RESUMO
OBJECTIVE: To compare the effects of glucosamine (GlcN), curcumin, and diacerein in immortalized human C-28/I2 chondrocytes at the cellular and the gene expression level. This study aimed to provide insights into the proposed beneficial effects of these agents and to assess the applicability of the C-28/I2 cell line as a model for the evaluation of chondroprotective action. METHODS: Interleukin-1beta (IL-1beta)-stimulated C-28/I2 cells were cultured in the presence of GlcN, curcumin, and diacerein prior to the evaluation of parameters such as viability, morphology and proliferation. The impact of GlcN, curcumin, and diacerein on gene expression was determined using quantitative real-time RT-PCR (qPCR). RESULTS: At the transcriptional level, 5 mM GlcN and 50 microM diacerein increased the expression of cartilage-specific genes such as aggrecan (AGC) and collagen type II (COL2), while reducing collagen type I (COL1) mRNA levels. Moreover, the IL-1beta-mediated shift in gene expression pattern was antagonized by GlcN and diacerein. These effects were associated with a significant reduction in cellular proliferation and the development of chondrocyte-specific cell morphology. In contrast, curcumin was not effective at lower concentrations but even damaged the cells at higher amounts. CONCLUSIONS: Both GlcN and diacerein promoted a differentiated chondrocytic phenotype of immortalized human C-28/I2 chondrocytes by altering proliferation, morphology, and COL2/COL1 mRNA ratios. Moreover, both agents antagonized inhibitory effects of IL-1beta by enhancing AGC and COL2 as well as by reducing COL1 mRNA levels.
Assuntos
Antraquinonas/farmacologia , Condrócitos/efeitos dos fármacos , Curcumina/farmacologia , Glucosamina/farmacologia , Osteoartrite/metabolismo , Substâncias Protetoras/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas/metabolismo , Expressão Gênica/efeitos dos fármacos , Glucosamina/genética , Humanos , Interleucina-1beta/genética , Modelos Biológicos , Osteoartrite/genética , Reação em Cadeia da PolimeraseRESUMO
The present study was performed to evaluate the applicability of plant lectins as mediators of bioadhesion in cartilage research using human chondrocyte cell lines C-28/I2 and T/C-28a2. The bioadhesive properties of fluorescein-labelled lectins with different carbohydrate specificities were investigated by flow cytometry. Specificity of the lectin-cell interactions was ascertained by competitive inhibition using complementary carbohydrates. As compared to that of other lectins, the interaction between wheat germ agglutinin (WGA) and chondrocytic cells was characterised by remarkable cytoadhesion, adequate binding strength and a high degree of specificity for N-acetyl-glucosamine as contained in hyaluronan chains. We therefore suggest WGA to be a promising candidate for mediating bioadhesion to low-adhesive scaffolds in cartilage tissue engineering. Moreover, the WGA-association rate of C-28/I2 and T/C-28a2 cells was dependent on temperature indicating cellular uptake of membrane-bound WGA. Intracellular enrichment was confirmed by confocal microscopy. Equilibration of intracellular pH gradients with monensin resulted in the reversal of quenching effects indicating accumulation of WGA within acid compartments of chondrocytic cells. Thus, WGA might be internalised into chondrocytes together with hyaluronan via the CD44 receptor-mediated endocytosis pathway and accumulated within lysosomes. This physiological process could represent a feasible pathway to target WGA-functionalised drug delivery devices into chondrocytes.
