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• W1982619130 abstract "Recent studies have demonstrated that covalent grafting of a single histidine residue into a twin-chain aliphatic hydrocarbon compound enhances its endosome-disrupting properties and thereby generates an excellent DNA transfection system. Significant increase in gene delivery efficiencies has thus been obtained by using endosome-disrupting multiple histidine functionalities in the molecular architecture of various cationic polymers. To take advantage of this unique feature, we have incorporated L-histidine (N,N-di-n-hexadecylamine) ethylamide (LH) in the membrane of hepatocyte-specific Sendai virosomes containing only the fusion protein (F-virosomes (Process for Producing a Targeted Gene (Sarkar, D. P., Ramani, K., Bora, R. S., Kumar, M., and Tyagi, S. K. (November 4, 1997) U. S. Patent 5,683,866))). Such LH-modified virosomal envelopes were four times more (p < 0.001) active in terms of fusion with its target cell membrane. On the other hand, the presence of LH in reconstituted influenza and vesicular stomatitis virus envelopes failed to enhance spike glycoprotein-induced membrane fusion with host cell membrane. Circular dichroism and limited proteolysis experiments with F-virosomes indicated that the presence of LH leads to conformational changes in the F protein. The molecular mechanism associated with the increased membrane fusion induced by LH has been addressed in the light of fusion-competent conformational change in F protein. Such enhancement of fusion resulted in a highly efficient gene delivery system specific for liver cells in culture and in whole animals. Recent studies have demonstrated that covalent grafting of a single histidine residue into a twin-chain aliphatic hydrocarbon compound enhances its endosome-disrupting properties and thereby generates an excellent DNA transfection system. Significant increase in gene delivery efficiencies has thus been obtained by using endosome-disrupting multiple histidine functionalities in the molecular architecture of various cationic polymers. To take advantage of this unique feature, we have incorporated L-histidine (N,N-di-n-hexadecylamine) ethylamide (LH) in the membrane of hepatocyte-specific Sendai virosomes containing only the fusion protein (F-virosomes (Process for Producing a Targeted Gene (Sarkar, D. P., Ramani, K., Bora, R. S., Kumar, M., and Tyagi, S. K. (November 4, 1997) U. S. Patent 5,683,866))). Such LH-modified virosomal envelopes were four times more (p < 0.001) active in terms of fusion with its target cell membrane. On the other hand, the presence of LH in reconstituted influenza and vesicular stomatitis virus envelopes failed to enhance spike glycoprotein-induced membrane fusion with host cell membrane. Circular dichroism and limited proteolysis experiments with F-virosomes indicated that the presence of LH leads to conformational changes in the F protein. The molecular mechanism associated with the increased membrane fusion induced by LH has been addressed in the light of fusion-competent conformational change in F protein. Such enhancement of fusion resulted in a highly efficient gene delivery system specific for liver cells in culture and in whole animals. Completion and annotation of the human genome sequence has significantly enhanced the possibility of using designer DNA for gene therapy. Nonetheless, in spite of the first gene therapy trial 14 years ago, and numerous clinical trials thereafter, successful gene therapy remains elusive. The three main hurdles of widespread belief have been bolstered as (i) delivery, (ii) delivery, and (iii) delivery (1Luo D. Trends Biotechnol. 