Porphyrins with applications in PDT of cancer
(Dr. R.-M. Ion)

Contact Person

  • Prof. Rodica-Mariana ION

  • INSTITUTE FOR CHEMICAL RESEARCH - ICECHIM
    Chemical Analysis Department, Photochemistry Group
    Splaiul Independentei 202
    Bucharest - 77208 (Romania)

  • Tel: (+40) 21 224 88 70
    Fax: (+40) 21 312 34 93

  • E-mail: irma@pcnet.ro
    Internet: in Romania

Topics of Interest

  • Porphyrins - synthesis and characterization
Structure and spectral characterization
Photophysical properties
Photochemical properties
    • singlet oxygen generation
    • photodegradation process
    • photoproducts formation

  • Applications of porphyrins in photo-neurosurgery

  • Ongoing projects

    • COST Projects
    • FPV project
    • Bilateral Cooperations

Introduction

From The Oxford English Dictionary, porphyrins are "a large class of deeply coloured red or purple, fluorescent crystalline pigments, with natural or synthetic origin, having in common a substituted aromatic macrocyclic ring consisting of four pyrrole-type residues, linked togheter by four methine bridging groups".

R

Name of the porphyrin

C6H5

5, 10,15,20-tetraphenylporphyrin (TPP)

C10 H8

5,10,15,20-tetra-1-naphthylporphyrin (TNP)

C6H4-SO3

5, 10,15, 20-tetra-p-sulfonatophenylporphyrin (TSPP)

C6H5-NH2

5,10,15,20-tetra(4-aminophenyl)porphyrin (TAPP)

C6H5-NO2

5,10,15,20-tetra(4-nitro-phenyl)porphyrin (TNPP)

C6H5-OH

5,10,15,20-tetra(4-hydroxyphenyl)porphyrin (THPP)

C6H5-CH3

5,10,15,20-tetra(4-methylphenyl)porphyrin (TMPP)

C6H5-OCH3

5,10,15,20-tetra(4-methoxyphenyl)porphyrin (TMOPP)

C10H7-SO3H

5,10,15,20-tetra(4-sulfonatonaphthyl)porphyrin (TSNP)

C10H7-OH

5,10,15,20-tetra(4-hydroxynaphthyl)porphyrin (THNP)

Figure 1. Structure of
the studied porphyrins

Table 1. Types of the different
meso-substituted porphyrins

Figure 2. Metals which can react with porphyrins
Photodynamic Therapy of Cancer

Photodynamic therapy (also called PDT, photoirradiation therapy, phototherapy, or photochemotherapy) as a new modality for cancer treatment is the combination of light with a photosensitizing drug in an oxygen-rich environment. Photosensitizing drugs (porphyrins, porphycenes and phthalocyanines) are used according to their efficiency to generate singlet oxygen, the phototoxic intermediary.

The photosensitization of oxygen is a slightly different process. The sensitizer molecule, which has a singlet electronic ground state, absorb light energy and becomes electronically excited. This process occurs very rapidly (10-15s) and produces a singlet excited state of a sensitizer, 1S*.

Figure 3. The Jablonski diagram

Toxic oxygen species such as singlet oxygen and free radicals are thus formed. These chemicals are very reactive and can damage proteins, lipids (including phospholipids and cholesterol), nucleic acids and other cellular components and with certain a-aminoacids side chains in proteins (Trp, His, Met); these are all vital components of membranes (external and internal to the cell) and membrane damage in this way is a plausible macroscopic mechanism for cell damage and death.

Singlet oxygen is a powerful, fairly indiscriminant, oxidant that reacts with a variety of biological molecules and assemblies.

Seven golden rules for a good PDT agent

  1. It must be a pure compound;
  2. It must be activated at l 650 nm to ensure better absorption of tissue-penetrating red light, and also sensitization by an external light source;
  3. It must be non-toxic in the absence of light;
  4. Its triplet excited state must be long-lived enough to enable it to photosensitize the production of singlet oxygen;
  5. It must localize specifically in the tumor;
  6. It must clear rapidly from the body after it has done its work;
  7. It must be soluble in the body´s tissue fluids so that it can be injected and transported to the tumor site.
Porphyrins for Photodynamic Therapy of Human Brain Tumors

Photodynamic therapy (PDT) offers a more selective approach to the treatment of brain malignancies compared with other standard available treatment modalities. PDT may be of advantage in the treatment of tumors of combining it with radiotherapy, hyperthermia, chemotherapy, sonotherapy, and hyperoxygenation.

In-vitro samples of brain tumoral tissues were prelevated from human patients and impregnated with different porphyrin-sensitizers solutions (including with metallic complexes of porphyrins). The radiation of the nitrogen pulsed laser is emitted at 337.1 nm with a power density ranging from 1 to 3.5 mW/cm2.

Photosensitizers preferentially accumulate in malignant tissue whether via increased uptake due to an accelerated cellular proliferation rate, decreased intratumoral pH favoring photosensitizer retention, increased phagocytosis capabilities, leaky vasculature, decreased lymph drainage tumor-associated macrophage engulfing photosensitizers or specific uptake via receptors.

The experiments were made in vitro on tumoral tissues obtained from different types of cerebral tumors prelevated from 10 patients on whom there were made previously surgical interventions. The samples were obtained by cutting pieces from tumor tissues. An aseptically excised brain tissue was cut on a sterile paraffin plate into pieces about 1 mm of diameter. These fragments were washed in MEM and then were places into wells. MEM with 10% FCS in total volume of 0.15 ml was added into each wells. The sample was irradiated by laser after 24-48 h incubation time. The dye impregnation was done immediately after operation.