Assuntos
Cartilagem/fisiologia , Condrócitos/metabolismo , Sistemas de Liberação de Medicamentos , Engenharia Tecidual , Acetilglucosamina , Ligação Competitiva/efeitos dos fármacos , Carboidratos/química , Cartilagem/citologia , Adesão Celular , Linhagem Celular , Sobrevivência Celular , Citometria de Fluxo , Humanos , Ácido Hialurônico/química , Lectinas , Microscopia Confocal , Aglutininas do Germe de TrigoRESUMO
In the management of bladder cancer, surgical resection of the tumour is usually followed by intravesical instillation of immunomodulatives and/or chemotherapeutics. The purpose of this local intravesical therapy is to eliminate residual malignant cells after surgical intervention. The main limitation of a localised adjuvant therapy is the insufficient concentration of the active pharmaceutical ingredient (API) in malignant cells due to the unique structure of the human urothelium making it an exclusively hard to overcome barrier in the human body. Different strategies such as electromotive drug administration or local hyperthermia are employed to ameliorate intravesical drug uptake. Previous studies on biorecognitive targeting showed promising results for lectin-, especially wheat germ agglutinin (WGA), mediated drug delivery. Here, we present a targeted conjugate that provides enough binding sites for a possible API as well as high cytoadhesive and cytoinvasive potential. The conjugate should comprise the following components: First WGA, as the targeting moiety, second poly-l-glutamic acid (PGA) as a polymeric backbone providing more than 300 possible binding sites for an API and third fluorescein cadaverine (Fc), a fluorescent dye we coupled to PGA rendering the conjugate traceable. After purification via size exclusion chromatography (SEC) the WGA containing and therefore binding conjugate was isolated from the reaction mix. In flow-cytometric and fluorimetric experiments with single cells and cell monolayers, respectively, binding and internalisation of the conjugate representing a high molecular weight (>100kDa) could be demonstrated. Fluorescent PGA without the WGA component showed neither binding nor internalisation potential. Microscopic colocalization studies with cell monolayers and single cells confirmed the cytoadhesive and cytoinvasive potential of the WGA containing conjugate. In accordance with the results of specificity studies the interaction between the conjugate and the cell surface depended solely on the WGA component of the conjugate. With the help of this targeted drug delivery system limiting factors of intravesical adjuvant therapy could eventually be overcome and thereby treatment efficacy of instillative chemotherapy could be improved. In addition, traceable drug conjugates might bear interesting advantages for theranostic purposes in the treatment of bladder cancer.
Assuntos
Ácido Glutâmico/química , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/química , Polímeros/química , Neoplasias da Bexiga Urinária/tratamento farmacológico , Aglutininas do Germe de Trigo/química , Administração Intravesical , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Humanos , Lectinas/química , Bexiga Urinária/efeitos dos fármacos , Urotélio/efeitos dos fármacosRESUMO
There is an increasing interest in cell-based microelectronic biosensors for high-throughput screening of new products from the biotech pipeline. This requires fundamental knowledge of the biocompatibility of the materials used as the growing support for the cells. Using monolayer-forming Caco-2 cells of human origin, the biocompatibility of silicon wafers coated with various metals, dielectrics and semiconductors was assessed. Besides microscopic inspection, proliferation of cells indicating viability as well as brush border enzyme activity indicating differentiation of adherent growing cells were chosen as parameters to estimate biocompatibility. The type of wafer used for deposition of the coating initially influences the biocompatibility of the final product. Whereas p-doped silicon was fully biocompatible, n-doped silicon reduced the proliferation of cells. Among the different coatings, Al and Ti even increased the cell growth as compared to glass. Culturing the cells for 6 days on coated wafers demonstrated that the differentiation of adhering cells on Ti- and ZrO2-coated wafers was comparable to glass, whereas coatings with Si3N4, Au, Al, and ITO reduced differentiation to 15-35%. In the cases of Au and Si3N4 this effect equilibrated with prolonged culturing. These results demonstrate the importance of a careful selection of the materials used for the production of cell-based biosensors.
