Worapaka Manosroi

Transdermal absorption enhancement through rat skin of gallidermin loaded in niosomes

Gallidermin (Gdm) loaded in anionic niosomes composed of Tween 61/CHL/DP (1:1:0.05 molar ratio) gave the highest entrapment efficiency (45.06%). This formulation gave antibacterial activity against Propionibacterium acnes and Staphylococcus aureus with the MIC and MBC of 3.75 and 7.5; 7.5 and 15 microg/microl, respectively. Gdm loaded in niosomes was more chemically stable than Gdm in aqueous solution of about 1.5 times. Gdm loaded and unloaded in niosomes were not found in the receiver solution investigated by vertical Franz diffusion cells at 37 degrees C for 6h. Gdm loaded in niosomes showed higher cumulative amounts in viable epidermis and dermis (VED) of rat skin of about 2 times more than unloaded Gdm. Gdm loaded in niosomes and incorporated in gel exhibited the highest cumulative amounts (82.42+/-9.28 microg cm(-2)) and fluxes (13.74+/-1.55 microg cm(-2)h(-1)) in stratum corneum (SC) and comparative cumulative amounts (183.16+/-30.32 microg cm(-2)) and fluxes (25.74+/-5.05 microg cm(-2)h(-1)) in VED to the unloaded Gdm incorporated in gel. This study has suggested that Gdm loaded in anionic niosomes and incorporated in gel is the superior topical antibacterial formulation because of the high accumulation in the skin with no risk of systemic effect.

Transdermal absorption and stability enhancement of salmon calcitonin by Tat peptide

Context: Highly organized structure of stratum corneum (SC) is the major barrier of the delivery of macromolecules such as proteins and peptides across the skin. Recently, cell penetrating peptides (CPPs) such as HIV1-trans-activating transcriptional (Tat) have been used to enhance the topical delivery of proteins and peptides. Objective: This study aimed to enhance the transdermal absorption and chemical stability of salmon calcitonin (sCT) by co-incubation with Tat. Materials and methods: Tat-sCT mixture at 1:1 molar ratio was prepared. Transdermal absorption and chemical stability of the mixture was evaluated in comparing with free sCT. Results: Tat-sCT mixture gave higher cumulative amounts and fluxes of sCT than free sCT. The maximum percentage of sCT of 58.36 ± 12.33% permeated into the receiving chamber was found in Tat-sCT mixture at 6 h which was 3.50 folds of free sCT. Tat-sCT mixture demonstrated better sCT stability than sCT solution after 1 month storage at 4°C, 25°C and 45°C. Discussion: The positively-charged arginine groups in Tat might be responsible for the binding of peptide complexes to negatively charged cell surfaces by electrostatic interactions and also the translocation of sCT through the excised skin. Conclusion: This study demonstrated the enhancements of transdermal absorption and stability of sCT by Tat peptide with potential for further application in transdermal delivery of other therapeutic peptides.

Enhancement of Transdermal Absorption, Gene Expression and Stability of Tyrosinase Plasmid (pMEL34)-Loaded Elastic Cationic Niosomes: Potential Application in Vitiligo Treatment

The pMEL34 was loaded in elastic cationic niosomes (Tween61/Cholesterol/DDAB at 1:1:0.5 molar ratio) by chloroform film method with sonication and rehydrated with 25% ethanol. The amount of pMEL34 was determined by gel electrophoresis and gel documentation. The maximum loading of pMEL34 in elastic cationic niosomes was 150 microg/16 mg of the niosomal compositions. At 8 weeks, the remaining plasmid in the elastic niosomes kept at 4 +/- 2 degrees C, 27 +/- 2 degrees C were 49.75% and 38.57%, respectively, whereas at 45 +/- 2 degrees C, all plasmids were degraded. For transdermal absorption through rat skin investigated by Franz diffusion cells at 6 h, the fluxes of pMEL34 loaded in elastic and nonelastic niosomes in viable epidermis and dermis (VED) were 0.022 +/- 0.00 and 0.017 +/- 0.01 microg/cm(2)/h, respectively, whereas only pMEL34 loaded in elastic cationic noisome was observed in the receiver solution. The pMEL34 loaded in elastic cationic niosomes showed the highest tyrosinase gene expression demonstrating higher tyrosinase activity than the free and the loaded plasmid in nonelastic niosomes of about four times. This study has suggested the potential application of elastic cationic niosomes as an efficient topical delivery for tyrosinase gene in vitiligo therapy.

