Timothy J. Smith

Chitosan‐membrane interactions and their probable role in chitosan‐mediated transfection

Recent studies in our laboratory have shown that chitosan, a polycationic polymer of glucosamine, can facilitate the transfection of HeLa cells with a plasmid that codes for β‐galactosidase. Although chitosan can bind to DNA and other polyanions, the kinetics of complexation might differ depending on the polyanion tested. This evidence suggests that, in addition to ionic interactions, the carbohydrate backbone of chitosan might have an important role in the process of transfection. Beads prepared by the complexation of chitosan with polyphosphate were used to investigate the nature of cellular interactions with chitosan. HeLa cells bound to chitosan‐polyphosphate beads could be readily displaced from the beads with methyl α‐D‐mannopyranoside but not with NaCl. Membrane proteins solubilized by CHAPS bound readily to chitosan‐polyphosphate beads. A major fraction of the membrane proteins could be eluted from the beads with methyl α‐D‐mannopyranoside. These results suggest that non‐ionic interactions between the carbohydrate backbone of chitosan and cell surface proteins might have an important role in the chitosanmediated transfection of HeLa cells.

A Microtiter Plate Assay for Sulfonamides

Abstract Sulfamethoxazole, sulfisoxazole, and sulfadiazine are sulfonamides used in the treatment of several infectious diseases. Several studies have demonstrated that the amino substituent plays an important role in both the toxicity and the therapeutic effect of these drugs. In view of these findings, a rapid and convenient method of analysis would be useful for monitoring selected patients receiving these drugs. With the increasing use of microtiter plate methodology in the clinical laboratory, an assay based upon the Bratton-Marshall reaction with the amino substituent was adapted to the microtiter plate format. The results indicate that the microtiter plate assay for sulfonamides retains the sensitivity and linearity necessary for analysis of sulfonamides in biological fluids at clinically relevant concentrations. The assay is simple, rapid, convenient, and suitable for monitoring procedures where only the measurement of the active drug concentration is required.

A Preliminary Investigation of Modified Alginates as a Matrix for Gene Transfection in a HeLa Cell Model

Previous reports have demonstrated the effectiveness of chitosan as a transfection agent. These studies have noted the importance of polysaccharide backbone interactions with the cell surface as well as cationic groups in the transfection process. The present study focuses upon the potential utility of another polysaccharide hydrogel, alginic acid, as a transfection agent. Alginic acid was modified by carbodiimide-mediated linkage of several heterocyclic and aromatic amines to the carboxyl group of the alginate, giving the alginate polycationic characteristics through which binding to nucleic acids could be facilitated. The amines used for this modification include diaminoacridine, thionin, basic fuchsin, acridine yellow, and diaminomethyltriazine. Of all the conjugates tested, basic fuchsin-modified alginate produced the greatest increase in the transfection of a plasmid coding for β-galactosidase into HeLa cells. These studies demonstrate that other polysaccharide hydrogels can be used as transfection agents, and the structural orientation of the cationic spacer arm is crucial for effective transfection.

Activation of diacetyldapsone and a preliminary evaluation of a cyclodextrin–diacetyldapsone complex in cultured lung cells

A diacetyldapsone–2‐hydroxypropyl‐β‐cyclodextrin complex (DADDS–CD) was evaluated with regard to the ability of cultured lung cells to activate DADDS to the active metabolite dapsone. The same system was used to assess the effect of the complex on cell growth. The complex was incubated with cells for periods of 24 to 72 h, followed by extraction of metabolites from the incubation medium and analysis by HPLC. In addition, the Trypan Blue exclusion technique was used to assess cell viability during this time period. Results indicated that lung cells could activate DADDS to dapsone and that, while the complex appeared to delay cell growth in the first 24 h period, no significant difference was seen between cells incubated in the presence and absence of the complex at 72 h. These results indicate that DADDS–CD has significant potential as a drug‐delivery system for DADDS in the lung based upon the ability of the cells to activate DADDS. The mixed effects of the complex on cell growth may have important implications when considering the frequency of administration of the complex to the lung.

Expression of alkaline phosphatases during embryonic development and immature stages of the earthworm, Eisenia andrei

Abstract Expression of alkaline phosphatases in developing embryo and mature stages of the earthworm, Eisenia andrei was investigated. The embryonic stages examined in this study appeared to have only one slow-moving form of alkaline phosphatase which had a different mobility from the intestinal alkaline phosphatases of the mature worm, suggesting that intestinal alkaline phosphatases of embryos may be different from mature forms. A surge in alkaline phosphatase activity after hatching is consistent with the expression of mature forms of intestinal alkaline phosphatase and this increase would be associated with postnatal differentiation of the intestine.

A preliminary evaluation of neural network analysis for pharmacodynamic modeling of the dosing of the hydroxymethylglutaryl coenzyme A-reductase inhibitors simvastatin and atorvastatin.

BACKGROUND Neural networks have been used in diagnosing and treating many diseases, including the diagnosis of myocardial infarction and insulin dosing in diabetes mellitus. OBJECTIVE The goal of this study was to develop a preliminary pharmacodynamic model for dosing of the hydroxymethylglutaryl coenzyme A (HMG-CoA)-reductase inhibitors simvastatin and atorvastatin using neural network analysis. METHODS Using NeuralSIM neural network software (NeuralWare, Carnegie, Pa), lipid panels from patients at a hospital lipid clinic were entered as inputs for the model, and the doses of simvastatin or atorvastatin that achieved those lipid results were used as outputs. The network was trained using 2 different data sets and was run using a subset of the data as inputs. The results of the neural network run (predictions) were compared with the actual doses used as directed by the hospital pharmacist in accordance with the lipid clinic protocol. RESULTS Complete data sets were available for 17 patients (11 men, 6 women). The mean age of these patients was 56.7 years (range, 40-67 years). The neural network model based on data set 1 predicted a dose that was within 95% of the actual dose 7 of 12 times and predicted use of the drug actually used 13 of 19 (68.4%) times. The neural network based on data set 2 predicted use of the drug actually used 10 of 17 (58.8%) times, but the predicted doses never approximated the actual doses by > or = 90%. CONCLUSIONS A neural network model for the dosing of the HMG-CoA-reductase inhibitors simvastatin and atorvastatin demonstrated an ability to predict appropriate dosing, but inclusion of other factors (eg, age, body weight, sex) and a larger sample size may be necessary for development of a more accurate model.

Citrate-mediated release of aurintricarboxylic acid from a calcium alginate complex: Implications for intravaginal HIV chemoprophylaxis and related applications

Factors associated with the intravaginal release of an anti-HIV agent from an alginate complex were considered. Among these is citrate associated with prostatic fluid. This study demonstrates that citrate, at a physiologically appropriate concentration, facilitates the release of an anti-HIV polymer from a calcium alginate complex. The release of the agent can be modified by the concentration of the calcium and alginate in the complex. These results suggest that seminal and prostatic fluid can be considered in the design of an intravaginal system for HIV chemoprophylaxis.