5-AMINOLEVULINIC ACID CONJUGATED QUANTUM DOT NANOPARTICLE

Abstract

A 5-aminolevulinic acid conjugated quantum dot nanoparticle is useful for treating cancer by administering the 5-aminolevulinic acid conjugated quantum dot nanoparticle in photodynamic therapy as a precursor of both a fluorescence label and a photosensitizer.

Claims

1. A functionalized quantum dot nanoparticle conjugated to 5-aminolevulinic acids.

2. The 5-ALA-nanoparticle conjugate of claim 1, wherein the nanoparticle is covalently linked to 5-ALA via an amide or an ester bond.

3. The functionalized quantum dot nanoparticle of claim 1, wherein the quantum dot nanoparticle is a core-shell nanoparticle.

4. The functionalized quantum dot nanoparticle of claim 1, further comprising a ligand capable of targeting a cancer cell.

5. The functionalized quantum dot nanoparticle of claim 1, wherein the ligand is PLZ4.

6. The functionalized quantum dot nanoparticle of claim 1, wherein the quantum dot nanoparticle is substantially cadmium free.

7. A method of preparing a 5-ALA-nanoparticle conjugate comprising the steps of: providing a nanoparticle comprising a molecular cluster compound, a core semiconductor material, and an outer layer; providing a coupling agent; providing 5-ALA, 5-ALA derivatives, or 5-ALA analogs; incubating the mixture to form crude 5-ALA-nanoparticle conjugate; purifying the crude 5-ALA-nanoparticle conjugate; and isolating the 5-ALA-nanoparticle conjugate.

8. The method of claim 7, wherein the coupling agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

9. The method of claim 7, further comprising the step of conjugating the 5-ALA-nanoparticle conjugate to a ligand capable of targeting a cancer cell.

10. A method of inducing apoptosis of a cell comprising the steps of: administering a functionalized nanoparticle conjugated to a plurality of 5-aminolevulinic acids to a mammal in thereof; allowing 5-aminolevulinic acids to form metabolites; and irradiating the metabolites.

11. The method of claim 10, wherein the metabolite is photoporphyrin IX.

12. The method of claim 10, wherein the step of irradiating is performed by the nanoparticle.

13. The method of claim 12, wherein the nanoparticle emits light in the range of 375-475 nm.

14. The method of claim 10, wherein the step of irradiating is sufficient to produces reactive oxygen species.

15. The method of claim 10, wherein the functionalized nanoparticle further comprises a ligand capable of targeting a cancer cell.

16. The method of claim 15, further comprising the step of the ligand binding to a cancer cell.

17. A method of detecting cancer cells comprising the steps of: administering a 5-ALA-nanoparticle conjugate in photodynamic diagnosis as a precursor of both a fluorescence label and a photosensitizer; allowing disassociation of 5-ALA from the nanoparticle; allowing 5-ALA to form PpIX; exciting a disassociated nanoparticle to emit blue light of 375-475 nm; activating the fluorescent properties of PpIX; and imaging the fluorescence.

18. The method of claim 17, wherein the administering step is performed by injection.

19. The method of claim 18, wherein the injection is performed intravenously.

20. The method of claim 19, wherein the nanoparticle is an alloyed quantum dot.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0024] FIG. 1 is a schematic diagram of a process of preparing a 5-ALA-nanoparticle conjugate.

[0025] FIG. 2 illustrates the conjugation with 5-ALA of a nanoparticle (represented by the filled circle) having surface-bound ligands attached thereto. In this representative illustration, X=a surface binding ligand (thiol, amine, phosphine, phosphine oxide, carboxylic acid, etc.), Y=a linking group (hydrocarbon chain comprising one or more of alkyls, alkenyls, alkynyls; polymers such as PEG, PPO, PEO, silicone rubber, polyethylene, acrylic resins, polyurethane, polypropylene, and polymethylmethacrylate; copolymers; block copolymers, etc.), and Z=a carboxylic acid, ester, acyl chloride, acid anhydride, or aldehyde.

[0026] FIG. 3 illustrates a metabolic pathway from the 5-ALA-nanoparticle conjugate of FIG. 2 to the photosensitizer protoporphyrin IX (PpIX or PROTO).

DETAILED DESCRIPTION OF THE INVENTION

[0027] In FIG. 1, a 5-ALA-nanoparticle conjugate is provided by reacting a nanoparticle with 5-ALA. As an example, the nanoparticle comprises a molecular cluster compound, a core semiconductor material, and an outer layer. The outer layer comprises a carboxyl group with which 5-ALA reacts to form a linkage. It should be understood that derivatives and analogs of 5-ALA could be used either alone or in combination. It should also be understood that an alloyed nanoparticle may be also be used. In addition, a combination of core-shell nanoparticles and alloyed nanoparticles may be used.

