The effect of tissue and cellular pH on the selective biodistribution of porphyrin-type photochemotherapeutic agents: a volumetric titration study

Volumetric titration of aqueous solutions of haematoporphyrin IX (HP) yields two inflexion points, whereas four pK values can be obtained via mathematical fitting of the experimental data. Assignment of all pK protonation sites is made. A zwitterion is proposed as the neutral species of HP. Evaluation of the pK values of HP in the presence of sodium dodecyl sulphate (SDS) leads to a drastically altered ionic species distribution. On the basis of the distribution diagrams obtained, a pH-sensitive twofold mechanism is proposed for the selective biodistribution of porphyrin-type photochemotherapeutic agents, one involving tissue distribution and the other involving cell membrane penetration by the neutral zwitterion.     

Photosensitized processes in vivo: proposed phototherapeutic applications

The use of photosensitizing dyes having intense absorption bands in the 600-900 nm spectral interval opens up new prospects in the field of photochemotherapy, because it allows the illumination of relatively large tissue volumes with no significant damage to photosensitizer-free tissues. Special interest is currently focused on the photodynamic therapy of solid tumors, because of the property of several dyes with a macrocyclic chemical structure (porphyrins, chlorins, phthalocyanines, xanthenes) to accumulate in significant amounts and be retained for prolonged periods of time by neoplastic lesions. Strategies are being developed for enhancing the selectivity of tumor targeting by photosensitizers through the exploitation of functional or biochemical differences between normal and malignant cells.     

Temporally and spatially heterogeneous distribution of mTHPC in a murine tumor observed by two-color confocal fluorescence imaging and spectroscopy in a whole-mount model

Efficient intratumor delivery of anticancer drugs and photosensitizers is an important factor in the success of chemotherapy and photodynamic therapy, respectively. Unfortunately, their adequate and uniform intratumor distribution is impeded by several physiological barriers and by binding to tissue components. Measurement of gross tumor drug accumulation is a routine method of investigating the uptake and clearance of chemotherapy agents and photosensitizers but tells little about their extravascular spatial distribution. We use whole-mount two-color confocal fluorescence imaging and imaging spectroscopy of unprocessed excised murine tumor fragments to investigate the intratumor distribution of the photosensitizer meso-tetrahydroxyphenyl chlorin (mTHPC) as a function of distance from blood vessels perfused with 0.2 mum diameter fluorescent microspheres. Significant mismatches between drug and perfused vasculature are caused by heterogeneities in tumor blood supply. We describe complex microscopic mTHPC gradients that reverse dramatically relative to the perfused vasculature with time after injection. This imaging technique can be applied to screen the dynamic intratumor distribution of other fluorescent photosensitizers and anticancer drugs.     

pH-Dependent Chalcogenopyrylium Dyes as Potential Sensitizers for Photodynamic Therapy: Selective Retention in Tumors by Exploiting pH Differences between Tumor and Normal Tissue

Ideal photosensitizers have long-wavelength absorption and strong tumor selectivity with rapid clearance from normal tissues. The telluroselenopyrylium dye 1 that absorbs light at 795 nm (epsilon = 285,000 M-1 cm-1) has a novel property that enhances the tumor specificity and normal tissue clearance. After intralesional injection to both tumors and surrounding skin, it disappeared from the normal skin of BALB/c mice faster than it did from subcutaneously implanted Colon 26 tumors, which resulted in therapeutic selectivity. In vivo reflectance spectroscopy showed that the half-life in tumor was about 50 min while in skin it was around 12 min. This phenomenon appears to be related to the pH differences in normal skin versus tumor, because the rates of drug hydrolysis in solution were shown to be sensitive to changes in pH. Inhibition of tumor regrowth following intratumoral photosensitizer administration depended on both light dose and drug dose, as well as the time interval between dye injection and irradiation; selectivity depended on the time interval. Although treatment parameters were not optimized efficacy was superior to systemic Photofrin under our standard conditions. We discuss how new, more optimal, photosensitizers can be designed that use rates of hydrolysis to exploit the differences in pH between normal tissue and tumor.     

