Multi-modal bioprobe for bladder cancer imaging and photodynamic therapy
09840522 ยท 2017-12-12
Assignee
Inventors
Cpc classification
A61K41/008
HUMAN NECESSITIES
A61K41/0071
HUMAN NECESSITIES
A61N5/062
HUMAN NECESSITIES
C07F5/00
CHEMISTRY; METALLURGY
International classification
C07F5/00
CHEMISTRY; METALLURGY
A61K41/00
HUMAN NECESSITIES
Abstract
The present invention relates to a new generation of PDT agents based on porphyrin-lanthanide complexes with specific functional groups which can specifically localize on particular tumors, and their PDT processes can be monitored via NIR emission from erbium. In particular, the present invention provides a multi-modal lanthanide-porphyrin PDT agent (ErR.sub.3) that are capable of killing the bladder tumor cells selectivity via .sup.1O.sub.2 from porphyrin moiety and affording the fluorescence imaging simultaneously upon ErR.sub.3 binding with the integrin .sub.v.sub.3 isoform in bladder cancer cells.
Claims
1. A composition for photodynamic therapy and imaging of cancer cells comprising an Erbium porphyrin based complex, Ytterbium porphyrin based complex, or Gadolinium porphyrin based complex represented by the molecular formula: ##STR00020## wherein Ln is Er, Yb, or Gd; and R.sub.n is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 2; and SEQ ID NO: 3; or a water-soluble porphyrin-based Gadolinium complex represented by a molecular formula selected from the group consisting of Gd.sub.1, Gd.sub.2, Gd.sub.3, Gd.sub.4, and Gd.sub.5: ##STR00021## ##STR00022## ##STR00023## or a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; and SEQ ID NO: 5 or a pharmaceutically acceptable salt thereof.
2. The composition according to claim 1 wherein the Erbium porphyrin based complexes are conjugated with integrin .sub.v.sub.3 isoform-specific peptides.
3. The composition according to claim 1, wherein the composition comprises the Erbium porphyrin based complex and R.sub.n is SEQ ID NO: 4.
4. The composition according to claim 1, wherein the composition comprises the Erbium porphyrin based complex and R.sub.n is SEQ ID NO: 5.
5. The composition according to claim 1, wherein the composition comprises the Erbium porphyrin based complex and R.sub.n is SEQ ID NO: 3.
6. The composition according to claim 1, wherein the composition comprises the Erbium porphyrin based complex represented by the molecular formula of: ##STR00024## wherein Ln is Er and R.sub.n is a polypeptide having an amino acid sequence of SEQ ID NO: 3.
7. The composition according to claim 1 wherein the cancer cells comprising of bladder cancer cells, cervical cancer cells and lung cancer.
8. A method of photodynamic therapy and imaging of cancer cells comprising administering to a subject in need thereof the composition according to claim 1 and irradiating the cancer cells in the subject in need thereof with a radiation source.
9. The method according to claim 8 wherein the administration of said composition is performed intravenously or by injection to site of said cancer cells.
10. The method according to claim 8, wherein said radiation source is a light source with a wavelength in the Q band of porphyrin.
11. The method according to claim 8, wherein said radiation source is a light source with a wavelength beyond 550 nm.
12. The method according to claim 8, wherein said radiation source is a light source with a wavelength is at 860 nm.
13. The method according to claim 8 wherein the imaging is performed using fluorescent imaging.
14. The method according to claim 8 wherein the imaging is performed using NIR imaging.
15. The method according to claim 8 wherein the imaging is performed using MRI imaging.
16. The method according to claim 8 wherein the composition comprises the Gadolinium porphyrin based complex and Ln is Gd; or the composition comprises a compound selected from the group consisting of Gd.sub.1, Gd.sub.2, Gd.sub.3, Gd.sub.4, and Gd.sub.5.
17. A method of synthesizing the composition according to claim 1 wherein Ln=Er or Ln=Yb comprising steps according to the following scheme: ##STR00025## ##STR00026## wherein said compound Por(THP-TMS) is synthesized via steps comprising: dissolving Pyrrole, pentafluorobenzaldehyde and 4-[2-(trimethylsilyl)ethynyl]benzaldehyde 6 in CH.sub.2Cl.sub.2 under an argon atmosphere to produce a first solution; leaving the first solution for at least 10 minutes; adding BF.sub.3.O(Et).sub.2 to the first solution; stirring the first solution for at least 1 hour at room temperature; adding DDQ (2,3-Dichloro-5,6-dicyano-1,4-benzoquinone) to the first solution; stirring the first solution for at least another 1 hour at room temperature; removing the solvent from the first solution in vacuo to produce a first mixture; passing the first mixture through a silica column (hexanes-CH.sub.2Cl.sub.2) concentrated under reduced pressure to produce 5,10,15-Tris(pentafluorophenyl)-20-[4-{2-(trimethylsilyl)ethynyl}phenylporphyrin] or Por(THP-TMS); said compound Ln-1 is synthesized via steps comprising: dissolving Ln[N(SiMe.sub.3)2].sub.3.x[LiCl(THF).sub.3]: HN(SiMe.sub.3).sub.2 in THF at about 0 degrees Celcius to produce a second solution; adding n-BuLi slowly over at least 30-minutes period to the second solution; stirring the second solution for at least 12 hours; transferring the second solution to a Schlenk flask with LnCl.sub.3 suspended in THF to produce a second mixture; stirring the second mixture for at least 24 hours until all of the solid LnCl.sub.3 disappeared to produce Ln[N(SiMe.sub.3)2].sub.3.x[Li(THF).sub.3Cl] (x=35) wherein Ln=Er or Ln=Yb; said compound Yb-1 is further synthesized via steps comprising: transferring Yb[N(SiMe.sub.3)2].sub.3.x[Li(THF).sub.3Cl] (x=35) to a Schlenk flask; removing the solvent from Yb[N(SiMe.sub.3)2].sub.3.x[Li(THF).sub.3Cl] (x=35) under vacuum to produce a first residue; adding CH.sub.2Cl.sub.2 to the first residue for the precipitation of LiCl to produce a third mixture; centrifuging the third mixture until a clear layer is produced; transferring the clear layer to another Schlenk flask with dry Por(THP-TMS) free base dissolved in toluene to produce a third solution; refluxing the third solution until most of the free base coordinated with the metal ion; adding dry NaLOMe [LOMe-((cyclopentadienyl)tris(dimethylphosphito)-cobaltate or an anionic tripodalligand) to the third solution to produce a fourth mixture; stirring the fourth mixture for at least another 12 hours; cooling down the fourth mixture to room temperature; removing the solvent from the fourth mixture in vacuum to produce a second residue; dissolving the second residue in CHCl.sub.3; filtering and chromatographing the dissolved second residue on silica gel using CHCl.sub.3/petroleum ether as eluent; further dissolving the output from chromatography in CH.sub.2Cl.sub.2; and filtering the solution to produce compound Yb-1; Said compound Er-1 is further synthesized via steps comprising: the same steps as for Yb-1, replacing Yb[N(SiMe.sub.3)2].sub.3.x[Li(THF).sub.3Cl] (x=35) with Er[N(SiMe.sub.3)2].sub.3.x[Li(THF).sub.3Cl] (x=35); said compound Ln-2 wherein Ln=Yr is synthesized via steps comprising: adding TBAF to a solution of Yb-1 in CH.sub.2C12 to produce a fifth solution; stirring the fifth solution for at least 30 minutes; monitoring the progress of the reaction of the fifth solution by TLC; after completion of the reaction, passing the fifth solution through a short of silica gel column; removing the solvent from the fifth solution to produce Yr-2; said compound Er-2 is further synthesized via steps comprising: the same steps as for Yb-2, replacing Yb-1 with Er-1; said compound Ln-4 wherein Ln=Yr is synthesized via steps comprising: mixing Pd(PPh.sub.3).sub.4, CuI, Yb-2 and 4-iodobenzoic acid in a dried flask under nitrogen to produce a fifth mixture; adding THF and Net.sub.3 to the fifth mixture and degassing said fifth mixture with nitrogen; stirring said fifth mixture at least 40 C. for at least 12 hours; removing the solvent from said fifth mixture under reduced pressure to produce a third residue; purifying the third residue by chromatography; eluting the purified third residue with CH.sub.2Cl.sub.2/Methanol to produce an eluted compound; mixing the eluted compound, EDCI, NHS in a dried flask and under nitrogen to produce a sixth mixture; adding dry DMF to the sixth mixture; stirring the sixth mixture at room temperature for at least 48 hours; removing the solvent from the stirred sixth mixture to produce a fourth residue; recrystallizing the fourth residue by diethyl ether and drying the crystals to produce Yb-4; said compound Er-4 is further synthesized via steps comprising: the same steps as for Yb-4, replacing Yb-2 with Er-2; said compound YbR.sub.1 is synthesized via steps comprising: mixing a stirred solution of Yb-4 in anhydrous DMF with N,N-diisopropylethylamine (DIPEA) to produce a seventh mixture; adding peptide R.sub.1 to the seventh mixture; leaving the seventh mixture to react at room temperature for at least 24 hours; removing the solvent from the seventh mixture under vacuum to produce a dry fifth residue; recrystallizing the dry fifth residue by diethyl ether for at least three times; drying the recrystallized dry fifth residue to produce YbR.sub.1; said compound YbR.sub.2 is further synthesized via steps comprising: the same steps as for YbR.sub.1, replacing R.sub.1 with R.sub.2; said compound YbR.sub.3 is further synthesized via steps comprising: the same steps as for YbR.sub.1, replacing R.sub.1 with R.sub.3; said compound ErR.sub.1 is further synthesized via steps comprising: the same steps as for YbR.sub.1, replacing Yb-4 with Er-4; said compound ErR.sub.2 is further synthesized via steps comprising: the same steps as for YbR.sub.2, replacing Yb-4 with Er-4; said compound ErR.sub.3 is further synthesized via steps comprising: the same steps as for YbR.sub.3, replacing Yb-4 with Er-4.
