- Email: [email protected]
Enhancement of TAT cell membrane penetration efficiency by dimethyl sulphoxide
Journal of Controlled Release 143 (2010) 64–70
Contents lists available at ScienceDirect
Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l
Enhancement of TAT cell membrane penetration efficiency by dimethyl sulphoxide Hu Wang a, Chun-Yan Zhong a,1, Jiang-Feng Wu a, Yu-Bin Huang b,⁎, Chang-Bai Liu a,⁎ a b
The Institute of Molecular Biology, Three Georges University, Yichang, 443002, P.R. China State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 130022, P.R. China
a r t i c l e
i n f o
Article history: Received 10 June 2009 Accepted 2 December 2009 Available online 16 December 2009 Keywords: Cell penetrating peptides (CPPs) Dimethylsulfoxide (DMSO) TAT Penetration
a b s t r a c t Cell penetrating peptides (CPPs) are promising tools for transducing presynthesized therapeutic molecules which possess low membrane permeability. The poor efficiency of cellular uptake and unexpected cellular localization are still the main obstacles to the development of drug delivery by using CPPs. In this study, we investigated the effect of a penetration enhancer, dimethylsulfoxide (DMSO), on the penetrating efficiency of a synthetic TAT peptide or the TAT fusion protein. FITC-labeled TAT and TAT-GFP were added to 10% DMSO or 100 μM chloroquine pretreated cells, fluorescence uptake into culturing cells was observed using fluorescence microscopy, FACS or quantitatively analyzed by a fluorescence spectrum. 10% DMSO treatment markedly increased internalization of TAT into cells and appeared in a well-distributed pattern throughout the cytosol and nucleus without membrane perforating or detectable cytotoxicity, the enhancement effect by 10% DMSO was reduced by endocytosis inhibitors including ammonium chloride and sodium azide. 10% DMSO also enhanced TAT-Apoptin induced apoptosis of carcinoma cells. These findings implicated that DMSO can be a novel delivery enhancer appropriate for CPP penetration. © 2009 Elsevier B.V. All rights reserved.
1. Introduction It was discovered that certain peptides, referred to as cell penetrating peptides (CPPs), can penetrate cells accompanied by a large molecular cargo. CPPs have found numerous applications in biology and medicine since the first synthetic cell-permeable sequence was identified two decades ago. Considerable research effort is currently focused on utilizing CPPs as peptide-based vehicle for intracellular drug delivery. Several types of drugs have been transported into cells using CPPs, including small molecule pharmaceuticals, therapeutic proteins, siRNA and antisense oligonucleotides [1–3]. Many CPPs are derived from the protein transduction domains (PTD) of viral proteins involved in interaction with cell membrane. A typical example of CPPs is TAT, a basic peptide segment corresponding to the positions 48–60 of Tat protein, a transcription regulator protein of HIV-1, is also one of the most frequently employed for intracellular delivery. It has been reported that CPPs including TAT are able to deliver various bioactive molecules into cells and have enormous ⁎ Corresponding authors. Liu is to be contacted at The Institute of Molecular Biology, Three Georges University, 8 Daxue Road, Yichang, 443002 China. Tel.:+86 717 639 7179. Huang, State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun City, Jilin Province, 130022, China. E-mail addresses: [email protected] (C.-B. Liu), [email protected] (Y.-B. Huang). 1 Current address: Clinical laboratory, The 3rd General Hospital of Hangzhou, Hangzhou, 310009, P.R. China. 0168-3659/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2009.12.003
potential to solve age-old problems in the delivery of macromolecular therapeutics [4]. Despite their broad acceptance as molecular vehicle, the mechanism of internalization of CPPs and the conjugated cargo is not well understood. The cellular uptake of TAT peptide had originally been described to be insensitive to low temperature and to inhibitors of endocytosis, but there are recent reports controversially declared that endocytosis may also be involved in TAT transduction [5–7]. Although accumulated research efforts are currently focused on CPPs as potential intracellular drug delivery vehicle, the application of this technology is limited because the transduction efficiency is often insufficient for therapeutic purposes, even using TAT. Some reports have observed that greater than 90% of the fluorescent signal from labeled TAT or the fusion proteins appears in punctate vesicles, not in the nucleus or cytosol [8,9]. Reports have also shown an improvement in transduction efficiency with endosomolytic agents like chloroquine, which indicates that much of the peptide remains in vesicles of undetermined fate [10,11]. This would appear to be a limitation of transduction if the CPPs remain in endosome/lysosome to be degraded rather than enter the cytosol or nucleus [12]. To use CPPs as effective intracellular drug delivery tool for clinical applications, it is necessary to search agents which could enhance the penetrating ability of CPPs. Dimethylsulfoxide (DMSO) has been commonly used for several years as a cryoprotectant agent for storage of culture cells or for hematopoietic stem cell transplantation in a variety of hematological disorders. Since the biological properties of DMSO were discovered in 1960 it has been used not only for laboratory but also clinical
H. Wang et al. / Journal of Controlled Release 143 (2010) 64–70
purposes. In 1978 it received approval by the US Food and Drug Administration for use in the treatment of interstitial cystitis by intravesical instillation [13]. DMSO has been used successfully in the treatment of various human diseases such as dermatological, urinary, pulmonary, rheumatic and renal manifestations of amyloidosis, gastrointestinal diseases, traumatic brain edema, musculoskeletal disorders, chronic prostatitis, dermatological diseases, schizophrenia, and Alzheimer's disease [14–33]. In more recent studies, DMSO was used as an efficient penetration enhancer for percutaneous drug [34– 37]. Li et al. [38] and Shen et al. [39] described that the transfection of exogenous DNA incubated with DMSO were more efficient than without any treatment. Based on these researches, the present study was undertaken to investigate the possibility of improving TAT or TAT fusion proteins penetrating efficiency into cytoplasma for future development as a drug delivery tool based on TAT peptide. As a vehicle to deliver bio-drugs into cells, it should have no any effect on the bioactivity of the drugs. We also analyzed that cancer cell apoptosis induced by Apoptin, a small protein derived from chicken anemia virus, can especially induce apoptosis in transformed or tumor cells, but not in normal cells, fused with TAT. We also found that DMSO increased the cell apoptosis caused by TAT-Apoptin. 2. Materials and methods 2.1. Chemicals, reagents and cells RPMI-1640 medium (Invitrogen, USA), FBS (Invitrogen, USA); the chemicals: sodium azide (Sigma-Aldrich), In Situ Cell Death Detection Kit (Roche Applied Science, USA), and CytoTox-ONETM Membrane integrity assay kit (Promega, USA) were purchased from commercial company. Caski, Siha, HepG2, and A549 cells were maintained in our laboratory. 2.2. Peptide internalization FITC-labeled peptides used in this study (TAT: YGRKKRRQRRRK and NCO, a nonsense peptide: KALGISYGRKK) were synthesized by SBS Genetech (Beijing, China). The peptides were purified using reversed phase analytical HPLC to more than 99% purity then diluted to 500 mM in PBS and were stored at −20 °C for further use. TAT-FITC and NCO–FITC internalized into culture cells were observed under fluorescence microscopy. Caski, Siha, or HepG2 cells were seeded into 12-well plates (Greiner, Germany) at a density of 5 × 105 cells per well and cultivated to semi-confluence in an RPMI1640 medium in a humid atmosphere supplemented with 5% CO2 for 24 h at 37 °C respectively. Cells were washed 3 times with PBS and then added different concentrations (v/v %) of DMSO supplemented with a serum-free medium, incubation for 1 h with 5% CO2 at 37 °C, then added 5 μM FITC-labeled peptides and incubated for another 1 h with 5% CO2 at 37 °C. The cells were further washed 5 times with PBS then the fluorescence was observed under fluorescence microscopy (Nikon, Japan) using a bandpass filter (detects FITC). 100 µM chloroquine was used as a positive control in all FITC-labeled peptide uptake experiments. TAT-GFP fusion protein (Supplementary data Fig. S1) internalized into culture cells was observed under fluorescence microscopy. When the cells were 70% confluent, the culture medium was replaced with a fresh serum-free medium containing indicated concentrations of DMSO for 1 h with 5% CO2 at 37 °C; the cells were washed 3 times with PBS and then TAT-GFP was added to the serum-free medium, with a final concentration of 5 μM. The cells were incubated for 3 h with 5% CO2 at 37 °C. TAT-GFP fusion protein treated cells were washed 5 times with PBS and then observed under microscopy. In some experiments, cell internalization of synthetic FITC-labeled peptides was analyzed using flow cytometry. Caski cells were pretreated with indicated concentrations of DMSO for 1 h in serum-
65
