Hematoporphyrin ethers--III. Cellular uptake and photosensitizing properties

1. The cellular uptake and the efficiency in sensitizing cells to photoinactivation were determined for hematoporphyrin (Hp) diphenyl ether, Hp dicyclohexyl ether and Hp dihexyl ether. 2. The phenyl diether was taken up by the cells to the same degree as was the clinically used porphyrin preparation photofrin II, while the dihexyl and notably the dicyclohexyl ether were taken up 3-4 times better. 3. Furthermore, the quantum yields for photoinactivation of cells were similar for the three diethers and twice as large as that for photofrin II. 4. Fluorescence- and absorption spectroscopy indicate that these findings are related to the fact that photofrin II is much more aggregated in the cells than are the three Hp diethers. 5. When cells loaded with the porphyrins are incubated with porphyrin-free medium containing serum a certain percentage of the cell-bound drug is removed: 14% for photofrin II, 28% for Hp diphenyl ether, 50% for Hp dicyclohexyl ether and 20% for Hp dihexyl ether. 6. With respect to cell uptake and retention of the dyes, the data did not show any uniform relationship to the polarity of the drugs, in contrast to what has been found earlier for Hp diethers of linear hydrocarbons.     

Tumor localization of newly developed hematoporphyrin (DHP) using a bladder tumor model: a novel hematoporphyrin derivative

Application of laser irradiation with porphyrin(s) or their derivatives for the destruction of tumors in humans requires preliminary studies of their localization in normal and malignant tissues. A novel derivative of hematoporphyrin (HP) was prepared. The newly developed hematoporphyrin (DHP) was administered to Fisher rats with bladder tumors and showed greater accumulation in the tumoral tissues. Comparative data on (HP) and (DHP) are presented and discussed in light of the enhanced tumor porphyrin uptake caused by these agents. The homogeneous intense fluorescence noted with DHP-treated animals suggests that total tumor kill curative therapy will be more feasible. The study paves the way to refining increased porphyrin augment phototherapy and laser application in the field of oncology.     

Formation of metal complexes of tumor-localizing porphyrins

Whereas the tumor localizer and photosensitizer hematoporphyrin derivative (Hpd) has its fluorescence emission maximum at 610-630 nm, several authors have reported that in aqueous solutions of hematoporphyrin (Hp) and Hpd, or in tumors after an injection of Hpd, a compound is formed which has its fluorescence emission maximum at 570-590 nm. This work (HPLC and fluorescence analysis) indicates that this peak is due to the formation of Zn-porphyrins either in vitro or in vivo. Cu- and Co-porphyrins may be formed as well, from traces of these metallic ions. In contrast to free porphyrins and Zn-porphyrins the latter complexes are non-fluorescent and do not act as photosensitizers.     

The effect of solution electrolytes on the uptake of photosensitizers by liposomal membranes: a salting-out effect

In this study we investigated, spectroscopically, the effect of electrolytes on the partitioning of hematoporphyrin IX (HP) and hypericin (Hy) into non-charged lipid vesicles. Our aim was to assess the salting-out effect of electrolytes on membrane-partitioning. We titrated aqueous solutions of HP and Hy with lecithin liposomes, at different concentrations of several monovalent and divalent electrolytes in the suspension. The partitioning constant of HP to lecithin liposomes increased from 3.3 (mL/mg) in water containing only 5mM buffer to 8.7 (mL/mg) at 0.36M KCl. KF had a similar effect. NaCl caused a 3-fold increase in the partitioning of Hy to liposomes. MgSO(4) and MgCl(2) also increased the partitioning of HP, by a factor of more than 4 and this occurred already at 0.03M concentration. We analyze the comparative effects of the electrolytes in relation to the Hofmeister series. The salting-out effect could be utilized to enhance the uptake of HP and Hy, and possibly other photosensitizers as well, by artificial and natural membranes.     

Hematoporphyrin diethers--V. Plasma protein binding and photosensitizing efficiency

1. Binding of added hematoporphyrin (HP) ethers to human plasma proteins and lipoproteins has been investigated by ultracentrifugation. 2. The binding to low density lipoproteins (LDL) has been discussed in terms of photosensitized tumor growth delay of tumors and HPLC-retention time, i.e. degree of polarity. 3. The LDL-binding data show a uniform relationship to sensitizing efficiency and degree of polarity, the only exception being HP-diamyl ether. No such uniform relationship exists for less related dyes, such as HP, tetraphenylporphyrin tetrasulfonate and HP-dimethyl ether.     

