Human Transbodies to HCV NS3/4A Protease Inhibit Viral Replication and Restore Host Innate Immunity.

A safe and effective direct acting anti-hepatitis C virus (HCV) agent is still needed. In this study, human single chain variable fragments of antibody (scFvs) that bound to HCV NS3/4A protein were produced by phage display technology. The engineered scFvs were linked to nonaarginines (R9) for making them cell penetrable. HCV-RNA-transfected Huh7 cells treated with the transbodies produced from four different transformed E. coli clones had reduced HCV-RNA inside the cells and in the cell spent media, as well as fewer HCV foci in the cell monolayer compared to the transfected cells in culture medium alone. The transbodies-treated transfected cells also had up-expression of the genes coding for the host innate immune response, including TRIF, TRAF3, IRF3, IL-28B, and IFN-ß. Computerized homology modeling and intermolecular docking predicted that the effective transbodies interacted with several critical residues of the NS3/4A protease, including those that form catalytic triads, oxyanion loop, and S1 and S6 pockets, as well as a zinc-binding site. Although insight into molecular mechanisms of the transbodies need further laboratory investigation, it can be deduced from the current data that the transbodies blocked the HCV NS3/4A protease activities, leading to the HCV replication inhibition and restoration of the virally suppressed host innate immunity. The engineered antibodies should be tested further for treatment of HCV infection either alone, in combination with current therapeutics, or in a mixture with their cognates specific to other HCV proteins.

Hypoglycemic activity and stability enhancement of human insulin-tat mixture loaded in elastic anionic niosomes.

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 = 4 h) loaded in the elastic anionic niosomes indicated the slower release rate than insulin in the mixture (T10 = 3 h) loaded in niosomes. At room temperature (30 ± 2 °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 100 IU/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 12 h, 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.

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

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 12 h 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.

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

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 (30 µg of plasmid per 1 g 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 0.31 ± 0.04 µg/cm(2) and 1.86 ± 0.24 µg/cm(2)/h (TPE solution); and 4.29 ± 0.40 µg/cm(2) and 25.73 ± 2.40 µg/cm(2)/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 6 h of 0.07 ± 0.01 µg/cm(2) and 0.40 ± 0.08 µg/cm(2)/h (PE solution); 0.10 ± 0.01 µg/cm(2) and 0.60 ± 0.06 µg/cm(2)/h (TPE solution); 0.88 ± 0.03 µg/cm(2) and 5.30 ± 0.15 µg/cm(2)/h (PE gel); and 1.02 ± 0.05 µg/cm(2) and 6.13 ± 0.28 µg/cm(2)/h (TPE gel), respectively. In stability studies, the plasmid still remained at 4 ± 2 °C and 25 ± 2 °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 45 ± 2 °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 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 gene expression and melanin production of human tyrosinase gene loaded in elastic cationic niosomes.

OBJECTIVES: 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 (B(16) F(10) ) cells. METHODS: Niosomes composed of Tween 61/cholesterol/dimethyl dioctadecyl ammonium bromide at 1:1:0.5 molar ratio were prepared by the freeze-dried empty liposomes method. The thin lipid film was redissolved in distilled water or 25% ethanol to obtain the non-elastic or elastic cationic niosomes, respectively. KEY FINDINGS: The maximum loading of the plasmid in non-elastic and elastic niosomes was 130 and 100 µg per 16 mg of the niosomal contents, respectively. The plasmid-loaded elastic cationic niosomes exhibited high specific tyrosinase activity of 1.66 and 1.50 fold in M5 cells and 6.81 and 4.37 fold in B(16) F(10) cells compared with the free plasmid and the plasmid-loaded non-elastic cationic niosomes, respectively. CONCLUSIONS: This study has demonstrated not only the enhancement of the expression of human tyrosinase gene by loading in elastic cationic niosomes, but also the potential application of this gene delivery system for the further development of vitiligo gene therapy.

Potent melanin production enhancement of human tyrosinase gene by Tat and an entrapment in elastic cationic niosomes: potential application in vitiligo gene therapy.

