Studies on Mefenamic Acid Microparticles: Formulation, In Vitro Release, and In Situ Studies in Rats

In this study, we investigated the in vitro characteristics of mefenamic acid (MA) microparticles as well as their effects on DNA damage. MA-loaded chitosan and alginate beads were prepared by the ionotropic gelation process. Microsponges containing MA and Eudragit RS 100 were prepared by quasi-emulsion solvent diffusion method. The microparticles were characterized in terms of particle size, surface morphology, encapsulation efficiency, and in vitro release profiles. Most of the formulation variables manifested an influence on the physical characteristics of the microparticles at varying degrees. We also studied the effects of MA, MA-loaded microparticles, and three different polymers on rat brain cortex DNA damage. Our results showed that DNA damage was higher in MA-loaded Eudragit microsponges than MA-loaded biodegradable chitosan or alginate microparticles.     

Nanostructures of complexes formed by calf thymus DNA interacting with cationic surfactants

Synchrotron small-angle X-ray scattering was used to study the nanostructures of the complexes formed by calf thymus DNA interacting with cationic lipids (or surfactants) of didodecyldimethylammonium bromide (DDAB), cetyltrimethylammonium bromide (CTAB), and their mixture with a zwitterionic lipid of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PHGPC). The effects of lipid/DNA ratios, DNA chain flexibility, lipid topology, and neutral lipid mixing on the nanostructures of DNA-lipid complexes were investigated. The complexes between double-stranded DNA (dsDNA) and double-tailed DDAB formed a bilayered lamellar structure, whereas the complexes between dsDNA and single-tailed CTAB preferred a structure of 2D hexagonal close packing of cylinders. With single stranded DNA (ssDNA) interacting with CTAB, the complexes showed a Pm3n cubic structure due to the different chain flexibility between dsDNA and ssDNA. The lipid molecules bound by rigid dsDNA like to form cylindrical micelles, whereas lipids bound to flexible ssDNA could form spherical or short cylindrical micelles. The addition of the neutral single-chained PHGPC lipids to the CTAB lipids could induce a structural transition of dsDNA-lipid complexes from a 2D hexagonal to a multi-bilayered lamellar structure. The parallel DNA strands were intercalated in the water layers of lamellar stacks of the mixed lipid bilayers. The DNA-DNA spacing depended on the ratios of charged lipid to neutral lipid, and charged lipid to DNA, respectively.     

Purification of influenza viral complementary RNA: its genetic content and activity in wheat germ cell-free extracts.

Influenza viral complementary RNA (cRNA) was purified free from any detectable virion-type RNA (vRNA), and its genetic content and activity in wheat germ cell-free extracts were examined. After phenol-chloroform extraction of cytoplasmic fractions from infected cells, poly(A)-containing viral cRNA is found in two forms: in single-stranded RNA and associated with vRNA in partially and fully double-stranded RNA. To purify single-stranded cRNA free of these double-stranded forms, it was necessary to employ, as starting material, RNA fractions in which cRNA was predominantly single stranded. Two RNA fractions were successfully employed as starting material: polyribosomal RNA and the total cytoplasmic RNA from infected cells treated with 100 mug of cycloheximide (CM) per ml at 3 h after infection. In WSN virus-infected canine kidney (MDCK) cells, the addition of CM at 3 h after infection stimulates the production of cRNA threefold and causes a very large increase in the proportion of the cytoplasmic cRNA which is single stranded; double-stranded RNA forms are greatly reduced in amount. Total cRNA was obtained by oligo(dT)-cellulose chromatography, and single-stranded cRNA was separated from double-stranded forms by Sepharose 4B chromatography. The cRNA preparation purified from polyribosomes consists of 95% single-stranded cRNA, with the remaining 5% apparently being double-stranded RNA forms. The cRNA preparation purified from CM-treated cells (CM cRNA) is even more pure: 100% of the radiolabeled RNA is single-stranded cRNA. Annealing experiments, in which a limited amount of 32P-labeled genome RNA was annealed to the cRNA, indicate that the purified cRNA contains at least 84 to 90% of the genetic information in the vRNA genome. Purified viral cRNA (CM cRNA) is very active in directing the synthesis of virus-specific proteins in wheat germ cell-free extracts.     

Newcastle Disease Virus-Specific RNA: Polyacrylamide Gel Analysis of Single-Stranded RNA and Hybrid Duplexes

Newcastle disease virus-specific [(3)H]uridine-labeled 18S RNA was resolved by polyacrylamide gel electrophoresis into several components with molecular weights from 450,000 to 840,000. The analysis of 35 and 24S virus-specific RNA also revealed several components in each sedimentational class. The conversion of 18S RNA into double-stranded form by hybridization with an excess of unlabeled virion RNA improved the resolution in polyacrylamide gels and revealed at least six distinct components. The same six classes of hybrid duplexes were revealed when (32)P-labeled 50S virion RNA was hybridized with an excess of 18S RNA. The applicability of polyacrylamide gel electrophoresis of hybrid duplexes to the analysis of viral genome structure is discussed.     

