Monitoring of intracellular molecules and drug screening can provide valuable insights into the biological conditions of cells and the therapeutic efficiency of drugs. Fluorescence light-up probes based on aggregation-induced emission (AIE) fluorogens and water-soluble peptides have been used for real-time monitoring of cellular proteins. The first generation of specific AIE probe design is limited to peptide based recognition elements with high water solubility. To extend the design principle to include more broad recognition elements (such as, for instance, hydrophobic molecules, small molecules, etc), it is necessary to develop a more general strategy.
This invention relates to the development of AIE fluorogens, including those with excited state intramolecular proton transfer (ESIPT) characteristics, AIE light-up probes and their applications in sensing, imaging and drug screening.
More specifically, herein a series of fluorescent light-up probes are described, which generally comprise an AIE fluorogen, a recognition moiety, a targeted ligand and hydrophilic units (e.g. five aspartic acids) to ensure good water-solubility of the probe. Due to the unique nature of the AIE fluorogen, the probes are non-fluorescent in aqueous media but become highly emissive when cleaved by intracellular molecules. The probe enables light-up monitoring of intracellular molecules and drug screening with high signal-to-noise ratio. Based on a similar design principle, replacing the traditional AIE fluorogens with fluorophores showing both AIE and ESIPT characteristics could simplify such design, as the probe is non-fluorescent regardless of the probe water-solubility. As the AIE probe design strategy can be generalized to perform various tasks by simply substituting the recognition moiety to other cleavable linkers in chemical biology, it opens new opportunities to design specific light-up probes for imaging of intracellular molecules and drug screening.
The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.
The present invention is directed to luminogens (AIE fluorogens and AIE-ESIPT fluorogens being subclasses thereof) and chemical compositions (e.g. light-up probes) comprising a target recognition motif, a hydrophilic moiety, a linking moiety, and at least one luminogen. The present invention is also directed to methods of assessing the conversion of a prodrug into its active form, assessing the therapeutic efficacy of a prodrug, detecting glutathione in a biological sample, detecting alkaline phosphatase in a sample, and conducting fluorescence imaging or magnetic resonance imaging with the use of said compositions comprising the luminogens.
All definitions of substituents set forth below are further applicable to the use of the term in conjunction with another substituent.
“Alkyl” means a saturated aliphatic branched or straight-chain monovalent hydrocarbon radical, typically C1-C10, preferably C1-C6. “(C1-C6) alkyl” means a radical having from 1-6 carbon atoms in a linear or branched arrangement. “(C1-C6)alkyl” includes methyl, ethyl, propyl, butyl, tert-butyl, pentyl and hexyl.
“Alkylene” means a saturated aliphatic straight-chain divalent hydrocarbon radical. Thus, “(C1-C6)alkylene” means a divalent saturated aliphatic radical having from 1-6 carbon atoms in a linear arrangement. “(C1-C6)alkylene” includes methylene, ethylene, propylene, butylene, pentylene and hexylene.
“Cycloalkyl” means saturated aliphatic cyclic hydrocarbon ring. Thus, “C3-C8 cycloalkyl” means (3-8 membered) saturated aliphatic cyclic hydrocarbon ring. C3-C3 cycloalkyl includes, but is not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Preferably, cycloalkyl is C3-C6 cycloalkyl.
The term “alkoxy” means —O-alkyl; “hydroxyalkyl” means alkyl substituted with hydroxy; “aralkyl” means alkyl substituted with an aryl group; “alkoxyalkyl” mean alkyl substituted with an alkoxy group; “alkylamine” means amine substituted with an alkyl group; “cycloalkylalkyl” means alkyl substituted with cycloalkyl; “dialkylamine” means amine substituted with two alkyl groups; “alkylcarbonyl” means —C(O)-A*, wherein A* is alkyl; “alkoxycarbonyl” means C(O) OA*, wherein A* is alkyl; and where alkyl is as defined above. Alkoxy is preferably O(C1-C6)alkyl and includes methoxy, ethoxy, propoxy, butoxy, pentoxy and hexoxy.
“Cycloalkoxy” means a —O-cycloalkyl, wherein the cycloalkyl is as defined above. Exemplary (C3-C7)cycloalkyloxy groups include cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexoxy and cycloheptoxy.
The term “aryl” used alone or as part of a larger moiety as in “arylalkyl”, “arylalkoxy”, “aryloxy”, or “aryloxyalkyl”, means carbocyclic aromatic rings. The term “carbocyclic aromatic group” may be used interchangeably with the terms “aryl”, “aryl ring” “carbocyclic aromatic ring”, “aryl group” and “carbocyclic aromatic group”. An aryl group typically has 6-16 ring atoms. A “substituted aryl group” is substituted at any one or more substitutable ring atom. The term “C6-C16 aryl” as used herein means a monocyclic, bicyclic or tricyclic carbocyclic ring system containing from 6 to 16 carbon atoms and includes phenyl (Ph), naphthyl, anthracenyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl and the like. In particular embodiments, the aryl group is (C6-C10)aryl. The (C6-C10)aryl(C1-C6)alkyl group connects to the rest of the molecule through the (C1-C6)alkyl portion of the (C6-C10)aryl(C1-C6)alkyl group.
“Hetero” refers to the replacement of at least one carbon atom member in a ring system with at least one heteroatom selected from N, S, and O. The heteroatom can optionally carry a charge. When N is the heteroatom of a ring system, it may be additionally substituted by one or more substituents including H, OH, O-, alkyl, aryl, heterocyclyl, cycloalkyl or alkenylene, wherein any of the alkyl, aryl, heterocyclyl, cycloalkyl or alkenylene may be optionally and independently substituted by one or more substituents selected from halo, cyano, nitro, hydroxyl, phosphate (PO43−) or a sulfonate (SO3−).
“Heterocycle” means a saturated or partially unsaturated (3-7 membered) monocyclic heterocyclic ring containing one nitrogen atom and optionally 1 additional heteroatom independently selected from N, O or S. When one heteroatom is S, it can be optionally mono- or di-oxygenated (—S(O)— or S(O)2). Examples of monocyclic heterocycle include, but not limited to, azetidine, pyrrolidine, piperidine, piperazine, hexahydropyrimidine, tetrahydrofuran, tetrahydropyran, morpholine, thiomorpholine, thiomorpholine 1,1-dioxide, tetrahydro-2H-1,2-thiazine, tetrahydro-2H-1,2-thiazine 1,1-dioxide, isothiazolidine, or isothiazolidine 1,1-dioxide. The heterocycle can be optionally fused to a carbocyclic ring, as in, for example, indole.
The term “heteroaryl”, “heteroaromatic”, “heteroaryl ring”, “heteroaryl group” and “heteroaromatic group”, used alone or as part of a larger moiety as in “heteroarylalkyl” or “heteroarylalkoxy”, refers to aromatic ring groups having five to fourteen total ring atoms selected from carbon and at least one (typically 1-4, more typically 1 or 2) heteroatoms (e.g., oxygen, nitrogen or sulfur). They include monocyclic rings and polycyclic rings in which a monocyclic heteroaromatic ring is fused to one or more other carbocyclic aromatic or heteroaromatic rings. The term “5-14 membered heteroaryl” as used herein means a monocyclic, bicyclic or tricyclic ring system containing one or two aromatic rings and from 5 to 14 total atoms of which, unless otherwise specified, one, two, three, four or five are heteroatoms independently selected from N, NH, N(C1-6alkyl), 0 and S. (C3-C10)heteroaryl includes furyl, thiophenyl, pyridinyl, pyrrolyl, imidazolyl, and in preferred embodiments of the invention, heteroaryl is (C3-C10)heteroaryl.
“Halogen” and “halo” are interchangeably used herein and each refers to fluorine, chlorine, bromine, or iodine.
“Cyano” means —C≡N.
“Nitro” means —NO2.
As used herein, an amino group may be a primary (NH2), secondary (NHRx), or tertiary (NRxRy), wherein Rx and Ry may be any alkyl, aryl, heterocyclyl, cycloalkyl or alkenylene, each optionally and independently substituted with one or more substituents described above. The Rx and Ry substituents may be taken together to form a “ring”, wherein the “ring”, as used herein, is cyclic amino groups such as piperidine and pyrrolidine, and may include heteroatoms such as in morpholine.
The terms “haloalkyl”, “halocycloalkyl” and “haloalkoxy” mean alkyl, cycloalkyl, or alkoxy, as the case may be, substituted with one or more halogen atoms. The term “halogen” means F, Cl, Br or I.
The term “acyl group” means —C(O)A*, wherein A* is an optionally substituted alkyl group or aryl group (e.g., optionally substituted phenyl).