Assuntos
Materiais Biocompatíveis , Técnicas Biossensoriais/métodos , Fosfatase Alcalina/metabolismo , Divisão Celular , Linhagem Celular Tumoral , Eletrônica , Humanos , Cinética , Magnetismo , Microquímica , Microscopia de Força AtômicaRESUMO
In this study transcriptomic alterations of bacterially induced pattern triggered immunity (PTI) were compared with other types of tobacco-Pseudomonas interactions. In addition, using pharmacological agents we blocked some signal transduction pathways (Ca(2+) influx, kinases, phospholipases, proteasomic protein degradation) to find out how they contribute to gene expression during PTI. PTI is the first defense response of plant cells to microbes, elicited by their widely conserved molecular patterns. Tobacco is an important model of Solanaceae to study resistance responses, including defense mechanisms against bacteria. In spite of these facts the transcription regulation of tobacco genes during different types of plant bacterial interactions is not well-described. In this paper we compared the tobacco transcriptomic alterations in microarray experiments induced by (i) PTI inducer Pseudomonas syringae pv. syringae type III secretion mutant (hrcC) at earlier (6 h post inoculation) and later (48 hpi) stages of defense, (ii) wild type P. syringae (6 hpi) that causes effector triggered immunity (ETI) and cell death (HR), and (iii) disease-causing P. syringae pv. tabaci (6 hpi). Among the different treatments the highest overlap was between the PTI and ETI at 6 hpi, however, there were groups of genes with specifically altered activity for either type of defenses. Instead of quantitative effects of the virulent P. tabaci on PTI-related genes it influenced transcription qualitatively and blocked the expression changes of a special set of genes including ones involved in signal transduction and transcription regulation. P. tabaci specifically activated or repressed other groups of genes seemingly not related to either PTI or ETI. Kinase and phospholipase A inhibitors had highest impacts on the PTI response and effects of these signal inhibitors on transcription greatly overlapped. Remarkable interactions of phospholipase C-related pathways with the proteasomal system were also observable. Genes specifically affected by virulent P. tabaci belonged to various previously identified signaling routes, suggesting that compatible pathogens may modulate diverse signaling pathways of PTI to overcome plant defense.
RESUMO
Serum-free growth of Ewing's sarcoma (ES) and primitive peripheral neuroectodermal tumour (pPNET) cell lines was achieved by supplementing a basal medium with insulin-like growth factor-I (IGF-I). These cultures were used to investigate the sensitivity of 3 ES (EW-2, RD-ES, SK-ES-1) and 3 pPNET (SIM-1, KAL, SAL) cell lines to a panel of anti-tumour agents in short-term (48-h) proliferation assays. Of the four cytostatic drugs included in the currently used multi-drug regimens, cyclophosphamide, doxorubicin and actinomycin-D inhibit the proliferation of the cell lines with high efficacy, whereas the vinca alkaloids were less effective. Cisplatin, etoposide, mitomycin-C and mitoxanthrone were also found to have a high inhibitory activity in this in vitro ES/pPNET system. The most remarkable effect was observed for cytosine arabinoside (ARA-C), which gave a half-maximal inhibition at drug concentrations approximately 5000 times below the clinical peak plasma concentrations (250 micrograms/ml). The ARA-C sensitivity of ES and pPNET cell lines is comparable with the established ARA-C sensitivities of leukaemia-derived cells. The different ES and pPNET cell lines showed a rather uniform response to the different cytostatic drugs with decreased sensitivity of individual pPNET cell lines to vinblastin, ARA-C and mitoxanthrone. Modulation of the IGF-I/IGF-I receptor/IGF-I binding protein system, which seems to constitute an important stimulator of cell growth in neuroectoderm-derived or -related tumours, can be used to enhance the drug sensitivity of the tumour cells in vivo or in vitro therapeutic procedures. According to our results, serum-free conditions for autologous bone marrow purification are expected to result in significantly increased chemosensitivity of ES and pPNET cells in response to anthracyclines and cisplatin.
Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Neoplasias Embrionárias de Células Germinativas/metabolismo , Sarcoma de Ewing/metabolismo , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Citarabina/farmacologia , Humanos , Mitose/efeitos dos fármacos , Neoplasias Embrionárias de Células Germinativas/tratamento farmacológico , Neoplasias Embrionárias de Células Germinativas/patologia , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/patologia , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
UNLABELLED: An expert system was developed that interprets ventilation-perfusion lung scans. The use of such scans for suspected pulmonary embolism is ideal for computer-assisted diagnosis by expert systems. The data are digital, only a single disease entity is diagnosed or excluded, and well-established diagnostic criteria already exist for visual interpretation that can be easily integrated into an expert system. METHODS: This expert system is divided into two modules. The first module is responsible for image analysis. Analysis was performed on the eight standard perfusion images and on single-breath, equilibrium and 3-min washout ventilation images. Each image was analyzed for the presence of regional perfusion or ventilation defects, as determined by pixel values that fell 2.2 s.d. below the mean (or above the mean in the case of washout images) compared with a database of normal studies. The defect size, segment involved and number of defects were determined. Ventilation and perfusion images were then compared to determine whether defects were matched or mismatched. The second program module applied the modified Biello's criteria to the data and categorized the scan as normal to low, intermediate or high probability. RESULTS: A total of 80 patients were prospectively studied. An 81% (65 of 80) correlation was obtained when the results of the expert system were compared with visual interpretations made by three experienced nuclear medicine physicians. CONCLUSION: This study shows that the interpretation of ventilation-perfusion lung scans by an expert system is possible. The technique holds the promise of reducing interobserver variability and assisting less experienced observers in the interpretation of such scans.