free media prior to addition of FITC-labeled peptides with a final concentration of 5 μM in a serum-free medium, then the cells were washed 5 times with PBS. The cells were de-adhered by incubating with trypsin–EDTA for 10 min, and then washed again 3 times. The suspended cells were then fixed with 1% paraformaldehyde and then washed with PBS twice. Analysis was conducted on a FACS Caliber flow cytometer (Beckman Coulter, Fullerton, CA, USA), equipped with a 488-nm air-cooled argon laser. The filter settings for excitation and emission were 480/530 nm bandpass (FL1) for FITC. The fluorescence of 5000 vital cells was acquired, and data were analyzed in a logarithmic mode. In order to quantify the intracellular fluorescence intensity at the conditions of DMSO pretreatment, FITC-labeled peptide uptake in various cells were quantitated by Multimode Spectrophotometry. After the incubation of FITC-labeled peptide, cells were washed with PBS 5 times and then lysed by adding 300 μl 0.1 M NaOH for 10 min at room temperature, the cell lysates were centrifuged (14000 g for 5 min) and the fluorescence intensity of the supernatant was measured at 494/518 nm in a Multimode Microplate Reader (Tecan 2000; Tecan, Mannedorf, Switzerland). The cellular protein content was determined by the method of Bradford protein assay with BSA as standard (Bio-Rad). The fluorescence of cellular uptake is expressed as fluorescence intensity per mg of total cellular protein. All experiments were repeated at least three times and always performed in triplicate. The uptake levels are reported as the arithmetic mean of all samples, whereas the error bars represent the average maximum and minimum data spread [40]. To evaluate the influence of various cells, characterized inhibiting drugs were selected for their ability to inhibit specific steps in the endocytosis pathway. 50 mM ammonium chloride and 40 μM sodium azide were added to the cell culture medium for 30 min followed by DMSO treatment and then added the FITC-labeled peptides. The protein uptake efficiencies were quantified after a 1 h incubation of the cells with the FITC-labeled peptide in the presence of the inhibitor by the Multimode Microplate Reader, as described above. 2.3. Hemolysis and LDH release assay To rule out that DMSO enhanced TAT penetration by perforating the plasma membrane, hemolysis and lactate dehydrogenase (LDH) release assays were employed. Red blood cell (RBC) membrane destabilization, or hemolysis, has been associated with endosomal escape [41]. In the present study, a hemolytic assay was used to detect whether DMSO could perforate the RBC membrane different concentrations. Briefly, whole blood was collected from BALB/c mice. RBCs were separated from whole blood by centrifugation for 20 min at 300 g, and then washed 3 times with fresh 150 mM NaCl and collected by centrifugation for 5 min at 200 g, then diluted to 2 × 108 cell/ml in 0.1 M PBS at the desired DMSO concentrations (5%, 7.5%, 10%, 15%, and 20%). As a positive control, 0.1% Triton X-100 (Sigma) was added to 0.1 M PBS before addition of RBCs in the microcentrifuge tube, then incubated for 1 h at 37 °C. Maximum release of hemoglobin was detected by incubation of the RBCs in 0.1% Triton-X 100 under the same conditions. The optical density (OD) at 414 nm allowed evaluation of the percentage of hemolysis by comparison with the values obtained in PBS, after correction of the absorbance values by the blank value. The OD value of untreated, DMSO or 0.1% Triton X-100 treated cell supernatants is indicated as ODB, ODT and ODM respectively, and the cell toxicity is calculated as: toxicity= 100 × ((ODT − ODB) / (ODM − ODB))%. LDH release assay [42] was performed by using a CytoTox-ONETM Membrane integrity assay kit (Promega), according to the instructions of the manufacturer of the kit. Caski cells were seeded at a density of 1 × 105 cells per well in a 12-well plate. 16–24 h after seeding, the cells were treated with concentrations of DMSO and incubated for 1 h with 5% CO2 at 37 °C, then 100 μl of the cell supernatants were transferred
66
H. Wang et al. / Journal of Controlled Release 143 (2010) 64–70
to a black 96-well-plate and 100 μl of the substrate mix was added to each well. Background value was obtained from untreated cells and maximum LDH release by treating cells with 2% NP-40. After 10 min of incubation at room temperature, the enzymatic reaction was stopped by stop solution and then measured at 560/590 nm in a Multiskan Spectrum plate-reader (Thermo, USA). 2.4. Detection of cell viability by using MTT assay 4 × 104 cells per well of Caski, Siha, HepG2 or A549 cells were seeded in 96-well plates and cultivated to semi-confluence in an RPMI-1640 medium in a humid atmosphere supplemented with 5% CO2 for 24 h at 37 °C respectively. Cells were washed 3 times with PBS and then different concentrations (v/v %) of DMSO supplemented with a serum-free medium were added, incubated for 2 h with 5% CO2 at 37 °C and washed with PBS 3 times then incubated for 24 h with fresh serum medium. Cell viability was then assessed using MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] (Sigma-Aldrich) assays. Briefly, 20 µl of 5.5 mg/ml MTT in a serumfree medium was added directly to each well giving a final concentration of 0.5 mg/ml. The cells were then incubated for 4 h at 37 °C with 5% CO2. The plates were centrifuged at 1000 g for 5 min before removing the supernatant and then adding 100 µl of DMSO. The samples were finally incubated at 37 °C for 30 min before quantifying the absorbance at 550 nm. 2.5. TUNEL analysis of cell apoptosis induced by TAT-Apoptin fusion protein Cell apoptosis induced by TAT-Apoptin (Supplementary data Fig. S1) was analyzed by the terminal dUTP-digoxigenin nick-end labeling (TUNEL) assay using the ApopTag Plus Peroxidase In Situ Cell Death Detection Kit (Roche Applied Science, Germany). The procedure of TAT-Apoptin fusion protein treatment was same as TAT-GFP
Fig. 1. FITC-labeled peptides uptaken by Caski cell pretreated with different concentrations of DMSO. Caski cells were exposed to 10% DMSO pretreatment for 1 h, followed by incubating with 5 μM FITC-labeled peptide for 1 h after washing. The fluorescence intensity in cell lysis was measured at 494/518 nm. A. Fluorescence intensity of FITC-labeled peptides uptaken by flow cytometry assay with the concentrations of DMSO from 5% to 20%, 100 μM chloroquine and 0.5 M sucrose treatment as the positive control; B: TAT-FITC distributed in Caski cell with or without 10% DMSO pretreatment observed under fluorescence microscopy; scale bars, 50 μm. C: Fluorescence quantitation of FITC-labeled peptides uptaken in Caski cell pretreated by 10% DMSO; means ± s.d. of three experiments are shown. D: Fluorescence quantitation of FITC-labeled peptides uptaken at different incubation time. Means±s.d. of three experiments are shown.
H. Wang et al. / Journal of Controlled Release 143 (2010) 64–70
67
could be penetrating into the cytosol and then degraded (Fig. 1D). Comparing the transduction enhancement by different concentrations
Fig. 2. Fluorescence intensity of FITC-labeled peptides uptaken in different cells pretreated with 10% DMSO and peptides at different concentrations used. A: Caski cells uptaken into the cytosol quantified with the incubation of synthetic peptide at concentration from 2.5 μM to 10 μM treated by 10% DMSO. Means ± s.d. of three experiments are shown. B: Culturing Siha, A549, and HepG2 cell lines were exposed to 10% DMSO pretreatment for 1 h, followed by incubation with 5 μM FITC-labeled peptides for 1 h after washing. The fluorescence intensity in cell lysis was measured at 494/518 nm.
treatment. The cell was incubated for 24 h in a fresh serum medium containing TAT-Apoptin (5 μM), and then fixed with 1% paraformaldehyde in PBS, subjected to TUNEL staining, followed by visualization under microscopy with diaminobenzidine according to the instruction of the TUNEL kit. As negative controls, terminal deoxynucleotidyl transferase was omitted. 3. Results 3.1. 10% DMSO enhanced TAT-FITC uptake and in a well-distributed pattern throughout the cytosol and nucleus By pretreating with 10% DMSO, the TAT-FITC uptake into cells was markedly increased (Fig. 1A) and appeared in a well-distributed pattern throughout the cytosol and nucleus, whereas as a negative control, NCO–FITC could not be detected using the same photomultiplier settings under fluorescent microscopy (Fig. 1B). TAT-FITC uptake into cells with same condition was quantified using a Multimode fluorescent Microplate Reader (Fig. 1C). The fluorescence in the cytosol became weaker over time, suggesting that the peptide Fig. 3. Cell hemolysis and LDH activity treated by different concentrations of DMSO and cell viability evaluated by MTT assay. A. RBCs were incubated with DMSO at various concentrations for 1 h at 37 °C. Hemolytic activity was determined by the amount of hemoglobin released from RBCs with measuring the absorbance at 541 nm. There was no hemolytic activity seen at 10% DMSO compared with positive control using 0.1% Triton X-100. B. Different cells pre-incubated with DMSO at different concentrations as shown in A for 1 h at 37 °C. LDH release quantity was determined by measuring the absorbance at 570 nm. DMSO did not lyse any kind of cells compared with positive control using 2% NP-40. C: Different cells pre-incubated with DMSO at different concentrations (2.5%, 5%, 10%, 15%, and 20%) for 2h at 37 °C, discarding the supernatant, washed 3 times with PBS and added with a fresh medium then cultured for 24 h; measurement procedure following the MTT protocol. D: Caski cells incubated with 10% DMSO for 2 h at 37 °C, discarding the supernatant, washed 3 times with PBS and added with a fresh medium then cultured 0 h, 1 h, 12 h, 24 h and 48 h; measurement procedure was same as C. Means ± s.d. of three experiments are shown.
68
H. Wang et al. / Journal of Controlled Release 143 (2010) 64–70
of DMSO pretreatment, the result of FACS analysis revealed multiple levels of increased transduction potential by increasing DMSO concentration (Fig. 1A). This enhancement effect also was observed in A549, a human lung cancer cell line, HepG2, a human hepatocellular carcinoma cell line, Siha cells (Fig. 2B), and in other cell lines (Supplementary data Fig. S2). At 10% (w/v) DMSO, penetrating efficiency became higher with TAT concentration from 2.5 μM to 10 μM (Fig. 2A). These results revealed that transduction potential of TAT could be enhanced by DMSO pretreatment, and the efficiency is much higher than that of chloroquine pretreatment.
production within the cell membrane [44–49]. As shown in Fig. 4, before the addition of FITC-labeled TAT, cells were pre-incubated with the energy depleter, anti-acidification agent as endocytic inhibitor; TAT transduction levels were moderately inhibited. This data indicated that endocytosis is not fully, but partially involved in TAT internalization. 10% DMSO pretreatment does not affect TAT transduction.
3.2. 10% DMSO enhanced TAT penetrating efficiency without cell membrane perforation or cytotoxicity
To assess the effect of DMSO pretreatment on transduction of TAT with large cargo, we determined the subcellular localization of TATGFP fusion protein with or without DMSO pretreatment. As shown in Fig. 5, the levels of TAT-GFP fusion protein uptake by cells pretreated with 10% DMSO were significantly higher than in untreated cells. Fusion proteins were detected in the nucleus as well as in the cytoplasm of the cells, whereas pretreatment by chloroquine detected faint diffuse fluorescence in the cytoplasm; this is consistent with the results from FITC-labeled TAT peptide experiments (Fig. 1).