2004; 22: 101-103Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). It turns out that the most up-hill task toward ensuring clinical success of gene therapy is to design safe, efficacious and target-specific transfection vectors. Toward designing such gene carriers, significant progress has been made on gene transfection by novel non-glycerol-based histidylated cationic amphiphiles, presumably via the endosome-disrupting properties of the histidine functionalities (2Kumar V.V. Pichon C. Refregiers M. Guerin B. Midoux P. Chaudhuri A. Gene Ther. 2003; 10: 1206-1215Crossref PubMed Scopus (97) Google Scholar). Histidine residues in amphipathic peptide antibiotics are also known to promote efficient DNA delivery into mammalian cells in vitro by favoring endosomal escape of the DNA molecules at low pH (3Kichler A. Leborgne C. März J. Danos O. Bechinger B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1564-1568Crossref PubMed Scopus (196) Google Scholar). Complexes of recombinant adenoviruses and cationic lipids/polymers have been tested for the enhancement of therapeutic index for the treatment of cystic fibrosis (4Fasbender A. Zabner J. Chillón M. Moninger T.O. Puga A.P. Davidson B.L. Welsh M.J. J. Biol. Chem. 1997; 272: 6479-6489Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). The beneficial role of such complexes was clearly envisaged by the amplification of binding and entry of adenovirus particles into host cells. However, despite all these encouraging results, its failure in targeted gene transfer in whole animal and inherent cytopathic/cytotoxic side effects limit its applications in liver gene therapy (5Cavazzana-Calvo M. Thrasher A. Mavilio F. Nature. 2004; 427: 779-781Crossref PubMed Scopus (214) Google Scholar). Because liver is an ideal organ for somatic gene delivery as well as the therapeutic target for hepatic diseases (6Yamada T. Iwasaki Y. Tada H. Iwabuki H. Chuah M.K. VandenDriessche T. Fukuda H. Kondo A. Ueda M. Seno M. Tanizawa K. Kuroda S. Nat. Biotechnol. 2003; 21: 885-890Crossref PubMed Scopus (235) Google Scholar), we had developed a virus-based liver-specific delivery system (F-virosomes (FV)) 4The abbreviations used are: FVF containing virosomesDPBSDulbecco's phosphate-buffered salineEGFPenhanced green fluorescent proteinFfusion factorFV(H)heat-treated F containing virosomesGVvesicular stomatitis virus G-glycoprotein containing virosomesHAinfluenza hemagglutininHNhemagglutinin neuraminidaseHNFVHN and F containing virosomesLCN,N-di-n-hexadecyl-N-2-aminoethylamineLCFV(H)heat-treated LCFVLCFVLC containing FVLHL-histidine (N,N-di-n-hexadecylamine) ethylamideLH/cholesterolliposomes of LH and cholesterol with a molar ratio 1:2LHFV(H)heat-treated LHFVLHFVLH containing FVLHGVLH containing GVLHHAVLH containing hemagglutinin containing virosomesNBD-PEN-4-nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamineRITC-lysozymerhodamine isothiocynate-labeled lysozymeRTreverse transcriptionWGAwheat germ agglutininPBSphosphate-buffered salineRBCred blood cellHRheptad repeatTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. 4The abbreviations used are: FVF containing virosomesDPBSDulbecco's phosphate-buffered salineEGFPenhanced green fluorescent proteinFfusion factorFV(H)heat-treated F containing virosomesGVvesicular stomatitis virus G-glycoprotein containing virosomesHAinfluenza hemagglutininHNhemagglutinin neuraminidaseHNFVHN and F containing virosomesLCN,N-di-n-hexadecyl-N-2-aminoethylamineLCFV(H)heat-treated LCFVLCFVLC containing FVLHL-histidine (N,N-di-n-hexadecylamine) ethylamideLH/cholesterolliposomes of LH and cholesterol with a molar ratio 1:2LHFV(H)heat-treated LHFVLHFVLH containing FVLHGVLH containing GVLHHAVLH containing hemagglutinin containing virosomesNBD-PEN-4-nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamineRITC-lysozymerhodamine isothiocynate-labeled lysozymeRTreverse transcriptionWGAwheat germ agglutininPBSphosphate-buffered salineRBCred blood cellHRheptad repeatTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. (34Sarkar, D. P., Ramani, K., Bora, R. S., Kumar, M., and Tyagi, S. K. (November 4, 1997) U. S. Patent 5,683,866Google Scholar) efficient in transferring foreign genes into human hepatoblastoma cells (HepG2) in culture and mouse/rat hepatocytes in vivo. F-virosomes exploit the membrane fusogenic property of Sendai virus F glycoprotein and the high affinity terminal β-galactose-containing ligands (present on F glycoproteins) for the asialoglycoprotein receptors on the hepatocyte surface (7Bagai S. Puri A. Blumenthal R. Sarkar D.P. J. Virol. 