Potent melanin production enhancement of human tyrosinase plasmid (pAH7/Tyr, P) in mouse melanoma cells (B(16)F(10)) by Tat peptide (T) and an entrapment in elastic cationic niosomes (E) was described. The E composed of Tween 61/cholesterol/dodecyl dimethyl ammonium bromide at 1:1:0.5 molar ratio was prepared by freeze-dried emptying liposomes method. PE at P/E ratio of 1:160 w/w and TPE at T/P/E ratio of 0.125:1:160, 0.25:1:160, and 0.5:1:160 w/w/w were prepared. The final concentration of the plasmid in the study was 4 ng/µL. By sulforhodamine B assay, PE and TPE complexes showed slight or no cytotoxic effect. The cells transfected with TPE (0.5:1:160) exhibited the highest enhancement of tyrosinase enzyme activity of 11.82-, 7.67-, 5.07-, and 6.29-folds of control, P, PE, and TP (0.5:1) and melanin production of 13.03-, 8.46-, 5.36-, and 6.58-folds of control, P, PE, and TP (0.5:1), respectively. The elastic cationic niosomes demonstrated an increase in thermal stability of P at 4 ± 2, 25 ± 2, and 45 ± 2 °C. The vesicular size and the zeta potential values of PE and TPE complexes were slightly increased but still in the range of stable dispersion (out of ±30 mV). These results indicated the high potential application of the TPE complexes for further investigation for vitiligo gene therapy.

Polioviral receptor binding ligand: a novel and safe peptide drug carrier from polioviral capsid.

Cellular uptake enhancement of green fluorescent protein (GFP) into human colon adenocarcinoma (HT-29) and human mouth epidermal carcinoma (KB) cells by a segment of VP1-BC loop polioviral capsid (V), a polioviral receptor binding peptide and HIV-I transactivator of transcription (Tat) was evaluated. HT-29 and KB cells were incubated with various molar concentrations of GFP, V, and Tat mixtures. Both V and Tat showed potent enhancement of GFP uptake into HT-29 and KB cells. In HT-29 cells, the V-GFP, Tat-GFP, and V-Tat-GFP mixtures enhanced the GFP cellular uptake efficiency with the maximum of 3.98-, 4.59-, and 4.08-folds of GFP at 1:3, 1:1/6, and 1/6:1:1/6 molar ratios, respectively. For KB cells, the V-GFP, Tat-GFP, and V-Tat-GFP mixtures enhanced the GFP cellular uptake efficiency with the maximum of 4.05-, 5.09-, and 4.91-folds of GFP at 1:1/6, 1:1, and 1:1:1 molar ratios, respectively. Both V and V-GFP mixtures showed a lower cytotoxicity effect than Tat and Tat-GFP mixture. These studies demonstrated the potential of polioviral capsid, a polioviral receptor binding peptide as a novel, low cytotoxicity carrier for the development of peptide drugs delivery system.

Cellular uptake enhancement of Tat-GFP fusion protein loaded in elastic niosomes.

This study has demonstrated the enhancement of cellular uptake of GFP when fused with Tat (Tat-GFP) and loaded in elastic niosomes. GFP and the GFP fused with Tat at C- and N-terminals were expressed in E. coli BL21 (DE3). The N-terminal Tat-GFP fusion protein (Tat-GFP) which showed the highest uptake of 5.2% in HT-29 cell line at 2.4 folds of GFP was selected to load in various charged non-elastic and elastic niosomes and liposomes. All niosomes showed higher entrapment efficiency (EE) of the fusion protein more than in liposomes with the highest EE of 100% in elastic cationic niosomes. However, the fusion protein loaded in elastic anionic niosomes (Tween 61/cholesterol/dicetyl phosphate at 1:1:0.05 molar ratio) which gave the EE of only 32.8% showed the highest cellular uptake of GFP at 6.7, 2.8 and 1.7 times of GFP, Tat-GFP and Tat-GFP loaded in elastic cationic niosomes, respectively. After the 3 month-storage at 30 +/- 2 degrees C, the percentages remaining of the fusion protein loaded in the elastic anionic niosomes (61.9 +/- 12.7%) were about 2 times higher than the non-loaded fusion protein (33.7 +/- 2.8%). Thus, the cellular uptake and the chemical stability of the fusion protein were enhanced when loaded in elastic niosomes, especially the elastic anionic niosomes which can be further developed as an efficient delivery system for many therapeutic proteins.

