DELIVERY SYSTEM IN MICELLAR FORM HAVING MODULAR SPECTRAL RESPONSE BASED ON ENZYME-RESPONSIVE AMPHIPHILIC PEG-DENDRON HYBRID POLYMERS

Abstract

The present invention relates to new molecular design that allows micelles to report their activation and disassembly by an enzymatic trigger. The molecular design is based on introduction of a labeling moiety selected from a fluorescent dye, a dark quencher, combinations of dyes or dyes/quenchers, and a fluorinated moiety (a .sup.19F-magenetic resonance (MR) probe for turn ON/OFF of a .sup.19F-MR signal) through covalent binding to the focal point of amphiphilic polymer-dendron hybrids with the labeling moiety. At the assembled micellar state, the dyes are closely packed and hence the probability for intermolecular interactions increases significantly, leading to alteration of the fluorescent properties (signal quench or shift) or the .sup.19F-MR signal (OFF state) of the micelles. Upon enzymatic cleavage of the hydrophobic end-groups from enzyme-responsive dendron, the polymers become hydrophilic and disassemble. This structural change is then translated into a spectral change as dye-dye interactions are halted and the dyes regain their intrinsic fluorescent properties, or alternatively by turn ON the .sup.19F-MR signal. The high modularity of the design allows the introduction of various types of dyes and thus enables rational adjustment of the spectral response. Two major types of responses are described: Turn-On/Off and spectral shift, depending on the type of labeling dye. The present invention further provides methods of use of the hybrid delivery system and to a kit comprising the same.

Claims

1. A hybrid polymer comprising: (i) a hydrophilic polyethylene glycol (PEG) polymer; (ii) a hydrophobic dendron, the dendron comprising at least one enzymatically cleavable hydrophobic end group that is covalently attached to the dendron; and (iii) at least one labeling moiety selected from a fluorescent dye, a dark quencher, and a fluorinated labeling moiety; wherein the PEG polymer, hydrophobic dendron and labeling moiety are covalently attached, directly or through a multi-functional moiety.

2. The hybrid polymer according to claim 1, wherein the labeling moiety is a fluorescent dye or a dark quencher.

3. The hybrid polymer according to claim 2, wherein the fluorescent dye or dark quencher is selected from the group consisting of a coumarin, a cyanine dye, an azo dye, an acridine, a fluorone, an oxazine, a phenanthridine, a naphthalimide, a rhodamine, a benzopyrone, a perylene, a benzanthrone, and a benzoxanthrone.

4. The hybrid polymer according to claim 3, wherein the fluorescent dye or dark quencher is or is the residue of a compound selected from the group consisting of Coumarin, Fluorescein, Cyanine 3 (Cy3), Cyanine 5 (Cy5), Cyanine 7 (Cy7), Alexa dyes, bodipy derivatives, (E)-2-(4-(phenyldiazenyl)phenoxy)acetic acid, 3-(3,3-dimethyl-6-nitrospiro[chromene-2,2-indolin]-1-yl)propanoate (Spiropyran), 3,5-dihydroxybenzoate, (E)-2-(4-(phenyldiazenyl)phenoxy)acetic acid, and combinations thereof.

5. The hybrid polymer according to claim 1, wherein the labeling moiety is a fluorinated labeling moiety, the fluorinated labeling moiety being a magnetic resonance (MR) probe capable of turning on a .sup.19F-MR signal.

6. The hybrid polymer according to claim 1, wherein the multi-functional moiety is a trifunctional moiety that is capable of attaching to the hydrophobic dendron, the PEG polymer, and the labeling moiety, preferably, wherein the trifunctional group is covalently attached to the labeling moiety and is present at a focal point between the PEG polymer and the dendron.

7. The hybrid polymer according to claim 6, wherein the trifunctional moiety is selected from the group consisting of an amino acid, a C1-C20 alkylene, a C2-C20 alkenylene, a C2-C20 alkynylene and an arylene, each comprising at least three functional groups selected from the group consisting of C(O)O, NH, O, S, C(O), OC(O)O, C(O)NH, NHC(O)NH, NHC(O)O, S(O), S(O)O, PO(O)O, and any combination thereof.

8. The hybrid polymer according to claim 7, wherein the trifunctional moiety is an amino acid capable of attaching to the hydrophobic dendron, the PEG polymer, and the fluorescent dye through its carboxyl group, amino group, and side chain.

9. The hybrid polymer according to claim 8, wherein the trifunctional moiety is an alpha-amino acid selected from the group consisting of lysine, aspartic acid, glutamic acid, tyrosine, asparagine, serine, homoserine, cysteine, homocysteine, glutamine, threonine, ornithine, citrulline and arginine, preferably wherein the alpha-amino acid is lysine.

10. The hybrid polymer according to claim 1, wherein the enzymatically cleavable hydrophobic end group is conjugated to the dendron through an enzymatically cleavable functional group selected from the group consisting of an ester, an amide, a carbamate, a carbonate, a urea, a sulfate, an amidine, an ether, a phosphate, a phosphoamide, sulfamates, and a trithionate.

11. The hybrid polymer according to claim 10, wherein the enzymatically cleavable hydrophobic end group is conjugated to the dendron through an ester which is cleavable by an esterase.

12. The hybrid polymer according to claim 11, wherein the esterase is selected from the group consisting of carboxylesterase, arylesterase, and acetylesterase.

13. The hybrid polymer according to claim 10, wherein the enzymatically cleavable hydrophobic end group is conjugated to the dendron through an amide which is cleavable by an amidase.

14. The hybrid polymer according to claim 13, wherein the amidase is selected from the group consisting of aryl-acylamidase, aminoacylase, alkylamidase, and phthalyl amidase.

15. The hybrid polymer according to claim 1, wherein the enzymatically cleavable hydrophobic end group is represented by the structure OC(O)R, C(O)ORNHC(O)R or C(O)NHR wherein R is C 1-C12 alkyl or an aryl.

16. The hybrid polymer according to claim 1, wherein the dendron comprises a plurality of enzymatically cleavable hydrophobic end groups, and wherein the enzymatically cleavable hydrophobic end groups are present at one or more terminal repeating units (terminal generation) of the hydrophobic dendron.

17. The hybrid polymer system according to claim 1, wherein the PEG has an average molecular weight between 0.5 and 70 kDa; or wherein the PEG has at least 10 repeating units of ethylene glycol monomers.

18. The hybrid polymer according to claim 1, wherein the PEG is linked to the dendron or the multi-functional moiety through a PEG terminal group selected from the group consisting of (CH.sub.2).sub.tX(CH.sub.2).sub.tX, X(CH.sub.2).sub.tX, (CH.sub.2).sub.t wherein X is independently at each occurrence selected from O, S and NH, and t is independently at each occurrence 1-10; O, S, NH, C(O), C(O)O, OC(O)O, C(O)NH, NHC(O)NH, NHC(O)O, S(O), S(O)O, PO(O)O, CC, CC, triazolyl, and any combination thereof, preferably, wherein the PEG terminal functional group is (CH.sub.2).sub.3S(CH.sub.2).sub.2NH.

19. The hybrid polymer according to claim 1, comprising: a trifunctional moiety having a first bond to a PEG polymer, a second bond to a labeling moiety selected from a fluorescent dye, a dark quencher and a fluorinated labeling moiety, and a third bond, directly or through a linker or branching unit, to a first generation dendron which comprises at least one functional group capable of binding to a further generation or to said enzymatically cleavable hydrophobic end group; and optionally, at least one additional generation which is covalently bound to said first generation or preceding generation, and optionally to a further generation, wherein each of said optional generations comprises at least one functional group capable of binding to said first generation, to a preceding generation, to a further generation, and/or to said enzymatically cleavable hydrophobic end group, each of said bonds being formed directly or through a linker or branching unit.

20. The hybrid polymer according to claim 1, wherein each generation of the hydrophobic dendron comprises a linear or branched C1-C20 alkylene, C2-C20 alkenylene, C2-C20 alkynylene or arylene moiety which is substituted at each end with a group selected from the group consisting of O, S, NH, C(O), C(O)O, OC(O)O, C(O)NH, NHC(O)NH, NHC(O)O, S(O), S(O)O, PO(O)O, and any combination thereof.

21. The hybrid polymer according to claim 20, wherein each generation of the dendron is derived from a compound selected from the group consisting of HXCH.sub.2CH.sub.2XH, HX(CH.sub.2).sub.1-3CO.sub.2 H and HXCH.sub.2CH(XH)CH.sub.2XH wherein X is independently at each occurrence NH, S or O.

22. The hybrid polymer according to claim 21, wherein each generation of the dendron is derived from a compound selected from the group consisting of HSCH.sub.2CH.sub.2OH, HS(CH.sub.2).sub.1-3CO.sub.2 H and HSCH.sub.2CH(OH)CH.sub.2OH.

23. The hybrid polymer according to claim 1, further comprising a linker or branching unit which connects the trifunctional moiety to the first generation dendron and/or which forms a part of the first generation dendron, and/or which connects between dendron generations.

24. The hybrid polymer according to claim 23, wherein the linker/branching unit moiety is selected from a group consisting of a substituted or unsubstituted acyclic, cyclic or aromatic hydrocarbon moiety, heterocyclic moiety, a heteroaromatic moiety or any combination thereof, preferably wherein the linker moiety/branching unit is a substituted arylene, a C 1-C20 alkylene, a C2-C20 alkenylene, or a C2-C20 alkynylene each comprising at least one functional group selected from the group consisting of O, S, NH, C(O), OC(O)O, C(O)O, C(O)NH, NHC(O)NH, NHC(O)O, S(O), S(O)O, PO(O)O, CC, CC, (CH.sub.2).sub.t wherein t is an integer of 1-10, and any combination thereof.

25. The hybrid polymer according to claim 1, which is represented by the structure of formula (I): ##STR00037## wherein Q is selected from the group consisting of OR wherein is H or a C1-C4 alkyl; NH.sub.2, SH and COOH; T is absent or is a functional group selected from the group consisting of (CH.sub.2).sub.tX(CH.sub.2).sub.tX, X(CH.sub.2).sub.tX, (CH.sub.2).sub.t wherein X is independently at each occurrence selected from O, S and NH, and t is independently at each occurrence 1-10; O, S, NH, C(O), C(O)O, OC(O)O, C(O)NH, NHC(O)NH, NHC(O)O, S(O), S(O)O, PO(O)O, CC, CC, any combination thereof. B is a trifunctional moiety comprising a labeling moiety covalently attached thereto; Y is independently at each occurrence absent or is a linker moiety/branching unit; Z is independently at each occurrence a dendron repeating unit selected from the group consisting of: ##STR00038## and any combination of the foregoing; wherein X.sup.1 is independently, at each occurrence, selected from the group consisting of O, S and NH; A represents a multiplicity of hydrophobic end groups conjugated to terminal units of the dendron through at least one enzymatically cleavable functional group selected from the group consisting of an ester, an amide, a carbamate, a carbonate, a urea, a sulfate, an amidine, an ether, a phosphate, a phosphoamide, sulfamates, and a trithionate; n is an integer in the range of 1 to 1,500; and m and z are each an integer of 1 to 15.

26. The hybrid polymer according to claim 25, wherein the trifunctional moiety B is lysine, which is linked to a labeling moiety through its side chain amino group: ##STR00039##

27. The hybrid polymer according to claim 25, wherein the terminal unit of said dendron is represented by any of the following structures: ##STR00040## wherein X.sup.2 has the same meaning as X.sup.1.

28. The hybrid polymer according to claim 25, wherein each hydrophobic end group A is conjugated to the dendron through an enzymatically cleavable functional group represented by the structure: ##STR00041## wherein X.sup.2 is a part of the terminal unit of said dendron and C(O) is part of hydrophobic end group; or wherein X.sup.2 is part of the hydrophobic end group and C(O) is a part of the terminal repeating unit of said dendron, or wherein X.sup.2C(O) are part of the hydrophobic end group, or wherein X.sup.2C(O) is part of the terminal unit of said dendron; and wherein X.sup.2 has the same meaning as X.sup.1.

29. The hybrid polymer according to claim 25, which is represented by the structure of formula (II): ##STR00042##

30. The hybrid polymer according to claim 29, which is represented by the structure of formula (IIa): ##STR00043##

31. The hybrid polymer according to claim 1, which is represented by the structure of any of formulae 1C, 1F, 8C, 8F, 12a, 12b, 13a, 13b, 14a, 14b, 15a, 15b, 16a, 16b, 18 or 20.

32. A self-assembled amphiphilic delivery system in micellar form, comprising at least one hybrid polymer according to claim 1, wherein the micelle disassembles upon enzymatic cleavage of the hydrophobic end group; and wherein the labeling moiety provides a different signal in the assembled vs. unassembled state of the micelle.

33. The delivery system according to claim 32, wherein the labeling moiety in 15 the hybrid polymer is a fluorescent dye, wherein the fluorescent dye is a self-quenching dye or an excimer forming dye, and wherein the fluorescence of the dye, when used alone or in combination with another dye or a dark quencher, is wholly or partially quenched or is shifted upon micelle assembly, and said dye regains its intrinsic fluorescence upon micelle disassembly.

34. The delivery system according to claim 32, comprising a multiplicity of hybrid polymers containing the same labeling moiety, or mixture of hybrid polymers containing different labeling groups.

35. The delivery system according to claim 32 comprising: (a) a hybrid polymer comprising a self-quenching fluorescent dye (FRET), wherein the fluorescence of the dye is wholly or partially quenched upon micelle assembly (TURN OFF); (b) a mixture of a hybrid polymer comprising a fluorescent dye and a hybrid polymer comprising dark quencher, wherein the fluorescence of the dye is wholly or partially quenched upon micelle assembly (TURN OFF); (c) a mixture of hybrid polymers comprising two or more different fluorescent dyes, wherein the fluorescence of the dyes is shifted upon micelle assembly (FRET) and disassembly (spectral shift); or (d) a hybrid polymer comprising an excimer forming fluorescent dye, wherein the fluorescence of the dye is shifted upon micelle assembly and disassembly (spectral shift).

36. The delivery system according to claim 32, wherein the labeling moiety is a fluorinated labeling moiety, the fluorinated labeling moiety being a magnetic resonance (MR) probe capable of turning on a .sup.19F-MR signal, and wherein the .sup.19F-MR signal is turned OFF upon micelle assembly, and turned ON upon enzymatic activation and micelle disassembly.

37. The delivery system according to claim 32, wherein the micelle has an average particle size of less than about 100 nm, preferably, between about 10 nm and 50 nm, more preferably about 10 nm or 20 nm.

38. A method of delivering the delivery system according to claim 32, comprising the step of contacting the delivery system with an enzyme to induce cleavage of the enzymatically cleavable hydrophobic end group,

39. A kit for delivering the delivery system according to claim 32, comprising in one compartment the delivery system, and in a second compartment an enzyme capable of cleaving the enzymatically cleavable hydrophobic end group so as to disassemble said micelle.

40. A method of monitoring enzymatic activity in a biological system, the method comprising the step of contacting the biological system with a delivery system according to claim 1, and monitoring said enzymatic activity by fluorescence or .sup.19F-magnetic resonance (MR).

41. Use of the delivery system according to claim 1 wherein the labeling moiety is a fluorinated labeling moiety, as .sup.19F-magnetic resonance (MR) probe for .sup.19F-MR imaging

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0062] FIG. 1A: Schematic illustration of labeled enzyme-responsive hybrids and their assembly into micelles with quenched (left) or shifted (right) fluorescence emissions. Upon enzymatic activation, the polymeric hybrids become hydrophilic and the structural changes are translated into spectral responses as the labeling dyes regain their intrinsic fluorescence.

[0063] FIG. 1B: Schematic illustration of several embodiments of self-assembly leading to altered supramolecular fluorescent properties.

[0064] FIG. 2: (a) DLS data for hybrids 1F and 1C (160 M) indicate the formation of micelles with diameters of 17nm that decreased to 6 nm after enzymatic activation by PLE. TEM images of micelles of (b) hybrid 1F and (c) hybrid 1C.

[0065] FIG. 3: (a) Fluorescence spectra of 1F (Ex 470 nm). (b) Comparison of the emission intensities (Ex 470 nm, Em 525 nm) of hybrids 1F and 7F (obtained by chemical hydrolysis) as a function of concentration. Photos of solutions of hybrids (160 M) excited by a standard 365 nm UV-lamp.

[0066] FIG. 4: (a) Fluorescence spectra of 1C (Ex 420 nm) and photos of solutions of hybrids (160 M) excited by a standard 365 nm UV-lamp. (b) Fluorescence spectra of 1C (Ex 420 nm) at concentrations below its CMC value. (c) Excitation spectra of hybrid 1C recorded at .sub.em monomer=480 and .sub.em excimer=540 mm (d) Comparison of the ratio of emission intensities at 540 nm and 480 nm (Ex 420 nm) of hybrids 1C and 7C (obtained by chemical hydrolysis) as a function of their concentrations.

[0067] FIG. 5: (a) Time-dependent fluorescence spectra of hybrid 1F (160 M) after the addition of PLE (27 nM) and photos of a solution of 1F before and after addition of PLE. (b) Overlay of the increase in fluorescence and HPLC analysis of enzymatic degradation of hybrid 1F.

[0068] FIG. 6: (a) Time-dependent fluorescence spectra of hybrid 1C (160 M) after the addition of PLE (270 nM). (b) Overlays of the changes in fluorescence and HPLC analysis of the enzymatic degradation of hybrid 1C.

[0069] FIG. 7: (a) Structure of amidase-responsive hybrid 8C bearing four cleavable amide bonds and labeled with a coumarin derived dye. (b) Time-dependent fluorescence spectra of hybrid 8C (160 M) after the addition of PGA (1 M). (c) Overlays of the changes in fluorescence and HPLC analysis of the enzymatic degradation of hybrid 1C.

[0070] FIG. 8A: .sup.1H-NMR spectrum of hybrid 1F in D.sub.2O before addition of PLE.

[0071] FIG. 8B: .sup.1H-NMR spectrum of hybrid 1C in D.sub.2O before addition of PLE.

[0072] FIG. 9: Micelle degradation of hybrid 8C in presence of 1 M PGA enzyme (after 24 hours).

[0073] FIG. 10: Absorbance spectra overlay of hybrid 1F (160 M), hybrid 7F (160 M) and hybrid 1F (160 M) after addition of 270 nM PLE enzyme (after 24 hours).

[0074] FIG. 11: Absorbance spectra overlay of hybrid 1C (160 M), hybrid 7C (160 M) and hybrid 1C (160 M) after addition of 270 nM PLE enzyme (after 24 hours).

[0075] FIG. 12: Absorbance spectra overlay of hybrid 8C (160 M) and hybrid 1C (160 M) after addition of 1 M PGA enzyme (after 48 hours).

[0076] FIG. 13: Fluorescence emission intensity spectra overlay of hybrid 1F (160 M), hybrid 7F (160 M) and of hybrid 1F (160 M) after addition of 27 nM PLE enzyme (after 24 hours).

[0077] FIG. 14: Fluorescence emission intensity spectra overlay of hybrid 1C (160 M), hybrid 7C (160 M) and of hybrid 1C (160 M) after addition of 270 nM PLE enzyme (after 24 hours).

[0078] FIG. 15: Fluorescence emission intensity spectra overlay of hybrid 8C (160 M) and of hybrid 8C (160 M) after addition of 1 M PGA enzyme (after 48 hours).

[0079] FIG. 16: HPLC monitoring of micelle degradation in presence of 27 nM PLE enzyme for hybrid 1F over time.

[0080] FIG. 17: HPLC monitoring of micelle degradation in presence of 270 nM PLE enzyme for hybrid 1C over time.

[0081] FIG. 18: HPLC monitoring of micelle degradation in presence of 1 M PGA enzyme for hybrid 8C over time. The overlay shows accumulation of partially cleaved hybrids.

[0082] FIG. 19: Schematic illustration of labeled enzyme-responsive hybrids as .sup.19F magnetic probes and their assembly into micelles. The hybrids contain non-cleavable fluorine containing probes on the polymer. At the assembled state, aggregation of the hydrophobic fluorinated groups at the core of the micelles results in extremely short T.sub.2 relaxation time, leading to an OFF state. Upon enzymatic activation, the mobility of the fluorinated hydrophilic polymer increases and its magnetic resonance signal is turned ON.

[0083] FIG. 20: Overlay of (a) HPLC chromatograms, (b) .sup.19F-NMR spectra; and (c) kinetic data (HPLC, .sup.19F-NMR and fluorescence) for the enzymatic-induced activation of hybrid 20.

[0084] FIG. 21: .sup.19F-NMR spectra obtained by spin-echo sequence (9.4T, 376 MHz, TE=80 ms) showing the micellar OFF states and ON states after the disassembly of hybrid 20 (aOFF; bON).

[0085] FIG. 22: Micelle degradation of hybrid 20 (640 M) in presence of 1.1 M PLE enzyme.

[0086] FIG. 23: Fluorescence emission spectra of Nile Red (1.25 M) in the presence of hybrid 20 (640 M) as a function of time after the addition of 1.1 M PLE. A decrease in the intensity was observed as Nile Red was released into solution due to micelles degradation.

[0087] FIG. 24: Fluorescence emission spectra of Nile Red (1.25 M) in the presence of hybrid 20 (640 M) in the absence of PLE enzyme over 8 hours.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

PEG-dendron Hybrids:

[0088] The present invention provides hybrid polymers comprising: (i) a hydrophilic polyethylene glycol (PEG) polymer; (ii) a hydrophobic dendron, the dendron comprising at least one enzymatically cleavable hydrophobic end group that is covalently attached to the dendron; and (ii) at least one labeling moiety selected from a fluorescent dye (or combinations of fluorescent dyes), a dark quencher, combinations of a fluorescent dye and a dark quencher, and a fluorinated labeling moiety. The PEG polymer, hydrophobic dendron and labeling moiety are covalently attached, directly or through a multi-functional moiety.

