EP3523312A1 - Multimodale biosonde für blasenkrebsbildgebung und fotodynamische therapie - Google Patents

Multimodale biosonde für blasenkrebsbildgebung und fotodynamische therapie

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Publication number
EP3523312A1
EP3523312A1 EP17857842.3A EP17857842A EP3523312A1 EP 3523312 A1 EP3523312 A1 EP 3523312A1 EP 17857842 A EP17857842 A EP 17857842A EP 3523312 A1 EP3523312 A1 EP 3523312A1
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Prior art keywords
mixture
produce
solution
residue
compound
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English (en)
French (fr)
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EP3523312A4 (de
Inventor
Ka Leung Wong
Wai Kwok Wong
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Hong Kong Baptist University HKBU
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Hong Kong Baptist University HKBU
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Priority claimed from US15/352,561 external-priority patent/US9840522B2/en
Application filed by Hong Kong Baptist University HKBU filed Critical Hong Kong Baptist University HKBU
Publication of EP3523312A1 publication Critical patent/EP3523312A1/de
Publication of EP3523312A4 publication Critical patent/EP3523312A4/de
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a new generation of photodynamic therapy agents based on porphyrin-lanthanide complexes, and the photodynamic therapy processes that can be monitored via NIR emission from erbium.
  • the present invention provides a multi-modal lanthanide-porphyrin photodynamic therapy agent that are capable of killing bladder tumor cells.
  • Photodynamic Therapy is emerging as a novel cancer treatment modality to help patients to live longer and to improve their quality of life without causing long-term side-effects.
  • PDT is still failing to obtain deserved popularity in society due to current technological constraints and absence of practical advances as it is only offered in some treatment center and studied with little clinical trials.
  • FDA United States Food and Drug Administration
  • ALA Aminolevulinic acid
  • conventional PDT still has from several limitations and drawbacks: (i) it is only able to treat diseased areas where light can be reached, that is, on or just under the skin; (ii) currently-used PDT drugs could leave people very sensitive to light, and therefore special precautions must be taken after the drugs are put in or on the body; (iii) adverse in-vitro/in-vivo reactions occur due to the variation in physiological conditions and notched distribution of cytotoxic singlet oxygen; and (iv) non-specific therapeutic nature may jeopardize normal cells during PDT treatment.
  • porphyrin-based moieties another novel class of promising PDT agents, have been investigated extensively by scientists worldwide to develop photodynamic therapy can be available and effective to other types of cancers and diseases particularly in the skin, bladder, mouth, and brain.
  • the light penetration depth for the singlet oxygen ( 1 O 2 ) generation is concerned, several porphyrin moieties have successfully showed the possibility to achieve near-infrared (NIR) excitation (via multi-photon/Second harmonic generation) .
  • NIR photons can penetrate deep and emit expeditiously from tissues without causing cell damage with their strong two-photon absorption properties being at ⁇ 860nm.
  • porphyrinato metal complex which can serve as an in-vivo anti-cancer torpedo equipped with visible-to-NIR emission for imaging and discriminating radar for tumor cell selectivity, and 1 O 2 explosive ammunition.
  • the cancer selectivity of these PDT agents are still not yet solved and there exists a need to provide PDT agents with better cancer selectivity.
  • composition for photodynamic therapy and imaging of cancer cells comprising gadolinium porphyrinate complex (Gd-N) of Formula (I) :
  • Ln Gd, or a pharmaceutically acceptable salt thereof.
  • a composition for photodynamic therapy and imaging of cancer cells wherein the cancer cells have anionic cell membranes.
  • a method of photodynamic therapy and imaging of cancer cells comprising administering to a subject in need thereof the composition according to the first aspect of the present invention and irradiating a radiation source to the cancer cells in the subject in need thereof.
  • the administration of said composition is performed intravenously or by injection to site of said cancer cells.
  • a method of photodynamic therapy and imaging of cancer cells comprising administering to a subject in need thereof a composition according to the first aspect of the present invention and irradiating the cancer cells in the subject in need thereof with a radiation source, wherein said radiation source is a light source of about 860 nm in wavelength.
  • Step b) Adding Tetrabutylammonium fluoride to a solution of the Gd-TMS in CH 2 Cl 2 , and stirring the Gd-TMS solution to create a chemical reaction; after completion of the chemical reaction, the solution is passed through column chromatography to form a fifth mixture; removing solvent from the fifth mixture to obtain an intermediate; dissolving the intermediate and 4-iodophenol in dry tetrahydrofuran and triethylamine to form a sixth mixture; mixing the sixth mixture with nitrogen to form a nitrogenized sixth mixture; adding Pd (PPh 3 ) 4 and CuI to said nitrogenized sixth mixture to form a seventh mixture; stirring the seventh mixture at least 35°C for at least 10 hours under a nitrogen atmosphere to produce a stirred seventh mixture; removing solvent from the stirred seventh mixture to produce a second residue; purifying the second residue using column chromatography with CH 2 Cl 2 /Methanol as eluent to produce Gd-OH;
  • Step c) Adding anhydrous K 2 CO 3 to a solution of Gd-OH and tetraethyleneglycol diiodide in dry N, N-Dimethylmethanamide to form an eighth mixture; heating said eighth mixture to at least 80 °C for at least 8 hours under a nitrogen atmosphere to form a heated eighth mixture; removing solvent from the heated eighth mixture to form a first crude product; purifying the first crude product using column chromatography eluented by CH 2 Cl 2 /CH 3 OH to produce Gd-I, and Step d) : Adding anhydrous Net 3 to a solution of Gd-I in dry DMF to form a ninth mixture; heating the ninth mixture to at least 85 °C for at least 24 hours under nitrogen atmosphere to form a heated ninth mixture; removing the solvent from the heated ninth mixture to obtain a second crude product; purifying the second crude product using column chromatography with CH 2 Cl 2 /CH 3 OH as the eluent to remove unreacted Gd-I and other im
  • a multi-modal lanthanide-porphyrin PDT agent (Er-R 3 ) that are capable of killing bladder tumor cells selectivity via 1 O 2 from porphyrin moiety and affording fluorescence imaging simultaneously upon Er-R 3 binding with integrin ⁇ v ⁇ 3 isoform in bladder cancer cells.
