CONJUGATES COMPRISING OCULAR ANGIOGENESIS GROWTH FACTOR APTAMERS AND USES THEREOF IN THE DETECTION AND TREATMENT OF OPHTHALMOLOGICAL ANGIOGENESIS INDICATIONS

Abstract

Provided is an antiangiogenic agent in the form of a vehicle, e.g., a nanoparticle associated (directly or indirectly) with at least one ocular angiogenesis growth factor aptamer, wherein said association labile to interaction between the aptamer and an ocular angiogenesis growth factor.

Claims

1. An antiangiogenic agent comprising a vehicle having a plurality of nucleic acids associated therewith, each nucleic acid in said plurality of nucleic acids having a sequence comprising at least 9 nucleic acid bases hybridized to at least 9 nucleic acid bases of a complementary nucleic acid sequence comprised within a sequence of at least one ocular angiogenesis growth factor (OAGF) aptamer, wherein said hybridization dissociates upon binding of at least one ocular angiogenesis growth factor to said at least one aptamer.

2.-3. (canceled)

4. The agent according to claim 1, wherein the vehicle is in the form of a nanoparticle selected from the group consisting of carbon quantum dots (C-dots), graphene oxide nanoparticles, DNA based nanoparticles, carbon nitride nanoparticles, metal organic framework nanoparticles, polymeric nanoparticles, polysaccharide nanoparticles and combinations thereof.

5. The agent according to claim 2, wherein the vehicle is C-dot.

6. The agent according to claim 5, wherein the C-dot is surface-associated with a plurality of nucleic acids, each nucleic acid in said plurality of nucleic acids having a sequence comprising at least 9 nucleic acid bases hybridized to at least 9 nucleic acid bases of a complementary nucleic acid sequence comprised within a sequence of at least one ocular angiogenesis growth factor (OAGF) aptamer.

7. The agent according to claim 6, wherein the C-dot is surface-associated with each of the nucleic acids via a covalent bond or the C-dot is surface associated with each of the nucleic acids via non-covalent interaction or the C-dot is functionalized with amine or carboxylic groups which associate to the plurality of nucleic acids.

8.-10. (canceled)

11. The agent according to claim 1, wherein at least a portion of the plurality of nucleic acids associated with the vehicle are single-stranded DNA having a sequence consisting or comprising 5-TCTACCCGGCCC-3 (SEQ ID NO: 1).

12.-15. (canceled)

16. The agent according to claim 11, wherein the single-stranded DNA having the structure 5-NH.sub.2-(CH.sub.2).sub.n-TCTACCCGGCCC-3 (SEQ ID NO:2), wherein n is an integer between 1 and 10.

17.-18. (canceled)

19. The agent according to claim 16, comprising a C-dot surface-associated with a plurality of single-stranded DNA sequences of SEQ ID NO:2, wherein n is 2 or 6.

20. The agent according to claim 1, wherein the vehicle is C-dot associated with a plurality of sequences having SEQ ID NO:2, wherein n is 6, each of the plurality of sequences being hybridized to the aptamer.

21. The agent according to claim 1, wherein the aptamer is of a sequence selected from the group consisting of: TABLE-US-00006 (SEQIDNO:3) 5-ACCTGGGGGAGTATTGCGGAGGAAGGTT-3, and (SEQIDNO:4) 5-TGTGGGGGTGGACGGGCCGGGTAGA-3.

22.-30. (canceled)

31. A composition comprising at least one antiangiogenic agent according to claim 1.

32.-38. (canceled)

39. A method of preventing or treating an ophthalmic disease or disorder associated with ocular angiogenesis in a subject, the method comprising topically administering to said subject an antiangiogenic agent according to claim 1.

40.-46. (canceled)

47. A conjugate comprising: (a) a nanoparticle having a plurality of nucleic acids associated therewith, each nucleic acid in said plurality of nucleic acids having a sequence comprising at least 9 nucleic acid bases hybridized to at least 9 nucleic acid bases of a complementary nucleic acid sequence comprised within a sequence of at least one ocular angiogenesis growth factor (OAGF) aptamer; (b) at least one fluorophore moiety, where the nanoparticle is not a fluorophore; and (c) at least one quencher moiety; wherein said at least one aptamer is associated with the at least one or both of fluorophore moiety and the at least one quencher moiety; and wherein upon binding of the aptamer to at least one ocular angiogenesis growth factor, said fluorophore moiety and/or quencher moiety are dissociated.