Assuntos
Sistemas Inteligentes , Processamento de Imagem Assistida por Computador , Embolia Pulmonar/diagnóstico por imagem , Relação Ventilação-Perfusão , Humanos , Estudos Prospectivos , Cintilografia , Software , Agregado de Albumina Marcado com Tecnécio Tc 99m , Radioisótopos de XenônioRESUMO
A patient with known aortoiliac occlusive disease and hypertension suffered a cerebrovascular accident 6.5 min after the administration of intravenous dipyridamole during a 201Tl myocardial study. Despite aminophylline administration, the patient developed a completed stroke. The mechanism most likely responsible for precipitating this patient's stroke is dipyridamole-induced vascular steal. Although dipyridamole-201Tl myocardial imaging is relatively free of major complications, the morbidity and mortality associated with a cerebrovascular accident is significant. The possibility of precipitating a cerebrovascular accident during dipyridamole-201Tl imaging should be considered in all patients with significant risk factors for stroke before performing a dipyridamole cardiac study.
Assuntos
Transtornos Cerebrovasculares/induzido quimicamente , Dipiridamol/efeitos adversos , Coração/diagnóstico por imagem , Tálio , Dipiridamol/administração & dosagem , Humanos , Injeções Intravenosas , Masculino , Pessoa de Meia-Idade , Cintilografia , Fatores de RiscoRESUMO
UNLABELLED: The purpose of this study was to determine the efficacy of 111In-polyclonal immunoglobulin (IgG) for the diagnosis of infection or inflammation. METHODS: Fifty-three patients with suspected infection were prospectively studied. Each underwent an 111In-polyclonal IgG study; biopsy, surgery, additional nuclear medicine scans and radiographic studies were used to confirm the IgG scan results. RESULTS: The polyclonal IgG scan had a sensitivity of 97.9% and a specificity of 94% for infection or inflammation. When only infection or severe inflammation such as bowel infarction was considered, the sensitivity remained the same but the specificity fell to 83%. Chronic infections were detected equally as well as acute infections. Antibiotics, steroids, anti-inflammatory agents, diabetes and diminished renal function did not affect scan sensitivity. There were no adverse reactions to the radiopharmaceutical. Three patients underwent extended imaging. Their scans stayed positive for an average of 8 days. Three patients treated for infection had their scans turn negative on repeat study, confirming the efficacy of their antibiotic therapy. CONCLUSION: Indium-111-polyclonal IgG is an effective imaging agent of infection and/or inflammation that is useful in a variety of infections and in severe inflammatory diseases. The ease of preparation and safety make it an attractive alternative to labeled leukocytes.
Assuntos
Imunoglobulina G , Radioisótopos de Índio , Infecções/diagnóstico por imagem , Inflamação/diagnóstico por imagem , Ácido Pentético/análogos & derivados , Radioimunodetecção , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Infecções/tratamento farmacológico , Inflamação/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
The pseudoautosomal encoded MIC2 glycoprotein is a tumor-associated antigen of Ewing's sarcoma (ES) and closely related tumors of unknown function. To investigate the use of this protein as selective drug carrier recombinant MIC2 was coupled to doxorubicin by a two step glutaraldehyde method (molar ratio DOX/MIC2 of 32 and 16). The conjugates showed dose-dependent cytostatic activity against the ES cell line SK-ES1, the peripheral neuroectodermal line KAL and the prostate cancer cell line PC-3 concurrent with reduced toxicity against normal lymphoblasts. In comparison to free doxorubicin the MIC2-doxorubicin conjugates exhibited highest activity against the PC-3 cell line. Confocal microscopy showed intracellular accumulation of MIC2 conjugates.