Gurtovenko et al. [43] report that high concentration of DMSO induced transient water pores on a chemical membrane in a simulating test. It was speculated that there is a correlation between the ability of penetration enhancer to lyse membranes under natural conditions and penetrating efficiency. In order to exclude membrane disturbance by DMSO, a hemolysis and a lactate dehydrogenase (LDH) leakage assay were performed in the present study. Both hemolysis assay and LDH release assay showed that DMSO treatment does not induce pores or lyse the cell membrane under physiological conditions (Fig. 3A and B). This result suggested that DMSO pretreatment enhanced cell membrane penetration of TAT was not through inducing pores on cell membrane. The mechanism needs to be explored in a further study. The effect of DMSO treatment on the cell growth of different cells was first evaluated by using various concentrations (2.5%, 5%, 10%, 15% and 20%) of DMSO at 2 h through an MTT assay. Repressive effect on the cell growth was observed at 20% DMSO treatment, whereas, 10% DMSO treatment up to 2 h has very little repressive effect on cell growth (Fig. 3C and D). The cytotoxicity of 10% DMSO to HeLa, NIH3T3 and CHO cells was also detected; DMSO suppressed only NIH3T3 growth to about 60% but not other cells (Supplementary data Fig. S3). These results here suggested that the sensitivity to DMSO maybe dependent on different cell lines, and there was minimal cytotoxicity to some cell lines up to 10% of DMSO treatment.
3.4. DMSO enhanced cellular uptake of TAT-GFP fusion protein
3.3. Endocytosis inhibitors suppressed the enhancement effect of DMSO on TAT penetration To analyze the effects of DMSO pretreatment on the internalization of TAT, different chemicals (ammonium chloride and sodium azide) were used to inhibit endocytosis of FITC-labeled peptides into the cells. Ammonium chloride is a weak base that increases the pH of acidic organelles, including endocytic vesicles. Sodium azide is an oxidative phosphorylation inhibitor commonly used to abolish ATP
Fig. 4. Suppressive effect of NaN3, NH4Cl exposure on TAT-FITC uptake with 10% DMSO pretreatment in Caski and Siha cells. Confluent monolayers of Caski or Siha cell was preincubated in a 10%DMSO serum-free medium for 1 h at 37 °C; cells were washed twice prior to addition of inhibitors, incubation with inhibitors for 30 min at 37 °C then addition of FITC-labeled peptides. Both ammonium chloride and sodium azide inhibited the enhancement effects of DMSO pretreatment on cell uptake FITC-labeled TAT peptide. Mean ± s.d. of three experiments are shown.
Fig. 5. TAT-GFP fusion proteins' transduction ability enhanced by 10% DMSO pretreatment. Analysis of TAT-GFP fusion protein uptaken into Caski cells under microscopy. 10% DMSO treatment for 1 h, 100 μM Chloroquine treatment as a control, washed 3 times with PBS then cells were incubated with 5 μM fusion protein, followed by 4 h incubation. Shown are observed fluorescence microscopy by imaging in bright field (left column) and corresponding image in fluorescence mode (green; right column). Exposure to 10% DMSO enhanced TAT-GFP uptake into cells significantly.
H. Wang et al. / Journal of Controlled Release 143 (2010) 64–70
69
Fig. 6. Caski cell apoptosis induced by TAT-Apoptin fusion protein was enhanced by 10% DMSO pretreatment. Detection of TAT-Apoptin fusion protein induced Caski cell apoptosis by using TUNEL assay. Caski cells were incubated with 5 μM fusion protein, the treatment was same as the procedure of TAT-GFP, following the TUNEL kit instruction on how to visualize by imaging in bright field.
3.5. TAT-Apoptin induced cell apoptosis was enhanced by DMSO To test whether TAT conjugation affects the bioactivity of Apoptin or not, we observed Caski cell apoptosis induced by TAT-Apoptin with or without DMSO pretreatment. Result was showing that the presence of DMSO enhanced TAT-Apoptin to enter into the cells and then induced apoptosis in Caski cells with a higher efficiency, compared with no DMSO pretreatment (Fig. 6). These findings suggested that 10% DMSO increased the efficiency of TAT-Apoptin penetration, but had no effect on the Apoptin anti-tumor activity. 4. Discussion The use of information rich macromolecules to be bio-drugs has great advantages in specificity and potency that most small molecule therapeutics cannot match. Poor membrane permeability of macromolecules such as proteins or siRNA, limits therapeutic applications. Previous studies described protocols for the delivery of CPPs to adherent culturing cells using photodynamic treatment (photochemical internalization), Ca2+ treatment or chloroquine treatment that increased release of CPP conjugates into the cytoplasm [50], but the improvement was still limited. To identify a way to enhance the transduction efficiency of HIV-1 Tat PTD, the present study essentially aimed at introducing DMSO as a penetration enhancer used in cellular uptake of synthetic TAT peptide or the fusion proteins. Here, we evaluated the ability of DMSO to facilitate TAT peptide with different concentrations of DMSO ranging from 5% to 20% (Fig. 1A), and it was
demonstrated that DMSO has the potential for enhancing TAT or TAT fusion protein to penetrate into the cytosol. Recently, proposed mechanisms for the cellular uptake of most CPPs were broadly categorized into two groups: energy-dependent endocytosis and energy-independent direct translocation across the membrane bilayer. These possible mechanisms are well summarized in earlier reviews on CPPs [51–54]. In this work, we expected to clarify the mechanisms by which TAT peptide enters cells pretreated with DMSO. As in the case of previous reports, TAT entry into several different primary cells is found to be ATP dependent, indicating the involvement of endocytosis. Judging from the effects of specific inhibitors, we have chosen to re-evaluate the mechanism of cellular uptake after treatment with DMSO. Our data showed that intracellular delivery of TAT could be decreased by inhibitors. As shown in Fig. 4, that would be the possible mechanism for DMSO treatment to enhance TAT transduction but did not affect the mechanism of penetration. Although it has been reported that high concentration of DMSO has such a cytotoxic effect and an effect to induce cell differentiation [55– 57], application of DMSO to be a penetrating enhancer is an alternative strategy to promote penetrating efficiency of CPPs. As we mentioned above, there are lot of clinical practices using DMSO to treat human diseases. It was also shown that DMSO, at least, 10% or lower concentration of DMSO is safe in vitro to most cell lines. Our data here showed that 10% DMSO markedly improved TAT or TAT fusion proteins penetrate into cells without membrane perforating. However, the precise mechanism of DMSO enhancing CPP penetrating efficiency and safety considerations for using DMSO still need further study.
70
H. Wang et al. / Journal of Controlled Release 143 (2010) 64–70
Acknowledgments We thank John R Hood, BSc (Hons), PhD (National Institute of Biological Standards and Control, South Mimms, Potters Bar, EN6 3QG, UK) and Prof. Yan-Lin Wang (The China Three Gorges University) for reading the manuscript and for the helpful suggestions. The authors would like to thank the financial support from the National Natural Science Foundation of China (No. 20774094, and No. 20874097), the Ministry of Science and Technology of China (973 Project, No. 2009CB930102), “100 Talents Program” of the Chinese Academy of Sciences (No. KGCX2-YW-802), and Jilin Provincial Science and Technology Department (No. 20082104). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jconrel.2009.12.003. References [1] J.S. Wadia, S.F. Dowdy, Modulation of cellular function by TAT mediated transduction of full length proteins, Curr. Protein Pept. Sci. 4 (2003) 97–104. [2] J.S. Wadia, S.F. Dowdy, Protein transduction technology, Curr. Opin. Biotechnol. 13 (2002) 52–56. [3] A. Prochiantz, Messenger proteins: homeoproteins, TAT and others, Curr. Opin. Cell Biol. 12 (2000) 400–406. [4] G.P. Dietz, P.C. Valbuena, B. Dietz, Application of a blood–brain-barrier penetrating form of GDNF in a mouse model for Parkinson's disease, Brain Res. 1082 (2006) 61–66. [5] Y. Yoshioka, R. Asavatanabodee, Y. Eto, H. Watanabe, T. Morishige, X. Yao, S. Kida, M. Maeda, Y. Mukai, H. Mizuguchi, K. Kawasaki, N. Okada, S. Nakagawa, Tat conjugation of adenovirus vector broadens tropism and enhances transduction efficiency, Life Sci. 83 (2008) 747–755. [6] T. Suzuki, S. Futaki, M. Niwa, Possible existence of common internalization mechanisms among arginine-rich peptides, J. Biol. Chem. 277 (2002) 2437–2443. [7] R. Fischer, K. Köhler, M. Fotin-Mleczek, A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides, J. Bio. Chem. 279 (2004) 12625–12635. [8] Y. Mukai, T. Sugita, T. Yamato, Creation of novel Protein Transduction Domain (PTD) mutants by a phage display-based high-throughput screening system, Biol. Pharm. Bull. 29 (2006) 1570–1574. [9] A. Chauhan, A. Tikoo, A. Kapur, M. Singh, The taming of the cell penetrating domain of the HIV Tat: myths and realities, J. Control. Release 117 (2007) 1148–1162. [10] N.J. Caron, S.P. Quenneville, J.P. Tremblay, Endosome disruption enhances the functional nuclear delivery of TAT-fusion proteins, Biochem. Biophys. Res. Commun. 319 (2004) 12–20. [11] A. Sloots, W.S. Wels, Recombinant derivatives of the human high mobility group protein HMGB2 mediate efficient non viral gene delivery, FEBS J. 272 (2005) 4221–4236. [12] L. Hyndman, J.L. Lemoine, L. Huang, HIV-1 Tat protein transduction domain peptide facilitates gene transfer in combination with cationic liposomes, J. Control. Release 99 (2004) 435–444. [13] J. Parkin, C. Shea, G.R. Sant, Intravesical dimethyl sulfoxide (DMSO) for interstitial cystitis — a practical approach, Urology 49 (1997) 105–107. [14] J.L. Burgess, A.P. Hamner, W.O. Robertson, Sulfhemoglobinemia after dermal application of DMSO, Int. J. Dermatol. 37 (1998) 949–954. [15] C.K. Wong, C.S. Lin, Remarkable response of lipoid proteinosis to oral dimethyl sulphoxide, Br. J. Dermatol. 119 (1988) 541–544. [16] S.D. Hsieh, R. Yamamoto, K. Saito, Y. Iwamoto, T. Kuzuya, S. Ohba, S. Kobori, K. Saito, Amyloidosis presented with whitening and loss of hair which improved after dimethyl sulfoxide (DMSO) treatment, Jpn. J. Med. 26 (1987) 393–395. [17] K.A. McCammon, N.A. Lentzner, R.P. Moriarty, P.F. Schellhammer, Intravesical dimethyl sulfoxide for primary amyloidosis of the bladder, Urology 52 (1998) 1136–1138. [18] T. Iwasaki, T. Hamano, K. Aizawa, K. Kobayashi, E. Kakishita, A case of pulmonary amyloidosis associated with multiple myeloma successfully treated with dimethyl sulfoxide, Acta Haematol. 91 (1994) 91–94. [19] P. Morassi, F. Massa, E. Mesesnel, D. Magris, B. D'Agnolo, Treatment of amyloidosis with dimethyl sulfoxide (DMSO), Minerva Med. 80 (1989) 65–70. [20] A.S. Salim, Role of oxygen-derived free radical scavengers in the management of recurrent attacks of ulcerative colitis: a new approach, J. Lab. Clin. Med. 119 (1992) 710–717. [21] A.S. Salim, Allopurinol and dimethyl sulfoxide improve treatment outcomes in smokers with peptic ulcer disease, J. Lab. Clin. Med. 119 (1992) 702–709. [22] A.S. Salim, Oxygen-derived free-radical scavengers prolong survival in colonic cancer, Chemotherapy 38 (1992) 127–134. [23] A.S. Salim, Role of oxygen-derived free radical scavengers in the treatment of recurrent pain produced by chronic pancreatitis. A new approach, Arch. Surg. 126 (1991) 1109–1114. [24] A.S. Salim, Protection against stress-induced acute gastric mucosal injury by free radical scavengers, Intensive Care Med. 17 (1991) 455–460.
