SEARCH

SEARCH BY CITATION

Keywords:

  • elastic cationic niosomes;
  • gene expression;
  • Tat;
  • tyrosinase plasmid;
  • vitiligo

Abstract

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments
  8. Conflict of Interest
  9. References

Potent melanin production enhancement of human tyrosinase plasmid (pAH7/Tyr, P) in mouse melanoma cells (B16F10) 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.

Gene can be delivered into the cell by viral or non-viral vector systems. For the viral vectors, retrovirus, adenovirus, and adeno-associated virus have been used to deliver the desired gene into the target cells. However, these vectors have several disadvantages such as host immune responses to viral proteins, difficulties in the large-scale production of recombinant viruses, and insertion of cellular oncogenes, by which the altering of their expression and/or biochemical properties can lead to unrestricted growth and cancer (1–3). Non-viral vectors, including liposomes and niosomes, exhibited several advantages for efficient non-viral gene delivery systems such as biocompatible, biodegradable, non-toxic, ease of preparation, and no induction of a strong host immune response after frequent administration (4–9). However, these vector systems have several limitations, including the low gene transfer efficiency (10) and the quick loss of gene expression in the fast-replicating cells (4). Elastic niosome is a non-ionic surfactant-based nanovesicle composes of nanovesicular fluidized compounds such as deoxycholate and ethanol (11). Novel non-ionic surfactant-based elastic niosomes containing ethanol as nanovesicular membrane fluidizers were first described by Manosroi et al. (12). These nanovesicles squeezed themselves and passed through a small pore in stratum corneum, which was smaller than their vesicular size. Hence, these types of nanovesicles were more efficient in delivering both low and high molecular weight drug both in quantity and depth (11). Elastic niosomes demonstrated the prolonged release and better biological activity of the entrapped substances comparing with conventional niosomes (10). Cell-penetrating peptides (CPPs) or protein transduction domains have been extensively studied. These peptides can translocate across almost all types of plasma membrane of eukaryotic cells via different forms of endocytosis. In the in vitro and in vivo studies, CPPs facilitated the delivery of broad range of functional hydrophilic macromolecular cargoes, including proteins, DNA, antibodies, oligonucleotides, imaging agents, liposomes, and niosomes, through the plasma membrane of diverse cells without altering the biological activities of the cargoes (13). One of the most widely studied CPPs is trans-activating transcriptional (Tat, T) peptide of human immunodeficiency virus type 1 (HIV-1). It is an essential factor for viral gene expression and plays a critical role for viral replication. For the protein transduction of T, the regarded domain sequence is residues 47–57. Currently, by using T-cargoes attachment technique, several drugs and gene delivery systems have been developed. Nitin et al. (14) demonstrated that T-attached streptavidin was concentrated in the cell nuclei after 90 min of delivery and suggested the significant implication in gene delivery. Multimers of the T peptide can also efficiently condense DNA and produce a six to eightfold increase in transfection (15). In this study, Tat/human tyrosinase plasmid (pAH7/Tyr, P)/elastic cationic niosomes (TPE) formulations were developed and the enhancement of tyrosinase gene transfection and expression determined as tyrosinase enzyme activity and melanin production in the melanoma (B16F10) cell line were investigated.

Methods and Materials

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments
  8. Conflict of Interest
  9. References

Materials

Human tyrosinase plasmid (pAH7/Tyr, P) was provided by Boehringer Ingelheim Company, Germany. The plasmid composed of 4986 bp with CMV promoter. The Tat peptide (GRKKRRQRRRPPQRKC) was purchased from Chengdu KaiJie Bio-pharmaceutical Co., Ltd. (Chengdu, China). Tween 61 (polyoxyethylene sorbitan monostearate), cholesterol, and DDAB (dimethyl dioctadecyl ammonium bromide) were from Sigma-Aldrich, St. Louis, MO, USA. Phenol/chloroform/isoamylalcohol (25:24:1) and ethanol were analytical grade reagents (Fluka, Buchs, Switzerland).

