Ivo Crnolatac - Academia.edu (original) (raw)
Papers by Ivo Crnolatac
Advanced Engineering Materials, 2011
Recently, various bio-medical applications of nanoporous silicon (np-Si) have been suggested. Thi... more Recently, various bio-medical applications of nanoporous silicon (np-Si) have been suggested. This work investigates the biocompatibility of np-Si particles taking into account hazardous residua confined in the pores after preparation. The emphasis is on the potential ...
International journal of oncology, 2007
Previous studies have shown that hypericin is an excellent diagnostic tool for the fluorescence d... more Previous studies have shown that hypericin is an excellent diagnostic tool for the fluorescence detection of carcinoma in situ in the human bladder. The present work was performed to get a better insight into the mechanism of cellular uptake of hypericin (HYP) using RT-112 human papillary TCC cells of the bladder. Using lipophilic hypericin acid amide derivatives like hypericin acid hexylamide (AM6), hypericin acid octylamide (AM8) and hypericin acid dodecylamide (AM12), the effect of increased lipophilicity on the binding to serum proteins was investigated, as well as the cellular accumulation and permeation, both in 2-D and 3-D cell conditions. Density-gradient ultracentrifugation of the compounds pre-incubated with fetal bovine serum (FBS) showed that HYP and to a lesser extent AM6 predominantly bind to LDL, whereas AM8 and especially AM12 preferably associate with HDL. The cellular accumulation of the compounds did not significantly differ when LDL or AcLDL was supplemented to m...
Photonic Therapeutics and Diagnostics IV, 2008
ABSTRACT Hypericin (HYP) is used after instillation as a diagnostic tool for the fluorescence det... more ABSTRACT Hypericin (HYP) is used after instillation as a diagnostic tool for the fluorescence detection of CIS in the human bladder. In this study the in vitro cellular accumulation and intraspheroidal distribution of HYP and three analogues (OH1, OH2, OH3) with gradually increasing hydrophilicity were studied. E-cadherin negative (T24-C1-) and E-cadherin positive (T24-H3++) human bladder cancer cells were used. We report that in the presence of FBS all compounds were taken up by the monolayer cells to the same limited extent, whereas the overall intracellular accumulation was substantially higher when the incubation of the different dyes took place using cell medium not supplemented with FBS. The results of this study therefore confirm the competition between cellular uptake of HYP and analogues and binding to FBS constituents. Investigating the permeation of the compounds in spheroids, it was found that all HYP analogues diffused dramatically better through the three-dimensional cell layers than HYP itself. This enhanced ability of hydrophilic HYP analogues to permeate through the cell layers in the presence of FBS can be explained in terms of a preferred binding to HDL as compared to LDL. The results further show that all compounds, including LDL-binding HYP, substantially permeated better in T24-C1- spheroids than in T24-H3++ spheroids. The data therefore support the hypothesis that a lowered cellular cohesion is the key to understand the selective uptake of hypericin and its analogues in malignant urothelial cells.
The Journal of Urology, 2008
To obtain unambiguous evidence for the putative role of E-cadherin in the selective accumulation ... more To obtain unambiguous evidence for the putative role of E-cadherin in the selective accumulation of hypericin after intravesical instillation in humans we investigated the accumulation of hypericin in spheroids from 3 clones of the human bladder carcinoma cell line T-24 that express different levels of E-cadherin, as determined by immunohistochemistry and reverse transcriptase-polymerase chain reaction. Clones of T-24 cells transfected with the E-cadherin gene were analyzed for E-cadherin expression and 3 cell lines with different expression levels were selected. Spheroids of these cell lines were incubated with 10 microM hypericin in cell culture medium supplemented or not with fetal calf serum for 2 hours. After the incubation period centrally cut sections were examined by fluorescence microscopy. An imaging software system was used to measure average fluorescence in concentric layers from rim to center. Data showed that in the presence of serum the accumulation of hypericin in spheroids was inversely associated with the level of E-cadherin expressed by the T-24 transfectants used, whereas in the absence of serum differential accumulation of the compound was completely abolished. Spheroids composed of cancer cell lines expressing variable levels of E-cadherin represent an excellent model in which to study the role of intercellular adhesion in bladder cancer. The outcome of this study strongly suggests that E-cadherin is the key mediator in the selective accumulation of hypericin in superficial bladder cancer after intravesical instillation in humans.