18. A method of synthesizing a composition comprising steps according to the following scheme: ##STR00027## ##STR00028## wherein said compound Por-TMS is synthesized via steps comprising: mixing 4-((trimethylsilyl)ethynyl)benzaldehyde with Pyridine-4-carboxaldehyde in propionic acid to produce an eighth mixture; stirring the eighth mixture for at least half an hour in at least 130 C.; adding pyrrole dropwise into the eighth mixture with the temperature increased to at least 140 C.; stirring the eighth mixture for at least 30 minutes in open air; cooling down the eighth mixture to room temperature; removing the solvent from the eighth mixture under reduce pressure to produce a crude product; dissolving the crude product in CH.sub.2Cl.sub.2 to produce a sixth solution; purifying the sixth solution by column chromatography on silica gel column CH.sub.2Cl.sub.2/Methanol to produce Por-TMS; said compound Gd[N(SiMe.sub.3).sub.2]3.x[LiCl(THF).sub.3] is synthesized via steps comprising: dissolving HN(SiMe.sub.3).sub.2 in THF at about 0 degrees Celcius to produce a seventh solution; adding n-BuLi to the seventh solution over at least a 30-minutes period; stirring the seventh solution at least for 12 hours until a clear pale yellow solution was obtained; transferring the seventh solution a Schlenk flask with GdCl.sub.3 suspended in THF to produce a ninth mixture; stirring the ninth mixture for at least 24 hours until all of the solid GdCl.sub.3 disappeared to produce the resultant solution Gd[N(SiMe.sub.3).sub.2].sub.3.x[LiCl(THF).sub.3] (x=35); said compound Gd-1-L1 is synthesized via steps comprising: transferring Gd[N(SiMe.sub.3).sub.2].sub.3.x[LiCl(THF).sub.3] (x=35) to a Schlenk flask and removing the solvent therein under vacuum to produce a sixth residue; adding CH.sub.2Cl.sub.2 to the sixth residue for the precipitation of LiCl to produce a tenth mixture; centrifuging the tenth mixture until a clear layer is produced; transferring the clear layer to another Schlenk flask with dry Por-TMS free base dissolved in toluene to produce an eighth solution; refluxing the eighth solution until most of the free base coordinated with the metal ion; adding dry NaL.sub.1 (0.1 g, 0.22 mmol) [L.sub.1-((cyclopentadienyl)tris(dimethylphosphito)-cobaltate, an anionic tripodal ligand) to the eighth solution to produce an eleventh mixture; stirring the eleventh mixture for at least another 12 hours; cooling down the eleventh mixture to room temperature; removing the solvent from the eleventh mixture in vacuum to produce a seventh residue; dissolving the seventh residue in CHCl.sub.3; filtering and chromatographing the dissolved second residue on silica gel using CHCl.sub.3/CH.sub.3OH ether as eluent; further dissolving the output from chromatography in CH.sub.2Cl.sub.2; and filtering the dissolved output to produce compound Gd-1-L1; Said compound Gd-1-L2 is synthesized via steps comprising: the same steps as for Gd-1-L1, replacing NaL.sub.1 with KL.sub.2 (potassium tris(1-pyrazolyl) borohydride); said compound Gd-3 is synthesized via steps comprising: adding TBAF to a solution of Gd-1-L1 in DCM to produce a ninth solution; stirring the ninth solution for at least 30 minutes; monitoring the reaction of the ninth solution by TLC; passing the ninth solution through a short of silica gel column using DCM to remove the solvent therein to produce a pure product; placing the pure product and Pd(PPh.sub.3).sub.4, CuI, 4-iodobenzoic acid in a dried flask and under nitrogen to produce a twelfth mixture; adding THF and Net.sub.3 to the twelfth mixture; degassing the twelfth mixture with nitrogen; stirring the twelfth mixture at a temperature at least 40 C. for at least 12 hours; removing the solvent from the twelfth mixture under reduced pressure to produce an eighth residue; purifying the eighth residue by chromatography; eluting the purified eighth residue with CH.sub.2Cl.sub.2/Methanol; placing the eluted purified eighth residue, EDCI, NHS in a dried flask and under nitrogen to produce a thirteenth mixture; adding dry DMF to the thirteenth mixture; stirring the thirteenth mixture at room temperature for at least 48 hours; removing the solvent from the thirteenth mixture to produce a ninth residue; recrystallizing the ninth residue by diethyl ether and dried said crystals to produce Gd-3; dissolving the Gd-3 in DMF; adding CH.sub.3I to the dissolved Gd-3; stirring the dissolved Gd-3 for at least 5 hours; removing the solvent from the stirred dissolved Gd-3 to produce a tenth residue; washing the tenth residue with ether .DCM. to produce pure Gd-3; said compound Gd-4 is synthesized via steps comprising: the same steps as for Gd-3, replacing Gd-1-L1 with Gd-1-L2; said compound Gd-3-Rn is synthesized via steps comprising: mixing a stirred solution of Gd-3 in anhydrous DMF with N,N-diisopropylethylamine (DIPEA) to produce a fourteenth mixture; adding peptide Rn to the fourteenth mixture; reacting the fourteenth mixture at room temperature for at least 24 hours; removing the solvent from the fourteenth mixture under vacuum to produce a dry eleventh residue; recrystallizing the dry eleventh residue by diethyl ether for at least three times and further dry the result to produce Gd-3-Rn; said compound Gd-4-Rn is synthesized via steps comprising: the same steps as for Gd-3-Rn, replacing Gd-3 with Gd-4.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, in which:
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(13) ) processes in the (A) gadolinium porphyrinate complex (GdN) and (B) ytterbium porphyrinate complex (YbN).
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DETAILED DESCRIPTION OF THE INVENTION
(53) The present invention is not to be limited in scope by any of the specific embodiments described herein. The following embodiments are presented for exemplification only.
(54) Without wishing to be bound by theory, the inventors of the present invention have developed gadolinium porphyrinate (GdN;
(55) Results and Discussion
(56) The detailed synthesis and characterization of GdN, which is the motif structure of the ytterbium complex (YbN) reported in the inventors' previous work, are shown in Scheme 1 and
(57) Investigations with relation to real PDT-applications of GdN in vitro and, particularly, in vivo had been accomplished in terms of tumor selectivity, cytotoxicity and photocytotoxicity, imaging, PDT efficiency, as well as biodistribution. The selectivity of GdN against tumor and normal cells are superiorly distinct. As shown in
(58) The concentration-dependent photocytotoxicity of GdN, YbN and GdRhB, ranging from 0.2 to 1 M dosage, are measured under varying light doses from 0.25 to 1 J/cm.sup.2 in cancer cells and normal cells. The light dose-response curves obtained are displayed in
(59) To demonstrate in vivo uptakes of complexes of the present invention, studies of biodistribution on these complexes' specialty towards infections of particular organs are carried out via xenograft mouse models and ICP-MS. Four complexes are classified into four groups. HeLa cells pre-incubated separately with GdN, YbN, GdRhB and YbRhB, and are subcutaneously injected into BALB/c nude mice and then irradiated the injected sites with 860 nm laser. Two weeks later, mice are pictured and the tumor volumes are measured (picture of the mice and measurement of tumor volumes are shown in
(60) This result is also confirmed by two-photon microscopic imaging of the tumor tissues extracted from GdN administrated BALB/c nude mice. There are obvious two-photon microscopic signal from GdN(image of GdN, circled points), while the control image (showed as background, imaged by bright field shows no specific signal. The merge image is the overlap photon of Background and GdN which is shown
(61) Alternatively, mice with xenograft tumor are caudal vein injected with GdN and GdRhB (2.0 mg/kg body weight) and allowed for full circulation for 6 hours. Then tumors are irradiated with 860 nm light similarly as above. The tumor with light untreated serves as a control. The treatments are repeated for three times in the following days in a one-time-per-day manner. Consistently, it is found that GdN plus light treated tumors are inhibited compared to their opposite flank controls of tumor or GdRhB groups. Pharmacokinetics analyses also show that GdN persisted in animals for longer time with a larger MRT (mean resistance time) value (12.50 hours), while GdRhB is fast cleared (with MRT of 5.04 hours) (results are illustrated in
(62) TABLE-US-00001 TABLE 1 Pharmacokinetic parameters of Gd-N and Gd-RhB in plasma after caudal vein injections of 20 nmol of GdN (37.34 ug) or GdRhB (44.28 g) to BALB/c nude mice (n = 3), respectively. Parameters GdN GdRhB Equation C.sub.(t) = 138.61 e .sup.0.08t C.sub.(t) = 176.08 e .sup.0.1986t AUC.sub.(0-t) (g/mL h) 1732.63 886.61 MRT.sub.(0-t) (h) 12.50 5.04 t.sub.1/2 (h) 8.66 3.49 V.sub.d (mL) 0.269 0.251 AUC, area under the concentration-time curve; MRT, mean residence time; t.sub.1/2, statistical half life; V.sub.d, volume of distribution.
(63) Molecular mechanism of PDT, protein levels of cellular surviving and inhibitors of apoptosis protein family (IAP) in protein lysates of PDT-treated HeLa cells are also investigated. Survivin and the IAP family members, c-IAP1, c-IAP2 and XIAP, are all remarkably expressed in GdN plus laser-treated samples. mTOR pathway is found to be involved in response to the PDT treatment of the cancer cells. The levels of two critical members, mTOR and GL, are obviously elevated upon GdN or GdRhB induced .sup.1O.sub.2 stress stimulus. These results demonstrate the successful cell killing effect of GdN promoted photodynamic therapy at the molecular level and may also shed new light on the design and improvement of current PDT agents (
(64) Conclusion
(65) The present invention provides theranostic gadolinium complex GdN for use as an anti-cancer agent which is equipped with visible-to-NIR emission for imaging, tumor cell selectivity, and .sup.1O.sub.2 generation. Through a string of in vitro and in vivo studies, the effectiveness and advantages of GdN of the present invention for use as smart dual-functional PDT agent is shown. The present invention also provides a method of tracking and imaging long-term live cancer cell, using GdN, as well as selective photodynamic therapy.
Experimentation Methods
(66) Linear Induced Photophysical Properties
(67) UV-Visible absorption spectra (ranging from 200 to 1100 nm) and single-photon luminescence spectra are recorded with an HP UV-8453 spectrophotometer and an Edinburgh Instrument FLS920 Combined Fluorescence Lifetime and Steady state spectrophotometer equipped with a UV-to-NIR-sensitive photomultiplier inside a nitrogen flow cooled housing. The Inventors had corrected all the spectra from the detector response and stray background light phosphorescence, measuring the quantum yields of the lanthanide complexes by a demountable 142 mm (inner) diameter barium sulphide-coated integrating sphere supplied with the two access ports in Edinburgh Instrument FLS920.
