Marin Alexandrov

Electrospun Nanofibrous Mats Containing Quaternized Chitosan and Polylactide with In Vitro Antitumor Activity against HeLa Cells

Nanofibrous materials containing the antitumor drug doxorubicin hydrochloride (DOX) were easily prepared using a one-step method by electrospinning of DOX/poly(L-lactide-co-D,L-lactide) (coPLA) and DOX/quaternized chitosan (QCh)/coPLA solutions. The pristine and DOX-containing mats were characterized by ATR-FTIR and X-ray photoelectron spectroscopy (XPS). The release rate of DOX from the prepared fibers increased with the increase in DOX content. The DOX release process was diffusion-controlled. MTT cell viability studies revealed that incorporation of DOX and QCh in the nanofibrous mats led to a significant reduction in the HeLa cells viability. It was found, that the antitumor efficacy of the DOX-containing mats at 6 h was higher than that of the free DOX. SEM, TEM, and fluorescence microscopic observations confirmed that the antitumor effect of QCh-based and DOX-containing fibrous mats was mainly due to induction of apoptosis in the HeLa cells.

Antitumor activity of quaternized chitosan-based electrospun implants against Graffi myeloid tumor

Nanofibrous implants containing quaternized chitosan (QCh), poly(L-lactide-co-D,L-lactide) (coPLA), and the antitumor drug doxorubicin (DOX) were fabricated by electrospinning. The surface chemical composition and the morphology of the implants were characterized by XPS and SEM. In vitro cell viability studies demonstrated that QCh- and DOX-based implants exhibited high cytotoxicity against Graffi tumor cells. The implants efficiently inhibited the growth of Graffi tumor in hamsters with minimum weight loss. Insertion of QCh/coPLA/DOX implants in the place of removed tumor led to an increase in the animal survival rate and to a decrease in the percentage of recurrences.

Quaternized chitosan-coated nanofibrous materials containing gossypol: preparation by electrospinning, characterization and antiproliferative activity towards HeLa cells.

Nanofibrous polylactide-based materials loaded with a natural polyphenolic compound gossypol (GOS) with antitumor properties were prepared by electrospinning. The nanofibrous materials were coated with a thin film of crosslinked quaternized chitosan (QCh). GOS incorporated in the nanofibrous mats was in the amorphous state. GOS release was diffusion-controlled and its in vitro release profiles depended on the mat composition. The nanofibrous materials exhibited high cytotoxicity towards HeLa tumor cells. Interestingly, it was particularly pronounced in the case of fibrous materials, which contain both QCh and GOS. The observed strong antiproliferative effect of the nanofibrous mats was mainly due to induction of cell apoptosis.

Antiproliferative activity of nanofibers containing quaternized chitosan and/or doxorubicin against MCF-7 human breast carcinoma cell line by apoptosis:

The antiproliferative activity of electrospun mats of poly(L-lactide-co-D,L-lactide) (coPLA) containing quaternized chitosan (QCh) and/or doxorubicin hydrochloride (DOX) was evaluated against the Michigan Cancer Foundation-7(MCF-7) human breast carcinoma cell line. QCh- and DOX-containing nanofibrous mats possess good antiproliferative activity and decrease considerably the viability of the MCF-7 cells for the different periods of cell incubation as confirmed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Fluorescent microscopy analyses and scanning electron microscopy observations revealed that apoptosis was one of the major mechanisms of MCF-7 cell death induced by the QCh- and DOX-containing mats.

Some pathological features of lungs from domestic and wild ruminants with single and mixed protostrongylid infections.

Lungs of 40 ruminants from Bulgaria with natural small lungworm (Nematoda: Protostrongylidae) infections were investigated, including 16 goats, 15 sheep, 7 mouflons, and 2 chamois. Muellerius capillaris, M. tenuispiculatus, Cystocaulus ocreatus, Neostrongylus linearis, and Protostrongylus brevispiculum infections were predominantly associated with nodular lesions, and Protostrongylus rufescens, Protostrongylus hobmaieri and Protostrongylus rupicaprae were associated with extensive lesions located mainly along the length of the large bronchi. The extent of lung abnormalities was most severe in the sheep. Alveolitis, parasite granulomas, damage of the alveolar septa, hyperplasia of the lung associated lymphoid tissue, and sclerosis of the parenchyma were found upon microscope examinations. In the goats compared to the sheep and mouflons, the terminal bronchi, bronchioles, and alveoli were more affected than the interstitium. Our research shows that the pathological lesions in the lungs of ruminants infected with protostrongylids depend on both the helminth and the host species. To our knowledge, this work is the first to provide data on the pathomorphological lesions in mouflon lungs infected with protostrongylids.

Electron- and immunoelectron-microscopic investigation on the rabbit haemorrhagic disease virus.