48. The conjugate according to claim 47, for use in a method of determining an amount of at least one ocular angiogenesis growth factor in a subject eye.

49. The conjugate according to claim 48, wherein the nanoparticle a fluorophore.

50.-60. (canceled)

61. The conjugate according to claim 48, wherein the at least one quencher moiety is selected from black hole quenchers, electron transfer quenchers and fluorescence resonance transfer quenchers.

62. The conjugate according to claim 48, wherein the at least one quencher moiety is selected from Iowa Black FQ and Black Hole Quencher (BHQ-3).

63.-65. (canceled)

66. A method of measuring levels of at least one OAGF in an eye of a subject, said method comprising topically administering to the eye of said subject a composition comprising at least one conjugate according to claim 48 and measuring fluorescence emitted following administration, wherein the level of fluorescence being indicative of the level of at least one OAGF in the eye.

67. (canceled)

68. A method of diagnosing at least one ophthalmic disease or disorder associated with increased level of at least one OAGF in a subject, said method comprising topically administering to the eye of said subject a composition comprising at least one conjugate according to claim 48 and measuring fluorescence emitted following administration, wherein the level of fluorescence being indicative of the level of the at least one OAGF in the eye and occurrence of said disease or disorder.

69. The method according to claim 68, for determining early stages of a disease, for monitoring success of drug treatment, or for determining suitability of existing treatment.

70.-74. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0146] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0147] FIGS. 1A-D depict various modes of association between a vehicle, e.g., a nanoparticle, and an aptamer, according to embodiments of the invention. When considered a therapeutic tool, the fluorophore (F) and quencher (Q) components may be ignored, while for the diagnostic aspect of the invention, either or both components are utilized. FIG. 1D provides a general scheme for chemically modifying a C-dot to permit association with an amine-associated aptamer.

[0148] FIGS. 2A-2B show the mean (SD) C-dot concentrations in the aqueous humor (AH), vitreous humor (VH), cornea and lens (FIG. 2A) and histological sections of the C-dot fluorescence in the eye structures including the retina (FIG. 2B).

[0149] FIGS. 3A-3B show the live imaging microscope (FIG. 3A) of the fluorescence of C-dots in the retina and the VH of live animals up to 24 hours following treatment and fluorescence measurements thereof (FIG. 3B).

[0150] FIG. 4 shows no evidence for C-dot toxicity for three cell types for different concentrations of C-dots, as presented in Example 3.

[0151] FIG. 5 shows sensing capabilities of the C-dot aptamer by fluorescence measurement after ATP injection.

[0152] FIG. 6 shows no evidence for in vivo toxicity as measured by ERG electroretinography test, as presented in Example 4.

[0153] FIG. 7 shows the plate reader readout of the extraction.

[0154] FIGS. 8A-8E show the fluorescence spectra for raw, quenched and post ATP/Cocaine.

[0155] FIG. 9 demonstrates the effect of C-dot aptamer on inhibition of choroidal blood vessels in vitro.

DETAILED DESCRIPTION OF EMBODIMENTS

[0156] FIGS. 1A-D depict general constructions of antiangiogenic agents and diagnostic tools according to the invention, as further explained herein. The experiments described herein and data provided have been mainly generated on C-dot systems, wherein the aptamer is associated to the C-dot as depicted, for general purposed, in FIG. 1D.

[0157] As may be realized from the data provided herein, the effect of C-dot-aptamer complex was shown to significantly inhibit growth of blood vessels in the choroid in an in vitro model of eye explants (FIG. 9). The effect was similar to common anti VEGF agents, which are given by intraocular injections. Thus, results presented herein may be extrapolated to other nanoparticles, aptamers and modes of conjugation.

[0158] The inventors of the technology disclosed herein have developed a breakthrough approach in which treatment of certain ophthalmological conditions is achieved by topically administering a drug, thereby eliminating the need for conventional repeated injections. The technology further provides the ability to maintain and monitor treatment efficacy by optical non-invasive measurement of ocular vascular endothelial growth factor (VEGF) levels, and other factors involved in the pathogenesis of blood vessels in the eye, thereby in fact, providing a sense and treat methodology. This approach has two main advantages over current available treatment modalities: first, it is capable of measuring the level of VEGF in the vitreous cavity, enabling treatment to be modified accordingly and customized per the patient's needs; and second, it can be applied topically, eliminating the need for repeated intraocular injections.

