Photoinactivation of amelanotic and melanotic melanoma cells sensitized by axially substituted Si-naphthalocyanines.

The photosensitizing activity of the new far-red absorbing naphthalocyanine SiNc [OSi (n-C10H21)3] [OSi(CH3)2(CH2)3N(CH3)2], (DAP-SiNc), and of its analogue SiNc [OSi(i-C4H9)2(n-C18H37)]2, (IsoBO-SiNc), was studied with two cell variants of B16 melanoma, the amelanotic clone B78H1 and the highly pigmented B16F1 cells. Upon excitation with a 776 nm diode laser, DAP-SiNc appeared to be a markedly more efficient photosensitizer than isoBO-SiNc. The higher photoefficiency of DAP-SiNc was likely to reflect its accumulation in significantly larger amounts by both cell types, as well as a much smaller tendency to undergo aggregation when bound to the cells. In any case, melanotic cells were less sensitive to the photoinactivating action of DAP-SiNc: the protective action of melanin was a consequence of an optical filtering of the 776 nm light and an appreciable shortening of the DAP-SiNc triplet lifetime (40 microseconds for the amelanotic vs. 17 microseconds for the melanotic cells). Functional and morphological studies on irradiated cells showed that cell death due to DAP-SiNc photosensitization was mainly correlated with the modification of targets located in the lysosomes and the cytoplasmic membrane.

Liposome-mediated delivery of photosensitizers: localization of zinc (II)-phthalocyanine within implanted tumors after intravenous administration.

CGP55847, liposomal zinc(II)-phthalocyanine (Zn-Pc), was administered by the intravenous route to Swiss mice bearing intramuscularly implanted Ehrlich carcinomas or to C57/BL6 mice bearing subcutaneously implanted B16 melanomas. Tumors were removed 3 h or 24 h after dosing the intratumoral distribution determined by fluorescence microscopy. Localization of the photosensitizer occurred more rapidly in the Ehrlich carcinoma than in the B16 melanoma; this difference in photosensitizer uptake may be related to a higher degree of vascularization of the carcinoma. The photosensitizer was found in association with blood vessels at 3 h but not 24 h after dosing and appeared to have a greater affinity for areas of tissue necrosis within the tumor compared to viable tumor tissue. Little or no Zn-Pc was detected in the muscle tissue invaded by the Ehrlich carcinoma and was associated with the membranes and the cytosol, but not the nucleus, of cells in both tumors.

Effect of photosensitizer delivery system and irradiation parameters on the efficiency of photodynamic therapy of B16 pigmented melanoma in mice.

Previous studies (Biolo et al., Photochem. Photobiol. 59, 362-365, 1994) showed that liposome-delivered Si(IV)-naphthalocyanine (SiNc) photosensitizes B16 pigmented melanoma subcutaneously transplanted in C57 mice to the action of 776 nm light. However, the efficacy of the phototreatment was limited by a lack of selectivity of tumor targeting by SiNc as well as by incomplete necrosis of the neoplastic mass. The present investigations show that the use of a different delivery system (Cremophor emulsion vs liposomes of dipalmitoylphosphatidylcholine) causes no significant increase in the selectivity of tumor targeting for three injected doses of SiNc (0.5, 1, 2 mg/kg). However, upon 776 nm light irradiation (300 mW/cm2; 520 J/cm2), the delay in the rate of tumor growth was maximal (7-8 days) for the highest naphthalocyanine dose. On the other hand, a remarkable improvement in the tumor response was obtained by inducing an intratumoral temperature increase to 44 degrees C immediately after PDT. The thermal effect appeared to be due to photoexcitation of melanin by 776 nm light (550 mW/cm2; 520 J/cm2) and subsequent partial conversion of absorbed energy into heat.

[Apoptosis of mouse MS-2 fibrosarcoma cells induced by photodynamic therapy with LDL-administered zinc-phthalocyanine].

Photodynamic destruction of MS-2 fibrosarcoma cells in mice induced by LDL-administered Zinc-phthalocyanine and red light irradiation was studied by electron microscopy. The pronounced structural changes such as chromatin condensation, disappearance of nuclear pores, karyopyknosis, karyorrhexis, leakage of chromatin aggregates, autophagocytosis, bleb formation on the cell surface, cytoplasmic vacuolization and cell fragmentation suggest that the tumor cell death was induced by apoptosis. However, its exact mechanism and regulating pathways remain to be further investigated.