Comparison of Release-Controlling Efficiency of Polymeric Coating Materials Using Matrix-type Casted Films and Diffusion-Controlled Coated Tablet

Polymeric coating materials have been widely used to modify release rate of drug. We compared physical properties and release-controlling efficiency of polymeric coating materials using matrix-type casted film and diffusion-controlled coated tablet. Hydroxypropylmethyl cellulose (HPMC) with low or high viscosity grade, ethylcellulose (EC) and Eudragit® RS100 as pH-independent polymers and Eudragit S100 for enteric coatings were chosen to prepare the casted film and coated tablet. Tensile strength and contact angle of matrix-type casted film were invariably in the decreasing order: EC> Eudragit S100> HPMC 100000> Eudragit RS100>HPMC 4000. There was a strong linear correlation between tensile strength and contact angle of the casted films. In contrast, weight loss (film solubility) of the matrix-type casted films in three release media (gastric, intestinal fluid and water) was invariably in the increasing order: EC < HPMC 100000 < Eudragit RS100 < HPMC 4000 with an exception of Eudragit S100. The order of release rate of matrix-type casted films was EC > HPMC 100000 > Eudragit RS100 > HPMC 4000 > Eudragit S100. Interestingly, diffusion-controlled coated tablet also followed this rank order except Eudragit S100 although release profiles and lag time were highly dependent on the coating levels and type of polymeric coating materials. EC and Eudragit RS100 produced sustained release while HPMC and Eudragit S100 produced pulsed release. No molecular interactions occurred between drug and coating materials using ¹H-NMR analysis. The current information on release-controlling power of five different coating materials as matrix carrier or diffusion-controlled film could be applicable in designing oral sustained drug delivery.Key words: diffusion-controlled coated tablet, drug release rate, matrix-type casted film, polymeric coating materials, release-controlling power     

Dissociation of double-stranded polynucleotide helical structures by eukaryotic initiation factors, as revealed by a novel assay

A new technique has been applied to the study of the RNA secondary structure unwinding activity of the eukaryotic initiation factors (eIFs) 4F, 4A, and 4B. Secondary structures were generated at the 5' ends of reovirus and globin mRNA molecules by hybridization with 32P-labeled cDNA molecules 15 nucleotide residues long. The dissociation of the labeled cDNAs from the mRNAs was assayed by a gel filtration chromatography procedure which separates the free cDNAs from mRNAs and mRNA/cDNA hybrids. When the three factors were tested alone, only eIF-4F stimulated dissociation of hybrids. The combination of eIF-4A plus eIF-4B also exhibited a strong hybrid dissociating activity, which was markedly temperature dependent. Under optimum conditions, up to 90% of the hybrid structures are disrupted in 60 min. These results demonstrate for the first time that stable double-stranded regions can be melted and dissociated by eIFs. They also characterize more precisely the first step in the structure unwinding reaction.     

Investigation of the Drug Release and Surface Morphological Properties of Film-Coated Pellets, and Physical, Thermal and Mechanical Properties of Free Films as a Function of Various Curing Conditions

The purpose of the present investigation was to elucidate the influence of curing on different physical properties of Eudragit NE and RS coating systems. Increased curing times resulted in decreased drug release rates from Eudragit NE-coated beads. However, an increase in drug release rates was noticed at longer curing times and higher temperatures for the Eudragit RS coating system. The surface morphological changes of the film-coated beads revealed that there were no visible macroscopic changes as a result of curing. The absence of any ibuprofen melting peak in the DSC thermograms of cured NE and RS coated beads confirmed that there was no surface crystallization of ibuprofen. These results indicated that the increase in drug release rates from RS coated pellets, when subjected to long curing times, resulted from loss of plasticizer. Free films of Eudragit NE exhibited an increase in tensile strength with increased curing times, whereas Eudragit RS free films showed a decrease in tensile strength beyond 4 h of curing at 70 and 90 degrees C. The film thicknesses and weights of free films of Eudragit RS prepared with triethyl citrate plasticizer were found to change more dramatically with curing than did free films of Eudragit RS prepared with ibuprofen as the plasticizer. An increase in pore volume was also observed with increased curing times for Eudragit RS free films. Such changes with curing were shown to be due to the loss of plasticizer molecules, leading to the formation of molecular-scale voids and channels.     