An “alkylene group” is represented by —[CH2]z, wherein z is a positive integer, preferably from one to eight, more preferably from one to four.
An “alkenylene group” is an alkylene in which at least a pair of adjacent methylenes are replaced with —CH═CH.
The term benzyl (Bn) refers to —CH2Ph.
The term “Alkenyl” means a straight or branched hydrocarbon radical including at least one double bond. The (C6-C10)aryl(C2-C6)alkenyl group connects to the remainder of the molecule through the (C2-C6)alkenyl portion of (C6-C10)aryl(C2-C6)alkenyl.
Pharmaceutically acceptable salts of the compounds of the present invention are also included. For example, an acid salt of a compound of the present invention containing an amine or other basic group can be obtained by reacting the compound with a suitable organic or inorganic acid, resulting in pharmaceutically acceptable anionic salt forms. Examples of anionic salts include the acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, and triethiodide salts.
Salts of the compounds of the present invention containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base. Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceutically acceptable cation, which includes alkali metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts, as well as salts made from physiologically acceptable organic bases such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N′-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, dehydroabietylamine, N,N′-bisdehydroabietylamine, glucamine, N-methylglucamine, collidine, quinine, quinoline, and basic amino acids such as lysine and arginine.
The term “luminogen”, as used herein, refers to a molecule that exhibits light-emission. If the light that is emitted is fluorescent light, the luminogen is alternately referred to as a fluorogen.
“Aggregation-induced emission” refers to a property in which a luminogen, when dispersed, for example in organic solvent, emits little or no light. Upon aggregation of luminogen molecules, however, for example in the solid state or in water due to the hydrophobicity of the luminogen, light emission from the luminogen is significantly enhanced.
A “target recognition motif” as used herein, is a chemical moiety having an affinity for a biological target such as a protein, a peptide, or a receptor in the cell membrane. A target recognition motif can comprise a peptide, a protein, an oligonucleotide, or an organic functional group having an affinity for a specific target structure.
A “linking moiety” as used herein, is a chemical moiety that links two or more groups through one covalent bond or through a series of covalent bonds. Example linking moieties include disulfide groups, amino groups, 2-nitrobenzyl derivatives, sulfones, hydrazones, vicinal diols, or simply one or more covalent bonds. Further examples of linking moieties can be found in Table 1 of Bioorg. Med. Chem., 2012, 20, 571-582.
Hydrophilic moieties for use in the compositions of the present invention can include water soluble polymers or alkyl chains functionalized by charged side groups. Examples of water soluble polymers for use with the present invention include polyethylene glycol or polyethylenimine. Charged side groups that may be used with the present invention include, for example SO33− or PO43−.
As used herein, “spectroscopy” encompasses any method by which matter reacts with radiated energy. This includes, but is in no way limited to, microscopy, fluorescence microscopy, UV/Vis spectrometry, and flow cytometry. A “microplate reader” as used herein, means a laboratory instrument that measures, for example, fluorescence, absorbance and luminescence of samples contained in a microplate.
A “prodrug” as used herein, is a therapeutic compound that is typically administered to a subject in its inactive form and is converted to its active form in the body of the subject. For example, a prodrug may include a platinum (IV) [Pt (IV)] complex that is converted to an active platinum (II) [Pt (II)] complex. In certain embodiments, such a conversion occurs via reduction with a chemical reagent. In certain other embodiments, such a conversion occurs via metabolic processes.
Tetraphenylethylene, or TPE, is:
Tetraphenylsilole, or TPS, is:
“Live target cells” as used herein, are live cells that are the target of a treatment or therapeutic regimen. In some embodiments, live target cells can be cancer cells that are the therapeutic target of a prodrug.
A description of various aspects of the invention follows.
In this first aspect of the invention are described luminogens. A luminogen is an atom, or groups of atoms, that luminesces. Similar to a luminogen, a fluorogen is an atom, or groups of atoms, that fluoresces. Luminescence is a process of emitting light. Types of luminescence include bioluminescence, chemiluminescence, electroluminescence, electrochemiluminescence, photoluminescence, and others. A type of photoluminescence is known as fluorescence. Thus, fluorogens are a subset of luminogens.
Within the family of fluorogens can be found aggregation-induced emission (AIE) fluorogens. One embodiment of this aspect of the present invention is directed to an AIE fluorogen having the structure of formula:
or a pharmaceutically acceptable salt thereof, wherein:
R1 is selected from H, (C1-C6)alkyl, (C3-C6)cycloalkyl, (C6-C10)aryl, (C3-C10)heteroaryl, or (C2-C6)alkenyl;
R2 is independently selected from H, NHR3, N(R3)2, (C1-C6)alkyl, (C3-C6)cycloalkyl, (C6-C10)aryl, (C3-C10)heteroaryl, —O(C1-C6)alkyl, (C2-C6)alkenyl, CH═CH((C3-C10)heteroaryl), or CH═CH((C6-C10)aryl); and
R3 is selected from H, (C1-C6)alkyl or (C3-C6)cycloalkyl.
The AIE fluorogen is also optionally and independently substituted with one or more substituents selected from: (C3-C10)heteroaryl,
wherein * indicates the point of attachment to the luminogen residue and ** indicates the point of attachment to either the prodrug, the target recognition motif or the hydrophilic peptide.
In another embodiment, the AIE fluorogen has the structure of formula:
wherein R1 is (C1-C6)alkyl. In a preferred embodiment, R1 is C2H5 or C6H13.
In yet another embodiment, the AIE fluorogen has the structure of formula:
The following schemes more specifically illustrate the design and synthesis of several exemplary AIE fluorogens.
More detailed synthetic routes for exemplary AIE fluorogens can be found in the Exemplification section of this application.
Within the family of AIE fluorogens can be found those with excited state intramolecular proton transfer (ESIPT) characteristics (AIE-ESIPT fluorogens).
One embodiment of this aspect of the present invention is drawn toward a fluorogen having the structure of formula:
or a pharmaceutically acceptable salt thereof, wherein:
M is selected from S, O or NH;
Q is selected from P(═O)(OH)2 or C(O)O(C1-C6)alkyl, wherein C(O)O(C1-C6)alkyl is optionally functionalized by one or more substituents selected from SH, OH, NH2, or (C6-C10)aryl optionally substituted with one or more substituents selected from OH, SH, or NH2;
R4 is selected from NHR6, N(R6)2, (C1-C6)alkyl, (C3-C6)cycloalkyl, (C6-C10)aryl, (C3-C10)heteroaryl, —O(C1-C6)alkyl, —O(C3-C6)cycloalkyl or (C2-C6)alkenyl;
R5 is (C0-C6)alkyl, optionally functionalized by a linking moiety; and
R6 is selected from H, (C1-C6)alkyl or (C3-C6)cycloalkyl.
In a preferred embodiment, the linking moiety, if present, is covalently attached to a target recognition motif. The target recognition motif preferably has an affinity for a cell membrane receptor. More preferably, the target recognition motif has a cyclic (Arg-Gly-Asp) (cRGD) residue having an affinity for integrin αvβ3.
In another embodiment, the AIE-ESIPT fluorogen has the structure of formula:
known as Phos-HC.
The following scheme more specifically illustrates the design and synthesis of an exemplary AIE-ESIPT fluorogen.
Use of this exemplary AIE fluorogen in the detection of alkaline phosphatase in a sample can be found in the Exemplification section of this application.
Both AIE fluorogens and AIE-ESIPT fluorogens can be used in the creation of light-up probes for conducting fluorescence imaging and magnetic resonance imaging as well as for assessing the conversion of a prodrug into its active form, assessing the therapeutic efficacy of a prodrug, detecting glutathione in a biological sample, and detecting alkaline phosphatase in a sample.
Targeted drug delivery to tumor cells with minimized side effects and real-time in-situ monitoring of drug efficacy is highly desirable for personalized medicine. More specifically, it is highly desirable if one could design and develop a system that can simultaneously deliver drugs and non-invasively evaluate the therapeutic responses in-situ. The most promising solution to this issue is to incorporate an apoptosis sensor into the system.
The inventors of the present invention have developed a strategy for real-time monitoring of cell apoptosis in-vitro and in-vivo based on probes containing fluorogens (light-up probes) with AIE characteristics. The composition of the light-up probes will be discussed first, followed by a description of the applications of the light-up probes.
In the light-up probe aspect of the present invention, the light-up probe is a chemical composition comprising at least one luminogen, a hydrophilic moiety, a linking moiety, and a target recognition motif, wherein the luminogen exhibits aggregation-induced emission properties, and further wherein the target recognition motif, the hydrophilic moiety, the linking moiety, and the at least one luminogen are linked by covalent linkages in a linear array.