A Twist‐Assisted Biphenyl Photosensitizer Passable Through Glucose Channel

While the development of low‐molecular‐weight drugs is saturating, agents for photodynamic therapies (PDTs) may become alternative seeds in pharmaceutical industry. Among them, orally administrative, cancer‐selective, and side effect‐free photosensitizers (PSs) that can be activated by tissue‐penetrative near‐infrared (NIR) lights are strongly demanded. We discovered such a PS from scratch by focusing on a twist‐assisted spin‐orbit charge transfer intersystem crossing (ISC) mechanism in a biphenyl derivative, which was demonstrated by thorough photophysical studies. The unique ISC mechanism enables the PS to be small and slim so as to pass through glucose transporters and exert a PDT effect selectively on a cancer cell line. The smallness will allow for oral administration and fast clearance, which have been agenda of approved PSs with larger molecular weights. We also demonstrated that our PS was able to be activated with an NIR pulse laser through two‐photon excitation.     

Partition of rose bengal anion from aqueous medium into a lipophilic environment in the cell envelope of Salmonella typhimurium: implications for cell-type targeting in photodynamic therapy

Photodynamic therapy employs photosensitizers for the selective destruction of tumor tissue while sparing the surrounding healthy tissue. Photosensitization may also be applied to the selective eradication of microorganisms. Photosensitized inactivation requires that the sensitizer bind to the target and therefore the factors that determine photosensitizer binding are critical to photosensitization selectivity. This paper reports the determination of some features of the binding site of the potent photosensitizer, Rose Bengal, in Salmonella bacteria and describes some of the factors that affect this binding. The shift in the wavelength of maximum fluorescence and experiments with the fluorescence quencher TNBS indicate that Rose Bengal is located in a non-aqueous compartment such as the outer membrane. The dye does not seem to significantly accumulate inside the cell, but rather to accumulate in the outer membrane. Time-dependent changes in sensitizer localization in two strains of Salmonella typhimurium that differ in cell wall formation, LT-2 and TA1975, correspond to their differences in susceptibility to photosensitized killing. Therefore these results provide clues to the factors that determine photosensitization selectivity. Understanding this phenomenon is essential for the efficient design of selective photosensitizers and for optimizing antitumor and antiviral photodynamic therapy.     

蛋白质在离子交换色谱上选择性和非吸附特性

对蛋白质在离子交换柱上选择民性和非吸附特性进行了研究。蛋白质在有机磷酸锆阳离子色谱柱上,其保留作用随流动相ｐＨ值在离子强度的增加而减小;蛋白质在强阳离子和强阴离子色谱柱上的保留作用,即是流动相中的ｐＨ值等于蛋白质的等当点,其净电荷为零。不册蛋白质仍有不同程度的保留,这主要是由于蛋白质的三维结构使电荷 密度的大小和分布的不均匀以及离子交换填料表面性质的影响。     

Biodistribution of tritiated benzoporphyrin derivative (3H-BPD-MA), a new potent photosensitizer, in normal and tumor-bearing mice

The biodistribution of a new and very potent photosensitizer, benzoporphyrin derivative-monoacid, ring A (BPD-MA), was determined in normal and P815 (mastocytoma) or M1 (rhabdomyosarcoma) tumor-bearing DBA/2J mice. A dose of 80 micrograms of 3H-BPD-MA was determined at 3, 24, 48, 72, 96 and 168 h post injection. The following tissues were tested: blood, brain, heart, intestine, kidney, lung, liver, muscle, skin, stomach, spleen, thymus and tumor. The biodistribution of 3H-BPD-MA in normal and tumor-bearing mice was comparable overall. 3H-BPD-MA localized in tumors better than in other tissues except kidney, liver and spleen. The tumor to tissue ratios were in the range 1.5-3 at 24 h post injection and increased further during the next 72 h. The highest levels of 3H-BPD-MA were observed in all tissues at 3 h post injection and decreased rapidly during the first 24 h. After 24 h the clearance from tissues was rather slow. The preliminary clearance data obtained in a group of five normal mice indicated that the majority of the injected dose (60%) cleared from the body via the bile and feces, while only about 4% cleared via kidneys and urine. Studies in which 3H-BPD-MA was extracted from tumor, kidney and liver 3 and 24 h after injection showed that, at 3 h, all the photosensitizing activity in tumor was retained. At 24 h only 39% of the activity was retained and considerably less active material was present in liver and kidney.     