[0089] The present invention further provides an amphiphilic delivery system in micellar form, comprising at least one hybrid polymer, as described above. According to the principles of the present invention, the micelle disassembles upon enzymatic cleavage of the hydrophobic end group to elicit a change in the signal afforded by the labeling moiety (either fluorescence or .sup.19F-magnetic resonance). Monitoring of the differentials signals in the assembled vs. unassembled state of the micelle by fluorescence imaging or magnetic resonance (MR) provides useful diagnostic information about the location of the hybrid delivery system (e.g., in a biological sample such as a human subject), as well as about the enzymatic activity that is responsible for the micellar disassembly. The hybrids of the present invention thus provide powerful tools, especially in the medical field as diagnostic/prognostic tools as well as in imaging technology. Furthermore, these hybrids can be self-assembled into therapeutic drug delivery platforms that can self-report their location and degree of activation.

[0090] In one aspect of the present invention, the modular design of the conjugates of the present invention is based on labeling enzyme responsive PEG-dendron hybrids with a labeling moiety which can either be a fluorescent labeling moiety or a fluorinated labeling moiety. In one embodiment, the labeling moiety is a fluorescent labeling moiety based on a single fluorescent dye (either self-quenching or excimer-forming), a combination of fluorescent dyes (FRET pairs), or a combination of a fluorescent dye and a dark quencher. In one embodiment, the labeling moiety is present at the focal point between the PEG and the dendron. It was hypothesized that in the micellar form the dyes will be closely packed at the interface between the PEG shell and the hydrophobic core. This spatial proximity should lead to supramolecular dye-dye interactions and hence the micelles are expected to have different fluorescence spectrum compared with the intrinsic properties of the dyes. Upon enzymatic cleavage of the hydrophobic end-groups, the micelles disassemble into hydrophilic hybrids that diffuse away from each other. As the distances between the labeled hybrids increase, the non-assembled dyes are expected to regain their intrinsic fluorescence, thus generating a spectral response.

[0091] Any fluorescent dyes/dark quenchers and combinations thereof may be used in the context of the present invention. For example, the fluorescent dye and/or dark quencher may be selected from the group consisting of a coumarin, a cyanine dye, an azo dye, an acridine, a fluorone, an oxazine, a phenanthridine, a naphthalimide, a rhodamine, a benzopyrone, a perylene, a benzanthrone, and a benzoxanthrone. Each possibility represents a separate embodiment of the present invention. In some representative embodiments, the fluorescent dye is or is the residue of a compound selected from the group consisting of Coumarin, Fluorescein, Cyanine 3 (Cy3), Cyanine 5 (Cy5), Cyanine 7 (Cy7), Alexa dyes, bodipy derivatives, (E)-2-(4-(phenyldiazenyl)phenoxy)acetic acid, 3-(3,3-dimethyl-6-nitrospiro [chromene-2,2-indolin]-1-yl)propanoate (Spiropyran), 3,5-dihydroxybenzoate and (E)-2-(4-(phenyldiazenyl)phenoxy)acetic acid.

[0092] A dark quencher is a substance that absorbs excitation energy from a fluorophore and dissipates the energy as heat. Dark quenchers are used in molecular biology in conjunction with fluorophores. When the two are close together, such as in an assembled micelle as described herein, the fluorophore's emission is suppressed.

[0093] Specific hybrids comprising fluorescent dyes or dark quenchers are represented by the structure of any of compounds 1C, 1F, 8C, 8F, 12a, 12b, 13a, 13b, 14a, 14b, 15a, 15b, 16a, 16b and 18, the structures of which are depicted in the Experimental Section hereinbelow. In another aspect, the labeling moiety is a fluorinated labeling moiety, which functions as a magnetic resonance (MR) probe capable of turning on a .sup.19F-MR signal. According to the principles of the present invention, the hybrids are designed such that the .sup.19F-MR signal is turned ON upon enzymatic activation and micelle disassembly, as the fluorinated derivatives become more hydrophilic and their mobility and T.sub.2 relaxation increase. Conversely, the hybrids are expected to be turned OFF or quenched upon micelle assembly. A specific hybrid comprising a .sup.19F-MR probe is a compound of formula 20, the structure of which is depicted in the Experimental Section hereinbelow.

[0094] A dendron is a hyper-branched monodisperse organic molecule defined by a tree-like or generational structure. In general, dendrons possess three distinguishing architectural features: a linker moiety; an interior area containing generations with radial connectivity to the linker moiety; and a surface region (peripheral region) of terminal moieties. According to certain embodiments, each generation of the hydrophobic dendron comprises a linear or branched C1-C20 alkylene, C2-C20 alkenylene, C2-C20 alkynylene or arylene moiety which is substituted at each end with a group selected from the group consisting of O, S, NH, C(O), C(O)O, OC(O)O, C(O)NH, NHC(O)NH, NHC(O)O, S(O), S(O)O, PO(O)O, and any combination thereof. Each possibility represents as separate embodiment of the present invention.

[0095] According to other embodiments, each generation of the dendron is derived from a compound selected from the group consisting of HXCH.sub.2CH.sub.2XH, HX(CH.sub.2).sub.1-3CO.sub.2H, and HXCH.sub.2CH(XH)CH.sub.2XH wherein X is independently at each occurrence NH, S or O. In one currently preferred embodiment, the dendron is derived from a compound selected from the group consisting of HSCH.sub.2CH.sub.2OH, HS(CH.sub.2).sub.1-3CO.sub.2H and HSCH.sub.2CH(OH)CH.sub.2OH. Each possibility represents as separate embodiment of the present invention.

[0096] The hydrophobic dendron of the present invention comprises a preferred number of generations in the range of 0 to 5, more preferably 0 to 3. In one embodiment, the hydrophobic dendron is a generation 0 (G0) dendron. In another embodiment, the hydrophobic dendron is a generation 1 (G1) dendron. In another embodiment, the hydrophobic dendron is a generation 2 (G2) dendron. In yet another embodiment, the hydrophobic dendron is a generation 3 (G3) dendron.

[0097] According to some embodiments, the hybrid polymers further comprises a linker moiety and/or a branching unit which connects the PEG polymer/labeling moiety to the first generation dendron, and/or forms a part of the first generation, and/or connects between dendron generations. In one embodiment, the linker moiety and/or the branching unit is selected from a group consisting of a substituted or unsubstituted acyclic, cyclic or aromatic hydrocarbon moiety, heterocyclic moiety, a heteroaromatic moiety or any combination thereof. Each possibility represents as separate embodiment of the present invention. In one currently preferred embodiment, the linker moiety/branching unit is a substituted arylene which may be positioned between the PEG and the first generation or may form a part of the first generation, or alternatively may be positioned at one or more intermediary generations of the dendron, or alternatively may be positioned between the moiety comprising the fluorescent dye and the first generation dendron. The branching unit may in some cases impart functionality (e.g., UV absorbance or other desired properties). Each possibility represents a separate embodiment of the present invention.

[0098] According to various embodiments, each of the linker moiety/branching unit may be connected through a functional group selected from the group consisting of O, S, NH, C(O), C(O)O, OC(O)O, C(O)NH, NHC(O)NH, NHC(O)O, S(O), S(O)O, PO(O)O, CC, CC, (CH.sub.2).sub.t wherein t is an integer of 1-10, and any combination thereof. One representative example of a functional group linking the PEG to the dendron is S(CH.sub.2).sub.tNH. Each possibility represents as separate embodiment of the present invention.

[0099] The modular design of the hybrid delivery systems of the present invention provides control over the disassembly of the micelle and release rate of the hydrophobic end groups and/or encapsulated cargo. This can be achieved by adjusting the structural features of the nanocarriers (such as length of PEG polymer, dendron generation, number of enzymatically cleavable moieties, linkage chemistry and polymer/dendron weight ratio) as well as enzymatic-tuning parameters (e.g., enzyme specificity, amount of enzyme and incubation time.

[0100] The architecture of the hybrids of the present invention requires three orthogonal functional groups to allow conjugation of the PEG, dendron, and labeling moiety. Thus, in some embodiments, the multi-functional moiety is a trifunctional moiety that is capable of attaching to the hydrophobic dendron, the PEG polymer, and the labeling moiety. According to some embodiments of the present invention, the trifunctional group comprises one functional group capable of bonding to the PEG moiety (directly or through a linker), a second functional moiety capable of bonding to the dendron (directly or through a linker), and a third functional group capable of bonding to the labeling moiety. In a currently preferred embodiment, the trifunctional group is advantageously situated at a focal group, between the PEG moiety and the dendron. As such, the hybrid of the invention, may generally be represented by the structure:

##STR00001##

[0101] wherein

##STR00002##

represents the core of the trifunctional moiety, and each independently represents a functional group on the trifunctional moiety linking the PEG, dendron and labeling moiety, each directly or through functional groups/linkers as described herein. The dendron comprises a multiplicity of enzymatically cleavable groups at its terminus, as described herein.

[0102] The hybrid polymer of the present invention may generally be represented by the structure of formula (I):

##STR00003##

[0103] wherein

[0104] Q is selected from the group consisting of OR wherein is H or a C1-C4 alkyl (e.g., methyl); NH.sub.2, SH and COOH;

[0105] T is absent or is a functional group selected from the group consisting of (CH.sub.2).sub.tX(CH.sub.2).sub.tX, X(CH.sub.2).sub.tX, (CH.sub.2).sub.t wherein X is independently at each occurrence selected from O, S and NH, and t is independently at each occurrence 1-10; O, S, NH, C(O), C(O)O, OC(O)O, C(O)NH, NHC(O)NH, NHC(O)O, S(O), S(O)O, PO(O)O, CC, CC, any combination thereof.

[0106] B is a trifunctional moiety comprising a labeling moiety covalently attached thereto;

[0107] Y is independently at each occurrence absent or is a linker moiety/branching unit;

[0108] Z is independently at each occurrence a dendron repeating unit selected from the group consisting of:

##STR00004##

[0109] and any combination of the foregoing;

[0110] wherein X.sup.1 is independently, at each occurrence, selected from the group consisting of O, S and NH;

[0111] A represents a multiplicity of hydrophobic end groups conjugated to terminal units of the dendron (as defined hereinbelow) through at least one enzymatically cleavable functional group selected from the group consisting of an ester, an amide, a carbamate, a carbonate, a urea, a sulfate, an amidine, an ether, a phosphate, a phosphoamide, sulfamates, and a trithionate;

[0112] n is an integer in the range of 1 to 1,500; and

[0113] m and z are each an integer of 1 to 15.

[0114] In some embodiments, n is an integer in the range of 1 to 1,000.

[0115] It is understood that the number of groups (A) will depend on the number of dendron terminal units and the number of functional groups on each dendron terminal group available to form an enzymatically cleavable functional group.

Trifunctional Moiety B

[0116] According to some embodiments, the hybrid polymer comprises a trifunctional moiety having a first bond to a PEG polymer, a second bond to a labeling moiety, and a third bond, directly or through a linker or branching unit, to a first generation dendron which comprises at least one functional group capable of binding to a further generation or to said enzymatically cleavable hydrophobic end group; and optionally, at least one additional generation which is covalently bound to said first generation or preceding generation, and optionally to a further generation, wherein each of said optional generations comprises at least one functional group capable of binding to said first generation, to a preceding generation, to a further generation, and/or to said enzymatically cleavable hydrophobic end group, each of said bonds being formed directly or through a linker or branching unit.

[0117] The nature of the functional groups on the trifunctional moiety B may vary, depending on the particular hybrid being constructed. According to non-limiting embodiments, the bifunctional moiety may comprise three functional groups selected from the group consisting of O, S, NH, C(O), C(O)O, OC(O)O, C(O)NH, NHC(O)NH, NHC(O)O, S(O), S(O)O, PO(O)O, and any combination thereof. Those functional groups may be linked to a core structure which can be, e.g., an amino acid, a C 1-C20 alkylene, a C2-C20 alkenylene, a C2-C20 alkynylene an arylene, etc.

[0118] In one currently preferred embodiment, the trifunctional moiety B is an amino acid capable of attaching to the hydrophobic dendron, the PEG polymer, and the labeling moiety through its carboxyl group, amino group, and side chain. Suitable amino acids are those that contain, in addition to the amino and carboxy functionalities, an additional functional group (e.g., on the side chain), together defining a trifunctional moiety. The amino acid may be an alpha-amino acid, a beta-amino acid, a gamma-amino acid, a delta-amino acid, etc. In some embodiments, the the bifunctional moiety is an alpha-amino acid selected from the group consisting of lysine, aspartic acid, glutamic acid, tyrosine, asparagine, serine, homoserine, cysteine, homocysteine, glutamine, threonine, ornithine, citrulline, and arginine

[0119] In one embodiment, the amino acid is an alpha amino acid represented by the structure:

##STR00005##

wherein G is the amino acid side chain, which comprises a functional group enabling its attachment to the labeling moiety.

[0120] In one currently preferred embodiment, the amino acid is lysine, and the trifunctional group may generally be represented by the structure:

##STR00006##

Linkers/Branching Moiety

[0121] The linking group T, when present, connects the PEG polymer to the functional group B. In one embodiment, T is absent and the PEG is bound directly to the trifunctional moiety B. In other embodiments, T is present and may be any functional moiety selected from the group consisting of (CH.sub.2).sub.tX(CH.sub.2).sub.tX, X(CH.sub.2).sub.tX, (CH.sub.2).sub.t wherein X is independently at each occurrence selected from O, S and NH, and t is independently at each occurrence 1-10; O, S, NH, C(O), C(O)O, OC(O)O, C(O)NH, NHC(O)NH, NHC(O)O, S(O), S(O)O, PO(O)O, CC, CC, any combination thereof.

[0122] In currently preferred embodiment, T is (CH.sub.2).sub.3S(CH.sub.2).sub.2NH. This group may be introduced by functionalizing OR-PEG-OH with an allyl derivative (e.g., allyl halide) followed by coupling with cystamine hydrochloride. In accordance with this embodiment, PEG-T is represented by the structure:

##STR00007##

[0123] wherein Q, R and n are as defined above. In one embodiment, OR is methoxy. This functional is sometimes abbreviated as: MeO-PEG-NH.

[0124] According to some embodiments, the hybrid delivery system further comprises a linker moiety and/or a branching unit (designated herein Y), or a multiplicity of such units, each of which connect(s) the trifunctional moiety B to the first generation dendron, and/or forms a part of the first generation, and/or connects between dendron generations. In one embodiment, the linker moiety and/or the branching unit is selected from a group consisting of a substituted or unsubstituted acyclic, cyclic or aromatic hydrocarbon moiety, heterocyclic moiety, a heteroaromatic moiety or any combination thereof. Each possibility represents as separate embodiment of the present invention. Specific examples of linker moieties/branching units, useful for this invention include but are not limited to, arylenes, which may be substituted with one or more hydroxyls (e.g., phenols), trimethylolpropane, glycerine, pentaerythritol, polyhydroxy phenols such as phloroglucinol, propylene glycol, tri-substituted alkylamines, diethylenetriamine, triethylenetetramine, diethanolamine, triethanolamine, amino carboxylic acids, such as ethylenediaminetetraacetic (EDTA) and porphyrin, ethylene glycol, ethylenediamine di-substituted alkylamines, diethylenetriamine, triethylenetetramine, diethanolamine, fumaric, maleic, phthalic, malic acid, 6-aminohexanol, 6-mercaptohexanol, 10-hydroxydecanoic acid, 1,6-hexanediol, beta-alanine, 2-aminoethanol, 2-aminoethanethiol, 5-aminopentanoic acid, and 6-aminohexanoic acid among others. Each possibility represents as separate embodiment of the present invention. In one currently preferred embodiment, the linker moiety/branching is an unsubstituted or substituted arylene or phenol which may be positioned between the PEG and the first generation or may form a part of the first generation, or alternatively may be positioned at one or more intermediary generations of the dendron. The linker/branching unit may further provide additional functionality to the hybrid delivery system (e.g., UV absorption). According to various embodiments, each of the linker moiety/branching unit may be connected to the PEG or to other dendron generations through a functional group selected from the group consisting of O, S, NH, C(O), C(O)O, OC(O)O, C(O)NH, NHC(O)NH, NHC(O)O, S(O), S(O)O, PO(O)O, CC, CC, (CH.sub.2).sub.t wherein t is an integer of 1-10, and any combination thereof. Each possibility represents as separate embodiment of the present invention.

[0125] The group Y may be present once at the focal point of the dendron, or may be present multiple times as branching units. In one embodiment, the branching group Y is an arylene which serves as a branching group for the dendron arms. One particular embodiment uses a linker moiety/branching unit derived from 3,5-dihydroxybenzoic acid.

Dendron Repeating Units:

[0126] According to other embodiments, the terminal repeating unit of said dendron is represented by any of the following structures:

##STR00008##

[0127] wherein X.sup.2 has the same meaning as X.sup.1.

[0128] According to yet other embodiments, the hydrophobic end group A is conjugated to the dendron through a functional group represented by the structure:

##STR00009##

[0129] wherein X.sup.2 is a part of the terminal repeating unit of said dendron and C(O) is part of hydrophobic end group; or wherein X.sup.2 is part of the hydrophobic end group and C(O) is a part of the terminal repeating unit of said dendron, or wherein X.sup.2-C(O) are part of the hydrophobic end group, or wherein X.sup.2C(O) is part of the terminal repeating unit of said dendron; and wherein X.sup.2 has the same meaning as X.sup.1.

Enzymatically Cleavable Hydrophobic End Groups (A)

[0130] According to the present invention, the dendron comprises a plurality of enzymatically cleavable hydrophobic end groups.

[0131] According to some embodiments, the enzymatically cleavable hydrophobic end group is present at one or more of the terminal repeating units (i.e., terminal generations) of the hydrophobic dendron, and/or in intermediary generations of the dendron. In other embodiments, the enzymatically cleavable hydrophobic end group is present only at the terminal repeating units of the hydrophobic dendron (i.e., the enzymatically cleavable hydrophobic end group is not present in intermediary generations of the dendron).

[0132] According to some embodiments, the enzymatically cleavable hydrophobic end group is conjugated to the dendron through an enzymatically cleavable functional group selected from the group consisting of an ester, an amide, a carbamate, a carbonate, a urea, a sulfate, an amidine, an ether, a phosphate, a phosphoamide, sulfamates, and a trithionate. Each possibility represents as separate embodiment of the present invention.

[0133] According to some embodiments, the enzymatically cleavable hydrophobic end group is conjugated to the dendron through an amide which is cleavable by an amidase. In one embodiment, the amidase is selected from the group consisting of aryl-acylamidase, aminoacylase, alkylamidase, and phthalyl amidase. Each possibility represents as separate embodiment of the present invention.

[0134] According to some embodiments, the enzymatically cleavable hydrophobic end group is conjugated to the dendron through an ester which is cleavable by an esterase. In one embodiment, the esterase is selected from the group consisting of carboxylesterase, arylesterase, and acetylesterase. Each possibility represents as separate embodiment of the present invention.

[0135] In other embodiments, the enzymatically cleavable hydrophobic end group is an ester OC(O)R or C(O)OR wherein R is an aliphatic chain of C 1-C12 carbon atoms, or an aryl, which is cleavable by esterases. Examples or esters based on octanoic acid and undecanoic acid as describe herein, with each possibility representing a separate embodiment of the present invention. In other embodiments, the enzymatically cleavable hydrophobic end group is an amide NHC(O)R or C(O)NHR wherein R is an aliphatic chain of C1-C12 carbon atoms, or an aryl, which is cleavable by amidases. In one currently preferred embodiment, the enzymatically cleavable hydrophobic end group is phenyl acetate, which is cleaved by esterases. In other currently preferred embodiments, the enzymatically hydrophobic end group is phenylacetamide, which is cleavable by amidases.

[0136] According to other embodiments, the enzymatically cleavable hydrophobic end group is cleaved by an enzyme which is (i) present in greater amount at; or (ii) produced in greater quantity at, or (iii) has higher activity in cells near or at a site of disease or infection. Each possibility represents as separate embodiment of the present invention.

[0137] As contemplated herein, the term enzymatic cleavage covers total or partial cleavage of the enzymatically cleavable hydrophobic end group, i.e., all of the cleavable hydrophobic end group may be hydrolyzed, or only a portion of such groups, by any of the enzymes described herein.

[0138] PEG Polymer

[0139] The hydrophilic PEG polymer is a currently preferred polymer to prepare the block co-polymer hybrid of the present invention as it is generally recognized as safe for use in food, cosmetics, medicines and many other applications by the US Food and Drug Administration. PEG has beneficial physical and/or chemical properties such as water-solubility, non-toxic, odorless, lubricating, nonvolatile, and non-intrusive which are particularly suitable for pharmaceutical utility.

[0140] There are many commercial available derivatives of PEG, all of which may be useful in the present invention. When the PEG is derivatized at both termini, a heterobifunctional PEG is preferably used, i.e., containing orthogonal functional groups that are reactive under different conditions, thereby allowing for selective reactivity on each side. Examples of PEG derivatives that may be used include, but not limited to methoxy PEG-OH (mPEG), amine-terminated PEG (PEG-NH.sub.2), carboxylated PEG (PEG-COOH), thiol-terminated PEG (PEG-SH), N-hydroxysuccinimide-activated PEG (PEG-NHS), NH.sub.2-PEG-NH.sub.2 or NH.sub.2-PEG-COOH. Additional non-limiting examples of PEG derivatives that may be used as starting materials are: PEG-azide (for Cu/azide/alkyne click chemistry), PEG-acrylate/acrylamide, PEG-alkyne (for Cu/azide/alkyne click chemistry), PEG-DBCO, PEG-epoxide glycidyl ether, PEG-halide; PEG-hydrazide, PEG-maleimide, PEG-nitrophenyl carbonate (NPC), PEG-orthopyridyl disulfide (PUSS), PEG-silane, PEG-sulfonate (e.g., tosyl, mesyl), PEG-COOR wherein R is an alkyl, etc. Each possibility represents as separate embodiment of the present invention.

