In vivo fluorescence and photodynamic activity of zinc phthalocyanine administered in liposomes.

Zinc(II) phthalocyanine, a hydrophobic photosensitiser, was incorporated in unilamellar liposomes and studied in vivo for fluorescence kinetics and photodynamic activity. An observation chamber mounted in a dorsal skinfold of female WAG/Rij rats was used as a model system. In the chamber, an isogeneic mammary carcinoma was transplanted in the subcutaneous tissue. Phthalocyanine fluorescence was excited at 610 nm with a power density of 0.25 mW cm-2 and was detected above 665 nm through a high-pass filter using a two-stage image intensifier coupled to a charge-coupled device (CCD) camera. Following i.v. administration of 0.14 mg kg-1 of the drug, the fluorescence pharmacokinetics of the dye in vasculature, normal tissue and tumour tissue was determined as a function of time. Tumour fluorescence increased slowly to a maximum about 3 h post injection (p.i.), and remained well above the normal tissue fluorescence till 24 h p.i. Fluorescence in the circulation was always stronger than in the tissues. A treatment light dose at a wavelength of 675 nm was delivered 24 h p.i. One group of six animals received a total light dose of 150 J cm-2 (100 mW cm-2). A second group of six animals received a total light dose of 450 J cm-2 at the same dose rate. Vascular damage resulting from treatment was observed only at the final stages of the irradiation, despite the relatively high levels of fluorescence in the circulation. Immediate post-treatment (re)transplantation of the content of the chamber into the flank always resulted in tumour regrowth, confirming the presence of viable tumour cells following photodynamic therapy (PDT). When the chamber was left intact, the light dose of 450 J cm-2 yielded complete tissue necrosis. The role of the dye-carrier complex in shielding the vascular surrounding from photoproducts was studied in a third group of animals. The presence of peroxides was demonstrated in the serum of these animals after PDT with zinc phthalocyanine in liposomes (ZnPc-lip) using a total light dose of 450 J cm-2. This ex vivo observation supports the previously reported observations in vitro that the carrier complex is able to quench the photoproducts resulting from photoactivation of the photosensitiser which is present in the circulation.

In vivo photodynamic effects of phthalocyanines in a skin-fold observation chamber model: role of central metal ion and degree of sulfonation.

Six sulfonated metallophthalocyanines, chelated with either aluminum or zinc and sulfonated to different degrees, were studied in vivo for their photodynamic activity in a rat skin-fold chamber model. The chamber, located on the back of female WAG/Rij rats, contained a syngeneic mammary carcinoma implanted into a layer of subcutaneous tissue. Twenty-four hours after intravenous administration of 2.5 mumol/kg of one of the dyes, the chambers received a treatment light dose of 600 J/cm2 with monochromatic light of 675 nm at a power density of 100 mW/cm2. During light delivery and up to a period of 7 days after treatment, vascular effects of tumor and normal tissue were scored. Tumor cell viability was determined by histology and by reimplantation of the chamber contents into the skin of the same animal, either 2 h after treatment or after the 7 day observation period. Vascular effects of both tumor and subcutaneous tissue were strongest with dyes with the lowest degree of sulfonation and decreased with increasing degree of sulfonation. Tumor regrowth did not occur with aluminum phthalocyanine mono- and disulfonate and with zinc phthalocyanine monosulfonate. With the protocol that was used, complete necrosis without recovery was only observed when reimplantation took place at the end of the 7 day follow-up period. Reimplantation 2 h after treatment always resulted in tumor regrowth. At this interval, the presence of viable tumor cells was confirmed histologically. In general tumor tissue vasculature was more susceptible to photodynamic damage than vasculature of the normal tissue. The effect on the circulation of both tumor and normal tissue increased with decreasing degree of sulfonation.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo fluorescence kinetics of phthalocyanines in a skin-fold observation chamber model: role of central metal ion and degree of sulfonation.