[25] Y. Ikeda, D.M. Long, Comparative effects of direct and indirect hydroxyl radical scavengers on traumatic brain oedema, Acta Neurochir. Suppl. (Wien) 51 (1990) 74–76. [26] E.D. Rosenstein, Topical agents in the treatment of rheumatic disorders, Rheum. Dis. Clin. North Am. 25 (1999) 899–918. [27] I. Goto, Y. Yamamoto-Yamagushi, Y. Honma, Enhancement of sensitivity of human lung adenocarcinoma cells to growth-inhibitory activity of interferon a by differentiation inducing agents, Br. J. Cancer 74 (1996) 546–554. [28] G.K. Abdullaeva, B.S. Shakimova, An evaluation of the efficacy of treating rheumatoid arthritis with preparations for local use, Revmatologiia (Mosk) 4 (1989) 35–39 in Russian. [29] I.V. Murav'ev, Treatment of rheumatoid synovitis by intra-articular administration of dimethyl sulfoxide and corticosteroid, Ter Arkh 58 (1986) 104–105. [30] S.W. Shirley, B.H. Steward, S. Mirelman, Dimethyl sulfoxide in treatment of inflammatory genitourinary disorders, Urology 11 (1978) 215–220. [31] B.N. Swanson, Medical use of dimethyl sulfoxide (DMSO), Rev. Clin. Basic Pharm. 5 (1985) 1–33. [32] P. Guerre, V. Burgat, F. Casali, Le diméthylsulfoxyde (DMSO) usages experimentaux et toxicité, Rev. Méd. Vét. 150 (1999) 391–412. [33] W. Regelson, S.W. Harkins, ‘Amyloid is not a tombstone’ — a summation. The primary role for cerebrovascular and CSF dynamics as factors in Alzheimer's disease (AD): DMSO, fluorocarbon oxygen carriers, thyroid hormonal, and other suggested therapeutic measures, Ann. N.Y. Acad. Sci. 826 (1997) 48–374. [34] X. Hui, P.G. Hewitt, N. Plblete, In vivo bioavailability and metabolism of topical diclofenac lotion in human volunteers, Pharm. Res. 15 (1998) 1589–1595. [35] P.G. Hewitt, N. Poblete, R.C. Wester, In vitro cutaneous disposition of a topical diclofenac lotion in human skin: effect of a multi-dose regimen, Pharm. Res. 15 (1998) 988–992. [36] E.C. Guillard, A. Tfayli, C. Laugel, Molecular interactions of penetration enhancers within ceramides organization: a FTIR approach, Eur. J. Pharm. Sci. 36 (2009) 192–199. [37] A. Mittal, U.V. Sara, A. Ali, The effect of penetration enhancers on permeation kinetics of nitrendipine in two different skin models, Biol. Pharm. Bull. 31 (2008) 1766–1772. [38] L. Li, W. Shen, L. Min, H. Dong, Y. Sun, Q. Pan, Human lactoferrin transgenic rabbits produced efficiently using dimethylsulfoxide sperm mediated gene transfer, Reprod. Fertil. Dev. 18 (2006) 689–695. [39] W. Shen, L. Li, Q. Pan, L. Min, H. Dong, J. Deng, Efficient and simple production of transgenic mice and rabbits using the new DMSO-sperm mediated exogenous DNA transfer method, Mol. Reprod. Dev. 73 (2006) 589–594. [40] H.L. Amand, K. Fant, B. Nordén, Stimulated endocytosis in penetratin uptake: effect of arginine and lysine, Biochem. Biophys. Res. Commun. 371 (2008) 621–625. [41] N.M. Moore, C.L. Sheppard, T.R. Barbour, S.E. Sakiyama-Elbert, The effect of endosomal escape peptides on in vitro gene delivery of polyethylene glycol-based vehicles, J. Gene Med. 10 (2008) 1134–1149. [42] C. Korzeniewski, D.M. Callewaert, An enzyme-release assay for natural cytotoxicity, J. Immunol. Methods 64 (1983) 313–320. [43] A.A. Gurtovenko, J. Anwar, Modulating the structure and properties of cell membranes: the molecular mechanism of action of dimethyl sulfoxide, J. Phys. Chem. B 111 (2007) 10453–10460. [44] B. Poole, S. Ohkuma, Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages, J. Cell Biol. 90 (1981) 665–669. [45] Y. Eto, Y. Yoshioka, R. Asavatanabodee, S. Kida, M. Maeda, Y. Mukai, H. Mizuguchi, K. Kawasaki, N. Okada, S. Nakagawa, Transduction of adenovirus vectors modified with cell-penetrating peptides, Peptides 30 (2009) 1548–1552. [46] J.S. Wadia, R.V. Stan, S.F. Dowdy, Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis, Nat. Med. 10 (2004) 310–315. [47] G. Drin, S. Cottin, E. Blanc, A.R. Rees, J. Temsamani, Studies on the internalization mechanism of cationic cell-penetrating peptides, J. Biol. Chem. 278 (2003) 31192–31201. [48] H. Hashida, M. Miyamoto, Y. Cho, Y. Hida, K. Kato, T. Kurokawa, S. Okushiba, S. Kondo, H. Dosaka-Akita, H. Katoh, Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool, Br. J. Cancer 90 (2004) 1252–1258. [49] T.B. Potocky, A.K. Menon, S.H. Gellman, Cytoplasmic and nuclear delivery of a TATderived peptide and a beta-peptide after endocytic uptake into HeLa cells, J. Biol. Chem. 278 (2003) 50188–50194. [50] T. Shiraishi, P.E. Nielsen, Enhanced delivery of cell-penetrating peptide–peptide nucleic acid conjugates by endosomal disruption, Nat. Protoc. 1 (2006) 633–636. [51] S.B. Fonseca, M.P. Pereira, S.O. Kelley, Recent advances in the use of cellpenetrating peptides for medical and biological applications, Adv. Drug Deliv. Rev. 61 (2009) 953–964. [52] C. Foerg, H.P. Merkle, On the biomedical promise of cell penetrating peptides: limits versus prospects, J. Pharm. Sci. 97 (2008) 144–162. [53] J.M. Gump, S.F. Dowdy, TAT transduction: the molecular mechanism and therapeutic prospects, Trends Mol. Med. 13 (2007) 443–448. [54] H. Brooks, B. Lebleu, E. Vivès, Tat peptide-mediated cellular delivery: back to basics, Adv. Drug Deliv. Rev. 57 (2005) 559–577. [55] J.L. Hanslick, K. Lau, K.K. Noguchi, J.W. Olney, C.F. Zorumski, S. Mennerick, N.B. Farber, Dimethyl sulfoxide (DMSO) produces widespread apoptosis in the developing central nervous system, Neurobiol. Dis. 34 (2009) 1–10. [56] W. Qi, R.J. Salvi, Cytotoxic effects of dimethyl sulphoxide (DMSO) on cochlear organotypic cultures, Hear Res. 236 (2008) 52–60. [57] H.N. Yu, Y.R. Lee, E.M. Noh, K.S. Lee, E.K. Song, M.K. Han, Y.C. Lee, C.Y. Yim, J. Park, B.S. Kim, S.H. Lee, S.J. Lee, J.S. Kim, Tumor necrosis factor-alpha enhances DMSOinduced differentiation of HL-60 cells through the activation of ERK/MAPK pathway, Int. J. Hematol. 87 (2008) 189–194.
