DOI 10.1007/s10517-014-2670-2
798
Bulletin of Experimental Biology and Medicine, Vol. 157, No. 6, October, 2014 NANOTECHNOLOGIES
Study of Photoinduced Antitumor Activity of PhthalocyaninBased Nanostructures as Pro-Photosensitizers in Photodynamic Therapy of Malignant Tumors In Vivo A. A. Pankratov, T. N. Andreeva, R. I. Yakubovskaya, B. Ya. Kogan*, A. V. Butenin*, R.K.-G. Feizulova*, V. A. Puchnova*, N. V. Novoseletsky*, A. V. Khromov*, E. A. Lukyanets*, and G. N. Vorozhtsov* Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 157, No. 6, pp. 771-777, June, 2014 Original article submitted June 6, 2013 Nanoparticles of aluminum and zinc phthalocyanin and metal-free phthalocyanin (AlPc, ZnPc, and H2Pc), whose molecular forms are photosensitizers, can serve as effective “prophotosensitizers” in photodynamic therapy for malignant tumors. Transition (stimulation) of photo-inert nanoparticles into a photoactive photosensitizer is realized locally in the tumor node by its exposure to potent laser pulses. Systemic injection of AlPc, ZnPc, and H2Pc nanoparticles has not led to accumulation of their photoactive form in the skin, which can lead to the development of skin phototoxicity. Effective protocols of photodynamic therapy with ZnPc nanoparticles are determined. The use of these protocols in mice with S-37 sarcoma led to 92-70% tumor growth inhibition, 48% improvement of survival, and cure in 84% cases. Key Words: photodynamic therapy; zinc phthalocyanin nanoparticles; aluminum phthalocyanin nanoparticles; pro-photosensitizers; photosensitizers Photodynamic therapy (PDT) is an effective method for conservative antitumor therapy used in clinical practice with good results for more than 25 years [1,7,9]. Phthalocyanin derivatives are promising compounds for the development of effective drugs for PDT of malignant tumors, due to their appropriate photophysical characteristics and the maximum absorption in the distant red and close IR spectra [2,3]. Active search for potential PDT photosensitizers with better photophysical and biological characteristics in this class of compounds is now in progress [3,4,10-14]. Photosense, a first-generation PDT agent, has been developed and licensed in Russia. It is a mixture of sodium salts of sulfated aluminum phthalocyanin [1,6-9]. P. A. Hertzen Moscow Oncological Institute, the Ministry of Health of the Russian Federation; *Institute of Organic Intermediate Products and Stains, Moscow, Russia. Address for correspondence: [email protected]. A. A. Pankratov
One of the pharmacological characteristics of water-soluble phthalocyanins is their slow pharmacokinetics. Long circulation of the photoactive form of the compound in vivo allows realization of several PDT sessions without additional photosensitizer charge. However, slow elimination of the photosensitizer from the body can lead to the development of phototoxic reactions, primarily skin phototoxicity, this limiting their use. These complications can be prevented by administration of the so-called “pro-drug” without photoactive properties, which should be stimulated directly in the focus. We studied photoinert nanoparticles of phthalocyanins with crystalline structure as compounds of this kind: aluminum (AlPc), zinc (ZnPc) phthalocyanins, and metal-free phthalocyanin (H2Pc). Transition of AlPc, ZnPc, and H2Pc in the photoactive (molecular) state was attained by exposing them to potent laser pulses with the generation wavelength in the nanoparticle absorption region.
0007-4888/14/15760798 © 2014 Springer Science+Business Media New York
A. A. Pankratov, T. N. Andreeva, et al.
799
We studied the photophysical (fluorescence) and biological (photoinduced antitumor efficiency in vivo) characteristics of AlPc, ZnPc, and H2Pc nanoparticles as “pro-photosensitizers” for PDT.