[0028] Derivatives of 5-ALA include, but are not limited to:

[0029] 5-ALA n-alkyl esters [0030] 5-ALA methyl ester (methylaminolevulinate, Trade name METVIV) [0031] 5-ALA ethyl ester [0032] 5-ALA propyl ester [0033] 5-ALA butyl ester [0034] 5-ALA pentyl ester [0035] 5-ALA hexyl ester (hexylaminolevulinate, Trade name HEXVIX) [0036] 5-ALA octyl ester

[0037] As well as: [0038] 5-ALA (hydroxymethyl)tetrahydrofuranyl ester; and, [0039] 5-ALA polyethylene glycol derivatives

[0040] Plus salts such as: [0041] 5-ALA.HCl

[0042] The types of core-shell nanoparticles include but are not limited to core material comprising the following types:

[0043] IIA-VIB (2-16) material, consisting of a first element from Group 2 of the periodic table and a second element from Group 16 of the periodic table and also including ternary and quaternary materials and doped materials. Nanoparticle material include but are not restricted to: MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe.

[0044] IIB-VIB (12-16) material consisting of a first element from Group 12 of the periodic table and a second element from Group 16 of the periodic table and also including ternary and quaternary materials and doped materials. Nanoparticle material includes but are not restricted to: ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe.

[0045] II-V material consisting of a first element from Group 12 of the periodic table and a second element from Group 15 of the periodic table and also including ternary and quaternary materials and doped materials. Nanoparticle material include but is not restricted to: Zn.sub.3P.sub.2, Zn.sub.3As.sub.2, Cd.sub.3P.sub.2, Cd.sub.3As.sub.2, Cd.sub.3N.sub.2, Zn.sub.3N.sub.2.

[0046] III-V material consisting of a first element from Group 13 of the periodic table and a second element from Group 15 of the periodic table and also including ternary and quaternary materials and doped materials. Nanoparticle material include but is not restricted to: BP, AlP, AlAs, AlSb; GaN, GaP, GaAs, GaSb; InN, InP, InAs, InSb, AlN, BN.

[0047] III-IV material consisting of a first element from Group 13 of the periodic table and a second element from Group 14 of the periodic table and also including ternary and quaternary materials and doped materials. Nanoparticle material include but is not restricted to: B.sub.4C, Al.sub.4C.sub.3, Ga.sub.4C.

[0048] III-VI material consisting of a first element from Group 13 of the periodic table and a second element from Group 16 of the periodic table and also including ternary and quaternary materials. Nanoparticle material include but is not restricted to: Al.sub.2S.sub.3, Al.sub.2Se.sub.3, Al.sub.2Te.sub.3, Ga.sub.2S.sub.3, Ga.sub.2Se.sub.3, GeTe; In.sub.2S.sub.3, In.sub.2Se.sub.3, Ga.sub.2Te.sub.3, In.sub.2Te.sub.3, InTe.

[0049] IV-VI material consisting of a first element from Group 14 of the periodic table and a second element from Group 16 of the periodic table, and also including ternary and quaternary materials and doped materials. Nanoparticle material include but is not restricted to: PbS, PbSe, PbTe, Sb.sub.2Te.sub.3, SnS, SnSe, SnTe.

[0050] Nanoparticle material consisting of a first element from any Group in the transition metal of the periodic table, and a second element from any group of the d-block elements of the periodic table and also including ternary and quaternary materials and doped materials. Nanoparticle material include but is not restricted to: NiS, CrS, CuInS.sub.2.

[0051] The term doped nanoparticle for the purposes of this specification and its claims refers to nanoparticles of the above and a dopant comprising one or more main group or rare earth elements. This most often is a transition metal or rare earth element, such as but not limited to zinc sulfide with manganese, such as ZnS nanoparticles doped with Mn.sup.+.

[0052] In one embodiment, cadmium-free nanoparticles are preferred.

[0053] In an embodiment, the nanoparticle includes a first layer including a first semiconductor material provided on the nanoparticle core. A second layer including a second semiconductor material may be provided on the first layer.

[0054] Standard conjugation chemistry may be used for conjugation. For example, a method preparing a 5-ALA-nanoparticle conjugate may include the steps of providing a nanoparticle, providing a coupling agent, providing 5-ALA, 5-ALA derivatives (such as, for example, its ester derivatives), 5-ALA analogs, incubating the mixture to form a crude 5-ALA-nanoparticle conjugate. The crude 5-ALA-nanoparticle conjugate may then be purified and isolated to obtain a 5-ALA-nanoparticle conjugate.

[0055] The incubations conditions may be chosen to allow for formation of either an amide or an ester. It should be understood that other bonds may be formed (e.g., both covalent and non-covalent). In one embodiment, 5-ALA is bonded to a nanoparticle. The 5-ALA may be conjugated with the nanoparticle either covalently, physically, ion pairing, or Van der Waals' interactions. The bond may be formed by an amide, ester, thioester, or thiol anchoring group directly on the inorganic surface of the quantum dot nanoparticle, or on the organic corona layer that is used to render the nanoparticles water soluble and biocompatible.