  • composition for photodynamic therapy and imaging of cancer cells comprising Erbium porphyrin based complexes or Ytterbium porphyrin based complexes or Gadolinium porphyrin based complexes represented by the molecular formula:
  • Ln is Er, Yb, or Gd
  • R 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 1 , Gd 2 , Gd 3 , Gd 4 , and Gd 5 :
  • a composition wherein the Erbium porphyrin based complexes are conjugated with both peptide RrRk (SEQ ID NO: 4) and integrin ⁇ v ⁇ 3 isoform specific peptide sequence (-cGRLKEKKc-) (SEQ ID NO: 5) .
  • composition comprising the Erbium porphyrin based complex represented by the molecular formula:
  • Ln is Er and R n is a polypeptide having an amino acid sequence of SEQ ID NO: 3.
  • composition wherein the cancer cells comprising of bladder cancer cells, cervical cancer cells and lung cancer.
  • a method of photodynamic therapy and imaging of cancer cells comprising administering to a subject in need thereof the composition and irradiating the cancer cells in the subject in need thereof with a radiation source.
  • a method wherein the administration of said composition is performed intravenously or by injection to site of said cancer cells.
  • said radiation source is a light source with a wavelength in the Q band of porphyrin.
  • said radiation source is a light source with a wavelength beyond 550 nm or is at 860nm.
  • a fourth embodiment of the sixth aspect of the present invention there is provided a method wherein the imaging is performed using fluorescent imaging, NIR imaging or MRI imaging
  • a method wherein the imaging is performed using MRI imaging when Ln is Gd, or Ln is Gd 1 , Gd 2 , Gd 3 , Gd 4 , or Gd 5 .
  • said compound Por (THP-TMS) is synthesized via steps comprising:
  • said compound Ln-1 is synthesized via steps comprising:
  • said compound Yb-1 is further synthesized via steps comprising:
  • Said compound Er-1 is further synthesized via steps comprising:
  • said compound Er-2 is further synthesized via steps comprising:
  • said compound Er-4 is further synthesized via steps comprising:
  • said compound Yb-R 1 is synthesized via steps comprising:
  • said compound Yb-R 2 is further synthesized via steps comprising:
  • said compound Yb-R 3 is further synthesized via steps comprising:
  • said compound Er-R 1 is further synthesized via steps comprising:
  • said compound Er-R 2 is further synthesized via steps comprising:
  • said compound Er-R 3 is further synthesized via steps comprising:
  • said compound Gd-1-L1 is synthesized via steps comprising:
  • Said compound Gd-1-L2 is synthesized via steps comprising:
  • said compound Gd-3 is synthesized via steps comprising:
  • said compound Gd-4 is synthesized via steps comprising:
  • said compound Gd-3-Rn is synthesized via steps comprising:
  • said compound Gd-4-Rn is synthesized via steps comprising:
  • the present invention includes all such variation and modifications.
  • the present invention also includes all of the steps and features referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.
  • Patent law e.g., they can mean “includes” , “included” , “including” , and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the present invention.
  • Figure 1A shows molecular structure of the cancer cells specific photodynamic therapy agent (Gd-N) and their control analogues Yb-N, Gd-RhB and Yb-RhB.
  • Figure 1B is 3D in vitro imaging of Gd-N after 15-hour incubation in HeLa cells.
  • Figure 1C and Figure 1D show difference in subcellular localization of Gd-N in cancer cells (HeLa) and normal cells. (WPMY-1) , respectively.
  • Yb-N and H 2 TPP are measured similarly as control.
  • Figure 3 shows in vitro imaging of Gd-N and Gd-RhB in tumor cells –HeLa and normal cells –MRC5 (as the controls) after 2-hour incubation. PDT effect is triggered upon 860 nm excitation.
  • Figure 4 shows photocytotoxicities of Gd-N, Gd-RhB (control) and Yb-N (control) towards (A) cancer cell (HeLa) and (B) normal cell (QSG 7701) .
  • Gd-N 1 O 2 available, tumor specific, strong photocytotoxicity in cancer cells, but no photocytotoxicity in normal cells
  • Gd-RhB control– 1 O 2 available, non tumor specific, strong cancer and normal cell photocytotoxicity
  • Yb-N control, 1 O 2 not available, no photocytotoxicity in both cancer and normal cells
  • Photocytotoxicity curves are obtained using 1 ⁇ M of conjugates and various light doses from 0 to 1 J/cm 2 ; MTT assays are carried out after incubation for 24 hours. (37 °C, 5%CO 2 ) .
  • Figure 6 shows in vivo studies of Gd-N as the cancer cell-specific PDT agent.