Chemical stability and cytotoxicity of human insulin loaded in cationic DPPC/CTA/DDAB liposomes.

Liposomes were prepared from DPPC (dipalmitoyl phosphatidyl choline) mixed with Chol (cholesterol) and CTA [cholest-5-en-3-ol(3beta)(trimethylammonio) acetate] or DDAB (dioctadecyl dimethyl ammonium bromide) at various molar ratios by chloroform film method with sonication. The most physical stable (no sedimentation with an average zeta potential value of 47.7+/-1.44 mV) liposomal formulation (DPPC/CTA/DDAB at 7:2:1 molar ratio) was selected to load with human insulin (0.45 mg/mL) by the freeze dried empty liposomes (FDELs) method with the entrapment efficiency of human insulin of 62.72% (determined by gel filtration). Liposomes were spherical shape with unilamellar structure and an average size of 2.26+/-0.87 microm determined by TEM. The percentages of insulin remaining in liposomes when stored at 4+/-2, 30+/-2 and 45+/-2 degrees C for 4 months were 26.21, 36.86 and 15.75% which were higher than human insulin solution of 6.13, 11.31 and 2.61 times, respectively. The percentages of entrapment of human insulin were 62.72 at initial and at 31.72, 64.10 and 8.10 when kept at 4+/-2, 30+/-2 and 45+/-2 degrees C, respectively, for 4 months. The synthesized cationic lipid, CTA, and the DPPC/Chol/CTA liposomes loaded with human insulin demonstrated no cytotoxicity on normal human skin fibroblast but some cytotoxic effects on mouth epidermal cancer cell line. This study has demonstrated the enhancement of chemical stability of human insulin with no cytotoxicity when loaded this protein in cationic DPPC/CTA/DDAB liposomes. The results indicated the potential application of this cationic liposomal formulation for topical therapeutic use.

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.

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.

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.

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.

Entrapment enhancement of peptide drugs in niosomes.

The objective of this study was to enhance the entrapment of various charged peptide drugs [(bacitracin (BCT), insulin and bovine serum albumin (BSA)] in niosomes by modifying the vesicular charge compositions. Cationic, anionic and neutral niosomes were prepared from sorbitan monostearate (Span 60) or polyoxyethylene sorbitan monostearate (Tween 61), cholesterol (CHL), dimethyldioctadecylammonium bromide (DDAB) and/or dicetyl phosphate (DP) in distilled water, by freeze dried empty liposome (FDEL) method. Morphology and vesicular sizes of the vesicles were investigated by optical microscope, TEM, X-ray diffractometry and dynamic light scattering. The entrapment efficiency of the peptides in niosomes was determined by gel electrophoresis and gel documentation. After reconstitution of the empty niosomal powder in phosphate buffer pH 7.0 containing the peptide drugs, they were oligolamellar membrane structure, with the sizes of 40-60 nm, except the neutral niosomes entrapped with insulin and cationic niosomes entrapped with BSA which showed the sizes of 0.1-1.3 microm and 100-150 nm, respectively. The zeta potential values of neutral, cationic and anionic niosomes entrapped with BSA, insulin and BCT were -22.3 +/- 1.52, -30.7 +/- 2.92 and +22.68+/- 1.31 mV, respectively. The entrapment efficiency of BSA, BCT and insulin in neutral niosomes (Tween 61/CHL at 1 : 1 molar ratio) was 72.94, 69.89 and 10.26%, in cationic niosomes (Tween 61/CHL/DDAB at 1 : 1 : 0.05 molar ratio) was 84.54, 32.85 and 87.15% and in anionic niosomes (Tween 61/CHL/DP at 1 : 1 : 0.05 molar ratio) was 50.13, 90.88 and 44.31%, respectively. The highest entrapment efficiency of BSA, BCT and insulin at 72.94, 90.88 and 87.15 was observed in neutral, anionic and cationic niosomes, respectively. The results from this study has suggested the appropriate niosomal formulation to entrap the peptides with different charges and polarity for pharmaceutical application.