[0141] These PEG derivatives may be subjected to further chemical modifications and substitutions. For example RU-PEG-OH may be functionalized by allylation followed by thiolation with cystamine to produce RO-PEG-(CH.sub.2).sub.3S(CH.sub.2).sub.2NH.sub.2 as a starting material, as described in Scheme 3A hereinbelow.

[0142] Thus, according to various embodiments, the PEG is linked to the dendron or the multi-functional moiety through a PEG terminal functional group selected from the group consisting of (CH.sub.2).sub.tX(CH.sub.2).sub.tX, X(CH.sub.2).sub.tX-, -(CH.sub.2).sub.t wherein X is independently at each occurrence selected from O, S and NH, and t is independently at each occurrence 1-10; O, S, NH, C(O), C(O)O, OC(O)O, C(O)NH, NHC(O)NH, NHC(O)O, S(O), S(O)O, PO(O)O, CC, CC, triazolyl, and any combination thereof. Preferably, the PEG is functionalized by the functional group(CH.sub.2).sub.3S(CH.sub.2).sub.2NH (i.e., the group T in formula (I)).

[0143] The PEG may be derivatized by a terminal group Q which is preferably selected from the group consisting of OR wherein is H or a C1-C4 alkyl (e.g., methyl); NH.sub.2, SH and COOH. However, other functional groups can advantageously be used, e.g., as targeting moieties to direct the hybrid delivery systems of the present invention to their biological targets.

[0144] According to some embodiments, the PEG has an average molecular weight between about 0.5 and 70 kDa. In one currently preferred embodiment, the hydrophilic PEG polymer is an mPEG. In another currently preferred embodiment, the PEG polymer has a molecular weight of about 2 kDa. In another currently preferred embodiment, the PEG polymer has a molecular weight of about 5 kDa. In yet another currently preferred embodiment, the PEG polymer has a molecular weight of about 10 kDa. In yet another currently preferred embodiment, the PEG polymer has a molecular weight of about 20 kDa. In yet another currently preferred embodiment, the PEG polymer has a molecular weight of about 30 kDa. Preferably, the PEG has at least 10 repeating units of ethylene glycol monomers.

[0145] In one embodiment, the hybrid polymer of the present invention may generally be represented by formula (II):

##STR00010## [0146] wherein each X.sup.1 and X.sup.2 is independently at each occurrence selected from the group consisting of O, S and NH;

[0147] Q is selected from the group consisting of OR wherein is H or a C1-C4 alkyl (e.g., methyl); NH.sub.2, SH and COOH;

[0148] A, alone or together with C(O) is a hydrophobic end group;

[0149] T is absent or is a functional group selected from the group consisting of

[0150] (CH.sub.2).sub.tX(CH.sub.2).sub.tX, X(CH.sub.2).sub.tX, (CH.sub.2).sub.t wherein X is independently at each occurrence selected from O, S and NH, and t is independently at each occurrence 1-10; O, S, NH, C(O), C(O)O, OC(O)O, C(O)NH, NHC(O)NH, NHC(O)O, S(O), S(O)O, PO(O)O, CC, CC, any combination thereof;

[0151] the labeling moiety is selected from a fluorescent dye, a dark quencher and a fluorinated moiety; and

[0152] n is an integer of 1 to 1,500, preferably 1 to 1,000.

[0153] One specific embodiment of formula (II) is represented by the structure of formula (IIa):

##STR00011##

[0154] Other examples of the hybrid polymers of formula (I) include, but are not limited to, any one or more of the following structures:

##STR00012##

##STR00013## [0155] wherein each X.sup.1 and X.sup.2 is independently at each occurrence selected from the group consisting of O, S and NH;

[0156] Q is selected from the group consisting of OR wherein is H or a C1-C4 alkyl (e.g., methyl); NH.sub.2, SH and COOH A, alone or together with C(O) is a hydrophobic end group;

[0157] B is a bifunctional moiety comprising a labeling moiety covalently attached thereto;

[0158] pis 1,2, 3, 4 or 5; and

[0159] n is an integer of 1 to1,500. 10

[0160] In some embodiments, n is an integer in the range of 1 to 1,000.

[0161] Each possibility represents as separate embodiment of the present invention.

[0162] Also contemplated are analogues of compounds of formulae (I), (II), (IIa), G0, G1 , G1, G2, G2, and G3 wherein the linkage of A to X.sup.2C(O) is reversed, i.e., the compounds incorporate the following moiety:

##STR00014##

[0163] wherein X.sup.2 is part of the hydrophobic end group A or part of the dendron.

[0164] In some embodiments, the micelle has an average particle size of less than about 100 nm, preferably about 50 nm or lower, more preferably about 10 nm to 50 nm, and most preferably about 10 nm to 20 nm. Each possibility represents as separate embodiment of the present invention.

[0165] A diagnostic agent refers to a chemical or biological molecule used to identify a disease, disorder or medical condition as well as monitor treatment effects. Diagnostic agents include radiopharmaceuticals, contrast agents for use in imaging techniques, allergen extracts, activated charcoal, different testing strips (e.g., cholesterol, ethanol, and glucose), pregnancy test, breath test with urea .sup.13C, and various stains/markers. Each possibility represents as separate embodiment of the present invention.

[0166] The term derived from as used herein means a moiety that is derived from an active compound (i.e., any of the biologically or diagnostically active compounds described herein) and that is incorporated into the hybrid systems of the present invention. A derivative of an active moiety may be formed, e.g., by removing one or more of the atoms of said compound or adding one or more atoms or functional groups so as to chemically conjugate it to the dendron.

[0167] Chemical Definitions

[0168] The term alkyl used herein alone or as part of another group denotes a saturated aliphatic hydrocarbon, including straight-chain and branched-chain alkyl groups. In one embodiment, the alkyl group has 1-12 carbons designated here as C 1-C12-alkyl. In another embodiment, the alkyl group has 1-4 carbons designated here as C1-C4-alkyl. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, and the like.

[0169] The term aryl used herein alone or as part of another groups denotes an aromatic ring system containing from 6-14 ring carbon atoms. The aryl ring can be a monocyclic, bicyclic, tricyclic and the like. Non-limiting examples of aryl groups are phenyl, naphthyl including 1-naphthyl and 2-naphthyl, and the like.

[0170] The term C1-C20 alkylene used herein alone or as part of another group denotes a bivalent radicals of 1 to 20 carbons, which is bonded at two positions connecting together two separate additional groups (e.g., CH.sub.2). Examples of alkylene groups include, but are not limited to (CH.sub.2), (CH.sub.2).sub.2, (CH.sub.2).sub.3, (CH.sub.2).sub.4, etc.

[0171] The term C2-C20 alkenylene used herein alone or as part of another group denotes a bivalent radical of 2 to 20 carbons which contains at least one double bond, which is bonded at two positions connecting together two separate additional groups (e.g., CHCH).

[0172] The term C2-C20 alkynylene used herein alone or as part of another group denotes a bivalent radicals of 2 to 20 carbons containing at least one triple bond, which is bonded at two positions connecting together two separate additional groups (e.g., CC).

[0173] The term arylene denotes a bivalent radicals of aryl, which is bonded at two positions connecting together two separate additional groups. The term acyclic hydrocarbon used herein denotes to any linear or branched, saturated and mono or polyunsaturated carbon atoms chain, or the residue of such compound after it has chemically bonded to another molecule. Preferred are acyclic hydrocarbon moieties containing from 1 to 20 carbon atoms. The acyclic hydrocarbon of the present invention may comprise one or more of an alkyl, an alkenyl, and an alkynyl moieties. Examples of acyclic hydrocarbon include, but are not limited to, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl, n-pentyl, n-hexyl, vinyl, allyl, butenyl, pentenyl, ropargyl, butynyl, pentynyl, and hexynyl. Each possibility represents as separate embodiment of the present invention.

[0174] The term cyclic hydrocarbon generally refers to a C3 to C8 cycloalkyl or cycloalkenyl which includes monocyclic or polycyclic groups. Non-limiting examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl. The cycloalkyl group can be unsubstituted or substituted with any one or more of the substituents defined above for alkyl.

[0175] The term aromatic hydrocarbon used herein denotes to an aromatic ring system containing from 6-14 ring carbon atoms. The aryl ring can be a monocyclic, bicyclic, tricyclic and the like. Non-limiting examples of aryl groups are phenyl, naphthyl including 1-naphthyl and 2-naphthyl, and the like. Each possibility represents as separate embodiment of the present invention. The aryl group can be unsubstituted or substituted through available carbon atoms with one or more groups defined hereinabove for alkyl.

[0176] The terms heterocyclic or heterocyclyl used herein alone denote a five-membered to eight-membered rings that have 1 to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen. These five-membered to eight-membered rings can be saturated, fully unsaturated or partially unsaturated. Preferred heterocyclic rings include piperidinyl, pyrrolidinyl, pyrrolinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, pyranyl, thiopyranyl, piperazinyl, indolinyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothiophenyl, tetrahydrothiophenyl, dihydropyranyl, tetrahydropyranyl, and the like. Each possibility represents as separate embodiment of the present invention. The heterocyclyl group can be unsubstituted or substituted through available atoms with one or more groups defined hereinabove for alkyl.

[0177] The term heteroaryl used herein denotes a heteroaromatic system containing at least one heteroatom ring atom selected from nitrogen, sulfur and oxygen. The heteroaryl generally contains 5 or more ring atoms. The heteroaryl group can be monocyclic, bicyclic, tricyclic and the like. Also included in this expression are the benzoheterocyclic rings. If nitrogen is a ring atom, the present invention also contemplates the N-oxides of the nitrogen containing heteroaryls. Non-limiting examples of heteroaryls include thienyl, benzothienyl, 1-naphthothienyl, thianthrenyl, furyl, benzofuryl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, isoindolyl, indazolyl, purinyl, isoquinolyl, quinolyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, carbolinyl, thiazolyl, oxazolyl, isothiazolyl, isoxazolyl and the like. Each possibility represents as separate embodiment of the present invention. The heteroaryl group may optionally be substituted through available atoms with one or more groups defined hereinabove for alkyl.

[0178] Any of the moieties described herein (e.g., alkylene, alkenylene, alkynylene, arylene, acyclic and cyclic hydrocarbons, heterocyclic and heteroaromatic moieties) may be unsubstituted, or substituted with one or more substituents selected from the group consisting of halogen, hydroxy, alkoxy, aryloxy, alkylaryloxy, heteroaryloxy, oxo, cycloalkyl, phenyl, heteroaryl, heterocyclyl, naphthyl, amino, alkylamino, arylamino, heteroarylamino, dialkylamino, diarylamino, alkylarylamino, alkylheteroarylamino, arylheteroarylamino, acyl, acyloxy, nitro, carboxy, carbamoyl, carboxamide, cyano, sulfonyl, sulfonylamino, sulfinyl, sulfinylamino, thiol, C.sub.1 to C.sub.4 alkylthio, arylthio, or C.sub.1 to C.sub.4 alkylsulfonyl groups. Any substituent can be unsubstituted or further substituted with any one of these aforementioned substituents. Each possibility represents as separate embodiment of the present invention.

[0179] All stereoisomers, optical and geometrical isomers of the compounds of the instant invention are contemplated, either in admixture or in pure or substantially pure form. The compounds of the present invention can have asymmetric centers at any of the atoms. Consequently, the compounds can exist in enantiomeric or diastereomeric forms or in mixtures thereof. The present invention contemplates the use of any racemates (i.e., mixtures containing equal amounts of each enantiomers), enantiomerically enriched mixtures (i.e., mixtures enriched for one enantiomer), pure enantiomers or diastereomers, or any mixtures thereof. The chiral centers can be designated as R or S or R,S or d,D, 1,L or d,1, D,L. In addition, several of the compounds of the invention contain one or more double bonds. The present invention intends to encompass all structural and geometrical isomers including cis, trans, E and Z isomers, independently at each occurrence.

[0180] One or more of the compounds of the invention, may be present as a salt. The term salt encompasses both basic and acid addition salts, including but not limited to phosphate, dihydrogen phosphate, hydrogen phosphate and phosphonate salts, and include salts formed with organic and inorganic anions and cations. Furthermore, the term includes salts that form by standard acid-base reactions of basic groups and organic or inorganic acids. Such acids include hydrochloric, hydrofluoric, hydrobromic, trifluoroacetic, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, cholic, pamoic, mucic, D-camphoric, phthalic, tartaric, salicyclic, methanesulfonic, benzenesulfonic, p-toluenesulfonic, sorbic, picric, benzoic, cinnamic, and like acids. Additional salts of the conjugates described herein may be prepared by reacting the parent molecule with a suitable base, e.g., NaOH or KOH to yield the corresponding alkali metal salts, e.g., the sodium or potassium salts. Additional basic addition salts include ammonium salts (NH.sub.4), substituted ammonium salts, Li, Ca, Mg, salts, and the like.

[0181] Uses

[0182] The molecular assemblies described herein are useful as diagnostic probes for monitoring specific enzymatic activity and/or as probes capable of reporting the degree and location of activation.

[0183] Potential uses of the hybrid delivery systems described herein include advanced enzymatically activated fluorescent imaging probes and smart drug delivery platforms, as well as or .sup.19F probes for turn-on of .sup.19F-magnetic resonance signal (.sup.19F-MR probes) for diagnostic and monitoring purposes.

[0184] In one aspect, the present invention provides a method of delivering the amphiphilic hybrid system of the invention comprising the step of contacting the amphiphilic hybrid delivery system with an enzyme to induce cleavage of the enzymatically cleavable hydrophobic end group, thereby disassembling the micelle.

[0185] In another aspect, the present invention provides a method of monitoring enzymatic activity in a biological system, the method comprising the step of contacting the biological system with a hybrid delivery system according to claims 1, and monitoring said enzymatic activity by fluorescence or .sup.19F-magnetic resonance (MR).

[0186] In another aspect, the present invention relates to the use of the hybrid delivery system according to claim 1 wherein the labeling moiety is a fluorescent labeling moiety, as .sup.19F-magnetic resonance (MR) probe for .sup.19F-MR imaging.

[0187] As used herein, the term contacting refers to bringing in contact with the amphiphilic hybrid delivery system of the present invention. Contacting can be accomplished to cells or tissue cultures, or to living organisms, for example humans. In one embodiment, the present invention encompasses contacting the amphiphilic hybrid delivery system of the present invention with a human subject.

[0188] As used herein, the term contacting the amphiphilic hybrid delivery system may be ex-vivo on a surface, on a device, in cell/tissue culture dish, in food and water, as well as in-vivo, among others. Alternatively, the contact may be in the body of a human or non-human subject.

[0189] Kits

[0190] In another aspect, the present invention provides a kit for delivering the amphiphilic hybrid system comprising in one compartment the amphiphilic hybrid system, and in a second compartment an enzyme capable of cleaving the enzymatically cleavable hydrophobic end group so as to disassemble the micelle.

[0191] The kit may further include appropriate buffers and reagents known in the art for administering/contacting the compartments listed above to a host cell or a host organism. The amphiphilic hybrid delivery system and the enzyme may be provided in solution and/or in lyophilized form. When the enzyme is in a lyophilized form, the kit may optionally contain a sterile and physiologically acceptable reconstitution medium such as water, saline, buffered saline, and the like.

[0192] According to some embodiments, associated with such compartments may be various written materials such as instructions for use.

[0193] The examples hereinbelow are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art may readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES 20

Example 1

Smart Micelles Containing Tunable Fluorescent Probes

[0194] The amino acid lysine with two orthogonal amine protecting groups was chosen as the tri-functional junction since selective deprotection should allow simple synthesis of the dendron from a-amine and fluorescent labeling of the 6-amine. Fluorescein (F) was chosen as the Turn-On dye, due to its small Stokes shift and known self-quenching at high loading. 7-Diethylamino-3-carboxy coumarin (C) was utilized as the spectral shift dye as coumarins can form excimers with red-shifted emission. Two esterase-responsive labeled hybrids, 1F and 1C, bearing four enzymatically cleavable hydrophobic end-groups and either fluorescein or coumarin dye, respectively, were chosen as model compounds (Scheme 1).

##STR00015##

Characterization of the self-assembled micelles: Dynamic light scattering (DLS) measurements showed diameters of about 17 nm for both hybrids 1F and 1C (FIG. 2a). Transmission electron microscopy (TEM) images confirmed the formation of spherical structures (FIGS. 2b and 2c). The critical micellar concentrations, determined using Nile red, were 6 M and 3 M for hybrids 1F and 1C, respectively. Further support for the micellar core-shell morphology was obtained from comparison of .sup.1H-NMR spectra in CDCl.sub.3 and D.sub.2O as peaks assigned to dendron were significantly broadened or disappeared in D.sub.2O, whereas the PEG signals remained unchanged (FIG. 8A-8B).

[0195] After the self-assembly of hybrids 1F and 1C into micelles was confirmed, their fluorescence was studied together with hydrophilic hybrids 7F and 7C as controls. As hypothesized, the emission intensities of hybrid 1F (FIG. 3a) were significantly weaker than those of hydrophilic hybrid 7F (FIG. 3b) due to self-quenching in the assembled state.

[0196] Comparison of the coumarin-labeled hybrids showed that hydrophilic hybrid 7C had an emission maximum at 480 nm, as expected for this coumarin derivative, whereas micelles based on hybrid 1C showed a significantly red-shifted maximum at 540 nm with a shoulder at 480 nm (FIGS. 4a and 4b). This 60 nm red-shift can be explained by formation of excimers by closely packed coumarin dyes at the interface between the PEG shell and the hydrophobic core, as confirmed by excitation measurements (FIG. 4c). Very interestingly, comparison of the ratio of emission intensities at 540 nm and 480 nm, which is indicative of the self-assembly process, reveals that the amphiphilic labeled hybrids start to self-assemble in concentrations well below their CMC value (FIG. 4d). The inherited ability of these new hybrids to report on their self- assembly, opens opportunities to deeper understanding of self-assembly as it allows a direct feedback from the polymers on their aggregation state.

[0197] Characterization of the enzymatically triggered disassembly: The significant differences between the fluorescence of the assembled and disassembled states motivated us to explore whether the enzymatically triggered structural change from micelles into hydrophilic hybrids could induce the spectral responses. Parallel HPLC analysis and fluorescence measurements of both hybrids in the presence and absence of the activating enzyme (porcine liver esterase, PLE) were used to study the enzymatic activation. The HPLC data indicated transformations of hybrids 1F and 1C into the corresponding hydrophilic tetra-hydroxy hybrids 7F and 7C, respectively, upon addition of PLE (FIGS. 16 and 17). The very weak fluorescence of the fluorescein-labeled hybrid 1F at 525 nm (excited at 470 nm) increased rapidly upon addition of the enzyme (FIG. 5a). The excellent correlation with the HPLC data (FIG. 5b) indicates that, as hypothesized, the enzymatic hydrolysis of the micelle-forming amphiphilic hybrids leads to the formation of hydrophilic hybrids that disassemble and diffuse away from each other. This physical separation halts the self-quenching of the dyes and thus turns on their strong intrinsic fluorescence.

[0198] A very promising spectral response was also observed for the micelles of hybrid 1C that showed a red-shifted spectrum relative to the hydrophilic-labeled hybrid 7C, with maximum emission at 540 nm due to the formation of excimers in the assembled state. Addition of the enzyme resulted in a decrease of emission at 540 nm and an increase in emission intensity at 480 nm (FIG. 6a). This change in fluorescence, which correlates very well with the HPLC analysis (FIG. 6b), resulted from enzymatic cleavage of the hydrophobic end-groups of hybrids 1C, which led to the formation of hydrophilic hybrids (7C) and disassembly of the micelles. As the spatial separation of the labeled hybrids increases, the coumarin dyes cannot form excimers and the emission at 540 nm decays whereas the expected intrinsic emission at 480 nm increases.

[0199] In order to demonstrate the high modularity of this molecular design and the ability to tune its enzymatic activity, an amidase-responsive hybrid MeO-PEG-Lys(C)dendron-(NHCOCH.sub.2Ph).sub.4, 8C was synthesized, with hydrophobic phenyl acetamide end-groups (FIG. 7a) that can be cleaved by the enzyme penicillin G amidase (PGA)..sup.2 Whereas the ester-based hybrids 1F and 1C showed complete transformation into fully hydrolyzed hybrids, the amidase-responsive hybrids showed accumulation of partially cleaved intermediates. With fluorescently labeled hybrids, this partial cleavage could lead to the formation of small aggregates such as dimers or trimers, which might result in significant dye-dye interactions also in the non-micellar state, thus limiting the magnitude of the desired spectral effect. HPLC analysis of the enzymatic degradation of 8C by PGA (FIG. 18) showed indeed the formation of partially cleaved intermediates, however, the fluorescence response (FIG. 7b) was as strong as the one observed for the esterase-responsive hybrids (FIG. 6b). Furthermore, a very good correlation was observed for the disappearance of the starting hybrid 8C and the spectral changes at both 540 nm and 480 nm (FIG. 7c).

[0200] In summary, the novel molecular design described herein enabled the synthesis of fluorescently labeled smart polymeric amphiphiles and their self-assembly into enzyme-responsive micelles with significantly altered fluorescent properties in the assembled compared to unassembled states due to dye-dye interactions in the micelles. Enzymatic cleavage of the hydrophobic end-groups of the dendrons increased the hydrophilicity of the hybrids, resulting in disassembly of the micelles. This supramolecular structural change was translated into a spectral response as the dyes diffuse away from each other and dye-dye interactions are diminished and the intrinsic fluorescence of the dyes is regained. Taking advantage of the high modularity of the supramolecular translation mechanism, micelles were designed with Turn-On or spectral switching of the emitted fluorescence, depending on the type of the labeling dye. The highly efficient synthesis and the ability to rationally adjust both the activating enzyme and spectral-response by installing suitable enzymatic substrates and labeling dye, respectively, make this platform highly promising for the fabrication of advanced enzymatically activated fluorescent imaging probes and smart drug delivery platforms. Furthermore, harnessing the structural responsiveness of this polymeric platform opens the way for simple transformation of non-responsive dyes into enzymatically activated smart fluorescent probes.