Contents lists available at ScienceDirect
Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l
Enhancement of TAT cell membrane penetration efficiency by dimethyl sulphoxide Hu Wang a, Chun-Yan Zhong a,1, Jiang-Feng Wu a, Yu-Bin Huang b,⁎, Chang-Bai Liu a,⁎ a b
The Institute of Molecular Biology, Three Georges University, Yichang, 443002, P.R. China State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 130022, P.R. China
a r t i c l e
i n f o
Article history: Received 10 June 2009 Accepted 2 December 2009 Available online 16 December 2009 Keywords: Cell penetrating peptides (CPPs) Dimethylsulfoxide (DMSO) TAT Penetration
a b s t r a c t Cell penetrating peptides (CPPs) are promising tools for transducing presynthesized therapeutic molecules which possess low membrane permeability. The poor efficiency of cellular uptake and unexpected cellular localization are still the main obstacles to the development of drug delivery by using CPPs. In this study, we investigated the effect of a penetration enhancer, dimethylsulfoxide (DMSO), on the penetrating efficiency of a synthetic TAT peptide or the TAT fusion protein. FITC-labeled TAT and TAT-GFP were added to 10% DMSO or 100 μM chloroquine pretreated cells, fluorescence uptake into culturing cells was observed using fluorescence microscopy, FACS or quantitatively analyzed by a fluorescence spectrum. 10% DMSO treatment markedly increased internalization of TAT into cells and appeared in a well-distributed pattern throughout the cytosol and nucleus without membrane perforating or detectable cytotoxicity, the enhancement effect by 10% DMSO was reduced by endocytosis inhibitors including ammonium chloride and sodium azide. 10% DMSO also enhanced TAT-Apoptin induced apoptosis of carcinoma cells. These findings implicated that DMSO can be a novel delivery enhancer appropriate for CPP penetration. © 2009 Elsevier B.V. All rights reserved.
1. Introduction It was discovered that certain peptides, referred to as cell penetrating peptides (CPPs), can penetrate cells accompanied by a large molecular cargo. CPPs have found numerous applications in biology and medicine since the first synthetic cell-permeable sequence was identified two decades ago. Considerable research effort is currently focused on utilizing CPPs as peptide-based vehicle for intracellular drug delivery. Several types of drugs have been transported into cells using CPPs, including small molecule pharmaceuticals, therapeutic proteins, siRNA and antisense oligonucleotides [1–3]. Many CPPs are derived from the protein transduction domains (PTD) of viral proteins involved in interaction with cell membrane. A typical example of CPPs is TAT, a basic peptide segment corresponding to the positions 48–60 of Tat protein, a transcription regulator protein of HIV-1, is also one of the most frequently employed for intracellular delivery. It has been reported that CPPs including TAT are able to deliver various bioactive molecules into cells and have enormous ⁎ Corresponding authors. Liu is to be contacted at The Institute of Molecular Biology, Three Georges University, 8 Daxue Road, Yichang, 443002 China. Tel.:+86 717 639 7179. Huang, State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun City, Jilin Province, 130022, China. E-mail addresses: [email protected] (C.-B. Liu), [email protected] (Y.-B. Huang). 1 Current address: Clinical laboratory, The 3rd General Hospital of Hangzhou, Hangzhou, 310009, P.R. China. 0168-3659/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2009.12.003
potential to solve age-old problems in the delivery of macromolecular therapeutics [4]. Despite their broad acceptance as molecular vehicle, the mechanism of internalization of CPPs and the conjugated cargo is not well understood. The cellular uptake of TAT peptide had originally been described to be insensitive to low temperature and to inhibitors of endocytosis, but there are recent reports controversially declared that endocytosis may also be involved in TAT transduction [5–7]. Although accumulated research efforts are currently focused on CPPs as potential intracellular drug delivery vehicle, the application of this technology is limited because the transduction efficiency is often insufficient for therapeutic purposes, even using TAT. Some reports have observed that greater than 90% of the fluorescent signal from labeled TAT or the fusion proteins appears in punctate vesicles, not in the nucleus or cytosol [8,9]. Reports have also shown an improvement in transduction efficiency with endosomolytic agents like chloroquine, which indicates that much of the peptide remains in vesicles of undetermined fate [10,11]. This would appear to be a limitation of transduction if the CPPs remain in endosome/lysosome to be degraded rather than enter the cytosol or nucleus [12]. To use CPPs as effective intracellular drug delivery tool for clinical applications, it is necessary to search agents which could enhance the penetrating ability of CPPs. Dimethylsulfoxide (DMSO) has been commonly used for several years as a cryoprotectant agent for storage of culture cells or for hematopoietic stem cell transplantation in a variety of hematological disorders. Since the biological properties of DMSO were discovered in 1960 it has been used not only for laboratory but also clinical
H. Wang et al. / Journal of Controlled Release 143 (2010) 64–70
purposes. In 1978 it received approval by the US Food and Drug Administration for use in the treatment of interstitial cystitis by intravesical instillation [13]. DMSO has been used successfully in the treatment of various human diseases such as dermatological, urinary, pulmonary, rheumatic and renal manifestations of amyloidosis, gastrointestinal diseases, traumatic brain edema, musculoskeletal disorders, chronic prostatitis, dermatological diseases, schizophrenia, and Alzheimer's disease [14–33]. In more recent studies, DMSO was used as an efficient penetration enhancer for percutaneous drug [34– 37]. Li et al. [38] and Shen et al. [39] described that the transfection of exogenous DNA incubated with DMSO were more efficient than without any treatment. Based on these researches, the present study was undertaken to investigate the possibility of improving TAT or TAT fusion proteins penetrating efficiency into cytoplasma for future development as a drug delivery tool based on TAT peptide. As a vehicle to deliver bio-drugs into cells, it should have no any effect on the bioactivity of the drugs. We also analyzed that cancer cell apoptosis induced by Apoptin, a small protein derived from chicken anemia virus, can especially induce apoptosis in transformed or tumor cells, but not in normal cells, fused with TAT. We also found that DMSO increased the cell apoptosis caused by TAT-Apoptin. 2. Materials and methods 2.1. Chemicals, reagents and cells RPMI-1640 medium (Invitrogen, USA), FBS (Invitrogen, USA); the chemicals: sodium azide (Sigma-Aldrich), In Situ Cell Death Detection Kit (Roche Applied Science, USA), and CytoTox-ONETM Membrane integrity assay kit (Promega, USA) were purchased from commercial company. Caski, Siha, HepG2, and A549 cells were maintained in our laboratory. 2.2. Peptide internalization FITC-labeled peptides used in this study (TAT: YGRKKRRQRRRK and NCO, a nonsense peptide: KALGISYGRKK) were synthesized by SBS Genetech (Beijing, China). The peptides were purified using reversed phase analytical HPLC to more than 99% purity then diluted to 500 mM in PBS and were stored at −20 °C for further use. TAT-FITC and NCO–FITC internalized into culture cells were observed under fluorescence microscopy. Caski, Siha, or HepG2 cells were seeded into 12-well plates (Greiner, Germany) at a density of 5 × 105 cells per well and cultivated to semi-confluence in an RPMI1640 medium in a humid atmosphere supplemented with 5% CO2 for 24 h at 37 °C respectively. Cells were washed 3 times with PBS and then added different concentrations (v/v %) of DMSO supplemented with a serum-free medium, incubation for 1 h with 5% CO2 at 37 °C, then added 5 μM FITC-labeled peptides and incubated for another 1 h with 5% CO2 at 37 °C. The cells were further washed 5 times with PBS then the fluorescence was observed under fluorescence microscopy (Nikon, Japan) using a bandpass filter (detects FITC). 100 µM chloroquine was used as a positive control in all FITC-labeled peptide uptake experiments. TAT-GFP fusion protein (Supplementary data Fig. S1) internalized into culture cells was observed under fluorescence microscopy. When the cells were 70% confluent, the culture medium was replaced with a fresh serum-free medium containing indicated concentrations of DMSO for 1 h with 5% CO2 at 37 °C; the cells were washed 3 times with PBS and then TAT-GFP was added to the serum-free medium, with a final concentration of 5 μM. The cells were incubated for 3 h with 5% CO2 at 37 °C. TAT-GFP fusion protein treated cells were washed 5 times with PBS and then observed under microscopy. In some experiments, cell internalization of synthetic FITC-labeled peptides was analyzed using flow cytometry. Caski cells were pretreated with indicated concentrations of DMSO for 1 h in serum-
65