(68) Singlet Oxygen Quantum Yield
(69) With phosphorescence at 1270 nm, the singlet oxygen with an InGaAs detector on the PTI QM4 luminescence spectrometer are detected, and the quantum yields (.sub.) of all compounds in CHCl.sub.3 through comparing the .sup.1O.sub.2 emission intensity of the sample solution to that of a reference material.sup.[4] (H.sub.2TPP, .sub.A=0.55 in CHCl.sub.3) are determined and as illustrated in the following equation:
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where .sub. denotes the singlet oxygen quantum yield, G.sub. indicates the integrated emission intensity, A represents the absorbance at the operation excitation wavelength, n reflects the solvent's refractive index, given that the Superscripts REF and S stand for the reference and sample respectively. In all cases, the inventors had measured the .sup.1O.sub.2 emission spectra upon due excitation. To reduce the impacts of re-absorption of the emitted light, all absorbance were set at 0.05 as well.
(71) Cell Culture
(72) Human HeLa (cervical carcinoma) and WPMY-1 (normal prostate stroma immortalized cell) cells are grown in DMEM medium; A549 (lung adenoma) are maintained in a mixture of Ham's F12K medium and L-glutamine (N3520, Sigma, St. Louis, Mo., USA); QSG 7701 (normal liver cell), HK-1, HONE1 (nasopharyngeal carcinoma) are grown in RMPI-1640 medium; MRC-5 (normal lung fibroblasts) and SK-N-SH (neuroblastoma) cells are grown in MEM medium. (i) 10% (v/v) fetal bovine serum (FBS), (ii) 100 g/ml streptomycin, and (iii) 100 units/ml penicillin are also added in the all the medium.
(73) In Vitro Imaging
(74) To test the suitability of the water-soluble complexes of the present invention as bioprobes, a commercial confocal laser scanning microscope, Leica TCS SP5, equipped with a Ti:Sapphire laser (Libra II, Coherent) as well as a 980 nm wavelength laser for excitation are used to in vitro image HeLa/WPMY-1/MRC-5 cells incubated with the five complexes of the present invention.
(75) MTT Cell Viability Assay
(76) After 24 hours, the water-soluble complexes and the treated cells are incubated further with 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (0.5 mg/ml) for 4 hours, so that formazan is formed along with the cell's metabolic pathways. Then, the formazan are extracted and dissolved by dimethyl sulfoxide (DMSO), with the absorbance of the subsequent solutions being measured in a Bio-Rad iMark microplate reader (490 nm). Quadruplicates are performed and the data are interpreted and analyzed by plottings using the GraphPad Prism 5 software.
(77) Photodynamic Treatment (PDT) Assay
(78) On a 96-well plate, cancer cells (210.sup.4/well) are first incubated overnight and then treated with complexes of the present invention and control analogues for 6 hours in dark. After the old medium being replaced with the fresh one, the cells are accordingly exposed to yellow light (1-8 J/cm.sup.2) generated from a 400 W tungsten lamp fitted with a heat-isolation filter and a 500 nm long-pass filter under the fluency rate of mW/cm2. After 24 hours, post-PDT cell viability is examined by MTT assay. Cell monolayers are rinsed with PBS prior to incubation with 250 g/mL MTT solution at 37 C. for 3 hours. The formazan crystals formed and dissolved in DMSO then undergo absorbance measurement at 540 and 690 nm by a 96-well plate reader (Elx800 Absorbance Microp late Reader).
(79) Animals:
(80) All the experiments entailing animal models are performed on athymic nude mice (BALB/c-nu/nu) which are all obtained from Guangdong Medical Lab Animal Center (license number: SCXK-2008-0002). Mice are raised and operated according to the strict protocol the National Standard of Animal Care and Use Procedures (20080820).
(81) Pharmacokinetics Analysis:
(82) GdN and GdRhB (1.0 mol/kg body weight each) are caudal vein injected into the mice. Then sera are collected at different time points from 0-20 hours as indicated. The concentrations of GdN and GdRhB are measured by PerkinElmer EnVision Multilabel Reader 2104 at 570 nm, and calculated using standard absorptions via concentration curve. Pharmacokinetic parameters (t.sub.1/2, Vd, MRT, AUC) are calculated by fitting with one compartment model.
(83) In Vivo Bio Distribution Via ICP-MS
(84) Biodistribution studies of In vivo uptakes of complexes of the present invention in particular organs/bacterial infections are carried out via ICP-MS. GdN and GdPhB are administered to mice at a dosage of 1.0 mol/kg body weight when tumor xenograft attains a size of approximately 0.1 cm.sup.3. 2 days later, around 0.02-0.04 gram of sample tissues are collected in tumor, liver, lung, kidney, spleen, brain, prostate, skin and blood (80-90 L). All samples are incubated with 500 L nitric acid at 37 C. for releasing the metal ions for further ICP-MS examinations, in addition to dissolving the interfering organic molecules.
(85) In Vivo Photodynamic Therapy Studies
(86) For the establishment of mouse tumor xenograft mode, cells are trypsinized, harvested and suspended in the culture medium. 110.sup.6 cells in 100 L, volume are s.c. injected into the flanks of female athymic nude mice (with 5-week old) and waited for 10-15 days. When the tumor volume reaches to size of 100-150 mm.sup.3, the animals are randomly divided into different groups for further experiments. Tumor volume is measured by calipers (accuracy of 0.02 mm) and then calculated independently on the basis of the equation V=(LW.sup.2)/2, where L and W correspond to the larger and smaller dimensions, respectively. One-way analysis of variance towards statistical significances between groups is assessed by the GraphPad Prism 5.0 software.
(87) Materials and Methods
(88) All chemicals used are of reagent-grade and are purchased from Sigma-Aldrich and used without further purification. Preparations of intermediates Yb[N(SiMe.sub.3).sub.2].sub.3.[LiCl(THF).sub.3].sup.1 and starting porphyrin free base TFP-TMS.sup.2 are performed according to the literature procedures. Preparations of the control compounds GdRhB, YbRhB.sup.4 and YbN.sup.5 are accomplished according to the inventors' previous procedures. All analytical-grade solvents are dried by standard procedures, distilled and deaerated before use. High-resolution mass spectra, reported as m/z, are obtained on a Bruker Autoflex MALDI-TOF mass spectrometer. Elemental analyses carried out at the School of Chemical Engineering, Northwest University, P. R. China. The synthetic route of intermediates and GdN is shown in Scheme 1:
(89) ##STR00013## ##STR00014## ##STR00015##
(90) GdOH:
(91) Tetrabutylammonium fluoride (TBAF, 1.0 M in THF, 200 L, 0.2 mmol) is added to a solution of Gd-TMS (182 mg, 0.1 mmol) in 10 ml CH.sub.2Cl.sub.2, and the solution is stirred for 30 minutes. The progress of the reaction is monitored by TLC. After completion of the reaction, the mixture is passed through a short column of silica gel. After removal of the solvent, the intermediate is obtained and used for the next step without further purification. Then the obtained intermediate and 4-iodophenol (33 mg, 0.15 mmol) are dissolved in dry tetrahydrofuran (THF, 15 ml) and triethylamine (Net.sub.3, 5 mL), and the mixture is bubbled with nitrogen for 30 minutes. After that, Pd(PPh.sub.3).sub.4 (12 mg 0.01 mmol) and CuI (3.8 mg, 0.02 mmol) are added to the above solution. The reaction mixture is stirred at least 35 C. for at least 10 hours under a nitrogen atmosphere. Then the solvent is removed under reduced pressure. The residue is purified by column chromatography on silica gel using CH.sub.2Cl.sub.2/Methanol (50:1) as eluent to afford the pure product as a red solid. Yield: 73% (Table 2); M. p.>300 C.; MALDI-TOF MS: calcd. For [M.sup.+]: 1607.0291. found: 1608.0308; Anal. Calc. For [C.sub.63H.sub.40CoF.sub.15N.sub.4O.sub.10P.sub.3Gd]: C, 47.08; H, 2.51; N, 3.49%. Found: C, 47.10; H, 2.49; N, 3.51%; UV/Vis (DMSO, 25 C.): .sub.max (log )=426 (5.70), 555 nm (4.48 dm.sup.3 mol.sup.1 cm.sup.1).
(92) TABLE-US-00002 TABLE 2 Yield in different cross-coupling reaction condition(%). Considering both time and temprature, 40 C. and 12 hours is selected as the major reaction condition. Time (hours) Temperature ( C.) 10 12 15 35 60 64 69 40 68 73 74 60 59 62 66
(93) GdI:
(94) To a solution of GdOH (161 mg, 0.1 mmol) and tetraethyleneglycol diiodide (207 mg, 0.5 mmol) in dry N,N-Dimethylmethanamide (DMF, 10 ml) is added anhydrous K.sub.2CO.sub.3 (69 mg, 0.5 mmol), and the mixture is heated to 80 C. for 8 hours under a nitrogen atmosphere. Then the solvent is removed under reduced pressure. The crude product is purified by column chromatography on silica gel eluented by CH.sub.2Cl.sub.2/CH.sub.3OH (v/v, 100:1) to afford the pure product as a red solid. Yield: 82%; M. p.>300 C.; MALDI-TOF MS: calcd. For [M.sup.+]: 1893.2210. found 1893.1038; Anal. Calc. for [C.sub.71H.sub.55CoF.sub.15IN.sub.4O.sub.13P.sub.3Gd]: C, 45.04; H, 2.94; N, 3.11%. Found: C, 45.21; H, 2.99; N, 3.06%; UV/Vis (DMSO, 25 C.): .sub.max (log )=425 (5.71), 555 nm (4.50 dm.sup.3 mol.sup.1 cm.sup.1).