  • A) the representative gross images of tumors after PDT using 860nm laser for excitation, and candidates are divided into four groups (Group 1: Yb-N; Group 2: Gd-N; Group 3: Yb-RhB; Group 4: Gd-RhB) ; b) the measurement of tumor volume in a) ; c) In vivo biodistribution of Gd-N via ICP-MS studies; d) Two-photon microscopic images of tumor samples in c) ; e) In vivo tumor inhibition assays of Gd-N; f) In vivo tumor inhibition via Gd-N induced 1 O 2 through caudal vein injection.
  • Figure 7 shows Gd-N and Gd-RhB induced 1 O 2 activated the inhibitor of apoptosis protein family and mTOR pathway.
  • A western blot of HeLa cells dosed with 1 ⁇ M Gd-N or Gd-RhB and irradiated with 0.5 J/cm 2 . Untreated or free of chemicals samples are served as the controls.
  • B Cellular protein changes are semi-quantitatively measured using Gel-Pro Analyzer software of western blotting bands in (A) and showed as the ratio to ⁇ -actin (loading control of total proteins) . P values are calculated between untreated and Gd-N or Gd-RhB plus laser groups by One-way Analysis of Variance.
  • Figure 8 shows A) High-resolution MALDI-TOF mass spectrum of Gd-N; B) Isotopic patterns for the molecular ion Gd-N; C) Calculated MS patterns of the molecular ion Gd-N (using the software: IsoPro 3.0) .
  • Figure 9 shows the absorption spectra of Gd-N and Gd-RhB.
  • Figure 10 shows the schematic representation of energy absorption, migration and emission (indicated by ) processes in the (A) gadolinium porphyrinate complex (Gd-N) and (B) ytterbium porphyrinate complex (Yb-N) .
  • Figure 11 shows the open-aperture Z-scan trace of Gd-N (351 GM) and Gd-RhB (418 GM) excited at 800 nm in DMSO (5 ⁇ M) .
  • the average power of the laser beam is 0.271 mW.
  • Figure 13 shows subcellular localization of Er-R n and Yb-R n porphyrin complexes in human bladder carcinoma (T24 and 5637) cells, normal lung fibroblast (MRC-5) cells, and Human cervical carcinoma (HeLa) cells.
  • Figure 14 shows the cellular uptake analyzed by flow cytometry of Er-R n and Yb-R n porphyrin complexes in 5637, T24, HeLa and MRC-5 cells incubated for 0, 3, 6, and 24 hours as indicated by arrows.
  • the y-axis and x-axis are corresponding to cell counts and fluorescence intensity in FL3 channel (wavelength >650 nm) .
  • Figure 15 shows the comparison of in vitro photo-cytotoxicity of Er-R n and Yb-R n porphyrin complexes with ALA in (A) T24, (B) HeLa and (C) MRC-5 cells irradiated at 10 J cm - 2 with 550 nm long-pass filter, D) Summary of IC 50 value of Er-R n and Yb-R n porphyrin complexes and ALA in the presence and absence of irradiation in T24, HeLa and MRC-5 cells.
  • Figure 16 shows HPLC chromatogram of the complexes.
  • Elution conditions column, Agilent ZORBAXSB-C18 (4.6 X 150 mm, particle size 5; flow rate, 1.0 mL/min; gradient elution; detection wavelength, 430 nm.
  • Retention time (A) Yb-4, 7.24min ; (B) Er-4, 7.23 min; (C) Yb-R 1 , 10.00 min; (D) Yb-R 2 , 10.21 min; (E) Yb-R 3 , 10.01 min; (F) Er-R 1 , 9.66min; (G) Er-R 2 , 10.09min; and (H) Er-R 3 , 9.80 min.
  • Figure 17 shows the 400 MHz- 1 H-NMR (CDCl 3 ) spectrum of Por (THP-TMS) .
  • Figure 18 shows the MALDI-TOF spectrum of Por (THP-TMS) .
  • Figure 19 shows the 400 MHz- 1 H-NMR (CDCl 3 ) spectrum of Yb-1.
  • Figure 20 shows the MALDI-TOF spectrum of Yb-1.
  • Figure 21 shows the 400 MHz- 1 H-NMR (CDCl 3 ) spectrum of Er-1.
  • Figure 22 shows the MALDI-TOF spectrum of Er-1.
  • Figure 23 shows the 400 MHz- 1 H-NMR (CDCl 3 ) spectrum of Yb-2.
  • Figure 24 shows the MALDI-TOF spectrum of Yb-2.
  • Figure 25 shows the 400 MHz- 1 H-NMR (CDCl 3 ) spectrum of Er-2.
  • Figure 26 shows the MALDI-TOF spectrum of Er-2.
  • Figure 27 shows the 400 MHz- 1 H-NMR (CDCl 3 ) spectrum of Yb-4.
  • Figure 28 shows the MALDI-TOF spectrum of Yb-4.
  • Figure 29 shows the 400 MHz- 1 H-NMR (CDCl 3 ) spectrum of Er-4.
  • Figure 30 shows the MALDI-TOF spectrum of Er-4.
  • Figure 31 shows the MALDI-TOF spectrum of Yb-R 1.
  • Figure 32 shows the MALDI-TOF spectrum of Yb-R 2.
  • Figure 33 shows the MALDI-TOF spectrum of Yb-R 3.