In one embodiment, the linking moiety is a prodrug, such as for example a platinum (IV) complex (“Pt”).
In another embodiment, the linking moiety is a cleavable linking group. Preferably, a cleavable linking moiety is a disulfide (“SS”). Alternatively, the cleavable linking group can be a hydrazone bond that can be cleaved in acidic conditions or an aminoacrylate (AA) linker that can be cleaved by reactive oxygen species.
In one embodiment, the hydrophilic moiety comprises a hydrophilic peptide, a self-assembling peptide, an oligonucleotide, a water-soluble polymer, or an alkyl chain functionalized by charged side groups. Preferably, the alkyl chain has greater than five carbon atoms and the charged side groups can be, for example, an amine group, a carboxyl group or a guanidinium group.
In one embodiment, the hydrophilic moiety is a hydrophilic peptide. For example, the hydrophilic peptide can comprise an amino acid residue sequence comprising at least one of Lys, Asp, Arg, His or Glu. Preferred hydrophilic peptides can be Asp-Asp-Asp-Asp-Asp (SEQ ID NO:1) (“D5”) or Asp-Glu-Val-Asp (SEQ ID NO:2) (“DEVD”).
Preferred self-assembling peptides are (Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys)2 (SEQ ID NO:3) or Phe-Phe.
The target recognition motif preferably has an affinity for a cell membrane receptor. Preferably, the target recognition motif has a cyclic (Arg-Gly-Asp) residue (“cRGD”) having an affinity for integrin αvβ3. Alternatively, the target recognition motif can be a lysosomal protein transmembrane 4 beta.
The luminogens of the light-up probes of the present invention comprise at least those fluorogens described herein, as well as tetraphenylsilole (“TPS”), tetraphenylethene pyridinium (“PyTPE”) and tetraphenylethylene (“TPE”).
The components of the light-up probe composition are covalently linked in a linear array. The determination of components and the order of linkage can be varied and are selected based on the desired application of the light-up probe. The following Table illustrates exemplary component selections as well as exemplary orders of linkage of said components (read from left to right).
This table is for illustration purposes only and does not reflect all possible component selections nor does it reflect all possible orders of linkage. For instance, when AIE-ESIPT fluorogens are used as the luminogen, the hydrophilic moiety can be removed, and when two luminogens are present, the second luminogen can be inserted after the third component (hydrophilic moiety or linking moiety) but before the fourth component (target recognition motif).
In another embodiment of the light-up probe aspect of the present invention, there are specific AIE light-up probes with dual imaging functionality. An example of such a probe is TPE-DEVD-DOTA/Gd (“DDT-Gd”).
Detailed synthetic routes as well as testing parameters and results for several exemplary probes (e.g. the probes of Table rows A, O and X, and the DDT-Gd probe) can be found in the Exemplification section of this application.
The light-up probes of the present invention can be used generally for conducting fluorescence imaging and magnetic resonance imaging and specifically for assessing the conversion of a prodrug into its active form, assessing the therapeutic efficacy of a prodrug, detecting glutathione in a biological sample, and detecting alkaline phosphatase in a sample. A more detailed description of the applications aspect of the invention follows.
While the light-up probes of the present invention can be used for conducting fluorescence imaging and magnetic resonance imaging generally, the following discussion will focus on uses for non-invasive early evaluation of therapeutic responses in-situ, selective and real-time monitoring of drug activation in-situ, targeted intracellular thiol imaging, and alkaline phosphatase (ALP) detection.
The inventors of the present invention have developed a strategy for real-time monitoring of cell apoptosis in-vitro and in-vivo based on light-up probes of the present invention that contain fluorogens with AIE (or AIE-ESIPT) characteristics.
An embodiment of this aspect of the present invention thus pertains to a method for assessing the therapeutic efficacy of a prodrug, comprising:
a) incubating a biological sample comprising live target cells with a chemical composition of the invention under conditions sufficient to convert the prodrug into its active form, to form an incubated mixture; and
b) analyzing the incubated mixture of step a) by fluorescence spectroscopy,
wherein an increase in fluorescence intensity as compared to the fluorescence intensity of the chemical composition not in the presence of the biological sample is indicative of the efficacy of the active drug.
With specific reference to the light-up probe TPS-DEVD-Pt-cRGD (see row A of Table), the method of the present invention for assessing the therapeutic efficacy of a prodrug will be explained and described in more detail as follows.
A targetable theranostic Pt(IV) prodrug was developed with a special focus on monitoring drug induced apoptosis in-situ. The theranostic system comprises a chemotherapeutic Pt(IV) prodrug which can be reduced to active Pt(II) intracellularly, an apoptosis sensor (TPS-DEVD) based on tetraphenylsilole (TPS) with AIE characteristic and a cyclic (RGD) peptide as a targeting ligand (Scheme 1). The prodrug can accumulate preferentially in cancer cells with overexpressed αvβ3 integrin and release the active drug Pt(II) and apoptosis sensor TPS-DEVD upon the intracellular reduction of Pt(IV) prodrug. The released Pt(II) can induce cell apoptosis and activate caspase-3 to cleave the DEVD peptide in TPS-DEVD and trigger fluorescence. The fluorescence turn-on response can be utilized in the theranostic system for real-time and noninvasive imaging of therapeutic responses of a specific anticancer drug. The cancer cells of the theranostic system include, for example, U87-MG, MDA-MB-231 and HT29. Contemplated anticancer drugs include, for instance, doxorubicin and paclitaxel.
The inventors of the present invention have developed a simple strategy for in situ monitoring of drug activation utilizing the light-up probes of the present invention that contain fluorogens with AIE (or AIE-ESIPT) characteristics.
An embodiment of this aspect of the present invention thus pertains to a method for assessing the conversion of a prodrug into its active form, the method comprising:
a) incubating a biological sample with the above-noted chemical composition under conditions sufficient to form an incubated mixture; and
b) analyzing the fluorescence of the incubated mixture of step a) using a microplate reader,
wherein an increase in fluorescence intensity as compared to the fluorescence intensity of the above-noted chemical composition not in the presence of the biological sample is indicative of the conversion of the prodrug into its active form.
Preferably this method is conducted in a live cell.
With specific reference to the light-up probe PyTPE-Pt-D5-cRGD (see row O of Table), the method of the present invention for assessing the conversion of a prodrug into its active form will be explained and described in more detail below.
The design and synthesis of a targeted theranostic platinum(IV) prodrug delivery system was developed. This system was based on an AIE luminogen for in situ monitoring of the platinum(IV) prodrug activation. The theranostic system, comprises a chemotherapeutic prodrug Pt(IV) that can be reduced to active Pt(II) inside the cells, a tetraphenylethene pyridinium (PyTPE) unit with AIE characteristics, a short hydrophilic peptide with five aspartic acid (D5) units to ensure its water solubility and a cyclic (RGD) peptide (cRGD) as a targeting ligand (Scheme 2). The prodrug can accumulate preferentially in cancer cells that overexpress αvβ3 integrin and can be utilized as an excellent guiding molecule to tumor cells, for example, U87-MG, MDA-MB-231 and HT29 cells. In aqueous media, the AIE moiety is non-fluorescent due to the high hydrophilicity of the D5-cRGD, but its emission is enhanced significantly after the reduction of the Pt(IV) complex, which releases the two axials. The fluorescent enhancement (“turn-on”) is attributed to the restriction of intramolecular rotations of the PyTPE phenyl rings in the cleaved residues, which populates the radiative decay channels. The prodrug design of the invention offers good opportunity for efficient targeted platinum drug delivery and real-time monitoring of the release and distribution of the drug with a high signal-to-noise ratio.
The inventors of the present invention have developed a strategy for cell specific intracellular thiol (e.g. glutathione) imaging utilizing the light-up probes of the present invention that contain fluorogens with AIE (or AIE-ESIPT) characteristics.
An embodiment of this aspect of the present invention thus pertains to a method of detecting glutathione in a biological sample, the method comprising:
a) incubating a biological sample thought to contain glutathione with the above-noted chemical composition under conditions sufficient to form an incubated mixture; and
b) analyzing the incubated mixture of step a) by fluorescence spectroscopy,
wherein an increase in fluorescence intensity as compared to the fluorescence intensity of the above-noted chemical composition not in the presence of the biological sample is indicative of the presence of glutathione.
In the method of detecting glutathione, the fluorescence intensity of the incubated mixture preferably increases with increased concentration of glutathione.