Preparation of elastic cationic niosomes

The 20 mm elastic cationic niosomes (E) were prepared by freeze-dried empty liposomes (FDELs) method. Briefly, Tween 61, cholesterol, and dodecyl dimethyl ammonium bromide (DDAB) at 1:1:0.5 molar ratios were mixed, placed in a clean, dry round bottom flask, and dissolved in chloroform. The solvent was then removed by a rotary evaporator under vacuum (R-124 Buchi, Flawil, Switzerland). The resulting film was dried in an evacuated desiccator at room temperature (27 ± 2 °C) under reduced pressure for over 12 h and rehydrated with distilled water at 50 ± 2 °C for 30 min. The dispersion was sonicated using a microtip probe sonicator (Vibra Cell; Sonics & Materials Inc., Newton, CT, USA) at pulse on 3.0, pulse off 1.0, and 33% amplitude for 15 min and centrifuged at 2190 × g, 4 °C for 1 min. The dispersion was further lyophilized overnight by a freeze-dryer (Alpha 1–2 LD Model, Christ, Germany) and kept at 4 °C until use. Cationic niosomal powder was reconstituted in 25% ethanol to form elastic cationic niosomal dispersion. The dispersion was further sonicated for 15 min at 4 °C in an ice bath. The niosomal dispersion was centrifuged at 2190 × g, 4 °C for 1 min. The supernatant was collected and kept at 4 °C.

Maximum loading amount of T in TP complexes

T and P were separately diluted with 3.125 mm phosphate buffer, pH 6.8, to obtain the final concentration of 0.05 and 0.1 mg/mL, respectively. The P was mixed with T peptide at the T/P ratio of 0.125:1, 0.25:1, 0.5:1, 1:1, and 2:1 w/w and incubated at room temperature (25 ± 2 °C) for 30 min to obtain the TP complexes. The complexes were analyzed by using SDS–polyacrylamide gel electrophoresis with 12% separating gel and 4% stacking gel at 100 V for 2 h. The gel was stained by Coomassie Brilliant Blue G-250 for 30 min and soaked to destain in 10% acetic acid for 24 h. The bands of free T at approximately 2.1 kDa for monomer and 6–14 kDa for multimers were observed. The highest amount of T that showed no free T band on the SDS–polyacrylamide gel indicated the maximum loading amount of the T in the TP complexes.

Preparation of human tyrosinase plasmid–loaded elastic cationic niosomes (PE) and Tat/human tyrosinase plasmid/elastic cationic niosomes (TPE) complexes

PE at P/E ratio of 1:160 w/w were prepared by incubating the plasmid with 20 mm elastic cationic niosomes at the ratio of 100 μg plasmid/16 mg niosomal lipids at room temperature (25 ± 2 °C) for 1 h. For TPE complexes, the TP that exhibited the highest complexation efficiency of T was incubated with elastic cationic niosomes at the same condition of PE. The entrapment efficiency of P and TP in elastic cationic niosomes was determined. The P and TP from PE and TPE complexes, respectively, were extracted by mixing 100 μL of PE or TPE complexes with 100 μL phenol/chloroform/isoamylalcohol, vortex for 1 min, centrifuged at 13 680 × g, 4 °C for 10 min, and run on agarose gel electrophoresis at 100 V for 40 min. The percentages of the entrapment efficiency were calculated as the following equation:

  • image

The percentages recovery of the P from TP, PE, and TPE complexes were determined by extracting PE and TPE with phenol/chloroform/isoamylalcohol. Then, T was hydrolyzed by trypsinization with trypsin according to Adami et al. (15). TP, PE, and TPE containing 2.5 μg of plasmid were mixed with 20 μL of trypsin solution (5 μg of trypsin in 50 mm HEPES, pH 7.4) and incubated at room temperature (25 ± 2 °C) for 24 h. The recovered plasmid was determined by agarose gel electrophoresis (1% agarose gel and run at 100 V for 40 min), and the band area was calculated by gel documentation software (Universal Hood; Biorad Laboratory, Milan, Italy).