A NEW ELECTRONIC ABSORBANCE BAND IN CONCENTRATED AQUEOUS SOLUTIONS OF HEMATOPORPHYRIN IX DETECTED BY PHOTOACOUSTIC SPECTROSCOPY

Abstract— Photoacoustic (PA) spectroscopy reveals a new electronic transition in concentrated aqueous solutions of hematoporphyrin IX (Hp). This new band has a maximum near 440 nm. Its intensity is sensitive to pH, concentration, PA chopping frequency, and the presence of sodium dodecyl sulfate (SDS). Data presented support the assignment of this new band to a dimeric form of Hp. The pH effects on this new band are discussed in terms of their implications for the selective biodistribution of certain porphyrin-based photochemotherapeutic agents.     

pH,serum proteins and ionic strength influence the uptake of merocyanine 540 by WiDr cells and its interaction with membrane structures

It has been suggested that selective uptake of photosensitizers is due to significantly lower pH of the interstitial fluid in tumors compared to normal tissue. Therefore, the cellular uptake of merocyanine 540 (MC 540) was examined at two pH values: 6.8+/-0.1 and 7.4+/-0.1. There was no difference in spectral properties (absorption and fluorescence maxima positions, fluorescence intensity) of the drug in the presence of increasing amounts of either human blood plasma or FCS (0-2%) at the two pH values investigated. Nevertheless, significantly higher amounts of the drug were taken up by WiDr cells at pH 6.8+/-0.1, both in the presence of 10% FCS and in the absence of FCS. The absorption spectra of MC 540 in the presence of egg phosphatidylcholine (PC) liposomes turned out to be NaCl concentration-dependent (0.00-0.30 mol l(-1)). Membrane fluidity, as measured by fluorescence anisotropy of diphenylhexatriene (DPH), was unchanged within the experimental error in the NaCl concentration range 0.01-0.30 mol l(-1). The spectral changes indicated an enhancement of the incorporation of MC 540 into lipid membranes with increasing ionic strength. Such a salt concentration dependence suggests a possible involvement of the surface potential in the interaction of MC 540 with lipid membranes. The results might provide an explanation of the pH dependency of the cellular uptake of MC 540 observed in this study.     

Strategies for selective cancer photochemotherapy: antibody-targeted and selective carcinoma cell photolysis

A principle objective in chemotherapy is the development of modalities capable of selectively destroying malignant cells while sparing normal tissues. One new approach to selective photochemotherapy, antibody-targeted photolysis (ATPL) uses photosensitizers (PS) coupled to monoclonal antibodies (MAbs) which bind to cell surface antigens on malignant cells. Selective destruction of human T leukemia cells (HBP-ALL) was accomplished by coupling the efficient PS chlorin e(6) to an anti-T cell MAb using dextran carriers. Conjugates with chlorin: MAb ratios of 30:1 retained > 85% MA b binding activity, and had a quantum yield for singlet oxygen production of 0.7 +/- 0.1, the same as that of free chlorin e(6). Cell killing was dependent on the doses of both MAb-PS and 630-670 nm light and occurred only in target cell populations which bound the MAb. On the order of 10(10) singlet oxygen molecules were necessary to kill a cell. A second approach to specific photochemotherapy, selective carcinoma cell photolysis (SCCP), relies on preferential accumulation of certain cationic PS by carcinoma cell mitochondria. We have evaluated several classes of cationic dyes, and in the case of N,N'-bis-(2-ethyl-1,3-dioxolane)-kryptocyanine (EDKC) and some of its analogs, have demonstrated highly selective killing of human squamous cell, bladder and colon carcinoma cells in vitro. In isolated mitochondria, EDKC uptake and fluorescence depended on membrane potential, and the dye specifically photosensitized damage to Complex I in the electron transport chain. N,N'-bis-(2-ethyl-1,3-dioxolane)-kryptocyanine and some of its analogs accumulated within subcutaneous xenografts of human tumors in nude mice with tumor:skin ratios > 8. Photoirradiation caused significant inhibition of tumor growth, without cutaneous phototoxicity.     