Functional role of soluble mitochondrial ATPase subunits.

A preparation of soluble mitochondrial ATPase (coupling factor F1) containing no gamma and delta minor subunits has been isolated. The minor-subunits-deficient F1 was found to be competent in ATP hydrolysis. However, it did not demonstrate a "coupling" effect in EDTA-submitochondrial particles. A portion of the ATPase activity of EDTA particles, stimulated by the minor-subunits-deficient F1, was insensitive to oligomycin. ATPase activity of Na+-particles was changed only slightly by this F1. It is suggested that gamma and delta subunits are necessary to form specific contacts between the F1 molecule and components of the mitochrondrial membrane.     

Volumetric properties for the monosaccharide (D-xylose, D-arabinose, D-glucose, D-galactose)-NaCl-water systems at 298.15 K

Densities have been measured for monosaccharide (D-xylose, D-arabinose, D-glucose and D-galactose)-NaCl-water solutions at 298.15 K. These data have been used to determine the apparent molar volumes of these saccharides and NaCl in the studied solutions. Infinite-dilution apparent molar volumes for the saccharides (V0(phi,S)) in aqueous NaCl and those for NaCl (V0(phi,E)) in aqueous saccharide solutions have been evaluated, together with the standard transfer volumes of the saccharides (delta(t) V0S) from water to aqueous NaCl and of NaCl (delta(t) V0E) from water to aqueous saccharide solutions. It is shown that the delta(t) V0S and delta (t) V0E values are positive and increase with increasing co-solute molalities. Volumetric parameters indicating the interactions of NaCl with saccharides in water have been obtained, respectively, by using transfer volumes of the saccharides and NaCl, and the resulting values are in good agreement with each other within experimental error. The interactions between saccharides and NaCl are discussed in terms of the structural interaction model and the stereochemistry of the saccharide molecules in water.     

Spectrofluorimetric study of porphyrin incorporation into membrane models--evidence for pH effects

The effects of hydrophobicity and charges of dicarboxylic porphyrins upon their interactions with membrane model systems are investigated. Four protonation steps are evidenced from fluorescence emission studies of hematoporphyrin IX and its more hydrophobic parent compound lacking of alcoholic chain, deuteroporphyrin IX. They are attributed to the successive protonations of the inner nitrogens of the porphyrin cycle (pK = 4.7 and 2.9 for hematoporphyrin and 4.4 and 2.7 for deuteroporphyrin) and successive deprotonations of propionic groups (pK approximately equal to 5.0 and 5.5 for hematoporphyrin and 5.4 and 6.0 for deuteroporphyrin). These porphyrins, as well as their dimethyl ester forms, are shown to incorporate as monomers into the hydrophobic bilayer of egg phosphatidylcholine small unilamellar vesicles, although the esterified forms are highly aggregated in aqueous solutions. In the case of the non-esterified forms, the incorporation of the porphyrins into the lipidic bilayer is reversible and strongly pH-dependent. A theoretical model is presented which takes into account the various protonation steps and the partition equilibria of the porphyrin between the vesicle lipidic phase and the water medium. The neutral form of the porphyrin (i.e., carboxylic groups protonated) presents the higher affinity, with constants of K approximately equal to 2 X 10(5) and K approximately equal to 6 X 10(6) M-1 (relative to lipid concentration) for hematoporphyrin and deuteroporphyrin, respectively. Protonation of one inner nitrogen leading to the monocationic form is sufficient to prevent incorporation into the hydrophobic bilayer. On the other hand, deprotonation of the peripheral propionic chains leading to anionic forms is less effective. These interactions between vesicles and porphyrins lead to shifts of the apparent pK of nitrogens and carboxylic groups, the latter one being now in the range of physiological pH. These results are discussed with regards to the hypothesis of a possible role of pH in the preferential uptake of porphyrins by tumors.     