MATERIALS AND METHODS Nanostructures based on AlPc, ZnPc, and H2Pc were used in the form of water dispersions in a concentration of 2 mg/ml (0.2% colloid solution; Institute of Organic Intermediate Products and Stains). The study was carried out on F1 female hybrid mice (20-25 g) from Center of Biomedical Technologies of the Russian Academy of Medical Sciences (Andreevka Affiliated Department). Experimental groups consisted of 9-12 animals, control groups of 10-12 animals. All manipulations on animals were carried out in accordance with the Order No. 708n of the Ministry of Health of the Russian Federation of 13.10.2010 “On Laboratory Practice Regulations” and the EC Directive of 24.11.1986 “On Convergence of th EC Countries Laws, Decisions, and Administrative Assumptions on the Protection of Animals Used for Experimental and Other Research Purposes” (86/609/EEC). Sarcoma S-37 was selected as the tumor model. The tumor strain was maintained in vivo in the ascitic variant. The tumor was transplanted subcutaneously in the outer surface of the hip by the common method [5]. The therapy was started on day 6 or 7 after tumor material injection. The tumor node volume on day 1 of therapy was ~71±11 mm3. Antitumor effect was evaluated by tumor growth inhibition (%), mean lifespan prolongation (%), and cure (the absence of continuing tumor growth for 90 days after treatment). Tumor growth inhibition by of 50% and lifespan prolongation by 25% were regarded as the minimum biologically significant effect [5]. The nanoparticles in water dispersion were injected in a single intravenous dose of 1 to 15 mg/kg. The nanoparticles were stimulated by a potent pulsed
ruby laser at =694 nm. Photodynamic therapy was carried out with a diode laser at =670 nm. Pulsed exposure (stimulation stage) was carried out 2-5 min after injection of the nanocomposites at the following parameters: pulse energy density 0.6 J/cm2, number of pulses 1 to 10 (summary energy density/ session 0.6-6.0 J/cm2). Continuous laser exposure of the focus (PDT) was carried out 2-5 min and 24 h after the stimulation stage, at the following parameters: power density 10 and 100 mW/cm2, energy density 40 and 90 J/cm2. Before the exposure the animals were narcotized by intramuscular droperidole (6.25 mg/kg). Controls (no laser exposure) were injected with 0.9% NaCl (15 ml/kg). The data were statistically processed by Statistica 7.0 software. If the hypothesis on the normal distribution were confirmed, the equality of intragroup dispersions was analyzed (Lӓwen test). Further comparisons of the studied parameters were carried out using the Student t test. If the parameters did not conform to the normal distribution, the Mann–Whitney U test was used. The differences were considered significant at p
798
Bulletin of Experimental Biology and Medicine, Vol. 157, No. 6, October, 2014 NANOTECHNOLOGIES
Study of Photoinduced Antitumor Activity of PhthalocyaninBased Nanostructures as Pro-Photosensitizers in Photodynamic Therapy of Malignant Tumors In Vivo A. A. Pankratov, T. N. Andreeva, R. I. Yakubovskaya, B. Ya. Kogan*, A. V. Butenin*, R.K.-G. Feizulova*, V. A. Puchnova*, N. V. Novoseletsky*, A. V. Khromov*, E. A. Lukyanets*, and G. N. Vorozhtsov* Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 157, No. 6, pp. 771-777, June, 2014 Original article submitted June 6, 2013 Nanoparticles of aluminum and zinc phthalocyanin and metal-free phthalocyanin (AlPc, ZnPc, and H2Pc), whose molecular forms are photosensitizers, can serve as effective “prophotosensitizers” in photodynamic therapy for malignant tumors. Transition (stimulation) of photo-inert nanoparticles into a photoactive photosensitizer is realized locally in the tumor node by its exposure to potent laser pulses. Systemic injection of AlPc, ZnPc, and H2Pc nanoparticles has not led to accumulation of their photoactive form in the skin, which can lead to the development of skin phototoxicity. Effective protocols of photodynamic therapy with ZnPc nanoparticles are determined. The use of these protocols in mice with S-37 sarcoma led to 92-70% tumor growth inhibition, 48% improvement of survival, and cure in 84% cases. Key Words: photodynamic therapy; zinc phthalocyanin nanoparticles; aluminum phthalocyanin nanoparticles; pro-photosensitizers; photosensitizers Photodynamic therapy (PDT) is an effective method for conservative antitumor therapy used in clinical practice with good results for more than 25 years [1,7,9]. Phthalocyanin derivatives are promising compounds for the development of effective drugs for PDT of malignant tumors, due to their appropriate photophysical characteristics and the maximum absorption in the distant red and close IR spectra [2,3]. Active search for potential PDT photosensitizers with better photophysical and biological characteristics in this class of compounds is now in progress [3,4,10-14]. Photosense, a first-generation PDT agent, has been developed and licensed in Russia. It is a mixture of sodium salts of sulfated aluminum phthalocyanin [1,6-9]. P. A. Hertzen Moscow Oncological Institute, the Ministry of Health of the Russian Federation; *Institute of Organic Intermediate Products and Stains, Moscow, Russia. Address for correspondence: [email protected]. A. A. Pankratov
One of the pharmacological characteristics of water-soluble phthalocyanins is their slow pharmacokinetics. Long circulation of the photoactive form of the compound in vivo allows realization of several PDT sessions without additional photosensitizer charge. However, slow elimination of the photosensitizer from the body can lead to the development of phototoxic reactions, primarily skin phototoxicity, this limiting their use. These complications can be prevented by administration of the so-called “pro-drug” without photoactive properties, which should be stimulated directly in the focus. We studied photoinert nanoparticles of phthalocyanins with crystalline structure as compounds of this kind: aluminum (AlPc), zinc (ZnPc) phthalocyanins, and metal-free phthalocyanin (H2Pc). Transition of AlPc, ZnPc, and H2Pc in the photoactive (molecular) state was attained by exposing them to potent laser pulses with the generation wavelength in the nanoparticle absorption region.