For transmission electron microscopy, a drop of PE or TPE complexes was applied on a 300-mesh Formvar copper grid and allowed to adhere for 10 min. The remaining dispersion was removed, and a drop of 2% ammonium molybdate was added for 4 min. The remaining solution was then removed, and the grid was air-dried overnight. The sample was examined with a transmission electron microscope (TEM, Philips Tecnai 10, FEI Company, Eindhoven, the Netherlands) with 80 kV acceleration voltage, objective diaphragm 4 (20 μm), and 100 μm condenser aperture. Vesicular size and zeta potential of PE, TP, and TPE complexes were determined by using dynamic light scattering technique using Zetasizer Nano ZS (Malvern Instrument, Malvern, UK), with dtsv5.0 software. All samples were diluted to 30-fold with freshly distilled water prior to both measurements. The measurement was performed at 25 °C for five individual runs. The medium used in these measurements was water, which has the viscosity, refractive index, and dielectric constant of 0.8872 cP, 1.330, and 78.5, respectively.

Cytotoxicity and melanin production

Cell culture

Mouse melanoma cells (B16F10) were cultured in complete Dulbecco’s modified Eagle medium (DMEM supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin). The culture was incubated at 37 °C in a humidified 5% CO2 incubator. The culture medium was changed 2–3 times per week.

Cytotoxicity

The cytotoxicity of P, PE, TP, and TPE complexes on B16F10 cells was determined by sulforhodamine B (SRB) assay. Briefly, 10,000 B16F10 cells in 180 μL of the complete medium were added into each well of 96-well plate and incubated for 24 h. The medium was replaced with DMEM (without fetal bovine serum) containing P, PE, TP, and TPE with the final concentration of P of 4 ng/μL and incubated for 1 h. The medium was then replaced with complete DMEM and further incubated for 24 h. The cells were fixed with 50 μL/well of 50% trichloroacetic acid (Merck, Darmstadt, Germany) and incubated at 4 °C for 1 h. The fixed cells were rinsed for five times with distilled water and dried at room temperature. After 24 h, the cells were stained with 100 μL/well of 0.4% SRB and incubated at room temperature for 30 min. The SRB was removed, and the cells were washed for 5–6 times with 1% acetic acid and dried at room temperature overnight. The remaining SRB in the cells was dissolved by 100 μL/well of 10 mm Tris–HCl with shaking for 30 min. The absorbance was measured at 540 nm, and the percentage of viable cells was determined.

Cell transfection, melanin production, and tyrosinase enzyme activity

Cell transfection: The B16F10 cells were placed in 24-well plate (1 × 105 cells/well) and incubated at 37 °C in a humidified 5% CO2 incubator until 90% confluent. The medium was replaced with incomplete DMEM containing P, PE, TP, and TPE complexes at the final plasmid concentration of 4 ng/μL and incubated at 37 °C for 1 h. Then, the medium was replaced with complete DMEM and further incubated for 24 h.

Determination of melanin production: The transfected B16F10 cells were incubated with 500 μL of trypsin/EDTA solution at 37 °C in a humidified 5% CO2 incubator for 5 min. Trypsinization was inactivated by adding 500 μL of the complete DMEM. The cells were harvested, centrifuged at 2660 × g, 4 °C for 10 min. The cell pellet was washed by phosphate-buffered saline (PBS), pH 6.8, and resuspended in 500 μL of 1 N sodium hydroxide (NaOH) solution. The solution was incubated in a water bath at 60 °C for 1 h, centrifuged at 2190 × g, collected the supernatant, and measured the absorbance at 450 nm. The melanin concentration was determined from the calibration curve of the plot between A450 and concentration of standard melanin (Sigma-Aldrich).