  • Figure 34 shows the MALDI-TOF spectrum of Er-R 1.
  • Figure 35 shows the MALDI-TOF spectrum of Er-R 2.
  • Figure 36 shows the MALDI-TOF spectrum of Er-R 3.
  • Figure 37 shows the subcellular localization of Er-R n and Yb-R n porphyrin complexes by staining with Lyso Tracker green in (A) 5637 cells, (B) T24 cells, (C) HeLa cells and (D) MRC-5 cells.
  • Figure 38A shows NIR emission of existing organelle/DNA specific lanthanide complex.
  • Meso-pyridinium-substituted porphyrin-based ytterbium complexes shows a responsive NIR emission upon the addition of DNA;
  • Figure 38B shows NIR emission of water-soluble and mitochondria specific porphyrin-based Yb (III) complex (Yb-2) .
  • Figure 39A shows affinity of Yb-N to phosphatidylserine and cancer cells; a strong binding to phosphatidylserine and the capability to differentiate of cancer cells via targeting the anionic phospholipid membrane;
  • Figure 39B shows the development of organometallic complexes as in vitro and vivo tumor specific PDT agents as PDT agents.
  • Figure 40 shows the molecular structures of multi-modal porphyrin based metal complexes A) Gd-1, B) Gd-2, C) Gd-3-R 1 and D) Gd-3-N which are used as PDT, optical and MRI agents.
  • FIG 41 shows flow cytometry analysis of cellular uptake of Gd-3-R 1 .
  • Gd-3-R 1 has the fastest uptake rate among the four porphyrin complexes in the cancer cells and also with the better selectivity towards (B) bladder cancer T24 cells rather than (A) normal MRC-5 cells.
  • Figure 42 shows emission intensities of Gd-3-R 1 complex and H 2 TPP irradiated at 430nm excitation; similar 1 O 2 and emission quantum yield upon protonation of the Gd-3-R 1 complex compared with H 2 TPP reported in PNAS, 2014, E5492–E5497, which are around ⁇ 70% 1 O 2 quantum yield and 46%emission quantum yield with the excitation at 430 nm respectively.
  • Figure 43 shows the t 1 relaxivity of Gd-3-R 1 and Gd-DOTA of various concentrations; t 1 relaxivity of Gd-3-R 1 is three times greater than Gd-DOTA.
  • Figure 44 shows concentration of Gd-1, Gd-2, Gd-3-R 1 and Gd-3-N in ppm level per gram of different tissues.
  • Figure 45 shows the structures (A) Gd-3-R n , (B) Gd-4-R n and (C) Gd-5-R n with the change of different substituent groups (improve water solubility) , and organometallic/molecular caps (for stability/relaxivity) .
  • Figure 46 shows the reaction scheme of the complexes Gd-3-R n and Gd-4-R n intermediates for Figure 40 and 45.
  • Figure 47 shows structures of (A) Gd-5-R n , (B) Gd-3-R n , (C) Gd-4-R n , (D) Gd-6-R n that achieve better MRI and PDT effects. (With better cellular permeability, t 1 relaxivity and NIR emission for optical imaging) .
  • Figure 48 shows the development of multi-modal water-soluble, lanthanide-based PDT agents for optical imaging and inhibition of bladder cancer.
  • Figure 49A shows the selectivity towards (T24 bladder cancer, xenograft tumor) by comparing the in vivo magnetic resonance images of Gd-DOTA and Gd-3-R 1 ( Figure 49B) .
  • Gd-N gadolinium porphyrinate
  • Figure 1A a PDT agent which is synthesized on the basis of Yb-N and shown 51%singlet oxygen quantum yield with characteristic NIR emission of porphyrin upon photoexcitation.
  • Figure 1B-D Comprehensive studies have revealed that Gd-N can recognize tumor cells by their anionic phosphotidylserine membrane in the first six hours after administration. Upon administration of Gd-N, laser-irradiation at certain wavelengths, Gd-N enters the tumor cells and produce 1 O 2 in addition to exhibiting TP-induced NIR emission.
  • results of the in vivo mouse models and biodistribution assays further illustrates that Gd-N is found to be located in the tumor after simple injection of Gd-N into the blood vessel. Upon 1 O 2 releasing from the porphyrin, the solid tumor is found to be reduced after 24-hour treatment.
  • the present invention provide a novel PDT agent, Gd-N, and the use thereof for practical cancer tracking, imaging and treatment.
  • Gd-N is the motif structure of the ytterbium complex (Yb-N) reported in the inventors’previous work, are shown in Scheme 1 and Figure 8.
  • Gd-N and Yb-N are structurally different in the lanthanide ion present in the complex (the vector ligated to Gd-N is also the same as Yb-N) .
  • porphyrin’s coordination with different lanthanides can cause changes not only in the NIR emission, but also the 1 O 2 generation.
  • Figure 2 and Figure 9 Such phenomena, in principle, arise from the fact that better orbital overlapping between the metal center and the ligand results in better energy transfer (i.e.
  • the bonding orbitals of Yb which consists of a smaller atomic radius than Gd is thus overlap more preferably and compatibly with the porphyrin’s orbitals) .
  • the heavy atom effect exerted by the lanthanide can also augment the triplet-state decay rate and lead to higher triplet-state quantum yields of the porphyrin system.
  • the singlet oxygen quantum yield of Yb-N is measured to be 0%and Gd-N is determined to be 51%.
  • Rhodamine B is a well-known mitochondria vector common for conjugation.