(95) GdN:
(96) To a solution of GdI (95 mg, 0.05 mmol) in dry (DMF, 10 ml), anhydrous Nets (1 ml, excess) is added, and the mixture is heated to 85 C. for 24 h under the nitrogen atmosphere. Then the solvent is removed under reduced pressure. The obtained crude product is purified by silica gel column chromatography using CH.sub.2Cl.sub.2/CH.sub.3OH (v/v, 80:1) as the eluent to remove unreacted GdI and other impurities, then using CH.sub.2Cl.sub.2/CH.sub.3OH (v/v, 10:1) to obtain the pure product as a red solid. Yield: 80%; M. p.>300 C.; MALDI-TOF MS: calcd. For [M.sup.+]: 1867.5095. found 1867.2538; Anal. Calc. For [C.sub.99H.sub.85CoF.sub.15N.sub.6O.sub.16P.sub.3Gd]: C, 46.37; H, 3.54; N, 3.51%. Found: C, 46.40; H, 3.59; N, 3.48%; UV/Vis (DMSO, 25 C.): .sub.max (log )=426 (5.74), 555 nm (4.53 dm.sup.3 mol.sup.1 cm.sup.1).
(97) Two-Photon-Absorption Measurements
(98) The two-photon-absorption spectra (i.e., Z-scan traces) are measured at 800 nm by the open-aperture Z-scan method using 100 fs laser pulses with a peak power of 276 GWcm.sup.2 from an optical parametric amplifier operating at a repetition rate of 1 kHz generated from a Ti:sapphire regenerative amplifier system. The laser beam is split into two parts by a beam splitter. One is monitored by a photodiode (D1) as the incident intensity reference, I.sub.0, and the other is detected as the transmitted intensity by another photodiode (D2). After passing through a lens with f=20 cm, the laser beam is focused and passed through a quartz cell. The position of the sample cell, z, is moved along the direction of the laser beam (z axis) by a computer-controlled translatable table so that the local power density within the sample cell could be changed under the constant incident intensity laser power level. Finally, the transmitted intensity from the sample cell is detected by the photodiode D2. The photodiode D2 is interfaced to a computer for signal acquisition and averaging. Each transmitted intensity datum represents the average of over 100 measurements. Assuming a Gaussian beam profile, the non-linear absorption coefficient, , can be obtained by curve-fitting to the observed open-aperture traces, T(z), with Equation (1).sup.6, where a.sub.0 is the linear absorption coefficient, l is the sample length (the 1 mm quartz cell) and z.sub.0 is the diffraction length of the incident beam. After obtaining the nonlinear absorption coefficient, , the 2 PA cross-section, .sup.(2), of the sample molecule (in units of 1 GM=10.sup.50 cm.sup.4 sphoton.sup.1) can be determined by using Equation (2), where N.sub.A is Avogadro.'s constant, d is the concentration of the sample compound in solution, h is Planck's constant and v is the frequency of the incident laser beam.
(99)
Further Embodiments of the Present Inventions
(100) In a further embodiment of the present invention, new generation of PDT agents based on porphyrin-lanthanide complexes with specific functional groups are provided which can specifically localize on particular tumors, and their PDT processes can be monitored via NIR emission from erbium (Er). The newly developed erbium porphyrin complexes are conjugated with integrin .sub.v.sub.3 isoform-specific peptides. The porphyrin and erbium emission from ErR.sub.3 show that ErR.sub.3 are able to significantly interrupt bladder cancer tumor growth that specific binds to integrin .sub.v.sub.3 isoform with responsive emission for imaging.
(101) The water solubility of Er porphyrin complexes are improved compared with previously reported analogues with conjugation of hydrophilic peptide RrRk (SEQ ID NO: 4). The integrin .sub.v.sub.3 isoform specific peptide sequence (-cGRLKEKKc-) (SEQ ID NO: 5) is chosen to conjugate with RrRk (SEQ ID NO: 4) in different positions for the estimation of binding selectivity to integrin .sub.v.sub.3 isoform in bladder cancer cells (Scheme 3). The amphiphilic character of the peptides is synthesized with the combination of hydrophilic RrRk (SEQ ID NO: 4) and hydrophobic cGRLKEKKc (SEQ ID NO: 5) to improve the cell permeability. The absorption coefficient (Porphyrin: Soret Band at 430 nm, 199,526 cm.sup.1) and emission quantum yield (Porphyrin: Soret Band and Er: .sup.2F.sub.5/2.fwdarw..sup.2F.sub.7/2) of ErR.sub.1, ErR.sub.2 and ErR.sub.3 are similar. The details of photophysical measurement of Ln-R.sub.n are shown in the Table 3. The Er moiety demonstrates stronger singlet oxygen quantum efficiency than Yb moiety due to the energy transfer from porphyrin to Yb for f-f emission which is much better than from porphyrin to Er f-f emission. All of the ErR.sub.n porphyrin complexes and YbR.sub.n porphyrin
(102) TABLE-US-00003 TABLE 3 Photophysical properties Summary of LnR.sub.n (Ln = Yb, Er, n = 1, 2, 3) Absorption (.sub.max)[nm] Excitation Emission(.sub.em) [nm].sup.a Compound Log([dm.sup.3mol.sup.1cm.sup.1]).sup.a (.sub.exc)[nm] (.sub.em, ).sup.b,c .sub..sup.d Yb-R.sub.1 425 (5.37), 554 (4.09) 430 656, 712 (0.012) Not Found 975 (29.86 s) Yb-R.sub.2 425 (5.34), 554 (4.16) 430 56, 712 (0.013) Not Found 975 (30.08 s) Yb-R.sub.3 425 (5.27), 554 (4.04) 430 656, 712(0.013) Not Found 975 (29.97 s) Er-R.sub.1 426 (5.32), 554 (4.05) 430 654, 715 (0.014), 1531 0.11 Er-R.sub.2 426 (5.50), 554 (4.53) 430 654, 715 (0.014), 1531 0.12 Er-R.sub.3 426 (5.36), 554 (4.24) 430 654, 715 (0.015), 1531 0.12 Complexes are characterized by .sup.1H NMR and mass spectrometry (FIG. 17-36). .sup.aAbsorption and Emission are measured in water (3% DMSO) at room temperature. .sup.bThe emission quantum yield standard used in this study is tetraphenylporphyrin (H.sub.2TPP) in anhydrous DCM (em = 0.120 at 298 K). .sup.clifetime were measured in water (3% DMSO) at room temperature .sup.dThe Singlet oxygen quantum yield standard used in this study was tetraphenylporphyrin (H.sub.2TPP) in anhydrous DCM ( = 0.62 at 298 K).
In
(103) To further prove that the selective uptake of ErRn and YbRn (n=1, 2 and 3) porphyrin complexes in bladder cancer cells was induced by the recognition of the .sub.v.sub.3 integrin on the surface of the T24 through the tailor-made cyclic peptide R1, R2 and R3, the flow cytometry analysis of erbium (Er) and ytterbium (Yb) complexes in three cell lines is performed and the results are shown in the
(104) Molecular docking simulates the present porphyrin complexes and provides great steric hindrance to the peptides and facilitates interaction with the .sub.v.sub.3 integrin. Zhang et al. (Urologic Oncol. 2012, 30, 635-645) have tested the peptides R.sub.1 and R.sub.2 of the complexes of the subject invention in different cell lines and screened through the OBOC combinatorial library to demonstrate the binding specificity towards blabber cancer. The amphiphilic peptide R.sub.3 is the modification of R.sub.2 by adding RrRk (SEQ ID NO: 4) to improve water solubility and cellular uptake. Therefore, as shown in
(105) TABLE-US-00004 TABLE 4 Summary of ErR.sub.n porphyrin complexes and Yb R.sub.n porphyrin complexes median fluorescence intensity in T24, HeLa and MRC-5 cells incubated for 24 hours. Median fluorescence intensity T24 HeLa MRC-5 ErR.sub.1 11.80 4.86 4.07 YbR.sub.1 6.68 4.31 3.56 ErR.sub.2 21.21 4.11 3.09 YbR.sub.2 14.07 4.21 3.02 ErR.sub.3 40.52 4.13 3.59 YbR.sub.3 26.07 3.36 3.33
(106) After verification of specific uptake of ErR.sub.n porphyrin complexes and YbR.sub.n porphyrin complexes into T24 cells, in vitro PDT in various cell lines are carried out. Low-dark and high-light cytotoxicity are the necessary properties of a photosensitizer for the application in PDT. The cytotoxicity of ErR.sub.n porphyrin complexes and YbR.sub.n porphyrin complexes to T24, HeLa and MRC-5 cells are examined in both presence (550 nm long-pass filter, 6 mW cm.sup.2, 28 minutes) and absence of irradiation using MTT assay. ErR.sub.n and YbR.sub.n exhibit high photo-cytotoxicity under irradiation of 10 Jcm.sup.2. Furthermore, the photo-cytotoxicity increased along with the concentration of ErR.sub.n porphyrin complexes and YbR.sub.n porphyrin complexes with a half lethal dose (IC.sub.50) calculated in
(107) In summary, the present invention provides a multi-modal lanthanide-porphyrin PDT agent that is capable of killing the tumor cells via .sup.1O.sub.2 from porphyrin moiety and affording the fluorescence imaging simultaneously. ErR.sub.3 porphyrin complexes are synthesized and shown to be highly selective to bladder cancer cells by specific targeting integrin .sub.v.sub.3 isoform in bladder cancer cells with strong NIR and .sup.1O.sub.2 emission. The cancer cells selectivity uptake property of the porphyrin complexes of the present invention is confirmed by flow cytometry and in-vitro imaging and is able to significantly interrupt the bladder cancer cells growth with specific binding to integrin .sub.v.sub.3 isoform of blabber cancer cells.
(108) General Information about the Compound Synthesis.
(109) All chemicals used are of reagent-grade and are purchased from Sigma-Aldrich and used without further purification. All analytical-grade solvents are dried by standard procedures, distilled and deaerated before use. NMR spectra are recorded on a Bruker Ultra shield 400 Plus NMR spectrometer. The .sup.1H NMR chemical shifts are referenced to tetramethylsilane, TMS (d=0.00). High-resolution mass spectra, reported as m/z, are obtained on a Bruker Autoflex MALDI-TOF mass spectrometer. The synthetic Route of intermediates and Ln-Rn (Ln=Yb,Er, n=1, 2, 3) are shown in Scheme 2. All the Ln-Rn (Ln=Yb or Er, n=1, 2, 3) complexes are purified by High Performance Liquid Chromatography. The solvent system was shown in Table 5.