Tyrosinase enzyme activity assay: The transfected B16F10 cells were trypsinized, harvested, and washed with PBS. The cell pellet was resuspended in 500 μL tyrosinase extraction buffer (0.1 mm phenylmethylsulfonyl fluoride and 0.5% Triton-X-100 in 50 mm phosphate buffer, pH 6.8). The cell dispersion was sonicated by microtip probe sonicator at 30% amplitude, pulse on 60.0, pulse off 30.0 for 6 cycles, and incubated at 4 °C for 1 h. The cell lysate was centrifuged at 13 680 × g, 4 °C for 30 min and collected the supernatant (16,17). Fifty microliters each of 2.5 mm l-tyrosine, 1.3 mm l-DOPA, 100 mm sodium phosphate buffer, pH 6.8, and cell lysate were mixed in a 96-well plate and measured the absorbance at 450 nm (A450 at T0). Then, the mixture was incubated at 37 °C for 60 min and measured the absorbance again at 450 nm (A450 at T60). The tyrosinase enzyme activity was determined from the calibration curve of the plot between A450 (A450 at T60-A450 at T0) and enzyme activity of standard mushroom tyrosinase (Sigma-Aldrich).

Stability study of P, PE, and TPE

An amount of 5 mL of P (0.05 μg/μL), PE (1:160), and TPE (0.5:1:160) complexes was transferred to a clear glass vial and kept at 4 ± 2, 25 ± 2, and 45 ± 2 °C in a dark chamber for 8 weeks. Samples were withdrawn at several time intervals (initial, 2, 4, 6, and 8 weeks). The P and TP were extracted from the PE and TPE complexes by mixing with phenol/chloroform/isoamylalcohol, centrifuged at 13 680 × g at 4 °C for 10 min, and the supernatant was collected. The remaining plasmid was hydrolyzed from T by trypsinization (15). Briefly, the supernatant containing 2.5 μg of plasmid was mixed with 20 μL of trypsin solution (5 μg of trypsin in 50 mm HEPES, pH 7.4) and incubated at room temperature (25 ± 2 °C) for 24 h. The remaining plasmid was determined by agarose gel electrophoresis and gel documentation as previously described. Quantitative determination of remaining tyrosinase plasmid was performed by using Quant-iT™ dsDNA BR assay kit (Invitrogen, Darmstadt, Germany). The vesicular size and zeta potential values of PE and TPE were also determined by ZetaSizer Nano ZS as previously described.

Statistical analysis

Statistical analysis was performed using Kruskal–Wallis test (Statview; Abacus Concepts, Piscataway, NJ, USA). A significance level was p < 0.05. All experiments were performed in triplicates.

Results

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments
  8. Conflict of Interest
  9. References

Physical properties of PE, TP, and TPE complexes

Table 1 shows zeta potential values of T, P, and E, which exhibited zeta potential values of 12.8 ± 4.0, (−)5.7 ± 1.9, and 45.9 ± 1.9 mV, respectively. TP (0.125:1), TP (0.25:1), and TP (0.5:1) showed negative zeta potential values of (−)16.8 ± 1.8, (−)11.4 ± 0.3, and (−)8.7 ± 1.5 mV, respectively, whereas the TP (1:1) and TP (2:1) were positive zeta potential of 4.5 ± 0.2 and 8.1 ± 2.8 mV, respectively. The blank E demonstrated vesicular size of 99.7 ± 0.1 nm, and the vesicular sizes were increased when loaded with both P and TP to obtain PE (163.5 ± 1.8 nm) and TPE (101.5 ± 0.5 to 151.4 ± 3.1 nm) complexes, respectively. In Figure 1, the excess T as determined by SDS–PAGE was observed at the T/P ratios of 0.5:1, 1:1, and 2:1 (w/w). This result indicated the maximum loading of T in TP complexes at the T/P ratio of 0.25:1. The agarose gel electrophoresis of various TP complexes (Figure 2) was correlated to the zeta potential values. In lanes 6 and 7, when the T/P ratio was >0.5:1, the plasmid band was not observed, indicating the complete interaction of P with the T peptide (T/P ratio of 1:1 and 2:1), resulting in the failure of the staining with ethidium bromide. When hydrolyzed the Tat peptide with trypsin, the plasmid DNA could be stained with ethidium bromide as shown in lane 8–12. The intact tyrosinase plasmid bands observed from all digested TP complexes also indicating the stability against enzymatic degradation of the plasmid in all TP complexes. From the results of SDS–PAGE, agarose gel electrophoresis, and zeta potential values, only the TP (0.125:1), TP (0.25:1), and TP (0.5:1) that exhibited negatively charged were selected to entrap in elastic cationic niosomes, whereas the TP (1:1) and TP (2:1) that showed positively charged were not selected. Agarose gel electrophoresis of PE and TPE complexes was shown in Figure 3. The free P was not observed in lanes 3 and 6–8 indicating 100% entrapment efficiency of the P in PE and TPE. When extracted with phenol/chloroform/isoamylalcohol, the PE and TPE complexes exhibited intact P at lanes 4 and 9–11, with percentages recovery of the plasmid of 70.80%, 89.86%, 93.89%, and 96.60%, respectively (Table 2). Negative staining of TEM (transmission electron microscope) images of PE and TPE complexes was demonstrated in Figure 4. The vesicles were irregular in shape, unilamella structure with 150–300 nm in size.

