Novel water-soluble photosensitizers from chitosan

Novel water-soluble polymeric photosensitizers based on the natural polymer chitosan were synthesized and studied. The modified chitosans contain covalently attached Rose Bengal. The polymers absorb light from the visible spectral region and generate singlet oxygen. They can serve as environmentally friendly, biodegradable polymeric photosensitizers, which can use light from a visible spectral region to initiate photooxidation of organic compounds in water.     

Photodynamic antimicrobial chemotherapy by liposome-encapsulated water-soluble photosensitizers

Photodynamic antimicrobial chemotherapy is an alternative method for killing bacterial cells in view of the increasing problem of multi-antibiotic resistance. We examined the effect of three water-soluble photosensitizers (PhS): methylene blue (MB), neutral red (NR) and rose bengal (RB) on Gram-positive and Gram-negative bacteria. We compared the efficacy of PhS in their free form and encapsulated in liposomal formulations against various bacterial strains, and determined conditions for the effective use of encapsulated PhS. We found that all three PhS were able to eradicate the Gram-positive microbes Staphylococcus aureus and Sarcina lutea; and MB and RB were effective against St. epidermidis. In the case of the Gram-negative species, MB and RB were cytotoxic against the Shigella flexneri, NR-inactivated Escherichia coli and Salmonella para B, and BR was effective in killing Pseudomonas aeruginosa. None of the examined PhS showed activity against Klebsiella pneumoniae. MB and NR enclosed in liposomes gave a stronger antimicrobial effect than free PhS for all tested prokaryotes, whereas encapsulation of RB led to no increase in its activity. We suggest that encapsulation of PhS can increase the photoinactivation of bacteria.     

Synthesis and in vitro photodynamic activities of water-soluble fluorinated tetrapyridylporphyrins as tumor photosensitizers

A series of water-soluble fluorinated cationic porphyrins were designed, synthesized, and characterized. In vitro photocytotoxicity of these compounds was evaluated by MTT assay on HeLa cells. Their photocytotoxicity was dependent on the positions of the cations and the fluorines in the pyridine ring, and 5,10,15,20-tetrakis-(N-methyl-2-fluoro-pyridin-3-yl)-porphyrin (8) showed the most potent photo-induced cytotoxicity without photobleaching. PDT-induced ROS inside HeLa cells was measured with flow cytometry using ROS-sensitive fluorometric probe, 2,7-dichlorofluororescin (DCF), which revealed high correlations of ROS with cellular cytotoxicity. FACS analysis shows that PDT with porphyrin 8 induced apoptosis in HeLa cells. In summary, efficient generation of ROS, biological effectiveness, and good photostability of porphyrin 8 indicate its potential application in photodynamic therapy (PDT) in the near future.     

Novel 3,6-bis(imidazolidine)acridines as effective photosensitizers for photodynamic therapy

The photoeffect of new proflavine derivatives with DNA-binding and antitumour activities, 3,6-bis((1-alkyl-5-oxo-imidazolidin-2-yliden)imino)acridine hydrochlorides (AcrDIMs), was studied to evaluate them as potential photosensitizers for photodynamic antitumor therapy. EPR measurements showed that superoxide radical anion and singlet oxygen were produced upon irradiation of AcrDIMs with UV-A light (>300 nm) in the presence of molecular oxygen. This indicates that AcrDIMs may act as photosensitizers. The most active pentyl-AcrDIM and hexyl-AcrDIM displayed photocytotoxic effect toward the mouse lymphocytic leukemia cell line L1210 and human ovarian cancer cells A2780. Antitumor activity of pentyl-AcrDIM increased as high as about 12 times (72 h incubation) after irradiation of A2780 cells (365 nm, 1.05 J/cm²). The photocytotoxicity seems to be associated with oxidative stress. Concerning the cell cycle, flow cytometry showed an arrest in the S-phase already 4 h after irradiation. In a comet assay, no genotoxicity of AcrDIMs was found. Typical morphologic changes and formation of DNA-ladders indicated induction of apoptotic cell death, though no activation of caspase-3 was observed. Investigation of intracellular localization of pentyl-AcrDIM confirmed its partial accumulation in mitochondria and lysosomes. After irradiation of the A2780 cells, colocalization of pentyl-AcrDIM with monodansylcadaverine, a lysosomal dye, was proven, suggesting that lysosomes in the irradiated cells may be involved in the cell death.     

