Biomedical subjects

M R Hamblin

Publications and source records attributed to M R Hamblin.

29 records · Page 2Linked to original sources

PubMed1996

Effect of charge on the interaction of site-specific photoimmunoconjugates with human ovarian cancer cells.

A marked effect of charge modification on the uptake and phototoxicity of a photoimmunoconjugate (PIC) was demonstrated. A site-specific conjugation strategy was developed to attach the photosensitizer chlorin(e6) (c(e6)) to the F(ab')2 fragment of the murine antiovarian cancer monoclonal antibody OC125. Poly-L-lysine linkers carrying c(e6) with a cationic charge or by polysuccinylation with an anionic charge were used and covalently attached to partially reduced antibody via a heterobifunctional reagent. PICs were purified by column chromatography and were also radiolabeled with 125I. PIC binding and uptake were studied with a human ovarian cancer cell line, NIH-OVCAR-5, and a nonantigen-expressing colon cancer cell line, SW1116, and the data were compared with the binding and uptake of nonspecific rabbit IgG PICs. PICs with both cationic and anionic charges preserved antigen binding as shown by competition studies with native antibody, but the cationic PIC had up to 17 times higher cellular uptake of c(e6), probably due to enhanced internalization. The ratio of c(e6) to 125I retained by the cells varied with the likelihood of internalization and lysosomal degradation. The phototoxicity of the PICs generally varied with their uptake, but a correlation was found between lysosomal hydrolysis as measured by an increased cellular ratio of c(e6):125I and increased relative phototoxicity. These data suggest cationic PICs may have advantages for photoimmunotherapy of disseminated intracavity cancer following local administration.

Antigens, Surface↗

PubMed1996

Intraperitoneal photodynamic therapy of human epithelial ovarian carcinomatosis in a xenograft murine model.

The objective of this investigation was to determine the efficacy of i.p. photodynamic therapy (PDT) against solid, multifocal ovarian carcinoma using a newly described NIH:OVCAR-5 induced murine model. PDT was initiated when diffuse microscopic disease and small multifocal tumor nodules were present, similar to the extent of residual carcinoma that may persist clinically after laparotomy and tumor debulking. The photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), was administered in a dose of 0.25 mg/kg body weight i.p. 90 min prior to light exposure. An argon-pumped dye laser was used to deliver low intensity light (20 J) i.p. through a cylindrically diffusing fiberoptic tip. Treatment regimens consisted of a series of three to five treatments at 3-7 day intervals, with the extent of macroscopic disease or death from disease being the evaluable outcome parameters for tumoricidal and survival studies, respectively. The mean tumor burden at necropsy for treated animals was 0.034 +/- 0.014 g compared to 0.379 +/- 0.065 g in untreated controls (P<0.001). Survival studies were initiated in two groups at day 7 and day 14 following cell inoculation. The first group received either three or five treatments at 5-day intervals, and both had a significant increase in median survival compared to untreated controls (57 and 53 days, respectively, compared to 43 days, P<0.05). The second group was treated every 7 days until death and also had a significant survival advantage over controls (57 days compared to 47 days, P<0.05). These studies suggest that benzoporphyrin derivative mono acid ring A-mediated PDT is a feasible, well-tolerated, experimental treatment approach that elicits a tumoricidal response against diffuse, solid i.p. disease in tumor-bearing mice, with concomitant prolongation of survival and needs careful optimization.

Animals↗

PubMed1994

Photosensitizer targeting in photodynamic therapy. II. Conjugates of haematoporphyrin with serum lipoproteins.

Conjugates between haematoporphyrin (HP) and human low-density lipoprotein (LDL), human high-density lipoprotein (HDL) and bovine HDL have been prepared, purified and characterized. HP-LDL is aggregated possibly via interparticle apoB protein cross-linking. HP-HDL human and bovine conjugates show different degrees of intraparticle apoA polypeptide cross-linking. Receptor-mediated endocytosis of HP-LDL by NIH 3T3 cells is inferred from the increased uptake observed when LDL receptors are upregulated. HP-LDL uptake into HT29 cells faces competition from unlabelled LDL, albeit at rather high doses. HP-HDL uptake is also inhibited by LDL, suggesting that both lipoprotein conjugates may have cell-surface binding sites in addition to the specific LDL (apoB) receptor. J774.2 macrophages avidly accumulate HP-LDL, retaining most of the fluorescence and some of the protein while degrading the remainder. Oxidized LDL species compete in these processes, with the major effect on protein degradation. Chloroquine has little effect on the fluorescence uptake but inhibits protein degradation (and hence enhances protein accumulation). HP-HDL is also avidly taken up by J774.2 cells, but in the case of the bovine material with a sigmoidal concentration dependence. This is consistent with prior aggregation before the particles can be endocytosed. P388.D1 cells, which appear to be less activated than the J774.2 line, take up less fluorescence and retain and degrade less protein, but still to higher extents than observed for non-phagocytic cells. We conclude that photosensitizer-lipoprotein conjugates can be taken up in large amounts by cells possessing scavenger receptors and/or phagocytic activity, and that this may be a means of targeting photodynamic therapy.