(110) ##STR00016## ##STR00017##
(111) ##STR00018## ##STR00019##
(112) Synthesis of the Intermediates and Ln-R.sub.n (Ln=Yb or Er, n=1, 2, 3)
(113) Preparation of Compound Por(THP-TMS)
(114) Pyrrole (280 uL, 4.0 mmol), pentafluorobenzaldehyde (588 mg, 0.3.0 mmol) and 4-[2-(trimethylsilyl)ethynyl]benzaldehyde 6 (202 mg, 1.0 mmol) are dissolved in 410 mL CH.sub.2Cl.sub.2 under an argon atmosphere. After 10 minutes BF.sub.3O(Et).sub.2 (0.60 mL of 2.65M stock solution, 1.32 mmol) is added via syringe with vigorous stirring. After addition is complete, the reaction is left to stir for 1 hour at room temperature. DDQ (2,3-Dichloro-5,6-dicyano-1,4-benzoquinone) (0.68 g, 3.0 mmol) is added and after 1 hour stirring at room temperature the solvent is removed in vacuo. The crude reaction mixture is passed through a short silica column (hexanes-CH.sub.2Cl.sub.2 (9:1)) concentrated under reduced pressure, to give product 5,10,15-Tris(pentafluorophenyl)-20-[4-{2-(trimethylsilyl)ethynyl}phenylporphyrin], a pink/purple solid (238 mg, 22.8%); .sup.1HNMR (CDCl.sub.3): 2.87 (2H, s, NH), 7.91 (2H, d, J 8.1 Hz, ArH), 8.17 (2H, d, J 8.1 Hz, ArH), 8.89 (2H, d, J 4.7 Hz, P-pyrrole), 8.932 (4H, s, P-pyrrole), 8.94 (2H, d, J 4.7 Hz, P-pyrrole); 0.387 (9H, s) MS (MALDI) for [M].sup.+, calcd. for C.sub.49H.sub.23F.sub.15N.sub.4Si 980.1513. found 981.1519.
(115) Preparation of Compound Ln-1
(116) Ln[N(SiMe.sub.3)2].sub.3.x[LiCl(THF).sub.3]: HN(SiMe.sub.3).sub.2 (Ln=Yb or Er, 10.8 ml 0.050 mol) is dissolved in 20 ml of THF in ice bath, then n-BuLi (1.6 M in hexane) is added slowly over 30-minutes period. The resulting solution is magnetically stirred for 12 hours until a clear pale yellow solution is obtained. Then the solution is transferred slowly to a Schlenk flask with LnCl.sub.3 (Ln=Yb or Er, 4.74 g, 0.017 mol) suspended in 20 ml THF. The resulting mixture is magnetically stirred for 24 hours until all of the solid LnCl.sub.3 (Ln=Yb or Er,) disappear. The resultant solution Ln[N(SiMe.sub.3)2].sub.3.x[Li(THF).sub.3Cl] (Ln=Yb or Er, x=35) is used for next synthetic step.
(117) Yb-1:
(118) Yb[N(SiMe.sub.3)2].sub.3.x[Li(THF).sub.3Cl] (2.5 ml, 0.52 mmol of Yb) as prepared above is transferred to a Schlenk flask and the solvent is removed under vacuum. Then 10 ml CH.sub.2Cl.sub.2 is added, for the precipitation of LiCl. The mixture is centrifuged and the clear layer is transferred to another Schlenk flask with dry Por(THP-TMS) (0.1 g, 0.16 mmol) dissolved in 15 ml toluene. The resulting solution is refluxed until most of the free base coordinated with the metal ion. Then, dry NaLOMe (0.1 g, 0.22 mmol) [LOMe-((cyclopentadienyl)tris(dimethylphosphito)-cobaltate, an anionic tripodalligand) is added and magnetically stirred for another 12 hours before the reaction solution is cooled down to room temperature. Upon completion of the reaction, the solvent is removed in vacuum and the residue dissolved in CHCl.sub.3, filtered and chromatographed on silica gel using CHCl.sub.3/petroleum ether (V/V 1:1) as eluent. The product is dissolved in CH.sub.2Cl.sub.2 (5 ml) and the solution is filtered.
(119) Yb-1:
(120) Yield: 81%; .sup.1HNMR (CDCl.sub.3): 5.02 (s, 5H), 0.93 (s, 9H), 6.37 (s, 18H), 8.70 (s, 1H), 8.97 (d, J=4.96 Hz, 1H), 10.88 (s, 1H), 14.65 (s, 2H), 14.89 (s, 2H), 15.18 (s, 2H), 15.58 (s, 2H), 17.40 (s, 1H); MALDI-TOF MS: calcd. For C.sub.60H.sub.44CoF.sub.15N.sub.4O.sub.9P.sub.3SiYb [M].sup.+ 1603.0571. found: 1603.0556.
(121) Er-1:
(122) The same procedure with Yb-1, replace Yb[N(SiMe.sub.3)2].sub.3.x[Li(THF).sub.3Cl] with Er[N(SiMe.sub.3)2].sub.3.x[Li(THF).sub.3Cl]; Yield: 80%. .sup.1HNMR (CDCl.sub.3): 35.54 (s, 5H), 3.48 (s, 9H), 14.09 (s, 1H), 13.50 (s, 1H), 21.73 (s, 18H), 21.16 (s, 1H), 31.22 (s, 2H), 32.93 (s, 2H), 36.37 (s, 2H), 37.76 (s, 2H), 46.77 (s, 1H); MALDI-TOF MS: calcd. For C.sub.60H.sub.44CoErF.sub.15N.sub.4O.sub.9P.sub.3Si [M].sup.+ 1597.1878. found 1597.2927.
(123) General Procedure for the Preparation of Ln-2
(124) Yb-2:
(125) TBAF (1.0 M in THF, 0.2 mL, 0.2 mmol) is added to a solution of Yb-1 (0.05 mmol 76.55 mg,) in 10 ml CH.sub.2Cl.sub.2, and the solution is stirred for 30 min. The progress of the reaction is monitored by Thin-layer Chromatography (TLC). After completion of the reaction, the mixture is passed through a short of silica gel column. After removal of solvent, pure product is obtained.
(126) Yb-2:
(127) Yield: 92%; .sup.1HNMR (CDCl.sub.3): 4.82 (s, 5H), 4.13 (s, 1H), 6.30 (s, 18H), 8.63 (s, 1H), 8.95 (d, J=4.44 Hz, 1H), 10.83 (s, 1H), 14.51 (s, 2H), 14.90 (s, 2H), 15.08 (s, 2H), 15.44 (s, 2H), 17.21 (s, 1H); MALDI-TOF MS: calcd. For C.sub.57H.sub.36CoF.sub.15N.sub.4O.sub.9P.sub.3Yb [M+Na+Cl].sup.+ 1587.0176. found 1587.0514.
(128) Er-2:
(129) The same procedure with Yb-2, replace Yb-1 with Er-1; Yield: 92%; .sup.1HNMR (CDCl3): 35.05 (s, 5H), 13.94 (s, 1H), 13.19 (s, 1H), 20.56 (s, 18H), 21.02 (s, 1H), 30.97 (s, 2H), 32.77 (s, 2H), 36.44 (s, 2H), 37.36 (s, 2H), 46.20 (s, 1H); MALDI-TOF MS: calcd. For C.sub.57H.sub.36CoErF.sub.15N.sub.4O.sub.9P.sub.3 [M+H].sup.+: 1525.0067. found: 1525.0143.
(130) General Procedure for the Preparation of Ln-4
(131) Yb-4:
(132) Pd(PPh.sub.3).sub.4 (22.16 mg 0.08 mmol), CuI (7.65 mg, 0.04 mmol), Yb-2 (30.62. mg, 0.02 mmol) and 4-iodobenzoic acid 5.087 mg are placed in a dried flask and under nitrogen. THF (15 mL) and Net.sub.3 (5 mL) are added and the reaction mixture degassed with nitrogen. The reaction mixture is stirred at 40 C. for 12 hours. After that, the solvent is removed under reduced pressure. The residue is purified by chromatography. Elution with CH.sub.2Cl.sub.2/Methanol (12:1). The eluted compound (26 mg, 0.0157 mmol), EDCI (6.04 mg, 0.031 mmol), NHS (3.57 mg, 0.031 mmol) are placed in a dried flask and under nitrogen, 10 mL dry DMF is added. Stirred at room temperature for 48 hours, then remove the solvent. The residue is recrystallized by diethyl ether and dried to give Yb-4.
(133) Yb-4:
(134) Yield: 72%; .sup.1HNMR (CDCl.sub.3): 4.82 (s, 5H), 4.16 (s, 1H), 6.39 (s, 18H), 8.68 (s, 1H), 8.98 (d, J=4.44 Hz, 1H), 8.47 (s, J=4.44 Hz, 2H), 8.45 (s, J=4.44 Hz, 2H) 10.91 (s, 1H), 14.63 (s, 2H), 14.92 (s, 2H), 15.24 (s; 2H), 15.61 (s, 2H), 17.39 (s, 1H); MALDI-TOF MS: calcd. For C.sub.68H.sub.43CoF.sub.15N.sub.5O.sub.13P.sub.3Yb [M].sup.+ 1748.0176. found 1748.0460. HPLC characterization: retention time=7.24 min (
(135) Er-4:
(136) The same procedure with Yb-4, only replace Yb-2 with Er-2; Yield: 80%; .sup.1HNMR (CDCl.sub.3): 35.94 (s, 5H), 6.04 (s, 1H), 8.64 (d, J=7.96 Hz, 2H), 10.80 (d, J=5.12 Hz, 2H), 13.12 (s, 1H), 13.76 (s, 1H), 20.67 (s, 18H), 20.90 (s, 1H), 31.06 (s, 2H), 32.94 (s, 2H), 36.39 (s, 2H), 37.62 (s, 2H), 46.54 (s, 1H); MALDI-TOF MS: calcd. For C.sub.68H.sub.43CoErF.sub.15N.sub.5O.sub.13P.sub.3 [M+Cl.sup.]: 1777.2035. found: 1777.4591. HPLC characterization: retention time=7.23 min(
(137) General Procedure for the Preparation of Ln-Rn
(138) YbR.sub.1:
(139) A stirred solution of Yb-4 (16 mg, 1 equiv.) in anhydrous DMF is mixed with N,N-diisopropylethylamine (DIPEA) (1 equiv.). The mixture solution is added into a vial which contains peptide R.sub.1 (1.3 equiv.) It was then reacted at room temperature overnight, after that, the solvent is removed under vacuum to get the dry compound. The residue is recrystallized by diethyl ether three times and dried to give YbR.sub.1.