Example 2

Synthesis Protocol of the Amphiphilic PEG-Dendron Hybrids Containing Fluorescent Probes

[0201] The two hybrids, 1F and 1C, were synthesized from MeO-PEG-Lys(Boc)-Fmoc (2) as illustrated in Scheme 2. Following selective deprotection of the Fmoc group, the amine was conjugated to di-acetylene 3 to give hybrid 4. The acetylene groups were then reacted with 2-mercaptoethanol through a thiol-yne reaction to yield hybrid 5. Esterification with phenyl acetic acid yielded hybrid 6 with four enzymatically cleavable end-groups. The Boc group was removed by trifluoroacetic acid, followed by conjugation of dye to the deprotected amine to yield amphiphilic hybrids 1F and 1C. The expected hydrolysis products, hydrophilic hybrids 7F and 7C, were also synthesized as reference compounds. All polymeric hybrids were characterized by .sup.1H-NMR, .sup.13C-NMR, IR, and GPC (1F, 1C, 7F, and 7C were also characterized by MALDI-MS) and the experimental data was found to be in good agreement with the theoretical one.

##STR00016## ##STR00017##

[0202] General Procedure for Preparing Precursor 2:

##STR00018##

[0203] MeO-PEG-Allyl precursors may be prepared by the process described in general Scheme 3A hereinabove. Poly (ethylene glycol) methyl ether was dissolved in toluene (10 mL per 1g) with KOH (10 eq.). The solution was refluxed for at least 1 hour using a Dean Stark water separation system. Solution was cooled down to 50 C. and then allyl bromide (10 eq.) was added slowly and the reaction was stirred overnight. The solution was filtered hot through celite, the celite was then washed with DCM. Solvents were evaporated in vacuum and the residue was re-dissolved in DCM (5 mL per 1 g PEG). MeO-PEG-Allyl product was precipitated by the dropwise addition of 1:1 v/v Ether:Hexane mixture (50 mL per 1 g PEG). Precipitate was filtered and washed with ether and then with hexane. The final white solid product was dried under high vacuum. Three different products were prepared using 3 PEG precursors (2 kDa PEG (2a), 5 kDa PEG (2b) and 10 kDa PEG (2c).

[0204] MeO-PEG2kDa-Allyl: 3.00g (1.5mmo1) Poly (ethylene glycol) methyl ether (M.sub.n=2 kDa) were reacted according to the general procedure (I) and the product was obtained as a white solid (2.42g) 80% yield. .sup.1H-NMR (CDC1.sub.3): 5.85-5.95 (m, 1H, vinyl CHCH.sub.2), 5.26 (dd, J=1.4 Hz, 17.2 Hz, 1H ,trans vinyl CH=CH.sub.2), 5.17 (dd, J=1.0 Hz, 10.4 Hz, 1H, cis vinyl CH=CH2), 4.01 (d, J=5.7 Hz, 2H, OCH.sub.2CHCH.sub.2), 3.44-3.82 (m, 206H, PEG backbone), 3.37 (s, 3H, H.sub.3CO); .sup.13C-NMR (CDC1.sub.3) 134.9, 117.2, 72.4, 72.1, 70.7, 69.6, 59.1; FT-IR, v(cm.sup.1) 2878, 1466, 1456, 1359, 1341, 1279, 1240, 1145, 1098, 1060, 957, 947, 842; GPC (DMF+LiBr) M.sub.n=1.8 kDa, PDI=1.04. MALDI-TOF MS: molecular ion centered at 2.0 kDa.

[0205] MeO-PEG5kDa-Allyl: 5.00 g (1 mmol) Poly (ethylene glycol) methyl ether (M.sub.n=5 kDa) were reacted according to the general procedure (I) and the product was obtained as a white solid (4.45g), 88% yield. .sup.1H-NMR (CDC1.sub.3): 5.86-5.95 (m, 1H, CHCH.sub.2), 5.26 (d, J=17.3 Hz, 1H, trans vinyl CHCH.sub.2), 5.17 (d, J=10.3 Hz, 1H, cis vinyl CHCH.sub.2), 4.01 (d, J=5.3 Hz, 2H, OCH.sub.2CHCH.sub.2), 3.44-3.82 (m, 553H, PEG backbone), 3.37 (s, 3H, H.sub.3CO); .sup.13C-NMR (CDC1.sub.3) 6 134.9, 117.2, 72.3, 72.0, 70.7, 69.5, 59.1; FT-IR, v(cm.sup.1) 2881, 1466, 1360, 1341, 1279, 1240, 1147, 1098, 1060, 959, 842; GPC (DMF+LiBr): M.sub.n=5.7 kDa, PDI=1.02.

[0206] MeO-PEG10kDa-Allyl: 2.00g (0.2mmo1) Poly (ethylene glycol) methyl ether (M.sub.n=10 kDa) were reacted according to the general procedure (I) and the product was obtained as a white solid (1.98g). .sup.1H-NMR (CDC1.sub.3): 5.82-5.95 (m, 1H, CHCH.sub.2), 5.25 (dd, J=1.4 Hz, 17.2 Hz, 1H, trans vinyl CHCH.sub.2), 5.15 (dd, J=1.1 Hz, 10.3 Hz, 1H, cis vinyl CHCH.sub.2), 4.00 (d, J=5.6 Hz , 2H, OCH.sub.2CHCH.sub.2), 3.43-3.81 (m, 956H, PEG backbone), 3.35 (s, 3H, H.sub.3CO); .sup.13C-NMR (CDC1.sub.3) 134.9, 117.2, 72.3, 72.0, 71.1, 70.7, 69.5, 59.1; FT-IR, v(cm.sup.1): 2881, 1467, 1454, 1360, 1341, 1279, 1240, 1147, 1098, 1060, 960, 948, 842; GPC (DMF+LiBr): M.sub.n=11.2 kDa, PDI=1.02.

[0207] General Procedure for Compounds 2a-c

[0208] MeO-PEG-Allyl was dissolved in MeOH (5 mL per 1g). Cystamine hydrochloride (40 eq.) and DMPA (0.2 eq.) were added. The solution was purged with nitrogen for minutes and then placed under UV light at 365 nm for 2 hours. MeOH was evaporated to dryness and the crude mixture was dissolved in NaOH 1N (100 mL per 1g). This aqueous phase was extracted with DCM (350 mL). The organic phase was filtered through celite and evaporated in vacuum. The residue was re-dissolved in DCM (5 mL per 1 g PEG) and product was precipitated by the dropwise addition of 1:1 v/v Ether:Hexane mixture (50 mL per 1 g PEG). The white precipitate was filtered and washed with ether and then with hexane and was dried under high vacuum.

[0209] 2a: 2.00 g (0.97 mmol) MeO-PEG2k-Allyl were reacted according to the general procedure (II) and the product was obtained as a white solid (1.70 g, 82% yield) .sup.1H-NMR (CDCl.sub.3): 6 3.44-3.82 (m, 225H, PEG backbone), 3.37 (s, 3H, H.sub.3CO), 2.86 (t, J=6.3 Hz, 2H, CH.sub.2NH.sub.2), 2.56-2.62 (m, 4H, CH.sub.2SCH.sub.2), 1.85 (qui, J=6.7 Hz, 2H, OCH.sub.2CH.sub.2CH.sub.2S); .sup.13C-NMR (CDCl.sub.3) 72.1, 70.7, 70.4, 69.8, 59.2, 41.3, 36.4, 30.0, 28.6; FT-IR v(cm.sup.1): 2883, 1467, 1456, 1360, 1343, 1280, 1241, 1146, 1115, 1061, 963, 947, 842; GPC (DMF+LiBr): M.sub.n=1.8 kDa, PDI=1.04.

[0210] 2b: 2.12 g (0.42 mmol) MeO-PEG5k-Allyl were reacted according to the general procedure (II) and the product was obtained as a white solid (2.02 g, 94% yield). .sup.1H-NMR (CDCl.sub.3): 3.45-3.83 (m, 590H, PEG backbone), 3.38 (s, 3H, H.sub.3CO), 2.87 (t, J=6.2 Hz, 2H, CH.sub.2NH.sub.2), 2.57-2.63 (m, 4H, CH.sub.2SCH.sub.2), 1.82-1.89 (m, 2H, OCH.sub.2CH.sub.2CH.sub.2S); .sup.13C-NMR (CDCl.sub.3): 72.1, 70.7, 70.3, 69.4, 59.2, 40.6, 36.4, 29.8, 28.5; FT-IR v(cm.sup.1): 2882, 1542, 1466, 1360, 1341, 1279, 1240, 1146, 1102, 1060, 959, 842; GPC (DMF+LiBr): M.sub.n=5.6 kDa, PDI=1.04.

[0211] 2c: 500 mg (0.05 mmol) MeO-PEG10k-Allyl were reacted according to the general procedure (II) and the product was obtained as a white solid (434 mg) 86% yield. .sup.1H-NMR (CDCl.sub.3): 3.43-3.81 (m, 1152H, PEG backbone), 3.36 (s, 3H, H.sub.3CO), 2.87 (t, J6.4=Hz, 2H, CH.sub.2NH.sub.2), 2.53-2.66 (m, 4H, CH.sub.2SCH.sub.2), 1.84 (qui, J=6.7 Hz, 2H, OCH.sub.2CH.sub.2CH.sub.2S); .sup.13C-NMR (CDCl.sub.3) 72.0, 71.2, 70.7, 69.7, 59.1, 41.1, 35.9, 29.9, 28.5; FT-IR v(cm.sup.1): 2880, 1467, 1454, 1359, 1341, 1279, 1240, 1146, 1096, 1060, 960, 947, 841; GPC (DMF+LiBr): M.sub.n=11.3 kDa, PDI=1.02.

##STR00019##

[0212] Hybrid 2 (MeO-PEG.sub.5kDa-Lys(Boc)-Fmoc):

[0213] Fmoc-Lys(Boc)CO.sub.2H (5 eq.) and HBTU (5 eq.) were dissolved in DCM:DMF 1:1 (1 mL) followed by addition of DIPEA (15 eq.) and allowed to stir for 1 hour. The solution was added to 200 mg (0.04 mmol) of MeO-PEG.sub.5kDa-NH.sub.2.sup.[1] dissolved in DCM (1 mL). The reaction was stirred for 3 hours and complete coupling was confirmed by a negative Kaiser test. The crude mixture was loaded on a MeOH based LH20 SEC column. The fractions that contained the product were unified, the MeOH was evaporated in vacuum, further purification was done by re-dissolving the oily residue in DCM (1 mL) followed precipitation by the dropwise addition of Ether (50 mL). The white precipitate was filtered, washed twice with Ether and dried under high vacuum. The product was obtained as a white solid (197 mg, 90% yield).

[0214] .sup.1H-NMR (CDCl.sub.3): 7.69 (d, J=7.5 Hz, 2H, Ar-H), 7.53 (d, J=7.2 Hz, 2H, Ar-H), 7.33 (t, J=7.4 Hz, 2H, Ar-H), 7.24 (d, J=7.4 Hz, 2H, Ar-H), 6.60 (m, 1H, CH.sub.2NHCOCH-), 5.59 (m, 1H, NH-Fmoc), 4.71 (m, 1H, NH-Boc), 4.35 (d, J=6.2 Hz, 2H, FmocCH.sub.2), 4.14 (t, J=6.6 Hz, 1H, FmocCHCH.sub.2), 4.05 (m, 1H, COCHNH), 3.57-3.31 (m, PEG backbone), 3.31 (s, 3H, CH.sub.3O-PEG), 3.10-2.86 (m, 2H, Boc-NHCH.sub.2), 2.64-2.49 (m, 4H, CH.sub.2SCH.sub.2), 1.88-1.49 (m, 4H, OCH.sub.2CH.sub.2CH.sub.2S+Boc-NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.36 (m, 13H, Boc-NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH+Boc); .sup.13C-NMR (CDCl.sub.3): 171.8, 156.3, 156.2, 143.9, 141.4, 127.8, 127.2, 125.1, 120.0, 79.1, 72.0, 70.6, 69.5, 66.9, 63.8, 59.1, 55.0, 47.3, 40.0, 38.6, 32.2, 31.6, 29.8, 29.7, 28.6, 28.3, 22.6; FT-IR, v (cm.sup.1): 2883, 1467, 1453, 1359, 1341, 1279, 1240, 1147, 1099, 1060, 959, 948, 842; GPC: Mn=5.3 kDa, PDI=1.05. Expected Mn=5.6 kDa.

[0215] Hybrid 4 (MeO-PEG.sub.5kDa-Lys(Boc)-di-vne):

[0216] 180 mg (0.03 mmol) of MeO-PEG.sub.5kDa-Lys(Boc)-Fmoc (2) were dissolved in 20% piperidine v/v in DMF (3 mL) and stirred for 1 hour. The deprotected product was precipitated by the dropwise addition of 1:1v/v Ether:Hexane mixture (50 mL). The white precipitate was filtered and washed with Hexane and Ether and dried under high vacuum. The deprotected product was obtained as a white solid. Compound 3.sup.[1] (5 eq.) and HBTU (5 eq.) were dissolved in DCM:DMF 1:1 (1 mL) followed by addition of DIPEA (15 eq.) and allowed to stir for 1 hour. The solution was added to 173 mg (0.03 mmol) of the deprotected hybrid 2 MeO-PEG.sub.5kDa-lys(BOC)-NH.sub.2 dissolved in DCM (1 mL). The reaction was stirred for 1 hour, complete coupling was confirmed by a negative Kaiser test. The crude mixture was loaded on a MeOH based LH20 SEC column. The fractions that contained the product were unified and the MeOH was evaporated in vacuum, further purification was done by re-dissolving the oily residue in DCM (1 mL) followed by precipitation by the dropwise addition of Ether (50 mL). The white precipitate was filtered and washed twice with Ether and dried under high vacuum. The product was obtained as a white solid (162 mg, 87% yield).

[0217] .sup.1H-NMR (CDCl.sub.3): 7.08 (m, 1H, CHNHCOAr), 7.00 (m, 2H, ArH), 6.90 (d, J=6.1 Hz, 1H, CH.sub.2NHCOCH), 6.66 (d, J=2.4 Hz, 1H, ArH), 4.78 (m, 1H, NHBoc), 4.64 (d, J=2.3 Hz, 4H, OCH.sub.2CCH), 4.48 (q, J=7.1 Hz, 1H, COCHNH), 3.82-3.33 (m, PEG backbone), 3.30 (s, 3H, CH.sub.3O-PEG), 3.01 (d, J=6.9 Hz, 2H, Boc-NHCH.sub.2), 2.54 (m, 6H, CH.sub.2SCH.sub.2+OCH.sub.2CCH), 1.94-1.59 (m, 4H, OCH.sub.2CH.sub.2CH.sub.2S+Boc-NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.44-1.24 (m, 13H, Boc-NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH+Boc); .sup.13C-NMR (CDCl.sub.3) 171.7, 166.8, 158.8, 156.2, 136.2, 107.1, 105.8, 82.1, 78.1, 76.3, 72.0, 70.6, 69.5, 59.1, 56.2, 53.7, 40.1, 38.8, 32.2, 31.5, 29.8, 29.7, 28.5, 28.3, 22.9; FT-IR, v (cm.sup.1): 2882, 1593, 1467, 1453, 1380, 1359, 1341, 1279, 1240, 1147, 1098, 1060, 960, 948, 842; GPC: Mn=5.8 kDa, PDI=1.08. Expected Mn=5.6 kDa.

##STR00020##

[0218] Hybrid 5 (MeO-PEG.sub.5kDa-Lvs(Boc)-dendron-(OH).sub.4):

[0219] 152 mg (0.03 mmol) of MeO-PEG.sub.5kDa-Lys(Boc)-di-yne (4) were dissolved in MeOH (1 mL), 2-mercaptoethanol (80 eq.) and DMPA (0.8 eq.) were added. The solution was purged with nitrogen for 15 minutes and then placed under UV light at 365 nm for hours. The crude mixture was then loaded on a MeOH based LH20 SEC column. The fractions that contained the product were unified and the MeOH was evaporated in vacuum to yield an oily residue. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1 mL) followed by addition of Hexane (3 mL). DCM and Hexane were evaporated to dryness and the off-white solid was dried under high vacuum. The product was obtained as an off-white solid (157 mg, quantitative yield).

[0220] .sup.1H-NMR (CDCl.sub.3): 7.42 (d, J=8.0 Hz, 1H, CHNHCOAr), 7.06 (d, J=2.8 Hz, 2H, ArH), 6.99 (m, 1H, CH.sub.2NHCOCH), 6.62 (d, J=2.6 Hz, 1H, ArH), 4.79 (m, 1H, NH-Boc), 4.58 (q, J=7.3 Hz, 1H, COCHNH), 4.33-4.12 (m, 4H, ArOCH.sub.2), 3.83-3.42 (m, PEG backbone), 3.36 (s, 3H, CH.sub.3O-PEG), 3.30 (q, J=6.1 Hz, 2H, CHS), 3.08 (m, 2H, Boc-NHCH.sub.2), 2.98 (dd, J=13.8, 6.4 Hz, 2H, CHCH.sub.2S), 2.89-2.77 (m, 6H, CHCH.sub.2S+CHSCH.sub.2), 2.74 (t, J=6.0 Hz, 4H, CHCH.sub.2SCH.sub.2), 2.61 (t, 6.9 Hz, 4H, CH.sub.2CH.sub.2SCH.sub.2), 1.83 (m, 4H, OCH.sub.2CH.sub.2CH.sub.2S+Boc-NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.51-1.39 (m, 13H, Boc-NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH-+Boc); .sup.13C-NMR (CDCl.sub.3) 172.2, 167.1, 167.0, 159.5, 156.3, 136.1, 106.4, 106.3, 79.4, 72.0, 70.6, 70.3, 70.2, 69.5, 62.1, 61.2, 59.1, 53.8, 45.4, 40.2, 38.9, 35.2, 35.1, 35.0, 34.9, 32.2, 31.5, 29.7, 28.5, 28.4, 23.0; FT-IR, v (cm.sup.1): 2885, 1591, 1467, 1452, 1359, 1342, 1278, 1241, 1146, 1101, 960, 948, 842; GPC: Mn=6.4 kDa, PDI=1.06. Expected Mn=5.9 kDa

[0221] Hybrid 6 (MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(Ph).sub.4):

[0222] 72 mg (0.01 mmol) of MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(OH).sub.4 (5) were dissolved in DCM (1 mL), Phenyl acetic acid (18 mg, 0.15 mmol, 3 eq. per OH) was added. The flask was cooled to 0 C. followed by the addition of DCC (30 mg, 0.15 mmol, 3 eq. per OH) and DMAP (0.1 eq. per OH) dissolved in DCM (1 mL). The reaction was heated to 30 C. and allowed to stir overnight. The crude mixture was filtered and the organic solution was evaporated to dryness. The crude mixture was dissolved in MeOH and loaded on a MeOH based LH20 SEC column. The fractions that contained the product were unified and the MeOH was evaporated in vacuum to yield an oily residue. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1 mL) followed by addition of Hexane (3 mL). DCM and Hexane were evaporated to dryness and the obtained solid was dried under high vacuum. The product was obtained as an off-white solid (68 mg, 87% yield).

[0223] .sup.1H-NMR (CDCl.sub.3): 7.30-7.15 (m, 20H, ArH), 7.03 (d, J=7.9 Hz, 1H, CHNHCOAr), 6.94 (d, J=2.2 Hz, 2H, ArH), 6.77 (s, 1H, CH.sub.2NHCOCH), 6.54 (t, J=2.2 Hz, 1H, ArH), 4.68(m, 1H, NH-Boc), 4.50 (q, J=7.6 Hz, 1H, COCHNH), 4.31-4.15 (m, 8H, CH.sub.2OCO), 4.15-4.00 (m, 4H, ArOCH.sub.2), 3.65-3.54 (m, PEG backbone), 3.33 (s, 3H, CH.sub.3O-PEG), 3.07 (m, 4H, Boc-NHCH.sub.2+CHS), 2.94-2.76 (m, 8H, CHCH.sub.2S+CHSCH.sub.2), 2.72 (t, J=6.8 Hz, 4H, CHCH.sub.2SC11.sub.2), 2.57 (t, 7.0 Hz, 4H, CH.sub.2SCH.sub.2), 2.05-1.59 (m, 4H, OCH.sub.2CH.sub.2CH.sub.2S-+Boc-NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.37 (m, 13H, Boc-NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH+Boc); *.sup.13C-NMR(CDCl.sub.3): 171.4, 166.8, 159.5, 156.1, 136.2, 133.79, 133.75, 129.3, 128.7, 127.23, 127.21, 106.3, 104.7, 81.6, 72.0, 69.7, 69.5, 64.1, 63.8, 59.1, 53.6, 45.5, 41.3, 38.7, 34.8, 32.1, 31.5, 30.3, 29.6, 28.5, 28.3, 22.9; FT-IR, v (cm.sup.1): 2884, 1736, 1599, 1466, 1454, 1359, 1341, 1279, 1240, 1147, 1102, 1060, 958, 948, 842; GPC: Mn=6.3 kDa, PDI=1.04. Expected Mn=6.3 kDa.

[0224] *Another digit was added to peaks that have very close chemical shift in order to distinguish between them.