[0056] Standard incubation conditions for coupling may be employed. For example, the coupling conditions may be a solution in the range of 0.5 to 4 hours. The temperature range of the coupling conditions may be in the range of 100 C. to 200 C. The coupling conditions may be constant or varied during the reaction. For example, the reaction conditions may be 130 C. for one hour then raised to 140 C. for three hours.

[0057] Linkers may be used to form an amide or an ester group between the carboxyl functions on the nanoparticles and either the carboxyl or the amine end groups on the 5-ALA. Linkers or coupling agents may include benzotriazolyloxy-tris(dimethylamino) phosphonium Hexafluorophosphate (BOP) and carbodiim ides such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), and 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). EDC is a preferred carbodiimide to use as the amide coupling agent.

[0058] In an example, the quantum dot nanoparticles bearing a carboxyl end group and 5-ALA may be mixed in a solvent. A coupling agent, such as EDC, may be added to the mixture. The reaction mixture may be incubated. The crude 5-ALA-QD nanoparticle conjugate may be subject to purification to obtain the 5-ALA-QD nanoparticle conjugate.

[0059] Standard solid state purification method may be used. Several cycles of filtering and washing with a suitable solvent may be necessary to remove excess unreacted 5-ALA and EDC.

[0060] In another embodiment, the 5-ALA-nanoparticle conjugate may further include a ligand capable of targeting a cancer cell. For example, a chemical compound or a peptide, such as, for example, an antibody may be conjugated to the 5-ALA-nanoparticle conjugate to further effect cellular uptake of the 5-ALA-nanoparticle conjugate for either photo-detection or phototherapy. An example of a peptide is PLZ4 (QDGRMGF), which is a peptide that may selectively bind to bladder cancer cells. The peptides may form amide or ester bonds with the functionalized nanoparticle by their amine or carboxylic acid groups.

[0061] Once selectively bound to the cancer cell, the 5-ALA-nanoparticle conjugate will be taken up by the cell. Once internalized, 5-ALA undergoes conversion to the natural photosensitizer photoporphyrin IX (PpIX). Subsequent illumination of the tumor site with light, for example, blue light in the range of 375-475 nm, activates PpIX, triggers the oxidative damage with the release of reactive oxygen species (ROS) and induces cytotoxicity or apoptosis.

[0062] Accordingly, embodiments disclosed herein may be used for methods of inducing apoptosis of a cell, for example, a mammalian cell, comprising the step of administering a 5-ALA-nanoparticle conjugate to a mammal in need thereof, allowing 5-ALA to form metabolites, such as PpIX, and irradiating the metabolites. The irradiating step may be done by excitation of a nanoparticle, such as a disassociated nanoparticle.

[0063] Embodiments also include methods of detecting cancer cells by imaging the mammal.

[0064] The administration of the 5-ALA-nanoparticle conjugate may be enteral or parenteral. For example, the 5-ALA-nanoparticle conjugate may be administered subcutaneously, intravenously, intramuscular, topically, and orally. Examples include bolus injections or IV infusions.

[0065] The 5-ALA-QD nanoparticle conjugate of the current invention has the following advantages over the free 5-ALA.

[0066] First, the 5-ALA-QD nanoparticle conjugate has enhanced cell permeability and may be taken up more efficiently by the cancer cells, especially by the very active cancer stem cells. Nanoparticles in general accumulate in cancer cells more than normal cells. The QD nanoparticles act as a vectorized delivery system.

[0067] Second, the QD emission may be tuned to overlap with PpIX absorption. Once the QD-5ALA particles are internalized into the cancer cell, the 5-ALA will be released and transformed into PpIX within a few hours. The QDs then may be used as a light or FRET donor to enhance the excitation of the produced PpIX. Because QD nanoparticles have 10-100 fold higher molecular extinction coefficient compared to small molecular dyes like PpIXs, more light may be absorbed, and a stronger signal may be generated, improving signal to noise detection ratio.

[0068] Third, the high light absorption intensity may also increase the efficacy of PpIX in generating singlet oxygen as a photodynamic therapeutic (PDT) agent.

[0069] Fourth, the tunability of the QD nanoparticles and the potential for multi-photon excitation (including two-photo excitation) may enable deeper tissue detection and deeper PDT, unlike 5-ALA alone where only a few millimeters of tissue depth may be accessed.

[0070] Fifth, two-photo excitation or multiphoton excitation provides a means for excitation wavelength at greater than 700 nm, and allows PDT with highly localized light dosage.

[0071] These and other advantages of the present invention will be apparent to those skilled in the art from the foregoing specification. Accordingly, it is to be recognized by those skilled in the art that changes or modifications may be made to the above-described embodiments without departing from the broad inventive concepts of the invention. It is to be understood that this invention is not limited to the particular embodiments described herein, but is intended to include all changes and modifications that are within the scope and spirit of the invention.