1993; 67: 3312-3318Crossref PubMed Google Scholar). Upon tight binding to the asialoglycoprotein receptors, F-virosomes undergo fusion both at the level of plasma membrane and endosomal membrane followed by receptor-mediated endocytosis and deliver the entrapped molecules into the cytosol (8Bagai S. Sarkar D.P. J. Biol. Chem. 1994; 269: 1966-1972Abstract Full Text PDF PubMed Google Scholar, 9Ramani K. Bora R.S. Kumar M. Tyagi S.K. Sarkar D.P. FEBS Lett. 1997; 404: 164-168Crossref PubMed Scopus (33) Google Scholar, 10Ramani K. Hassan Q. Venkaiah B. Hasnain S.E. Sarkar D.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11886-11890Crossref PubMed Scopus (53) Google Scholar). We also observed that fusion efficiency of Sendai virus F protein is much reduced in the absence of its native attachment glycoprotein, hemagglutinin neuraminidase (HN) (7Bagai S. Puri A. Blumenthal R. Sarkar D.P. J. Virol. 1993; 67: 3312-3318Crossref PubMed Google Scholar). To overcome such impediments, we tested the efficacy of histidine functionality in disrupting endosomes. Thus, we have incorporated LH molecules in the F-virosomal membrane and systematically assessed its ability to enhance the binding and fusion with the target cells (cultured hepatocytes and mouse liver) as monitored by transgene expression. We report for the first time that histidine functionality of a cationic amphiphile specifically enhances F glycoprotein-induced membrane fusion and results in efficient gene delivery. 5Patent applied. 5Patent applied. The possible mechanisms of LH-induced activation of F-virosome-target membrane fusion have also been investigated. F containing virosomes Dulbecco's phosphate-buffered saline enhanced green fluorescent protein fusion factor heat-treated F containing virosomes vesicular stomatitis virus G-glycoprotein containing virosomes influenza hemagglutinin hemagglutinin neuraminidase HN and F containing virosomes N,N-di-n-hexadecyl-N-2-aminoethylamine heat-treated LCFV LC containing FV L-histidine (N,N-di-n-hexadecylamine) ethylamide liposomes of LH and cholesterol with a molar ratio 1:2 heat-treated LHFV LH containing FV LH containing GV LH containing hemagglutinin containing virosomes N-4-nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine rhodamine isothiocynate-labeled lysozyme reverse transcription wheat germ agglutinin phosphate-buffered saline red blood cell heptad repeat N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. F containing virosomes Dulbecco's phosphate-buffered saline enhanced green fluorescent protein fusion factor heat-treated F containing virosomes vesicular stomatitis virus G-glycoprotein containing virosomes influenza hemagglutinin hemagglutinin neuraminidase HN and F containing virosomes N,N-di-n-hexadecyl-N-2-aminoethylamine heat-treated LCFV LC containing FV L-histidine (N,N-di-n-hexadecylamine) ethylamide liposomes of LH and cholesterol with a molar ratio 1:2 heat-treated LHFV LH containing FV LH containing GV LH containing hemagglutinin containing virosomes N-4-nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine rhodamine isothiocynate-labeled lysozyme reverse transcription wheat germ agglutinin phosphate-buffered saline red blood cell heptad repeat N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. N-4-Nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine (NBD-PE) was purchased from Avanti Polar Lipids. SM2 Bio-Beads were obtained from Bio-Rad. Triton X-100 was obtained from Aldrich. Lysozyme (chicken egg white, IUB 3.2.1.17), trypsin (type III), dithiothreitol, wheat germ agglutinin (WGA), sodium