Selective complex formation of saccharides with europium(III) and iron(III) ions at alkaline pH studied by ligand-exchange chromatography

At high pH, saccharides become negatively charged by deprotonation of one or several hydroxylic groups and they are highly and selectively retained by ligand-exchange chromatography. The systems consist of a sulphonated polystyrene strong cation exchanger in europium(III) or iron(III) form and sodium hydroxide as mobile phase. The degree of complex formation is dependent on solute character and concentration, metal ion and pH, the reaction being of second order as confirmed by breakthrough studies. Rapid desorption of the solutes is performed by the introduction of an acidic mobile phase. Monosaccharides, and especially sugar alcohols, are selectively retained by a column in Fe(III) form whereas all saccharides are strongly retained as Eu(III) complexes, e.g., the capacity factor for the breakthrough of 10 μM glucose, in 0.1 M NaOH as mobile phase, was ca. 3500 The systems are proposed to be highly selective for the analysis of sugars.     

SYSTEMIC IMMUNOSUPPRESSION INDUCED BY PHOTODYNAMIC THERAPY (PDT) IS ADOPTIVELY TRANSFERRED BY MACROPHAGES

Some derivatives of hematoporphyrins are strongly retained by tumor tissue as compared to normal tissue, and exposure of these photosensitizers to radiation in the visible spectrum can cause serious biological damage. These properties have been exploited in the development of a new treatment for cancer termed photodynamic therapy (PDT). However, recent studies have also demonstrated that PDT can also induce a state of systemic immunosuppression. The purpose of this study was to determine whether PDT-induced suppression of contact hypersensitivity (CHS) responses was an active phenomenon that could be adoptively transferred by viable splenocytes from PDT-treated mice. Although induction of adoptively transferable suppressor cells in PDT-treated mice required exposure to antigen, the suppressor cells were found to be antigen nonspecific in their function. Furthermore, splenocytes from PDT-treated mice were capable of generating levels of allospecific cytotoxic T lymphocyte (CTL) activity which were comparable to those generated by normal control mice, but the ability of irradiated spleen cells from PDT-treated mice to stimulate a mixed lymphocyte response (MLR) was dramatically impaired. Finally, chromatographic separation of T cells, B cells and macrophages showed that the cell type which mediates adoptively transferable suppression of CHS responsiveness is in the macrophage lineage.     

A dual-column HPLC method for the simultaneous determination of DHPG (9-[(1,3-dihydroxy-2-propoxy)methyl]guanine) and its mono and diesters in biological samples

A convenient method for the simultaneous determination of various DHPG species present in biological samples is presented. This method utilizes a cation exchange column (25 cm X 4.6 mm i.d.) coupled in series to a short reversed-phase column (5 cm X 4.6 mm i.d.). The mobile phase consists of methanol:0.005M ammonium phosphate buffer, pH 2.5. There is a large polarity difference between DHPG and its esters due to the non-polar side chain of the ester moiety. The simultaneous determination of the diesters, monoesters, and DHPG in these samples using only the cation exchange or the reversed-phase column is not possible without time-consuming gradient elution. In the reversed-phase mode alone, the esters are highly retained relative to DHPG, whereas the esters are only slightly retained on a cation exchange column and are insensitive to changes in pH and ionic strength of the mobile phase. However, a combination of these two columns provides interesting selectivity for these compounds and offers a unique way of controlling the retention times of these species relative to each other. The retention time of esters can be selectively altered (with respect to DHPG) by changing the composition of methanol in the mobile phase. In contrast, the retention time of DHPG is controlled by changing the buffer strength and pH of the mobile phase.     

SITES OF PHOTODAMAGE in vivo and in vitro BY A CATIONIC PORPHYRIN

Abstract— Localization and photodynamic efficacy of a monocationic porphyrin (MCP) were assessed using murine leukemia cells in culture. This sensitizer localized at surface membrane loci and catalyzed selective photodamage to membrane structures. Although both cationic and hydrophobic, this porphyrin was not recognized by the multidrug transporter, which excludes many cationic agents from cells that express multidrug resistance. Photodynamic studies with the murine radiation-induced fibrosarcoma tumor model indicated moderate photosensitization of neoplastic lesions in vivo at 3 h, but not at 24 h after sensitizer administration. Pharmacokinetic studies indicate that plasma levels, not tissue levels were the major determinant of photodynamic therapy (PDT) response. Consistent with this observation, vascular damage and disturbances of tissue perfusion followed PDT. These effects were more pronounced in tumor-bearing skin than in normal skin. The therapeutic response to MCP appeared to be related mainly to secondary, probably vascular, effects.     