(140) YbR.sub.1:
(141) Yield: 69%. MALDI-TOF MS: calculated for C.sub.109H.sub.109CoF.sub.15N.sub.19O.sub.23P.sub.3S.sub.3Yb [M+H].sup.+: 2760.4878. found: 2760.6458. HPLC characterization: retention time=10.00 min (
(142) YbR.sub.2:
(143) The same procedure with YbR.sub.1, peptide R.sub.2 is used; Yield: 69% MALDI-TOF MS: calculated for C.sub.113H.sub.129CoF.sub.15N.sub.21O.sub.22P.sub.3S.sub.2Yb [M+H].sup.+ 2808.6835. found: 2808.6715. HPLC characterization: retention time=10.21 min (
(144) YbR.sub.3:
(145) The same procedure with YbR.sub.1, peptide R.sub.3 is used; Yield: 65% MALDI-TOF MS: calculated for C.sub.143H.sub.187CoF.sub.15N.sub.35O.sub.28P.sub.3S.sub.2Yb [M+H].sup.+ 3520.2985. found: 3520.2543. HPLC characterization: retention time=10.01 min (
(146) ErR.sub.1:
(147) The same procedure with YbR.sub.1, replace Yb-4 with Er-4; Yield: 75% MALDI-TOF MS: calculated for C.sub.109H.sub.109CoErF.sub.15N.sub.19O.sub.23P.sub.3S.sub.3 [M+K].sup.+: 2791.4826. found: 2791.3747. HPLC characterization: retention time=9.66 min (
(148) ErR.sub.2:
(149) The same procedure with YbR.sub.2, replace Yb-4 with Er-4; Yield: 72% MALDI-TOF MS: calculated for C.sub.113H.sub.129CoErF.sub.15N.sub.21O.sub.22P.sub.3S.sub.2 [M+K].sup.+: 2839.6015. found: 2839.2967. HPLC characterization: retention time=10.09 min (
(150) ErR.sub.3:
(151) The same procedure with YbR.sub.3, replace Yb-4 with Er-4; Yield: 70% MALDI-TOF MS: calculated for C.sub.143H.sub.187CoErF.sub.15N.sub.35O.sub.28P.sub.3S.sub.2 [M].sup.+: 3511.4955. found: 3511.5162. HPLC characterization: retention time=9.80 min (
(152) Cell Culture
(153) Human bladder carcinoma (T24) and (5637) cells are cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and antibiotics (penicillin, 50 gmL.sup.1; streptomycin, 50 gmL.sup.1). Human cervical carcinoma (HeLa) cells are cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) and antibiotics (penicillin, 50 gmL.sup.1; streptomycin, 50 gmL.sup.1). Human normal lung fibroblast (MRC-5) cells are maintained in minimum essential medium (MEM) supplemented with 10% FBS and 1% 50 gmL.sup.1 penicillin; 50 gmL.sup.1 streptomycin. All cells are incubated at 37 C. in a humidified environment with 5% CO.sub.2.
(154) Dark Cytotoxicity
(155) T24, HeLa and MRC-5 cells (110.sup.5) are treated with ErR.sub.n porphyrin complexes and YbR.sub.n porphyrin complexes for 24 hours at six concentrations (1, 5, 10, 50, 100, 500M). The cell monolayers are rinsed once with phosphate-buffered saline (PBS) and incubated with 500 gmL.sup.1 3-(4, 5-dimethylthiazol-2-yl)-2 and 5-diphenyltetrazolium bromide (MTT) solution. The cellular inhibitory potency of the complexes is examined by treating the cells with MTT for 3 hours to allow formazan production during cell metabolism. After that, the formazan crystals are fully dissolved in DMSO with oscillation. Finally, the absorbance of solution is measured with Biotek PowerWave XS microplate reader at the wavelengths of 570 and 690 nm.
(156) Photo-Cytotoxicity
(157) T24, HeLa and MRC-5 cells (110.sup.5) are treated with ErR.sub.n porphyrin complexes and YbR.sub.n porphyrin complexes for 24 hours at four concentrations (1, 5, 10, 50M). Then, the cells are irradiated at 6 mWcm.sup.2 (equipped with 550 nm long pass filter) for about 27 minutes and further incubated for 24 hours. The cells are then treated according to the same protocol as the previous MTT assay.
(158) In Vitro Confocal Microscopy
(159) To investigate the suitability of the obtained complexes as bioprobes, T24, 5637, HeLa and MRC-5 cells (110.sup.5) are imaged. After incubation with the complexes at 5M for 24 hours, the cells are washed with PBS for three times before imaging. LysoTracker Green DND-26 was used as containing dye. Images were acquired on a Leica TCS SPE confocal laser-scanning microscope. The samples and LysoTracker were excited at wavelength of 561 and 488 nm respectively.
(160) Flow Cytometry Measurements of Cellular Uptake
(161) 5637, T24, HeLa and MRC-5 cells (110.sup.5 per sample) are seeded onto 35 min Petri dishes and incubated overnight. Then the cells are incubated with the ErR.sub.n and YbR.sub.n porphyrin complexes (5M) for 3, 6 and 24 hours. Cells are harvested with trypsin and washed twice with PBS. The uptake of the complexes by the 5637, T24, HeLa and MRC-5 cells is analyzed by flow cytometry. The cells are excited with a 488 nm argon laser and emission is collected in the FL-3 channel (with a 650 nm long-pass filter); 10000 events are analyzed.
(162) HPLC Characterization of the Complexes.
(163) TABLE-US-00005 TABLE 5 Solvent gradient for HPLC Time/min 0.05% TFA in water/% MeOH/% 0 50 50 5 20 80 20 0 100
(164) Another further embodiment of the present invention.
(165) i. Development and Comprehensive Studies of Various Organometallic/Lanthanide Complexes for Biological Studies
(166) Another series of organometallic complexes are provided in the present invention and their structure-photophysical property relationship in multi-photon and non-linear processes are studied. These complexes are applicable for biological imaging. Water-soluble lanthanide(III) porphyrinate complexes and meso-pyridinium-substituted porphyrin are obtained by methylation of the corresponding pyridyl complexes with methyl iodide and unambiguously characterized. The binding interactions and photocleavage activities of these water-soluble lanthanide(III) porphyrinate complexes towards DNA are investigated (
(167) ii. Development of Organometallic Complexes as In Vivo Tumor Specific PDT Agents
(168) The present invention provides another set of novel organelle specific markers (for lysosome, mitochondria, Golgi apparatus). These complexes in-vitro simultaneously trigger the generation of .sup.1O.sub.2 in-vitro and give luminescent images of the organelles upon irradiation by visible/NIR excitation. Such behavior affords spatial control using dual laser excitations to damage selected cell compartments/components. Previously, the inventors reported a porphyrinato ytterbium complex which shows a strong binding to phosphatidylserine and the capability to differentiate of cancer cells via targeting the anionic phospholipid membrane (
(169) In the present invention, another set of multi-modal porphyrinato lanthanide-based complexes for biological imaging are providedNIR optical and t.sub.1 magnetic resonance imagingthat bind strongly to the targets, phospholysation anionic membrane/integrin .sub.v.sub.3 isoform, and generates .sup.1O.sub.2 as anticancer agents. The present invention provides (1) complexes for use as dual probe for optical and MRI imaging and cancer specific PDT effect, via the bio-conjugation with the known functional groups and peptides, and .sup.1O.sub.2 from porphyrin moieties; (2) in vitro anticancer effects via optical imaging and other typical protocols; (3) in vivo pharmacokinetics and bio-distribution of these complexes (with anticancer effects) by MRI imaging/ICPMS.
(170) Herein, the inventors have designed and synthesized 4 water-soluble, cell-permeable porphyrin-based gadolinium complexes, Gd-1, Gd-2, Gd-3-R1, Gd-3-N (
(171) (a) The Stability, Solubility and Uptake Profile of Gadolinium Complexes, Gd-1, Gd-2, Gd-3-R.sub.1, Gd-3-N
(172) The protonation of the substituent groups on the porphyrin of Gd-1, Gd-2, Gd-3-R.sub.1, Gd-3-N demonstrates an improvement of the water solubility. All the complexes have been purified by HPLC. The organometallic system Gd-2, Gd-3-R.sub.1 and Gd-3-N show better stability than Gd-1, with the P.sub.m values of Gd-2. Gd-3-R.sub.1 and Gd-3-N being around 8.15. P.sub.m value is the negative log of the concentration of free metal ion left uncomplexed by a given chelator, pM=log [M].sub.free. The cellular uptake profile from flow cytometry also shows that Gd-3-R.sub.1 has the fastest uptake rate among the four complexes in the cancer cells and also with the better selectivity towards bladder cancer T24 cells rather than normal MRC-5 cells (
(173) (b) The .sup.1O.sub.2 and NIR Emission Quantum Yield
(174) Upon protonation of the complexes, similar .sup.1O.sub.2 and emission quantum yield are shown and compared with the existing complex, H.sub.2TPP, which are around 70% .sup.1O.sub.2 quantum yield (
(175) I the t.sub.1 Relaxivity and Ex Vivo Toxicity/Distribution of Gd-1, Gd-2, Gd-3-R.sub.1, Gd-3-N
(176) With an aim to develop them as MR contrast agents, the water exchange rate of the complexes is one of key preliminary studied. The t.sub.1 relaxivity of Gd-1, Gd-2, Gd-3-R.sub.1, Gd-3-N are evaluated and Gd-3-R.sub.1 has three times t.sub.1 relaxivity greater than Gd-DOTA (
(177) (d) The In Vivo Magnetic Resonance Imaging (MRI)
(178) After tail vein injection of Gd-3-R.sub.1 and Gd-DOTA into the mice, MRI experiment is conducted immediately. As shown in
(179) Multi-Modal Lanthanide-Based Bio-Probes with Integrin .sub.v.sub.3 Isoform, Specific Peptide Coatings as a Simultaneous Imaging (Optical and MR) and Anti-Cancer Agent In Vitro and In Vivo.