Dyes and Pigments, 2012
ABSTRACT Six asymmetric monomethine cyanine dyes have been synthesized and their spectral charact... more ABSTRACT Six asymmetric monomethine cyanine dyes have been synthesized and their spectral characteristics and interaction with double stranded (ds)DNA have been investigated for their prospective use as fluorescent markers in molecular biology. Therefore, the non-covalent binding of the compounds with dsDNA was explored. Apart from the fluorescence spectroscopy, the study includes UV/Vis spectrophotometry and circular dichroism spectroscopy, as well as the thermal melting experiments. Although the compounds show relatively low binding affinitiy towards dsDNA, do not have intrinsic fluorescence, however in the presence of dsDNA they exhibited enhancement in fluorescence intensity. Therefore the studied dyes show interesting platform for future modifications directed towards more sequence selective derivatives. The compound with the highest affinity towards dsDNA showed interesting anti proliferative potential and specificity
Food technology and …, 1999
The whole oxytetracycline (OTC) gene cluster was cloned from the strain Streptomyces rimosus R6. ... more The whole oxytetracycline (OTC) gene cluster was cloned from the strain Streptomyces rimosus R6. Its restriction map is indistinguishable from that of the strain S. rimosus M15883, which means that results from the two strains can probably be combined. Constructions to induce gene disruptions of otcD1 and otcC were undertaken within the chromosome of S. rimosus R6. OtcD1 is thought to be involved in the folding, cyclization and aromatization of the hypothetical nonaketide intermediate in OTC biosynthesis (pre-polyketide), whereas OtcC hydroxylates the completed tetracyclic nucleus at C-6 (post-polyketide). The disrupted strains no longer produced OTC. OtcC::gmr produced three novel compounds, while otcD1::ermE produced a number of compounds, not detectable in the wild-type strain. These novel compounds contained 9, 15 and 17 C-atoms in their backbones instead of 19, as with OTC. The disrupted strains did not have the expected DNA structures, but carried DNA amplifications and deletions. This is probably due to the fact that the otc cluster is located within an amplifiable unit of DNA (AUD) which undergoes frequent spontaneous DNA amplifications and deletions. Spontaneous mutants (Class II) that have deleted the otc cluster are potential hosts for expressing new hybrid polyketide clusters. However, it was possible to isolate derivatives (Class IIR) that produce an antibiotic activity. It was shown that this compound was oxytetracycline and that the Class II mutants themselves also produce very small amounts of oxytetracycline.
Chemistry - A European Journal, 2012
BJU International, 2012
Recently, we investigated the in vitro tumouritropic characteristics of several compounds with a ... more Recently, we investigated the in vitro tumouritropic characteristics of several compounds with a very high absorption coeffi cient under visible light. The underlying aim was the development of a diagnostic
BJU International, 2007
To investigate the influence of a glycosaminoglycan (GAG) layer on the specific location of hyper... more To investigate the influence of a glycosaminoglycan (GAG) layer on the specific location of hypericin in superficial urothelial carcinoma lesions of the bladder after intravesical instillation. Fisher rat bladders were incubated with 15 or 30 microm hypericin for 2 h. To examine the influence of the GAG layer on the permeation of hypericin, bladders were pre-treated with chondroitinase ABC, n-dodecyl-beta-d-maltoside (DDM) or sodium dodecyl sulphate (SDS) to disrupt, or protamine to neutralise the GAG layer before incubating with hypericin. After incubation, the photosensitizer permeation was examined quantitatively in cryostat sections of the bladders, using fluorescence microscopy and image analysis. Disrupting or neutralising the GAG layer in the bladder had no influence on the permeation of hypericin. Pre-treatment of the bladder with chondroitinase, DDM or SDS resulted in a significantly lower accumulation of hypericin, whereas neutralising the GAG layer in rats with protamine had no significant effect on the biodistribution of hypericin. The GAG matrix causes no obstacle to the permeation of hypericin in the urothelium of the bladder, and modification of this GAG layer cannot explain the enhanced accumulation of hypericin in superficial bladder tumours.