Potent enhancement of GFP uptake into HT-29 cells and rat skin permeation by coincubation with tat peptide

The delivery enhancements of green fluorescent protein (GFP), a model reporter protein into/transepithelial colon adenocarcinoma (HT-29) cells and excised rat skin by coincubation, by simple mixing or as fusion protein with HIV1-trans-activating transcriptional (Tat), a cell-penetrating peptide, have been described. By simple mixing, Tat/GFP mixture could increase the cellular uptake of GFP into HT-29 cells by 4.25-fold and 1.79-fold of GFP and Tat-GFP fusion protein, respectively. The incubation time showed no effect on the cellular uptake of Tat/GFP and Tat-GFP. In transepithelial study, Tat-GFP demonstrated the highest ability to penetrate through the HT-29 cells of about 1.3-fold and 1.2-fold of GFP and Tat/GFP, respectively. Only Tat/GFP gave lower cytotoxicity than Tat or GFP alone. In transdermal delivery study, Tat/GFP showed better transdermal delivery profile with higher cumulative amount than Tat-GFP in stratum corneum (SC), viable epidermis and dermis, and the receiver compartment of the diffusion cell with the highest fluxes of 7.42 and 35.6; 8.87-fold and 5.57-fold of GFP and Tat-GFP in SC and receiver compartment, respectively. This study demonstrated an efficient enhancement of GFP uptake into cells and through excised rat skin by simple mixing with Tat peptide, which can be further applied for the development of protein drug delivery systems.

Potent enhancement of transdermal absorption and stability of human tyrosinase plasmid (pAH7/Tyr) by Tat peptide and an entrapment in elastic cationic niosomes.

Abstract Enhancement of transdermal absorption through rat skin and stability of the human tyrosinase plasmid (P) using Tat (T) and an entrapment in elastic cationic niosomes (E) were described. E (Tween61:cholesterol:DDAB at 1:1:0.5 molar ratio) were prepared by the freeze-dried empty liposomes (FDELs) method using 25% ethanol. TP was prepared by a simple mixing method. TPE was prepared by loading T and P in E at the T:P:E ratio of 0.5:1:160 w/w/w. For gel formulations, P, TP, PE and TPE were incorporated into Carbopol 980 gel (30u2009µg of plasmid per 1u2009g of gel). For the transdermal absorption studies, the highest cumulative amounts and fluxes of the plasmid in viable epidermis and dermis (VED) were observed from the TPE of 0.31u2009±u20090.04u2009µg/cm2 and 1.86u2009±u20090.24u2009µg/cm2/h (TPE solution); and 4.29u2009±u20090.40u2009µg/cm2 and 25.73u2009±u20092.40u2009µg/cm2/h (TPE gel), respectively. Only plasmid from the PE and TPE could be found in the receiving solution with the cumulative amounts and fluxes at 6u2009h of 0.07u2009±u20090.01u2009µg/cm2 and 0.40u2009±u20090.08u2009µg/cm2/h (PE solution); 0.10u2009±u20090.01u2009µg/cm2 and 0.60u2009±u20090.06u2009µg/cm2/h (TPE solution); 0.88u2009±u20090.03u2009µg/cm2 and 5.30u2009±u20090.15u2009µg/cm2/h (PE gel); and 1.02u2009±u20090.05u2009µg/cm2 and 6.13u2009±u20090.28u2009µg/cm2/h (TPE gel), respectively. In stability studies, the plasmid still remained at 4u2009±u20092u2009°C and 25u2009±u20092u2009°C of about 48.00–65.20% and 27.40–51.10% (solution); and 12.34–38.31% and 8.63–36.10% (gel), respectively, whereas at 45u2009±u20092u2009°C, almost all the plasmid was degraded. These studies indicated the high potential application of Tat and an entrapment in elastic cationic niosomes for the development of transdermal gene delivery system.

Transdermal Absorption Enhancement of N-Terminal Tat–GFP Fusion Protein (TG) Loaded in Novel Low-Toxic Elastic Anionic Niosomes

Elastic anionic niosomes (Tween 61/cholesterol/dicetyl phosphate at 1:1:0.05 molar ratio of 20 mM) with various concentrations of ethanol and edge activators sodium cholate (NaC) and sodium deoxycholate (NaDC) showed larger vesicular size (171.94 ± 63.52 - 683.17 ± 331.47 nm) and higher negative zeta potential (-6.45 ± 2.76 to - 17.40 ± 2.51 mV) than the nonelastic anionic niosomes. The elasticity (deformability index) and entrapment efficiency of all elastic vesicles except the NaDC vesicles were higher than the nonelastic vesicles. The morphology, under transmission electron microscope, of elastic and nonelastic niosomes loaded and not loaded with Tat-green fluorescent protein fusion protein (TG) were in large unilamellar structure. TG loaded in elastic (1 mol% NaC) anionic niosomes gave the highest cell viability both in HT-29 (92.32 ± 3.82%) and KB cells (96.62 ± 5.96%), the highest cumulative amounts (62.75 ± 2.68 μg/cm(2) ) and fluxes (10.46 ± 3.45 μg/cm(2) h) in receiving chamber in rat skin transdermal study by Franz diffusion cells. This study has not only indicated the synergistic enhancement effects of the Tat peptide and the niosomal delivery system on the cellular uptake and transdermal absorption of TG but also 1 mol% NaC as an edge activator to obtain a novel low-toxic elastic anionic niosomes for topical use of therapeutic macromolecules such as proteins, as well.

Enhancement of gene expression and melanin production of human tyrosinase gene loaded in elastic cationic niosomes

Objectivesu2002 Disturbance in the synthesis of tyrosinase might be one of the major causes of vitiligo. The enhancement of tyrosinase gene expression and melanin production by loading the plasmid in elastic cationic niosomes was investigated in tyrosinase gene knocked out human melanoma (M5) cells and in tyrosine‐producing mouse melanoma (B16F10) cells.