  • the inventors find Gd-RhB’s emission in both normal and cancer cells’mitochondria, and this very observation becomes the clear, cognizant, and convincing evidence of the tumor-specific property of the Gd-N. ( Figure 3) Through the MTT assays, the cytotoxicity of the three complexes, Gd-N, Yb-N and Gd-RhB in dark can be subsequently determined against the two kinds of cell lines.
  • the IC 50 values of them are 0.78, 0.80, and 0.65 mM in cancer cells (HeLa) and 0.70, 0.70, and 0.45 mM in normal cells (MRC-5) respectively.
  • the underlying reason of the vast difference in the dark cytotoxicity of Gd-RhB towards cancer/normal cells compared with Gd-Nand Yb-N can be largely due to Gd-RhB’s non-selectivity.
  • Gd-N of the present invention exhibits crucial tumor selectivity.
  • the in vitro PDT effect of the three complexes is evaluated using in vitro confocal microscopy and photocytotoxicity assays.
  • Gd-N, Yb-N and Gd-RhB complexes are dosed in HeLa cells and MRC-5 cells for 6 hours, and then subjected to excitation at 860 nm for triggering any PDT effect.
  • Three complexes are all available for TP-induced in vitro imaging with TP cross-section ⁇ 351 GM; given the limitation of the confocal spectroscope, the emission from porphyrin had only been monitored from 600 nm to 750 nm only
  • the emission of Gd-RhB can be noticed in the mitochondria.
  • Upon suitable laser-induction only small quantities of 1 O 2 are produced but the cancer cells are killed within a few minutes; in effect, the normal cells are also killed rapidly under the same conditions.
  • Gd-RhB The PDT effect of Gd-RhB is therefore efficient enough but obviously non-selective and undesirable; it accumulates inside the mitochondria of cancer and normal cells, annihilating them unselectively.
  • Yb-N is cancer-specific, its incapability to produce any 1 O 2 imposes a restriction on any PDT practice.
  • the red emissive Gd-N not only it recognizes and localizes on the anionic membrane of tumor cells, but also access to certain parts of cytoplasm and induce cancer apoptosis via 1 O 2 upon 9-minutes light dose flashing 5 seconds per minute. More time is required to trigger cancer cell death by Gd-N after definite laser irradiation; however, there is no significant cell death in the normal cells, far outweighing its slow-response drawback.
  • the concentration-dependent photocytotoxicity of Gd-N, Yb-N and Gd-RhB are measured under varying light doses from 0.25 to 1 J/cm 2 in cancer cells and normal cells.
  • the light dose-response curves obtained are displayed in Figure 4.
  • Gd-RhB and Gd-N exhibit strong photocytotoxicity, whereas Yb-N (without singlet oxygen) has no photocytotoxcity (Figure 4 (A) ) .
  • Figure 4 (B) in normal cell QSG 7701, no photocytotoxicty is found from Gd-N, while Gd-RhB gives very similar results as it behaved in the cancer cells.
  • the tumors are found effectively inhibited in the groups of Gd-N and Gd-RhB, compared with their counterparts Yb-N and Yb-RhB; Gd-N, among the four complexes, is the best in vivo PDT agent that devastate the tumor with 100%efficiency.
  • Gd-N among the four complexes
  • BALB/c nude mice with tumor xenograft attaining a size of approximately 0.1 cm 3 are caudal vein injected with Gd-N (1.0 mg/kg) .
  • concentrations of Gd-N and Gd-RhB in different tissues or circulating blood are examined using ICP-MS.
  • tumors have the largest enrichment of Gd-N (4.84 ppm/g) , demonstrating the specific recognition of the Gd-N towards tumor cells.
  • mice with xenograft tumor are caudal vein injected with Gd-N and Gd-RhB (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 Gd-N plus light treated tumors are inhibited compared to their opposite flank controls of tumor or Gd-RhB groups.
  • the present invention provides theranostic gadolinium complex Gd-N for use as an anti-cancer agent which is equipped with visible-to-NIR emission for imaging, tumor cell selectivity, and 1 O 2 generation.
  • Gd-N gadolinium complex
  • the present invention also provides a method of tracking and imaging long-term live cancer cell, using Gd-N, as well as selective photodynamic therapy.
  • 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.
  • ⁇ ⁇ denotes the singlet oxygen quantum yield
  • G ⁇ 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.
  • the inventors had measured the 1 O 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.
  • 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.
  • FBS fetal bovine serum
  • streptomycin 100 ⁇ g/ml streptomycin
  • penicillin 100 units/ml penicillin
  • 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.
  • MTT 5-diphenyltetrazolium bromide
  • 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 analyzedby plottings using the GraphPad Prism 5 software.
  • cancer cells (2 x 10 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 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 Microplate Reader) .
  • mice 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) .
  • Gd-N and Gd-RhB 1.0 ⁇ mol/kg body weight each
  • Gd-N and Gd-RhB 1.0 ⁇ mol/kg body weight each
  • the concentrations of Gd-N and Gd-RhB are measured by PerkinElmer EnVision Multilabel Reader 2104 at 570 nm, and calculated using standard absorptions via concentration curve.
  • Pharmacokinetic parameters (t 1/2 , Vd, MRT, AUC) are calculated by fitting with one compartment model.
  • Gd-OH 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 2 Cl 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.
  • TAF Tetrabutylammonium fluoride
  • 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.