RESUMO
In order to take advantage of the biorecognition between lectins and specific carbohydrates for targeted drug delivery, fluorescein-labelled lectins of different carbohydrate specificities were screened for binding to human colorectal carcinoma cell lines by flow cytometry and confocal microscopy. The lectin-binding rate increased as follows: Dolichos biflorus agglutinin, DBAAssuntos
Adesão Celular
, Mucosa Intestinal/metabolismo
, Lectinas/metabolismo
, Células CACO-2
, Células HT29
, Humanos
, Intestinos/citologia
, Microscopia Confocal
, Microscopia de Fluorescência
, Lectinas de Plantas
, Plantas/metabolismo
, Ligação Proteica
, Células Tumorais Cultivadas
RESUMO
As the mucous layer represents the first barrier to peroral lectin-mediated drug delivery, the influence of mucin on the cytoadhesive properties of lectins was studied in vitro by establishing a rapid and simple microplate format assay using pig gastric mucin (PGM) for coating the wells. The lectin-binding capacity of mucin followed the order WGA>>UEA-I>>LCA=STL>PNA>DBA. The PGM-binding of wheat germ agglutinin (WGA) was strongly dependent on pH being highest at pH 5.0. In comparison, PGM-binding of WGA was about 15% at gastric pH and 60-70% at intestinal pH. This points to unimpeded gastric transit of WGA-grafted formulations and favorable conditions within the intestine for binding to mucus coated enterocytes. Moreover the WGA-PGM interaction was concentration-dependent, specific and fully reversible. According to a competitive assay in the presence of Caco-2 monolayers, the PGM-binding of WGA was saturated and influenced by the lectin-concentration yielding 28% Caco-2 bound WGA (125 ng WGA/0.29 cm(2) monolayer) and 68% Caco-2 bound WGA (4 microg WGA/0.29 cm(2) monolayer), respectively. Following on from these results, lectins are expected to suffer at least partially from premature inactivation by shed off mucus like bioadhesives of the first generation, however initial but reversible mucus-binding of lectins offers partititioning to the cell membrane followed by uptake into the enterocyte.
Assuntos
Adesivos/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Lectinas/fisiologia , Mucinas/fisiologia , Aglutininas do Germe de Trigo/farmacocinética , Animais , Sítios de Ligação/fisiologia , Células CACO-2/metabolismo , Células CACO-2/fisiologia , Adesão Celular/fisiologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Concentração de Íons de Hidrogênio , SuínosRESUMO
Camptothecin (CPT) and its water-insoluble derivatives are known as topoisomerase-I inhibitors exhibiting high antitumoral activity against a wide spectrum of human malignancies. Until now clinical application of CPT is restricted by insolubility and instability of the drug in its active lactone form resulting in less antitumor potency and poor bioavailability. For these reasons CPT-loaded-microspheres were prepared by the solvent evaporation method using the H-series of poly(D,L-lactide-co-glycolide) (H-PLGA), which contain more carboxylic acid end chains and hydrate faster than the non-H-series. At 1.2% CPT-payload the drug was molecular dispersed throughout the matrix whereas at higher CPT-payload the amount of crystalline CPT-islets increased with the CPT content. The release pattern of CPT was biphasic comprising a first burst effect delivering 20-35% of the payload and increasing with drug-loading. This phase was followed by sustained delivery of CPT releasing 40-75% of the payload within 160 h. In comparison to PLGA-microspheres, the CPT-release rate from H-PLGA was twofold higher and accelerated. The active CPT-lactone was maintained during preparation, storage and release due to hindered diffusion of acidic oligomers among other mechanisms. Thus stabilization and sustained release of CPT from PLGA-microspheres might reduce local toxicity combined with prolonged efficacy offering new perspectives in CPT chemotherapy.