Novel boronated chlorin e6-based photosensitizers: Synthesis,binding to albumin and antitumour efficacy

Chlorins, a class of plant porphyrins, are perspective as photosensitizing agents due to light absorption in the long wavelength spectral region and deeper photodamage of tissues. Aiming at optimization of antitumour properties of chlorins, we synthesized a series of boronated derivatives of chlorin e₆ and their complexes containing Zn(II), Pd(II) or Sn(IV). The compounds were synthesized by alkylation of amino or hydroxy derivatives of chlorin e₆ with 1-trifluoromethanesulfonylmethyl-o-carborane. Chlorin e₆ 13(1)-N-{2-[N-(o-carboran-1-yl)methyl]aminoethyl}amide-15(2), 17(3)-dimethyl ester (compound 5) formed complexes with serum albumin, a major porphyrin carrier. The binding constant of these complexes was ～4 times bigger than the respective value for the complexes of albumin with boron-free aminochlorin e₆. Compound 5 potently sensitized rat fibroblasts to illumination with monochromatic red light: >98% of cells were necrotic by 24 h post-illumination with 1 μM of 5. This compound demonstrated high efficacy in photodynamic therapy of rat M-1 sarcoma. After PDT with 25 mg/kg of 5 the residual tumours were significantly smaller than in animals subjected to PDT with equal concentration of boron-free aminochlorin e₆. No signs of general toxicity were detectable after PDT with 5. Thus, boronation can enhance the potency of chlorins in PDT, in particular, due to an increased binding to albumin. Our data expand the therapeutic applicability of boronated chlorins beyond boron neutron capture therapy; these agents emerge as dual efficacy photoradiosensitizers.     

Novel water-soluble analogues retaining potent antitumor activity of RA-VII, a cyclic hexapeptide from Rubia plants

Deoxybouvardin (3) was reacted with 2-dialkylaminoethyl chloride salts to produce analogues 4a-h. All analogues retained antitumor activity against P388 leukemia in mice, and analogue 4c showed more promising antitumor activity than RA-VII (1) against the P388 leukemia, B16 melanoma and colon adenocarcinoma 26 murine tumor models. The hydrochloride salt of 4c is soluble in water.     

Thin layer gel chromatography of dextrans

Microgram quantities of soluble dextran fractions have been separated by thin layer gel chromatography. The dextrans are first combined with a triazine dye to render them visible during the chromatography and to facilitate the densitometric evaluation of the chromatograms. The method allows the determination of dextran molecular weights up to approximately 100 000 and can also be used in studying the polydispersity of the fractions.     

Passage of uncharged dextrans from blood to lung lymph in awake sheep

To examine how molecular size alone influences the passage of macromolecules from the pulmonary microcirculation into lymph collected from the caudal mediastinal lymph node of the sheep, we infused polydisperse uncharged [3H]dextrans intravenously at a constant rate over a period of 7.5 h in nine awake sheep with lung lymph fistulas. Lymph and plasma were collected during hours 5.5-7.5 of the infusions, and the [3H]dextrans were separated by molecular sieve chromatography into fractions that ranged from 1.6 to 8.4 nm in effective molecular (Stokes-Einstein) radius. Lymph-to-plasma (L/P) ratios for [3H]dextrans were near 1.0 at 1.6-nm radius, decreased with increasing molecular size, and approached zero at radii above 5.0 nm. We confirmed that these L/P ratios represented steady-state values by extending the duration of the infusion to approximately 30 h in two of the nine sheep and finding that the L/P ratios remained unchanged. These results were consistent with molecular sieving through a homoporous membrane with cylindrical pores of 5.0-nm radius. We also found that the L/P ratio for albumin [0.76 +/- 0.13 (SE)] in five of the same sheep was much higher than that for the [3H]dextran fraction of the same effective molecular radius [0.11 +/- 0.02 (SE)]. These results suggest that the movement of macromolecules from the pulmonary microcirculation into pulmonary lymph collected from the caudal mediastinal node of the sheep is influenced by both molecular size and molecular charge and that, compared with uncharged dextrans, the steady-state passage of anionic endogenous proteins from plasma to lymph is enhanced.     