##STR00021##

[0225] Hybrid 1F (MeO-PEG.sub.5kDa-Lys(Fluorescein)-dendron-(Ph).sub.4):

[0226] 60 mg (0.01 mmol) of MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(Ph).sub.4 (6) were dissolved in DCM (1 mL) and TFA was added (1 mL). After 30 minutes the solution was evaporated to dryness and dried in vacuum. The deprotected hybrid 6 (MeO-PEG.sub.5kDa-Lys(NH.sub.2)dendron-(Ph).sub.4) was re-dissolved in DMF (1 mL). Fluorescein isothiocyanate (2 eq.) was added followed by addition of DIPEA (20 eq.) and allowed to stir over night. The crude mixture was loaded on a DCM:MeOH 1:1 v/v based LH20 SEC column. The fractions that contained the product were unified and the DCM and MeOH were evaporated in vacuum to yield an oily residue. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1 mL) followed by addition of Hexane (3 mL). DCM and Hexane were evaporated to dryness and the obtained orange solid was dried under high vacuum. The product was obtained as an orange solid (50 mg, 80% yield).

[0227] .sup.1H NMR (CDCl.sub.3): 8.84 (s, 1H, ArOH), 8.10 (s, 1H, ArH), 7.91 (d, J=7.8 Hz, 1H, ArH), 7.35-7.11 (m, 21H, Ar-H), 7.06-6.93 (m, 4H, Ar-H +CHNHCOAr), 6.72 (s, 2H, ArH), 6.66-6.46 (m, 5H, ArH), 4.57 (m, 1H, COCHNH), 4.30-3.95 (m, 12H, CH.sub.2OCO+ArOCH.sub.2), 3.78-3.43(m, PEG backbone), 3.35 (s, CH.sub.3O-PEG), 3.15-3.03 (m, 2H, CHS), 2.95-2.43 (m, 12H, CHSCH.sub.2+CHCH.sub.2SCH.sub.2), 1.95-1.36 (m, 8H, SCNHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH+OCH.sub.2CH.sub.2CH.sub.2S); .sup.13C-NMR (CDCl.sub.3) 181.1, 171.70, 171.5, 169.4, 167.0, 159.7, 152.6, 140.8, 136.1, 133.6, 129.4, 128.7, 127.3, 112.8, 110.5, 106.5, 103.2, 70.7, 70.6, 69.5, 64.2, 63.8, 59.1, 45.6, 44.3, 41.3, 39.0, 35.0, 32.3, 31.6, 30.4, 29.7, 28.4, 23.2; FT-IR, v (cm.sup.1): 2884, 1737, 1592, 1466, 1454, 1359, 1341, 1279, 1240, 1146, 1101, 1060, 1030, 960, 948,842; GPC: Mn=11 kDa, PDI=1.09. Expected Mn=6.6KDa. MALDI-TOF MS: molecular ion centered at 6.6 kDa.

[0228] Hybrid 1C (MeO-PEG.sub.5kDa-Lys(Coumarin)-dendron-(Ph).sub.4)

[0229] 31 mg (4.9mol) of MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(Ph).sub.4 (6) were dissolved in DCM (1 mL) and TFA was added (1 mL). After 30 minutes the solution was evaporated to dryness and dried in vacuum. 7-(diethylamino)-3-carboxy coumarin (3 eq.) and HBTU (3 eq.) were dissolved in DCM:DMF 1:1 (1 mL) followed by addition of DIPEA (15 eq.) and allowed to stir for 1 hour. The solution was added to the deprotected hybrid 6 (MeO-PEG.sub.5kDa-Lys(NH.sub.2)-dendron-(Ph).sub.4) dissolved in DCM (1 mL). The reaction was stirred for 1 hours, complete coupling was confirmed by a negative Kaiser test. The crude mixture was loaded on a DCM:MeOH 1:1v/v based LH20 SEC column. The fractions that contained the product were unified and the DCM and MeOH were evaporated in vacuum, further purification was done by resolving the oily residue in DCM (1 mL) followed precipitation by the dropwise addition of Ether (50 mL). The yellow precipitate was filtered and washed twice with Ether and dried under high vacuum. The product was obtained as a yellow solid (29 mg, 91% yield).

[0230] .sup.1H-NMR (CDCl.sub.3): 8.89-8.77 (m, 1H, CH.sub.2CH.sub.2NHCO), 8.55 (s, 1H, ArH), 7.35 (d, J=9.0 Hz, 1H, ArH), 7.25 (m, 20H, ArH), 7.01 (m, 3H, ArH +CHNHCOAr) 6.77 (m, 1H, CH.sub.2NHCOCH), 6.64-6.52 (m, 2H, ArH), 6.46 (d, J=2.5 Hz, 1H, ArH), 4.53 (m, 1H, COCHNH), 4.35-3.96 (m, 12H, CH.sub.2OCO+AROCH.sub.2), 3.78-3.47(m, PEG backbone), 3.36 (s, 3H, CH.sub.3O-PEG), 3.11 (m, 2H, CHS), 2.85 (m, 8H, CHCH.sub.2S+CHSCH.sub.2), 2.74 (t, J=6.8 Hz, 4H, CHCH.sub.2SCH.sub.2), 2.60 (t, 7.0 Hz, 4H, CH.sub.2CH.sub.2SCH.sub.2), 2.04-1.88 (m, 1H, OCNHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.86-1.69 (m, 3H, OCH.sub.2CH.sub.2CH.sub.2S+OCNHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.66-1.57 (m, 2H, OCNHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.42 (m, 2H, OCNHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.27-1.09 (m, 6H, NCH.sub.2CH.sub.3); .sup.13C-NMR (CDCl.sub.3): 171.8, 171.5, 167.1, 163.5, 162.8, 159.7, 159.6, 157.7, 152.6, 148.1, 133.9, 133.8, 131.3, 129.4, 128.7, 127.3, 110.1, 106.5, 96.7, 72.1, 70.9, 70.7, 69.9, 69.6, 64.2, 63.9, 59.1, 45.6, 45.2, 41.3, 38.8, 35.0, 31.7, 31.6, 30.4, 29.8, 29.7, 29.5, 29.4, 28.4; FT-IR, v (cm.sup.1): 2884, 1736, 1694, 1614, 1584, 1535, 1513, 1466, 1454, 1359, 1341, 1279, 1240, 1146, 1102, 1060, 958, 842; GPC: Mn=6.2 kDa, PDI=1.05. Expected Mn=6.5 kDa. MALDI-TOF MS: molecular ion centered at 6.5 kDa.

##STR00022##

[0231] Hybrid 7F (MeO-PEG.sub.5kDa-Lvs(Fluorescein)-dendron-(OH).sub.4):

[0232] 35 mg (5.3 mol) of MeO-PEG.sub.5kDa-Lys(Fluorescein)-dendron-(Ph).sub.4 (1F) were dissolved in MeOH (1 mL) followed by the addition of a drop of water (cat.) and about 10 l of NaOH 1N (2 eq.) was added. The mixture was allowed to stir for 20 minutes at 40 C. Complete hydrolysis was confirmed by HPLC. The crude mixture was loaded on a DCM:MeOH 1:1 v/v based LH20 SEC column. The fractions that contained the product were unified and the DCM and MeOH were evaporated in vacuum. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1 mL) followed by addition of Hexane (3 mL). DCM and Hexane were evaporated to dryness and the obtained orange solid was dried under high vacuum. The product was obtained as an orange solid (30 mg, 92% yield).

[0233] .sup.1H NMR (MeOD): 8.03 (s, 1H, ArH), 7.70 (d, J=9.0 Hz, 1H, ArH), 7.11 (m, 3H, ArH), 6.86 (s, 1H, ArH), 6.76 (s, 1H, ArH), 6.68 (s, 2H, ArH), 6.60 (m, 2H, ArH), 4.54 (m, 1H, COCHNH), 4.42-4.08 (m, 4H, ArOCH.sub.2), 3.81-3.45 (m, PEG backbone), 3.35 (s, 3H, CH.sub.3O-PEG), 2.98 (m, 4H, CHCH.sub.2S), 2.84-2.76 (m, 4H, CHSCH.sub.2), 2.75-2.57 (m, 8H, CHCH.sub.2SCH.sub.2+CH.sub.2CH.sub.2SCH.sub.2), 2.10-1.68 (m, 6H, OCH.sub.2CH.sub.2CH.sub.2S+SCNHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.59 (m, 2H, SCNHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH); .sup.13C-NMR (MeOD): 176.4, 173.1, 168.2, 159.9, 136.0, 130.0, 129.6, 115.9, 106.4, 104.9, 102.4, 81.8, 77.6, 71.7, 69.8, 69.2, 63.4, 61.7, 61.3, 57.8, 54.2, 45.5, 38.9, 35.2, 35.0, 34.6, 33.8, 33.4, 30.7, 29.6, 29.4, 29.3, 29.1, 29.0, 28.9, 27.9, 26.8, 25.6, 23.3, 22.9, 22.4, 13.1; FT-IR, v (cm.sup.1): 2883, 1591, 1466, 1454, 1359, 1341, 1279, 1241, 1147, 1099, 1060, 961, 947, 841; GPC: Mn=10.5 kDa, PDI=1.18. Expected Mn=6.2 kDa. MALDI-TOF MS: molecular ion centered at 6.2 kDa.

[0234] Hybrid 7C (MeO-PEG.sub.5kDa -Lys(Coumarin)-dendron-(OH).sub.4):

[0235] 20 mg (3.1mol) of MeO-PEG.sub.5kDa-Lys(Coumarin)-dendron-(Ph).sub.4 (1C) were dissolved in MeOH (1 mL) and followed by addition of DMAP (30 eq.) and a drop of water (cat.). The mixture was allowed to stir for 7 days at 40 C. Complete hydrolysis was confirmed by HPLC. The crude mixture was loaded on a DCM:MeOH 1:1 v/v based LH20 SEC column. The fractions that contained the product were unified and the DCM and MeOH were evaporated in vacuum. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1 mL) followed by addition of Hexane (3 mL). DCM and Hexane were evaporated to dryness and the obtained orange solid was dried under high vacuum. The product was obtained as a yellow solid (16 mg, 86% yield).

[0236] .sup.1H-NMR (CDCl.sub.3): 8.89 (m, 1H, CH.sub.2CH.sub.2NHCO), 8.53 (s, 1H, ArH), 7.38 (d, J=9.0 Hz, 1H, ArH), 7.22-7.04 (m, 3H, CHNHCOAr+ArH), 6.98 (m, 1H, CH.sub.2NHCOCH), 6.75-6.57 (m, 2H, ArH), 6.47 (s, 1H, ArH), 4.56 (m, 1H, COCHNH), 4.38-4.14 (m, 4H, CH.sub.2OCO), 3.82-3.44 (m, PEG backbone), 3.37 (s, 7H, CH.sub.3O-PEG+CHS), 2.97 (dd, J=13.7, 6.2 Hz, 2H, CHCH.sub.2S), 2.92-2.70 (m, 10H, CHCH.sub.2SCH.sub.2+CHSCH.sub.2), 2.62 (t, 6.9 Hz, 4H, CH.sub.2CH.sub.2SCH.sub.2), 1.88-1.41 (m, 8 H OCH.sub.2CH.sub.2CH.sub.2S+OCNHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.37-1.21 (m, 28H, NCH.sub.2CH.sub.3 +Hexane); .sup.13C-NMR (CDCl.sub.3): 172.4, 162.8, 159.4, 157.7, 154.7, 152.7, 148.6, 136.4, 131.3, 130.1, 129.8, 127.5, 110.1, 108.4, 106.4, 99.5, 96.6, 89.0, 72.0, 70.2, 69.5, 63.7, 62.1, 61.1, 59.1, 57.1, 53.9, 52.2, 45.3, 45.2, 42.5, 38.8, 37.0, 36.3, 36.0, 35.1, 35.0, 33.8, 32.0, 31.5, 30.7, 29.8, 29.7, 29.4, 29.2, 28.4, 27.8, 27.3, 25.6, 23.6, 23.1, 22.8, 16.4, 14.2, 13.1, 12.53, 10.94; FT-IR, v (cm.sup.1): 2884, 1612, 1586, 1467, 1451, 1359, 1342, 1279, 1240, 1146, 1102, 1061, 961, 947, 842; GPC: Mn=5.6 kDa, PDI=1.04. Expected Mn=6.0 kDa. MALDI-TOF MS: molecular ion centered at 6.0 kDa.

##STR00023## ##STR00024##

[0237] Hybrid 9 MeO-PEG.sub.5kDa-L :

[0238] 150 mg (0.03mmol) of MeO-PEG.sub.5kDa-Lys(Boc)-di-yne (4) were dissolved in DCM (1 mL) and TFA was added (1 mL). After 30 minutes the unprotected product MeO-PEG.sub.5kDa-Lys(NH.sub.2)-di-yne was precipitated by the dropwise addition of Ether (50 mL). The white precipitate was filtered and washed twice with Ether and dried under high vacuum. The product was dissolved in MeOH (1 mL). 2-(Boc-amino)-ethanethiol (80 eq.) and DMPA (0.8 eq.) were added. The solution was purged with nitrogen for minutes and then placed under UV light at 365 nm for 2 hours. The crude mixture was loaded on a MeOH based LH20 SEC column. The fractions that contained the product were unified and the MeOH was evaporated in vacuum to yield an oily residue. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1 mL) followed by addition of Hexane (3 mL). DCM and Hexane were evaporated to dryness and the off-white solid was dried under high vacuum. The product was obtained as an off-white solid (162 mg, quantitative yield).

[0239] .sup.1H-NMR (CDCl.sub.3): 7.61 (s, 1H, NH.sub.2), 7.07 (s, 2H, ArH), 6.58 (d, J=2.2 Hz, 1H, ArH), 5.04-5.22 (m, 4H, NH-Boc), 4.62 (q, J=7.8 Hz, 1H, COCHNH), 4.07-4.29 (m, 4H, ArOCH.sub.2), 3.40-3.80 (m, PEG backbone), 3.35 (s, 3H, CH.sub.3-0-PEG), 3.31 (m, 8H, CH.sub.2NH-Boc), 3.17-3.08 (m, 2H, CHS), 2.90 (m, 6H, CHCH.sub.2S+NH.sub.2CH.sub.2), 2.75 (t, J=6.9 Hz, 4H, CHCH.sub.2SCH.sub.2), 2.70-2.60 (m, 6H, CHSCH.sub.2+CH.sub.2CH.sub.2SCH.sub.2), 2.57 (t, J=7.1 Hz, 2H, CH.sub.2CH.sub.2SCH.sub.2), 2.05-1.60 (m, 6H, OCH.sub.2CH.sub.2CH.sub.2S+NH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.40 (m, 40H, Boc+NH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH); .sup.13C-NMR (CDCl.sub.3): 171.5, 166.2, 159.0, 155.4, 135.7, 105.9, 104.7, 81.6, 78.9, 76.9, 76.8, 76.6, 76.3, 73.5, 71.4, 71.2, 70.0, 69.9, 69.5, 69.2, 68.9, 66.6, 63.2, 58.5, 53.1, 44.6, 40.0, 39.6, 38.5, 33.9, 32.4, 31.5, 30.9, 29.1, 27.9, 27.8, 22.1; FT-IR, v (cm.sup.1): 2884, 1666, 1593, 1466, 1454, 1360, 1341, 1279, 1240, 1147, 1100, 1060, 960, 948, 842; GPC: Mn=5.0 kDa, PDI=1.13. Expected Mn=6.2 kDa.

[0240] Hybrid 10C (MeO-PEG.sub.5kDa-Lys(Coumarin)-dendron-(NH-Boc).sub.4):

[0241] 7-(diethylamino)-3-carboxy coumarin.sup.[2] (3 eq.) and HBTU (3 eq.) were dissolved in DCM:DMF 1:1 (1 mL) followed by addition of DIPEA (20 eq.) and allowed to stir for 20 minutes. The solution was added to 80 mg (0.0 lmmol) of hybrid 9 (MeO-PEG.sub.5kDa-Lys(NH.sub.2)-dendron-(NH-Boc).sub.4) dissolved in DCM (1 mL). The reaction was stirred for 1 hours, complete coupling was confirmed by a negative Kaiser test. The crude mixture was loaded on a DCM:MeOH 1:1v/v based LH20 SEC column. The fractions that contained the product were unified and the DCM and MeOH were evaporated in vacuum, further purification was done by resolving the oily residue in DCM (1 mL) followed precipitation by the dropwise addition of Ether (50 mL). The yellow precipitate was filtered and washed twice with Ether and dried under high vacuum. The product was obtained as a yellow solid (80 mg, quantitative yield).

[0242] .sup.1H-NMR (CDCl.sub.3): 8.84 (t, J=5.7 Hz, 1H, CH.sub.2CH.sub.2NHCO), 8.53 (s, 1H, ArH), 7.36 (d, J=9.0 Hz, 1H, ArH), 7.05 (m, 2H, ArH +CHNHCOAr), 6.82 (m, 1H, CH.sub.2NHCOCH), 6.70-6.55 (m, 2H, ArH), 6.47 (d, J=2.3 Hz, 1H, ArH), 5.18 (m, 4H, NH-Boc), 4.52 (m, 1H, COCHNH), 4.28-4.05 (m, 4H, ArOCH.sub.2), 3.81-3.62 (m, PEG backbone), 3.35 (s, 3H, CH.sub.3O-PEG), 3.35-3.20 (m, 8H, CH.sub.2NH-Boc), 3.18-3.07 (m, 2H, CHS), 2.89 (m, 4H, CHCH.sub.2S), 2.75 (t, J=6.5 Hz, 4H, CHCH.sub.2SCH.sub.2), 2.71-2.60 (m, 6H, CHSCH.sub.2+CH.sub.2CH.sub.2SCH.sub.2), 2.57 (t, J=7.2 Hz, 2H, CH.sub.2CH.sub.2SCH.sub.2), 2.06-1.74 (m, 4H, OCH.sub.2CH.sub.2CH.sub.2S+NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH-), 1.72-1.56 (m, 2H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH-), 1.41 (s, 44H, Boc+NH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH-), 1.31-1.16 (m, 16H, -NCH.sub.2CH.sub.3 +Hexane); *.sup.13CNMR (CDCl3): 171.3, 166.6, 163.0, 162.2, 159.0, 157.1, 155.4, 152.1, 147.5, 135.8, 130.7, 109.5, 107.9, 105.8, 105.0, 96.0, 88.5, 81.6, 78.9, 78.5, 76.9, 76.8, 76.6, 76.3, 73.5, 71.4, 71.2, 70.0, 69.7, 69.3, 69.0, 66.6, 63.2, 58.5, 53.3, 51.7, 44.6, 40.95, 39.57, 38.2, 33.9, 32.5, 31.5, 31.0, 29.1, 28.8, 27.9, 27.8, 22.3, 12.0; FT-IR, v (cm.sup.1): 2883, 1693, 1614, 1585, 1564, 1547, 1530, 1513, 1466, 1454, 1359, 1341, 1279, 1240, 1146, 1100, 1060, 960, 948, 841; GPC: Mn=6.3 kDa, PDI=1.09. Expected Mn=6.4 kDa.

[0243] *Another digit was added to peaks that have very close chemical shift in order to distinguish between them.

[0244] Hybrid 8C (MeO-PEG.sub.5kDaLvs(Coumarin)-dendron-(NHCOPh).sub.4) :

[0245] 30 mg (4.6 mol) of MeO-PEG.sub.5kDa-Lys(Coumarin)-dendron-(NH-Boc).sub.4 (10C) were dissolved in DCM (1 mL) and TFA was added (1 mL). After 30 minutes the solution was evaporated to dryness and dried in vacuum to afford dendron (8C). The unprotected product MeO-PEG.sub.5kDa-Lys(Coumarin)-dendron-(NH.sub.2).sub.4 was re-dissolved in DMF (1.5 mL). 4-nitrophenyl 2-phenylacetate (12 eq.) and DIPEA (40 eq.) were added and the reaction was allowed to stir overnight. The crude mixture was loaded on a MeOH based LH20 SEC column. The fractions that contained the product were unified and the MeOH was evaporated in vacuum to yield an oily residue. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1 mL) followed by addition of Hexane (3 mL). DCM and Hexane were evaporated to dryness and the yellow solid was dried under high vacuum. The product was obtained as a yellow solid (30 mg, quantitative yield).

[0246] .sup.1H-NMR (CDCl.sub.3): 8.80 (t, J=5.6 Hz, 1 H CH.sub.2CH.sub.2NHCO), 8.53 (s, 1H, ArH), 7.69 (s, 1H, CHNHCOAr), 7.35 (d, J=9.0 Hz, 1H, ArH), 7.32-7.13 (m, 21H, ArH), 7.08 (q, J=2.3 Hz, 2H, ArH), 6.99 (m, 1H, CH.sub.2NHCOCH), 6.61 (d, 2.4 Hz, 1H, ArH), 6.55 (s, 1H, ArH), 6.45-6.35 (m, 4H, CH.sub.2NHCOCH.sub.2Ar +ArH), 4.63-4.42 (m, 1H, COCHNH), 4.25-3.98 (m, 4H, ArOCH.sub.2), 3.81-3.62 (m, PEG backbone), 3.35 (m, CH.sub.3O-PEG), 3.04 (t, J=5.9 Hz, 2H, CHS), 2.87-2.64 (m, 8H, CHCH.sub.2SCH.sub.2), 2.57 (m, 8H, CHSCH.sub.2+CH.sub.2CH.sub.2SCH.sub.2), 1.98 (m, 1H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.79 (m, 3H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH+OCH.sub.2CH.sub.2CH.sub.2S), 1.62 (q, J=6.7 Hz, 2H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.54-1.38 (m, 2H, NH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.20 (t, J=7.1 Hz, 10H, NCH.sub.2CH.sub.3 +Hexane); .sup.13C-NMR (CDCl.sub.3): 171.4, 170.8, 166.7, 162.9, 162.2, 158.9, 157.0, 152.0, 147.5, 134.4, 130.6, 128.9, 128.4, 126.7, 109.5, 107.9, 106.0, 104.9, 96.0, 76.9, 76.8, 76.6, 76.3, 71.4, 70.1, 69.6, 69.3, 69.0, 58.5, 53.5, 44.6, 44.2, 43.1, 38.9, 38.5, 33.7, 31.8, 31.0, 30.7, 29.1, 28.8, 27.8, 22.6, 12.0; FT-IR, v (cm.sup.1): 2884, 1693, 1643, 1614, 1583, 1563, 1547, 1536, 1513, 1466, 1454, 1415, 1359, 1341, 1279, 1240, 1146, 1100, 1060, 961, 948, 841; GPC: Mn=6.4 kDa, PDI=1.12. Expected Mn=6.5 kDa. MALDI-TOF MS: molecular ion centered at 6.5 kDa.

