Narrow Results

The spectroscopic and biological properties of the new photosensitizer lutetium texaphyrin (Lu-Tex) were assessed in vitro and in vivo on a C26 colon carcinoma model, in comparison with hematoporphyrin (Hp), photofrin II (PII) and chlorin e₆(Chl). Strong binding of Lu-Tex to lipid bilayer membranes was observed. The results of confocal fluorescence microscopy on C26 cells showed that Lu-Tex was localized in small vesicles in the cytoplasm, possibly in the lysosomes, while Chl and Hp were distributed in larger cytoplasmic vesicles attributed to mitochondria. Scanning electron microscopy and X-ray microanalysis revealed that photodynamic therapy with Lu-Tex induced only slight damage to the cell membrane, leading to a delayed cell response. Chl and Hp caused significant structural damage to the outer cell membrane, resulting in ionic imbalance and fast cell death. The in vitro quantitative assessment of the relative efficiency per absorbed photon of the sensitizers revealed that Lu-Tex was less effective than Chl and Hp. However, the results of our in vivo study showed that at the same light and drug doses the anti-tumor efficiency of the agents was in the following order: Lu-Tex > Chl > PII. The strong in vivo anti-tumor effect of Lu-Tex can be explained by its higher integrated absorption in the long-wavelength range.

Chlorin e6 (Ce6) was conjugated with anti-tumor monoclonal antibody (IgG2b) to increase its binding affinity for tumors. The Ce6 was activated by N-hydroxysuccinimide and directly conjugated with IgG2b (IgG2b–Ce6) via peptide bonds. An average of 11 Ce6 molecules were coupled to each IgG2b molecule. IgG2b–Ce6 showed high cellular uptake in tumor cells and high photocytotoxicity. These results indicate IgG2b–Ce6 is a good photosensitizer with high binding affinity to tumor cells.

Chlorin e6(Ce6) was conjugated with anti-tumor monoclonal antibody (IgG) to increase its binding affinity for tumors. Ce6 was activated by N-hydroxysulfosuccinimide and conjugated with IgG via peptide bonds, and Ce6 molecules were conjugated with IgG(IgG-Ce6) and their binding affinities to tumor cells were investigated. Intracellular localization of IgG-Ce6 was observed, and IgG-Ce6 was accumulated in tumor cells much higher than Ce6, indicating that the IgG-Ce6 has specific binding affinity to tumor cells. The effective photocytotoxicity of the cells with IgG-Ce6 is caused by the high accumulation of IgG-Ce6 in tumor cells.

Glucose-, galactose- and lactose-containing photosensitizers based on derivatives of chlorophyll a and bacteriochlorophyll a were synthesized with the use of [3+2] cycloaddition between sugar azides and triple bond derivatives of chlorins and bacteriochlorins. Unlike bacteriochlorin cycloimide, chlorin was detected to form a Cu-complex during the click reaction. An approach to the synthesis of metal-free glycosylated chlorins was developed with the use of "protection" by Zn²⁺ cation and subsequent demetalation. It is based on the action of alkynyl chlorin e₆ derivative Zn-complex, which is resistant to the substitution by copper cation. Bacteriochlorin p cycloimide conjugate with per-acetylated β-D-lactose was obtained and shown to become water-soluble after unblocking of the lactose hydroxy functions. NMR studies allowed for the elucidation of structure, tautomeric form and conformation of the obtained compounds.

Syntheses of three new chlorin e₆ conjugates for PDT of tumors are reported. One of the new compounds 17 is conjugated with lysine at the 13¹-position, but the others are mono-conjugated 14 or diconjugated 15 with the non-amino acid species ethanolamine. Cellular experiments with the three new compounds and previously synthesized non-amino acid 15²-conjugates (7–10), 13¹-monoconjugates 14, 16, and a 13¹,15²-diconjugate 12 are reported. In vitro cytotoxicity experiments show that the 13¹-conjugates are more toxic than the 15²-conjugates, and the most toxic derivative (dark- and photo-toxicity) is the 13¹-ethylenediamine conjugate 11. The most useful PDT photosentitizers appear to be the ethanolamine derivatives, conjugated at the 15²- and the 13¹,15²-positions; these show high phototoxicity but relatively low dark toxicity compared with 11, and also the highest dark/photo cytotoxicity ratios.

Prostate cancer is an extremely common cancer among older men. Conventional chemotherapy has proven to be not effective enough in battling it due to its high systemic toxicity and low selectivity. An alternative method of cancer treatment known as photodynamic therapy (PDT) has been shown to be effective. It is not without its faults either: one of the issues it’s been known to have is the insufficient selectivity of photosensitizer accumulation in tumor tissues. Recent studies, however, seem to indicate that introducing a PSMA-targeted moiety into photosensitizer might prove to be a solution to this problem. The present paper is concerned with synthesis of PSMA-targeted 13¹- and 15²-substituted chlorin e₆ conjugates and their biological characteristics. Our data suggests that the developed conjugates show potential as targeted agents for photodynamic therapy.

Chlorins (dihydroporphyrins) are considered, due to their ideal photophysical properties, as attractive photosensitizers for photodynamic therapy (PDT) of cancer and other therapeutic and diagnostic applications. Chlorophyll $a$, as a naturally occurring chlorin, forms an almost unlimited renewable resource for preparation of potential biologically active chlorin photosensitizers and fluorescence markers. To achieve amphiphilic photosensitizers which might be selectively enriched in tumor cells, we addressed linkage of per se lipophilic chlorophyll derivatives with carbohydrate based hydrophilic aminopolyols.