A novel pH-responsive polysaccharidic ionic complex for proapoptotic D-(KLAKLAK)2 peptide delivery

We report charge-switching ionic nanocomplexes comprised of glycol chitosan grafted with 2,3-dimethylmaleic acid (DMA) (denoted as 'GCS-g-DMA' hereafter) and a proapoptotic peptide. This system allowed for improved peptide delivery to tumor sites via a mechanism of selective peptide release when the pH was dropped from 7.4 to 6.8.     

High-performance liquid chromatographic quantitation of rhodamines 123 and 110 from tissues and cultured cells

Rhodamine 123 is a fluorescent vital dye which has potential for therapeutic use in cancer treatment. The dye concentrates in mitochondria of normal and neoplastic cells but accumulates in and is toxic to neoplastic cells. When dye-treated cells are irradiated with blue laser light at 514 nm, mitochondrial injury or cell death results. Rhodamine concentration in cultured cells and tumor tissue was quantitated to correlate cell or tumor death with drug dose. A reversed-phase separation of rhodamine 123 was accomplished using a gradient of 0.05 M phosphate buffer pH 2.85 (mobile phase A) and acetonitrile (mobile phase B), 10-80% B in 15 min with a DuPont Golden Series C8 column. Effluent was monitored with a fluorescence detector at 295 nm excitation and 520 nm emission. Stock rhodamine 123 contained approximately 6-8% of rhodamine 110, the parent compound, which eluted at 9.8 min whereas rhodamine 123 eluted at 11.7 min. Structural verification of both compounds by field desorption mass spectrometry was performed. This is the first report of the chemical separation and quantitation of rhodamine 123 from cultured tumor cells or tumor tissue.     

Acid-Base Properties of Some Penicillins

The acidity constants of benzylpenicillin, carbenicillin, and ampicillin were determined by pH-metric titration at 20°C and an ionic strength of 0.1 M (KCl). Diagrams of distribution of ionic and molecular forms of these compounds vs. pH were calculated.     

pH-dependent spectral properties of HpIX, TPPS2a, mTHPP and mTHPC

Lower extracellular pH in tumors as compared to normal tissues has been proposed to be a factor contributing to the tumor selective uptake of several photosensitizers. Therefore, the pH dependence of absorption and fluorescence spectral properties of four different drugs relevant for photodynamic therapy (hematoporphyrin IX [HpIX], disulfonated meso-tetraphenylporphine [TPPS2a], meso-tetra(3-hydroxyphenyl)porphine [mTHPP] and meso-tetra(3-hydroxyphenyl)chlorin [mTHPC]) has been examined. Spectral analysis of the dyes dissolved in phosphate buffered saline (PBS) indicates pH-dependent modification in the physiologically important region (6.0-8.0) only in the case of HpIX. This modification is probably related to the protonation of carboxylic groups. Spectral changes of HpIX in PBS observed at acidic pH values < 5, as well as those of the rest of the drugs (inflection points of titration curves occurred at about 5.1, 3.8 and 2.4 for TPPS2a, mTHPP and mTHPC, respectively), are likely to be due to the protonation of imino nitrogens. The tumor localizing properties of mTHPP and mTHPC reported in the literature appear to be due to factors other than pH-dependent changes in the lipophilicity of the drugs.     

pH-dependent modification of lipophilicity of porphyrin-type photosensitizers

Structural modifications of photosensitizers (changes in protonation, ionic state and aggregation state) under different environmental conditions should be precisely determined to understand the interaction of the photosensitizers with biological systems. In the present study partition coefficients of hematoporphyrin IX (HpIX), disulfonated meso-tetraphenylporphine, meso-tetra(3-hydroxyphenyl)porphine (mTHPP) and meso-tetra(3-hydroxyphenyl)chlorin in the 1-octanol-phosphate buffer system were determined in the pH region 4.0-8.0. Only the partition coefficients of HpIX and mTHPP were found to be pH dependent. Computer processing of fluorimetric titration data was applied to estimate pKa values of the imino nitrogens of mTHPP. Monoprotonated species of mTHPP seem to be unstable or nonexistent. The possibility that both imino nitrogens of this dye are protonated according to a common pKa is proposed. The pKa value of the imino nitrogens of mTHPP was found to be 2.99 +/- 0.04 after the application of a model taking aggregation of the drug into account. The contributions of various aqueous ionic species of mTHPP as functions of pH were calculated and compared with partition coefficients.