With specific reference to the light-up probe TPE-SS-D5-cRGD (see row X of Table), the method of the present invention for detecting glutathione will be explained and described in more detail below.
An integrin αvδ3 targeted light-up probe was designed for cell specific intracellular thiol imaging. The probe comprises a targeted cyclic RGD (cRGD) peptide, a highly water soluble peptide with five aspartic acids (Asp, D5), a TPE fluorogen and a thiol-specific cleavable disulfide linker. cRGD exhibits high binding affinity to αvβ3 integrin which is a unique molecular biomarker for early detection and treatment of rapidly growing solid tumors comprising, for example, U87-MG, MDA-MB-231 and HT29 cancer cells. The probe is highly water soluble and is almost non-fluorescent in aqueous media. The cleavage of the disulfide group by thiols leads to enhanced fluorescence signal output (Scheme 3). This probe can thus be used for real-time monitoring of thiol (glutathione) level in specific tumor cells.
The inventors of the present invention have developed a strategy for detecting alkaline phosphatase utilizing the light-up probe Phos-HC that has AIE-ESIPT characteristics.
An embodiment of this aspect of the present invention thus pertains to a method for the detection of alkaline phosphatase in a sample, comprising:
a) incubating a sample thought to comprise alkaline phosphatase with Phos-HC under conditions sufficient to form an incubated media; and
b) analyzing the incubated media of step a) by fluorescence spectroscopy,
wherein an increase in fluorescence intensity of a fluorescence signal at about 641 nm is indicative of the presence of alkaline phosphatase.
The sample of the method is preferably a live cell.
A more detailed description of this method of the present invention for detecting glutathione, including detection scheme and optical results, can be found in the Exemplification section of this application.
Detailed Synthetic Routes
To a solution of (4-aminophenyl)(phenyl)methanone (1.970 g, 10 mmol) in THF (30 mL) was added sodium hydride (1.200 g, 30 mmol, 3 equiv, 60% suspension in oil) slowly at 0° C., the reaction was kept for 2 h, then bromoethane (2.24 mL, 30 mmol, 3 equiv) was injected. The reaction mixture was warmed slowly to room temperature and stirred overnight. The solution was diluted with dichloromethane and washed with aq. NaHCO3 solution and brine. The organic layer was dried over sodium sulfate. The filtrate was concentrated and purified by silica gel column chromatography (ethyl acetate/hexane=1/10) to yield the yellow solid (2.302 g, 91%).
To a solution of (4-aminophenyl)(phenyl)methanone (0.986 g, 5 mmol) in THF (30 mL) was added sodium hydride (0.600 g, 15 mmol, 3 equiv, 60% suspension in oil) slowly at 0° C., the reaction was kept for 2 h, then n-bromohexane (2.476 g, 15 mmol, 3 equiv) was injected. The reaction mixture was warmed slowly to room temperature and stirred overnight. The solution was diluted with dichloromethane and washed with aq. NaHCO3 solution and brine. The organic layer was dried over sodium sulfate. The filtrate was concentrated and purified by silica gel column chromatography (ethyl acetate/hexane=1/10) to yield the yellow solid (1.605 g, 88%).
Under an Ar (g) atmosphere, a two-necked flask equipped with a magnetic stirrer was charged with zinc powder (1.308 g, 20 mmol) and 40 mL THF. The mixture was cooled to −5 to 0° C., and TiCl4 (1.09 mL, 10 mmol) was slowly added by a syringe with the temperature kept under 10° C. The suspending mixture was warmed to room temperature and stirred for 0.5 h, then heated at reflux for 2.5 h. The mixture was again cooled to −5 to 0° C., charged with pyridine (0.5 mL, 6 mmol) and stirred for 10 min. The solution of A (506 mg, 2 mmol)+B (570 mg, 2 mmol) in 15 mL THF was added slowly. After addition, the reaction mixture was heated at reflux for overnight. The reaction was quenched with 10% K2CO3 aqueous solution and taken up with CH2Cl2. The organic layer was collected and concentrated. The crude material was purified by silica gel column chromatography (EA/DCM=2:5) to give the desire yellow products (0.360 g, 36%).
Under an Ar (g) atmosphere, a two-necked flask equipped with a magnetic stirrer was charged with zinc powder (1.308 g, 20 mmol) and 40 mL THF. The mixture was cooled to −5 to 0° C., and TiCl4 (1.09 mL, 10 mmol) was slowly added by a syringe with the temperature kept under 10° C. The suspending mixture was warmed to room temperature and stirred for 0.5 h, then heated at reflux for 2.5 h. The mixture was again cooled to −5 to 0° C., charged with pyridine (0.5 mL, 6 mmol) and stirred for 10 min. The solution of A (731 mg, 2 mmol)+B (570 mg, 2 mmol) in 15 mL THF was added slowly. After addition, the reaction mixture was heated at reflux for overnight. The reaction was quenched with 10% K2CO3 aqueous solution and taken up with CH2Cl2. The organic layer was collected and concentrated. The crude material was purified by silica gel column chromatography (EA/DCM=2:5) to give the desire yellow products (0.360 g, 29%).
Under an Ar (g) atmosphere, a two-necked flask equipped with a magnetic stirrer was charged with C2-TPE-Py (100 mg, 0.197 mmol) and 1,3-Propanesultone (241 mg, 1.97 mmol) in methanol (10 mL). The reaction mixture was refluxed for 24 h, then solvent was removed under vacuum and the residue was subjected for silica gel column chromatography (methanol/DCM=1/3 to pure MeOH) to yield the yellow solid (91 mg, 73%).
Under an Ar (g) atmosphere, a two-necked flask equipped with a magnetic stirrer was charged with C6-TPE-Py (62 mg, 0.1 mmol) and 1,3-Propanesultone (122 mg, 1.0 mmol) in methanol (10 mL). The reaction mixture was refluxed for 24 h, then solvent was removed under vacuum and the residue was subjected for silica gel column chromatography (methanol/DCM=1/3 to pure MeOH) to yield the yellow solid (61 mg, 82%).
To a solution of (4-aminophenyl)(phenyl)methanone (1.970 g, 10 mmol) in THF (30 mL) was added sodium hydride (1.200 g, 30 mmol, 3 equiv, 60% suspension in oil) slowly at 0° C., the reaction was kept for 2 h, then iodomethane (1.87 mL, 30 mmol, 3 equiv) was injected. The reaction mixture was warmed slowly to room temperature and stirred overnight. The solution was diluted with dichloromethane and washed with aq. NaHCO3 solution and brine. The organic layer was dried over sodium sulfate. The filtrate was concentrated and purified by silica gel column chromatography (ethyl acetate/hexane=1/5) to yield the yellow solid (2.010 g, 89%).
Under an Ar (g) atmosphere, a two-necked flask equipped with a magnetic stirrer was charged with zinc powder (2.616 mL, 20 mmol) and 40 mL THF. The mixture was cooled to −5 to 0° C., and TiCl4 (2.16 mL, 20 mmol) was slowly added by a syringe with the temperature kept under 10° C. The suspending mixture was warmed to room temperature and stirred for 0.5 h, then heated at reflux for 2.5 h. The mixture was again cooled to −5 to 0° C., charged with pyridine (1.0 mL, 12 mmol) and stirred for 10 min. The solution of A (900 mg, 4 mmol)+B (805 mg, 4 mmol) in 30 mL THF was added slowly. After addition, the reaction mixture was heated at reflux for overnight. The reaction was quenched with 10% K2CO3 aqueous solution and taken up with CH2Cl2. The organic layer was collected and concentrated. The crude material was purified by silica gel column chromatography (EA/DCM=2:5) to give the desired yellow products (0.280 g, 23%).
Under an Ar (g) atmosphere, a two-necked flask equipped with a magnetic stirrer was charged with A (2.616 g, 10 mmol), 4-vinylpyridine (1.25 mL, 11 mmol), Pd(OAc)2 (90 mg, 4% mmol), P(o-Tolyl)3 (426 mg, 14% mmol), Et3N (36 mL), DMF (24 mL), were heated to 110° C. for 30 h. The reaction was then diluted with water and the aqueous phase was washed with CH2Cl2 and extracted with CHCl3. Collecting all the organic layers, and evaporate the solvents, a yellow crude product was collected, then recrystallization (EA/CHCl3) was performed, and the title product was achieved as yellow powder (2.600 g, 91%).