[0247] An additional hybrid 8F, containing a Fluorescein moiety instead of the Coumarin moiety was prepared according to the same method:

##STR00025##

[0248] Compounds 8c and 8F may be converted by cleavage of the amide bond to the corresponding hybrids 8(i)C and 8(i)F.

##STR00026##

Example 3

Critical Micelle Concentration (CMC) Measurements

[0249] Instrument Method:

[0250] Excitation: 550 nm

[0251] Emission intensity scan: 580-800 nm

[0252] Diluent solution preparation: Into 10 ml Phosphate buffer solution (pH 7.4), 4.5 L of Nile red stock solution (0.88 mg/m1 in Ethanol) were added and mixed to give a final concentration of 1.25 M.

[0253] CMC measurement for compound 1F, 1C and 8C: A 800 M solution was prepared in diluent and sonicated for 15 minutes. This solution was repeatedly diluted by a factor of 1.5 with diluent. 150 L of each solution were loaded onto a 96 wells plate. The fluorescence emission intensity was scanned for each well. Maximum emission intensity was plotted vs. hybrid concentration in order to determine the CMC. All measurements were repeated 3 times. The CMC for hybrids 1F, 1C and 8C were 61 M, 31 M and 61 M, respectively.

Example 4

Dynamic Light Scattering (DLS)

[0254] General Sample Preparation:

[0255] Hybrids 1F, 1C and 8C were dissolved in phosphate buffer (pH 7.4) to give a final concentration of 160 M. Solution was sonicated for 15 minutes and filtered through a 0.22 m nylon syringe filter. 700 L of this solution were accurately transferred into a polystyrene cuvette and a measurement was performed (t=0).

[0256] For micelle degradation in the presence of 27 nM PLE enzyme: 4.20 L of PLE enzyme stock solution (14.0 M in phosphate buffer pH 7.4) was added to 2.20 mL solution of hybrid 1F (160 M). Measurement was performed after 24 hours.

[0257] For micelle degradation in the presence of 270 nM PLE enzyme: 21.5 L of PLE enzyme stock solution (28.1 M in PBS buffer pH 7.4) were added to 2.20 mL solution of hybrid 1C (160 M). Measurement was performed after 24 hours.

[0258] For micelle degradation in the presence of 1 M PGA enzyme: 44.9 L of PGA enzyme stock solution (50 M in phosphate buffer pH 7.4) was added to 2.20 mL solution of hybrid 8C (160 M). Measurement was performed after 24 hours (FIG. 9).

Example 5

Absorbance Measurements

[0259] Instrument Method:

[0260] Spectra were recorded on TECAN Infinite M200Pro plate reader device.

[0261] Absorbance intensity scan: 350-700 nm

[0262] Diluted solution preparation of hybrids 1F, 1C, 7F and 7C:

[0263] A 160 M solution was prepared in diluent. Solution was sonicated for 15 minutes and then filtered through a 0.22 m nylon syringe filter. This solution was repeatedly diluted by a factor of 2 with diluent. 100 L of each solution were loaded onto a 96 wells plate. The absorbance intensity was scanned for each well. Maximum absorbance intensities were plotted vs. concentrations in order to show the similarity of the molar absorption coefficient.

[0264] For micelle degradation in the presence of 27 nM PLE enzyme:

[0265] 4.2 L of PLE enzyme stock solution (14.004 in phosphate buffer pH 7.4) was added to 2.20 mL solution of hybrid 1F (160 M). Measurement was performed after 24 hours. (FIG. 10).

[0266] For micelle degradation in the presence of 270 nM PLE enzyme:

[0267] 21.5 L of PLE enzyme stock solution (28.1 M in PBS buffer pH 7.4) were added to 2.20 mL solution of hybrid 1C (160 M). Measurement was performed after 24 hours. (FIG. 11).

[0268] For micelle degradation in the presence of 1 M PGA enzyme:

[0269] 44.9 L of PGA enzyme stock solution (50 M in phosphate buffer pH 7.4) was added to 2.20 mL solution of hybrid 8C (160 M). Measurement was performed after 48 hours. (FIG. 12).

Example 6

Fluorescence Measurements

[0270] Instrument Method of Hybrids 1F and 7F:

[0271] Spectra were recorded on TECAN Infinite M200Pro plate reader device:

[0272] Excitation: 470 nm

[0273] Emission intensity scan: 490-700 nm

[0274] Gain: 70

[0275] Instrument Method of Hybrids 1C and 7C:

[0276] Fluorescence measurements were performed using an Agilent Technologies Cary Eclipse Fluorescence Spectrophotometer:

[0277] Excitation: 420 nm

[0278] Emission scan: 440-700 nm

[0279] Excitation and Emission slits width: 5 nm and 10 nm for the lower concentrations overlay.

[0280] Scan rate: 600 nm/min and 700 nm/min for the lower concentrations overlay.

[0281] Excitation measurements were performed using Fluorolog 3.22 fluoremeter (Horiba) Measurements were taken in front-face mode using a 2mm cuvette.

[0282] Instrument Method of Hybrid 8C:

[0283] Spectra were recorded on TECAN Infinite M200Pro plate reader device:

[0284] Excitation: 420 nm

[0285] Emission intensity scan: 440-700 nm

[0286] Gain: 90

[0287] Diluted solution preparation of hybrids 1F,1C, and 8C:

[0288] A 160 M solution was prepared in diluent. Solution was sonicated for 15 minutes and then filtered through a 0.22 m nylon syringe filter. This solution was repeatedly diluted by a factor of 2 with diluent. 100 L of each solution were loaded onto a 96 wells plate. The emission intensity was scanned for each well.

[0289] For micelle degradation in the presence of 27 nM PLE enzyme:

[0290] 4.2 L of PLE enzyme stock solution (14.0 M in phosphate buffer pH 7.4) was added to 2.20 mL solution of hybrid 1F (160 M). Measurement was performed after 24 hours.

[0291] For micelle degradation in the presence of 270 nM PLE enzyme:

[0292] 21.5 L of PLE enzyme stock solution (28.1 M in phosphate buffer pH 7.4) were added to 2.20 mL solution of hybrid 1C (160 M). Measurement was performed after 24 hours.

[0293] For micelle degradation in the presence of l.sub.iuM PGA enzyme:

[0294] 44.9 L of PGA enzyme stock solution (50 M in phosphate buffer pH 7.4) was added to 2200 L solution of hybrid 8C (160 M). Measurement was performed after 48 hours.

[0295] FIG. 13 depicts the fluorescence emission intensity spectra overlay of hybrid 1F (160 M), hybrid 7F (160 M) and of hybrid 1F (160 M) after addition of 27 nM PLE enzyme (after 24 hours).

[0296] FIG. 14 depicts the fluorescence emission intensity spectra overlay of hybrid 1C (160 M), hybrid 7C (160 M) and of hybrid 1C (160 M) after addition of 270 nM PLE enzyme (after 24 hours).

[0297] FIG. 15 depicts the fluorescence emission intensity spectra overlay of hybrid 8C (160 M) and of hybrid 8C (160 M) after addition of 1 M PGA enzyme (after 48 hours).

[0298] Fluorescence monitoring of enzymatic degradation:

[0299] Instrumentation:

[0300] Monitoring of micelle disassembly rate by enzymes was performed using an Agilent Technologies Cary Eclipse Fluorescence Spectrophotometer.

[0301] Instrument Method Hybrid 1F:

[0302] Excitation: 488 nm

[0303] Emission scan: 495-700 nm

[0304] Excitation and Emission slits width: 5 nm

[0305] Scan rate: 480 nm/min

[0306] Instrument Method Hybrid 1C and 8C:

[0307] Excitation: 420 nm

[0308] Emission scan: 440-700 nm

[0309] Excitation and Emission slits width: 5 nm

[0310] Scan rate: 600 nm/min

[0311] Sample Preparation and Measurement:

[0312] Hybrid 1F was dissolved in phosphate buffer (pH 7.4) to give a concentration of 160 M. Solution was sonicated for 15 minutes and then filtered through a 0.22 m nylon syringe filter. 700 L were accurately transferred to a quartz cuvette. A fluorescence emission scan was performed (t=0). 4.2 L of PLE enzyme stock solution (14.0 M in phosphate buffer pH 7.4) were added to 2.20 mL solution of hybrid 1F (160 M) and mixed for 10 seconds (vortex mixer) to give final PLE concentration of 27 nM. 700 L were accurately transferred to a quartz cuvette. Repeating fluorescence scans were performed every 15 minutes for 16 hours. All measurements were repeated 3 times.

[0313] Hybrid 1C was dissolved in phosphate buffer (pH 7.4) to give a concentration of 160 M. Solution was sonicated for 15 minutes and then filtered through a 0.22 m nylon syringe filter. 700 L were accurately transferred to a quartz cuvette. A fluorescence emission scan was performed (t=0). 21.5 L of PLE enzyme stock solution (28.1 M in phosphate buffer pH 7.4) were added to 2.20 mL solution of hybrid 1C (160 M) and mixed for 10 seconds (vortex mixer) to give final PLE concentration of 270 nM. 700 L were accurately transferred to a quartz cuvette. Repeating fluorescence scans were performed every 15 minutes for 15 hours. All measurements were repeated 3 times.

[0314] Hybrid 8C was dissolved in phosphate buffer (pH 7.4) to give a concentration of 160 M. Solution was sonicated for 15 minutes and then filtered through a 0.22 m nylon syringe filter. 700 L were accurately transferred to a quartz cuvette. A fluorescence emission scan was performed (t=0). 44.9 L of PGA enzyme stock solution (50 M in phosphate buffer pH 7.4) were added to 2.20 mL solution of hybrid 8C (160 M) and mixed for 10 seconds (vortex mixer) to give final PGA concentration of 1 M. 700 L were accurately transferred to a quartz cuvette. Repeating fluorescence scans were performed every 15 minutes for 13 hours. All measurements were repeated 3 times.

Example 7

HPLC Monitoring of Enzymatic Degradation

[0315] Instrument Method:

[0316] Column: Phenomenex, Aeris WIDEPORE, C4, 1504.6 mm, 3.6 m.

[0317] Column Temperature: 30 C.

[0318] Mobile Phase: Solution A: 0.1% HClO.sub.4 in H.sub.2O:Acetonitrile 95:5 V/V. [0319] Solution B: 0.1% HClO.sub.4 in H.sub.2O:Acetonitrile 5:95 V/V. [0320] Solution C: THF.

[0321] Gradient Program:

TABLE-US-00001 Time % Sol. % Sol. % Sol. [min] A B C 0.0 95 0 5 1.0 95 0 5 20.0 0 95 5 23.0 0 95 5 23.1 95 0 5 30.0 95 0 5

[0322] Injection volume: 30 L.

[0323] Detector: UV at 295 nm, 2 Hz detection rate.

[0324] Needle Wash: MeOH.

[0325] Seal wash solution: H.sub.2O:MeOH 90:10 V/V.

[0326] Diluent: phosphate buffer pH 7.4. 5

[0327] General Sample Preparation:

[0328] All hybrids were dissolved in phosphate buffer to give a concentration of 160 M. Solution was sonicated for 15 minutes and then filtered through a 0.22 m nylon syringe filter.

[0329] For enzymatic cleavage of hybrid 1F in the presence of 27 nM PLE enzyme:

[0330] 300 L of the 160 M 1F solution were transferred to a proper vial. 30 L were injected to the HPLC as t=0 injection. 2.20 mL of the 160 M 1F solution were transferred to a proper vial. 4.41 L of PLE (14.0 M) stock solution in phosphate buffer (pH 7.4) were added and mixed manually. 1.50 mL were transferred to a proper vial (700 L were accurately transferred to a quarts cuvette for monitoring enzymatic degradation of micelles by fluorescence measurements). Enzymatic degradation was monitored by repeating 30 L injections from the same vial over 16 hours. All measurements were repeated 3 times.

[0331] For enzymatic cleavage of hybrids 1C in the presence of 270 nM PLE enzyme:

[0332] 300 L of the 160 M 1C solution were transferred to a proper vial. 30 L were injected to the HPLC as t=0 injection. 2.20 mL of the 160 M 1C solution were transferred to a proper vial. 21.5 L of PLE (28.1 M) stock solution in phosphate buffer (pH 7.4) were added and mixed manually. 1.50 mL were transferred to a proper vial (700 L were accurately transferred to a quarts cuvette for monitoring enzymatic degradation of micelles by fluorescence measurements). Enzymatic degradation was monitored by repeating 30 L injections from the same vial over 15 hours. All measurements were repeated 3 times.

[0333] For enzymatic cleavage of hybrids 8C in the presence of l.sub.iuM PGA enzyme:

[0334] 300 L of the 160 M 8C solution were transferred to a proper vial. 30 L were injected to the HPLC as t=0 injection. 2.2 omL of the 160 M 8C solution were transferred to a proper vial. 44.9 L of PGA (50 M) stock solution in phosphate buffer (pH 7.4) were added and mixed manually. 1.5 omL were transferred to a proper vial (700 L were accurately transferred to a quarts cuvette for monitoring enzymatic degradation of micelles by fluorescence measurements). Enzymatic degradation was monitored by repeating 30 L injections from the same vial over 13 hours. The overlay shows accumulation of partially cleaved hybrids with the enzymatic degradation. All measurements were repeated 3 times.

[0335] FIG. 16 depicts the HPLC monitoring of micelle degradation in presence of 27 nM PLE enzyme for hybrid 1F over time.

[0336] FIG. 17 depicts the HPLC monitoring of micelle degradation in presence of 270 nM PLE enzyme for hybrid 1C over time.

[0337] FIG. 18 depicts the HPLC monitoring of micelle degradation in presence of 1 M PGA enzyme for hybrid 8C over time. The overlay shows accumulation of partially cleaved hybrids.

Example 8

Preparation of Additional MeO-PEG.SUB.5kDa.-Lys(Labeled)-dendron-(Aliphatic).SUB.4 .Hybrids Containing Fluorescent Probes

[0338] Additional hybrids were synthesized from MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(OH).sub.4.sup.1 (5) as generally described in Scheme 8. Esterification with an aliphatic carboxylic acid yielded hybrid (11) with four enzymatically cleavable end-groups. The BOC group was removed with trifluoroacetic acid, followed by conjugation of the dye to the protected amine to yield amphiphilic hybrids. Embodiments of hybrids with specific labeling groups are depicted in Schemes 9-12 hereinbelow.

##STR00027## ##STR00028##

[0339] In some specific embodiments of Scheme 8, m=6 (i.e., the carboxylic acid is octanoic acid), and the product is hybrid 12a. In other specific embodiments of Scheme 8, m=9 (i.e., the carboxylic acid is undecanoic acid), and the product is hybrid 12b.

[0340] General Procedure for MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(Aliphatic).sub.4:

[0341] MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(OH).sub.4.sup.1 (5) was dissolved in DCM (1 mL, per 0.1 g). Aliphatic acid (20 eq.) were added. The flask was cooled to 0 C. followed by the addition of DCC (20 eq.) and DMAP (0.1 eq.) dissolved in DCM (1 mL). The reaction was stirred for 1 hour at room temperature. The crude mixture was filtered and the organic solution was evaporated to dryness. The residue was re-dissolved in DCM (5 mL per 1 g) and the product was precipitated by the drop wise addition of Ether (50 mL per 1 g). The precipitate underwent centrifugation and was separated from the organic solvent. The precipitate was collected and residual of solvents were evaporated under vacuum. The residue was dissolved in MeOH and loaded on a MeOH based LH20 SEC column. The fractions that contained the product were unified and the MeOH was evaporated in vacuum to yield an oily residue. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1 mL) followed by addition of Hexane (3 mL). DCM and Hexane were evaporated to dryness and the obtained solid was dried under high vacuum.

[0342] Hybrid 11a MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(Oct).sub.4: 245 mg (0.042 mmol) of MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(OH).sub.4 (5) and Octanoic acid (0.830 mmol, 20 eq.) were reacted according to the general procedure and the product was obtained as an off-whit solid (240 mg, 90% yield).

[0343] .sup.1H NMR (400 MHz, Chloroform-d): 7.10-6.86 (m, 3H, CHNHCOAr+ArH), 6.73-6.49 (m, 2H, CH.sub.2NHCOCH+ArH), 4.66 (m, 1H, NH-Boc), 4.53 (q, J=7.5 Hz, 1H, COCHNH), 4.31-4.06 (m, 12H, CH.sub.2OCO+ArOCH.sub.2), 3.88-3.39 (m, 514H, PEG backbone), 3.34 (s, 3H, CH.sub.3O-PEG), 3.16 (q, J=6.1 Hz, 2H, CHS), 3.06 (m, 2H, Boc-NHCH.sub.2), 3.01-2.82 (m, 8H, CHCH.sub.2S+CHSCH.sub.2), 2.81-2.71 (m, 4H, CHCH.sub.2SCH.sub.2), 2.59 (dt, J=22.0, 7.0 Hz, 3H, CH.sub.2SCH.sub.2), 2.27 (td, J=7.5, 1.9 Hz, 8H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3), 2.02-1.65 (m, 4H, OCH.sub.2CH.sub.2CH.sub.2S+Boc-NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.59 (m, 10H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3 +Boc-NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.37 (m, 11H, Boc-NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH+Boc), 1.31-1.11 (m, 34H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3), 0.91-0.73 (m, 12H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3). .sup.13C NMR (101 MHz, CDC13): 173.1, 171.0, 166.3, 159.1, 155.7, 149.1, 135.6, 105.8, 104.3, 71.5, 70.5, 69.8, 69.7, 69.3, 68.9, 62.9, 62.6, 58.5, 53.0, 45.1, 39.6, 38.2, 34.4, 33.7, 31.6, 31.2, 31.1, 29.9, 29.2, 29.1, 28.6, 28.5, 28.4, 27.9, 27.9, 24.4, 22.4, 22.1, 13.6.

[0344] Hybrid 11b MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(Und).sub.4: 267 mg (0.045mmol) of MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(OH).sub.4 (5) and Undecanoic acid (0.910 mmol, 20 eq.) were reacted according to the general procedure and the product was obtained as an off-whit solid (268 mg, 90% yield).

[0345] .sup.1H NMR (400 MHz, Chloroform-d): 7.06-6.90 (m, 3H, CHNHCOAr+ArH), 6.72-6.50 (m, 2H, CH.sub.2NHCOCH+ArH), 4.66 (m, 1H, NH-Boc), 4.53 (q, J=7.6 Hz, 1H, COCHNH-), 4.33-4.06 (m, 12H, CH.sub.2OCO+ArOCH.sub.2), 3.77-3.44 (m, 498H, PEG backbone), 3.34 (s, 3H, CH.sub.3O-PEG), 3.17 (q, J=5.9, 2H, CHS), 3.06 (q, J=6.7 Hz, 2H, Boc-NHCH.sub.2), 3.03-2.80 (m, 8H, CHCH.sub.2S+CHSCH.sub.2), 2.82-2.71 (m, 4H, CHCH.sub.2SCH.sub.2), 2.59 (dt, J=21.9, 7.0 Hz, 4H, CH.sub.2SCH.sub.2), 2.27 (td, J=7.5, 2.0 Hz, 8H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.7CH.sub.3), 2.03-1.65 (m, 4H, OCH.sub.2CH.sub.2CH.sub.2S+Boc-NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH-), 1.64-1.44 (m, 10H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.7CH.sub.3 +Boc-NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.38 (m, 11H, Boc-NH-CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH+Boc), 1.22 (m, 59H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.7CH.sub.3), 0.84 (t, J=6.9 Hz, 12H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.7CH.sub.3). .sup.13C NMR (101 MHz, CDCl.sub.3): 173.1, 171.0, 166.3, 159.1, 155.6, 135.6, 105.8, 104.3, 71.4, 70.3, 69.87, 69.7, 69.3, 68.9, 62.9, 62.6, 58.5, 53.0, 45.0, 39.6, 38.2, 34.4, 33.7, 31.6, 31.4, 31.1, 31.0, 29.9, 29.2, 29.1, 29.0, 28.9, 28.8, 28.7, 28.6, 28.0, 27.8, 24.4, 22.4, 22.2, 13.6.

[0346] General procedure for MeO-PEG.sub.5kDa-Lys(Labeled)-dendron-(Aliphatic).sub.4:

[0347] MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(Aliphatic).sub.4 (11a or 11b) was dissolved in DCM (1 mL) and TFA was added (1 mL). After 30 minutes the solution was evaporated to dryness and dried in vacuum. The labeling moiety with a carboxylic acid functional group (2 eq.) and HBTU (2 eq.) were dissolved in DCM: DMF 1:1 (1 mL) followed by addition of DIPEA (20 eq.). The solution was added to the deprotected hybrid (MeO-PEG.sub.5kDa-Lys(NH.sub.2)-dendron-(Aliphatic).sub.4) dissolved in DCM (1 mL, per 0.1 g). The reaction was stirred for 1 hour in room temperature. The crude mixture was concentrated under vacuum and loaded on a MeOH based LH20 SEC column. The fractions that contained the product were unified and the DCM and MeOH were evaporated in vacuum to yield an oily residue. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1 mL) followed by addition of Hexane (3 mL). DCM and Hexane were evaporated to dryness and the obtained solid was dried under high vacuum.