Assuntos
Antineoplásicos Fitogênicos/química , Materiais Biocompatíveis/química , Camptotecina/química , Ácido Láctico/química , Ácido Poliglicólico/química , Polímeros/química , Antineoplásicos Fitogênicos/administração & dosagem , Materiais Biocompatíveis/administração & dosagem , Camptotecina/administração & dosagem , Preparações de Ação Retardada , Estabilidade de Medicamentos , Cinética , Ácido Láctico/administração & dosagem , Microesferas , Ácido Poliglicólico/administração & dosagem , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros/administração & dosagemRESUMO
The aim of this study was to evaluate the feasibility and in vitro activity of ligand-conjugates based on the use of poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA or Gantrez AN). Fluorescently labelled PVM/MA nanoparticles were prepared by desolvation and cross-linkage with 1,3-diaminopropane (DP). Conjugates were obtained by incubation between the carriers and Sambucus nigra agglutinin (SNA) for 1 h in an aqueous medium. The lectin binding to the surface of nanoparticles was increased by both increasing the bulk ligand concentration and decreasing the amount of cross-linker. However, a concentration of about 0.3-0.4 mg DP per mg polymer was necessary to obtain maximum agglutination activity. Under optimal conditions, the amount of fixed ligand was 46 microg/mg nanoparticle (binding efficiency of 86%); although the activity of SNA conjugates was 13.3 microg/mg particle. The activity of nanoparticles, measured by the association to Caco-2 monolayers, was higher when SNA was covalently bound onto their surface. The lectin-conjugate interaction was 6-fold higher than conventional nanoparticles. Moreover, energy-dependent mechanisms were only observed in SNA-PVM/MA particles. Finally, the decrease in association in the presence of lactose demonstrates that both SNA- and SNA-conjugate-binding was due to a true lectin-sugar interaction.
Assuntos
Maleatos/síntese química , Nanotecnologia/métodos , Polivinil/síntese química , Animais , Células CACO-2 , Bovinos , Humanos , Ligantes , Maleatos/metabolismo , Lectinas de Plantas/metabolismo , Polietilenos/metabolismo , Polivinil/metabolismo , Proteínas Inativadoras de Ribossomos , Soroalbumina Bovina/metabolismoRESUMO
A novel system for the packaging of drugs as well as vaccines is presented. Bacterial ghosts are intact, non-denatured bacterial envelopes that are created by lysis of bacteria through the expression of cloned phage PhiX174 gene E. Inhibition of induced E-mediated lysis by MgSO(4), harvesting of cells by centrifugation, and resuspension in low-ionic-strength buffers leads to rapid, violent lysis and results in empty bacterial envelopes with large (approximately 1 microm in diameter) openings. The construction of plasmid pAV1, which encodes a streptavidin fusion protein with an N-terminal membrane anchor sequence, allows the loading of the inner side of the cytoplasmic membrane with streptavidin. The functionality and efficacy of binding of even large biotinylated compounds in such streptavidin ghosts (SA-ghosts) was assessed using the enzyme alkaline phosphatase. The successful binding of biotinylated fluorescent dextran, as well as fluorescent DNA complexed with biotinylated polylysine, was demonstrated microscopically. The display by bacterial ghosts of morphological and antigenic surface structures of their living counterparts permits their attachment to target tissues such as the mucosal surfaces of the gastrointestinal and respiratory tract, and their uptake by phagocytes and M cells. In consequence, SA-ghosts are proposed as drug carriers for site-specific drug delivery.
Assuntos
Bactérias/metabolismo , Portadores de Fármacos , Veículos Farmacêuticos , Fosfatase Alcalina/metabolismo , Sítios de Ligação , Biotinilação , Membrana Celular , Fagocitose , Estreptavidina/metabolismoRESUMO
We investigated the efficiency and the mechanism of action of two--one symmetrically and one asymmetrically substituted--glycoconjugated tetraphenyl porphyrins in their photoreaction with T7 phage as a model of nucleoprotein (NP) complexes. A correlation was found between the dark inactivation of T7 and the binding of porphyrins determined by fluorescence spectroscopy. Both types of porphyrin sensitized the photoinactivation of T7, but the slopes of inactivation kinetics were markedly different. There was no correlation between the dark binding and the photosensitizing efficacy of the two derivatives. Inactivation was moderated by 1,3-diphenylisobenzofuran and 1,3-dimethyl-2-thiourea; however, neither of them inhibited T7 inactivation completely. This result suggests that both Type-I and Type-II reactions play a role in the virus inactivation. Optical melting studies revealed structural changes in the protein part but not in the DNA of the photochemically treated NP complex. Polymerase chain reaction analysis of a 555 bp segment of gene 1 and a 3826 bp segment of genes 3 and 4 failed to demonstrate any DNA damage.