Under an Ar (g) atmosphere, a two-necked flask equipped with a magnetic stirrer was charged with zinc powder (2.616 mL, 20 mmol) and 40 mL THF. The mixture was cooled to −5 to 0° C., and TiCl4 (2.16 mL, 20 mmol) was slowly added by a syringe with the temperature kept under 10° C. The suspending mixture was warmed to room temperature and stirred for 0.5 h, then heated at reflux for 2.5 h. The mixture was again cooled to −5 to 0° C., charged with pyridine (1.0 mL, 12 mmol) and stirred for 10 min. The solution of A (729 mg, 4 mmol)+B (805 mg, 4 mmol) in 30 mL THF was added slowly. After addition, the reaction mixture was heated at reflux for overnight. The reaction was quenched with 10% K2CO3 aqueous solution and taken up with CH2Cl2. The organic layer was collected and concentrated. The crude material was purified by silica gel column chromatography (EA/DCM=2:5) to give the desired yellow products (0.230 g, 22%).
Under an Ar (g) atmosphere, a two-necked flask equipped with a magnetic stirrer was charged with A (100 mg, 0.33 mmol), B (1.00 g, 10 eq) in CH3CN (10 mL), the reaction mixture was refluxed for at least 36 h. The reaction was quenched until the consumption of the starting material A. The solvent was evaporated and subjected for column chromatography (DCM, CH3OH), the salt was achieved as a red solid (120 mg, 71%).
Under an Ar (g) atmosphere, a two-necked flask equipped with a magnetic stirrer was charged with A (50 mg, 0.10 mmol), B (52 mg, 2 eq) in a mixture of MeOH (10 mL) and THF (5 mL), the reaction mixture was refluxed for at least 48 h. The reaction was quenched until the consumption of the starting material A. The solvent was evaporated and subjected for column chromatography (DCM, CH3OH), the salt was achieved as a red solid (the yield was not calculated).
Under an Ar (g) atmosphere, a two-necked flask equipped with a magnetic stirrer was charged with zinc powder (1.308 g, 20 mmol) and 40 mL THF. The mixture was cooled to −5 to 0° C., and TiCl4 (1.09 mL, 10 mmol) was slowly added by a syringe with the temperature kept under 10° C. The suspending mixture was warmed to room temperature and stirred for 0.5 h, then heated at reflux for 2.5 h. The mixture was again cooled to −5 to 0° C., charged with pyridine (0.5 mL, 6 mmol) and stirred for 10 min. The solution of A (870 mg, 2 mmol)+B (680 mg, 2 mmol) in 15 mL THF was added slowly. After addition, the reaction mixture was heated at reflux for overnight. The reaction was quenched with 10% K2CO3 aqueous solution and taken up with CH2Cl2. The organic layer was collected and concentrated. The mixed crude material was purified by silica gel column chromatography a yellow mixture. The mixture was placed in a sealed tube, and then charged with 4-vinylpyridine (0.62 mL, 5.5 mmol), Pd(OAc)2 (45 mg, 4% mmol), P(o-Tolyl)3 (213 mg, 14% mmol), Et3N (12 mL), DMF (8 mL). The reaction was heated to 110° C. for 24 h. The reaction was then diluted with water and the aqueous phase was washed with CH2Cl2 and extracted with CHCl3. The organic layer was collected and concentrated. The crude material was purified by silica gel column chromatography (EA/DCM=2:5) to give the desired yellow products (0.260 g, 16% in 2 steps).
AIE Behavior of DMA-HC
Upon addition of water to the THF solution of DMA-HC, the solution fluorescence red-shifts and intensifies upon aggregation formation. See
ALP Detection
The probe-1 gives distinct optical response to ALP in solution. The emission maximum of the solution changes from 520 nm to 640 nm in the presence of ALP. See
The same probe can also be used for cellular ALP detection. See
The probe thus demonstrates a new strategy for AIE based light-up sensing and imaging.
General Information
Cisplatin, N,N-diisopropylethylamine (DIEA), N-hydroxysuccinimide (NHS), 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), copper(II) sulfate (CuSO4), sodium ascorbate, ascorbic acid, succinic anhydride, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), anhydrous dimethyl sulfoxide (DMSO), anhydrous dimethylformamide (DMF), lithium wires, naphthalene, 4-bromobenzene, 4-bromobenzyl bromide, sodium azide, dichlorobis(triphenylphosphine)palladium(II), ZnCh.TMEDA, piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), diethyldithiocarbamate (DDTC), bovine serum albumin (BSA), lysozyme, pepsin, trypsin and other chemicals were all purchased from Sigma-Aldrich and used as received without further purification. Hexane and tetrahydrofuran (THF) purchased from Fisher Scientific were distilled from sodium benzophenoneketyl immediately prior to use. Dichloromethane (DCM) was distilled over calcium hydride. Deuterated solvents with tetramethylsilane (TMS) as internal reference were purchased from Cambridge Isotope Laboratories Inc. Alkyne-functionalized DEVD (Asp-GluVal-Asp-Pra) and amine-functionalized cRGD (cyclic(Arg-Gly-Asp-D-Phe-Lys)) were customized from GL Biochem Ltd. cis,cis,trans-Diamminedichlorodisuccinatoplatinum(IV) was synthesized following a literature method. [1]
Dulbecco's modified essential medium (DMEM) was a commercial product of National University Medical Institutes (Singapore). Milli-Q water was supplied by Milli-Q Plus System (Millipore Corporation, Breford, USA). Piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer containing 50 mM PIPES, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% w/v 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic and 25% w/v sucrose (pH=7.2). Recombinant human caspase-3 was purchased from R&D Systems. Caspase-3 inhibitor 5-[(S)-(+)-2-(methoxymethyl)pyrrolidino]sulfonylisatin was purchased from Calbiochem. Fetal bovine serum (FBS) and trypsin-EDTA solution were purchased from Gibco (Lige Technologies, AG, Switzerland). Staurosporine (STS) was purchased from Biovision. DRAQ5 was purchased from Biostatus. Cleaved caspase-3 (Asp 175) (5A1E) rabbit mAb (#9664) was purchased from Cell Signaling. Mouse anti-rabbit IgG-TR (sc-3917) was purchased from Santa Cruz.
Characterization
NMR spectra were measured on a Bruker ARX 400 NMR spectrometer. Chemical shifts were reported in parts per million (ppm) referenced with respect to residual solvent (CDCl3=7.26 ppm, (CD3)2SO=2.50 ppm or tetramethylsilane Si(CH3)4=0 ppm). Particle size and size distribution were determined by laser light scattering (LLS) with a particle size analyzer (90 Plus, Brookhaven Instruments Co., USA) at a fixed angle of 90° at room temperature. HPLC profiles and mass spectra were acquired using a Shimadzu IT-TOF. A 0.1% TFA/H20 and 0.1% TFA/acetonitrile were used as eluents for the HPLC experiments. High resolution mass spectra (HRMS) were recorded on a Finnigan MAT TSQ 7000 Mass Spectrometer. UV-vis absorption spectra were taken on a Milton Ray Spectronic 3000 array spectrophotometer. Photoluminescence (PL) spectra were measured on a Perkin-Elmer LS 55 spectrofluorometer. The cells were imaged by confocal laser scanning microscope (CLSM, Zeiss LSM 410, Jena, Germany) with imaging software (Fluoview FV500). The images were analyzed by Image J 1.43 x program (developed by NIH, http://rsbweb.nih.gov/ij/).
Into a flask equipped with a magnetic stirrer were added 4-bromobenzyl bromide (7.5 g, 30 mmol), sodium azide (7.8 g, 120 mmol) and 40 mL of DMSO. After stirring at 70° C. for 12 h, the solution was poured into 150 mL of water and extracted with DCM. The crude product was purified by silica-gel chromatography using hexane as eluent to give a colorless viscous liquid in 96% yield (6.12 g). 1H NMR (CDCl3, 400 MHz), δ (TMS, ppm): 7.47 (d, 2H), 7.15 (d, 2H), 4.26 (s, 2H). 13C NMR (CDCl3, 100 MHz), δ (TMS, ppm): 134.3, 131.8, 129.6, 122.1, 53.9. HRMS (MALDI-TOF): m/z 210.9640 (M+, calcd 210.9745).