The effect of hematoporphyrin derivative and human erythrocyte ghost encapsulated hematoporphyrin derivative on a mouse myeloma cell line

The effect of hematoporphyrin derivative (HPD) in combination with HeNe laser radiation on a mouse myeloma cell line, P3X63Ag8U1, has been examined. The effects of varying doses of radiation and photosensitizer were assessed using both dye exclusion and clonogenic assay systems. Development of photosensitivity by those cells in the presence of hematoporphyrin (HP) and HPD has been examined. In addition, with a view towards assessing erythrocyte encapsulation as a vehicle for an immunotargeting system for HPD, the effect of HPD encapsulated in such a manner, on the mouse myeloma cell line has been examined. From the results obtained from these in vitro experiments the authors believe that this may be a convenient means of packaging HPD for antibody-mediated delivery to tumour cells.     

Spectrofluorimetric study of porphyrin incorporation into membrane models - evidence for pH effects

The effects of hydrophobicity and charges of dicarboxylic porphyrins upon their interactions with membrane model systems are investigated. Four protonation steps are evidenced from fluorescence emission studies of hematoporphyrin IX and its more hydrophobic parent compound lacking of alcoholic chain, deuteroporphyrin IX. They are attributed to the successive protonations of the inner nitrogens of the porphyrin cycle (pK = 4.7 and 2.9 for hematoporphyrin and 4.4 and 2.7 for deuteroporphyrin) and successive deprotonations of propionic groups (pK ≈ 5.0 and 5.5 for hematoporphyrin and 5.4 and 6.0 for deuteroporphyrin). These porphyrins, as well as their dimethyl ester forms, are shown to incorporate as monomers into the hydrophobic bilayer of egg phosphatidylcholine small unilamellar vesicles, although the esterified forms are highly aggregated in aqueous solutions. In the case of the non-esterified forms, the incorporation of the porphyrins into the lipidic bilayer is reversible and strongly pH-dependent. A theoretical model is presented which takes into account the various protonation steps and the partition equilibria of the porphyrin between the vesicle lipidic phase and the water medium. The neutral form of the porphyrin (i.e., carboxylic groups protonated) presents the higher affinity, with constants of K ≈ 2 · 10⁵ and K ≈ 6 · 10⁶ M⁻¹ (relative to lipid concentration) for hematoporphyrin and deuteroporphyrin, respectively. Protonation of one inner nitrogen leading to the monocationic form is sufficient to prevent incorporation into the hydrophobic bilayer. On the other hand, deprotonation of the peripheral propionic chains leading to anionic forms is less effective. These interactions between vesicles and porphyrins lead to shifts of the apparent pK of nitrogens and carboxylic groups, the latter one being now in the range of physiological pH. These results are discussed with regards to the hypothesis of a possible role of pH in the preferential uptake of porphyrins by tumors.     

31P NMR of Mg-ATP in dilute solutions: complexation and exchange.

1. Monovalent-cation [(CH3)4N+, K(I), Na(I)] ATP, 1 mM in nucleotide, in aqueous solutions at pH 7.2, 24 degrees C, generates 2 different 31P NMR spectra, depending upon the salt content of the solution. At salt concentrations below 10 mM, the 31P NMR signals are chemically-shifted upfield (Na salt: alpha, -11.44 delta; beta, -22.91 delta; gamma, -8.36 delta) and the beta- and gamma-groups are broadened (at half-height: alpha, 3.5 Hz; beta, 9.6 Hz; gamma, 69 Hz). Above 10 mM salt, the signals are shifted downfield and are narrow (Na salt: alpha, -11.09 delta, 1.9 Hz; beta, -21.75 delta, 3.3 Hz; gamma, -6.30 delta, 3.9 Hz). 2. The Na-Mg-ATP complex, corresponding to the composition Na6Mg1ATP2, yields a single set of 31P resonances at concentrations of nucleotide of 100 mM, that upon dilution to 0.2 mM, resolve into 2 sets of ATP resonances characterized by low-field and high-field beta- and gamma-group resonance pairs. This set of ATP resonances, in contrast to the resonance set at 100 mM ATP, are broad (100 mM in ATP: alpha, -10.7 delta, 3.7 Hz; beta, -20.1 delta, 15 Hz; gamma, -5.7 delta, 7.3 Hz. 0.2 mM in ATP: alpha, -10.7 delta, 47 Hz; beta, -18.8 and -21.6 delta, 316 and 274 Hz; gamma, -5.5 and -8.7 delta, 460 and 374 Hz). 3. This new data, in combination with data derived from a survey of metal-ion-ATP studies, are interpreted in terms of ATP dimers, incorporating 2 molecules of ATP and 2 metal cations, that exist in water under the physiological conditions of neutral pH, high salt content [135 mM K(I)] and ATP concentrations in the range of 3 mM. 4. A compilation of 31P in vivo and ex vivo data compared to a reference Mg-ATP chemical shift vs Mg/ATP ratio plot indicates that ATP is not fully Mg-saturated in living systems and that 41% exists as the Mg(ATP)2 complex.     