azide, EDTA (disodium salt), rhodamine isothiocyanate (RITC), bafilomycin A1 were purchased from Sigma. L-Histidine (N,N-di-n-hexadecylamine) ethylamide (LH) (molecular formula, C40H81N5Cl2O, molecular weight, 752.5) and N,N-di-n-hexadecyl-N-2-aminoethylamine (LC) (molecular formula, C34H74N2Cl2, molecular weight, 581) were synthesized (Fig. 1) and stored as described earlier (2Kumar V.V. Pichon C. Refregiers M. Guerin B. Midoux P. Chaudhuri A. Gene Ther. 2003; 10: 1206-1215Crossref PubMed Scopus (97) Google Scholar). Dulbecco's modified Eagle's medium, DPBS, fetal bovine serum, and antibiotics were obtained from Invitrogen. Monoclonal antibody against GFP was procured from BD Bioscience Clontech. VITROGEN® was purchased from COHESION Technologies Inc. (Palo Alto, CA). Other reagents used were also of the highest grade commercially available. Sendai virus (Z strain) and influenza virus X:31 (A/Aichi/68;H3N2) were grown in the allantoic sac of 10–11-day-old embryonated chicken eggs. The virus was harvested and purified following standard procedures (7Bagai S. Puri A. Blumenthal R. Sarkar D.P. J. Virol. 1993; 67: 3312-3318Crossref PubMed Google Scholar). Vesicular stomatitis virus (Indiana) was grown on monolayer cultures of baby hamster kidney (BHK-21) cells and purified as described elsewhere (11Paternostre M.-T. Lowy R.J. Blumenthal R. FEBS Lett. 1989; 243: 251-258Crossref PubMed Scopus (20) Google Scholar). HepG2 cells (human hepatoblastoma cell line) and HeLa cells were obtained from American Type Culture Collection and were grown as described earlier (7Bagai S. Puri A. Blumenthal R. Sarkar D.P. J. Virol. 1993; 67: 3312-3318Crossref PubMed Google Scholar). Fresh red blood cells were prepared from healthy Swiss albino mice in accordance with the norms of the Animal Ethics Committee, University of Delhi South Campus laws and regulations. F-virosomes were prepared following published/patented procedures. Cationic amphiphiles (LH and LC) were incorporated in reconstituted viral membrane as described earlier for making NBD-PE-labeled virosomes (7Bagai S. Puri A. Blumenthal R. Sarkar D.P. J. Virol. 1993; 67: 3312-3318Crossref PubMed Google Scholar). Briefly, the LH and LC compounds dissolved in solvents (chloroform:methanol, 2:1, v/v) were dried in a glass vial separately under nitrogen to form a thin film. The supernatant from the detergent extract of pure Sendai virus (50 mg of protein), containing only the viral F protein and the lipids, was added to the respective LH and LC films and incubated at 20 °C for 30 min with gentle shaking. The detergent was removed by SM2 Bio-Beads to form the virosomes. The amount of amphiphiles incorporated into the virosomal membranes (LHFV and LCFV) was analyzed by high performance liquid chromatography after purifying the virosomal preparations through a continuous sucrose gradient as described elsewhere (12Hsu M. Scheid A. Choppin P.W. J. Biol. Chem. 1981; 256: 3557-3563Abstract Full Text PDF PubMed Google Scholar). NBD-PE-labeled influenza virosomes (HA containing virosomes) with LH and LC were made according to earlier protocols with modifications as above (13Bagai S. Sarkar D.P. FEBS Lett. 1994; 353: 332-336Crossref PubMed Scopus (8) Google Scholar). Similarly, labeled vesicular stomatitis virus virosomes (GV) with LH and LC were prepared following a published procedure (11Paternostre M.-T. Lowy R.J. Blumenthal R. FEBS Lett. 1989; 243: 251-258Crossref PubMed Scopus (20) Google Scholar). The plasmid pEGFP-N1 (Clontech, isolated using a Qiagen Megaprep unit) coding for enhanced green fluorescent protein (EGFP) under the control of cytomegalovirus promoter and RITC-lysozyme were entrapped in various virosomal preparations following our patented protocol (8Bagai S. Sarkar D.P. J. Biol. Chem. 1994; 269: 1966-1972Abstract Full Text PDF PubMed Google Scholar, 10Ramani K. Hassan Q. Venkaiah B. Hasnain S.E. Sarkar D.