  • Gd-I To a solution of Gd-OH (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 2 CO 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 2 Cl 2 /CH 3 OH (v/v, 100: 1) to afford the pure product as a red solid. Yield: 82%; M. p.
  • Gd-N To a solution of Gd-I (95 mg, 0.05 mmol) in dry (DMF, 10 ml) , anhydrous Net 3 (1ml, excess) is added, and the mixture is heated to 85 °C for 24h 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 2 Cl 2 /CH 3 OH (v/v, 80: 1) as the eluent to remove unreacted Gd-I and other impurities, then using CH 2 Cl 2 /CH 3 OH (v/v, 10: 1) to obtain the pure product as a red solid. Yield: 80%; M. p.
  • the two-photon-absorption spectra are measured at 800 nm by the open-aperture Z-scan method using 100 fs laser pulses with a peak power of 276 GWcm –2 from an optical parametric amplifier operating at a repetition rate of 1 kHz generated from a Ti:sapphire regenerative amplifier system.
  • 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.
  • 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.
  • the non-linear absorption coefficient, ⁇ can be obtained by curve-fitting to the observed open-aperture traces, T (z) , with Equation (1) 6 , where a 0 is the linear absorption coefficient, l is the sample length (the 1 mm quartz cell) and z 0 is the diffraction length of the incident beam.
  • 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 ⁇ v ⁇ 3 isoform–specific peptides.
  • the porphyrin and erbium emission from Er-R 3 show that Er-R 3 are able to significantly interrupt bladder cancer tumor growth that specific binds to “integrin ⁇ v ⁇ 3 isoform” with responsive emission for imaging.
  • 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 ⁇ v ⁇ 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 ⁇ v ⁇ 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 -1 ) and emission quantum yield (Porphyrin: Soret Band and Er: 2 F 5/2 ⁇ 2 F 7/2 ) of Er-R 1 , Er-R 2 and Er-R 3 are similar.
  • the details of photophysical measurement of Ln-R 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 Yb for f-f emission which much better than from porphyrin to Er f-f emission. All of the Er-R n porphyrin complexes and Yb-R n porphyrin complexes are characterized by 1 H NMR and mass spectrometry ( Figure 17-36) .
  • the in vitro fluorescent intensity of three erbium porphyrin complexes is higher than its ytterbium motif analogue due to efficient energy transfer from the porphyrin molecules to the Yb3+ ion and emits Yb near-infrared fluorescence.
  • red porphyrin emission from Er-R1 is found only on the cell membrane, however, the red emission of Er-R2 and Er-R3 are found inside the cells.
  • Ytterbium analogues also show the same subcellular localization; emission of porphyrin Yb-R1 is found in the cell membrane.
  • the peptides sequence in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 can recognize the ⁇ v ⁇ 3 integrin.
  • the lack of ⁇ v ⁇ 3 integrin in HeLa and MRC-5 cells should limit the uptake of Er-Rn and Yb-Rn .
  • the T24 cells display significant fluorescence within a 6 hours incubation of Er-R n porphyrin complexes and Yb-R n porphyrin complexes in FL3 channel (Emission filter: 670 long-pass filter) , whereas the HeLa and MRC-5 (cell surface ⁇ v ⁇ 3 integrin receptor-negative) incubated with Er-R n porphyrin complexes and Yb-R n porphyrin complexes show little fluorescence signal under similar experimental condition.
  • the cellular uptake increases along with the incubation time in T24 cells which is quantified as median fluorescence intensity after 24-hour (Table 4) .
  • Table 4 Summary of Er-R n porphyrin complexes and Yb-R n porphyrin complexes median fluorescence intensity in T24, HeLa and MRC-5 cells incubated for 24 hours.
  • Er-R n and Yb-R n exhibit high photo-cytotoxicity under irradiation of 10 Jcm -2 . Furthermore, the photo-cytotoxicity increased along with the concentration of Er-R n porphyrin complexes and Yb-R n porphyrin complexes with a half lethal dose (IC 50 ) calculated in Figure 15 after incubation for 24 hours.
  • the IC 50 of Er-R n porphyrin complexes and Yb-R n porphyrin complexes to T24 is 8 to 10 times lower than HeLa and MRC-5 which demonstrates Er-R n porphyrin complexes and Yb-R n porphyrin complexes selectively kill bladder cancer.
  • the cellular uptake of them is higher than Er-R 1 porphyrin complexes, Er-R 2 porphyrin complexes, Yb-R 1 porphyrin complexes and Yb-R 2 porphyrin complexes which lead to higher photo-cytotoxicity.
  • the excitation wavelength 550nm is located in the Q band of porphyrin which would provide better tissue penetration in practice. However, it cannot trigger efficient PDT effect comparable to the FDA approved Aminolevulinic acid, (ALA) . ALA is excited at 400-450 nm.
  • Er-R n porphyrin complexes and Yb-R n porphyrin complexes cause more intense photo-cytotoxic effect than ALA when excited beyond 550 nm.
  • Er-R 3 porphyrin complexes kill bladder cancer cells mostly effectively (IC 50 as low as 31 ⁇ M can be reached) due to brightest in vitro fluorescence and highest cellular uptake.
  • IC 50 over 1000 ⁇ M Based on the results above, Er-R 3 porphyrin complexes is of the preferred embodiment for PDT agent to selectively kill bladder cancer.
  • the present invention provides a multi-modal lanthanide-porphyrin PDT agent that is capable of killing the tumor cells via 1 O 2 from porphyrin moiety and affording the fluorescence imaging simultaneously.