Dimethylbis(phenylethynyl)silane was prepared according to our published procedures. [2] A mixture of lithium (0.056 g, 8 mmol) and naphthalene (1.04 g, 8 mmol) in 8 mL of THF was stirred at room temperature under nitrogen for 3 h to form a deep dark green solution of LiNaph. A solution of dimethylbis(phenylethynyl)silane (0.52 g, 2 mmol) in 5 mL of THF was then added dropwise to LiNaph solution at room temperature. After stirring for 1 h, the mixture was cooled to 0° C. and then diluted with 25 mL of THF. A black suspension was formed upon addition of ZnCl2.TMEDA (2 g, 8 mmol). After stirring for an additional hour at room temperature, a solution containing 4-bromobenzene (0.34 g, 2.2 mmol), 4-bromobenzyl azide (0.47 g, 2.2 mmol) and PdCl2(PPh3)2 (0.08 g, 0.1 mmol) in 25 mL of THF was added. The mixture was refluxed overnight. After cooling down to room temperature, 100 mL of 1M HCl solution was added and the mixture was extracted with DCM several times. The organic layer was combined and washed with brine and water and then dried over magnesium sulfate. After solvent evaporation under reduced pressure, the residue was purified by a silica-gel column using hexane as eluent. The product was obtained as a yellow solid in 36% yield (0.34 g). 1H NMR (400 MHz, CDCl3), δ (TMS, ppm): 7.15-7.06 (m, 6H), 7.02-6.99 (m, 5H), 6.95-6.93 (m, 4H), 6.82-6.79 (m, 4H), 4.23 (s, 2H), 0.48 (s, 6H). 13C NMR (CDCl3, 100 MHz), δ (TMS, ppm): 154.5, 153.9, 142.1, 141.2, 140.1, 139.8, 138.7, 132.5, 130.0, 129.2, 128.9, 128.0, 127.5, 126.4, 126.3, 125.7, 54.7, −3.80. HRMS (MALDI-TOF): m/z 469.1959 (M+, calcd 469.1974).
A mixture of platinum(IV) complex cis,cis,trans-diamminedichlorodisuccinatoplatinum(IV) (32.1 mg, 0.06 mmol), EDC (23.0 mg, 0.12 mmol) and NHS (13.8 mg, 0.12 mmol) in anhydrous DMF (1 mL) was stirred at room temperature overnight. After that, the mixture was purified by HPLC (solvent A: water with 0.1% TFA, solvent B: CH3CN with 0.1% TFA) and quickly lyophilized to yield the desired product as a white powder in 78% yield (34.1 mg). 1H NMR (400 MHz, DMF-d7), δ (TMS, ppm): 6.92-6.68 (m, 6H), 2.94-2.91 (m, 8H), 2.89-2.84 (m, 4H), 2.72-2.68 (m, 4H). 13C NMR (DMF-d7, 100 MHz), δ (TMS, ppm): 178.5, 170.6, 168.8, 30.0, 27.1, 25.9. IT-TOF-MS: m/z [M+H]+ calc. 728.026. found 728.021.
Alkyne-functionalized DEVD (10.2 mg, 20 μmol) and TPS-CH2N3 (9.4 mg, 20 μmol) were dissolved in a mixture of DMSO/H2O solution (v/v=1/1; 1.0 mL). The “click” reaction was initiated by sequential addition of catalytic amounts of CuSO4 (9.6 mg, 6 mop and sodium ascorbate (2.4 mg, 12 μmol). The reaction was continued with shaking at room temperature for another 24 h. The final product was purified by HPLC and lyophilized under vacuum to yield the probe as white powders in 45% yield (9.4 mg). 1H NMR (DMSO-d6, 400 MHz): 12.24 (s, 3H), 8.49 (d, 1H), 8.32 (d, 1H), 8.05 (d, 1H), 7.92 (d, 1H), 7.86 (s, 1H), 7.22-7.06 (m, 6H), 7.02-6.99 (m, 5H), 6.95-6.88 (m, 4H), 6.82-6.79 (m, 4H), 5.45 (s, 2H), 4.54-4.49 (m, 1H), 4.38 (m, 2H), 4.17-4.13 (m, 2H), 3.10-3.05 (m, 1H), 2.92-2.88 (m, 1H), 2.71-2.65 (m, 2H), 2.26-2.21 (m, 2H), 2.01-1.85 (m, 3H), 0.84-0.74 (m, 6H), 0.43 (s, 6H); IT-TOF-MS: m/z [M+H]+ calc. 1040.426. found 1040.866.
TPS-DEVD-NH2 (9.0 mg, 8.7 mmol) and amine-functionalized cRGD (5.2 mg, 8.7 mmol) were dissolved in anhydrous DMSO (1.0 mL) with a catalytic amount of DIEA (1.0 μL). The mixture was stirred at room temperature for 10 min. Then N-hydroxysuccinimide-activated platinum(IV) complex (6.3 mg, 8.7 mmol) in DMSO (0.5 mL) was added quickly to the above mixture. The reaction was continued with stirring at room temperature for another 24 h. The final product was purified by HPLC and lyophilized under vacuum to yield the prodrug as white powders in 40% yield (7.4 mg). 1H NMR (DMSO-d6, 400 MHz): 12.24 (s, 3H), 8.55 (d, 1H), 8.51 (d, 1H), 8.31 (d, 1H), 8.15-8.05 (m, 6H), 7.91 (d, 1H), 7.86 (s, 1H), 7.62 (d, 2H), 7.56 (d, 1H), 7.45 (m, 1H), 7.22-7.06 (m, 6H), 7.02-6.99 (m, 5H), 6.95-6.88 (m, 4H), 6.82-6.79 (m, 4H), 6.60-6.35 (m, 6H), 5.45 (s, 2H), 4.65-4.60 (m, 1H), 4.54-4.48 (m, 1H), 4.40-4.32 (m, 3H), 4.17-4.10 (m, 3H), 4.05-4.02 (m, 1H), 3.95-3.91 (m, 1H), 3.10-3.06 (m, 4H), 2.95-2.87 (m, 2H), 2.85-2.78 (m, 2H), 2.75-2.62 (m, 6H), 2.50-2.45 (m, 8H), 2.27-2.23 (m, 4H), 1.93-1.89 (m, 4H), 1.72 (m, 3H), 1.41-1.38 (m, 2H), 1.37-1.32 (m, 2H), 0.84-0.74 (m, 6H), 0.43 (s, 6H); ESI-MS: m/z [M+H]+ calc. 2141.719. found 2141.689.
General Procedure for Enzymatic Assay
DMSO stock solutions of TPS-DEVD-Pt-cRGD were diluted with a mixture of DMSO and PIPES (v/v=1/199) to 10 μM. Next, each probe was incubated with ascorbic acid or caspase-3 at room temperature and the change of fluorescence intensity was measured. The PL spectra were collected from 420 to 650 nm under an excitation wavelength at 365 nm.
Cell Culture
U87-MG human glioblastoma cancer cells, MCF-7 breast cancer cells and 293T normal cells were provided by American Type Culture Collection (ATCC). The cells were cultured in DMEM (Invitrogen, Carlsbad, Calif.) containing 10% heat-inactivated FBS (Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin (Thermo Scientific) and maintained in a humidified incubator at 37° C. with 5% C02. Before experiment, the cells were pre-cultured until confluence was reached.
Confocal Imaging
U87-MG, MCF-7 and 293T cells were cultured in the chambers (LAB-TEK, Chambered Coverglass System) at 37° C. After 80% confluence, the culture medium was removed and washed twice with PBS buffer. The probe in DMSO stock solution was then added to the chamber to reach a final concentration of 5 μM. In some experiments, the cells were pre-incubated with media containing cRGD (50 μM) or inhibitor (5 μM) prior to prodrug incubation. After incubation the prodrug at 37° C. for 2 h, the medium was replaced with fresh medium, after that the cells were washed twice with ice-cold PBS and the cell nucleus was living stained with DRAQ5 (Biostatus) following the standard protocol of the manufacturer. For co-localization with active caspase-3 antibody, the cells were first fixed for 15 min with 3.7% formaldehyde in 1×PBS at room temperature, washed twice with cold PBS again, and permeabilized with 0.1% Triton X-100 in PBS for 10 min. The cells were then blocked with 2% BSA in 1×PBS for 30 min and washed twice with PBS. The cells were subsequently incubated with a mixture of anti-caspase-3 antibody/PBS (v/v=1/99) for 1 h at, room temperature, washed once with PBS buffer, and then incubated with mouse anti-rabbit IgG-TR (0.8 μg mL−1) in PBS for I h, following by washing with PBS again. The cells were then imaged immediately by confocal laser scanning microscope (CLSM, Zeiss LSM 410, Jena, Germany). The images were analyzed by Image J 1.43 x program (developed by NIH, http://rsbweb.nih.gov/ij/).
Quantification of Cell Apoptosis by Fluorescence Microplate Reader
U87-MG and MCF-7 cells were seeded in 96-well plates (Costar, USA) at an intensity of 4×104 cells mL−1. After confluence, the medium was replaced by different concentrations of TPS-DEVD-Pt-cRGD in fresh PBS-free DMEM medium. After the determined incubation time at 37° C., the adherent cells were washed twice with 1×PBS buffer followed by fluorescence measurement using a T-CAN microplate reader. The excitation and emission wavelengths are 365 and 480 nm, respectively.