Assuntos
Bacteriófago T7/efeitos dos fármacos , Bacteriófago T7/efeitos da radiação , Luz , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacologia , Escherichia coli/virologia , Fármacos Fotossensibilizantes/química , Porfirinas/química , Espectrometria de FluorescênciaRESUMO
The 3-5 year survival rates of patients with disseminated Ewing's sarcoma (ES) or the closely related peripheral primitive neuroectodermal tumors (PNET) remain low, even under aggressive treatment involving highly toxic multidrug chemotherapeutic regimens. ES and PNET are sensitive to doxorubicin, but may escape treatment by expression of the multidrug-resistant phenotype and/or other mechanisms. In this study, we have identified albumin as growth supporting factor for ES and PNET cells in IGF-I-supplemented serum-free tissue culture medium. To investigate the specificity and toxicity of albumin-based drug conjugates, doxorubicin was coupled to bovine serum albumin (BSA) by either a two step glutaraldehyde or carbodiimide-C4-spacer technique, yielding monomeric DOX-albumin conjugates with conjugation numbers ranging from 3-20 moles DOX/mole BSA. Cellular uptake of fluorescein-isothiocyanate-(FITC)-labeled albumin and DOX-albumin conjugates could be demonstrated by flow cytometric measurements of cell-associated fluorescence and confocal microscopy. The cytostatic activity of these conjugates against ES/PNET cell lines, a neuroblastoma (LAN-1) and prostate cancer carcinoma cell line (PC-3) and normal lymphoblasts was tested in short-term proliferation assays (48 h). The results show a high selectivity of the DOX-albumin conjugates for ES/PNET cell lines, with highest growth inhibition by conjugates with low DOX conjugation numbers (n = 3) in serum-supplemented medium (17-32 fold loss of activity compared to free DOX), followed by 20-DOX-C4-albumin in serum-free medium and low activity of the other conjugates. In conclusion, DOX-albumin conjugates inhibit the growth of ES/PNET cell lines selectively, showing low activity against the unrelated carcinoma line PC-3 and sparing normal lymphoblasts. The inverse correlation of activity and conjugation number demonstrates a low cytotoxic activity of DOX in acid-stable binding to monomeric albumin, pointing to a selective cytostatic activity of the modified albumin against ES and PNET cells, even in the presence of a 100 fold excess of unmodified serum albumin.
Assuntos
Doxorrubicina/farmacologia , Tumores Neuroectodérmicos Primitivos/tratamento farmacológico , Sarcoma de Ewing/tratamento farmacológico , Soroalbumina Bovina/farmacologia , Carbodi-Imidas , Divisão Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacocinética , Portadores de Fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Glutaral , Substâncias de Crescimento/farmacologia , Humanos , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Tumores Neuroectodérmicos Primitivos/metabolismo , Tumores Neuroectodérmicos Primitivos/patologia , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Soroalbumina Bovina/administração & dosagem , Soroalbumina Bovina/farmacocinética , Selenito de Sódio/farmacologia , Transferrina/farmacologia , Células Tumorais CultivadasRESUMO
For the potential use of Wheat germ agglutinin (WGA) and Solanum tuberosum lectin (STL) as auxiliary excipients for targeting drugs to colonocytes, the number of Caco-2 and HT-29-bound lectins was determined by fluorimetry using fluorescein-labelled derivatives of the N-acetylglucosamine-specific lectins. After 1 h of incubation, the WGA-binding capacity of 5 x 10(4) Caco-2 cells was 26.9 +/- 0.5 pmol at 4 degrees C and 27.2 +/- 1.0 pmol at 37 degrees C respectively. In comparison, 19.5 +/- 2.9 pmol (37 degrees C) and 16.7 +/- 0.9 pmol (4 degrees C) WGA were bound within 1 h to 5 x 10(4) HT-29 cells referring to about 80% of the total amount of WGA bound within 4 h of incubation. In contrast, binding of STL to the colon carcinoma cell lines was independent of incubation times and temperatures tested exhibiting a binding rate of 8.4 +/- 0.6 pmol (HT-29) and 9.9 +/- 0.8 pmol (Caco-2) STL/5 x 10(4) cells. As determined by flow cytometry, non-specific binding is lower than 1.0% (WGA) and 3.4% (STL). Uptake and intracellular accumulation of the lectins were investigated by confocal laser scanning microscopy at 4 degrees C and 37 degrees C respectively. A decrease of initially membrane-bound lectins concurrent with increasing cytoplasmic enrichment by time was observed by digital cell image analysis. Due to specific and numerically sufficient adhesion as well as internalization, WGA and STL are anticipated as targeting tools in lectin-mediated drug delivery systems.