Photodynamic therapy of B16 pigmented melanoma with liposome-delivered Si(IV)-naphthalocyanine.

The possibility of extending photodynamic therapy to the treatment of highly pigmented neoplastic lesions was tested by using Si(IV)-naphthalocyanine (SiNc) as a tumor-localizing agent. Si(IV)-naphthalocyanine displays intense absorbance at 776 nm (epsilon = 5 x 10(5) M-1 cm-1), where melanin absorption becomes weaker. As an experimental model we selected B16 pigmented melanoma subcutaneously transplanted to C57BL mice. Upon injection of 0.5 or 1 mg kg-1 of liposome-incorporated SiNc, maximal accumulation of the photosensitizer in the tumor was observed at 24 h with recoveries of 0.35 and 0.57 microgram g-1, respectively. However, the tumor targeting by SiNc shows essentially no selectivity, since the photosensitizer concentrations in the skin (peritumoral tissue) were very similar to those found in the tumor at all postinjection times examined by us. Irradiation of SiNc-loaded melanoma with 776 nm light from a diode laser at 24 h postinjection induces tumor necrosis and delay of tumor growth. The effect appears to be of purely photochemical nature at dose rates up to 260 mW cm-2; at higher dose rates, thermal effects are likely to become important.

Laser diode coupled with optical fiber for applications in photodynamic therapy.

A diode laser with roughly 480 mW/cm2 emission at 776 nm was developed and engineered to obtain a uniform illumination of tumour cells deposited on Petri dishes having an external diameter of 5 cm. The efficacy of the apparatus in inducing photocytotoxic effects was successfully checked by irradiation of pigmented melanoma cells in the presence of a photosensitizer, Si(IV)-naphthalocyanine, having intense absorption bands at 776 nm. Our results open interesting prospects for extending photodynamic therapy to highly pigmented tumours.

A comparison of fluorescence methods used in the pharmacokinetic studies of Zn(II)phthalocyanine in mice.

The pharmacokinetics of Zn phthalocyanine (ZnPc) encapsulated in dipalmitoyl-phosphatidylcholine (DPPC) liposomes, injected intravenously in Skh:HR-1 nude mice, was monitored by two in vitro techniques and one in vivo technique, all based on fluorescence spectroscopy. The in vitro methods involve either fluorescence measurements on thin tissue sections or on extracts from these tissues. The in vivo method involves the fluorescence measurement at the skin surface. Both in vitro techniques gave similar results which are consistent with previous findings on the pharmacokinetic behavior of ZnPc. The liver and spleen showed rapid ZnPc concentration increases, reaching a maximum level in 30 min. or less post drug administration. Relatively little ZnPc was detected in the skin, fat or muscle, the maximum concentration occurring at 12 h. In vivo fluorescence reached a maximum intensity approx. 6 h post injection at the mid-chest analysis site and at 12 h in the thigh. The in vivo measurements at two different anatomical sites showed pharmacokinetic behavior that reflects an overall integrated fluorescence originating from several tissue sites.

Zn(II)-phthalocyanine as a photodynamic agent for tumours. II. Studies on the mechanism of photosensitised tumour necrosis.

The mechanism of tumour necrosis photosensitised by liposome-delivered Zn(II) phthalocyanine (Zn-Pc) has been studied in mice bearing a transplanted MS-2 fibrosarcoma. Ultrastructural analyses of tumour specimens obtained at different times after red light-irradiation (300 J cm-2, dose-rate 180 mW cm-2) indicate an early (3 h) photodamage of malignant cells especially at the level of the mitochondria and rough endoplasmic reticulum. The cellular damage becomes more evident between 6 h and 15 h after photodynamic therapy. On the other hand, the capillaries supplying the tumour tissue are modified at a much slower rate and appear to be severely damaged only after 15 h from irradiation, when the whole tissue becomes necrotic. Occasionally, mildly damaged capillaries are observed even at 72 h after irradiation. These findings support the hypothesis that low density lipoproteins (LDL) play a major role in the delivery of Zn-Pc to the tumour tissue; the photosensitiser is released specifically to malignant cells as a consequence of a receptor-mediated endocytosis of LDL.