0007-4888/14/15760798 © 2014 Springer Science+Business Media New York
A. A. Pankratov, T. N. Andreeva, et al.
799
We studied the photophysical (fluorescence) and biological (photoinduced antitumor efficiency in vivo) characteristics of AlPc, ZnPc, and H2Pc nanoparticles as “pro-photosensitizers” for PDT.
MATERIALS AND METHODS Nanostructures based on AlPc, ZnPc, and H2Pc were used in the form of water dispersions in a concentration of 2 mg/ml (0.2% colloid solution; Institute of Organic Intermediate Products and Stains). The study was carried out on F1 female hybrid mice (20-25 g) from Center of Biomedical Technologies of the Russian Academy of Medical Sciences (Andreevka Affiliated Department). Experimental groups consisted of 9-12 animals, control groups of 10-12 animals. All manipulations on animals were carried out in accordance with the Order No. 708n of the Ministry of Health of the Russian Federation of 13.10.2010 “On Laboratory Practice Regulations” and the EC Directive of 24.11.1986 “On Convergence of th EC Countries Laws, Decisions, and Administrative Assumptions on the Protection of Animals Used for Experimental and Other Research Purposes” (86/609/EEC). Sarcoma S-37 was selected as the tumor model. The tumor strain was maintained in vivo in the ascitic variant. The tumor was transplanted subcutaneously in the outer surface of the hip by the common method [5]. The therapy was started on day 6 or 7 after tumor material injection. The tumor node volume on day 1 of therapy was ~71±11 mm3. Antitumor effect was evaluated by tumor growth inhibition (%), mean lifespan prolongation (%), and cure (the absence of continuing tumor growth for 90 days after treatment). Tumor growth inhibition by of 50% and lifespan prolongation by 25% were regarded as the minimum biologically significant effect [5]. The nanoparticles in water dispersion were injected in a single intravenous dose of 1 to 15 mg/kg. The nanoparticles were stimulated by a potent pulsed
ruby laser at =694 nm. Photodynamic therapy was carried out with a diode laser at =670 nm. Pulsed exposure (stimulation stage) was carried out 2-5 min after injection of the nanocomposites at the following parameters: pulse energy density 0.6 J/cm2, number of pulses 1 to 10 (summary energy density/ session 0.6-6.0 J/cm2). Continuous laser exposure of the focus (PDT) was carried out 2-5 min and 24 h after the stimulation stage, at the following parameters: power density 10 and 100 mW/cm2, energy density 40 and 90 J/cm2. Before the exposure the animals were narcotized by intramuscular droperidole (6.25 mg/kg). Controls (no laser exposure) were injected with 0.9% NaCl (15 ml/kg). The data were statistically processed by Statistica 7.0 software. If the hypothesis on the normal distribution were confirmed, the equality of intragroup dispersions was analyzed (Lӓwen test). Further comparisons of the studied parameters were carried out using the Student t test. If the parameters did not conform to the normal distribution, the Mann–Whitney U test was used. The differences were considered significant at p