[0159] Carbon nanoparticles (C-dots) (2-3 nm) or graphene oxide (GO) nanoparticles are biocompatible and exhibit high luminescence properties. Single-stranded nucleic acids adsorb onto the graphitic nanomaterials, and upon using quencher-modified single-stranded nucleic acids, quenching of the C-dot or GO nanoparticles proceed.

[0160] The aptamer-based sensing platforms of VEGF are exemplified in FIGS. 1A-C. In one configuration shown in FIG. 1A, the aptamer sequence is caged by duplexes that include a fluorophor-functionalized nucleic acid and a quencher-modified nucleic acid. The fluorophore is quenched in the supramolecular structure. The formation of the aptamer-VEGF complex releases the fluorophore-labeled nucleic acid, resulting in the triggered-on fluorescence. Alternatively, a hairpin structure is modified at its 5 and 3 ends with a fluorophore quencher pair. The hairpin includes a caged sequence of the anti-VEGF aptamer, and the fluorescence of the fluorophore is quenched in the hairpin configuration. In the presence of VEGF, the formation of the aptamer-VEGF complex opens the hairpin, resulting in the switched-on fluorescence of the fluorophore. In a second sensing platform, shown in FIG. 1B, a supramolecular structure consisting of a nucleic acid (1) that includes the caged sequence of the VEGF aptamer is hybridized with the nucleic acid (2)-stabilized nanocluster, such as C-dot, and with a quencher functionalized nucleic acid, (3), to yield the sensing platform. In the presence of VEGF, the aptamer is uncaged, through the formation of the aptamer-ligand complex, leading to the release of the quencher-modified nucleic acid (3) and to the triggered-on luminescence of the nanocluster. The use of carbon nanomaterials (C-dots or graphene oxide GO nanoparticles) for the development of a luminescent VEGF sensing platform is shown in FIG. 1C. The quencher-functionalized aptamer sequence (8) is adsorbed on the surface of C-dots or GO nanoparticles, leading to the quenching of the luminescence of the nanostructures. The VEGF-stimulated release of the VEGF-aptamer complexes triggers-on the luminescence of the two different carbon-based nanomaterials.

[0161] In all sensing platforms, the resulting VEGF-induced luminescence relates directly to the concentration of VEGF. Accordingly, the development of the different sensing platforms includes evaluation of the resulting luminescence intensities as a function of VEGF concentration and the extraction of the respective calibration curves and detection limits for analysis of VEGF. All of the sensing platforms are based on the uncaging of the aptamer sequence or its desorption from the vehicle via the formation of aptamer-VEGF complexes. The time-interval for uncaging the aptamer sequences and the content of the released aptamer-VEGF complex are used to control the sensitivity of the sensing platform. Accordingly, the sensitivity of the sensing platforms, such as those shown in FIG. 1, may be optimized by structural alteration of the nucleic acids involved with the sensing systems.

EXAMPLE 1

In Vivo Model for C-Dot Penetration to the Eye

[0162] Methods: C-dots NPs were applied on rat eyes for 1 hour at different concentrations. The aqueous humour (AH), vitreous humour (VH), lens and cornea were collected from each eye and. Fluorescence intensity in the samples was measured by a plate reader and the concentration (mg/ml) of the NP was calculated according to calibration curves tested previously. In another set of experiments the corneal epithelium of the eye was scraped before the NP were instilled. In addition, histological sections were prepared from treated and untreated eyes the florescence of the section was photographed using a florescence microscope.

[0163] Results: In the C-dot treated eye, the mean (SD) C-dot concentrations in the AH, VH, cornea and lens were 8.9 (5.7), 8.9 (5.7), 6.2 (3.6) and 18.2 (8.0) ng/ml, respectively (FIG. 2A). Mechanical removal of this layer before instilling the NP, resulted in almost a tenfold increase in C-dot ocular concentration, compared to eyes with intact epithelium (FIG. 2, C-dot WO epithelium), indicating the great impact of the corneal epithelium on the NP penetration. Histological sections showed the distribution of the C-dot fluorescence in the eye structures including the retina (FIG. 2B).