Cytotoxicity of the Prodrug
3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were used to assess the metabolic activity of U87-MG and MCF-7 cancer cells. The cells were seeded in 96-well plates (Costar, Ill., USA) at an intensity of 4×104 cells mL−1. After 24 h incubation, the medium was replaced by the probe suspension at different concentration of the prodrug and incubated at 37° C. After the designated time intervals, the wells were washed twice with 1×PBS buffer, and 100 μL of freshly prepared MTT (0.5 mg mL−1) solution in culture medium was added into each well. The MTT medium solution was carefully removed after 3 h incubation in the incubator at 37° C. DMSO (100 μL) was then added into each well and the plate was gently shaken to dissolve all the precipitates formed. The absorbance of MTT at 570 nm was monitored by the microplate reader (Genios Tecan). Cell viability was expressed by the ratio of absorbance of the cells incubated with probe suspension to that of the cells incubated with culture medium only.
Azide-functionalized tetraphenylsilole (TPS-CH2N3) was synthesized by the heterobifunctional modification of dimethylbis(phenylethynyl)silane with 4-bromobenzene and 4-bromobenzyl azide. Detailed synthesis and characterization of TPS-CH2N3 and the intermediates are shown in the Experimental Section and Supporting Information. The coupling between TPS-CH2N3 and alkyne-functionalized DEVD via “click” reaction using CuS04/sodium ascorbate as the catalyst in DMSO/water (v/v=1/1) afforded the apoptosis sensor TPS-DEVD-NH2 in 45% yield after HPLC purification. The purity and identity of the probe was well characterized by analytical HPLC, NMR, and HRMS. Commercially available anticancer drug cisplatin was modified to be used as the linker between TPS-DEVD-NH2 and the amine functionalized cRGD. In the first step, cisplatin was oxidized by hydrogen peroxide to produce cis, cis, trans-diaminedichlorodihydroxyplatinum(IV) complex. Next, the Pt(IV) complex was reacted with succinic anhydride in DMSO at 70° C. for 12 h to yield cis, cis, trans-diamminedichlorodisuccinatoplatinum(IV) complex. The activated Pt(IV) complex was subsequently obtained by reacting the carboxylic acid groups with NHS in anhydrous DMF using EDC as the coupling reagent. The activated Pt(IV) linker was purified by HPLC and lyophilized as white powder with a yield of 78%. Asymmetric functionalization of activated Pt(IV) linker with TPS-DEVD-NH2 and aminefunctionalized cRGD in the presence of N,N-diisopropylethylamine (DIEA) in anhydrous DMSO afforded the desired product, TPS-DEVD-Pt-cRGD in 40% yield after HPLC purification (Scheme 4). The purity and identity of the probe was well characterized by HPLC, NMR, and HRMS.
For any prodrug, it is essential that it can be easily transformed into its original form to restore its therapeutic ability after the modification. To evaluate our prodrug as a potential drug delivery system, we studied the nature of the formed Pt(II) species upon reduction of the synthesized prodrug. It is reported that diethyldithiocarbamate (DDTC) can react with Pt(II) complexes to yield the adducts Pt(DDTC)2 but does not react with stable Pt(IV) complexes. [3][4] In this work, we use HPLC-MS system to monitor the adducts formation of Pt(IV) complexes before and after the reduction with ascorbic acid in the presence of DDTC. We choose ascorbic acid as a reduction agent because it is highly abundant in the cells (1 mM), which has been demonstrated to be a major substance for the reduction of Pt(IV). [5] As shown in
We next studied the optical properties of our prodrug. The UV-vis absorption spectra of TPS-CH2N3 in THF and TPS-DEVD-Pt-cRGD in DMSO/PIPES (v/v=1/199) buffer were obtained. Both have similar absorption profiles with an obvious absorbance in the 320-440 nm range with a little blue shift after the modification of the AIE fluorogen. It is known that AIE fluorogen is non-fluorescent in good solvents but emits intensely in solid or as aggregates in poor solvents. [6][7] As can be seen from the photoluminescence (PL) spectra shown in
First, we demonstrated that the fluorescence of TPS-DEVD-NH2 in DMSO/PIPEs (v/v=1/199) increased upon the addition of caspase-3 for 60 min.
The fluorescence change of the TPS-DEVD-Pt-cRGD solution (10 μM) upon addition of ascorbic acid and caspase-3 enzyme was also monitored over time in a mixture of DMSO/PIPEs (v/v=1/199) buffer. As shown in
To study the selectivity of the prodrug, we incubated ascorbic acid pretreated TPS-DEVD-Pt-cRGD (10 μM) with several proteins, including lysozyme, pepsin, bovine serum albumin (BSA) and trypsin under identical conditions. As shown in
To explore the capability of using TPS-DEVD-Pt-cRGD as a targeted drug delivery system and a drug induced apoptosis imaging probe in cancer cells. We first incubated TPS-DEVD-NH2 with U87-MG cells at 37° C. After 2 h of incubation, the cells were treated with cisplatin or staurosporine which both can activate the cell apoptosis and monitored with confocal microscopy. It was seen that the normal, un-induced cells show very low fluorescence intensity, indicating little or no caspase-3 activity. In sharp contrast, strong fluorescence signals are collected from the cells treated with cisplatin or staurosporine. These results demonstrate that TPS-DEVD-NH2 can be used as an indicator of cell apoptosis. We next incubated the prodrug TPS-DEVD-Pt-cRGD with U87-MG human glioblastoma, MCF-7 breast cancer cell lines and normal cell line 293T cells, the confocal imaging results are shown in
We next compared the relationship between the apoptosis induced fluorescence intensity change in cells and the cytotoxicity profile of the prodrug using both U87-MG and MCF-7 cells as an example. The fluorescence intensities at 480 nm are monitored upon excitation at 365 nm after incubation the cells for 6 h. The cytotoxicity of the cells was evaluated by a standard MTT method after the incubation for 72 h. In this experiment, we studied this effect using both U87-MG and MCF-7 cells. As shown in
In summary, we report the synthesis and biological application of a theranostic Pt(IV) prodrug for targeted drug delivery and early evaluation of its therapeutic response in situ. The prodrug can be reduced to active Pt(II) inside the cells and simultaneously release the cell apoptosis sensor on its axial position. The reduced Pt(II) can induce the apoptosis of the cancer cell and activate the caspase-3. The activated caspase-3 further cleaves DEVD sequence of the apoptosis sensor and triggers the AIE effect of TPS residue, thus enabling early evaluation of its therapeutic response in cells with high signal-to noise ratios. In addition, we found that the fluorescence intensity induced by apoptosis when incubated with our prodrug for 6 h shows a good correlation with that of the cell viability of the cells determined by MTT assay for 72 h, that is, with lower fluorescence intensity will indicate higher cell viability and vice versa. These results indicate that the theranostic drug delivery system with a build-in apoptosis sensor allows one to evaluate the drug therapeutic response quickly, which is essential to guide therapeutic decisions such as whether the treatment works well or the therapeutic regimes should stop.
Amine-functionalized PyTPE (PyTPE-NH2) was synthesized by reducing azide-PyTPE (PyTPE-N3) in methanol. Pentafluorophenol-activated Pt(IV) complex was prepared from commercially available anticancer drug cisplatin and was used as the linker. The synthetic route for the prodrug PyTPE-Pt-D5-cRGD is shown in Scheme 5. Asymmetric functionalization of activated Pt(IV) complex with PyTPE-NH2 and amine-functionalized peptide D5-cRGD in the presence of N,N-diisopropylethylamine afforded the prodrug in 42% yield. A control prodrug PyTPE-Pt-D5 with a similar structure but without cRGD moiety was also synthesized in 44% yield. In addition, a non-activatable control PyTPE-C6-D5-cRGD was prepared in 46% yield by using disuccinimidyl suberate to replace activated Pt(IV) complex in the coupling reaction. The NMR and MS characterization confirmed the right structures of the compounds with high purity.
