Laura Polo

Low-density lipoprotein receptors in the uptake of tumour photosensitizers by human and rat transformed fibroblasts

Low-density lipoproteins (LDL) increase the selectivity of tumour targeting by drugs, including sensitisers for photodynamic therapy, because of the enhanced expression of specific LDL receptors in many types of transformed as compared with non-transformed cells. This investigation aims at gaining more information on the role of LDL receptors in the accumulation of photosensitizer-LDL complexes by human and rat transformed fibroblasts, and the interference of the photosensitizer with LDL recognition by the specific receptors. Both an amphiphilic hematoporphyrin IX (Hp) and a hydrophobic Zn(II)-phthalocyanine (ZnPc) photosensitizers bind to human LDL with molar ratios of 5-6:1 and 10-12:1, respectively. The hematoporphyrin-LDL complex is accumulated by human HT1080 fibroblasts mainly through the high affinity LDL receptors, while the Zn-phthalocyanine-LDL complex is internalised through non specific endocytosis because of changes in the apoB LDL structure induced by phthalocyanine association, as suggested by spectroscopic studies. The uptake of LDL-delivered hematoporphyrin, but not Zn-phthalocyanine, is about 4-fold higher in HT1080 cells stimulated for maximal expression of LDL receptors as compared with non-stimulated cells. This difference is abolished by LDL acetylation. Human LDL-bound hematoporphyrin and Zn-phthalocyanine are up taken by stimulated and non-stimulated 4R rat fibroblasts with similar efficiency. Scatchard plot analysis of human (125)I-LDL binding to 4R cells shows the presence of only low affinity receptors while 350,000 high affinity receptors are expressed per HT1080 cell. It is concluded that a careful evaluation of the lack of conformational changes of LDL is critical for guaranteeing the selectivity and efficiency of photosensitizer delivery to tumour cells.

Polylysine–porphycene conjugates as efficient photosensitizers for the inactivation of microbial pathogens

Porphycenes are electronic isomers of porphyrins which, when neutral, display no appreciable photosensitizing action towards Gram-negative bacteria. The covalent binding of oligomeric polylysine moieties, which are cationic at physiological pH values, endows porphycenes with a significant phototoxic activity against Gram-negative bacteria while retaining their photoefficiency against a variety of microbial pathogens, including Gram-positive bacteria, fungi and mycoplasmas. The effect of the polylysine moiety is dependent on both the polylysine concentration and the degree of oligomerization. A suitable interplay among the various parameters opens the possibility to obtain either a broad spectrum of antimicrobial activity or a selective action toward a specific pathogen while minimizing the damage to human fibroblasts.

Novel approaches towards a detailed control of the mechanism and efficiency of photosensitized processes in vivo

Abstract The photoexcitation of an endogenous or exogenous biological chromophore can result in the re-emission of light as fluorescence or phosphorescence; this may be used for photodiagnostic applications or in the conversion of at least part of the absorbed radiation into thermal or chemical energy. The last two processes generate photothermal and photochemical sensitization respectively leading to the irreversible damage of cells and tissues; hence they can be used for phototherapeutic purposes. The efficacy and scope of photodiagnostic and phototherapeutic techniques are improving as we increase our knowledge about the influence of biological microenvironments on the mechanisms of photoprocesses and as new approaches are developed for controlling the biodistribution of photosensitizing agents in vivo and the depth/diffusion of incident light into tissues. In particular, even the localized and sublethal damage of cells can often be modulated to generate intracellular signalling in order to either induce a programmed cell death (apoptosis) or an acceleration of cell metabolism (photostimulation).

The distribution of the tumour photosensitizers Zn(II)-phthalocyanine and Sn(IV)-etiopurpurin among rabbit plasma proteins.

Zn-phthalocyanine (ZnPc) and Sn-etiopurpurin (SnET2) incorporated in unilamellar liposomes or solubilized in a Cremophor-EL emulsion have been incubated in vitro with rabbit plasma or intravenously administered to rabbits. Ultracentrifugation and chromatographic analysis of the plasma showed that ZnPc and SnET2 are mainly released to lipoproteins; within the lipoprotein family, both dyes are preferentially bound by low-density (LDL) and high-density (HDL) lipoproteins. The amount of dye bound with these two lipoprotein classes was related to their relative concentration in the plasma; in most cases a larger amount of photosensitizer was bound to HDL as compared to LDL on a protein concentration basis.

Effect of the delivery system on the biodistribution of Ge(IV) octabutoxy-phthalocyanines in tumour-bearing mice

The pharmacokinetic properties of the Ge(IV)-octabutoxy-phthalocyanines (GePc) with two axially ligated triethylsiloxy (GePcEt) or trihexyl-siloxy (GePcHex) chains were studied in BALB/C mice bearing a transplanted MS-2 fibrosarcoma. The GePcs were delivered to mice after incorporation into unilamellar liposomes of dipalmitoyl phosphatidylcholine (DPPC) or in an emulsion of Cremophor-EL. The Cremophor delivered GePcs were cleared from the blood circulation at a much slower rate than the liposome-delivered GePcs. At the same time, Cremophor induced a slower and reduced uptake of the GePcs in the liver and spleen while it greatly enhanced the uptake in the tumour as compared to liposomes. Maximum tumour uptake was observed at 24 h post-injection and was equivalent to 0.67 and 0.50 nmol/g, respectively, for the Cremophor delivered GePcHex and GePcEt. The corresponding values for the liposome-delivered drugs were approximately one fourth of that observed with Cremophor.