Table 1.   Vesicular size, zeta potential and cytotoxicity of Tat (T), tyrosinase plasmid DNA (P), elastic cationic niosomes (E), Tat/tyrosinase plasmid (TP), tyrosinase plasmid–loaded elastic cationic niosomes (PE), and Tat/tyrosinase plasmid/elastic cationic niosomes (TPE) complexes
SampleInitial8-Week storageCytotoxicity
Zeta potential (mV)aVesicular size (nm)aZeta potential (mV)aVesicular size (nm)a% Cell viability
4 ± 2 °C25 ± 2 °C45 ± 2 °C4 ± 2 °C25 ± 2 °C45 ± 2 °C
  1. ND, not done.

  2. aValues represented as mean ± SD (n = 3).

Tat peptide (T)(+) 12.8 ± 4.0
Tyrosinase plasmid DNA (P)(−) 5.7 ± 1.979.25 ± 5.86
Elastic cationic niosomes (E)(+) 45.9 ± 1.999.7 ± 0.1
TP complexes
 TP (0.125:1)(−) 16.8 ± 1.8209.9 ± 10.695.92 ± 4.37
 TP (0.25:1)(−) 11.4 ± 0.3252.9 ± 26.2103.90 ± 9.85
 TP (0.5:1)(−) 8.7 ± 1.5217.7 ± 19.0101.31 ± 2.40
 TP (1:1)(+) 4.5 ± 0.21697.3 ± 80.7ND
 TP (2:1)(+) 8.1 ± 2.8258.7 ± 8.8ND
PE complexes
 PE (1:160)(+) 37.0 ± 0.6163.5 ± 1.8(+) 31.8 ± 2.1(+) 46.0 ± 1.7(+) 45.5 ± 0.9170.3 ± 4.6218.5 ± 3.2190.7 ± 2.389.03 ± 2.75
TPE complexes
 TPE (0.125:1:160)(+) 39.2 ± 5.0109.2 ± 2.995.08 ± 3.65
 TPE (0.25:1:160)(+) 38.7 ± 0.9151.4 ± 3.192.57 ± 10.13
 TPE (0.5:1:160)(+) 36.1 ± 7.1101.5 ± 0.5(+) 30.6 ± 2.5(+) 35.6 ± 1.9(+) 36.2 ± 3.7135.1 ± 0.9158.7 ± 9.4200.1 ± 8.491.36 ± 5.22
image

Figure 1.  SDS–PAGE of Tat/tyrosinase plasmid (TP) complexes. Lane 1: std. polypeptide; lane 2: Tat (1 μg); and lane 3–7: TP (0.125:1), TP (0.25:1), TP (0.5:1), TP (1:1), and TP (2:1), respectively.

Download figure to PowerPoint

image

Figure 2.  Agarose gel electrophoresis of Tat/tyrosinase plasmid (TP) complexes. Lane 1: DNA ladder 1 kb; lane 2: tyrosinase plasmid 0.25 μg; lane 3–7: TP (0.125:1), TP (0.25:1), TP (0.5:1), TP (1:1), and TP (2:1), respectively, and lane 8–12: TP (0.125:1), TP (0.25:1), TP (0.5:1), TP (1:1), and TP (2:1), respectively, which trypsinized by 0.2 units trypsin.