To evaluate our prodrug as a potential anticancer drug, we studied the nature of the formed Pt(II) species upon reduction. It is reported that diethyldithiocarbamate (DDTC) can react with Pt(II) complexes to yield the adducts Pt(DDTC)2 but does not react with stable Pt(IV) complexes. [10] HPLC-Mass system was used to monitor the adduct formation of Pt(IV) complexes with DDTC after the reduction by ascorbic acid. We choose ascorbic acid as a reduction agent because of its high abundance inside the cells, which has been demonstrated to be a major compound for the reduction of Pt(IV). [11] As shown in
The UV-vis absorption spectra of PyTPE-NH2 in THF and PyTPE-Pt-D5-cRGD in DMSO/PBS (phosphate buffered saline) mixtures (v/v=1/199) were obtained. Both have a similar absorption profile in 348-500 nm with a maximum at 405 nm. The photoluminescence (PL) spectra of PyTPE-NH2 and PyTPE-Pt-D5-cRGD in DMSO/PBS (v/v=1/199) are shown in
To study the response of the prodrug upon reduction, we incubated PyTPE-Pt-D5-cRGD (10 μM) with ascorbic acid (1 mM) in DMSO/PBS (v/v=1/199), and the fluorescence spectra were measured at different time points. As shown in
Next, we incubated different concentrations of prodrug with ascorbic acid (1 mM) and the fluorescence intensities of the prodrug were monitored. The plot of the PL intensities at 605 nm against the pro drug concentration gives a perfect linear line (
The cell lysates of breast cancer cells MDA-MB-231 were directly incubated with PyTPE-Pt-D5-cRGD (10 μM) and the fluorescence intensity at 605 nm was monitored over time. The fluorescence intensity increases quickly in a similar way to that of the solution study with ascorbic acid in image C of
To explore the capability of using PyTPE-Pt-D5-cRGD to monitor targeted intracellular drug reduction in cancer cells, the prodrug was incubated with MDA-MB-231 and MCF-7 breast cancer cell lines. The confocal imaging results are shown in
We next studied the cytotoxicity profile of the prodrug to MDA-MB-231 and MCF-7 cells by a MTT assay. As shown in
In conclusion, we report the synthesis and biological applications of a fluorescent light-up prodrug based on an AIE luminogen for real-time monitoring of drug activation inside the cells. Thanks to the unique nature of the AIE luminogen, the prodrug is non-fluorescent in aqueous media but becomes highly emissive when reduced inside the cells. The cRGD functionalized peptide allows for selective targeting of αvβ3 integrin on many angiogenic cancers using MDA-MB-231 as an example, which opens new opportunity for specific drug delivery. The prodrug design thus opens new avenues for specific tumor targeting and which permits the concentration of activated drug to be monitored by fluorescence signaling changes.
Amine-functionalized TPE (TPE-CH2NH2) was synthesized by reducing azide-TPE (TPE-CH2N3) in methanol. Asymmetric functionalization of DSP linker with TPE-CH2NH2 and NH2 terminated D5-cRGD in the presence of N,N-diisopropylethylamine (DIEA) in anhydrous dimethyl sulfoxide (DMSO) afforded the probe TPE-SS-D5-cRGD in 45% yield (Scheme 6). A control probe TPE-SS-D5 with a similar structure but without cRGD moiety was also synthesized in 49% yield. In addition, a non-activatable control probe TPE-CC-D5 was prepared in 44% yield by using disuccinimidyl suberate to replace DSP in the coupling reaction. The NMR and MS characterizations confirmed the right structures with high purity of the three probes.
The photoluminescence (PL) spectra of TPE-CH2NH2 and TPE-SS-D5-cRGD in DMSO and phosphate buffered saline (PBS, pH=7.4) mixtures (v/v=1/199) are shown in
To study the response of the probe to free thiols, GSH was chosen as the representative thiol due to its high concentration in the human cellular system. [13] GSH (1 mM) was used to incubate with 10 μM TPE-SS-D5-cRGD in DMSO/PBS mixtures (v/v=1/199), and the fluorescence spectra were measured at different time points. As shown in
Next, we investigated the effect of GSH concentration on the probe emission. Different concentrations of GSH ranging from 3.9 μM to 1.0 mM were incubated with TPE-SS-D5-cRGD for 3 h, and the corresponding spectra are shown in
To monitor the GSH-induced fluorescence activation of TPE-SS-D5-cRGD, reverse-phase HPLC and MS analyses were used to follow the exposure of the probe to GSH. After incubation of TPE-SS-D5-cRGD with GSH for 3 h, the mixture was subjected to HPLC analysis. In addition to the TPE-SS-D5-cRGD peak eluted at 10.83 min, two new peaks at 10.68 min for GSS-TPE and 11.58 min for TPE-SH are observed and the peaks show mass-to-charge ratios (m/z) of 755.217 and 472.164 analyzed by IT-TOF, respectively. The fragments of TPE-SH and GSS-TPE tend to aggregate in DMSO/PBS (v/v=1/199), which show blue fluorescence with quantum yields of 19±1% and 12±1%, respectively, using quinoline sulfate as reference. These results clearly demonstrate that the observed GSH-induced fluorescence intensity change of TPE-SS-D5-cRGD is due to cleavage of the disulfide bond, which leads to solubility difference between the probe and the fragment. Further titration of TPE-SS-D5-cRGD with cysteine (Cys), glycine (Gly) and glutamate (Giu), the three amino acids contained in GSH, reveals that the fluorescence turn-on is due to the interaction of free thiol in Cys with the disulfide bond.
To explore the capability of using TPE-SS-D5-cRGD as a specific bioprobe for monitoring intracellular thiol levels in cancer cells, the probe is incubated with U87-MG human glioblastoma and MCF-7 breast cancer cell lines. The confocal imaging results are shown in
To provide further evidence for thiol-induced disulfide bond cleavage as the trigger of fluorescence turn-on, the U87-MG cells were also pretreated with buthionine sulfoximine (BSO) before incubation with TPE-SS-D5-cRGD. BSO is an inhibitor of g-glutamylcysteine synthetase which can inhibit the cells from synthesizing GSH. [14] The fluorescence of TPE-SS-D5-cRGD treated U87-MG cells decreases as the concentration of BSO increases from 25 to 100 μM. The significantly reduced fluorescence as compared to that in image A of
In conclusion, we report the synthesis and biological applications of a light-up GSH responsive AIE probe. Thanks to the unique nature of the AIE luminogen, the probe is nonfluorescent in aqueous media but becomes highly emissive when cleaved by thiols. The probe enables light-up monitoring of free thiols in solution and in cells with a high signal-to-noise ratio. The cRGD functionalized peptide allows for selective targeting of αvβ3 integrin of many angiogenic cancers using U87-MG as an example, which opens new opportunity for specific intracellular thiol imaging. Our AIE probe strategy can be generalized to perform various tasks by simply changing the disulfide groups with other cleavable linkers in chemical biology.
The synthetic route to TPE-DEVD-DOTA/Gd (DDT-Gd) is as follows:
Excess amount of alkyne bearing peptide (DEVD-alkyne, 22 mg, 38.7 μmol) and azide-functionalized TPE (TPE-CH2N3, 10 mg, 25.8 μmol) are dissolved in 0.8 mL of DMSO and vortexed to obtain a clear solution. CuSO4 (0.5 mg, 9.6 μmol) and sodium ascorbate (2.5 mg, 38.7 μmol) dissolved in 0.2 mL of Milli-Q water were subsequently added into the mixture to initiate click chemistry. The reaction was allowed to proceed at 37° C. under shaking for ˜2 days. The product TPE-DEVD was then purified by HPLC with a yield of 60% and further characterized by LC-MS and NMR.
The as-synthesized DEVD-TPE (10 mg, 10.4 μmol) and excess amount of DOTA-NHS ester (10.4 mg, 20.8 mop were dissolved in a total of 0.6 mL of DMSO and mixed thoroughly by vortexing. The reaction was allowed to proceed at room temperature for ˜2 days under shaking. The product DDT was then purified by HPLC with a yield of 70% and further characterized by LC-MS and NMR. IT-TOF-MS: m/z [M+2H]2+ calc. 672.799. found 672.782.
The as-synthesized DDT product (10 mg, 7.4 μmol) was dissolved in 0.4 mL of DMSO. GdCl3 (9.8 mg, 37 mop was dissolved in 0.4 mL of Milli-Q water with pH adjusted to 5 using NaOH. GdCl3 solution was then added into DDT and the mixture was mixed thoroughly by vortexing. The reaction mixture was shaken at room temperature to further react for ˜4 days. The product DDT-Gd was then purified by HPLC with a yield of 60% and further characterized by LC-MS and NMR. IT-TOF-MS: m/z [M+2H]2+ calc. 750.249. found 750.222.
The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.
While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
This application claims the benefit of U.S. Provisional Application No. 61/932,007, filed on Jan. 27, 2014. The entire teachings of the above application are incorporated herein by reference.
Filing Document | Filing Date | Country | Kind |
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PCT/SG2015/000022 | 1/27/2015 | WO | 00 |
Number | Date | Country | |
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61932007 | Jan 2014 | US |