free media prior to addition of FITC-labeled peptides with a final concentration of 5 μM in a serum-free medium, then the cells were washed 5 times with PBS. The cells were de-adhered by incubating with trypsin–EDTA for 10 min, and then washed again 3 times. The suspended cells were then fixed with 1% paraformaldehyde and then washed with PBS twice. Analysis was conducted on a FACS Caliber flow cytometer (Beckman Coulter, Fullerton, CA, USA), equipped with a 488-nm air-cooled argon laser. The filter settings for excitation and emission were 480/530 nm bandpass (FL1) for FITC. The fluorescence of 5000 vital cells was acquired, and data were analyzed in a logarithmic mode. In order to quantify the intracellular fluorescence intensity at the conditions of DMSO pretreatment, FITC-labeled peptide uptake in various cells were quantitated by Multimode Spectrophotometry. After the incubation of FITC-labeled peptide, cells were washed with PBS 5 times and then lysed by adding 300 μl 0.1 M NaOH for 10 min at room temperature, the cell lysates were centrifuged (14000 g for 5 min) and the fluorescence intensity of the supernatant was measured at 494/518 nm in a Multimode Microplate Reader (Tecan 2000; Tecan, Mannedorf, Switzerland). The cellular protein content was determined by the method of Bradford protein assay with BSA as standard (Bio-Rad). The fluorescence of cellular uptake is expressed as fluorescence intensity per mg of total cellular protein. All experiments were repeated at least three times and always performed in triplicate. The uptake levels are reported as the arithmetic mean of all samples, whereas the error bars represent the average maximum and minimum data spread [40]. To evaluate the influence of various cells, characterized inhibiting drugs were selected for their ability to inhibit specific steps in the endocytosis pathway. 50 mM ammonium chloride and 40 μM sodium azide were added to the cell culture medium for 30 min followed by DMSO treatment and then added the FITC-labeled peptides. The protein uptake efficiencies were quantified after a 1 h incubation of the cells with the FITC-labeled peptide in the presence of the inhibitor by the Multimode Microplate Reader, as described above. 2.3. Hemolysis and LDH release assay To rule out that DMSO enhanced TAT penetration by perforating the plasma membrane, hemolysis and lactate dehydrogenase (LDH) release assays were employed. Red blood cell (RBC) membrane destabilization, or hemolysis, has been associated with endosomal escape [41]. In the present study, a hemolytic assay was used to detect whether DMSO could perforate the RBC membrane different concentrations. Briefly, whole blood was collected from BALB/c mice. RBCs were separated from whole blood by centrifugation for 20 min at 300 g, and then washed 3 times with fresh 150 mM NaCl and collected by centrifugation for 5 min at 200 g, then diluted to 2 × 108 cell/ml in 0.1 M PBS at the desired DMSO concentrations (5%, 7.5%, 10%, 15%, and 20%). As a positive control, 0.1% Triton X-100 (Sigma) was added to 0.1 M PBS before addition of RBCs in the microcentrifuge tube, then incubated for 1 h at 37 °C. Maximum release of hemoglobin was detected by incubation of the RBCs in 0.1% Triton-X 100 under the same conditions. The optical density (OD) at 414 nm allowed evaluation of the percentage of hemolysis by comparison with the values obtained in PBS, after correction of the absorbance values by the blank value. The OD value of untreated, DMSO or 0.1% Triton X-100 treated cell supernatants is indicated as ODB, ODT and ODM respectively, and the cell toxicity is calculated as: toxicity= 100 × ((ODT − ODB) / (ODM − ODB))%. LDH release assay [42] was performed by using a CytoTox-ONETM Membrane integrity assay kit (Promega), according to the instructions of the manufacturer of the kit. Caski cells were seeded at a density of 1 × 105 cells per well in a 12-well plate. 16–24 h after seeding, the cells were treated with concentrations of DMSO and incubated for 1 h with 5% CO2 at 37 °C, then 100 μl of the cell supernatants were transferred
66
H. Wang et al. / Journal of Controlled Release 143 (2010) 64–70
to a black 96-well-plate and 100 μl of the substrate mix was added to each well. Background value was obtained from untreated cells and maximum LDH release by treating cells with 2% NP-40. After 10 min of incubation at room temperature, the enzymatic reaction was stopped by stop solution and then measured at 560/590 nm in a Multiskan Spectrum plate-reader (Thermo, USA). 2.4. Detection of cell viability by using MTT assay 4 × 104 cells per well of Caski, Siha, HepG2 or A549 cells were seeded in 96-well plates and cultivated to semi-confluence in an RPMI-1640 medium in a humid atmosphere supplemented with 5% CO2 for 24 h at 37 °C respectively. Cells were washed 3 times with PBS and then different concentrations (v/v %) of DMSO supplemented with a serum-free medium were added, incubated for 2 h with 5% CO2 at 37 °C and washed with PBS 3 times then incubated for 24 h with fresh serum medium. Cell viability was then assessed using MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] (Sigma-Aldrich) assays. Briefly, 20 µl of 5.5 mg/ml MTT in a serumfree medium was added directly to each well giving a final concentration of 0.5 mg/ml. The cells were then incubated for 4 h at 37 °C with 5% CO2. The plates were centrifuged at 1000 g for 5 min before removing the supernatant and then adding 100 µl of DMSO. The samples were finally incubated at 37 °C for 30 min before quantifying the absorbance at 550 nm. 2.5. TUNEL analysis of cell apoptosis induced by TAT-Apoptin fusion protein Cell apoptosis induced by TAT-Apoptin (Supplementary data Fig. S1) was analyzed by the terminal dUTP-digoxigenin nick-end labeling (TUNEL) assay using the ApopTag Plus Peroxidase In Situ Cell Death Detection Kit (Roche Applied Science, Germany). The procedure of TAT-Apoptin fusion protein treatment was same as TAT-GFP
Fig. 1. FITC-labeled peptides uptaken by Caski cell pretreated with different concentrations of DMSO. Caski cells were exposed to 10% DMSO pretreatment for 1 h, followed by incubating with 5 μM FITC-labeled peptide for 1 h after washing. The fluorescence intensity in cell lysis was measured at 494/518 nm. A. Fluorescence intensity of FITC-labeled peptides uptaken by flow cytometry assay with the concentrations of DMSO from 5% to 20%, 100 μM chloroquine and 0.5 M sucrose treatment as the positive control; B: TAT-FITC distributed in Caski cell with or without 10% DMSO pretreatment observed under fluorescence microscopy; scale bars, 50 μm. C: Fluorescence quantitation of FITC-labeled peptides uptaken in Caski cell pretreated by 10% DMSO; means ± s.d. of three experiments are shown. D: Fluorescence quantitation of FITC-labeled peptides uptaken at different incubation time. Means±s.d. of three experiments are shown.
H. Wang et al. / Journal of Controlled Release 143 (2010) 64–70
67
could be penetrating into the cytosol and then degraded (Fig. 1D). Comparing the transduction enhancement by different concentrations
Fig. 2. Fluorescence intensity of FITC-labeled peptides uptaken in different cells pretreated with 10% DMSO and peptides at different concentrations used. A: Caski cells uptaken into the cytosol quantified with the incubation of synthetic peptide at concentration from 2.5 μM to 10 μM treated by 10% DMSO. Means ± s.d. of three experiments are shown. B: Culturing Siha, A549, and HepG2 cell lines were exposed to 10% DMSO pretreatment for 1 h, followed by incubation with 5 μM FITC-labeled peptides for 1 h after washing. The fluorescence intensity in cell lysis was measured at 494/518 nm.
treatment. The cell was incubated for 24 h in a fresh serum medium containing TAT-Apoptin (5 μM), and then fixed with 1% paraformaldehyde in PBS, subjected to TUNEL staining, followed by visualization under microscopy with diaminobenzidine according to the instruction of the TUNEL kit. As negative controls, terminal deoxynucleotidyl transferase was omitted. 3. Results 3.1. 10% DMSO enhanced TAT-FITC uptake and in a well-distributed pattern throughout the cytosol and nucleus By pretreating with 10% DMSO, the TAT-FITC uptake into cells was markedly increased (Fig. 1A) and appeared in a well-distributed pattern throughout the cytosol and nucleus, whereas as a negative control, NCO–FITC could not be detected using the same photomultiplier settings under fluorescent microscopy (Fig. 1B). TAT-FITC uptake into cells with same condition was quantified using a Multimode fluorescent Microplate Reader (Fig. 1C). The fluorescence in the cytosol became weaker over time, suggesting that the peptide Fig. 3. Cell hemolysis and LDH activity treated by different concentrations of DMSO and cell viability evaluated by MTT assay. A. RBCs were incubated with DMSO at various concentrations for 1 h at 37 °C. Hemolytic activity was determined by the amount of hemoglobin released from RBCs with measuring the absorbance at 541 nm. There was no hemolytic activity seen at 10% DMSO compared with positive control using 0.1% Triton X-100. B. Different cells pre-incubated with DMSO at different concentrations as shown in A for 1 h at 37 °C. LDH release quantity was determined by measuring the absorbance at 570 nm. DMSO did not lyse any kind of cells compared with positive control using 2% NP-40. C: Different cells pre-incubated with DMSO at different concentrations (2.5%, 5%, 10%, 15%, and 20%) for 2h at 37 °C, discarding the supernatant, washed 3 times with PBS and added with a fresh medium then cultured for 24 h; measurement procedure following the MTT protocol. D: Caski cells incubated with 10% DMSO for 2 h at 37 °C, discarding the supernatant, washed 3 times with PBS and added with a fresh medium then cultured 0 h, 1 h, 12 h, 24 h and 48 h; measurement procedure was same as C. Means ± s.d. of three experiments are shown.
68
H. Wang et al. / Journal of Controlled Release 143 (2010) 64–70
of DMSO pretreatment, the result of FACS analysis revealed multiple levels of increased transduction potential by increasing DMSO concentration (Fig. 1A). This enhancement effect also was observed in A549, a human lung cancer cell line, HepG2, a human hepatocellular carcinoma cell line, Siha cells (Fig. 2B), and in other cell lines (Supplementary data Fig. S2). At 10% (w/v) DMSO, penetrating efficiency became higher with TAT concentration from 2.5 μM to 10 μM (Fig. 2A). These results revealed that transduction potential of TAT could be enhanced by DMSO pretreatment, and the efficiency is much higher than that of chloroquine pretreatment.
production within the cell membrane [44–49]. As shown in Fig. 4, before the addition of FITC-labeled TAT, cells were pre-incubated with the energy depleter, anti-acidification agent as endocytic inhibitor; TAT transduction levels were moderately inhibited. This data indicated that endocytosis is not fully, but partially involved in TAT internalization. 10% DMSO pretreatment does not affect TAT transduction.
3.2. 10% DMSO enhanced TAT penetrating efficiency without cell membrane perforation or cytotoxicity
To assess the effect of DMSO pretreatment on transduction of TAT with large cargo, we determined the subcellular localization of TATGFP fusion protein with or without DMSO pretreatment. As shown in Fig. 5, the levels of TAT-GFP fusion protein uptake by cells pretreated with 10% DMSO were significantly higher than in untreated cells. Fusion proteins were detected in the nucleus as well as in the cytoplasm of the cells, whereas pretreatment by chloroquine detected faint diffuse fluorescence in the cytoplasm; this is consistent with the results from FITC-labeled TAT peptide experiments (Fig. 1).