3T3 Cells↗

PubMed1994

Photosensitizer targeting in photodynamic therapy. I. Conjugates of haematoporphyrin with albumin and transferrin.

Conjugates of haematoporphyrin (HP) with serum albumin and transferrin were prepared, purified by gel filtration and characterized by high performance liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE) and spectroscopy. Although the fluorescence was somewhat quenched, the conjugates had similar singlet oxygen quantum yields to free porphyrin. The albumin conjugate (HP-BSA) could be divided into monomeric and cross-linked fractions. In NIH 3T3 and HT29 cells, native albumin could not compete with the uptake of HP-BSA and the uptake was greatly enhanced in the absence of serum and in the presence of poly-L-lysine. We infer that the conjugate was mostly associated with the plasma membrane in these cells. The uptake of HP-transferrin showed evidence of a receptor-mediated component in that it was partially inhibited by native protein and increased when transferrin receptors were upregulated by an iron chelator. J774 macrophage-like cells accumulated fluorescence from HP-BSA to a much higher degree than HT29 cells, even though the protein was extensively degraded (HT29 cells did not appear to degrade the protein). The time course of the photocytotoxicity of HP-BSA was prolonged in J774 cells, although their response to free porphyrins was similar to that seen in HT29 cells. Chloroquine inhibited protein degradation without having an effect on the fluorescence uptake. J774 cells acquired more fluorescence and degraded more protein when supplied with cross-linked HP-BSA compared with monomeric fraction. For a given fluorescence uptake, the cross-linked fraction was also more photocytotoxic. We conclude that macrophages can acquire photosensitizer-protein conjugates avidly and that these are delivered to the lysosomes. These types of conjugate may have applications in targeting fluorescent molecules for diagnostic imaging and for the photodynamic treatment of macrophage malignancies.

3T3 Cells↗

PubMed1994

On the mechanism of the tumour-localising effect in photodynamic therapy.

The proposed mechanisms by which tumours concentrate photosensitisers are reviewed. Tumour-associated macrophages have been shown by others to accumulate up to nine times the level of porphyrins as do tumour cells. Macrophages also take up and degrade oxidised or otherwise modified low-density lipoprotein (LDL). We propose that the interaction of photosensitisers with LDL is an important factor, leading to accumulation in macrophages. Uptake into these cells via liposomes and high-density lipoprotein is also possible. There may be three separate mechanisms for tumour destruction in photodynamic therapy: (i) direct damage to tumour cells; (ii) damage to the endothelial cells of the tumour microvasculature; and (iii) macrophage-mediated immune infiltration of the tumour. The association of photosensitisers with lipoproteins may accentuate the latter two (endothelial cells can also accumulate modified lipoproteins). Accumulation in macrophages may also largely explain the high porphyrin retention observed in atheromatous plaques.

Animals↗

PubMed1987

myo-Inositol phosphorothioates, phosphatase-resistant analogues of myo-inositol phosphates. Synthesis of DL-myo-inositol 1,4-bisphosphate and DL-myo-inositol 1,4-bisphosphorothioate.

Syntheses of a metabolite of the second messenger myo-inositol 1,4,5-trisphosphate, myo-inositol 1,4-bisphosphate, and an analogue, the 1,4-bisphosphorothioate, are reported, by using phosphite chemistry on (+/-)-1,2:4,5-di-isopropylidene-myo-inositol. The synthesis of (+/-)-1,2:4,5-di-isopropylidene 3,6-bis[di-(2-cyanoethyl)]phosphite provides an intermediate that can be oxidized to either the corresponding bisphosphate or bisphosphorothioate. myo-Inositol phosphorothioates are proposed as novel analogues of myo-inositol phosphates; their resistance to phosphatase-catalysed breakdown is reported.

Alkaline Phosphatase↗

PubMed1987

Mung bean (Phaseolus aureus) nuclease. A mechanistic investigation of the DNA-cleavage reaction using a dinucleoside phosphorothioate.

Mung-bean (Phaseolus aureus) nuclease has been found to cleave the Sp diastereoisomer of 5'-O-thymidyl 3'-O-(2'-deoxyadenosyl)phosphorothioate, (Sp)-d[Ap(S)T], in 18O-labelled water with inversion of configuration at phosphorus to give (Sp)-thymidine 5'-[16O, 18O]phosphorothioate, the stereochemistry of which was deduced by methylation to (Rp,Sp)-thymidine 5'-S-methyl-O-methyl-[16O,18O]phosphorothioate and 31P-n.m.r. analysis. This result is consistent with a mechanism involving a direct 'in-line' attack of water on DNA for the nuclease-catalysed reaction without the involvement of a covalent nucleotidylated-enzyme intermediate.