Liposome- or LDL-administered Zn (II)-phthalocyanine as a photodynamic agent for tumours. I. Pharmacokinetic properties and phototherapeutic efficiency.

The pharmacokinetics of Zn-phthalocyanine (Zn-Pc) in mice bearing a transplanted MS-2 fibrosarcoma has been studied using dipalmitoyl-phosphatidylcholine (DPPC) liposomes and low density lipoproteins (LDL) as drug delivery systems. LDL induce a higher Zn-Pc uptake by the tumour and improve the selectivity of tumour targeting as compared to DPPC liposomes. Experimental photodynamic therapy (PDT) of the MS-2 fibrosarcoma has been performed using liposome-delivered Zn-Pc and the efficiency of tumour necrosis has been measured following four different irradiation protocols. We found that Zn-Pc doses as low as 0.07-0.35 mg kg-1 are sufficient for inducing an efficient tumour response that is linearly dependent on the injected dose. The amount of Zn-Pc in the tumour decreases very slowly as a function of time, hence PDT gives satisfactory results even if performed at relatively long time intervals after administration.

Delivery of the tumour photosensitizer zinc(II)-phthalocyanine to serum proteins by different liposomes: studies in vitro and in vivo.

Zn-phthalocyanine (Zn-Pc) incorporated into liposomes of different phospholipids has been incubated in vitro with human serum and administered i.v. to rabbits. In both cases, chromatographic and density gradient ultracentrifugation studies indicate that Zn-Pc is almost exclusively bound by the 3 major lipoprotein components of the plasma (VLDL, LDL and HDL). The amounts of Zn-Pc recovered from the different lipoprotein fractions reflect their relative concentration in the serum. The presence of 20% moles of cholesterol in liposomes of dipalmitoyl phosphatidylcholine (DPPC) optimizes the release of Zn-Pc to LDL. This fact is important for enhancing the selectivity of drug delivery to tumors since LDL display a preferential interaction with neoplastic cells.

Tetra-n-propylporphycene as a tumour localizer: pharmacokinetic and phototherapeutic studies in mice.

The porphin isomer tetra-n-propyl-porphycene (TPP) was incorporated into unilamellar liposomes of dipalmitoylphosphatidylcholine and intravenously injected at a dose of 2 mg/kg to BALB/c mice bearing a MS-2 fibrosarcoma. Pharmacokinetic studies show that TPP is selectively transported by serum lipoproteins and delivered to the tumour tissue with good efficiency (approx. 1 microgram of the TPP per g of tissue at 24 h after injection) and selectivity (ratio of TPP concentration in the tumour to the peritumoural tissue 16.7 at 24 h). Large doses of TPP are also accumulated by the liver, in agreement with the elimination of the drug via the biliary route, while no TPP is recovered from the brain. Red light-irradiation (300 J/cm2) of the tumour area caused extensive necrosis, while only little cutaneous photosensitivity was observed. Since TPP has a large absorbance in the 630-640 nm region, can be synthesized with a high degree of purity and is an efficient generator of singlet oxygen, this drug represents a potential candidate as a phototherapeutic agent for tumours.

Pharmacokinetic studies with zinc(II)-phthalocyanine in tumour-bearing mice.

Zn(II)-phthalocyanine (Zn-Pc) incorporated into unilamellar liposomes of dipalmitoylphosphatidylcholine has been injected intraperitoneally (0.5 mg kg-1) to BALB/c mice bearing a transplanted MS-2 fibrosarcoma. The drug is specifically transported by serum lipoproteins and cleared from the serum via the bile-gut pathway in a biphasic process: approximately 60% of Zn-Pc is eliminated with a serum half-life of approximately 9 hours, while the remaining aliquot is eliminated at a very slow rate. Several normal tissues take up the drug within 3 hours after administration but release it almost completely after 24-48 hours. On the other hand, the tumour shows a maximum concentration of Zn-Pc (approximately 0.6 microgram g-1 of tissue) after 18-24 hours; at this time, the ratio between the Zn-Pc levels in the tumour and the muscle (which represents the surrounding normal tissue) is approximately 7.5. The results are discussed in terms of a possible use of Zn-Pc as a photosensitizer in the photodynamic therapy of tumours.