Gurtovenko et al. [43] report that high concentration of DMSO induced transient water pores on a chemical membrane in a simulating test. It was speculated that there is a correlation between the ability of penetration enhancer to lyse membranes under natural conditions and penetrating efficiency. In order to exclude membrane disturbance by DMSO, a hemolysis and a lactate dehydrogenase (LDH) leakage assay were performed in the present study. Both hemolysis assay and LDH release assay showed that DMSO treatment does not induce pores or lyse the cell membrane under physiological conditions (Fig. 3A and B). This result suggested that DMSO pretreatment enhanced cell membrane penetration of TAT was not through inducing pores on cell membrane. The mechanism needs to be explored in a further study. The effect of DMSO treatment on the cell growth of different cells was first evaluated by using various concentrations (2.5%, 5%, 10%, 15% and 20%) of DMSO at 2 h through an MTT assay. Repressive effect on the cell growth was observed at 20% DMSO treatment, whereas, 10% DMSO treatment up to 2 h has very little repressive effect on cell growth (Fig. 3C and D). The cytotoxicity of 10% DMSO to HeLa, NIH3T3 and CHO cells was also detected; DMSO suppressed only NIH3T3 growth to about 60% but not other cells (Supplementary data Fig. S3). These results here suggested that the sensitivity to DMSO maybe dependent on different cell lines, and there was minimal cytotoxicity to some cell lines up to 10% of DMSO treatment.
3.4. DMSO enhanced cellular uptake of TAT-GFP fusion protein
3.3. Endocytosis inhibitors suppressed the enhancement effect of DMSO on TAT penetration To analyze the effects of DMSO pretreatment on the internalization of TAT, different chemicals (ammonium chloride and sodium azide) were used to inhibit endocytosis of FITC-labeled peptides into the cells. Ammonium chloride is a weak base that increases the pH of acidic organelles, including endocytic vesicles. Sodium azide is an oxidative phosphorylation inhibitor commonly used to abolish ATP
Fig. 4. Suppressive effect of NaN3, NH4Cl exposure on TAT-FITC uptake with 10% DMSO pretreatment in Caski and Siha cells. Confluent monolayers of Caski or Siha cell was preincubated in a 10%DMSO serum-free medium for 1 h at 37 °C; cells were washed twice prior to addition of inhibitors, incubation with inhibitors for 30 min at 37 °C then addition of FITC-labeled peptides. Both ammonium chloride and sodium azide inhibited the enhancement effects of DMSO pretreatment on cell uptake FITC-labeled TAT peptide. Mean ± s.d. of three experiments are shown.
Fig. 5. TAT-GFP fusion proteins' transduction ability enhanced by 10% DMSO pretreatment. Analysis of TAT-GFP fusion protein uptaken into Caski cells under microscopy. 10% DMSO treatment for 1 h, 100 μM Chloroquine treatment as a control, washed 3 times with PBS then cells were incubated with 5 μM fusion protein, followed by 4 h incubation. Shown are observed fluorescence microscopy by imaging in bright field (left column) and corresponding image in fluorescence mode (green; right column). Exposure to 10% DMSO enhanced TAT-GFP uptake into cells significantly.
H. Wang et al. / Journal of Controlled Release 143 (2010) 64–70
69
Fig. 6. Caski cell apoptosis induced by TAT-Apoptin fusion protein was enhanced by 10% DMSO pretreatment. Detection of TAT-Apoptin fusion protein induced Caski cell apoptosis by using TUNEL assay. Caski cells were incubated with 5 μM fusion protein, the treatment was same as the procedure of TAT-GFP, following the TUNEL kit instruction on how to visualize by imaging in bright field.
3.5. TAT-Apoptin induced cell apoptosis was enhanced by DMSO To test whether TAT conjugation affects the bioactivity of Apoptin or not, we observed Caski cell apoptosis induced by TAT-Apoptin with or without DMSO pretreatment. Result was showing that the presence of DMSO enhanced TAT-Apoptin to enter into the cells and then induced apoptosis in Caski cells with a higher efficiency, compared with no DMSO pretreatment (Fig. 6). These findings suggested that 10% DMSO increased the efficiency of TAT-Apoptin penetration, but had no effect on the Apoptin anti-tumor activity. 4. Discussion The use of information rich macromolecules to be bio-drugs has great advantages in specificity and potency that most small molecule therapeutics cannot match. Poor membrane permeability of macromolecules such as proteins or siRNA, limits therapeutic applications. Previous studies described protocols for the delivery of CPPs to adherent culturing cells using photodynamic treatment (photochemical internalization), Ca2+ treatment or chloroquine treatment that increased release of CPP conjugates into the cytoplasm [50], but the improvement was still limited. To identify a way to enhance the transduction efficiency of HIV-1 Tat PTD, the present study essentially aimed at introducing DMSO as a penetration enhancer used in cellular uptake of synthetic TAT peptide or the fusion proteins. Here, we evaluated the ability of DMSO to facilitate TAT peptide with different concentrations of DMSO ranging from 5% to 20% (Fig. 1A), and it was
demonstrated that DMSO has the potential for enhancing TAT or TAT fusion protein to penetrate into the cytosol. Recently, proposed mechanisms for the cellular uptake of most CPPs were broadly categorized into two groups: energy-dependent endocytosis and energy-independent direct translocation across the membrane bilayer. These possible mechanisms are well summarized in earlier reviews on CPPs [51–54]. In this work, we expected to clarify the mechanisms by which TAT peptide enters cells pretreated with DMSO. As in the case of previous reports, TAT entry into several different primary cells is found to be ATP dependent, indicating the involvement of endocytosis. Judging from the effects of specific inhibitors, we have chosen to re-evaluate the mechanism of cellular uptake after treatment with DMSO. Our data showed that intracellular delivery of TAT could be decreased by inhibitors. As shown in Fig. 4, that would be the possible mechanism for DMSO treatment to enhance TAT transduction but did not affect the mechanism of penetration. Although it has been reported that high concentration of DMSO has such a cytotoxic effect and an effect to induce cell differentiation [55– 57], application of DMSO to be a penetrating enhancer is an alternative strategy to promote penetrating efficiency of CPPs. As we mentioned above, there are lot of clinical practices using DMSO to treat human diseases. It was also shown that DMSO, at least, 10% or lower concentration of DMSO is safe in vitro to most cell lines. Our data here showed that 10% DMSO markedly improved TAT or TAT fusion proteins penetrate into cells without membrane perforating. However, the precise mechanism of DMSO enhancing CPP penetrating efficiency and safety considerations for using DMSO still need further study.
70
H. Wang et al. / Journal of Controlled Release 143 (2010) 64–70
Acknowledgments We thank John R Hood, BSc (Hons), PhD (National Institute of Biological Standards and Control, South Mimms, Potters Bar, EN6 3QG, UK) and Prof. Yan-Lin Wang (The China Three Gorges University) for reading the manuscript and for the helpful suggestions. The authors would like to thank the financial support from the National Natural Science Foundation of China (No. 20774094, and No. 20874097), the Ministry of Science and Technology of China (973 Project, No. 2009CB930102), “100 Talents Program” of the Chinese Academy of Sciences (No. KGCX2-YW-802), and Jilin Provincial Science and Technology Department (No. 20082104). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jconrel.2009.12.003. References [1] J.S. Wadia, S.F. Dowdy, Modulation of cellular function by TAT mediated transduction of full length proteins, Curr. Protein Pept. Sci. 4 (2003) 97–104. [2] J.S. Wadia, S.F. Dowdy, Protein transduction technology, Curr. Opin. Biotechnol. 13 (2002) 52–56. [3] A. Prochiantz, Messenger proteins: homeoproteins, TAT and others, Curr. Opin. Cell Biol. 12 (2000) 400–406. [4] G.P. Dietz, P.C. Valbuena, B. Dietz, Application of a blood–brain-barrier penetrating form of GDNF in a mouse model for Parkinson's disease, Brain Res. 1082 (2006) 61–66. [5] Y. Yoshioka, R. Asavatanabodee, Y. Eto, H. Watanabe, T. Morishige, X. Yao, S. Kida, M. Maeda, Y. Mukai, H. Mizuguchi, K. Kawasaki, N. Okada, S. Nakagawa, Tat conjugation of adenovirus vector broadens tropism and enhances transduction efficiency, Life Sci. 83 (2008) 747–755. [6] T. Suzuki, S. Futaki, M. Niwa, Possible existence of common internalization mechanisms among arginine-rich peptides, J. Biol. Chem. 277 (2002) 2437–2443. [7] R. Fischer, K. Köhler, M. Fotin-Mleczek, A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides, J. Bio. Chem. 279 (2004) 12625–12635. [8] Y. Mukai, T. Sugita, T. Yamato, Creation of novel Protein Transduction Domain (PTD) mutants by a phage display-based high-throughput screening system, Biol. Pharm. Bull. 29 (2006) 1570–1574. [9] A. Chauhan, A. Tikoo, A. Kapur, M. Singh, The taming of the cell penetrating domain of the HIV Tat: myths and realities, J. Control. Release 117 (2007) 1148–1162. [10] N.J. Caron, S.P. Quenneville, J.P. Tremblay, Endosome disruption enhances the functional nuclear delivery of TAT-fusion proteins, Biochem. Biophys. Res. Commun. 319 (2004) 12–20. [11] A. Sloots, W.S. Wels, Recombinant derivatives of the human high mobility group protein HMGB2 mediate efficient non viral gene delivery, FEBS J. 272 (2005) 4221–4236. [12] L. Hyndman, J.L. Lemoine, L. Huang, HIV-1 Tat protein transduction domain peptide facilitates gene transfer in combination with cationic liposomes, J. Control. Release 99 (2004) 435–444. [13] J. Parkin, C. Shea, G.R. Sant, Intravesical dimethyl sulfoxide (DMSO) for interstitial cystitis — a practical approach, Urology 49 (1997) 105–107. [14] J.L. Burgess, A.P. Hamner, W.O. Robertson, Sulfhemoglobinemia after dermal application of DMSO, Int. J. Dermatol. 37 (1998) 949–954. [15] C.K. Wong, C.S. Lin, Remarkable response of lipoid proteinosis to oral dimethyl sulphoxide, Br. J. Dermatol. 119 (1988) 541–544. [16] S.D. Hsieh, R. Yamamoto, K. Saito, Y. Iwamoto, T. Kuzuya, S. Ohba, S. Kobori, K. Saito, Amyloidosis presented with whitening and loss of hair which improved after dimethyl sulfoxide (DMSO) treatment, Jpn. J. Med. 26 (1987) 393–395. [17] K.A. McCammon, N.A. Lentzner, R.P. Moriarty, P.F. Schellhammer, Intravesical dimethyl sulfoxide for primary amyloidosis of the bladder, Urology 52 (1998) 1136–1138. [18] T. Iwasaki, T. Hamano, K. Aizawa, K. Kobayashi, E. Kakishita, A case of pulmonary amyloidosis associated with multiple myeloma successfully treated with dimethyl sulfoxide, Acta Haematol. 91 (1994) 91–94. [19] P. Morassi, F. Massa, E. Mesesnel, D. Magris, B. D'Agnolo, Treatment of amyloidosis with dimethyl sulfoxide (DMSO), Minerva Med. 80 (1989) 65–70. [20] A.S. Salim, Role of oxygen-derived free radical scavengers in the management of recurrent attacks of ulcerative colitis: a new approach, J. Lab. Clin. Med. 119 (1992) 710–717. [21] A.S. Salim, Allopurinol and dimethyl sulfoxide improve treatment outcomes in smokers with peptic ulcer disease, J. Lab. Clin. Med. 119 (1992) 702–709. [22] A.S. Salim, Oxygen-derived free-radical scavengers prolong survival in colonic cancer, Chemotherapy 38 (1992) 127–134. [23] A.S. Salim, Role of oxygen-derived free radical scavengers in the treatment of recurrent pain produced by chronic pancreatitis. A new approach, Arch. Surg. 126 (1991) 1109–1114. [24] A.S. Salim, Protection against stress-induced acute gastric mucosal injury by free radical scavengers, Intensive Care Med. 17 (1991) 455–460.
