bioavailability, NPs do not only have to carry appropriate surface ligands but must also present them in an orchestrated fashion that is suitable for the respective targeting strategy. Moreover, NP accumulation in the mesangium also proved that the adenovirus-mimetic system of sterically controlled particle-cell interaction is highly effective.
[0231] Example 11: Cinaciguat-Loaded EXPcRGD NPs Show High Efficiency
[0232] Nanoparticles (NPs) using either an influenza A mimetic or adenovirus mimetic target cell recognition concept were detected to be efficiently accumulating within mesangial cells in an in vivo setting. In a next step, the experimental drug cinaciguat (BAY 58-2667) was encapsulated in adenovirus-mimetic EXPcRGD NPs. Cinaciguat (CCG) is a potent activator of the soluble guanylate cyclase (sGC) and has been shown to significantly decrease mesangial fibrosis and reduce glomerular damage in a diabetes animal model. By encapsulating CCG in the inventors' promising NP species, cell-selective delivery of CCG to pathological mesangial sites can be considerably increased, leading to an enhanced therapeutic effect with minimized off-target effects (
[0233] In the inventors' experimental set-up, CCG was initially encapsulated in hetero-multivalent EXPcRGD NPs. Resulting NPs carried approximately 500-700 CCG molecules per NP (data not shown). In all following experiments, the administration of free cinaciguat at a concentration of 2 μM was compared to CCG-loaded EXPcRGD NPs at a concentration of approximately 0.5 nM (equaling 0.2 μM of CCG) and drug-free EXPcRGD control NPs (
[0234] To assess the effect of CCG-loaded EXPcRGD NPs on target sGC, mesangial cells were initially incubated for 24 h and protein quantity was assessed using Western Blot analysis. Interestingly, the overall amount of sGC was thereby gradually increasing both after incubation with free drug and CCG-loaded NPs, indicating not only the previously shown activating but also a stabilizing effect of cinaciguat on the sGC (
[0235] Finally, the anti-fibrotic and anti-proliferative potential of a NP-assisted CCG delivery was analyzed. In that regard, mesangial cells were initially incubated for 4 h either with free CCG, CCG-loaded EXPcRGD NPs or control NPs without encapsulated drug. After 4 h, 10 ng mL.sup.−1 of transforming growth factor β (TGF-β) were added for 48 h to induce a fibrotic and hyperproliferative remodeling. While administration of TGF-β lead to a considerable increase in mesangial cell proliferation, pre-incubation with both free CCG and CCG-loaded NPs could significantly reverse this effect (
[0236] Taken together, these results revealed two major outcomes:
[0237] 1. Both free and NP-encapsulated cinaciguat showed a significant effect on its target enzyme sGC, leading to a considerable activation of described anti-fibrotic pathway (
[0238] 2. Throughout all experiments, cinaciguat-loaded EXPcRGD NPs showed effects that were comparable to the administration of free CCG even though the overall amount of encapsulated CCG was only 10% of the free drug dose (0.2 μM vs. 2 μM). This indicates the considerable potential of described NPs to more efficiently deliver the pharmaceutical agent to its intended intracellular target.
REFERENCES
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[0243] The features of the present invention disclosed in the specification, the claims, and/or in the accompanying figures may, both separately and in any combination thereof, be material for realizing the invention in various forms thereof