Depletion of IL-10- and TGF-beta-producing regulatory gamma delta T cells by administering a daunomycin-conjugated specific monoclonal antibody in early tumor lesions augments the activity of CTLs and NK cells.

It has been demonstrated that gamma delta T cells accumulating in early tumor lesions and those purified from spleen cells of tumor-bearing mice attenuate the activity of CTLs and NK cells. We, therefore, investigated whether depletion of gamma delta T cells from early lesions of tumors results in restoration of CTL and NK cell activities and subsequent regression of tumors. A daunomycin-conjugated anti-gamma delta TCR mAb UC7-13D5 (Dau-UC7) was prepared to efficiently deplete gamma delta T cells. An in vitro study revealed that Dau-UC7 specifically lysed gamma delta TCR+ cells and effectively inhibited splenic gamma delta T cells from tumor-bearing mice to produce cytotoxic cell-suppressive factors. Furthermore, intralesional injections of Dau-UC7 at an early stage of tumor development led to augmentation of tumor-specific CTL as well as NK cell activities and to the resultant regression or growth inhibition of the tumors. On analysis of cytokine profile, gamma delta T cells transcribed mRNAs for IL-10 and TGF-beta, but not IL-4 or IFN-gamma, suggesting the T regulatory 1-like phenotype. Finally, a blocking study with mAbs showed that the inhibitory action of gamma delta T cells on CTLs and NK cells was at least partly mediated by IL-10 and TGF-beta. These results clearly demonstrated the novel mechanism by which T regulatory 1-like gamma delta T cells suppress anti-tumor CTL and NK activities by their regulatory cytokines in early tumor formation.     

Photofrin Based Photodynamic Therapy and miR-99a Transfection Inhibited FGFR3 and PI3K/Akt Signaling Mechanisms to Control Growth of Human Glioblastoma In Vitro and In Vivo

Glioblastoma is the most common malignant brain tumor in humans. We explored the molecular mechanisms how the efficacy of photofrin based photodynamic therapy (PDT) was enhanced by miR-99a transfection in human glioblastoma cells. Our results showed almost similar uptake of photofrin after 24 h in different glioblastoma cells, but p53 wild-type cells were more sensitive to radiation and photofrin doses than p53 mutant cells. Photofrin based PDT induced apoptosis, inhibited cell invasion, prevented angiogenic network formation, and promoted DNA fragmentation and laddering in U87MG and U118MG cells harvoring p53 wild-type. Western blotting showed that photofrin based PDT was efficient to block the angiogenesis and cell survival pathways. Further, photofrin based PDT followed by miR-99a transfection dramatically increased miR-99a expression and also increased apoptosis in glioblastoma cell cultures and drastically reduced tumor growth in athymic nude mice, due to down regulation of fibroblast growth factor receptor 3 (FGFR3) and PI3K/Akt signaling mechanisms leading to inhibition of cell proliferation and induction of molecular mechanisms of apoptosis. Therefore, our results indicated that the anti-tumor effects of photofrin based PDT was strongly augmented by miR-99a overexpression and this novel combination therapeutic strategy could be used for controlling growth of human p53 wild-type glioblastomas both in vitro and in vivo.     

Analysis of the ATPase subassembly which initiates processive DNA synthesis by DNA polymerase III holoenzyme.

The gamma complex (gamma delta delta' chi psi) subassembly of DNA polymerase III holoenzyme transfers the beta subunit onto primed DNA in a reaction which requires ATP hydrolysis. Once on DNA, beta is a "sliding clamp" which tethers the polymerase to DNA for highly processive synthesis. We have examined beta and the gamma complex to identify which subunit(s) hydrolyzes ATP. We find the gamma complex is a DNA dependent ATPase. The beta subunit, which lacks ATPase activity, enhances the gamma complex ATPase when primed DNA is used as an effector. Hence, the gamma complex recognizes DNA and couples ATP hydrolysis to clamp beta onto primed DNA. Study of gamma complex subunits showed no single subunit contained significant ATPase activity. However, the heterodimers, gamma delta and gamma delta', were both DNA-dependent ATPases. Only the gamma delta ATPase was stimulated by beta and was functional in transferring the beta from solution to primed DNA. Similarity in ATPase activity of DNA polymerase III holoenzyme accessory proteins to accessory proteins of phage T4 DNA polymerase and mammalian DNA polymerase delta suggests the basic strategy of chromosome duplication has been conserved throughout evolution.     