EXAMPLE 2

In Vivo Monitoring of C-Dot Florescence in Rat Eyes

[0164] Methods: C-dot NPs were instilled on rat's eyes for 1 hour. Using live imaging microscope (micron IV, Phoenix Research Laboratories) we measured the fluorescence of the C-dot in the retina and the VH of live animals up to 24 hours following treatment.

[0165] Results: The florescence of the C-dot is detectable in the live animal (images in FIG. 3A). The results show a characteristic clearance during the first three hours after treatment (FIG. 3B).

EXAMPLE 3

In Vitro Toxicity of C-Dot Solution

[0166] C-dots are not showing any signs for toxicity to cells in culture.

[0167] Methods: In order to evaluate the C-dot solution toxicity to the eye, we used MTT assay on several cell types (fibroblasts, ARPE-19 and MeWo cells). Cells were cultured in the presence of C-dot solution in various concentrations and the viability of the cells was evaluated by measuring the absorbance at =570nm.

[0168] Results: Absorbance for the three cell types for different concentrations is presented in FIG. 4. Cells viability was not affected even at the highest C-dot concentration.

EXAMPLE 4

In Vivo Toxicity

[0169] Methods: 5 Wistar male rats were taken for this experiment. Treatment for treated eyes (n=5) was done by topical administration of c-dot solution (3mg/ml) for 15 minutes a day for 5 days. Eyes in the control group (n=5) same as treated eyes but with saline instead of c-dot solution. After treatment session, all eyes went through a test for SPKs (Superficial Punctate Keratitis) and Electroretinography measurements. Electroretinography measurements were done for each eye in two intensities (12.5 cd.Math.s/m.sup.2 and 50 cd.Math.s/m.sup.2)

[0170] Results: No difference is SPKs was observed between treated and control groups, suggesting no corneal toxicity of the aptamer. Results of the ERG test were presented in FIG. 6 suggesting no retinal toxicity of the compound.

EXAMPLE 6

Ex Vivo Penetrability

[0171] Methods: Porcine eyes were taken for one hour of topical administration of c-dot solution (3mg/ml). Aqueous humor, vitreous humor, lens and cornea were extracted from each eye. Extraction was done in several period of the after topical administration: 0 hrs (n=4), 1 hrs (n=4) and 4 hrs (n=5). Eyes in the control group were topically administrated with saline instead c-dot solution. Fluorescence readout of the extraction was done by a plate reader (excitation at A=420nm, measurement at A=520nm).

[0172] Results: Plate reader readout of the extraction is shown in FIG. 7. C-dots shows good penetrability to the aqueous humor.

EXAMPLE 7

In Vitro No Aptamer Complementary C-Dot Testing

[0173] Methods: C-dot with quencher were synthesized in a new method without the use of aptamer complementary segment. To types were preparedATP sensitive C-dots and Cocaine sensitive C-dots. ATP/Cocaine solution at different concentrations was added. Fluorescence spectra (excitation at 470 nm) was measured two hours after ATP/Cocaine was added.

[0174] Results: Fluorescence spectra for raw, quenched and post ATP/Cocaine is shown in FIG. 8. Fluorescence change versus ATP/Cocaine is also shown.

EXAMPLE 8

Establishment of C-Dot Synthesis Procedure

[0175] Synthesis route towards water-soluble luminescent C-dots: First, citric acid (3 g) and urea (3 g) were added to distilled water (10 mL) to form a transparent solution. The solution was then heated in a domestic 750 W microwave oven for 4-5 mins, during which the solution changed from being a colorless liquid to a brown and finally dark-brown clustered solid, indicating the formation of C-dots. This solid was then transferred to a vacuum oven and heated at 60 C. for 1 hour to remove the residual small molecules. An aqueous solution of the C-dots was purified in a centrifuge (3000 r.Math.min.sup.1, 20 min) to remove large or agglomerated particles. The resulting colored (brown) aqueous solution remained indefinitely stable at various concentrations. The dilute aqueous solution of the C-dots exhibits excitation-wavelength-dependent photoluminescence properties with emission peaks ranging from 440 nm (blue) to 570 nm (yellow) at excitation from 340 nm to 500 nm. The strongest fluorescence emission band, located at 540 nm, is observed under 470 nm excitation.