Bioorganic & Medicinal Chemistry, 2005
Hypericin, a naturally occurring hydroxylated phenanthroperylene dione, is used as a powerful pho... more Hypericin, a naturally occurring hydroxylated phenanthroperylene dione, is used as a powerful photosensitizer for photodynamic therapy as well as a diagnostic tool for the fluorescence detection of flat neoplastic lesions in the bladder of patients. Both applications are based on the tumouritropic characteristics of the compound. To get more insight into some of the physicochemical properties of hypericin affecting its tumouritropic characteristics, we set out to synthesize a series of more lipophilic hypericins. For this purpose, a synthetic pathway to hypericin acid amides with hydrocarbon chains of different lengths stably attached by an amide bond at position C10 was explored. Hypericin acid proved inert in amide forming reactions, whereas the precursor protohypericin acid showed higher reactivity and resulted in the desired amide derivatives, which afterwards can be easily converted into their phenanthroperylene dione form. Hexyl-, octyl-, decyl- and dodecylamides of hypericin acid were successfully synthesized in this way. In vitro cellular uptake and photo-induced antiproliferative effects of the compounds were evaluated, using the human moderately differentiated non-invasive papillary transitional carcinoma RT-112 cell line. Whereas the more lipophilic amides were taken up limitedly, the hexylamide accumulated approx. as well as hypericin itself. From the antiproliferative data it can further be concluded that not only the cellular uptake, but also the light-induced activity, is affected by the introduced structural changes.
Advanced Engineering Materials, 2011
Recently, various bio-medical applications of nanoporous silicon (np-Si) have been suggested. Thi... more Recently, various bio-medical applications of nanoporous silicon (np-Si) have been suggested. This work investigates the biocompatibility of np-Si particles taking into account hazardous residua confined in the pores after preparation. The emphasis is on the potential ...
ChemMedChem, 2013
To explore in more detail recently reported rare kinetic differentiation between homo-and alterna... more To explore in more detail recently reported rare kinetic differentiation between homo-and alternating AT-DNA by sterically restricted phosphonium dyesforming dimers within DNA minor groove, new analogues were prepared, whereby the quinolone-phosphonium part was kept constant while size, as well as hydrogen-binding properties of the rest of molecule were varied. Structure-activity relationship revealed that slight increase in length by additional methyl-resulted in loss of kinetic AT-selectivity but yielded AT-selective fluorescence response. These DNA/RNA groove bound dyes combined very low cytotoxicity with efficient cellular uptake and intriguingly specific fluorescent marking of mitochondria. At variance to longer analogues, decrease in length (by methyl-removal) and rearrangement of positive charge resulted in dyes switching to the intercalative binding mode to GC-DNA / ds-RNA but still forming dimers in minor groove AT sequences, consequently yielding significantly different chirooptical response. Latter dyes also revealed strongly selective antiproliferative activity toward HeLa cancer cell lines.
Advanced Engineering Materials, 2011
Recently, various bio-medical applications of nanoporous silicon (np-Si) have been suggested. Thi... more Recently, various bio-medical applications of nanoporous silicon (np-Si) have been suggested. This work investigates the biocompatibility of np-Si particles taking into account hazardous residua confined in the pores after preparation. The emphasis is on the potential ...
International journal of oncology, 2007
Previous studies have shown that hypericin is an excellent diagnostic tool for the fluorescence d... more Previous studies have shown that hypericin is an excellent diagnostic tool for the fluorescence detection of carcinoma in situ in the human bladder. The present work was performed to get a better insight into the mechanism of cellular uptake of hypericin (HYP) using RT-112 human papillary TCC cells of the bladder. Using lipophilic hypericin acid amide derivatives like hypericin acid hexylamide (AM6), hypericin acid octylamide (AM8) and hypericin acid dodecylamide (AM12), the effect of increased lipophilicity on the binding to serum proteins was investigated, as well as the cellular accumulation and permeation, both in 2-D and 3-D cell conditions. Density-gradient ultracentrifugation of the compounds pre-incubated with fetal bovine serum (FBS) showed that HYP and to a lesser extent AM6 predominantly bind to LDL, whereas AM8 and especially AM12 preferably associate with HDL. The cellular accumulation of the compounds did not significantly differ when LDL or AcLDL was supplemented to m...
Photonic Therapeutics and Diagnostics IV, 2008
ABSTRACT Hypericin (HYP) is used after instillation as a diagnostic tool for the fluorescence det... more ABSTRACT Hypericin (HYP) is used after instillation as a diagnostic tool for the fluorescence detection of CIS in the human bladder. In this study the in vitro cellular accumulation and intraspheroidal distribution of HYP and three analogues (OH1, OH2, OH3) with gradually increasing hydrophilicity were studied. E-cadherin negative (T24-C1-) and E-cadherin positive (T24-H3++) human bladder cancer cells were used. We report that in the presence of FBS all compounds were taken up by the monolayer cells to the same limited extent, whereas the overall intracellular accumulation was substantially higher when the incubation of the different dyes took place using cell medium not supplemented with FBS. The results of this study therefore confirm the competition between cellular uptake of HYP and analogues and binding to FBS constituents. Investigating the permeation of the compounds in spheroids, it was found that all HYP analogues diffused dramatically better through the three-dimensional cell layers than HYP itself. This enhanced ability of hydrophilic HYP analogues to permeate through the cell layers in the presence of FBS can be explained in terms of a preferred binding to HDL as compared to LDL. The results further show that all compounds, including LDL-binding HYP, substantially permeated better in T24-C1- spheroids than in T24-H3++ spheroids. The data therefore support the hypothesis that a lowered cellular cohesion is the key to understand the selective uptake of hypericin and its analogues in malignant urothelial cells.
