Photodynamic effect of deuteroporphyrin IX derivatives on

isolated nerve cell

A.B.Uzdensky¹, A.V.Ivanov², A.V.Reshetnikov³, G.V.Ponomarev³,
O.Yu.Dergacheva¹, A.A.Zhavoronkova¹,

¹⁾Rostov State University, Department of Biophysics, Rostov-on-Don, 344090, Russia. uzd@krinc.ru
²⁾N.N. Blokhin Oncological Scientific Center, Russian Academy of Medical Sciences,20, Kashirskoye shosse, Moscow, 115478, Russia
³⁾Institute of Biomedical Chemistry, Russian Academy of Medical Sciences,10, Pogodinskaya str., 119832, Moscow, Russia

ABSTRACT

Deuteroporphyrin IX derivatives are prospective PDT porphyrin photosensitizers (PS). The photodynamic effects of 6 new amphiphilic deuteroporphyrin derivatives with different hydrophobicity, as well as effects of known photosensitizers Photoheme and Photosens (used for comparison) on the firing of isolated crayfish mechanoreceptor neuron have been studied. After 30 min photosensitization, neurons were irradiated with He-Ne laser (632,8 nm, 0,3 W/cm²), and changes in neuron firing frequency were recorded. It has been shown that neuron firing is very sensitive to photodynamic impact and can serve as a sensitive indicator of cell photodamage. The comparison of dependencies of neuron lifetime on photosensitizer concentrations has provided comparison of their photodynamic efficiencies. The studied deuteroporphyrin IX derivatives have been found to be very potent PS. They induced irreversible firing abolition at pikomolar concentrations while Photoheme and Photosens were effective in the nanomolar range. The most effective PS were 4-(1-methyl-3-hydroxybutyl)- and 4-(1-methyl-2-acetyl-3-oxobutyl)-deuteroporphyrins. High photodynamic efficiencies of deuteroporphyrin derivatives were related to a weak dependence of photodynamic effect on sensitizer concentration, indicating that an initiation of several (3-5) chains of secondary processes such as free radical membrane damage by absorption of photon by photosensitizer molecule could take place. The main photosensitizer feature determining its intracellular localization and photodynamic efficiency has been amphiphilicity.

Keywords: photodynamic therapy, photodynamic effect, photosensitizers, deuteroporphyrin IX, porphyrin, Photosens, Photoheme, neuron, firing

1. INTRODUCTION

Photodynamic therapy (PDT) includes selective accumulation of photosensitizing substances (PS) in tumor and subsequent photogeneration of singlet oxygen ¹O₂ and other cytotoxic products causing malignant tissue destruction upon illumination^1,2. Different compounds - porphyrins, chlorins, phthalocyanines, etc. are under examination in the search of the optimal PS^1,3,4. Intracellular localization of PS which depends on its hydrophobicity and amphiphilicity is very important for its anti-cancer efficiency. An attractive feature of deuteroporphyrin IX derivatives (DP) is their high lipophilicity and, hence, the ability to photosensitize cellular membrane systems. In order to study the possible role of the position and character of side substituents endowing DP molecules with polarity or lipophilicity in their PD efficacy, we synthesized a number of 4-monosubstituted or 2,4-disubstituted DPs and studied their PD effect on the model systems^5,6. The study is in progress now for 2-monosubstituted porphyrins as well as for 2-devinylchlorin e6 derivatives.

Recently isolated crayfish mechanoreceptor neuron has been proposed as a sensitive model for comparison of PSs and investigation of some mechanisms of PD effect at the cellular level^7,8 . The structure, biochemical, electrophysiological, and photobiological features of this classic neurophysiological object are well studied^9-12. This neuron is able to fire with a nearly constant rate during several hours, and at this stable background one can continuously record the dynamics of cell response to external impact from initial threshold shifts to terminal events leading to the cell death. In the present paper we have studied the dynamics of neuron responses to different 4-monosubstituted or 2,4-disubstituted DPs in order to compare their PD efficiencies.

2. METHODS

Slowly adapting muscle receptor organs of the crayfish Astacus leptodactilus were isolated as described by Wiersma et al.¹³. These were placed into a plexiglass chamber with van Harreveld saline (mM: NaCl - 205; KCL -5.4; NaHCO₃ - 0.24; MgCl₂ - 5.4; CaCl₂ - 13.5; pH 7.2-7.4). In this preparation, stretch receptor neurons were capable of regular firing at a nearly constant rate for up to 8-12 hours. Neuron spikes were derived extracellularly from axons by the glass pipette suction electrodes, amplified (amplifier UU-90, Institute of Experimental Medicine, St.Petersbourg, Russia), with their frequency being converted into voltage by analog frequency meter (MFU-1, Institute of Experimental Medicine, St.Petersbourg, Russia) and continuously recorded by the chart-recorder (N-390, ZIP, Krasnodar, Russia). To test the irreversibility of neuron activity abolition we recorded neuron potentials 30-60 min after cessation of spikes and then additionally stimulated SRN by receptor muscle extension. The absence of spikes indicated that neuron had lost the ability to fire.