Download figure to PowerPoint

image

Figure 3.  Agarose gel electrophoresis of tyrosinase plasmid loaded in elastic cationic niosomes (PE) and Tat/tyrosinase plasmid/elastic cationic niosomes (TPE) complexes. Lane 1: DNA ladder 1 kb; lane 2: tyrosinase plasmid 0.25 μg; lane 3: intact PE (1:160); lane 4: PE (1:160) extracted with phenol/chloroform/isoamylalcohol; lane 5: PE (1:160) extracted by phenol/chloroform/isoamylalcohol and trypsinized by 0.2 units trypsin; lane 6–8: intact TPE (0.125:1:160), TPE (0.25:1:160), and TPE (0.5:1:160), respectively; lane 9–11: TPE (0.125:1:160), TPE (0.25:1:160), and TPE (0.5:1:160), respectively, extracted with phenol/chloroform/isoamylalcohol; and lane 12–14: TPE (0.125:1:160), TPE (0.25:1:160), and TPE (0.5:1:160), respectively, extracted by phenol/chloroform/isoamylalcohol and trypsinized by 0.2 units trypsin.

Download figure to PowerPoint

Table 2.   Percentage recovery of the plasmid in Tat/tyrosinase plasmid (TP) complexes, tyrosinase plasmid–loaded elastic cationic niosomes (PE), and Tat/tyrosinase plasmid/elastic cationic niosomes (TPE) complexes
Sample% Recovery
Tat/tyrosinase plasmid (TP) complexes
 TP (0.125:1)82.44
 TP (0.25:1)85.63
 TP (0.5:1)90.16
Tyrosinase plasmid–loaded elastic cationic niosomes (PE) complexes
 PE (1:160)70.80
Tat/tyrosinase plasmid/elastic cationic niosomes (TPE) complexes
 TPE (0.125:1:160)89.86
 TPE (0.25:1:160)93.89
 TPE (0.5:1:160)96.60
image

Figure 4.  Negative staining TEM images of (A) tyrosinase plasmid–loaded elastic cationic niosomes [PE (1:160)] and (B) Tat/tyrosinase plasmid/elastic cationic niosomes complexes [TPE (0.5:1:160)] at the magnification of ×20 000.

Download figure to PowerPoint

Cytotoxicity

The percentages of cell viability after over 1 h of incubation with samples as determined by SRB assay demonstrated were <90% (data not shown). Thus, the incubation period of 1 h was selected for the cytotoxicity and cell transfection studies. The percentages of cell viability are given in Table 1. The percentages of viability of the cells transfected with TP (0.25:1) and TP (0.5:1) were comparable with the control, which were 103.90 ± 9.85 and 101.31 ± 2.40% respectively. For P, PE, TP, and TPE complexes, slight decrease in cell viability (79.25 ± 5.86–95.92 ± 4.37% of control) was observed.

Tyrosinase gene expression and melanin production

Figure 5 shows the tyrosinase gene expression determined as tyrosinase enzyme activity and melanin production of P, TP, PE, and TPE in the B16F10 cells. The cells transfected with TPE (0.5:1:160) demonstrated 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), respectively 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 enzyme activity and melanin production of the B16F10 cells transfected with P, TP (0.125:1), TP (0.25:1), and TP (0.5:1) were not significantly different from the untreated cells.

image

Figure 5.  Folds of tyrosinase enzyme activity (□) and melanin production (▪) comparing with control of B16F10 cells transfected with various ratios of tyrosinase plasmid (P), Tat/tyrosinase plasmid (TP), tyrosinase plasmid loaded in elastic cationic niosomes (PE), and Tat/tyrosinase plasmid/elastic cationic niosomes (TPE) complexes. The control (untreated cells) was indicated as 1 (*p < 0.05).