[25] Y. Ikeda, D.M. Long, Comparative effects of direct and indirect hydroxyl radical scavengers on traumatic brain oedema, Acta Neurochir. Suppl. (Wien) 51 (1990) 74–76. [26] E.D. Rosenstein, Topical agents in the treatment of rheumatic disorders, Rheum. Dis. Clin. North Am. 25 (1999) 899–918. [27] I. Goto, Y. Yamamoto-Yamagushi, Y. Honma, Enhancement of sensitivity of human lung adenocarcinoma cells to growth-inhibitory activity of interferon a by differentiation inducing agents, Br. J. Cancer 74 (1996) 546–554. [28] G.K. Abdullaeva, B.S. Shakimova, An evaluation of the efficacy of treating rheumatoid arthritis with preparations for local use, Revmatologiia (Mosk) 4 (1989) 35–39 in Russian. [29] I.V. Murav'ev, Treatment of rheumatoid synovitis by intra-articular administration of dimethyl sulfoxide and corticosteroid, Ter Arkh 58 (1986) 104–105. [30] S.W. Shirley, B.H. Steward, S. Mirelman, Dimethyl sulfoxide in treatment of inflammatory genitourinary disorders, Urology 11 (1978) 215–220. [31] B.N. Swanson, Medical use of dimethyl sulfoxide (DMSO), Rev. Clin. Basic Pharm. 5 (1985) 1–33. [32] P. Guerre, V. Burgat, F. Casali, Le diméthylsulfoxyde (DMSO) usages experimentaux et toxicité, Rev. Méd. Vét. 150 (1999) 391–412. [33] W. Regelson, S.W. Harkins, ‘Amyloid is not a tombstone’ — a summation. The primary role for cerebrovascular and CSF dynamics as factors in Alzheimer's disease (AD): DMSO, fluorocarbon oxygen carriers, thyroid hormonal, and other suggested therapeutic measures, Ann. N.Y. Acad. Sci. 826 (1997) 48–374. [34] X. Hui, P.G. Hewitt, N. Plblete, In vivo bioavailability and metabolism of topical diclofenac lotion in human volunteers, Pharm. Res. 15 (1998) 1589–1595. [35] P.G. Hewitt, N. Poblete, R.C. Wester, In vitro cutaneous disposition of a topical diclofenac lotion in human skin: effect of a multi-dose regimen, Pharm. Res. 15 (1998) 988–992. [36] E.C. Guillard, A. Tfayli, C. Laugel, Molecular interactions of penetration enhancers within ceramides organization: a FTIR approach, Eur. J. Pharm. Sci. 36 (2009) 192–199. [37] A. Mittal, U.V. Sara, A. Ali, The effect of penetration enhancers on permeation kinetics of nitrendipine in two different skin models, Biol. Pharm. Bull. 31 (2008) 1766–1772. [38] L. Li, W. Shen, L. Min, H. Dong, Y. Sun, Q. Pan, Human lactoferrin transgenic rabbits produced efficiently using dimethylsulfoxide sperm mediated gene transfer, Reprod. Fertil. Dev. 18 (2006) 689–695. [39] W. Shen, L. Li, Q. Pan, L. Min, H. Dong, J. Deng, Efficient and simple production of transgenic mice and rabbits using the new DMSO-sperm mediated exogenous DNA transfer method, Mol. Reprod. Dev. 73 (2006) 589–594. [40] H.L. Amand, K. Fant, B. Nordén, Stimulated endocytosis in penetratin uptake: effect of arginine and lysine, Biochem. Biophys. Res. Commun. 371 (2008) 621–625. [41] N.M. Moore, C.L. Sheppard, T.R. Barbour, S.E. Sakiyama-Elbert, The effect of endosomal escape peptides on in vitro gene delivery of polyethylene glycol-based vehicles, J. Gene Med. 10 (2008) 1134–1149. [42] C. Korzeniewski, D.M. Callewaert, An enzyme-release assay for natural cytotoxicity, J. Immunol. Methods 64 (1983) 313–320. [43] A.A. Gurtovenko, J. Anwar, Modulating the structure and properties of cell membranes: the molecular mechanism of action of dimethyl sulfoxide, J. Phys. Chem. B 111 (2007) 10453–10460. [44] B. Poole, S. Ohkuma, Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages, J. Cell Biol. 90 (1981) 665–669. [45] Y. Eto, Y. Yoshioka, R. Asavatanabodee, S. Kida, M. Maeda, Y. Mukai, H. Mizuguchi, K. Kawasaki, N. Okada, S. Nakagawa, Transduction of adenovirus vectors modified with cell-penetrating peptides, Peptides 30 (2009) 1548–1552. [46] J.S. Wadia, R.V. Stan, S.F. Dowdy, Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis, Nat. Med. 10 (2004) 310–315. [47] G. Drin, S. Cottin, E. Blanc, A.R. Rees, J. Temsamani, Studies on the internalization mechanism of cationic cell-penetrating peptides, J. Biol. Chem. 278 (2003) 31192–31201. [48] H. Hashida, M. Miyamoto, Y. Cho, Y. Hida, K. Kato, T. Kurokawa, S. Okushiba, S. Kondo, H. Dosaka-Akita, H. Katoh, Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool, Br. J. Cancer 90 (2004) 1252–1258. [49] T.B. Potocky, A.K. Menon, S.H. Gellman, Cytoplasmic and nuclear delivery of a TATderived peptide and a beta-peptide after endocytic uptake into HeLa cells, J. Biol. Chem. 278 (2003) 50188–50194. [50] T. Shiraishi, P.E. Nielsen, Enhanced delivery of cell-penetrating peptide–peptide nucleic acid conjugates by endosomal disruption, Nat. Protoc. 1 (2006) 633–636. [51] S.B. Fonseca, M.P. Pereira, S.O. Kelley, Recent advances in the use of cellpenetrating peptides for medical and biological applications, Adv. Drug Deliv. Rev. 61 (2009) 953–964. [52] C. Foerg, H.P. Merkle, On the biomedical promise of cell penetrating peptides: limits versus prospects, J. Pharm. Sci. 97 (2008) 144–162. [53] J.M. Gump, S.F. Dowdy, TAT transduction: the molecular mechanism and therapeutic prospects, Trends Mol. Med. 13 (2007) 443–448. [54] H. Brooks, B. Lebleu, E. Vivès, Tat peptide-mediated cellular delivery: back to basics, Adv. Drug Deliv. Rev. 57 (2005) 559–577. [55] J.L. Hanslick, K. Lau, K.K. Noguchi, J.W. Olney, C.F. Zorumski, S. Mennerick, N.B. Farber, Dimethyl sulfoxide (DMSO) produces widespread apoptosis in the developing central nervous system, Neurobiol. Dis. 34 (2009) 1–10. [56] W. Qi, R.J. Salvi, Cytotoxic effects of dimethyl sulphoxide (DMSO) on cochlear organotypic cultures, Hear Res. 236 (2008) 52–60. [57] H.N. Yu, Y.R. Lee, E.M. Noh, K.S. Lee, E.K. Song, M.K. Han, Y.C. Lee, C.Y. Yim, J. Park, B.S. Kim, S.H. Lee, S.J. Lee, J.S. Kim, Tumor necrosis factor-alpha enhances DMSOinduced differentiation of HL-60 cells through the activation of ERK/MAPK pathway, Int. J. Hematol. 87 (2008) 189–194.