(180) Task 1Synthesis of Smart Organometallic Complexes that can Selectively Differentiate Tumor Cells for Effective PDT and Available for MR and Optical Imaging
(181) There are two major problems associated with commercially or literature available photosensitizers in photodynamic treatment: (i) the recognition of cancer cells and (ii) the monitoring of their effectiveness. Studies show that newly developed lanthanide complex, Gd-3-R.sub.1, is able to identify cancer cells through its anionic PS membrane, generate .sup.1O.sub.2 with certain laser wavelength, and display two-photon induced NIR emissions and MR availability. The inventors extend their previous findings and run a comprehensive program to obtain new lanthanide complexes (on top of cancer cell selectivity with better .sup.1O.sub.2 yield) as a new generation of PDT agents specifically for cancer disease, especially for bladder cancer. The new development agents are able to treat tumors that are deeper under the skin or in body tissues, more selective for cancer cells as opposed to normal cells and removed from the body more quickly, reducing the time people need to worry about photosensitivity reactions. Selection criteria for the best photosensitizers. There are a number of criteria for the selection of photosensitizers. First of all, it must be water soluble. The photosensitizer(s) should be able to be excited in the near infrared region, especially between 800 nm to 900 nm. Also, the .sup.1O.sub.2 quantum yield of the best photosensitizers should be >20% and with specific mitochondria subcellular localization. The photocytotoxicity of valid bladder cancer specific photosensitizer (i.e. LC.sub.50 in 1J laser dosed is 1M) must be 100 times lower than dark cytotoxicity. (IC50 have to be >0.1 mM).
(182) Absorption and Fluorescence Details of Porphyrin-Based Compounds
(183) Porphyrin is a highly conjugated molecule with 11 delocalized double bonds. The electronic absorption bands of metalloporphyrins are found at 410-430 nm (the Soret band or B band) and 550-650 nm (Q bands) with strong one photon absorption coefficients (>100K M.sup.1 cm.sup.1). The emission band of porphyrin is always located at 650 to 700 nm. As a result, the emission and excitation bands of metalloporphyrins are always located within biological windows. In addition, the strong two photon absorption cross section of metalloporphyrins are recorded with >100 GM, which indicates that the porphyrin can be excited at 860 nm and give two photon induced emission at 650 nm to 700 nm for molecular imaging, as well as generation of .sup.1O.sub.2 and PDT.
(184) 1.1 Synthesis of Water-Soluble, Highly Emissive, MR and .sup.1O.sub.2 Available Porphyrin-Based Gadolinium (III) Complexes which Target Anionic Phospholipid Membrane
(185) Water-soluble porphyrin-based gadolinium complex (GdN) exhibits strong selectivity toward the cancer cell membrane, emits strong visible-to-NIR emission, and undergoes high metabolism that can be removed from the body quickly. To improve the relaxivity and stability of the complexes as multi-modal agents, the present invention provides two more types of organometallic complexes in which the Gd ions are stabilized by other organometallic compounds (Gd-4-R.sub.n) or with carboxylic pendant arm (Gd-5-R.sub.n) (
(186) 1.2 Synthesis of Water-Soluble, Highly Emissive, MR and .sup.1O.sub.2 Available Porphyrin-Based Gadolinium (III) Complexes which Target Bladder Cancer Cell/Tumor
(187) For bladder cancer diagnosis, the physical examination on the detection of bladder cancer has high risk to damage the bladder function. When it comes to specific treatment, PDT is a newer treatment method that is now being studied to see if it is useful in treating especially early stage bladder cancers. The limitations of using the traditional PDT are the poor penetration power of light and the difficulty to monitor the performance in real time. The present invention provides multi-modal PDT agents for comprehensive diagnosis and treatment on bladder cancerMR for diagnosis, NIR induced .sup.1O.sub.2 for PDT and NIR induced NIR emission for real time monitoring the effectiveness of PDT. In the inventors' publication (PNSA, 2014, E5492-E5497) and the above examples, the stability, relaxivity, NIR-induced emission, .sup.1O.sub.2 ability and selectivity of the present porphyrin based complexes in cancer cells are shown. The design of the theranostic complexes targeting bladder cancer is shown in the
(188) Preparation of Compound Por-TMS
(189) 4-((trimethylsilyl)ethynyl)benzaldehyde (2.02 g, 10 mmol) was mixed with Pyridine-4-carboxaldehyde (3.21 g, 30 mmol) in propionic acid (700 mL) and the mixture was stirred for half an hour in 130 C. Then pyrrole (2.64 g, 40 mmol) was added dropwise into the reaction mixture with the temperature increased to 140 C. Then the mixture continued to be stirred for 30 minutes in open air. After cooled down to room temperature, the solvent was removed under reduce pressure to afford black solid. The crude product was dissolved in minimum amount of CH.sub.2Cl.sub.2 and purified by column chromatography on silica gel column CH.sub.2Cl.sub.2/Methanol (12:1) to give purple color solid. Yield 8%.
(190) Gd[N(SiMe.sub.3).sub.2].sub.3.x[LiCl(THF).sub.3]:
(191) HN(SiMe.sub.3).sub.2 (10.8 ml, 0.050 mol) was dissolved 20 ml of THF in ice bath, then n-BuLi (1.6M in hexane) was added slowly over 30-min period. The resulting solution was magnetically stirred for 12 hours until a clear pale yellow solution was obtained. Then the solution was transferred slowly to a Schlenk flask with GdCl.sub.3 (4.47 g, 0.017 mol) suspended in 20 ml THF. The resulting mixture was magnetically stirred for 24 h until all of the solid GdCl.sub.3 was disappeared. The resultant solution Gd[N(SiMe.sub.3).sub.2].sub.3.x[LiCl(THF).sub.3] (x=35) was referred to as solution C
(192) Gd-1-L1:
(193) Solution C (2.5 ml, 0.52 mmol of Gd) prepared above was transferred to a Schlenk flask and the solvent was removed under vacuum. Then 10 ml CH.sub.2Cl.sub.2 was added for the precipitation of LiCl. The mixture was centrifuged and the clear layer was transferred to another Schlenk flask with dry Por-TMS (0.099 g, 0.14 mmol) dissolved in 15 ml toluene. The resulting solution was refluxed until most of the free base coordinated with the metal ion. Dry NaL.sub.1 (0.1 g, 0.22 mmol) [L.sub.1-((cyclopentadienyl)tris(dimethylphosphito)-cobaltate, an anionic tripodal ligand) was then added and magnetically stirred for another 12 hours before the reaction solution was cooled down to room temperature. Upon completion of the reaction, the solvent was removed in vacuum and the residue dissolved in CHCl.sub.3, filtered and chromatographed on silica gel using CHCl.sub.3/CH.sub.3OH ether (V/V 200:1) as eluent. The product was dissolved in CH.sub.2Cl.sub.2 (5 ml) and the solution was filtered. Yield: 61%
(194) Gd-1-L2:
(195) The similar procedure with Gd-1-L1, replace NaL.sub.1 with KL.sub.2 (potassium tris(1-pyrazolyl) borohydride, 0.055 g, 0.22 mmol). Yield: 50%.
(196) Gd-3:
(197) TBAF (1.0M in THF, 0.2 mL, 0.2 mmol) was added to a solution of Gd-1-L1 (0.133 mg, 0.1 mmol) in 10 ml DCM, and the solution was stirred for 30 min. The progress of the reaction was monitored by TLC. After completion of the reaction, the mixture was passed through a short of silica gel column using DCM After removal of solvent, pure product was obtained, the pure product (33.2. mg, 0.02 mmol) and Pd(PPh.sub.3).sub.4 (2.2 mg 0.008 mmol), CuI (0.77 mg, 0.004 mmol), 4-iodobenzoic acid 5.1 mg were placed in a dried flask and under nitrogen. THF (15 mL) and Net.sub.3 (5 mL) were added and the reaction mixture degassed with nitrogen. The reaction mixture was stirred at 40 C. for 12 h. After that, the solvent was removed under reduced pressure. The residue was purified by chromatography. Elution with CH.sub.2Cl.sub.2/Methanol (10:1) afforded the pure product, the pure product (30 mg, 23.75 mmol), EDCI (9.02 g, 0.048 mmol), NHS (5.52 mg, 0.048 mmol) were placed in a dried flask and under nitrogen, 10 mL dry DMF was added. Stirred at room temperature for 48 h. then remove the solvent, The residue was recrystallized by diethyl ether and dried to give the title product, the product (33.37 mg, 0.025 mol) was dissolved in DMF (10 ml), Then CH.sub.3I (0.25 mmol) was added and stirred for 5 h, After completion of the reaction, solvent was removed. The residue was washed with ether .DCM. afforded the pure product. Yield 52%.
(198) Gd-4:
(199) The similar procedure with Gd-3, replace Gd-1-L1 with Gd-1-L2. Yield 50%.
(200) Gd-3-Rn:
(201) A stirred solution of Gd-3 (20 mg, 1 equiv) in anhydrous DMF was mixed with N,N-diisopropylethylamine (DIPEA) (1 equiv.). the mixture solution was added into a vial which containing peptide (1.3 equiv.) (R.sub.n). It was then reacted at RT overnight, after that, the solvent was removed under vacuum to get the dry compound. The residue was recrystallized by diethyl ether three times and dried to give the titled product. Yield 70%.
(202) Gd-4-Rn:
(203) The same procedure with Gd-3-Rn, replace Gd-3 with Gd-4. Yield 68%.