[0176] Fluorescence Quenching and Detection of ATP: Adsorption of a quencher labeled aptamer against ATP (SEQ ID NO: 3) onto the C-dots, leading to the fluorescence quenching of the C-dots. For the detection of ATP, 10 L of SEQ. ID. 1, 100 M, was first added to 100 L HEPES buffer solution, 20 mM, pH=7.4, 50 mM NaCl, that included 0.03 mg C-dots. Then, the C-dots solution with the aptamer was cleaned by sucrose solution, 3M, to remove access of aptamer. Then, different concentrations of ATP were added to the C-dots solution and left for different time-intervals at room temperature, after which, fluorescence of the mixture was measured.

TABLE-US-00003 (SEQIDNO:3) 5-ACCTGGGGGAGTATTGCGGAGGAAGGTT/FQ/-3

EXAMPLE 9

Preparation of C-Dot=V7t1 Complex

[0177] All nucleic acid strands were provided by Integrated DNA Technologies Inc. (Coralville, Iowa). The detailed DNA sequences used in the present study are:

TABLE-US-00004 Cap-VEGF (SEQIDNO:2) 5-NH.sub.2-(CH.sub.2).sub.6-TCTACCCGGCCC-3 Anti-VEGFaptamer (SEQIDNO:4) 5-TGTGGGGGTGGACGGGCCGGGTAGA-3.

[0178] Synthesis of C-dots. Citric acid and urea were mixed in water and heated for 4-5 min in a domestic 750W microwave. An aqueous solution of the C-dots was purified in a centrifuge (14000 rpm for 20 min) to remove large agglomerated particles. The resulting solution was further purified from large particles using a microcon (Millipore) spin filter unit (MWCO 10 kDa).

[0179] Synthesis of succinic anhydride-functionalized carbon dots (SA-C-dots): First, 25 mg of C-dots were dispersed in 5 mL of water and the pH was adjusted to 7.0 with NaOH. Subsequently, 500 mg of succinic anhydride was added to the C-dots solution, followed by stirring overnight at 60 C. and then, 1254, of 2 Molar NaCl solution and 45mL of ethanol were added to the reaction solution. The resulting SA-C-dots were collected by centrifugation (14000 rmp for 5 min) and washed using 50 mM NaCl solution and 90% ethanol for two times. The product was dried under vacuum at room temperature.

[0180] Preparation of cap-VEGF(1)/anti-VEGF aptamer (SEQ ID NO 4)-functionalized C-dots. Firstly, 0.5 mg of SA-C-dots were dispersed in 200 L of MES buffer (2.5 mM, pH=5.5) and bubbled with argon for 15 min 13.37 mg of N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and 8.05 mg of N-Hydroxysulfosuccinimide sodium salt (NHS) were added to the reaction solution, and mixed for 15 min. And then, 200 L of 1 mM cap-VEGF (SEQ ID NO 2) in PB buffer (0.2 M, pH=7.4) was added to the reaction solution. After an overnight incubation under room temperature, 3.6 mL of ethanol was added, and the mixture was set for a few minutes and then centrifuged at 14 000 rpm for 5 min. The resulting functionalized C-dots (functionalized with SEQ ID NO 2 were subjected to two rinsing steps: (1) 0.4 mL of 50 mM NaCl solution and 3.6 mL of ethanol and (2) 0.4 mL of HEPES buffer (10 mM, 150 mM NaCl, pH=7.2) and 3.6 mL of ethanol. The product was collected by centrifugation (14000 rmp for 5 min) and dried under vacuum at room temperature for 2 h. The loading of functionalized C-dots was determined by UV-Vis spectrophotometry.

[0181] To prepare aptamer associated product with the functionalized C-dots, 0.07 mol anti-VEGF aptamer (SEQ ID NO 4) was added to 200 L of 0.35 mg/mL functionalized C-dots in HEPES buffer (10 mM, 150 mM NaCl, pH=7.2), heated at 95 C. for 5 min and incubated overnight under 37 C. Then, 200 L of the reaction solution was mixed with 1.8 mL of ethanol under 4 C. and centrifuged (14000 rpm, 5 min). The product was dried under vacuum at room temperature. The loading of C-dots with the aptamer sequence was determined by UV-Vis spectrophotometry.

EXAMPLE 10

Preparation of VEGF Sensitive MOFs

[0182] The detailed DNA sequences used in the present study are:

TABLE-US-00005 Bindingaptamer (SEQIDNO2) 5-NH.sub.2-(CH.sub.2).sub.6-TCTACCCGGCCC-3 VEGFaptamer(2) (SEQIDNO4) 5-TGTGGGGGTGGACGGGCCGGGTAGA-3

[0183] Synthesis of azide-functionalized NMOFs (NMOF-N3): First, the organic ligand, amino-triphenyl dicarboxylic acid (amino-TPDC), was synthesized according to He et al. 2014. Afterwards, NMOFs were prepared by heating ZrCl.sub.4 with amino-TPDC at 80 C. for 5 days. The resulting NMOFs were collected by centrifugation and washed with DMF, triethylamine/ethanol (1:20, V/V), and ethanol gradually. For the preparation of azide modified NMOFs (NMOF-N.sub.3), 10 mg of the dried NMOFs were dispersed in 3 mL of THF, followed by adding 1.0 mL of the tert-butyl nitrite (tBuONO) and 0.9 mL of the azidotrimethylsilane (TMSN3). And then, the reaction mixture was stirred at the room temperature overnight to obtain NMOF-N.sub.3.

[0184] Synthesis of nucleic acid functionalized NMOFs: NMOF-N3 (10 mg, 2 mL) were added to an aqueous solution of DBCO-modified nucleic acid (1) (200 nmol, 1 mL). The mixture was incubated at 40 C. for 72 h, and three portions of a NaCl solution were added to the reaction mixture every two hours within the first 6 hours to reach a final concentration of 0.5 M. Thereafter, the obtained nucleic acid (1)-functionalized NMOFs were washed three times with HEPES buffer (10 mM, pH=7.4) to remove unbound DNA. The UV absorbance of the wash was measured at 260 nm to evaluate the amount of DNA loading on the NMOFs.

[0185] Loading of nucleic acid functionalized NMOFs: The nucleic acid functionalized NMOFs, 5 mg, were incubated with Rhodamine 6G (0.5 mg/mL) overnight in 2 mL of HEPES buffer solution (10 mM, pH=7.4). Subsequently, the NMOFs were separated and transferred to a HEPES buffer solution (10 mM, pH=7.4) that contained NaCl, 20 mM, and NMOFs were hybridized with the nucleic acid (2), leading to the locked state of the duplex DNA-functionalized NMOFs loaded with Rhodamine 6G. After 12 h, the resultant NMOFs were washed several times to remove the excess and nonspecifically bound Rhodamine 6G.

[0186] VEGF-induced unlocking of the NMOFs and the release of the encapsulated loads: Experiments were performed using solutions of the respective Rhodamine 6G-loaded aptamer-functionalized -locked NMOFs at a concentration corresponding to 1 mg/mL. Then, the NMOFs solutions, 30 L, were treated with 10 L of variable concentrations of VEGF for a fixed time-interval of 30 minutes. Other proteins, e.g. thrombin, hemoglobin, BSA, were used as controls to demonstrate the selective uncapping of the NMOFs by VEGF. After incubation, the respective samples were centrifuged at 10000 rpm for 10 min to precipitate the NMOFs, and the fluorescence of the released loads in the supernatant solution was measured using a Cary Eclipse Fluorescence Spectrophotometer.

[0187] Synthesis of Carbon dots (C-dots). The C-dots were synthesized according to Wang et Al. (ref1) Citric acid and urea were mixed in water and heated at 750 W for 4-5 min in a domestic microwave. An aqueous solution of the C-dots was purified in a centrifuge (14000 rpm, 20 min) to remove large agglomerated particles. The resulting solution was further purified from larger particles using microtron (Millipore) spin filter unit (MWCO 10 kDa).

[0188] Synthesis of succinic anhydride-functionalized Carbon dots (SA-C-dots): 25 mg of C-dots (1) were dispersed in 5 mL of water and the pH was adjusted to 7.0 with concentrated NaOH. Subsequently, 0.5 g of succinic anhydride was added to the C-dots solution, followed by stirring overnight at 60 C. The C-dots were washed 3 times from unreacted succinic anhydride by centrifugation in 90% v/v Ethanol and 10% v/v aqueous 50 mM NaCl (14000 rpm, 5min) and dried under vacuum at room temperature.