The fluorescence pharmacokinetics of a series of metallosulfophthalocyanines, chelated with either aluminum or zinc and sulfonated to different degrees, was studied by fluorescence measurements in vivo. Dyes were administered systemically to female WAG/RIJ rats with an isogeneic mammary carcinoma transplanted into the subcutis in a transparent observation chamber located on their backs. Following an intravenous injection of 2.5 mumol/kg of the dye, fluorescence dynamics was observed up to 7 h postinjection. The phthalocyanines were excited at 610 nm with a power density of 0.1 mW/cm2 without causing photodynamic damage to the vasculature. Fluorescence was detected above 665 nm using a fluorescence imaging system based on an image intensifier. Dye retention in the blood vessels and tumor tissue was expressed as ratios relative to the fluorescence signal of the surrounding subcutaneous tissue. Phthalocyanines chelated with aluminum gave the highest fluorescence signal with tumor-over-subcutis ratios of up to a value of 4. The zinc complexes exhibited the highest vascular-over-subcutis ratios with maximum values exceeding a value of 6. They also displayed the longest retention times in the vascular system of well over 7 h. Overall, decreasing the degree of sulfonation of the metallophthalocyanines results in lower tumor-over-normal tissue fluorescence ratios, and furthermore aluminum-based dyes seem superior tumor localizers over zinc-based dyes. The advantages of phthalocyanines over porphyrins with respect to tumor localization and photodynamic therapy are discussed.

Measurement of the humoral immune response against Streptococcus pneumoniae type 14-derived antigens by an ELISA and ELISPOT assay based on biotin-avidin technology.

A Streptococcus pneumoniae type 14-specific ELISA and ELISPOT assay have been developed based on the use of biotinylated type 14 capsular polysaccharide (S14PS-biotin). A major advantage of this application over other methods is the use of 10-100-fold less antigen than that reported in the literature for other similar assays. Moreover, the prepared biotinylated polysaccharides are very stable and it is possible to use the same procedures for other pneumococcal polysaccharide antigens (e.g., S6BPS) with no major changes necessary in the ELISA and ELISPOT protocols. Furthermore, a simple thin layer chromatography method has been developed as a method for quality control of the biotinylated polysaccharide. Immunization with the thymus-independent antigen S14PS resulted in the induction of IgM spot-forming cells (SFC) and antibodies while S14PS-protein conjugates induced a thymus-dependent response. The immune response to the conjugates was enhanced by the addition of the adjuvant Quil A resulting in high levels of both IgG SFC and antibodies at day 14 after immunization. The developed assays are reliable and reproducible tools for studying the humoral immune response against Streptococcus pneumoniae type 14 capsular polysaccharide derived antigens.

Modulation of the immune response to pneumococcal type 14 capsular polysaccharide-protein conjugates by the adjuvant Quil A depends on the properties of the conjugates.

Streptococcus pneumoniae type 14 capsular polysaccharide-bovine serum albumin (S14PS-BSA) conjugates were prepared by water-soluble-carbodiimide-mediated condensation with or without the use of N-hydroxy-sulfosuccinimide. The immunogenicities of the capsular polysaccharide (S14PS) and of the conjugates were studied in (CBA/N x BALB/c)F1 mice and in female BALB/c mice. The response in these mice indicates that S14PS could be classified as a thymus-independent type 2 antigen. Coupling of S14PS to BSA improved the immunogenicity of this polysaccharide, and an immunoglobulin G memory response was evoked. Conjugation with N-hydroxysulfosuccinimide resulted in a product with a higher polysaccharide/protein ratio. This conjugate induced a greater immune response than did the classical conjugate. Quil A enhanced the immune response to S14PS and to most S14PS-BSA conjugates. The enhancement of the immune response to the conjugates seemed to depend on the coupling procedure. Our results indicate that for the construction of immunostimulating complexes based on polysaccharide or oligosaccharide-protein conjugates, attention should be paid to the degree of cross-linking of the antigens involved.

Late effects in the central nervous system. A study of biochemical alterations after local exposure of the rat brain to 2 krd.

The brain area of female rats three months of age was exposed to 2 krd of X-rays, and various biochemical parameters were retermined as well as NAD(H) in vivo fluorescence of the brain surface after time intervals from one day to 18 months. During the early period, an increase in the uptake of alpha-aminobutyrate (AIB) and a temporary depression in beta-glucuronidase and cathepsin activity followed by an activation at one month was seen. Somewhat later, acid phosphatase increases. During the intermediate period, DNA and serotonin content and AIB uptake by brain increase, whereas AIB uptake by heart and muscle decreases. A fall in sialic acid content is also noted at this time. During the late phase collagen increases, AIB uptake by brain and liver decreases. No changes were found with respect to NAD(H) fluorescence and its response to breathing of low oxygen concentrations.