The Journal of Urology, 2008
To obtain unambiguous evidence for the putative role of E-cadherin in the selective accumulation ... more To obtain unambiguous evidence for the putative role of E-cadherin in the selective accumulation of hypericin after intravesical instillation in humans we investigated the accumulation of hypericin in spheroids from 3 clones of the human bladder carcinoma cell line T-24 that express different levels of E-cadherin, as determined by immunohistochemistry and reverse transcriptase-polymerase chain reaction. Clones of T-24 cells transfected with the E-cadherin gene were analyzed for E-cadherin expression and 3 cell lines with different expression levels were selected. Spheroids of these cell lines were incubated with 10 microM hypericin in cell culture medium supplemented or not with fetal calf serum for 2 hours. After the incubation period centrally cut sections were examined by fluorescence microscopy. An imaging software system was used to measure average fluorescence in concentric layers from rim to center. Data showed that in the presence of serum the accumulation of hypericin in spheroids was inversely associated with the level of E-cadherin expressed by the T-24 transfectants used, whereas in the absence of serum differential accumulation of the compound was completely abolished. Spheroids composed of cancer cell lines expressing variable levels of E-cadherin represent an excellent model in which to study the role of intercellular adhesion in bladder cancer. The outcome of this study strongly suggests that E-cadherin is the key mediator in the selective accumulation of hypericin in superficial bladder cancer after intravesical instillation in humans.
Dyes and Pigments, 2012
ABSTRACT Six asymmetric monomethine cyanine dyes have been synthesized and their spectral charact... more ABSTRACT Six asymmetric monomethine cyanine dyes have been synthesized and their spectral characteristics and interaction with double stranded (ds)DNA have been investigated for their prospective use as fluorescent markers in molecular biology. Therefore, the non-covalent binding of the compounds with dsDNA was explored. Apart from the fluorescence spectroscopy, the study includes UV/Vis spectrophotometry and circular dichroism spectroscopy, as well as the thermal melting experiments. Although the compounds show relatively low binding affinitiy towards dsDNA, do not have intrinsic fluorescence, however in the presence of dsDNA they exhibited enhancement in fluorescence intensity. Therefore the studied dyes show interesting platform for future modifications directed towards more sequence selective derivatives. The compound with the highest affinity towards dsDNA showed interesting anti proliferative potential and specificity
Food technology and …, 1999
The whole oxytetracycline (OTC) gene cluster was cloned from the strain Streptomyces rimosus R6. ... more The whole oxytetracycline (OTC) gene cluster was cloned from the strain Streptomyces rimosus R6. Its restriction map is indistinguishable from that of the strain S. rimosus M15883, which means that results from the two strains can probably be combined. Constructions to induce gene disruptions of otcD1 and otcC were undertaken within the chromosome of S. rimosus R6. OtcD1 is thought to be involved in the folding, cyclization and aromatization of the hypothetical nonaketide intermediate in OTC biosynthesis (pre-polyketide), whereas OtcC hydroxylates the completed tetracyclic nucleus at C-6 (post-polyketide). The disrupted strains no longer produced OTC. OtcC::gmr produced three novel compounds, while otcD1::ermE produced a number of compounds, not detectable in the wild-type strain. These novel compounds contained 9, 15 and 17 C-atoms in their backbones instead of 19, as with OTC. The disrupted strains did not have the expected DNA structures, but carried DNA amplifications and deletions. This is probably due to the fact that the otc cluster is located within an amplifiable unit of DNA (AUD) which undergoes frequent spontaneous DNA amplifications and deletions. Spontaneous mutants (Class II) that have deleted the otc cluster are potential hosts for expressing new hybrid polyketide clusters. However, it was possible to isolate derivatives (Class IIR) that produce an antibiotic activity. It was shown that this compound was oxytetracycline and that the Class II mutants themselves also produce very small amounts of oxytetracycline.