The effect of different liposomal formulations on the interaction of Zn(II)-phthalocyanine with isolated low and high density lipoproteins

Several phthalocyanines exhibit a high affinity for tumour tissues and upon red-light irradiation originate a photosensitized process leading to tumour necrosis. This study was designed to define the role of different liposomal formulations in modulating the affinity of Zn(II)-phthalocyanine (ZnPc) for isolated low- and high-density lipoproteins (LDL, HDL). This information is important as the uptake of hydrophobic photosensitizers by tumour tissues can be enhanced by their association with LDL. The kinetics and efficiency of ZnPc association with LDL and HDL as well as the redistribution of ZnPc between the lipoproteins were studied by salt gradient ultracentrifugation and spectrophotometric analyses of the isolated lipoproteins. The formation of the photosensitizer-lipoprotein complexes in the plasma is affected by the vehicle utilized for the drug delivery. ZnPc in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-snglycero-3-L-serine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes is transferred to lipoproteins within a few minutes, while the transfer of ZnPc from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposomes reaches a steady state value in a time scale of 5–6 hr. The binding capacity of LDL for ZnPc can be as high as about 60 phthalocyanine molecules per protein particle, although the final value is affected by the phospholipid composition of the liposomes and the liposome/lipoprotein ratio. HDL have a lower binding capacity (max. about 3 ZnPc/protein) as shown by studies of interlipoprotein transfer of the photosensitizer. The present findings indicate that the association of photosensitizers with lipid-based delivery systems, besides being necessary for water-insoluble compounds, affects their distribution among lipoproteins. Liposomes which are in a fluid state at the temperature of 37°C enhance the binding of ZnPc to LDL, which should increase the selectivity of tumour targeting by the phthalocyanine owing to the efficient receptor-mediated endocytosis of LDL by several types of malignant cells.

UNUSUALLY HIGH AFFINITY OF Zn(II)‐TETRADIBENZOBARRELENOOCTABUTOXY‐ PHTHALOCYANINE FOR LOW‐DENSITY LIPOPROTEINS IN A TUMOR‐BEARING MOUSE

Abstract— An important factor in determining the efficacy of photosensitizing compounds in photodynamic therapy of tumors is the level to which tumors take up the photosensitizers after systemic injection. This parameter seems to be related to the transport modalities of the photosensitizer in the bloodstream. In this work the photosensitizer Zn(II)‐tetradibenzobarreleno‐octabutoxyphthalocyanine was shown to have an unprecedentedly high association with low‐density lipoproteins (71% of the phthalocyanine in the plasma) when delivered in Cremophor micelles to tumor‐bearing mice. This was accompanied by a particularly high tumor uptake at 24 h post‐injection.

Effects of low power laser-irradiation on differential blood count and body temperature in endotoxin-preimmunized rabbits

Low power laser irradiation has been shown to have various immune-modulatory effects under in vitro conditions but little is known about such effects in animal models. Escherichia coli endotoxin-preimmunized rabbits were used to determine the influence of transcutaneously applied low power laser light on differential blood count and rectal temperature. After three initial immunizations animals were either boostered with 5 ng/kg of endotoxin or injected with pyrogen-free saline and subsequently underwent irradiation using two different wavelengths of red laser light and sham irradiation, respectively. Differential blood count of laser-treated animals was characterized by significantly higher lymphocyte values and lower neutrophil values at twenty hours (boostered rabbits) and twenty-three hours (non-boostered rabbits) after irradiation. Differential blood cell counts returned to baseline values within 23 hours in the boostered animals, whereas in the non-boostered rabbits lymphocytes showed a trend to further increase. Recording of rectal temperature revealed a further rise after laser application, changes being of greater magnitude and longer duration in the non-boostered animals. These results seem to indicate that a single low power laser irradiation can modulate immune-responses depending on the immunological status of the organism.

Liposome-delivered 131I-labelled Zn(II)-phthalocyanine as a radiodiagnostic agent for tumours

131I-Zn(II)-phthalocyanine (ZnPc) incorporated into unilamellar liposomes has been systemically injected to mice bearing a transplanted MS-2 fibrosarcoma. Biodistribution studies show that the pharmacokinetic behaviour of 131I-ZnPc is very similar to that defined for the parent molecule ZnPc including a serum half-life of ca. 12 h, a high recovery from liver and spleen and minimal accumulation in kidney and brain. The most important pharmacokinetic parameter is represented by the high tumour/ muscle ratio of 131I-ZnPc concentration (ca. 9 at 24 h post-injection). These results suggest the possible use of the radiolabelled derivative for a real-time non-invasive monitoring of the ZnPc concentration in the tumour and peritumoural tissue during photodynamic therapy.

Phthalocyanines as phototherapeutic agents for tumors

Zn(II)-phthalocyanine (ZnPc) is a tetraazaisoindole pigment which can be prepared by chemical synthesis with a high degree of purity, and it efficiently absorbs 680 nm of light. These properties, associated with its ability to generate activated oxygen species (e.g., singlet oxygen) upon photoexcitation, make ZnPc a potential phototherapeutic agent. Actually, upon delivery of ZnPc to tumor-bearing animals after incorporation into liposomal vesicles, the dye was uptaken and retained by the tumor tissue in significant amounts and with a good degree of selectivity. Irradiation of the ZnPc-loaded tumor area with ca. 680 nm of light caused tumor necrosis to an extent which was related to the dye concentration in the tumor.