Electron- and immunoelectron-microscopic methods were used for detection and investigation of rabbit haemorrhagic disease virus (RHDV). The observed virus particles were uncoated, with an icosahedral symmetry, with a 35-40 nm dia and capsids built up by 32 capsomers with a central hollow part and a 3.5-5 nm dia. Particles with empty capsids could also be seen among them. Single virions visible in 5-fold symmetry, exhibited 10 projections on the surface. Virions with a 33-34 nm dia and cup-shaped surface depressions were a rare finding. Some of these particles were indistinguishable from the caliciviruses, but in most of them the cup-shaped depressions were single or no more then 2-3 in number, localized predominantly unilaterally or asymmetrically. Particles 25-29 nm large with full or empty capsids without capsomers could also be seen. Most probably they were some immature forms in the process of development of the RHDV.

Fluorescent and electron-microscopy immunoassays employing polyclonal and monoclonal antibodies for detection of goose parvovirus infection

Polyclonal antibodies (PAbs) raised in geese and eight mice hybridomas secreting monoclonal antibodies (MAbs) against the goose parvovirus (GPV) were prepared. They were used for development of immunofluorescence (IF) and immunoelectron-microscopic (IEM) techniques to demonstrate the GPV infection in infected organs and biological fluids. The GPV antigens were established by immunofluorescence within the nuclei and the cytoplasm of many infected cells of the chorioallantoic membrane of goose and Peckin duck embryos, liver and heart of mortally diseased goslings. By means of IEM it was possible to detect the GPV in native organ homogenate supernatants and allantoic fluids. All techniques used in the study could be successfully applied for rapid diagnosis of the GPV infection. The test systems on the basis of MAbs should, however, be preferred. By means of immunoblotting (IB) using PAbs and MAbs four viral proteins (VP) with MW 88, 77, 65 and 60 kDa were demonstrated. Contrary to the others the VP with MW 65 kDa was the most antigenically reactive though invisible in the SDS-PAGE and Coomassie-blue dye-stained preparations.

Morphometric characteristics of first stage Elaphostrongylus cervi (Nematoda: Protostrongylidae) larvae from Bulgaria

A description of the morphology of first stage Elaphostrongylus cervi (Nematoda: Protostrongylidae) larvae on materials from Bulgaria is presented. The larvae were obtained from a red deer (Cervus elaphus L.) from the wildlife-breeding station “Vitinya” in the Balkan Mountains. The metric results about the larvae in micrometers are as follows: body length −423 (377–473), maximum body width −20 (18–24), esophagus length −193 (172–217), distance from the anterior body end to the excretory pore −110 (85–134), distance from the anterior body end to the genital premordium −281 (218–324), distance from the anus to the tail tip −37 (32–42), and tail length −11 (10–14). The present data were compared with previous reports on the same species in West and Central Europe. This is the first morphometric description of first stage E. cervi larvae from Bulgaria and Southeast Europe.

Cytostatic and cytotoxic properties of monensic acid and its biometal(II) complexes against human tumor / non-tumor cell lines

AbstractThe anticancer activity of monensic acid (MonH) and its biometal(II) complexes [M(Mon)2(H2O)2](M = Mg, Ca, Mn, Co, Ni, Zn) was evaluated against cultured human permanent cell lines established from glioblastoma multiforme (8MGBA) and cancers of the lung (A549), breast (MCF-7), uterine cervix (HeLa) and liver (HepG2). The viability and proliferation of the non-tumor human embryonic cell line Lep3 was also tested. The investigations were carried out using a thiazolyl blue tetrazolium bromide test, neutral red uptake cytotoxicity assay, crystal violet staining, colony forming method and double staining with acridin orange and propidium iodide. The results obtained reveal that the compounds applied at concentrations of 0.5–25 µg mL−1 for 24–72 h decrease the viability and proliferation of the treated cells in a time- and concentration-dependent manner. The metal(II) complexes studied (especially those of Co(II), Ni(II) and Zn(II)) have been found to express stronger cytotoxic and cytostatic activities than the non-coordinated monensic acid. The non-tumor human cell line showed strong chemosensitivity towards compounds tested comparable to that of cultured human tumor cell lines.

Quaternized chitosan-coated nanofibrous implants loaded with gossypol prepared by electrospinning and their efficacy against Graffi myeloid tumor

Nanofibrous poly(L-lactide-co-D,L-lactide) (coPLA) or coPLA/poly(ethylene glycol) implants loaded with plant polyphenolic compound gossypol (GOS) with anti-tumor activity were fabricated by electrospinning. Implants containing quaternized chitosan (QCh) were prepared by coating of the obtained fibrous materials with a thin film of cross-linked QCh. The morphology of the implants and chemical composition of the implant surface were studied by means of scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). In vitro cytotoxicity assay showed that GOS-loaded nanofibrous implants, both non-coated and QCh-coated displayed about two-fold higher inhibitory activity against Graffi tumor cells than that of free GOS at the 72nd h of incubation. As evidenced by the performed fluorescence microscopy analyses and SEM observations, the anti-tumor activity of the fibrous implants was mainly due to induction of apoptosis. The experiments in which the implants containing both QCh and GOS were placed locally into the tumor site after the tumor extirpation showed an increase in the survival rate and a lower rate of recurrence in the operative field and of metastases in regional lymph nodes. In this case, 40% of hamsters were alive on the 45th day of implantation and they did not show any clinical sign of recurrence in the operative field and metastases in the regional lymph nodes.