  • Er-R 3 porphyrin complexes are synthesized and shown to be highly selective to bladder cancer cells by specific targeting integrin ⁇ v ⁇ 3 isoform in bladder cancer cells with strong NIR and 1 O 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 ⁇ v ⁇ 3 isoform” of blabber cancer cells.
  • Yb-1 Yb [N (SiMe 3 ) 2] 3 ⁇ x [Li (THF) 3 Cl] (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 10ml CH 2 Cl 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) (0.1g, 0.16mmol) dissolved in 15ml toluene. The resulting solution is refluxed until most of the free base coordinated with the metal ion.
  • THP-TMS dry Por
  • Yb-2 TBAF (1.0 M in THF, 0.2 mL, 0.2 mmol) is added to a solution of Yb-1 (0.05 mmol 76.55mg, ) in 10 ml CH 2 Cl 2 , and the solution is stirred for 30min. 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.
  • TLC Thin-layer Chromatography
  • Yb-4 Pd (PPh 3 ) 4 (22.16 mg 0.08 mmol) , CuI (7.65 mg, 0.04 mmol) , Yb-2 (30.62. mg, 0.02mmol) and 4-iodobenzoic acid 5.087mg are placed in a dried flask and under nitrogen. THF (15 mL) and Net 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 2 Cl 2 /Methanol (12: 1) .
  • the eluted compound (26mg, 0.0157mmol) , EDCI (6.04mg, 0.031mmol) , NHS (3.57mg, 0.031mmol) are placed in a dried flask and under nitrogen, 10mL 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.
  • Yb-R 1 A stirred solution of Yb-4 (16mg, 1equiv. ) in anhydrous DMF is mixed with N, N’-diisopropylethylamine (DIPEA) (1 equiv. ) . The mixture solution is added into a vial which contains peptide R 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 Yb-R 1 .
  • DIPEA N, N’-diisopropylethylamine
  • 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, 50gmL -1 ; streptomycin, 50gmL -1 ) .
  • Human cervical carcinoma (HeLa) cells are cultured in DMEM (Gibco) supplemented with 10%FBS (Gibco) and antibiotics (penicillin, 50gmL -1 ; streptomycin, 50gmL -1 ) .
  • Human normal lung fibroblast (MRC-5) cells are maintained in minimum essential medium (MEM) supplemented with 10%FBS and 1%50gmL -1 penicillin; 50gmL -1 streptomycin. All cells are incubated at 37°C in a humidified environment with 5%CO 2 .
  • HeLa and MRC-5 cells (1 x 10 5 ) are treated with Er-R n porphyrin complexes and Yb-R 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 500gmL -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.
  • HeLa and MRC-5 cells (1 x 10 5 ) are treated with Er-R n porphyrin complexes and Yb-R n porphyrin complexes for 24 hours at four concentrations (1, 5, 10, 50M) . Then, the cells are irradiated at 6mWcm -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.
  • T24, 5637, HeLa and MRC-5 cells (1 x 10 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 costaining 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.
  • 5637, T24, HeLa and MRC-5 cells (1 x 10 5 per sample) are seeded onto 35 mm Petri dishes and incubated overnight. Then the cells are incubated with the Er-R n and Yb-R 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.
  • 5M Er-R n and Yb-R n porphyrin complexes
  • 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 (Figure 38A) .
  • 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 1 O 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.
  • 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 (Figure 39A) and recently, its motif structure (Gd-N) has demonstrated the availability as in vivo tumor specific PDT agents. ( Figure 39B) .
  • NIR optical and t 1 magnetic resonance imaging that bind strongly to the targets, phospholysation anionic membrane/integrin ⁇ v ⁇ 3 isoform, and generates 1 O 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 1 O 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.
  • the inventors have designed and synthesized 4 water-soluble, cell-permeable porphyrin-based gadolinium complexes, Gd-1, Gd-2, Gd-3-R 1 , Gd-3-N ( Figure 40) , which has the same general chemical formula as Figure 12 a) .
  • These complexes for use as multi-modal PDT agents are examined (1. Bio-stability –P M ; 2. PDT and in-situ imaging– 1 O 2 and emission quantum yield; 3. MR imaging–t 1 relaxivity; and 4. Cancer or bladder cells specific –ex vivo toxicity)
  • the protonation of the substituent groups on the porphyrin of Gd-1, Gd-2, Gd-3-R 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 1 and Gd-3-N show better stability than Gd-1, with the P m values of Gd-2.
  • Gd-3-R 1 and Gd-3-N being around 8.15.
  • the cellular uptake profile from flow cytometry also shows that Gd-3-R 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 ( Figure 41) .
  • 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 1 O 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 50 in 1J laser dosed is 1M
  • IC50 have to be > 0.1mM
  • 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 -1 cm -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.
  • 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 1 O 2 and PDT.
  • Gd-N Water-soluble porphyrin-based gadolinium complex
  • Gd-N Water-soluble porphyrin-based gadolinium complex
  • the present invention provides two more types of organometallic complexes in which the Gd ions are stabilized by other organometallic compounds (Gd-4-R n ) or with carboxylic pendant arm (Gd-5-R n ) ( Figure 45 and 46) .
  • the present invention provides multi-modal PDT agents for comprehensive diagnosis and treatment on bladder cancer –MR for diagnosis, NIR induced 1 O 2 for PDT and NIR induced NIR emission for real time monitoring the effectiveness of PDT.