Functional and morphologic characterization of human T lymphocytes expressing the TCR gamma /delta

A minor subset of T lymphocytes express a TCR composed of gamma and delta chains. This subset differs from conventional T cells for a number of phenotypic and functional characteristics. TCR gamma/delta+ cells simultaneously lack both CD4 and CD8 antigens. Cloning of CD4-8- peripheral blood lymphocytes, under limiting dilution conditions, revealed that they are homogeneously composed of cytolytic cells which efficiently lyse tumor target cells. Formal proofs have been provided that TCR gamma/delta+ cells are able to recognize antigens. For example, they proliferated in response to allogeneic mixed lymphocyte culture (MLC); in addition, MLC-derived TCR gamma/delta+ cells specifically lysed PHA-induced blast cells bearing the stimulating alloantigens. The selection of monoclonal antibodies specific for TCR gamma/delta molecules allowed to identify two distinct subsets of TCR gamma/delta+ cells. Both of these mABs, termed BB3 and delta TCS-1 respectively, induced specific activation of cloned cells expressing the corresponding antigenic determinants (as assessed by measurements of intracellular Ca++ and/or lymphokine production or cytolytic activity). Analysis of the distribution of subsets expressing different forms of TCR gamma/delta, showed that the BB3-reactive form is prevalent in the peripheral blood. In contrast, delta-TCS-1-reactive cells are relatively unfrequent in peripheral blood but represent the majority of TCR gamma/delta+ cells in tissues.     

Isolation of enzymes from mixed reversed micelles of surface-active agents

The catalytic activities of lysozyme, horseradish peroxidase (HP), catalase, glucose-6-phosphate dehydrogenase (G6PDH) and lactate dehydrogenase (LDH) were studied in aqueous solutions and after isolation of the enzymes from mixed reversed micelles of Aerosol OT and Triton X-45 by organic solvents (acetone, ethanol, isopropanol), by acetone-water mixtures, as well as by aqueous solutions containing urea, glycerol, polyethylene glycol 6000 and ammonium sulphate. The isolation conditions were found for catalase with retaining all the activity and for HP and lysozyme with retaining 72 and 84% of the catalytic activity, respectively. The G6PDH isolation from micelles by aqueous solutions of urea (6%) and glycerol (10%) resulted in retaining only 43% of the enzyme activity and led to almost complete inactivation of LDH. Stability of the enzymes after their entrapment in micelles and isolation from those is compared with thermostability of the same enzymes in aqueous solutions.     

Suppression of cytotoxic T lymphocyte activity by γ/δ T cells in tumor-bearing mice

Spleen cells derived from tumor-bearing mice prove useful for the elucidation of the mechanism determining how tumor cells evade cytotoxic T lymphocytes (CTL) in tumor-bearing hosts. Our data indicate that inactive CTL or precursor CTL specific for tumor antigens are present among lymphocytes of tumor-bearing mice. However, their activity is inhibited by a soluble factor produced by other cells present in the same source. Inhibition of the cytolytic reaction was also detected in the culture supernatant of spleen cells obtained from normal mice, precultured in the presence of tumor cell culture supernatant and interleukin-2 (IL-2). Cell-depletion and cell-purification studies let us conclude that cells that produced the CTL-inhibitory factor (CTL-IF) were / T cells. The / T cells that were activated in vivo in tumor bearers were able to produce CTL-IF after isolation and in vitro culture. Maximum activation of / T cells was achieved by antigenic stimulation and by suppression of cells that interfered with the activation of / T cells. CTL-IF, which was assayed by use of CTL clones, did not show antigen specificity. Inhibition depended on a relatively heat- and acidstable, but alkali-labile molecule with a molecular mass of less than 10 kDa. The latter characteristics imply that CTL-IF does not resemble any of the known lymphokines produced by / T cells. These observations emphasize the crucial role of the / T cells in the escape of tumor cells from the attack of tumorspecific CTL.