[0189] Preparation of cap-VEGF/anti-VEGF aptamer-functionalized C-dots: Firstly, 0.5 mg of SA-C-dots were dispersed in 200 uL of MES buffer (2.5 mM, pH 5.5) and bubbled with argon for 15 min 13.37 mg of N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and 8.05 mg of N-Hydroxysulfosuccinimide sodium salt (NHS) were added to the reaction solution, and mixed for 15 min. 200 uL of 1 mM cap-VEGF in PB buffer (0.2 M, pH 7.4) was added to the reaction solution. After overnight incubation at room temperature, 3.6 mL of ethanol was added, the mixture was set for a few minutes and centrifuged at 14000 rpm for 5 min. The resulting (1)-functionalized C-dots were subjected to two rinsing steps: (1) 0.4 mL of 50 mM NaCl solution and 3.6 mL of ethanol and (2) 0.4 mL of HEPES buffer (10 mM, 150 mM NaCl, pH 7.2) an 3.6 mL of ethanol. The product was collected by centrifugation (14000 rpm for 5 min) and dried under vacuum at room temperature for 2 hours. The loading of C-dots with (2) was determined by UV-Vis spectrophotometry.

EXAMPLE 11

In Vivo Dynamics

[0190] Methods: Fluorescence was measured for 120 minutes after c-dot administration was completed. Concentration was calculated and normalized to t=0min concentration.

[0191] Results: C-dot concentration half time was about 20 minutes.

EXAMPLE 12

In Vivo Penetration

[0192] Methods: C-dot solution (3 mg/ml) was topically administrated for 14 eyes. Seven eyes were administrated for 60 minutes while seven other eyes were administrated for five minutes. Saline solution was administrated for 60 minutes for six eyes as control. Fluorescence was measured right after administration and c-dot concentration was calculated based on our calibration experiments.

[0193] Results: Mean c-dot concentration was 15 g/ml for five minutes administration and 60 g/ml for 60 minutes administration.

EXAMPLE 13

TreatmentIn Vitro Model (1)

[0194] Methods: Rat's eyes were enucleated; the choroid layer was separated from the retina, cut into approximately 2 mm pieces and seeded in matrigel, with medium and different concentrations of VEGF. The cultures were monitored for blood vessel formation for 8 days.

[0195] Results: Sprouting growth tends to increase as VEGF concentration increases. This is validating our model for the following VEGF inhibition experiments.

EXAMPLE 14

TreatmentIn Vitro Model (2)

[0196] Methods: Rat's eyes were enucleated; the choroid layer was separated from the retina, cut into approximately 2 mm pieces and seeded in matrigel, with medium containing either common anti-VEGF agents, c-dot=aptamer complex, aptamer only and c-dot only. 2.6 nM VEGF were added to all samples. The cultures were monitored for blood vessel formation for 8 days.

[0197] Results: A similar effect was observed between common anti-VEGF agents and V7t1 aptamer with c-dot aptamer complex showing only slightly lesser effect.

EXAMPLE 15

TreatmentIn Vitro Model (3)

[0198] Methods: Rat's eyes were enucleated; the choroid layer was separated from the retina, cut into approximately 2 mm pieces and seeded in matrigel, with medium containing c-dot=aptamer complex in different concentrations. 2.6 nM VEGF were added to all samples. The cultures were monitored for blood vessel formation for 8 days.

[0199] Results: The results support the trend for which sprouting growth decreases as c-dot=aptamer complex concentration increases.

EXAMPLE 16

MOFs In Vivo Sensing

[0200] Methods: 5 L sensitive VEGF MOFs (1 mg/ml), were injected to four eyes with Rhodamine 6G fluorescence measured for 30 minutes after injection. Then, 5 L (10 M) of VEGF were injected to three of those eyes and no injection of one eye. Rhodamine 6G was measured for 60 minutes.

[0201] Results: For the three VEGF injected eyes, elevation of the fluorescence signal in observed for the first 15 minutes. The fluorescence signal seems to decay through 60 minutes after VEGF injection. For the no injection eye, the fluorescence signal is stable for the entire measurement session.