The effect of chain length on the melting temperature and size of dialkyldimethylammonium bromide vesicles

Differential scanning calorimetry (DSC) and dynamic light scattering (DLS) were used to obtain the gel to liquid-crystalline phase transition temperature (Tm) and the apparent hydrodynamic radius (Rh) of spontaneously formed cationic vesicles of dialkyldimethylammonium bromide salts (CnH2n+1)2(CH3)2N+.Br-, with varying chain lengths. The preparation of cationic vesicles from aqueous solution of these surfactants, for n=12, 14, 16 and 18 (DDAB, DTDAB, DHDAB and DODAB, respectively), requires the knowledge of the surfactant gel to liquid-crystalline phase transition temperature, or melting temperature (Tm) since below this temperature these surfactants are poorly or not soluble in water. That series of cationic surfactants has been widely investigated as vesicle-forming surfactants, although C12 and C18, DDAB and DODAB are by far the most investigated from this series. The dependence of Tm of these surfactants on the number n of carbons in the surfactant tails is reported. The Tm obtained by DSC increases non-linearly with n, and the vesicle apparent radius Rh is about the same for DHDAB and DODAB, but much smaller for DDAB.     

Isolation and characterization of DNA-DNA and DNA-RNA.

A simple method for the isolation and characterization of DNA-DNA and DNA-RNA hybrid molecules formed in solution was developed. It was based on the fact that, in appropriate salt concentration, such as 5% Na2HPO4, DNA in either double-stranded (DNA-DNA or DNA-RNA) or single-stranded forms, but not free nucleotides, can bind to diethylaminoethylcellulose disc filters (DE81). Thus tested samples were treated with the single-strand-specific nuclease S1 and then applied to DE81 filters. The free nucleotides, resulting from degrading the single-stranded molecules, were removed by intensive washing with 5% Na2HPO4, leaving only the hybrid molecules on the filters. The usefulness of this method was illustrated in dissociation and reassociation studies of viral (SV40) or cellular (NIH/3T3) DNAs and DNA-RNA hybrid molecules. Using this technique the reassociation of denatured SV40 DNA was found to be a very rapid process. Dissociation studies revealed that the melting curves of tested DNAs were dependent on salt concentration. Thus the melting temperatures (tm) obtained for SV40 DNA were 76 degrees C at 1 X SSC (0.15 M NaCl-0.015 M sodium citrate) and 65 degrees C at 0.1 X SSC, and for NIH/3T3 DNA 82 degrees C at 1 X SSC and 68 degrees C at 0.1 X SSC. MuLV DNA-RNA hybrid molecules were formed by annealing in vitro synthesized MuLV DNA with 70S MuLV RNA at 68 degrees C. The melting temperature of this hybrid in the annealing solution was 87 degrees C. Another important feature of this procedure was that, after being selectively bound to the filters, the hybrid molecules could efficiently be recovered by heating the filters for 5 min at 60 degrees C in 1.5-1.7 M KCl. The recovered molecules were intact hybrids as they were found to be completely resistant to S1 nuclease.     

Characterization of cationic liposomes. Influence of the bilayer composition on the kinetics of the liposome breakdown

The cationic large unilamellar mixed liposomes from 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and didodecyldimethylammonium bromide (DDAB) or dioctadecyldimethylammonium bromide (DODAB) were prepared. The influence of the addition of Triton X-100 (TX-100) or octaethylene glycol mono-n-dodecylether (C₁₂E₈) on the membrane integrity was investigated turbidimetrically. The stability of the liposomal systems was estimated by monitoring fluorimetrically at 25 °C the rate of spontaneous and surfactant-induced release of entrapped 5(6)-carboxyfluorescein (CF). In order to evaluate the interaction of the cationic DODAB guest with the host POPC membrane, the main phase transition temperatures (T_m) were determined by electron paramagnetic resonance spectroscopy (EPR). All the results obtained show that the presence of DODAB and DDAB stabilizes the POPC liposomes. The extent of stabilization depends on the concentration and nature of the cationic guest.     

Expression of luciferase plasmid (pCMVLuc) entrapped in DPPC/Cholesterol/DDAB liposomes in HeLa cell lines