  • the stability, relaxivity, NIR-induced emission, 1 O 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 Figure 45 (Gd-3-R 1 /Gd-4-R 1 /Gd-5-R 1 ) and the several bladder cancer specific peptide (s) as vector (s) (Gd-3-R n /Gd-4-R n /Gd-5-R n /Gd-6-R n ) in Figure 47 for the ⁇ v ⁇ 3 isoform of integrin protein will be conjugated to the multi-modal complexes in the section 1.1.
  • Gd-1-L1 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 2 Cl 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.
  • Gd-1-L2 The similar procedure with Gd-1-L1, replace NaL 1 with KL 2 (potassium tris (1-pyrazolyl) borohydride , 0.055g, 0.22mmol) . Yield: 50%.
  • Gd-3 TBAF (1.0M in THF, 0.2mL, 0.2mmol) was added to a solution of Gd-1-L1 (0.133mg, 0.1mmol) in 10ml 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, 0.02mmol) and Pd (PPh 3 ) 4 (2.2 mg 0.008 mmol) , CuI (0.77 mg, 0.004 mmol) , 4-iodobenzoic acid 5.1mg were placed in a dried flask and under nitrogen.
  • Gd-4 The similar procedure with Gd-3, replace Gd-1-L1 with Gd-1-L2. Yield 50%.
  • Gd-3-Rn A stirred solution of Gd-3 (20mg, 1equiv) 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 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%.
  • DIPEA N, N’-diisopropylethylamine
  • Gd-4-Rn The same procedure with Gd-3-Rn, replace Gd-3 with Gd-4. Yield 68%.
  • 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 ⁇ v ⁇ 3 isoform and the present invented porphyrin complexes Gd-3-R 1 is the prime factor for developing the multi-modal probe for monitoring ⁇ v ⁇ 3 isoform bladder cancer.
  • the binding affinities of the present invented complexes for ⁇ v ⁇ 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 ⁇ v ⁇ 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 ( Figure 48) .
  • the linear, multi-photon photophysical properties (i.e. emission spectra, emission lifetime, quantum yield and two-photon absorption cross section) and 1 O 2 yield and also photo-bleaching quantum yield (compared with standard, such as uroporphyrin) of Gd-3-R 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 M and P 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 31 P 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.
  • HSA human serum albumin
  • Electrophoretic Mobility Shift Assay is a powerful method for determining the binding affinity of the inventors’gadolinium porphyrin complexes to ⁇ v ⁇ 3 isoform cancer cell which is specific for bladder cancer.
  • the ⁇ v ⁇ 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 ⁇ v ⁇ 3 isoform would not be altered by the binding of the lanthanide bio-probes.
  • the binding affinity of the complexes and ⁇ v ⁇ 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.
  • ITC isothermal titration calorimetry
  • the binding affinity (K 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.
  • the relaxivity of the inventors’synthesized complexes will be calculated from the relaxation time obtained by a Bruker DPX300 NMR spectrometer in D 2 O solutions. An inversion-recovery pulse sequence is used and a ten x T 1 delay is maintained between successive pulses.
  • the relaxivity (r 1 ) is obtained by a plot of the inverse of longitudinal time (1/T 1 ) versus Gd concentrations:
  • T 1obs and T 1b are the longitudinal relaxation times of the sample and the solvent background respectively.
  • 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, (2 x 10 4 /well) are incubated in 96-well plates overnight. In vitro imaging for selective binding-the cells are treated with Gd-3-R 1 (Task 1) for 6, 12 and 24 hours in the dark.
  • Gd-3-R 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 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 1 in cells are carried out and their in vitro subcellular localization are compared.
  • the subcellular localization of Gd-3-R 1 are different in bladder cell T24 and other non-bladder cancer cell lines, such as HeLa, C666-1 and SK-N-SH.
  • 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 1 O 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.
  • the photocytotoxicity of valid bladder cancer specific photosensitizer i.e. LC 50 in 1J laser dosed is 1M
  • IC50 have to be > 0.1mM
  • Task 3 Structure and biological activity (In vitro/-vivo imaging and specific PDT effect)
  • 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.
  • bladder cancer cells T24 or non-bladder cancer cells (HeLa) are trypsinized, harvested and suspended in serum-free culture medium. 5 ⁇ 10 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 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, Gd-N low dose treatment group; group 4, Gd-N high dose treatment group. Treatments is administered via intratumoral injection once every 5 days, for 21-28 days. The experiment is repeated three times.
  • 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 Institution University.
  • One-way analysis of variance towards statistical significances between groups was assessed by the GraphPad Prism 5.0 software.
  • xenograft mice 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 MRI experiments are carried out on the xenograft in a Co-I institute with a Bruker Biospec 4.7 T/30 cm scanner (Bruker Inc., MA) .
  • the tumor sizes will be measured weekly.
  • Gd-3-R 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 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 ( Figure 44) .
  • 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.
  • the present invention provides a multi-modal lanthanide-porphyrin PDT agent (Er-R 3 ) that are capable of killing the bladder tumor cells selectivity via 1 O 2 from porphyrin moiety and affording the fluorescence imaging simultaneously upon Er-R 3 binding with the integrin ⁇ v ⁇ 3 isoform in bladder cancer cells.
  • Er-R 3 multi-modal lanthanide-porphyrin PDT agent
  • 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.

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