Origin of branches in native dextrans

According to the proposed mechanism for the enzymatic formation of dextran, branches of different length are formed in a reaction between a so-called acceptor dextran molecule and an active reaction complex; propagation of the dextran chain occurs by an insertion-type polymer growth. To investigate this branching reaction, radioactive labeled acceptor dextran of relatively low molecular weight was added to a dextran synthesizing system and the native high molecular weight dextran separated by a fractionation method. From the specific radioactivity of the isolated native dextran, and assuming a molecular weight of 2.5 × 10⁵, the average number of acceptor molecules incorporated into native dextran is calculated to be about unity, as predicted by the proposed mechanism. Analogous results have been obtained in the reaction of two different dextran sucrases from Leuconostoc mesenteroides B512F and B1307, respectively.     

Novel branched poly(ethylenimine)-cholesterol water-soluble lipopolymers for gene delivery

A novel water-soluble lipopolymer was synthesized by linking cholesteryl chloroformate to the secondary amino groups of branched poly(ethylenimine) (PEI) of 1,800 and 10,000 Da. Conjugation through PEI secondary amines gives this newly synthesized lipopolymer (abbreviated as PEI-Chol) special advantage over our previously synthesized lipopolymers, which utilized the primary amino groups for conjugation, as the primary amino groups have a significant role in DNA condensation. Also, significantly, only one cholesterol molecule was grafted onto each PEI molecule (confirmed by (1)H NMR and MALDI-TOF mass spectrometry), leaving enough space for the steric interactions of the PEI's primary amines with the DNA. The PEI-Chol lipopolymer was characterized for the critical micellar concentration (cmc), buffer capacity, DNA condensation (by band retardation and circular dichroism), in vitro transfection efficiency, and cell viability. The cmcs of PEI-Chol 1,800 and PEI-Chol 10,000 were 496.6 and 1,330.5 microg/mL, respectively. The acid-base titration indicated high buffering capacity of the polymers around the pH range of 5-7, which indicated their potential for buffering in the acidic pH environment of the endosomes. The band retardation studies indicated that efficient condensation of the plasmid DNA could be achieved using these lipopolymers. The circular dichroism spectra indicated a change in DNA conformation and adoption of lower energy state upon condensation with these lipopolymers when an N/P ratio of 2.5/1 or above was formulated. The mean particle size of these complexes was in the range 110-205 nm, except for the complexes prepared using PEI of 1,800 Da, which had a mean particle size of 384 +/- 300 nm. The zeta potential of DNA complexes prepared using PEI-Chol 1,800, PEI-Chol 10,000 and PEI of 1,800, 10,000, and 25,000 Da at an N/P ratio of 15/1 was in the range 23-30 mV and was dependent on the N/P ratios. The in vitro transfection of PEI-Chol/pCMS-EGFP complexes in Jurkat cells showed high levels of expressed Green Fluorescent Protein (GFP) with little toxicity as determined by flow cytometry. These novel water-soluble lipopolymers provided good transfection efficiency with other desirable characteristics such as water solubility, free primary amino groups for efficient DNA condensation and high buffer capacity that indicated the possibility of efficient endosomal release.