Chromatography, High Pressure Liquid↗

PubMed1985

E. coli Ada regulatory protein repairs the SP diastereoisomer of alkylated DNA.

Using HPLC and 31P NMR spectroscopy on a chemically synthesized asymmetric mixture of the diastereoisomers of thymidyl(3'----5')thymidyl-O-methyl phosphate absolute configuration has been correlated with chromatographic mobility. The methyl phosphotriester system in alkylated DNA which is repaired by the Ada regulatory protein of E. coli has consequently been established to possess the Sp configuration.

Alkylation↗

PubMedDate not supplied

Macrophage-targeted photodynamic therapy.

Photodynamic therapy (PDT) uses light activatable molecules that after illumination produce reactive oxygen species and unwanted tissue destruction. PDT has dual selectivity due to control of light delivery and to some extent selective photosensitizer (PS) accumulation in tumors or other diseased tissue, additional targeted selectivity of PS for disease is necessary. The delivery of drugs to selected lesions can be enhanced by the preparation of targeted macromolecular conjugates that employ cell type specific targeting by ligand-receptor recognition. Macrophages and monocytes express a scavenger-receptor that is a high-capacity route for delivering molecules into endocytic compartments in a cell-type specific manner. We have shown that by attaching PS to scavenger-receptor ligands it is possible to get three logs of selective cell killing in macrophages while leaving non-macrophage cells unharmed. The capability to selectively kill macrophages has applications in treating cancer and in the detection and therapy of vulnerable atherosclerotic plaque and possibly for autoimmune disease and some infections.

Animals↗

PubMedDate not supplied

Photodynamic therapy targeted to pathogens.

Photodynamic therapy (PDT) employs a non-toxic dye termed a photosensitizer (PS) together with low intensity visible light, which, in the presence of oxygen, produce cytotoxic species. PS can be targeted to its destination cell or tissue and, in addition, the irradiation can be spatially confined to the lesion giving PDT the advantage of dual selectivity. This promising approach can be used for various applications including microbial inactivation and the treatment of infections. Resistance to PDT has not been shown and multiantibiotic-resistant strains are as easily killed as naive strains. It is known that Gram (+) bacteria are more sensitive to PDT as compared to Gram (-) species. However, the use of cationic PS or agents that increase the permeability of the outer membrane allows for the effective killing of Gram (-) organisms. Some PS have an innate positive charge, but our approach is to link PS to a cationic molecular vehicle such as poly-L-lysine. This modification dramatically increases PS binding to and penetrating through the negatively charged bacterial permeability barrier. Due to focused light delivery the use of PDT is possible only for localized infections. Nonetheless numerous diseases can be treated. Selectivity of the PS for microbes over host cells, accurate delivery of the PS into the infected area, and PDT dose adjustment help minimize side effects and give PDT an advantage over conventional therapy. There are only a few reports about the use of antimicrobial PDT in animal models and clinical trials. We have used genetically modified bioluminescent bacteria to follow the effect of PDT in infected wounds, burns, and soft tissue infections in mice. Not only were bacteria infecting wounds, burns, and abscesses killed, but mice were saved from death due to sepsis and wound healing was improved.

Animals↗

PubMedDate not supplied

Macrophage-targeted photodynamic therapy: scavenger receptor expression and activation state.

Macrophage-targeted photodynamic therapy (PDT) may have applications in the selective killing of cells involved in atherosclerosis, inflammation and tumor. We have previously shown that a conjugate between the photosensitizer chlorin(e6) (ce6) and maleylated bovine serum albumin (BSA-mal) gives highly selective targeting to macrophages. In this report we examine the effect of macrophage activation and scavenger receptor class A (SRA) expression on this targeting in two murine macrophage tumor cell lines (RAW264.7 and P388D1) and a control murine mammary sarcoma cell line (EMT-6). Cells were pretreated with interferon gamma (IFNgamma) and/or lipopolysaccharide (LPS) followed byBSA-ce6-mal addition, and SRA expression, tumor necrosis factor alpha (TNFalpha) release, conjugate uptake and PDT killing were measured. Both macrophage cell lines expressed SRA and took up conjugate specifically in an SRA-dependent manner, but differences were observed in their response to activation. RAW264.7 expressed increasingly more SRA and took up increasingly more BSA-ce6-mal in response to IFNgamma, LPS, and IFNgamma+LPS, respectively. The PDT killing did not follow the same pattern as the uptake of the photosensitizer. The increase in uptake in the IFNgamma treated cells did not lead to an increase in PDT killing, while stimulation with LPS or IFNgamma + LPS resulted in a significant protection against PDT, despite a significant increase in photosensitizer uptake. P388D1 was responsive to neither IFNgamma, nor to LPS, or to IFNgamma +LPS with respect to SRA expression, conjugate uptake, and PDT killing. These data may have implications for the use of PDT to target physiologically undesirable macrophage subtypes implicated in disease, and on how manipulation of the activation status of the macrophage will influence the PDT effect.

Animals↗