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 x 10(6) 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 zeta 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 x 10(6) 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.

Transdermal enhancement through rat skin of luciferase plasmid DNA loaded in elastic nanovesicles.

Transdermal absorption of luciferase plasmid (pLuc) was enhanced by loading in elastic cationic liposomes and niosomes and the application of iontophoresis or the stratum corneum (SC) stripping method. Cationic liposomes (DPPC/Chol/DDAB at a 1:1:1 molar ratio) and niosomes (Tween61/Chol/DDAB at a 1:1:0.5 molar ratio) were prepared by the freeze-dried empty liposomes method. The elastic vesicles were prepared by hydrating the lipid or surfactant film by 25% of ethanol instead of distilled water. Gel electrophoresis of all nanovesicles showed the 100% pLuc entrapment efficiency. All nanovesicles loaded with pLuc showed larger vesicular sizes than the nonloaded vesicles of about 1.4 times for liposomes and 1.7 times for niosomes. The nanovesicles loaded with pLuc demonstrated less positive zeta potential than the nonloaded vesicles. The pLuc loaded in elastic vesicles kept at 4 +/- 2 and 27 +/- 2 degrees C for 8 weeks gave the remaining pLuc of about 70 and 60% for liposomes and 85 and 73% for niosomes, respectively. For nonelastic vesicles kept at 4 +/- 2 degrees C, 56 and 61% of the remaining pLuc were observed for liposomes and niosomes, respectively, while at 27 +/- 2 degrees C, all pLuc were degraded. The deformability indices of the elastic liposomes and niosomes loaded with the pLuc were 16.64 +/- 2.92 and 20.72 +/- 0.82, whereas the nonelastic vesicles gave 9.35 +/- 0.09 and 10.08 +/- 0.12, respectively. Transdermal absorption through rat skin pretreated with SC stripping or treated with iontophoresis of pLuc loaded in nanovesicles by vertical Franz diffusion cells was investigated at 37 degrees C. The cells were stopped and the skin and the receiving solution were withdrawn at 1, 3, and 6 hours and the pLuc contents in the stripped SC, whole skin (viable epidermis and dermis; VED), and the receiving solution were assayed by the modified gel electrophoresis and gel documentation. Without the SC stripping technique or iontophoresis, the pLuc loaded and nonloaded in nonelastic cationic liposomes or niosomes were not found in SC, VED, and receiving solution. The fluxes in the whole skin of pLuc loaded in nonelastic liposomes and niosomes with SC stripping and iontophoresis at 6 hours gave 2.73 +/- 0.46 and 3.83 +/- 0.73, and 7.01 +/- 1.22 and 9.60 +/- 1.31 g/cm(2)/h, respectively, while pLuc loaded in elastic liposomes and niosomes without the SC stripping and iontophoresis at 6 hours showed 2.79 +/- 0.09 and 2.84 +/- 0.04 g/cm(2)/h, respectively. The pLuc loaded in elastic niosomes or in nonelastic niosomes with iontophoresis was found in the receiving solution with a higher amount than that loaded in elastic liposomes or nonelastic liposomes with iontophoresis. The fluxes in the receiving solution of pLuc loaded in nonelastic liposomes and niosomes with iontophoresis at 6 hours were 6.71 +/- 0.31 and 8.82 +/- 0.28 g/cm(2)/h, respectively. For elastic liposomes and niosomes, the fluxes of the loaded pLuc in the receiving solution were the same, at about 1.9 g/cm(2)/h. Although pLuc loaded in nonelastic niosomes with iontophoresis gave the highest delivery of the plasmid in VED and receiving solution, a more promising applicable approach for gene delivery has been suggested to be the elastic niosomal systems, since no equipment is required.

Development of highly stable and low toxic cationic liposomes for gene therapy.

Cationic liposomes with high stability and low cytotoxicity for gene therapy have been developed. Luciferase plasmid DNA (pLuc) was used as a model gene. The empty liposomes and niosomes were prepared by freeze dried empty liposomes (FDEL) method. The entrapment of pLuc in the liposomes was by reconstitution of the lyophilized dried vesicles with the plasmid solution. The morphology of the vesicles showing multilamellar structure was characterized by transmission electron microscope (TEM) and cryo-TEM. Cytotoxicity of the vesicular formulations was investigated on mouse melanoma cell lines (B16F10) by MTT assay. Cationic lposomes and niosomes containing the cationic lipid DDAB were less cytotoxic than other bilayer vesicular formulations. The pLuc entrapped in the cationic DPPC/Chol/DDAB liposomes (at 1:1:1 molar ratio) exhibited higher stability than other vesicular formulations and the pLuc in solution when stored at 4, 30 and 50 degrees C for 8 weeks. The entrapment efficiency determined by gel electrophoresis and gel documentation of the pLuc in this liposomal formulation was 100%. Luciferase gene expression of pLuc-loaded in cationic liposomes (lipoplexes) in HeLa cell lines was evaluated from luciferase activity determined by a luminometer at 24 and 48 h incubation. Percentages of cell proliferation of the lipoplexes on HeLa cell line at 24 and 48 h incubation were evaluated by the WST-1 assay. When the amount of DPPC or cholesterol was increased in the lipoplexes, the higher amount of DDAB was needed to protect pLuc from enzymatic degradation. However, DPPC and cholesterol exceeded 33 and 50% mol, respectively gave no gene expression. The DPPC/Chol/DDAB (at 1:1:1 molar ratio) lipoplex has demonstrated moderately lower luciferase gene expression and low cytoxicity. This lipoplex with the DDAB/pLuc weight ratio of 14:1 was the most desirable formulation for gene therapy because of its high stability, high luciferase gene expression and low cytotoxicity.

Glycosylation of therapeutic proteins in different production systems.

UNLABELLED: Glycosylation plays an important role in a number of therapeutic proteins, including monoclonal antibodies. The enzymatic activity of a therapeutic protein is mainly determined by the protein structure, whereas the pharmacokinetics, pharmacodistribution, solubility, stability, enhancement of effector function and receptor binding are all influenced by the carbohydrate moiety. Hyperglycosylated proteins show increased serum half-life, are less sensitive to proteolysis and more heat-stable compared with the non-glycosylated forms. Molecular engineering of the TNK-tissue plasminogen activator molecule results in a more complex type of glycosylation and increases the half-life of the protein, which allows a single bolus injection at a lower dose for the treatment of acute myocardial infarction. Antibody-dependent cell cytotoxicity (ADCC) is determined partially by the specific N-glycosylation of the Fc domain of the monoclonal antibody. Specific glycoforms of monoclonal antibodies, which interact solely with the FcgammaRIIIa receptor of natural killer cells, result in superior ADCC compared with heterogeneous glycoforms that interact with different Fc receptors. This demonstrates that glycoengineering for directed glycosylation of therapeutic proteins can improve the therapeutic effect. While the amino acid sequence of the therapeutic protein is determined by the nucleotide sequence of the inserted gene, glycosylation depends on the glycosylating enzymes in the endoplasmatic reticulum and the Golgi apparatus of the eukaryotic host cell. In addition, the glycosylation of the therapeutic protein is affected by the culture medium used, the efficiency of protein expression and the physiological status of the host cell. CONCLUSION: For a given protein, changes in the type of host cell, composition of the culture media and fermentation conditions during process development will most likely result in changes in the site occupation and heterogeneity of glycosylation. This, of course, can influence the therapeutic profile. Therefore, the early selection of the host cell and selection of upstream parameters are key in the process development of a product.

Economic aspects of commercial manufacture of biopharmaceuticals.

Sustained approvals of new biopharmaceuticals supported by a sparkling pipeline are the drivers for the above-market growth of biopharmaceuticals. Due to usually high therapeutic dose of monoclonal antibodies, they are demanding for high capacity needs. This requires significant capital investment and stimulates innovation for process improvement to decrease cost of goods and to save capital investments. Such process improvements are either ongoing along the learning curve or result from significant process changes through regulatory authorities impact. Both approaches require extensive protein analytical guidance to maintain product quality, safety and equivalency. In addition to second generation processes, second generation products have the feature of optimizing the physiological principle of biopharmaceuticals to the therapeutic need and to decrease the therapeutic dose, which goes along with investment savings and lower cost of goods.