The experimental protocol was as follows: at the beginning of each experiment the initial neuron frequency level was set near 10-15 Hz by application of the appropriate receptor muscle extension. After 30 min 'control' recording of spike generation, the PS solution was added into the chamber. After the next 30 min, cells were irradiated with helium-neon laser (632,8 nm, 0.3 W/cm², LGN-111, 'Polyaron', L'vov, Republic of Belarus) until the irreversible firing cessation. The irradiation power was measured by laser dosimeter (IMO-2N, 'Etalon', Volgograd, Russia). The irradiation exposures were as long as the neuron lifetimes.

Table 1. The main physical and chemical features of deuteroporphyrin IX derivatives
 

Fig. 1. Chemical formulae of deuteroporphyrin IX derivatives.

All experiments were accomplished without special thermostating at room temperature of 303 ^oC. Standard statistical methods including correlation and regression analysis¹⁴ were used. To compare efficiencies of different photosensitizers, we studied concentration dependencies of neuron lifetime(T). Functions T(C) were approximated by the power functions: T(C) = a*C^b, which are linear in double logarithmic coordinates: lg T= lg a + b*l g C. Parameters a, and b were determined by the method of least squares.

3. RESULTS

3.1. Hydrophobicity and amphiphilicity of deuteroporphyrin IX derivatives

According to Table 1 2,4diOHbu appeared to be the most hydrophilic DP photosensitizer. Its R_f=0.05 was, however, higher than for Photosens. Other DPs were markedly more lipophilic (R_f=0.27-0.56). The least amphiphilic PS among them was Photosens (K_p=0.05), Photoheme was more amphiphilic (K_p=1.5). All DPs were much more amphiphilic and could be subdivided into three groups: 4Ac and 2,4diAc (K_p=9.1 and 10.0), then 2,4diOHbu and 2,4diAcAc (K_p=17.0 and 19.4, respectively), and 4AcAc and 4OHbu were the most amphiphilic PSs (K_p=28.0).

3.2. Dynamics of neuron response to photodynamic effect of deuteroporphyrin IX derivatives

The unstained SRN was found to be insensitive tothe He-Ne laser irradiation lasting several hours nor to the addition of PS in the dark. However, they were very sensitive to the combination of these factors, i.e. to the PD effect. Neuron response dynamics included phases of firing acceleration or inhibition. Prolonged irradiation caused irreversible firing cessation that was considered to be the functional sign of the cell death. The following two main ways of firing abolition were observed: (a) firing acceleration followed by its abrupt abolition, or (b) gradual firing inhibition resulting in the irreversible cessation of spike generation. In both cases firing did not resume neither spontaneously, nor under additional adequate stimulation (receptor muscle extension). The dynamics of the cell response to PD impact (the alternating of firing excitation (E) and inhibition (I) phases) depended on PS type and concentration (Fig.2, Table 2).

Table 2. The main types of neuron response dynamics to PD effect
 

As Table 3 shows, the initial response phase was acceleration of firing (E-, EI-, or EIE-responses) in 70-100 % neurons at the relatively high PS concentrations (> 10^-7-10^-6 M). More prominent difference between the neuron responses to various PSs was observed at the lesser concentrations ( < 10^-7-10^-9 M). In this case the neuron sensitization with 4AcAc, 4Ac, 4OHbu, or Photosens caused initial firing acceleration in 60-70 % cells. The sensitization with 2,4diOHbu or 2,4diAc caused, on the contrary, the initial inhibition of firing in 60-70 % neurons. 2,4diAcAc and Photoheme induced firing acceleration and inhibition in approximately one half of experiments.

Considering the terminal phases of the neuron response to PD effect (Table 3) one can subdivide all studied PSs into the following groups: (i) 71-75 % responses to sensitization with 4AcAc, 4Ac and Photosens were excitatory (E or EIE types) and culminated with abrupt firing abolition at both high and low PS concentrations. (ii) About 65 % responses to sensitization with 4OHbu were of I, EI, or IEI types with the irreversible firing cessation after prolonged gradual inhibition phase. This was observed both at high and at low PS concentrations. (iii) Excitatory final phases were dominant at high PS concentrations while inhibitory ones - at low concentrations of 2,4diOHbu; 2,4diAcAc; 2,4diAc or Photoheme.

Fig. 2. The main types of neuron responses to photodynamic effect. A - E-response; B - EIE-response; C - EI-response; D - I-response; E - IE-response; F - IEI- response. Ordinate - firing frequency, Hz; abscissa - time, min.

3.3. Concentration dependencies

In order to compare PD efficiencies of different PSs we studied dependencies of neuron lifetimes T on PS concentrations C. These were approximated by the power functions: T(C) = a*C^b linear in the double logarithmic coordinates: lg T= lg a + b*lg C (Fig. 3). Parameters a and b determined by the least squares method are presented in the Table 3. The most effective PS occupy the left lower corner in the Fig. 3. These data show that PD efficiency is increased in the following series:

Photoheme 2,4diOHbu < Photosens < 2,4diAcAc< 2,4di Ac 4Ac < 4OHbu < 4AcAc
 

Table 3. The main types of neuron responses to photodynamic effect of different photosensitizers