Chemistry - A European Journal, 2012
BJU International, 2012
Recently, we investigated the in vitro tumouritropic characteristics of several compounds with a ... more Recently, we investigated the in vitro tumouritropic characteristics of several compounds with a very high absorption coeffi cient under visible light. The underlying aim was the development of a diagnostic
BJU International, 2007
To investigate the influence of a glycosaminoglycan (GAG) layer on the specific location of hyper... more To investigate the influence of a glycosaminoglycan (GAG) layer on the specific location of hypericin in superficial urothelial carcinoma lesions of the bladder after intravesical instillation. Fisher rat bladders were incubated with 15 or 30 microm hypericin for 2 h. To examine the influence of the GAG layer on the permeation of hypericin, bladders were pre-treated with chondroitinase ABC, n-dodecyl-beta-d-maltoside (DDM) or sodium dodecyl sulphate (SDS) to disrupt, or protamine to neutralise the GAG layer before incubating with hypericin. After incubation, the photosensitizer permeation was examined quantitatively in cryostat sections of the bladders, using fluorescence microscopy and image analysis. Disrupting or neutralising the GAG layer in the bladder had no influence on the permeation of hypericin. Pre-treatment of the bladder with chondroitinase, DDM or SDS resulted in a significantly lower accumulation of hypericin, whereas neutralising the GAG layer in rats with protamine had no significant effect on the biodistribution of hypericin. The GAG matrix causes no obstacle to the permeation of hypericin in the urothelium of the bladder, and modification of this GAG layer cannot explain the enhanced accumulation of hypericin in superficial bladder tumours.
Bioorganic & Medicinal Chemistry, 2005
Hypericin, a naturally occurring hydroxylated phenanthroperylene dione, is used as a powerful pho... more Hypericin, a naturally occurring hydroxylated phenanthroperylene dione, is used as a powerful photosensitizer for photodynamic therapy as well as a diagnostic tool for the fluorescence detection of flat neoplastic lesions in the bladder of patients. Both applications are based on the tumouritropic characteristics of the compound. To get more insight into some of the physicochemical properties of hypericin affecting its tumouritropic characteristics, we set out to synthesize a series of more lipophilic hypericins. For this purpose, a synthetic pathway to hypericin acid amides with hydrocarbon chains of different lengths stably attached by an amide bond at position C10 was explored. Hypericin acid proved inert in amide forming reactions, whereas the precursor protohypericin acid showed higher reactivity and resulted in the desired amide derivatives, which afterwards can be easily converted into their phenanthroperylene dione form. Hexyl-, octyl-, decyl- and dodecylamides of hypericin acid were successfully synthesized in this way. In vitro cellular uptake and photo-induced antiproliferative effects of the compounds were evaluated, using the human moderately differentiated non-invasive papillary transitional carcinoma RT-112 cell line. Whereas the more lipophilic amides were taken up limitedly, the hexylamide accumulated approx. as well as hypericin itself. From the antiproliferative data it can further be concluded that not only the cellular uptake, but also the light-induced activity, is affected by the introduced structural changes.
Advanced Engineering Materials, 2011
Recently, various bio-medical applications of nanoporous silicon (np-Si) have been suggested. Thi... more Recently, various bio-medical applications of nanoporous silicon (np-Si) have been suggested. This work investigates the biocompatibility of np-Si particles taking into account hazardous residua confined in the pores after preparation. The emphasis is on the potential ...
ChemMedChem, 2013
To explore in more detail recently reported rare kinetic differentiation between homo-and alterna... more To explore in more detail recently reported rare kinetic differentiation between homo-and alternating AT-DNA by sterically restricted phosphonium dyesforming dimers within DNA minor groove, new analogues were prepared, whereby the quinolone-phosphonium part was kept constant while size, as well as hydrogen-binding properties of the rest of molecule were varied. Structure-activity relationship revealed that slight increase in length by additional methyl-resulted in loss of kinetic AT-selectivity but yielded AT-selective fluorescence response. These DNA/RNA groove bound dyes combined very low cytotoxicity with efficient cellular uptake and intriguingly specific fluorescent marking of mitochondria. At variance to longer analogues, decrease in length (by methyl-removal) and rearrangement of positive charge resulted in dyes switching to the intercalative binding mode to GC-DNA / ds-RNA but still forming dimers in minor groove AT sequences, consequently yielding significantly different chirooptical response. Latter dyes also revealed strongly selective antiproliferative activity toward HeLa cancer cell lines.