d(GC) - Association of Biotechnology and Pharmacy

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Current Trends in Biotechnology and Pharmacy Vol. 6 (2) 166-172 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online) delivery of some hydrophobic cytotoxic drugs to solid tumors. In a previous report, the LDL-drug complex was prepared for selective delivery of the cytostatic agent to tumors. However rapid dissociation of the complex in plasma resulted in stability issues preventing further development of these systems (9). In our study we explored lipoprotein approach for delivery of PADRE to tumors by physical entrapment of the peptide within the lipoprotein systems. Materials and Methods The peptide PADRE was synthesized by Genemed Synthesis Inc., San Francisco, USA. Low-density lipoproteins (LDL) and enzyme inhibitors were obtained from Sigma, USA. Caco- 2 cells were obtained from ATCC (Manassas, VA, USA). The Caco-2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM,Invitrogen Co., Carlsbad, CA) containing 10% heat inactivated fetal bovine serum and 1% penicillin-streptomycin (5000 I.U./ml, Cellgro, Mediatech Inc, VA). The cells were incubated in an environment of 37 °C and 5% CO 2 . The cells were grown up to 80-90% confluency before further passage for experiments. Fig. 1. Stability of native PADRE in Caco-2 cells Delivery Strategies to improve PADRE 168 Effect of peptidase inhibitors on stability of PADRE: Caco-2 cells were cultured at a density of 1 x 10 6 cells. The cells were trypsinized and suspended in growth media. For the control studies, 500 ìL of 100 ìg/mL of PADRE was incubated in PBS with Caco-2 cells in culture tubes for 0.5, 1, 2, and 4 hours at 37ºC. The cells were separated from the supernatant by centrifugation at 10,000 rpm for 5 minutes, digested, and the amount of peptide in supernatant and cell pellets was estimated by the HPLC method described previously (5). To determine effect of enzyme inhibitors on the stability, Caco-2 cells were pretreated with enzyme inhibitors (Anti-trypsin – 10 µM, Bestatin – 100 µM, Diprotinin A – 100 µM , Phospharidon –1 µM and Phenanthroline –100 µM) for one hour followed by treatment with PADRE (Table 1). At the end of 2 hour incubation, samples were deproteinated and the concentration of PADRE was analyzed by HPLC. Preparation of lipoprotein delivery systems: Ten mg of LDL was triple washed with 5 mL of heptane to extract the lipids. The apoprotein B was suspended in 3 mL of PBS using a laboratory
Current Trends in Biotechnology and Pharmacy Vol. 6 (2) 166-172 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online) vortex. To this, aqueous solution of peptide at 1 mg/mL was added, vortexed, and lyophilized at –50 °C for 12 hours to remove water. The heptane extract of lipids were then added to the powder mixture of apoprotein B and peptide and vortexed for 5 minutes. Heptane was slowly removed using rotoevaporator at 40 °C for 30 minutes under vacuum. Lipoprotein delivery systems were also prepared using exactly the same protocol except that 0.1% sodium lauryl sulphate was used during the suspension of the peptide in the apoprotein B. Stability of PADRE-lipoprotein system in Caco-2 cells: Caco-2 cells were cultured at a density of 1 x 10 6 cells. The cells were trypsinized and suspended in growth media. PADRElipoprotein equivalent to 50 mcg/mL of peptide, was added to Caco-2 cells in culture tubes and incubated for 4 hours at 37 degrees. The cells were separated from the supernatant by centrifugation at 10,000 rpm for 5 minutes, 169 digested, and the amount of peptide was estimated by the HPLC method. Native PADRE solution of 50 mcg/mL in water was used as control. CD4 proliferation assay to determine immunogenicity of PADRE-lipoprotein system: CD4 positive T-lymphocytes were derived from immunized mice and subsequently selected via in vitro stimulation by peptide and dendritic cells. PADRE specific, CD4 cell proliferation assay was performed by incubating T-lymphocyte cells in triplicate with PADRE 10 and 20 µg/mL or equivalent amount of PADRE incorporated lipoprotein delivery systems, and dendritic cells in the presence of IL2 at 37°C for 48 hours. At the end of 48 hours, the cells were incubated with 3H-Thymidine for 4 hours and harvested. Thymidine incorporation was measured on a scintillation counter. CD4 proliferation was taken as a measure of immunogenicity of PADRE. Naïve lymphocytes served as a negative control. Fig. 2. Improvement in PADRE stability upon pretreatment with peptidase inhibitors determined in Caco-2 cells. Srinivas Poondru et al
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6th Annual Convention of Associatio
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