Assuntos
Aglutininas/química , Sistemas de Liberação de Medicamentos/métodos , Lectinas/farmacologia , Acetilglucosamina/química , Células CACO-2 , Sobrevivência Celular , Fluorometria , Células HT29 , Humanos , Microscopia Confocal , Lectinas de Plantas , Ligação Proteica , Solanum tuberosum/química , Temperatura , Fatores de Tempo , Aglutininas do Germe de TrigoRESUMO
To take advantage of the cytoadhesive characteristics of Wheat germ agglutinin (WGA) for improved particulate drug delivery, the interaction between WGA-grafted poly(D,L-lactic-co-glycolic acid)-microspheres and Caco-2 monolayers was investigated using bovine serum albumin (BSA) or glycine coated microspheres as a control. Covalent immobilization of WGA by the carbodiimide/N-hydroxysuccinimide-method on 4 microm microspheres yielded a surface density of 9.67+/-1.21x10(6) molecules/particle, whereas 0.22+/-0.04x10(6) WGA-molecules were bound by physical adsorption. After storage for 21 days in HEPES-buffer and treatment of the particles with 5 M urea, 86% of covalently linked lectin was still attached to the particles. At 4 degrees C the Caco-2 binding rate of both, WGA- and BSA-modified particles increased with addition of increasing numbers of particles until saturation was reached at 38150+/-1740 (WGA) or 12066+/-1195 (BSA) microspheres bound/mm(2) Caco-2 monolayer. Inhibition of Caco-2 binding of WGA-functionalized microspheres by chitotriose indicated for specificity of the interaction. As observed by confocal laser scanning microscopy, the fluorescein-loading of the particles was accumulated intracellularly after incubation of Caco-2 monolayers with WGA-modified microspheres contrary to glycine-grafted microspheres. Additionally, in case of WGA-functionalized microspheres the amount of cell associated fluorescein was 200-fold higher than that of the free solution. In conclusion, WGA-modified microspheres are expected to enhance intestinal transport of incorporated drugs due to cytoadhesion provided by the lectin coating.
Assuntos
Células CACO-2/metabolismo , Ácido Láctico/farmacocinética , Ácido Poliglicólico/farmacocinética , Polímeros/farmacocinética , Aglutininas do Germe de Trigo/farmacocinética , Animais , Bovinos , Portadores de Fármacos , Humanos , Ácido Láctico/síntese química , Lectinas/síntese química , Lectinas/farmacocinética , Microesferas , Ácido Poliglicólico/síntese química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros/síntese química , Aglutininas do Germe de Trigo/síntese químicaRESUMO
Lectin-mediated drug delivery may become a promising strategy to improve the efficacy of poorly permeable drugs by utilising active high-capacity transport pathways of epithelial tissues. This requires the elucidation of the basic mechanisms of lectin uptake prior to their practical use. We studied the interaction between the dietary lectin wheat germ agglutinin (WGA) and Caco-2 cells (single cells and monolayers) by a newly established assay design that is able to discriminate between cellular binding and uptake as well as by confocal microscopy: (i) All binding sites available for WGA at the cell membrane were occupied within 10 min of incubation. (ii) Cytoadhesion was followed by immediate uptake. After 20 min, 60% (single cells) or 30% (monolayers) of the membrane bound lectin were internalised. However, regardless of cell arrangement, 80% of the surface bound lectin was taken up into the cells during the course of the experiment. (iii) About 50% of the internalised lectin accumulated within the lysosomes after 1 h. This was confirmed by assays in the presence of monensin, an inhibitor of endosomal acidification, and by colocalisation with lysosomal cathepsin followed by semiquantitative image analysis. Further analysis by immunocytochemistry suggested that the trans-Golgi complex and the caveoli were not involved. Due to cytoadhesion, cytoinvasion and partial lysosomal accumulation, WGA-mediated drug delivery may provide for improved intracellular availability of conjugated drugs or colloidal carrier systems.