##STR00029##

[0348] Hybrid 13a MeO-PEG.sub.5kDa-Lys(Coumarin)-dendron-(Oct).sub.4: 104 mg (0.016 mmol) of MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(04. (11a) were deprotected to yield MeO-PEG.sub.5kDa-Lys(NH.sub.2)-dendron-(Oct).sub.4 that were reacted with 7-(diethylamino)-3-carboxy coumarin (0.032 mmol, 2 eq.) according to the general procedure and the product was obtained as a yellow solid (98 mg, 92% yield).

[0349] .sup.1H NMR (400 MHz, Chloroform-d) 8.82 (m, 1H, CH.sub.2CH.sub.2NHO), 8.52 (s, 1H, ArH), 7.35 (d, J=9.0 Hz, 1H, ArH), 7.19 (d, J=6.8 Hz, 1H, CHNHCOAr), 7.02 (m, 2H, ArH), 6.78 (m, 1H, CH.sub.2NHCOCH), 6.66-6.55 (m, 2H, ArH), 6.44 (s, 1H, ArH), 4.51 (m, 1H, COCHNH), 4.35-4.06 (m, 12H, CH.sub.2OCO+ArOCH.sub.2), 3.76-3.39 (m, 535H, PEG backbone), 3.33 (s, 3H, CH.sub.3O-PEG), 3.16 (t, J=5.9 Hz, 2H, CHS), 3.03-2.70 (m, 12H, CHCH.sub.2SCH.sub.2+CHSCH.sub.2), 2.58 (dt, J=25.5, 6.9 Hz, 3H, CH.sub.2SCH.sub.2), 2.26 (t, J=7.7 Hz, 8H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3), 2.04-1.74 (m, 4H, OCH.sub.2CH.sub.2CH.sub.2S+NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.68-1.37 (m, 12H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3 +NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.22 (q, J=7.0, 6.4 Hz, 44H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3 +-NCH.sub.2CH.sub.3), 0.90-0.74 (m, 13H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3). .sup.13C NMR (101 MHz, CDCl.sub.3): 173.7, 171.7, 167.1, 163.5, 162.8, 159.5, 157.6, 152.6, 148.2, 136.3, 110.2, 108.4, 96.6, 71.8, 70.6, 69.5, 63.4, 63.1, 45.5, 45.1, 34.3, 31.7, 30.4, 29.6, 29.2, 29.0, 25.0, 22.7, 14.2, 12.6.

[0350] Hybrid 13b MeO-PEG.sub.5kDa-Lys(Coumarin)-dendron-(Und).sub.4: 81 mg (0.012 mmol) of MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(Und).sub.4 (11a) were deprotected to yield MeO-PEG.sub.5kDa-Lys(NH.sub.2)-dendron-(Und).sub.4 that were reacted with 7-(diethylamino)-3-carboxy coumarin (0.024 mmol, 2 eq.) according to the general procedure and the product was obtained as a yellow solid (81 mg, quantitative yield).

[0351] .sup.1H NMR (400 MHz, Chloroform-d): 8.84 (t, J=5.9 Hz, 1H, CH.sub.2CH.sub.2NHCO), 8.55 (s, 1H, ArH), 7.37 (dt, J=9.0, 1.2 Hz, 1H, ArH), 7.18 (d, J=7.8 Hz, 1H, CHNHCOAr), 7.02 (d, J=2.3 Hz, 2H, ArH), 6.78 (t, J=5.7 Hz, 1H, CH.sub.2NHCOCH), 6.69-6.52 (m, 2H, ArH), 6.47 (d, J=2.3 Hz, 1H, ArH), 4.54 (t, J=6.5 Hz, 1H, COCHNH), 4.35-4.08 (m, 11H, CH.sub.2OCO+ArOCH.sub.2), 3.79-3.44 (m, 522H, PEG backbone), 3.35 (s, 3H, CH.sub.3O-PEG), 3.23-3.13 (m, 2H, CHS), 3.03-2.81 (m, 8H, CHCH.sub.2S+CHSCH.sub.2), 2.80-2.72 (m, 4H, CHCH.sub.2SCH.sub.2), 2.61 (dt, J=26.0, 7.0 Hz, 4H, CH.sub.2SCH.sub.2), 2.37-2.24 (m, 9H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.7CH.sub.3), 2.06-1.76 (m, 4H, OCH.sub.2CH.sub.2CH.sub.2S+NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.70-1.42 (m, 11H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.7CH.sub.3 +NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.23 (q, J=4.3, 3.3 Hz, 63H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.7CH.sub.3 +-NCH.sub.2CH.sub.3), 0.85 (t, J=6.9 Hz, 12H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.7CH.sub.3)..sup.13C NMR (101 MHz, CDCl.sub.3): 173.1, 171.7, 171.1, 166.5, 162.9, 162.2, 159.0, 157.1, 152.0, 147.5, 135.8, 130.6, 129.2, 109.5, 107.9, 105.9, 104.5, 96.1, 81.6, 71.5, 70.1, 69.3, 69.0, 68.8, 62.9, 62.6, 58.5, 53.2, 50.5, 49.1, 45.1, 44.6, 38.2, 34.4, 33.7, 31.4, 31.1, 29.9, 29.2, 29.1, 29.0, 28.9, 28.7, 27.8, 24.4, 22.6, 22.2, 13.6, 12.0, 0.5.

##STR00030##

[0352] Hybrid 14a MeO-PEG.sub.5kDa-Lys(Cy5)-dendron-(Oct).sub.4: 150 mg (0.024 mmol) of MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(Oct).sub.4 (11a) were deprotected to yield MeO-PEG.sub.5kDa-Lys(NH).sub.2)-dendron-(Oct).sub.4 that were react1ed with carboxylic acid Cy5 acid (0.048 mmol, 2 eq.) according to the general procedure and the product was obtained as a blue solid (160 mg, quantitative yield).

[0353] .sup.1H NMR (400 MHz, Chloroform-d) 8.59 (m, 1H, CH.sub.2CONHCH.sub.2), 8.05 (m, 1H, CHNHCOAr+CH.sub.2NHCOCH), 7.76 (dt, J=26.3, 13.0 Hz, 2H, ArH), 7.41-7.16 (m, 8H, ArH), 7.14 (d, J=2.3 Hz, 2H, ArH), 7.09-6.96 (m, 1H, CCHCH), 6.65-6.50 (m, 2H, CHCHCHCHCH), 6.44 (d, J=13.7 Hz, 1H, CCHCH), 6.07 (d, J=13.4 Hz, 1H, CCHCH), 4.72-4.51 (m, 1H, COCHNH), 4.38 (t, J=7.0 Hz, 1H, NCH.sub.2CH.sub.2CO), 4.30-4.07 (m, 12H, CH.sub.2OCO+ArOCH.sub.2), 3.79-3.44 (m, 522H, PEG backbone), 3.35 (s, 4H, CH.sub.3O-PEG), 3.17 (m, 3H, CHS), 3.03-2.69 (m, 14H, CHCH.sub.2SCH.sub.2+CHSCH.sub.2), 2.58 (dt, J=23.2, 7.6 Hz, 3H, CH.sub.2SCH.sub.2), 2.06-1.73 (m, 4 H OCH.sub.2CH.sub.2CH.sub.2S+NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.66 (d, J=3.9 Hz, 9H, CCH.sub.3), 1.58 (t, J=7.2 Hz, 12H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3+NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.34-1.15 (m, 39H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3), 0.91-0.78 (m, 14H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3). .sup.13C NMR (101 MHz, CDCl.sub.3): 173.4, 173.0, 172.1, 171.7, 168.9, 166.1, 158.9, 153.0, 151.6, 142.3, 141.1, 140.0, 136.1, 128.4, 128.2, 125.7, 125.2, 124.3, 121.5, 121.5, 111.0, 109.6, 105.9, 104.4, 102.4, 71.4, 70.6, 70.3, 70.0, 69.6, 69.3, 69.1, 68.9, 62.8, 62.6, 58.5, 53.9, 49.0, 45.1, 41.3, 38.8, 38.1, 34.4, 33.7, 31.1, 31.0, 30.4, 29.8, 29.1, 28.5, 28.4, 27.8, 27.6, 27.5, 27.4, 27.3, 24.4, 22.5, 22.1, 22.0, 13.5.

[0354] A corresponding compound of formula 14b comprising an ester based on undecanoic acid was prepared following the procedure for compound 14a.

##STR00031##

[0355] Hybrid 15a MeO-PEG.sub.5kDa-Lys(Azo)-dendron-(Oct).sub.4: 130 mg (0.020 mmol) of MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(Oct).sub.4 (11a) were deprotected to yield MeO-PEG.sub.5kDa-Lys(NH).sub.2)-dendron-(Oct).sub.4 that were reacted with azo acid.sup.2 (0.04 mmol, 2 eq.) according to the general procedure and the product was obtained as an orange solid (131 mg, quantitative yield).

[0356] .sup.1H NMR (400 MHz, Chloroform-d) 7.98-7.74 (m, 4H, ArH), 7.49-7.31 (m, 3H, ArH), 7.11 (d, J=7.0 Hz, 1H, CHNHCOAr), 7.03-6.89 (m, 4H, ArH), 6.86-6.62 (m, 2H, CH.sub.2NHCOCH), 6.57 (t, J=2.2 Hz, 1H, ArH), 4.56-4.31 (m, 3H, COCHNH+OCH.sub.2CO), 4.26-4.09 (m, 12H, CH.sub.2OCO+ArOCH.sub.2), 3.8.0-3.38 (m, 509H, PEG backbone), 3.31 (s, 6H, CH.sub.3O-PEG), 3.21-3.07 (m, 3H, CHS), 3.00-2.63 (m, 13H, CHCH.sub.2SCH.sub.2+CHSCH.sub.2), 2.56 (dt, J=22.5, 7.0 Hz, 9H, CH.sub.2SCH.sub.2), 2.32-2.13 (m, 8H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3), 2.02-1.69 (m, 4H, OCH.sub.2CH.sub.2CH.sub.2S+NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.53 (dd, J=11.0, 4.0 Hz, 10H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3 +NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.43-1.31 (m, 3H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.31-1.08 (m, 35H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3), 0.80 (t, J=6.9 Hz, 12H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3). .sup.13C NMR (101 MHz, CDCl.sub.3): 173.0, 171.0, 167.5, 166.4, 159.1, 158.7, 152.1, 147.3, 135.5, 130.2, 128.5, 128.4, 124.3, 122.9, 122.2, 119.4, 114.5, 114.1, 105.8, 104.4, 71.4, 70.3, 69.8, 69.7, 69.3, 68.9, 66.9, 63.2, 62.9, 62.5, 58.5, 53.0, 45.0, 38.2, 37.7, 34.3, 33.7, 31.1, 31.0, 30.8, 29.9, 29.1, 28.6, 28.5, 28.4, 27.8, 24.4, 22.1, 13.6.

[0357] A corresponding compound of formula 15b comprising an ester based on undecanoic acid was prepared following the procedure for compound 15a.

##STR00032##

[0358] Hybrid 16a MeO-PEG.sub.5kDa-Lys(Spiropyrin)-dendron-(Oct).sub.4: 130 mg (0.020 mmol) of MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(Oct).sub.4 (11a) were deprotected to yield MeO-PEG.sub.5kDa-Lys(NH.sub.2)-dendron-(Oct).sub.4 that were reacted with azo acid (0.04 mmol, 2 eq.) according to the general procedure and the product was obtained as an orange solid (131 mg, quantitative yield).

[0359] .sup.1H NMR (400 MHz, Chloroform-d) 8.03-7.86 (m, 2H, ArH), 7.20-7.10 (m, 1H, ArH), 7.04 (d, J=7.1 Hz, 2H, ArH), 6.98 (d, J=2.3 Hz, 2H, ArH), 6.94-6.78 (m, 2H, ArH+CHCH), 6.69 (d, J=7.9 Hz, 1H, ArH), 6.63-6.57 (m, 2H, ArH+CH.sub.2NHCOCH), 5.80 (dd, J=10.5, 2.3 Hz, 1H, CHCH), 4.49 (d, J=7.5 Hz, 1H, COCHNH), 4.32-4.11 (m, 14H, CH.sub.2OCO+ArOCH.sub.2), 3.81-3.39 (m, 563H, PEG backbone), 3.35 (s, 5H, CH.sub.3O-PEG), 3.16 (q, J=6.0 Hz, 8H, CHS+NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 3.03-2.71 (m, 18H, CHCH.sub.2SCH.sub.2+CHSCH.sub.2), 2.59 (dt, J=22.6, 7.0 Hz, 5H, CH.sub.2SCH.sub.2), 2.52-2.42 (m, 2H, NCH.sub.2CH.sub.2CO), 2.39-2.16 (m, 19H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3), 2.04-1.64 (m, 4H, OCH.sub.2CH.sub.2CH.sub.2S+NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.57 (d, J=6.9 Hz, 8H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3), 1.44 (d, J=6.7 Hz, 2H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.35-1.14 (m, 40H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3+NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH+CHCH.sub.3), 1.10 (s, 3H, CHCH.sub.3), 0.93-0.75 (m, 12H, OCOCH.sub.2CH.sub.2(CH.sub.2).sub.4CH.sub.3). .sup.13C NMR (101 MHz, CDCl.sub.3): 173.1, 171.0, 166.4, 159.1, 135.5, 127.9, 127.3, 125.3, 122.3, 121.3, 119.2, 114.9, 106.3, 105.8, 71.5, 70.4, 70.1, 69.7, 69.3, 68.9, 62.9, 62.6, 58.5, 52.4, 45.1, 39.5, 38.5, 38.2, 35.2, 34.4, 33.7, 31.2, 31.1, 29.9, 29.2, 28.6, 28.4, 27.8, 25.3, 24.4, 22.1, 19.3, 13.6.

[0360] A corresponding compound of formula 16b comprising an ester based on undecanoic acid was prepared following the procedure for compound 16a.

Example 9

Preparation of MeO-PEG.SUB.5kDa.-Lys(3,5-dihydroxybenz)-dendron-(Ph).SUB.4 .aromatic Hybrid 18

[0361] ##STR00033##

[0362] 130 mg (0.020 mmol) of MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(Ph).sub.4.sup.1,3 (6) were dissolved in DCM (1 mL) and TFA was added (1 mL). After 30 minutes the solution was evaporated to dryness and dried in vacuum. Compound 17 (0.100 mmol, 5 eq.) and HBTU (0.100 mmol, 5 eq.) were dissolved in DCM:DMF 1:1 (1 mL) followed by addition of DIPEA (20 eq.). The solution was added to the deprotected hybrid (MeO-PEG.sub.5kDa-Lys(NH).sub.2)-dendron-(Ph).sub.4) dissolved in DCM (1 mL, per 0.1 g). The reaction was stirred for 1 hour in room temperature. The crude mixture was concentrated under vacuum and loaded on a MeOH based LH20 SEC column. The fractions that contained the product were unified and the DCM and MeOH were evaporated in vacuum to yield an oily residue. The oily product was re-dissolved in THF (1 mL, per 0.1 g) and tetra-n-butylammonium fluoride (TBAF) was added (0.06 mmol, 3 eq.). After 30 minutes the solution was concentrated under vacuum and loaded on a MeOH based LH20 SEC column. The fractions that contained the product were unified and the DCM and MeOH were evaporated in vacuum to yield an oily residue. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1 mL) followed by addition of Hexane (3 mL). DCM and Hexane were evaporated to dryness and the obtained off-white solid (compound 18) was dried under high vacuum (110 mg, 86% yield).

[0363] .sup.1H NMR (400 MHz, Chloroform-d) 8.05 (s, 1H, OH), 7.34-7.17 (m, 25H, ArH), 7.14 (d, J=5.4 Hz, 1H, CHNHCOAr), 7.00 (m, 3H, ArH +NHCOAr), 6.89 (m, 1H, CH.sub.2NHCOCH), 6.77 (d, J=2.2 Hz, 2H, ArH), 6.60-6.43 (m, 2H, ArH), 4.57 (d, J=6.4 Hz, 1H, COCHNH), 4.33-3.99 (m, 13H, CH.sub.2OCO+ArOCH.sub.2), 3.87-3.42 (m, 597H, PEG backbone), 3.35 (s, 6H, CH.sub.3O-PEG), 3.10 (t, J=5.7 Hz, 2H, CHS), 2.99-2.65 (m, 14H, CHCH.sub.2SCH.sub.2+CHSCH.sub.2), 2.55 (dt, J=22.2, 7.0 Hz, 8H, CH.sub.2SCH.sub.2), 2.04-1.68 (m, 4H, OCH.sub.2CH.sub.2CH.sub.2S+NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.62-1.39 (m, 4H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH).

Example 10

Fluorinated Hybrids as .SUP.19.F-magnetic Resonance Probes

[0364] Hybrid 20 (Scheme 14) was chosen as model compounds that can be activated by Porcine Liver Esterase (PLE)..sup.16 4-(Trifluoromethyl)phenylacetic acid was chosen as labeling group as it contains three equivalent fluorine atoms and a carboxylic acid that could be easily used for both esterification or amidation based labeling steps, as desired. The hybrid was synthesized directly on the PEG through accelerated dendritic growth approach.sup.17 using amidation and thiol-yne.sup.18,19 chemistries to build the dendrons. The synthesized hybrid and its fully hydrolyzed derivative 20a was obtained in high yields and characterized by .sup.1H-NMR, .sup.13C-NMR, .sup.19F-NMR, IR, GPC, HPLC and MALDI-MS) and the experimental data was found to be in good agreement with the theoretical one (see Example 17).

##STR00034##

[0365] Characterization of the Self-assembled Micelles

[0366] The critical micelle concentrations (CMC) were determined using Nile red.sup.23 and hybrid 20 was found to have CMC values at the low micro-molar range (4+/1 M). Dynamic light scattering (DLS) measurements showed a diameters of 16+/3 nm (FIG. 23), which fit micellar assemblies. The translation of the supramolecular structural changes into MR spectral responses depend on the differences in the spin-spin T.sub.2 relaxation times of the labeled polymers at the assembled and disassembled state, after their enzymatic activation. Hence, once the self-assembly of both hybrids into micelles was confirmed, their T.sub.1 and T.sub.2 relaxation times were measured and compared them to the values of synthetically obtained hydrophilic hybrid 20a (Table 1). The hybrid showed significant increase in both T.sub.1 and T.sub.2 values going from the amphiphilic hybrid to the hydrophilic one.

TABLE-US-00002 TABLE 1 .sup.19F-NMR relaxation times T.sub.1 and T.sub.2 of hybrid 20 before and after enzymatic activation. Hybrids 20a in the presence of the enzyme were used as control. (9.4 T, 376 MHz, TE = 80 ms Hybrid 20 20 + PLE 20a + PLE T.sub.1 (ms) 860 +/ 90 1090 +/ 50 1090 +/ 30 T.sub.2 (ms) 16 +/ 3 320 +/ 40 450 +/ 15

Structural and Spectral Responses

[0367] To study the structural and spectral responses of the micelles, their disassembly by DLS measurements were initially studied. The results show a clear disappearance of the larger micellar structures and formation of smaller spices with diameters of around 4-7 nm (FIG. 23), which fit well with the disassembly of the micelles into soluble hydrophilic hybrids. To get kinetic information on the disassembly process, fluorescence spectroscopy, HPLC and .sup.19F-NMR tools were combined, using fluorine atoms at a concentration of 640 M. The concentration of the enzyme was set to 1.1 M. Fluorescence spectroscopy was used to study the release of encapsulated Nile red upon enzymatic activation as upon its release from the disassembling micelles into the aqueous environment its fluorescence decreases. The obtained spectra for hybrid 20 showed a decrease in the fluorescence of the released Nile red molecules in the presence of the enzyme (FIG. 23) while no change was observed in the absence of the enzyme (FIG. 24), further supporting the enzymatic-induced disassembly. Both the DLS and the fluorescence spectra gave clear indication that the micelles break down upon enzymatic activation to release their molecular cargo. However these techniques do not reveal the exact degree of activation. In order to obtain direct analysis of the polymeric components during the disassembly process, HPLC was used to follow the enzymatic degradation. The HPLC data of hybrid 20 showed direct enzymatic transformation into the corresponding hydrophilic hybrid 20a (FIG. 20a).

[0368] Next, the enzymatic activation was studied using .sup.19F-NMR, and the results were correlated with the fluorescence and HPLC data. The acquisition parameters were set so ensure that the peaks of the labeled hydrophilic hybrid 20a were visible and could be integrated (sodium fluoride was used as internal reference and its chemical shift was set to zero). Kinetic .sup.19F-NMR measurements clearly showed the formation of labeled hydrophilic hybrid 20a (FIGS. 20b). Excellent correlations were observed when plotting the HPLC and .sup.19F-NMR peaks areas for both hybrids as a function of time and these results correlated well with the fluorescence data for Nile red, which is indicative of the presence of micelles (FIG. 20c).

[0369] After the disassembly of both types of micelles upon enzymatic activation was confirmed, their ability to show clear OFF/ON states was investigated. To do so, a spin-echo .sup.19F-NMR sequence was used (see Example XX) to measure the spectra before the addition of the enzyme and after the disassembly was completed (FIG. 21). This sequence allows one to utilize the significant differences in T.sub.2 relaxation times between the assembled and disassembled states. The obtained spectra didn't show any signal for the assembled state of hybrid 20, while the reference peak of NaF was clearly observed, indicating the OFF state of the assembled micelles (FIG. 21a). The spectra of the disassembled hybrids clearly showed the peaks correlating hydrophilic hybrid 20a, demonstrating the enzymatic-turn ON of the .sup.19F-MR signals (FIGS. 21b).