Novel application of polioviral capsid: development of a potent and prolonged oral calcitonin using polioviral binding ligand and Tat peptide

Abstract Context: Poor absorption and proteolytic degradation are major obstacles of orally administered peptide drugs including calcitonin. Cell penetrating peptides (CPPs) and receptor binding ligands are interesting tools for the application in the delivery of these drugs. Objective: To investigate the enhancements of in vitro and in vivo salmon calcitonin (sCT) activity by Tat, a trans-activating transcriptional peptide and VP1 peptide (V) from polioviral capsid. Materials and methods: Tat/sCT, V/sCT and V/Tat/sCT mixtures at various molar ratios were prepared and investigated for in vitro and in vivo activities of sCT. Results: Tat could increase in vitro sCT activity both in colon adenocarcinoma (HT-29) and mouth epidermal carcinoma (KB) cells. V/sCT (6:1) showed significant increase of intracellular calcium in HT-29 cells. V/Tat/sCT (6:1:1) gave highest increase of intracellular calcium in both cells. Oral administered Tat/sCT (1:1) showed comparable hypocalcemic effect to sCT injection with prolonged action. V/Tat/sCT (6:1:1) demonstrated hypocalcemic effect at 12u2009h after administration but no hypocalcemic effect was observed from V/sCT. Discussion: Positive charge from Tat might facilitate sCT uptake and absorption. Increasing of intracellular calcium in HT-29 cells by V but lacking of hypocalcemic effect from V/sCT in mice indicated the ligand-receptor mediated delivery of sCT by the interaction between V and PVR. Conclusion: Potential application of V and Tat in oral calcitonin delivery system was demonstrated. Further study in a proper PVR bearing host is still needed to provide more useful information for the application of V in the development of drug delivery systems.

Hypoglycemic Activity and Stability Enhancement of Human Insulin-Tat Mixture Loaded in Elastic Anionic Niosomes

Abstract This study aimed to investigate the synergistic effect of trans-activator of transcription (Tat) and niosomes for the improvement of hypoglycemic activity of orally delivered human insulin. The elastic anionic niosomes composing of Tween 61/cholesterol/dicetyl phosphate/sodium cholate at 1:1:0.05:0.02 molar ratio loaded with insulin–Tat mixture (1:3 molar ratio) was prepared. Deformability of the elastic anionic niosomes decreased after loaded with the mixture of 1.35 times. For the in vitro release, the insulin (T10 = 4u2009h) loaded in the elastic anionic niosomes indicated the slower release rate than insulin in the mixture (T10 = 3u2009h) loaded in niosomes. At room temperature (30u2009±u20092u2009°C), the mixture loaded in elastic anionic niosomes was more chemical stable than the free mixture of 1.3, 1.4 and 1.7 times after stored for 4, 8 and 12 weeks, respectively. Oral administration in the alloxan-induced diabetic mice of the mixture loaded in elastic anionic niosomes with the insulin doses at 25, 50 and 100u2009IU/kg body weight indicated significant hypoglycemic activity with the percentage fasting blood glucose reduction of 1.95, 2.10 and 2.10 folds of the subcutaneous insulin injection at 12u2009h, respectively. This study has demonstrated the synergistic benefits of Tat and elastic anionic niosomes for improving the hypoglycemic activity of the orally delivered human insulin as well as the stability enhancement of human insulin when stored at high temperature. The results from this study can be further developed as an effective oral insulin delivery.

Potent and prolonged hypoglycemic activity of an oral insulin - Tat mixture in diabetic mice

Trans-activator of transcription (Tat) is a cell penetrating peptide which can translocate and carry macromolecular cargoes through cell membranes. This study investigated the hypoglycemic activity of orally delivered insulin - Tat mixture in alloxan-induced diabetic mice. The mixtures of insulin and Tat at 1:1, 1:3 and 1:6 molar ratios were given orally at the insulin doses ranging from 1-200 IU/kg. The fasting blood glucose (FBG) levels were measured at initial, 1, 2, 4, 6, and 12 h after administration. At 1:3 molar ratio of the mixture and after 12 h of administration, insulin at 200 IU/kg showed the highest with prolonged hypoglycemic activity of 74.0±10.3% FBG reduction (2.18 folds of subcutaneously injected (SC) insulin). Free insulin administered orally did not show any hypoglycemic activity. The mixtures at the insulin doses of 100 and 50 IU/kg also showed potent FBG reduction of 73.8±8.2 and 71.3±16.9% at 12 h after administration (2.18 and 2.10 folds of SC insulin, respectively). After incubation with Mono-Mac-6 cells, only the -mixtures but not the free insulin showed intra-cellular insulin uptake, indicating the insulin penetration through the cell membranes via Tat. In simulated gastric fluid, the insulin content in the mixture was not found, demonstrating the degradation of insulin in the gastric environments. Insulin may be absorbed at upper gastrointestinal tract facilitated by Tat. The potent and prolonged hypoglycemic activity of insulin co-administered orally with Tat can be further developed as an effective oral insulin delivery system.