Download figure to PowerPoint

Stability of P, PE (1:160), and TPE (0.5:1:160)

Agarose gel electrophoresis of the remaining P of P, PE, and TPE kept at 4 ± 2, 25 ± 2, and 45 ± 2 °C for 8 weeks are shown in Figure 6. All P-loaded nanovesicular formulations demonstrated physical stability without sedimentation. After 8 weeks, the remaining P of P, PE, and TPE kept at 4 ± 2 °C and 25 ± 2 °C were 35.00 ± 0.53, 40.35 ± 1.41, and 44.71 ± 0.72% and 23.20 ± 0.87, 29.69 ± 0.29, and 31.91 ± 0.17%, respectively. At 45 ± 2 °C, the remaining P was observed only from PE and TPE complexes of 27.64 ± 0.16 and 28.32 ± 0.36%, respectively, whereas P was almost degraded at 6 weeks. Vesicular size and zeta potential of PE and TPE kept at various temperatures for 8 weeks are shown in Table 1. The vesicular size of PE and TPE kept was slightly increased up to 220 nm after 8 weeks of storage. The zeta potential values of PE and TPE were higher than +30 mV indicating the stability of the complexes after 8 weeks storage.

image

Figure 6.  Agarose gel electrophoresis of tyrosinase plasmid (P) solution, tyrosinase plasmid loaded in elastic cationic niosomes [PE (1:160)], and Tat/tyrosinase plasmid/elastic cationic niosomes [TPE (0.5:1:160)] complexes kept at 4 ± 2, 25 ± 2, and 45 ± 2 °C for 8 weeks. Initial (A): lane 1 = DNA ladder 1 kb; lane 2 = standard pAH7/Tyr 0.25 μg; lane 3–4 = PE and TPE, respectively; lane 5 = P; and lane 6–7 = PE and TPE, respectively, extracted by phenol/chloroform/isoamyl alcohol. Week 2 (B), week 4 (C), week 6 (D), and week 8 (E): lane 1 = DNA ladder 1 kb; lane 2 = standard pAH7/Tyr 0.25 μg; lane 3–5 = P at 4 ± 2, 25 ± 2, and 45 ± 2 °C, respectively; lane 6–8 = PE extracted by phenol/chloroform/isoamylalcohol at 4 ± 2, 25 ± 2, and 45 ± 2 °C, respectively; and lane 9–11 = TPE extracted by phenol/chloroform/isoamylalcohol at 4 ± 2, 25 ± 2, and 45 ± 2 °C, respectively.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments
  8. Conflict of Interest
  9. References

