Roberta Biolo

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.

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 (ɛ= 5 × 105 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 μg 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.

Effect of Photosensitizer Delivery System and Irradiation Parameters on the Efficiency of Photodynamic Therapy of B16 Pigmented Melanoma in Mice

Abstract— Previous studies (Biolo et al., Photochem. Photobiol. 59, 362‐365, 1994) showed that liposome‐delivered Si(IV)‐na‐phthalocyanine (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°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.

ULTRASTRUCTURAL STUDIES ON THE MECHANISM OF THE PHOTODYNAMIC THERAPY OF TUMORS

Abstract Balb/c mice bearing a transplanted MS‐2 fibrosarcoma were injected with 2.5 mg kg 1 of either tetra(4‐sulfonatophenyl/porphine (TPPS) in phosphate‐buffered saline or 0.5 mg kg−1 of Zn2+‐phthalocyanine (Zn‐Pc) incorporated into unilamellar liposomes of dipalmitoyl‐phosphatidylcholine. Chromatographic studies showed that TPPS is mainly transported in the serum by globulins and albumin, while Zn‐Pc is specifically bound by lipoproteins. Exposure of the injected mice to red light (300 J cm−2) caused extensive tumor necrosis. The ultrastructural analysis of tumor specimens taken from mice at 15 h after PDT showed that TPPS photoinduces a preferential necrosis of the neoplastic cells, while Zn‐Pc causes severe photodamage to both the vascular system and the neoplastic cells. The different modes of tumor photosensitization by TPPS and Zn‐Pc are discussed on the basis of the transport mechanism of the two dyes.

Liposome‐Mediated Delivery of Photosensitizers: Localization of Zinc (11)‐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 C57BL6 mice bearing subcutaneously implanted B16 melanomas. Tumors were removed 3 h or 24 h after dosing and the intratumoral distribution determined by fluorescence microscopy. Localization of the photosensi‐tizer occurred more rapidly in the Ehrlich carcinoma than in the B16 melanoma; this difference in photosen‐sitizer uptake may be related to a higher degree of vas‐cularization 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 membranes and the cytosol, but not the nucleus, of cells in both tumors.

A COMPARISON OF FLUORESCENCE METHODS USED IN THE PHARMACOKINETIC STUDIES OF Zn(II)PHTHALOCYANINE IN MICE

Abstract— The pharmacokinetics of Zn phthalocyanine (ZnPc) encapsulated in dipalmitoyl‐phosphatidyl‐choline (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 12h. 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.

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- or LDL-administered Zn(II)-phthalocyanine as a photodynamic agent for tumours III. Effect of cholesterol on pharmacokinetic and phototherapeutic properties

The pharmacokinetic behaviour of Zn-phthalocyanine (Zn-Pc) has been studied in mice bearing a transplanted MS-2 fibrosarcoma using liposomes of dipalmitoyl-phosphatidylcholine containing 15% moles of cholesterol as drug delivery systems. The presence of cholesterol optimizes the Zn-Pc localization in the tumour inducing a significantly larger uptake as well as a better selectivity. Experimental photodynamic therapy of the MS-2 fibrosarcoma has been performed using two different protocols. The data obtained show that an efficient tumour necrosis can be photosensitized by very low Zn-Pc doses (0.035–0.3 mg kg−1) at both short and long time intervals after injection of the dye.

Experimental photodynamic therapy with tetrapropyl-porphycene: Ultrastructural studies on the mechanism of tumour photodamage

Red light irradiation of a transplanted MS-2 fibrosarcoma in mice at 24 h after injection of liposome-zbound tetra-n-propyl-porphycene (TPP, 2mg kg−1 b.w.) caused an efficient tumour necrosis. Electron microscopy analysis of tumour specimens taken at different times after the phototherapeutic treatment showed the development of direct damage of malignant cells between 3 and 6 h; the earliest detectable alterations occurred at the level of mitochondria. The endocellular damage gradually progressed with extensive vacuolization of the cytoplasm and, at later stages, formation of pyknotic nuclei. On the other hand, the vascular system of the tumour appeared to be well preserved up to about 9 h, when several endothelial alterations were detected. The damage of the tumour tissue was essentially complete 24 h after the phototreatment. The pattern of tumour modification is consistent with a preferential transport and tumour release of the liposome-bound TPP by low-density lipoproteins.