[0370] Conclusions

[0371] In summary, demonstrated herein is the rational design of highly modular enzyme-responsive MR probes for turn-on of .sup.19F-MR signal based on smart fluorinated amphiphilic hybrids. A molecular approach using non-cleavable labeling of the polymeric backbone was used. This design was studied by combination of DLS, Fluorescence spectroscopy, HPLC and NMR and were shown to be OFF at the assembled micellar state. Upon enzymatic activation and cleavage of the hydrophobic end-groups, the micelles disassembled and the MR signals were turned ON. The obtained results clearly prove the great potential of enzyme-responsive smart polymers to serve as an innovative and modular platform for the rational design of responsive MR probes.

Example 11

Synthesis of Fluorinated Hybrids as .SUP.19.F-magnetic Resonance probes

[0372] Hybrid 20 (MeO-PEG.sub.5kDa-Lys(Ph-CF.sub.2)-dendron-(Ph).sub.4: 160 mg (0.03 mmol) of

##STR00035##

[0373] MeO-PEG.sub.5kDa-Lys(Boc)-dendron-(Ph).sub.4 (hybrid 6, prepared in accordance with Example 2), were dissolved in DCM (1.5 mL) and TFA was added (1.5 mL). After 30 minutes the solution was evaporated to dryness and dried in vacuum. 2-(4-(trifluoromethyl)phenyl) acetic acid (5 eq.) and HBTU (5 eq.) were dissolved in DCM:DMF 1:1 (2 mL) followed by addition of DIPEA (20 eq.) and allowed to stir for 0.5 hour. The solution was added to 160 mg (0.03mmol) of the deprotected hybrid (MeO-PEG.sub.5kDa-Lys(NH.sub.2)-dendron-(Ph).sub.4) dissolved in DCM (1 mL). The reaction was stirred for 1 hour. The crude mixture was purified by a silica column using 1% Acetic Acid in EtOAc followed by 20% MeOH in DCM. The fractions that contained the product were unified and the solvents were evaporated in vacuum to yield an oily residue. In order to facilitate the removal of residual solvents and solidification of the product, the oily residue was re-dissolved in DCM (2 mL) followed by addition of Hexane (6 mL). The white precipitate was filtered and washed twice with Ether and dried under high vacuum. The product was obtained as a white solid (160 mg, quantitative yield).

[0374] .sup.1H NMR (CDCl.sub.3): 7.50 (d, J=8.1 Hz, 2H, CF.sub.3ArH), 7.31 (d, J=8.0 Hz, 2H, CF.sub.3ArH), 7.28 (m, 22H, ArH), 7.14 (d, J=7.6 Hz, 1H, CHNHCOAr), 6.98 (d, J=2 Hz, 2H, ArH), 6.82 (t, J=5.7 Hz, 1H, CH.sub.2NHCOCH), 6.55 (s, 1H, ArH), 6.01 (m, 1H, CF.sub.3-ArCONH), 4.51 (q, J=7.3 Hz, 1H, COCHNH), 4.29-4.17 (m, 8H, CH.sub.2OCO), 4.14-3.97 (m, 4H, ArOCH.sub.2), 3.80-3.43(m, PEG backbone), 3.33 (s, 3H, CH.sub.3O-PEG), 3.17 (m, 2H, CF.sub.3ArCONHCH.sub.2), 3.13-3.01 (m, 2H, CHS), 2.92-2.75 (m, 8H, CHSCH.sub.2+CHCH.sub.2SCH.sub.2), 2.71 (t J=6.8 Hz, 8H, CHCH.sub.2SCH.sub.2), 2.62-2.51 (m, 4H, CH.sub.2CH.sub.2SCH.sub.2), 1.98-1.65 (m, 4H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH+OCH.sub.2CH.sub.2CH.sub.2S), 1.53-1.41 (m, 2H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.35 (m, 2H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH); .sup.13C-NMR (CDCl.sub.3) 171.8, 171.5, 170.3, 167.0, 159.7, 136.2, 133.8, 129.8, 129.4, 128.7, 127.3, 125.7,125.3, 106.5, 105.0, 72.0, 70.7, 69.9, 69.5, 64.2, 63.9, 59.1, 53.6, 45.6, 44.9, 43.4, 43.3, 41.3, 39.0, 38.8, 34.9, 31.8, 31.6, 30.4, 29.8, 29.7, 28.7, 28.4, 22.7; .sup.19F-NMR (NaF as internal reference, CDCl.sub.3): 58.2 (-ArCF.sub.3); FT-IR, v (cm.sup.1): 2883, 1738, 1728, 1591, 1467, 1453, 1359, 1341, 1327, 1279, 1240, 1147, 1100, 1060, 960, 948, 842; GPC: Mn=6.4 kDa, PDI=1.04. Expected Mn=6.4 kDa. MALDI-TOF MS: molecular ion centered at 6.4 kDa.

[0375] Hybrid 20a (MeO-PEG.sub.5kDa-Lys(Ph-CF.sub.3)-dendron-(OH).sub.4):

##STR00036##

[0376] 145 mg (0.02 mmol) of MeO-PEG.sub.5kDa-Lys(Ph-CF.sub.3)-dendron-(Ph).sub.4 (20) were dissolved in MeOH (1 mL) followed by the addition of a drop of water and about 40 L of NaOH 1N (2 eq.) was added. The mixture was allowed to stir over night at 40 C. Complete hydrolysis was confirmed by HPLC. The pH of the mixture was neutralized (pH 7). The crude mixture was loaded on a DCM:MeOH 1: lv/v based LH20 SEC column. The fractions that contained the product were unified and the DCM and MeOH were evaporated in vacuum. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1 mL) followed by addition of Hexane (3 mL). DCM and Hexane were evaporated to dryness and the obtained orange solid was dried under high vacuum. The product was obtained as an orange solid (130 mg, quantitative yield).

[0377] .sup.1H NMR (CDCl.sub.3): 7.80 (s, 1H, CHNHCOAr), 7.49 (d, J=8.2 Hz, 2H, CF.sub.3ArH), 7.34 (d, J=8.3 Hz, 2H, CF.sub.3ArH), 7.16-7.04 (m, 2H, CHNHCOAr +ArH), 6.63-6.49 (m, 1H, CH.sub.2NHCOCH+ArH), 4.55 (s, 1H, COCHNH), 4.32-4.10 (m, 4H, ArOCH.sub.2), 3.83-3.40(m, PEG backbone), 3.34 (s, 3H, CH.sub.3O-PEG), 3.30-3.11 (m, 5H, CF.sub.3ArCONHCH.sub.2+CHS), 2.92-2.88 (m, 2H, CHCH.sub.2SCH.sub.2), 2.87-2.74 (m, 6H, CHSCH.sub.2+CHCH.sub.2SCH.sub.2), 2.70 (t, J=6.1 Hz, 4H, CHCH.sub.2SCH.sub.2) 2.64-2.50 (m, 4H, CH.sub.2CH.sub.2SCH.sub.2), 1.78 (m, 4H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH+OCH.sub.2CH.sub.2CH.sub.2S), 1.53-1.32 (m, 4H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH); .sup.13C-NMR (CDCl.sub.3) 171.9, 170.0, 166.7, 159.0, 139.0, 135.6, 129.2, 125.1, 125.0, 125.0, 106.0, 105.5, 98.9, 72.0, 70.7, 69.9, 63.2, 61.5, 61.4, 60.7, 58.5, 53.4, 44.8, 44.7, 42.6, 38.5, 38.4, 35.6, 34.5, 34.3, 34.3, 31.2, 31.1, 30.9, 29.1, 28.2, 27.9, 22.3; .sup.19F-NMR (NaF as internal reference, CDCl.sub.3): 57.7 (ArCF.sub.3); FT-IR, v (cm.sup.1): 2882, 1588, 1467, 1451, 1444, 1359, 1342, 1327, 1279, 1240, 1147, 1100, 1060, 960, 948, 842; GPC: Mn=6.4 kDa, PDI=1.16. Expected Mn=6.0 kDa. MALDI-TOF MS: molecular ion centered at 5.9 kDa.

Example 12

Critical Micelle Concentration (CMC) Measurements

[0378] For instrumentation and sample preparation, see Example 3.

[0379] CMC measurement for compound 1F, 1C and 8C: A 800 M solution was prepared in diluent and sonicated for 15 minutes. This solution was repeatedly diluted by a factor of 1.5 with diluent. 150 L of each solution were loaded onto a 96 wells plate. The fluorescence emission intensity was scanned for each well. Maximum emission intensity was plotted vs. hybrid concentration in order to determine the CMC. All measurements were repeated 3 times. The CMC for hybrid 20 was 41 M.

Example 13

Dynamic Light Scattering (DLS)

[0380] General Sample Preparation:

[0381] Hybrid 20 was dissolved in phosphate buffer (pH 7.4) to give a final concentration of 640M. The solution was sonicated for 15 minutes and filtered through a 0.22 m nylon syringe filter. Measurements were performed (t=o before addition al f PLE enzyme). All measurements were repeated 3 times.

[0382] For Micelle Degradation in the Presence of 1.1M PLE Enzyme:

[0383] 20.0 L of PLE enzyme stock solution (28.4 M in phosphate buffer pH 7.4) were added 500 L solution of hybrid 20 (640 M). Measurements were performed after 24 hours. All measurements were repeated 3 times. FIG. 22 depicts micelle degradation of hybrid 20 (640 M) in the presence of 1.1 M PLE enzyme.

Example 14

Monitoring Micelle Disassembly with Nile Red Fluorescence

[0384] Instrumentation: Monitoring of micelle disassembly rate by enzymes was performed using an Agilent Technologies Cary Eclipse Fluorescence Spectrophotometer.

[0385] Instrument Method: [0386] (a) Excitation: 550 nm [0387] (b) Emission scan: 580-800 nm [0388] (c) Excitation and Emission slits width: 10 nm [0389] (d) Scan rate: 620 nm/min [0390] (e) Temperature control: 27 C. [0391] (f) Sample preparation and measurement: Hybrid 20 was separately dissolved in phosphate buffer (pH 7.4) to give a concentration of 640 M. 4 mL of the solution were accurately measured and 1.8 L of Nile Red stock solution (0.88 mg/mL in Ethanol) were added to give a final concentration of 1.25 M. To each solution 40.4 L of NaF stock solution (64 mM in Phosphate buffer pH 7.4) were added to give final concentration of 640 M. 700 L of the hybrid solution containing Nile Red and NaF were accurately transferred to separate quartz cuvettes for reference measurements without PLE enzyme and also for t=0 measurements.

[0392] For Micelle Degradation in the Presence of 1.1 M PLE Enzyme:

[0393] 128 L of PLE enzyme stock solution (28.4 M in phosphate buffer pH 7.4) were added to 3.2 mL of a 640 M solution of hybrid 20 containing Nile Red and NaF and mixed manually to give final PLE concentration of 1.1 M. 700 L of the solution were accurately transferred to separate quartz cuvettes (1.2 mL were accurately transferred to a proper HPLC vail for monitoring enzymatic degradation by HPLC measurements and 500 L were transferred to NMR tube for .sup.19F NMR analysis). Repeating fluorescence scans were performed every 20 minutes for 8 hours. All measurements were repeated twice.

[0394] FIG. 23 depicts fluorescence emission spectra of Nile Red (1.25 M) in the presence of hybrid 20 (640 M) as a function of time after the addition of 1.1 M PLE. A decrease in the intensity was observed as Nile Red was released into solution due to micelles degradation. As control, FIG. 24 depicts fluorescence emission spectra of Nile Red (1.25 M) in the presence of hybrid 20 (640 M) in absence of PLE enzyme over 8 hours. No decrease in intensity was observed.

Example 15

HPLC Monitoring of Enzymatic Degradation

[0395] For instrumentation, solutions and general reaction conditions, see Example 7.

[0396] Injection volume: 10 L.

[0397] Sample preparation: Hybrid 20 was dissolved in phosphate buffer (pH 7.4) to give concentrations of 640 M. 4 mL of the solution were accurately measured and 1.8 L of Nile Red stock solution (0.88 mg/mL in Ethanol) were added to give a final concentration of 1.25 M. To the solution 40.4 L of NaF stock solution (64 mM in Phosphate buffer pH 7.4) were added to give final concentration of 640 M. 100 L of the hybrid solution containing Nile Red and NaF were accurately transferred to separate HPLC vials. 10 L of the hybrid 20 solution were injected to the HPLC as t=0 injection.

[0398] For Micelle Degradation in the Presence of 1.1 M PLE Enzyme:

[0399] 128 L of PLE enzyme stock solution (28.4 M in phosphate buffer pH 7.4) were added to 3.2 mL of 0 the tested hybrid solution (640 M) containing Nile Red and NaF and mixed manually to give final PLE concentration of 1.1 M. 1.2 mL of each solution were accurately transferred to separate HPLC vials (700 L were accurately transferred to a quartz cuvette for monitoring enzymatic degradation by fluorescence measurements and 500 L were transferred to NMR tube for .sup.19F NMR analysis). Enzymatic degradation was monitored by repeating 10 L injections from the same vial over 8 hours by 10 L injections from the same vail over 8 hours. All measurements were repeated twice.

[0400] FIG. 20a depicts the HPLC monitoring of micelle degradation in presence of 1.1 M PLE enzyme over time. The HPLC data showed the direct enzymatic transformation of hybrid 20 into the corresponding hydrophilic hybrid 20a.

Example 16

.SUP.19.F NMR Spectroscopy

[0401] Instrumentation: .sup.19F NMR experiments were conducted at 376 MHz using a Bruker Avance III instrument equipped with a BBFO probe. NaF (640 M) was used as an internal reference added directly to the solution and its chemical shift was set to zero.

[0402] Samples preparation: Hybrid 20 was dissolved in phosphate buffer (pH 7.4) and 10% D.sub.2O (v/v) to give a concentration of 640 M. 4 mL of the solution were accurately measured and 1.8 L of Nile Red stock solution (0.88 mg/mL in Ethanol) were added to give a final concentration of 1.25M. To the solution 40.4 L of NaF stock solution (64 mM in Phosphate buffer pH 7.4) were added to give final concentration of 640M. 500 L of each hybrid solution containing Nile Red and NaF were accurately transferred to separate NMR tubes.

[0403] For Micelle Degradation in the Presence of 1.1M PLE Enzyme:

[0404] 128 L of PLE enzyme stock solution (28.4 M in phosphate buffer pH 7.4) were added to 3.2 mL of the tested hybrid solution (640 M) containing Nile Red and NaF and mixed manually to give final PLE concentration of 1.1M. 500 L of each solution were accurately transferred to separate NMR tubes for .sup.19F NMR analysis (1.2 mL were transferred to a proper HPLC vial for monitoring enzymatic degradation by HPLC measurements and 700 L were accurately transferred to a quartz cuvette for monitoring enzymatic degradation by fluorescence measurements).

[0405] Sample Preparation of Hybrid 20a as a Reference Product of Enzymatic Degradation:

[0406] Hybrid 20a was dissolved in phosphate buffer (pH 7.4) and 10% D.sub.2O (v/v) to give a concentration of 640M. 1.0 mL of the solution were accurately measured. To the solution of hybrid 20a (640 M) 40.4 L of phenyl acetic acid stock solution (64 mM in phosphate buffer pH 7.4) were added to give final concentration of 2.56 mM. To the solution 10.1 L of NaF stock solution (64 mM in Phosphate buffer pH 7.4) were added to give final concentration of 640M. 20 L of PLE enzyme stock solution (28.4 M in phosphate buffer pH 7.4) were added to 500 L of each solution of the tested hybrid (in order to imitate the same conditions as in the micelle degradation solution). 500 L of the hybrid solution containing the degradation products, Nile Red, NaF and PLE were accurately transferred to separate NMR tubes.

[0407] .sup.19F NMR Monitoring of Enzymatic Degradation:

[0408] Monitoring of micelles disassembly rate by PLE enzyme was performed by 1D .sup.19F NMR spectra with repetition of 8 seconds of samples containing solution of hybrid 20 separately without addition of PLE enzyme (samples were prepared as described earlier) (t=0). With addition of PLE enzyme to each of the tested solution of hybrid 20 (samples were prepared as described earlier) repeating 1D .sup.19F NMR experiments in which a delay of 1608 sec was embedded were performed every 30 min for 8 hours. Chemical shift of NaF was set to zero and its integration was set to 1. The kinetic rates were achieved by plotting the normalized integral intensities rations of the two signals in the spectra over time. All measurements were repeated twice

[0409] T.sub.1 and T.sub.2 Measurements:

[0410] T.sub.1 of each sample was determined using an inversion recovery pulse sequence with repetition time of 8 seconds. T.sub.1 values were obtained by fitting the .sup.19F signal intensities vs according to equation Mz=Mo [1-2 exp(/T1)]. The analyzed samples were separate solutions of hybrid 20 without PLE enzyme, hybrid 20 24 hours after addition of PLE and hybrid 20a (all samples were prepared as described earlier). All measurements were repeated 3 times.

[0411] T.sub.2 of each sample was analyzed using a Carr Purcell Meiboom Gill (CPMG) sequence with repetition of 8 seconds. T.sub.2 values were obtained by fitting the .sup.19F signal integral intensities vs to a single exponential decay. The analyzed samples were separate solutions of hybrid 20 without PLE enzyme, hybrid 20 24 hours after addition of PLE and hybrid 20a (all samples were prepared as described earlier). All measurements were repeated 3 times.

[0412] ON/OFF Spectra:

[0413] The measurements were acquired using CPMG 1D sequence with repetition of 8 seconds and echo time TE=80 ms. The analyzed samples were separate solutions of hybrid 20 without PLE enzyme and 24 hours after addition of PLE enzyme (all samples were prepared as described earlier).

Example 17

Materials and Methods

[0414] HPLC: All measurements were recorded on a Waters Alliance e2695 separations module equipped with a Waters 2998 photodiode array detector. All solvents were purchased from Bio-Lab Chemicals and were used as received. All solvents are HPLC grade.

[0415] .sup.1H and .sup.13C NMR: spectra were recorded on Bruker Avance I and Avance III 400MHz and 100 MHz (.sup.13C) spectrometers. Chemical shifts are reported in ppm and referenced to the solvent. The molecular weights of the PEG-dendron hybrids were determined by comparison of the areas of the peaks corresponding to the PEG block (3.63 ppm) and the protons peaks of the dendrons.

[0416] .sup.19F NMR: spectra were collected on a Bruker Avance III 376 MHz spectrometer by using sodium fluoride as the internal reference.

[0417] .sup.19F MRI experiments: all measurements were conducted on an Avance-III 14.1T wide-bore NMR/MRI scanner (Bruker, Germany), equipped with a micro2.5 gradient system, capable of producing gradient pulses of 300 gauss/cm in the x, y, z-directions.

[0418] GPC: All measurements were recorded on Viscotek GPCmax by Malvern using refractive index detector and PEG standards (purchased from Sigma-Aldrich) were used for calibration. DMF +25mM NH.sub.4Ac was used as mobile phase.

[0419] Infrared spectra: All measurements were recorded on a Bruker Tensor 27 equipped with a platinum ATR diamond.

[0420] Absorbance and fluorescence spectra (including CMC measurements): Spectra were recorded on an Agilent Technologies Cary Eclipse Fluorescence Spectrometer using quartz cuvettes or on TECAN Infinite M200Pro plate reader device.

[0421] MALDI-TOF MS: Analysis was conducted on a Bruker AutoFlex MALDI-TOF MS (Germany) and also on a Waters MALDI synapt (USA). DHB matrix was used.

[0422] TEM: Images were taken by a Philips Tecnai F20 TEM at 200 kV.

[0423] DLS: All measurements were recorded on a Malvern Zetasizer NanoZS (for fluorescence experiments) or VASCO-3 Particle Side Analyzer (Cordouan) for .sup.19F-MR experiments.

[0424] Materials: Poly (Ethylene Glycol) methyl ether 5 kDa, 2-(Boc-amino)-ethanethiol (97%), 2,2-dimethoxy-2-phenylacetophenone (DMPA, 99%), Penicillin G Amidase from Escherichia coli (PGA), Esterase from porcine liver (PLE), Allyl bromide (99%), 4-Nitrophenol (99.5%), N,N-dicyclohexylcarbodiimide (DCC, 99%), Fmoc-Lys(Boc)-OH (98%), Fluorescein 5-isothiocyanate (FITC, 90%), Sephadex LH20 and 4-(Trifluoromethyl)Phenylacetic Acid (97%) were purchased from Sigma-Aldrich. Cystamine hydrochloride (98%), potassium hydroxide and DIPEA were purchased from Merck. O-(Benzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HBTU, 99.9%) was purchased from Chem-Impex. Trifluoroacetic acid (TFA) and 2-Mercaptoethanol (99%) was purchased from Alfa Aesar and phenyl acetic acid was purchased from Fluka. Sodium hydroxide and all solvents were purchased from Bio-Lab and were used as received. Deuterated solvents for NMR were purchased from Cambridge Isotope Laboratories, Inc.

[0425] Gel permeation chromatography (GPC) was performed according to the following conditions: [0426] (a) Columns. 2PSS GRAM 1000 +PSS GRAM 30 [0427] (b) Columns Temperature: 50 C. [0428] (c) Flow rate: 0.5 ml/min [0429] (d) Mobile phase: DMF+25 mM NH.sub.4Ac [0430] (e) Detector: Refractive index detector at 50 C. [0431] (f) Injection Volume: 50 L [0432] (g) General sample preparation: Hybrids were dissolved in mobile phase to give final concentrations of 10 mg/ml. Solution was filtered through a 0.22 m PTFE syringe filter.

[0433] Transmission Electron Microscopy (TEM):

[0434] General Sample Preparation:

[0435] 5 L sample solution were dropped cast onto carbon coated copper grids and inspected in a transmission electron microscope (TEM), operated at 200 kV (Philips Tecnai F20). The excessive solvent of the droplet was wiped away using a solvent-absorbing filter paper after 1 min and the sample grids were ft to dry in air at room temperature for 5 minutes. This procedure was repeated 3 times. After the third cycle the sample grids were left to dry in air at room temperature overnight.

[0436] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

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