ET AAS evaluation of the stability and pH-sensitivity of, pH-sensitive stealth liposomes containing cisplatin in mouse plasma.

In this work, stability and the pH-sensitivity of pH-sensitive stealth liposomes containing cisplatin exposed to plasma medium and their subsequent responses to pH modifications were evaluated. A method to determine platin in mouse plasma by electrothermal atomic absorption spectroscopy (ET AAS) was developed and validated. At first, a comparative study of sample preparation treatments with basic, acidic, and acidic added with Triton X-100 as a modifier was done. The best treatment was obtained with HCl 3% (v/v). The ET AAS method with acid treatment presented linearity at a range of 10-160 ng Pt/mL. The limits of detection (LOD) was 3.1 ng/mL Pt for acid treatment, while the limit quantification (LOQ) was 10 ng/mL Pt. The acid treatment presented good repeatability (VC<15.0%) and recovery close to 100%. This treatment was chosen for subsequent studies due to its best value of repeatability, recovery, LOD and lowest cost. pH-sensitive stealth liposomes, containing cisplatin, demonstrated low stability and poor response to pH variation after plasma incubation. These findings suggest that further studies are needed to improve liposome formulation i.e., to reduce its size.

Preparation and cytotoxicity of cisplatin-containing liposomes.

We encapsulated cisplatin into stealth pH-sensitive liposomes and studied their stability, cytotoxicity and accumulation in a human small-cell lung carcinoma cell line (GLC4) and its resistant subline (GLC4/CDDP). Since reduced cellular drug accumulation has been shown to be the main mechanism responsible for resistance in the GLC4/CDDP subline, we evaluated the ability of this new delivery system to improve cellular uptake. The liposomes were composed of dioleoylphosphatidylethanolamine (DOPE), cholesteryl hemisuccinate (CHEMS), and distearoylphosphatidylethanolamine-polyethyleneglycol 2000 (DSPE-PEG2000) and were characterized by determining the encapsulation percentage as a function of lipid concentration. Among the different formulations, DOPE/CHEMS/DSPE-PEG liposomes (lipid concentration equal to 40 mM) encapsulated cisplatin more efficiently than other concentrations of liposomes (about 20.0%, mean diameter of 174 nm). These liposomes presented good stability in mouse plasma which was obtained using a 0.24-M EDTA solution (70% cisplatin was retained inside the liposomes after 30 min of incubation). Concerning cytotoxic effects, they are more effective (1.34-fold) than free cisplatin for growth inhibition of the human lung cancer cell line A549. The study of cytotoxicity to GLC4 and GLC4/CDDP cell lines showed similar IC50 values (approximately 1.4 microM), i.e., cisplatin-resistant cells were sensitive to this cisplatin formulation. Platinum accumulation in both sensitive and resistant cell lines followed the same pattern, i.e., approximately the same intracellular platinum concentration (4.0 x 10-17 mol/cell) yielded the same cytotoxic effect. These results indicate that long-circulating pH-sensitive liposomes, also termed as stealth pH-sensitive liposomes, may present a promising delivery system for cisplatin-based cancer treatment. This liposome system proved to be able to circumvent the cisplatin resistance, whereas it was not observed when using non-long-circulating liposomes composed of phosphatidylcholine, phosphatidylserine, and cholesterol.

Preparation and cytotoxicity of cisplatin-containing liposomes

We encapsulated cisplatin into stealth pH-sensitive liposomes and studied their stability, cytotoxicity and accumulation in a human small-cell lung carcinoma cell line (GLC4) and its resistant subline (GLC4/CDDP). Since reduced cellular drug accumulation has been shown to be the main mechanism responsible for resistance in the GLC4/CDDP subline, we evaluated the ability of this new delivery system to improve cellular uptake. The liposomes were composed of dioleoylphosphatidylethanolamine (DOPE), cholesteryl hemisuccinate (CHEMS), and distearoylphosphatidylethanolamine-polyethyleneglycol 2000 (DSPE-PEG2000) and were characterized by determining the encapsulation percentage as a function of lipid concentration. Among the different formulations, DOPE/CHEMS/DSPE-PEG liposomes (lipid concentration equal to 40 mM) encapsulated cisplatin more efficiently than other concentrations of liposomes (about 20.0 percent, mean diameter of 174 nm). These liposomes presented good stability in mouse plasma which was obtained using a 0.24-M EDTA solution (70 percent cisplatin was retained inside the liposomes after 30 min of incubation). Concerning cytotoxic effects, they are more effective (1.34-fold) than free cisplatin for growth inhibition of the human lung cancer cell line A549. The study of cytotoxicity to GLC4 and GLC4/CDDP cell lines showed similar IC50 values (approximately 1.4 æM), i.e., cisplatin-resistant cells were sensitive to this cisplatin formulation. Platinum accumulation in both sensitive and resistant cell lines followed the same pattern, i.e., approximately the same intracellular platinum concentration (4.0 x 10-17 mol/cell) yielded the same cytotoxic effect. These results indicate that long-circulating pH-sensitive liposomes, also termed as stealth pH-sensitive liposomes, may present a promising delivery system for cisplatin-based cancer treatment. This liposome system proved to be able to circumvent the cisplatin resistance, whereas...

Reversion by polyclonal antibodies of alpha effects of Tityus serrulatus venom on frog sciatic nerve.

Fraction T2 from Tityus serrulatus venom produced a marked lengthening of action potentials recorded with the single sucrose-gap technique, a characteristic effect of alpha scorpion toxins. This effect was not reversed by thorough washing of the nerve. On the other hand, T2 fraction deactivated by complete iodination did not cause any alteration of the compound action potential, even if applied in concentration as high as 4000 times the half saturation dose of the unmodified T2 fraction. This high dose of deactivated T2 did not hinder the onset of the full effect of a single dose of T2 fraction applied subsequently. Polyclonal antibodies against native or against iodinated venom reverted the action of T2 fraction, restoring normal electrical response. We conclude that both types of antibodies may remove the effect of alpha toxins on sodium channel.

A nontoxic derivative of Bothrops jararaca venom suitable to generate antibodies against the native venom.

Bothrops jararaca venom was toxoided by stepwise iodination with cold iodine, and doses up to 30 LD50 were non-lethal by i.p. route (BICALHO et al., 1990). Groups of mice injected chronically with the native, or the iodinated venom, have been subjected to histological examination. In the native group, in the spleen, around the white pulp, an acellular, amorphous eosinophilic substance, metachromatic to Methyl Violet, PAS positive, and dichroic to Congo Red under polarized light, was present. Strong congestion in the liver, kidneys and lungs was found. The salivary glands were replenished with an amorphous substance in the serosal acini. The groups injected with the iodinated venom only show discrete alterations, more akin to the control group. The anavenin was immunogenic. Antibody generation in mice and rabbits was detected by ELISA. In mice, active protection against challenge with native venom was presented. The iodinated venom generated a rabbit antiserum with strong lines in gel immunoprecipitation against the lethal venom. A minimum neutralization titer of 2.3 mg ml-1 against the native venom was attained in the first cycle (28 days) of immunization. After 3 cycles (100 days), the protection rose to 5.1 mg ml-1.