The encapsulation of five derivatives of chlorin e6 with different hydrophobicity and aggregation properties into a series of five poloxamer-type triblock copolymer micelles (BCMs) with varying numbers of polyethylene and polypropylene glycol (PEG, PPG) units was monitored using ¹H NMR spectroscopy. NMR chemical shift and line shape analysis, as well as dynamic methods including diffusion ordered spectroscopy (DOSY) and T₁ and T₂ relaxation time measurements of the chlorin and the polymer resonances, proved useful to assess the chlorin–BCM compatibility. The poloxamers had high capability to break up aggregates formed by chlorins up to intermediate hydrophobicity. Physically entrapped chlorins were always localized in the BCM core region. The loading capacity correlated with chlorin polarity for all poloxamers among which those with the lowest number of PPG units were most efficient. DOSY data revealed that relatively weakly aggregating chlorins partition between the aqueous bulk and micellar environment whereas more hydrophobic chlorins are well retained in the BCM core region, rendering these systems more stable. T₁ and T₂ relaxation time measurements indicated that motional freedom in the BCM core region contributes to encapsulation efficiency. The BCM corona dynamics were rather insensitive towards chlorin entrapment except for the poloxamers with short PEG chains. The presented data demonstrate that ¹H NMR spectroscopy is a powerful complementary tool for probing the compatibility of porphyrinic compounds with polymeric carriers such as poloxamer BCMs, which is a prerequisite in the development of stable and highly efficient drug delivery systems suitable for medical applications like photodynamic therapy of tumors.

Chlorin e6 (Ce6) and its derivatives are among the most important photosensitizers in photodynamic therapy. Due to their intense fluorescence, chlorins may also be used for diagnostics. However, low solubility in water and high tendency to aggregation restrict their medical use. Here we demonstrate that apo-myoglobin, by reinserting Ce6 in its heme binding pocket, can be used to monomolecularly disperse it. The reconstructed myoglobin-Ce6 adduct presents noticeable changes in the photophysical properties of the chromophore. A red-shift, in particular in the transparency window, can be observed in the absorption and in the emission spectra of the adduct compared to the spectra of the free chlorin in PBS. The adduct presents a higher quantum yield and an increased excited-state lifetime with respect to the free Ce6. The binding of Ce6 to apo-myoglobin determines a decrease of the ¹O₂ generation but a three-fold increase of peroxides production, determining globally an increase in the performance of Ce6 as a photosensitizer and imaging agent.

Three derivatives of chlorin e6 (1–3) were synthesized by introduction of maleimide, cysteine and glutathione at C-13 carboxyl of the chlorin scaffold. The evaluation of their PDT effects showed that compound 1, the derivative with a maleimide group, exhibited more potent photocytotoxicity against HepG2 cells (IC${}_{50}$ 3.2 $\mu$M) than 2 (IC${}_{50}$ 6.7 $\mu$M) and 3 (IC${}_{50}$ 10.2 $\mu$M), although the cellular uptake of 1 was slightly lower than that of 2 and 3. The high PDT effect of 1 was found to be in agreement with the high level of intracellular singlet oxygen. Further investigation of the mechanism revealed that 1 can significantly lower the GSH level in HepG2 cells due to the addiction reaction of maleimide and GSH, thus resulting in the reduction of ROS scavenging and the enhancement of cellular oxidative stress. This approach to improve PDT effects of photosensitizers by means of interfering with the cellular redox system and enhancing cellular oxidative stress offers a new strategy for development of photosensitizers in cancer therapy.

We have shown the possibility of intramolecular cyclization with the participation of the carboxyl group at position 15 of the chlorin macrocycle with a primary or secondary amide group at position 13 through the formation of an imide bond under the action of 2-Chloro-1-methylpyridinium iodide; the imide bond can be used to form a seven-membered exocycle. Using this reaction, we synthesized a number of cycloimide derivatives of chlorophyll a. The proposed synthetic route can be carried out if the substituent at the amide nitrogen atom has a hydroxyl, dimethylaminomethyl or vinyl group, which opens up possibilities for further chemical modification of the obtained derivatives.

The encapsulation of five derivatives of chlorin e6 with different hydrophobicity and aggregation properties into a series of five poloxamer-type triblock copolymer micelles (BCMs) with varying numbers of polyethylene and polypropylene glycol (PEG, PPG) units was monitored using ¹HNMR spectroscopy. NMR chemical shift and line shape analysis, as well as dynamic methods including diffusion ordered spectroscopy (DOSY) and T₁ and T₂ relaxation time measurements of the chlorin and the polymer resonances, proved useful to assess the chlorin–BCM compatibility. The poloxamers had high capability to break up aggregates formed by chlorins up to intermediate hydrophobicity. Physically entrapped chlorins were always localized in the BCM core region. The loading capacity correlated with chlorin polarity for all poloxamers among which those with the lowest number of PPG units were most efficient. DOSY data revealed that relatively weakly aggregating chlorins partition between the aqueous bulk and micellar environment whereas more hydrophobic chlorins are well retained in the BCM core region, rendering these systems more stable. T₁ and T₂ relaxation time measurements indicated that motional freedom in the BCM core region contributes to encapsulation efficiency. The BCM corona dynamics were rather insensitive towards chlorin entrapment except for the poloxamers with short PEG chains. The presented data demonstrate that 1H NMR spectroscopy is a powerful complementary tool for probing the compatibility of porphyrinic compounds with polymeric carriers such as poloxamer BCMs, which is a prerequisite in the development of stable and highly efficient drug delivery systems suitable for medical applications like photodynamic therapy of tumors.