Patient

Sensitizer / solvent

(mg / ml)

Irradiation conditions

time / dose / power density

(h) / (J/cm2) / (mW/cm2)

Incubation time

(h)

Tumor type

A

TSPP

0.1 (1)

4 / 14.3 / 1.05

24

Parasagial meningiom

B

TSPP

0.1 (2)

1 / 6.3 / 1.75

1

Right temporal

C

TSPP

0.1 (1)

2 / 12.6 / 1.75

2

Left parentheral

D

TPP

0.05 (2)

1 / 6.3 / 1.75

24

Left frontal

E

TNP

0.1 (1)

3 / 19 / 1.75

48

Malignant meningioma

F

TNP

0.1 (2)

3 / 19 / 1.75

48

Intraventricular

G

ZnTSPP

0.5

1 / 6.3 / 1.75

24

Left frontal

Measurement types:

Absorption spectra;

Fluorescence spectra;

The 3-fluorescence flow cytometry (Cytron Absolute, Ortho) equipped with an argon ion laser was used and with a peak fitting program using Gauss type functions on a PC computer program.

Optical microscopy for video recordings of the vascular effects for cells during the light irradiation, obtained by means of a Leitz microscope and a Leitz Aristoplan microscope.

Irradiation procedure with a nitrogen pulsed laser radiation.

Figure 4. Experimental set-up for tumor irradiation (in vitro)

The efficiency of the incorporation of these dyes into human blood cells changed in the same manner as the singlet oxygen generation: the sulfonated porphyrins was efficiently incorporated into leukocytes than into granulocytes and only in monomeric form.

The light led to a rapid necrosis of tumor which was not the result of the direct killing of tumor cells, but destruction of tumor microvasculature. It has been shown that the vascular damage expressed by a decreased blood flow stasis are immediate and major consequences of the photodynamic treatment with photodynamic systems introduced in these experiments.

The photodegradation reaction of porphyrinic agents

The porphyrins can be modified by light and oxygen, this process being called photodegradation or photobleaching.

It should be noted that the photobleaching does not decribe a "simple" photodegradation of the photosensitizer. It includes a chemical modification of the porphyrin, i.e. formation of photoproducts.

The main factors influencing the photodegradation rate:

  • the meso-substituents;
  • the central metal;
  • the axial ligand attached to the central metal;
  • the aggregation and ionization processes;
  • the medium temperature ;
  • the solvent or binary mixture of solvents.

Figure 5.
The photodegradation
scheme of porphyrins
Some References
  • M. L. Pascu, A. Popescu, L. Danaila, N. Carp, R. M. Ion, M. Pascu, A. Staicu
    Photodynamic therapy studies on brain tumors using nitrogen pulsed lasers
    Proc. SPIE, 4166, 140 (2000)

  • L. Danaila, M.L. Pascu, A. Popescu, M. Pascu, R. M. Ion
    Spectrophotometric characterization of useful dyes in laser photodynamic therapy of cancer
    Proc. SPIE, 4068, 712 (2000)

  • R. M. Ion, M. Grigorescu, F. Scarlat, V.I.R. Niculescu, K. Gunaydin
    Light, electron and photons beam effects on TSPP used in PDT
    J. Balkan Union Oncology, 3 (2) 129 (2000)

  • R. M. Ion
    Spectral analysis of the porphyrin into human blood cells
    J. Biomed. Opt., 4 (3), 319 (1999)

  • M. L. Pascu, L. Danaila, A. Popescu, M. Pascu, R.M. Ion
    Researches concerning the application of laser photodynamic therapy in neurosurgery
    Rom. Reports Phys., 2 (1999)

  • R. M. Ion, A. Planner, K. Wiktorowicz, D. Frackowiak
    Incorporation of various porphyrins into human blood cells measured using the flow-cytometry, the absorption and emission spectroscopy
    Acta Biochimica Polonica, 45 (30), 833 (1998)

  • R. M. Ion, A. Stirbet, C. Mandravel
    The analysis of the porphyrins purity
    Rev.Chim. (Bucharest), 49 (2), 121 (1998)

  • D. Frackowiak, A. Planner, R. M. Ion, K. Wiktorowicz
    Incorporation of dyes in resting and stimulated leukocytes, in Synthesis, properties and applications of near-infrared dyes in high technology fields
    Ed.S. Daehne, Kluwer Acad. Publ., NATO ASI Series (1998)

  • R. M. Ion
    The photophysical properties of some porphyrins in binary mixtures of solvents
    Rom. J. Biophys., 2, (1998)

  • R. M. Ion
    The photodynamic therapy of cancer -a photosensitization or a photocatalytic process
    Progr. Catal., 1, 55 (1997)

    A. Planner, R. M. Ion, K. Wictorowicz, D. Frackowiak
    The incorporation of porphyrins in human leucocytes measured by flow cytometry absorption and emission spectroscopy
    First Internet Conference on Photochem. Photobiol. (1997); Poster abstract - Conference home page

  • R. M. Ion
    Spectral studies of TSPP and TSNP used in PDT. I. Monomer-dimer equilibrium
    Rom. J. Biophys., 6 (3-4), 213-218 (1996)

  • R. M. Ion
    Photochemical production and quenching of singlet oxygen by the porphyrins used in PDT
    Rom. J. Biophys., 6 (3-4), 205-212 (1996)
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