The applications of nanovesicular formulations and CPPs including Tat for drugs, proteins, and gene delivery have been reported (18,19). In this study, the Tat-incorporated elastic nanovesicles exhibited an enhancement effect on cell transfection of the tyrosinase harboring plasmid. PE and TPE exhibited larger vesicular size and lower positive zeta potential values than the non-loaded elastic cationic niosomes, which might be due to the entrapment of P in the aqueous part between the vesicular bilayers and the adsorption of the negatively charged P on the vesicular membrane of the cationic niosomes. The increase in vesicular size might be also resulting from the charge repulsion effect between the T and P molecules (10) inside the bilayer nanovesicles. The TPE complexes gave high percentages recovery of the plasmid of 96.60% indicating the stability of the plasmid after complexation. Oxidation of the thiol groups (-SH) presence in cysteine residues of the Tat peptide led to interpeptide disulfide (S-S) bond formation, resulting in higher plasmid stability of the TPE complexes over the PE (15). Higher cytotoxicity of the PE and TPE complexes comparing to the TP complexes might be due to the effect of cationic lipid (20,21) and ethanol in the elastic cationic niosomal formulation. Cationic lipids can cause cytotoxicity by interacting with critical enzyme such as protein kinase C (PKC), the key enzyme family in signal transduction pathways (20). Ethanol, an amphipathic molecule, can alter the function of membrane-bound enzyme by interacting with the enzyme itself or by interacting with other components of the membrane resulting in cell lysis and cell death (4). The cells transfected with TPE complexes (0.5:1:160) demonstrated the highest tyrosinase gene expression as determined by tyrosinase enzyme activity and melanin production. The electrostatic interaction between the positively charged TPE complexes and the negatively charged cell membrane might increase the cell transfection resulting in an increase in gene expression and melanin production. After internalization by endocytosis, the niosomes might fuse with the endosomal membrane and allowed the plasmid DNA in the endosome to penetrate into the cytoplasm. Although the TPE (0.5:1:160) exhibited the highest tyrosinase activity and melanin production, the zeta potential value of the complexes was lower than the other PE and TPE complexes. A higher tyrosinase activity of cells transfected with TPE than PE might be due to the synergistic effect of E and T in TPE complexes. Although the PE showed a slight increase in cell transfection resulting in a slight increase in tyrosinase activity and melanin content, this probably might be due to the cationic behavior of E which interacted with the negative charge of the cell membrane. However, strong enhancement in cell transfection was observed when T together with E was incorporated. The result indicating that the E, which was participated in electrostatic interaction with the cell surface, might not be efficient enough to exert the proper internalization of the loaded P (22). T peptide in TPE might play an important role after the attachment of the complexes with the cell membrane. Tat peptide could adapt an α–helix structure at endosomal pH leading to hydrophobic and hydrophilic faces that could interact with and cause disruption and pore formation of the endosomal membrane leading to an effective release of the loaded DNA from the endosome followed by the internalization into the nucleus of the DNA (13,23). Moreover, no significant difference of the tyrosinase enzyme activity and melanin production between untreated cells and cells transfected with P, TP (0.125:1), TP (0.25:1), and TP (0.5:1) was observed. These might be due to their negative charge and the charge repulsion effect between them and the negatively charged cell membrane. The results of the stability study indicated the enhancement of thermal stability of the plasmid DNA by loading in elastic cationic niosomes. For the elastic cationic niosomes, the plasmid could be entrapped inside the hydrophilic region of the vesicles and the vesicular membrane might act as a shield to protect the loaded plasmid from thermal and stress of the environment (24). Furthermore, the interpeptide disulfide (S-S) bond formation via oxidation of cysteine thiol (-SH) groups of the Tat peptide was also resulting in the higher plasmid stability of the TPE complexes (23). The zeta potential values of the PE and TPE complexes after 8 weeks of storage were in the range of 31.8 ± 2.1 to 45.5 ± 0.9 mV for PE and 30.6 ± 2.5 to 36.2 ± 3.7 mV for TPE, indicating the physical stability of the complexes because the zeta potential out of the range of ± 30 mV is required to achieve a stable dispersion (25). These results indicated the potential application of the TPE complexes for further investigation for vitiligo gene therapy especially in the tyrosinase gene deficient patients.

Conclusion

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments
  8. Conflict of Interest
  9. References

The B16F10 cells transfected with the TPE complexes at the T/P/E ratio of 0.5:1:160 w/w/w demonstrated the highest enhancement of the tyrosinase enzyme activity and melanin production of 11.82- and 13.03-; 7.67- and 8.46-; 5.07- and 5.36-; and 6.29- and 6.58-folds of the untreated cells and cells transfected with P, PE, and TP (0.5:1), respectively. All TPE complexes showed a slight or no cytotoxic effect on the cell line. The PE and TPE complexes exhibited high stability of the loaded plasmid when kept at various temperatures for 8 weeks. This study indicated the enhancement of human tyrosinase gene expression and melanin production with relatively low cytotoxicity by Tat and entrapment in elastic cationic niosomes, and also the enhancement of stability of the plasmid loaded in the TPE complexes. The results of this work can be applied for further developments for vitiligo gene therapy.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments
  8. Conflict of Interest
  9. References

This work was supported by the Thailand Research Fund (TRF) under the RGJ-PhD program, Natural Products Research and Development Center (NPRDC), Science and Technology Research Institute (STRI), Chiang Mai University, Chiang Mai 50200, Thailand. The authors are gratefully acknowledged for supporting, as are Boehringer Ingelheim and University of Tübingen, Germany for providing tyrosinase (pAH7/Tyr) plasmid used in this study.

References

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments
  8. Conflict of Interest
  9. References