The luciferase gene expression of lipoplexes, a liposome containing luciferase plasmid (pCMVLuc), in HeLa cell lines, was investigated. Cationic liposomes were prepared by the chloroform film method with sonication. The lipoplex was formed by loading the liposome with pCMVLuc. The lipoplex with an optimal weight ratio of dimethyl dioctadecyl ammonium bromide (DDAB)/pCMVLuc protected from DNaseI was determined by an agarose gel electrophoresis. The selected lipoplexes were assayed for luciferaase activity by using a luminometer. The effect on cell proliferation was evaluated by WST-1 assay. The highest luciferase activity of 1.5 × 10⁶ RLU was observed in the cholesterol (Chol)/DDAB (2:1 molar ratio) lipoplex at the DDAB/pCMVLuc weight ratio of 10:1 at 48 hours, which was about 10, 100, and 1,000 times higher than the DDAB, L-alpha-dipalmitoyl phosphatidylcholine (DPPC)/Chol/DDAB (1:2:1 molar ratio), and DPPC/Chol/DDAB (2:2:1 molar ratio) lipoplexes, respectively. The liposome with the smallest particle size was obtained from the cationic liposome composed of DPPC/Chol/DDAB (7:1:1 molar ratio) with the ζ potential of 7.17 ± 0.73. The optimal weight ratio of DDAB/pCMVLuc that protected pCMVLuc from DNaseI digestion was 4:1 in the DDAB formulation. The Chol/DDAB (2:1 molar ratio) lipoplex with the DDAB/pCMVLuc of 10:1 showed the highest luciferase activity of 1.5 × 10⁶ RLU and the highest cytotoxicity as well. DPPC/Chol/DDAB (1:1:1 molar ratio)-lipoplex (DDAB/pCMVLuc = 14:1), which had the amount of DPPC and cholesterol not exceeding 33 and 50% mol, respectively, gave the lower gene expression of about 4 times, but lower cytoxicity of about 14 times, than the Chol/DDAB lipoplex (2:1 molar ratio) and was considered to be the most suitable formulation. The results from this study can be applied as a model for the development of a gene-therapeutic dosage form.     

Conformational transition of DNA induced by cationic lipid vesicle in acidic solution: spectroscopy investigation

The conformational transition of DNA induced by the interaction between DNA and a cationic lipid vesicle, didodecyldimethylammonium bromide (DDAB), had been investigated by circular dichroism (CD) and UV spectroscopy methods. We used singular value decomposition least squares method (SVDLS) to analyze the experimental CD spectra. Although pH value influenced the conformation of DNA in solution, the results showed that upon binding to double helical DNA, positively charged liposomes induced a conformational transition of DNA molecules from the native B-form to more compact conformations. At the same time, no obvious conformational changes occurred at single-strand DNA (ssDNA). While the cationic lipid vesicles and double-strand DNA (dsDNA) were mixed at a high molar ratio of DDAB vesicles to dsDNA, the conformation of dsDNA transformed from the B-form to the C-form resulting in an increase in duplex stability (DeltaT(m)=8+/-0.4 degrees C). An increasing in T(m) was also observed while the cationic lipid vesicles interacted with ssDNA.     

Unidirectional pulsed-field electrophoresis of single- and double-stranded DNA in agarose gels: analytical expressions relating mobility and molecular length and their application in the measurement of strand breaks

Unidirectional pulsed-field electrophoresis improves the separation of single-stranded DNA molecules longer than 20 kilobases (kb) in alkaline agarose gels compared to static-field electrophoresis. The greatest improvement in separation is for molecules longer than 100 kb. The improved resolution of long molecules with unidirectional pulsed-field electrophoresis makes possible the measurement of lower frequencies of single-strand breaks. The analytical function that relates the length and mobility of single-stranded DNA electrophoresed with a static field also applies to unidirectional pulsed field separations. Thus, the computer programs used to measure single-strand breaks are applicable to both undirectional pulsed- and static-field separations. Unidirectional pulsed-field electrophoresis also improves the separation of double-stranded DNA in neutral agarose gels. The function relating molecular length and mobility for double-stranded DNA separated by unidirectional pulsed-field electrophoresis is a superset of the function for single-stranded DNA. The coefficients of this function can be determined by iterative procedures.     

Dipstick-type biosensor for visual detection of DNA with oligonucleotide-decorated colored polystyrene microspheres as reporters

In recent years, there is a continuously growing interest in the development of biosensors for rapid, simple and inexpensive DNA tests suitable for the small laboratory or for on-site testing. Detection is accomplished through electrochemical, optical or gravimetric transduction. We report on the development of disposable dipstick-type DNA biosensors that employ oligonucleotide-decorated colored polystyrene microspheres as reporters and enable visual detection of DNA sequences without the use of instrumentation. The biosensors have been designed to detect DNA molecules that contain both, a biotin moiety and a segment that is complementary to the oligonucleotide attached on the surface of blue or red microspheres. Capture of the hybrids by immobilized streptavidin at the test zone results in the formation of a colored line. The biosensors were applied to: (a) detection of single-stranded DNA, (b) detection of PCR-amplified double-stranded DNA and (c) genotyping of single nucleotide polymorphisms (SNP). The results were compared with sensors based on gold nanoparticle reporters. It is also demonstrated that the microspheres offer the potential for multicolor detection of specific DNA sequences.