(204) Task 2: Examination of the Selectivity and Relaxivity of Gd-3-R.sub.1 with Bladder Cancer
(205) The general photophysical properties, such as emission quantum efficiency and emission lifetime are examined. The magnetic properties of these gadolinium porphyrin complexes are measured. The binding affinity between the .sub.v.sub.3 isoform and the present invented porphyrin complexes Gd-3-R.sub.1, is the prime factor for developing the multi-modal probe for monitoring .sub.v.sub.3 isoform bladder cancer. The binding affinities of the present invented complexes for .sub.v.sub.3 isoform bladder cancer are determined by enthalpy changes and electrostatic interactions. The cell-permeable and water-soluble porphyrin-based gadolinium porphyrin complexes are to be bioconjugated with a cancer-specific vector (peptide). The tailored peptide can be used to trace the integrin .sub.v.sub.3 isoform in bladder cancer cell membrane. Bladder cancer (T24) model are under studied and other cancer cell models such as HeLa, SK-N-SH, A549, C666-1 and normal cells: MRC-5 are served as control (
(206) 2.1 Photophysical Properties and Stability of the Newly Synthesized Gadolinium (III) Complex, Gd-3-R.sub.1
(207) The linear, multi-photon photophysical properties (i.e. emission spectra, emission lifetime, quantum yield and two-photon absorption cross section) and .sup.1O.sub.2 yield and also photo-bleaching quantum yield (compared with standard, such as uroporphyrin) of Gd-3-R.sub.1 is measured following the literature protocols. Titration experiments are conducted to investigate the stabilities of the synthesized porphyrin complexes toward several common biological anions and human serum albumin (HSA); P.sub.M and P.sub.Ka are determined. Liquid-concentrated stock solutions of each anion, as well as HSA, are added gradually to a solution of the complexes concerned separately. Absorption, fluorescence as well as .sup.31P NMR spectroscopy are used to monitor the stability of the complexes in aqueous solution upon the addition of various biological small molecules such as HSA, citrate etc.
(208) 2.2 Binding Affinity Via Electrophoretic Mobility Shift Assay
(209) Electrophoretic Mobility Shift Assay is a powerful method for determining the binding affinity of the inventors' gadolinium porphyrin complexes to .sub.v.sub.3 isoform cancer cell which is specific for bladder cancer. The .sub.v.sub.3 isoform cell are expressed in an E. coli system and further purified by glutathione affinity chromatography before carrying out agarose gel electrophoresis. Experiments are to confirm physical structure of the .sub.v.sub.3 isoform would not be altered by the binding of the lanthanide bio-probes.
(210) 2.3 Evaluation the Binding Affinity Via Emission and Isothermal Titration Calorimetric
(211) The binding affinity of the complexes and .sub.v.sub.3 isoform is studied by isothermal titration calorimetry (ITC), a solution state method that measures the interactions between molecules, e.g. macro-proteins and ligands. The binding affinity (K.sub.a), binding stoichiometry (N) and the enthalpy changes (H) of the interaction could all be determined by ITC experiments directly. From the enthalpy change, the Gibbs energy and entropy change are determined by established equations. Advantages of ITC include a real-time observation of inter-molecule interactions without limitation on molecular weight in, most importantly, a nondestructive manner.
(212) 2.4 Relaxivity Determination of Gadolinium Porphyrin Complex, Gd-3-R.sub.1
(213) The relaxivity of the inventors' synthesized complexes will be calculated from the relaxation time obtained by a Bruker DPX300 NMR spectrometer in D20 solutions. An inversion-recovery pulse sequence is used and a tenT.sub.1 delay is maintained between successive pulses. The relaxivity (r1) is obtained by a plot of the inverse of longitudinal time (1/T.sub.1) versus Gd concentrations:
(214)
(215) where T.sub.1obs and T.sub.1b are the longitudinal relaxation times of the sample and the solvent background respectively.
(216) 2.5 Tumour Models or Cell Lines Will be Used, and Details of Biological Studies
(217) Bladder cancer (T24) model is under studied and other cancer cell models such as HeLa, SK-N-SH, A549, C666-1 and normal cells: MRC-5 are served as control. Cancer/normal cells (Cancer cells: T24-bladder cancer, HeLa, SK-N-SH, A549, C666-1 and normal cells: MRC-5, (210.sup.4/well) are incubated in 96-well plates overnight. In vitro imaging for selective bindingthe cells are treated with Gd-3-R.sub.1 (Task 1) for 6, 12 and 24 hours in the dark. The culture medium is replaced by fresh medium and the cells are exposed to light (1-8 J/cm.sup.2) produced from a laser (linear and multi-photon femtosecond Ti:sapphire laser) in the multi-photon confocal microscope. The time-lapse confocal images of Gd-3-R.sub.1 in cells are carried out and their in vitro subcellular localization are compared. The subcellular localization of Gd-3-R.sub.1 are different in bladder cell T24 and other non-bladder cancer cell lines, such as HeLa, C666-1 and SK-N-SH.
(218) In Vitro Photo-Cytotoxicity
(219) Bladder cancer T24 cells are treated with several concentrations of complex and incubated for 12 hours. The free complex in the medium will be removed by changing the medium several times. The cells will be irradiated by laser to initiate the release of .sup.1O.sub.2 from the complex and MTT assay is performed to measure the cell viability after a number of incubation time points. Control experiment is performed with the same experimental condition, such as light dosed amount, incubation time and concentration of proposed complexes in non-bladder cancer cell lines.
(220) The in vitro dark toxicity of the invented complexes are tested. After 24 hours, the water-soluble complexes and the targeted cells are incubated further with 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (0.5 mg/ml), otherwise known as MTT, for 4 hours, so that formazan can be formed along with the cell's metabolic pathways. Then, the formazan is extracted and dissolved by dimethyl sulfoxide (DMSO), with the absorbance of the subsequent solutions measured in a Bio-Rad iMark microplate reader (490 nm). Quadruplicates are performed and the data are interpreted and analyzed using the GraphPad Prism 5 software.
(221) The photocytotoxicity of valid bladder cancer specific photosensitizer (i.e. LC.sub.50 in 1J laser dosed is 1M) must be 100 times lower than its dark cytotoxicity. (IC50 have to be >0.1 mM).
(222) Task 3Structure and Biological Activity (In Vitro/-Vivo Imaging and Specific PDT Effect)
(223) In this section, effectiveness of the Gd-3-R.sub.1 in vivo are evaluated. Comprehensive in vitro and in vivo examinations are employed, such as multi-confocal in vivo real time studies, MR imaging and metabolism studies (
(224) 3.1 Determine the Stability of Lanthanide Complexes for Biological Assays
(225) There is great challenge of the coordination stability lanthanide complexes for cellular studies. Therefore, the aqueous/tissue culture medium stability must be carried out. The aqueous stability of the present complexes in the presence of various biomolecules, including citrate and human serum albumin (HSA), and in varies pH are examined as well by simple UV-vis absorption/fluorescence titration via the aforementioned procedures. Liquid concentrated stock solutions of each anion, as well as HSA, are added individually and gradually to a solution of the complex concerned. Addition is ceased either when the volume of added anion totaled 5% of the complex solution or the influence on complex absorption/luminescence was saturated.
(226) 3.2 In Vitro Cytotoxicity Studies and Rate of Cellular Uptake
(227) For the establishment of the mouse tumor xenograft mode, bladder cancer cells (T24) or non-bladder cancer cells (HeLa) are trypsinized, harvested and suspended in serum-free culture medium. 510.sup.6 cells in a 100 L volume is injected subcutaneously into the flanks of female athymic nude mice (5-week old) When the tumor volume reaches the size of around 100 mm.sup.3, animals are divided randomly into four experimental group with SEVEN mice in each group, as follows: group 1, vehicle control group; group 2, cisplatin treatment group; group 3, GdN low dose treatment group; group 4, GdN high dose treatment group. Treatments is administered via intratumoral injection once every 5 days, for 21-28 days. The experiment is repeated three times. The tumor volume is measured every 2 days with electronic calipers (accuracy of 0.02 mm) and then calculated independently on the basis of the equation V=(LW.sup.2)/2, where L and W correspond to the larger and smaller dimensions respectively. All animal experiments is carried out in accordance with the guidelines of the Committee on Use of Human and Animal Subjects in Teaching and Research, Hong Kong Baptist University. One-way analysis of variance towards statistical significances between groups was assessed by the GraphPad Prism 5.0 software.
(228) 3.3 Real Time Analysis of .sub.v.sub.3 Isoform and Evaluation of Effectiveness (Tumorigenically Assays) of Tumor Inhibition-Days/Week Tracing of Tumor Development of Xenograft Mouse Via Multi-Photon Confocal and Magnetic Resonance Imaging for Pharmacokinetic Studies
(229) The development of xenograft mice is achieved by transplanting human bladder tumor cells (T24) to mice which are allowed to grow. The complexes is injected at the tail vein, the peritoneum or buccally delivered and after 24 to 48 hours, the xenograft are surgically extracted for two-photon confocal microscopy and MRI experiments, with the peritumor cells extracted being the control (no lanthanide complexes signals should be obtained). In vivo MM experiments are carried out on the xenograft in a CoI institute with a Bruker Biospec 4.7 T/30 cm scanner (Bruker Inc., MA). Moreover, the tumor sizes will be measured weekly.
(230) 3.4 In Vivo Bio-Distribution Evaluation of Proposed Gadolinium Complexes
(231) Gd-3-R.sub.1 is injected intravenously to BALB/c athymic mice bearing xenografted cancer tumors. After 24 hours of incubation, the mice are sacrificed and its main organs including the tumor is removed and fixed in 10% PBS buffered formalin. Control models are athymic mice with only the buffered formalin injected. The tissue samples are frozen and lyophilized for 24 h before being digested by conc. HNO.sub.3 at 70 C. for 4 hours. The gadolinium content, reflective of the quantity of the complexes, are determined by ICP-MS. The gadolinium content in urine of the mouse are evaluated to confirm the metabolism of these complexes in vivo. The results in 3.3 and 3.4 should be correlated (
INDUSTRIAL APPLICABILITY
(232) The present invention relates to a new generation of PDT agents based on porphyrin-lanthanide complexes with specific functional groups which can specifically localize on particular tumors, and their PDT processes can be monitored via NIR emission from erbium. In particular, the present invention provides a multi-modal lanthanide-porphyrin PDT agent (ErR.sub.3) that are capable of killing the bladder tumor cells selectivity via .sup.1O.sub.2 from porphyrin moiety and affording the fluorescence imaging simultaneously upon ErR.sub.3 binding with the integrin .sub.v.sub.3 isoform in bladder cancer cells.
(233) If desired, the different functions discussed herein may be performed in a different order and/or concurrently with each other. Furthermore, if desired, one or more of the above-described functions may be optional or may be combined.
(234) While the foregoing invention has been described with respect to various embodiments and examples, it is understood that other embodiments are within the scope of the present invention as expressed in the following claims and their equivalents. Moreover, the above specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety.