Carla Milanesi

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.

Morphological aspects of an experimental tumour photosensitized with a meso-substituted cationic porphyrin

The meso-substituted cationic porphyrin, meso-tetra(4N-methyl-pyridyl)porphine (T4MPyP) appears to be a selective tumour localizer on the basis of pharmacokinetic studies. Irradiation (at 600-680 nm) of an intramuscularly implanted MS-2 fibrosarcoma in Balb/c mice at 24 h after injection of T4MPyP causes tumour necrosis: histological and ultrastructural analyses of tumour specimens taken at different times after phototherapy indicate slowly-appearing tissue damage which involves both malignant cells and the vascular endothelium. At the subcellular level, the membranous systems and nuclei are the main targets of the photoprocess. The tumour necrosis is particularly extensive upon injection of 4.1 mg kg-1 T4MPyP.

AN ULTRASTRUCTURAL COMPARATIVE EVALUATION OF TUMORS PHOTOSENSITIZED BY PORPHYRINS ADMINISTERED IN AQUEOUS SOLUTION, BOUND TO LIPOSOMES OR TO LIPOPROTEINS

Abstract— Balb/c mice bearing a transplantedMS–2 fibrosarcoma have been injected with 2 mg kg‐1 hematoporphyrin either dissolved in phosphate‐buffered saline (Hp‐aq), or incorporated into unilamellar liposomes of dipalmitoyl‐phosphatidylcholine (Hp‐lip), or precomplexed with low density lipoproteins (Hp‐LDL). At 24 h after injection, the mice received 150 J cm‐2 of600–680 nm light irradiation. Electron microscopic studies performed on tumor tissues taken from mice sacrificed at different times after the phototreatment showed that, in the presence of Hp‐aq. tumor necrosis is largely the consequence of vascular damage. On the other hand, in the presence of Hp‐lip and Hp‐LDL, the response of the tumor to the phototreatment occurs at a faster rate and is mainly determined by direct damage of neoplastic cells. These findings are correlated with the different distribution of the various hematoporphyrin forms (Hp‐aq, Hp‐lip, Hp‐LDL) among the serum proteins and the modalities of hematoporphyrin delivery to tissues by the possible carriers.

Apoptosis of mouse MS-2 fibrosarcoma cells induced by photodynamic therapy with Zn (II)-phthalocyanine.

The destructive process of mouse MS-2 fibrosarcoma induced by photodynamic therapy (PDT) with liposome-administered Zn(II)-phthalocyanine (ZnPc) was studied by electron microscopy. Pronounced ultrastructural changes characteristic of apoptosis were observed for several tumour cells, including early occurrence of condensation and margination of chromatin, disappearance of nuclear pores, karyopyknosis, karyorrhexis, protuberance formation at the cell surface and cell fragmentation. The findings indicate that apoptosis was involved in the process of tumour cell death induced by ZnPc-PDT. The detailed mechanism and pathways controlling this phenomenon need to be elucidated further.

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.

CGP 55398, a liposomal Ge(IV) phthalocyanine bearing two axially ligated cholesterol moieties: a new potential agent for photodynamic therapy of tumours.

Ge(IV) phthalocyanine (GePc) with two axially ligated cholesterol moieties was prepared by chemical synthesis and incorporated in a monomeric state into small unilamellar liposomes (CGP 55398). Upon photoexcitation with light wavelengths around its intense absorption peak at 680 nm, GePc shows an efficient photosensitising activity towards biological substrates through a mechanism which largely involves the intermediacy of singlet oxygen. GePc injected systemically into mice bearing an intramuscularly implanted MS-2 fibrosarcoma is quantitatively transferred to serum lipoproteins and localises in the tumour tissue with good efficiency: at 24 h post injection the GePc content in the tumour is 0.74 and 1.87 micrograms per g of tissue with a tumour/peritumoral ratio of 4.35 and 5.67 for injected doses of 0.76 and 1.52 mg kg-1 respectively. At this time the red-light irradiation of the GePc-loaded fibrosarcoma causes a fast and massive tumour necrosis involving both malignant cells and blood vessels.

Fine structure of the gastric epithelium of the ascidian Botryllus schlosseri. Mucous, endocrine and plicated cells

SummaryThe following five cell types have been recognized and defined on the basis of their fine structure in the gastric epithelium of B. schlosseri: vacuolated and zymogenic cells (described in a previous paper); ciliated mucous, endocrine and plicated cells. The ciliated mucous cells are distributed at the apex and the bottom of the gastric folds and along the dorsal groove. The mucus droplets appear to form from the Golgi complex as secretory granules of variable density and texture, which are released from the cell after fusion of their membranes with the apical plasma membrane. Holocrine or apocrine secretion has not been observed. The endocrine cells are scattered and are characterized by electron dense granules, especially numerous in the basal region of the cell. Finally, the plicated cells, present in the pyloric caecum, show rod-like microvilli, a well developed Golgi complex and abundant, deep infoldings of the basal plasma membrane, which are associated with numerous mitochondria. The possible role of the gastric cell types is discussed taking into account information concerning morphologically similar cells in other animals, as well as previously reported data on the biochemistry and physiology of digestion and excretion in ascidians.

Fine structure of the gastric epithelium of the ascidian botryllus schlosseri vacuolated and zymogenic cells

SummaryVacuolated and zymogenic cells, which are two of five cell types identified by electron microscopy in gastric epithelium of B. schlosseri, are described in detail. The vacuolated cells are characterized by one, or a few, supranuclear vacuoles containing myelin figures. A peculiar Golgi apparatus is consistently found at the base of the vacuoles; it consists of cisternae frequently containing small vesicles and tubules of constant diameter and/or a strong electron-opaque material. A variety of vesicles and multivesicular bodies are visible in the apical cytoplasm below long ribbon-like microvilli. The se findings suggest that the vacuolated cells are involved in absorptive and perhaps secretory activity. The zymogenic cells are characterized by a highly developed RER, numerous apical secretory granules and a well developed supranuclear Golgi apparatus. At the apical end, autophagosomes are frequently encountered, some of which contain also zymogen granules. Both cell types contain numerous lipid droplets, which are interpreted as an energy reserve available for the cells and for the entire colony during the change of generation. Correlation between structure and function in both cell types is discussed by taking into account the peculiar life cycle of B. schlosseri, as well as previously reported data on similar cells in other ascidians.

Tumour-localising and -photosensitising properties of a novel zinc(II) octadecylphthalocyanine.

1,4,8,11,15,18,22,25-Octadecylphthalocyaninato zinc(II), ZnODPc, incorporated into a Cremophor emulsion, was assayed for its pharmacokinetic and phototherapeutic properties in Balb/c mice bearing an intramuscularly transplanted MS-2 fibrosarcoma. The phthalocyanine was injected intravenously (i.v.) in three doses, i.e. 1.46, 0.73 and 0.37 mumol kg-1 body weight. In all cases, the octadecyl-substituted phthalocyanine showed an unusually high affinity for serum low-density lipoproteins (LDLs) and a high efficiency and selectivity of tumour targeting: the maximum accumulation in the tumour occurred at 24 h after injection, whereas no detectable amount of phthalocyanine was recovered from the muscle, i.e. the peritumoral tissue, between 1 h and 1 week after injection. At the same time, low amounts of phthalocyanine were recovered from skin and then only at short times after injection, with skin photosensitivity rapidly disappearing and the phthalocyanine present in the serum only. Tumour photosensitisation studies were carried out at 24 h after administration of 1.46 mumol kg-1 ZnODPc and showed that this phthalocyanine has a very high phototherapeutic efficiency; this is probably a consequence of the multiple mechanisms by which the phthalocyanine induces tumour damage, involving both direct modification of malignant cells and impairment of blood flow, as well as the alteration of a variety of subcellular components, such as mitochondria, the rough endoplasmic reticulum, the perinuclear membrane and, occasionally, cell nuclei. Tumour necrosis appears to be the consequence of both random cell death and apoptosis.

Fine structure of the intestinal epithelium of the colonial ascidian Botryllus schlosseri

SummaryIn the intestine of the filtering zooids of B. schlossen three segments can be distinguished. In the intermediate segment the epithelium, which is encrusted by the ampullae of the pyloric gland, shows marked aspects of alteration. In the proximal and distal segments, ciliated mucous, vacuolated and endocrine-like cells are recognizable.Ciliated mucous cells, widely distributed along the intestine, possess the apical region filled with numerous mucous granules, which are extruded with merocrine modality. Variations in morphology of the granules are visible especially between cells of different regions. Vacuolated cells appear involved in absorptive function. They are characterized by developed microvilli, numerous apical small vesicles and great supranuclear vacuoles containing heterogeneous material. The vacuolated cells of the proximal segment resemble the gastric vacuolated cells of B. schlossen. The vacuolated cells of the distal segment show many morphological similarities with protein absorbing cells of various animals for the presence of a giant vacuole and an apical network of vesicles and tubules with fuzzy coating on the luminal face.The intestinal endocrine-like cells are rare and characterized by strongly electron dense granules distributed in all the cytoplasm, but predominantly in the basal region.