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11886-11890Crossref PubMed Scopus (53) Google Scholar). Liposomes containing LH/cholesterol and LC/cholesterol were prepared as reported earlier (2Kumar V.V. Pichon C. Refregiers M. Guerin B. Midoux P. Chaudhuri A. Gene Ther. 2003; 10: 1206-1215Crossref PubMed Scopus (97) Google Scholar). The virosome preparations were passed through 26-gauge needle 20 times and their particle size analyzed by a Photon Correlation Spectrometer (photon correlation spectrometry model: Photocor FC) using the software Flexcor/DynaLS provided by the Photocor company. The system was calibrated using 200-nm silica/latex particles (supplied by Photocor FC). Trypsinization, limited proteolysis by proteinase K, heat treatment (56 °C for 30 min), and DNase I digestion of virosome samples were carried as described elsewhere (8Bagai S. Sarkar D.P. J. Biol. Chem. 1994; 269: 1966-1972Abstract Full Text PDF PubMed Google Scholar, 9Ramani K. Bora R.S. Kumar M. Tyagi S.K. Sarkar D.P. FEBS Lett. 1997; 404: 164-168Crossref PubMed Scopus (33) Google Scholar, 14Kumar M. Hassan M.Q. Tyagi S.K. Sarkar D.P. J. Virol. 1997; 71: 6398-6406Crossref PubMed Google Scholar). Hemolysis and lipid mixing (fluorescence dequenching of NBD-PE) assay for virosome-cell fusion were carried out as described earlier (7Bagai S. Puri A. Blumenthal R. Sarkar D.P. J. Virol. 1993; 67: 3312-3318Crossref PubMed Google Scholar). Binding of NBD-PE-labeled virosomes and fusion-mediated delivery of RITC-lysozyme to the target cells were performed following our published protocol (7Bagai S. Puri A. Blumenthal R. Sarkar D.P. J. Virol. 1993; 67: 3312-3318Crossref PubMed Google Scholar, 8Bagai S. Sarkar D.P. J. Biol. Chem. 1994; 269: 1966-1972Abstract Full Text PDF PubMed Google Scholar). Targeted cytosolic delivery of pEGFP-N1 DNA to HepG2 cells in culture was essentially carried out as described elsewhere (9Ramani K. Bora R.S. Kumar M. Tyagi S.K. Sarkar D.P. FEBS Lett. 1997; 404: 164-168Crossref PubMed Scopus (33) Google Scholar). For fusion-mediated delivery (of entrapped RITC-lysozyme and DNA) experiments to target cells in culture with bafilomycin A1 (175 nm, a potent inhibitor of endosome acidification), the drug was mixed with loaded virosomes after dilution with the medium. The gene expression was assessed by recording EGFP fluorescence in a Nikon Eclipse TE 300 epifluorescent microscope attached to a digital camera (Digital sight DS-5M). For immunofluorescence studies, cells were washed with PBS twice and fixed in 2% paraformaldehyde in PBS at room temperature, followed by permeabilization with 0.1% Triton X-100 containing 0.5 m NH4Cl in PBS for 10 min. The cells were blocked in 5% fetal bovine serum containing PBS at room temperature for 30 min and incubated with anti-GFP A.v. monoclonal antibody (JL-8) overnight at 4 °C followed by washing with PBS and incubation with fluorescein isothiocyanate-labeled goat anti-mouse IgG (Sigma). The cells were then visualized in a Nikon microscope for fluorescein isothiocyanate fluorescence as above. In a parallel assay, cell extracts were processed for evaluation of EGFP expression by Western analysis using the ECL detection system (Santa Cruz Biotechnology) and also by fluorometric quantitation (15Dandekar D.H. Kumar M. Ladha J.S. Ganesh K.N. Mitra D. Anal. Biochem. 2005; 342: 341-344Crossref PubMed Scopus (21) Google Scholar). Total DNA/RNA isolation and PCR/RT-PCR amplification from the cells in culture were performed as described below for in vivo studies with mice. All experiments were independently repeated at least three times. Twelve-week-old female Balb/c mice (∼18 g) were injected intravenously into the tail vein (in accordance with the guidelines of the Animal Ethics Committee, University of Delhi South Campus), with DNA-loaded virosomes (0.8 mg F protein containing 4 μg of pEGFP-N1) in a final volume of 0.2 ml of DPBS containing 2 mm Ca2+. Injection of the equivalent amount of free DNA and heat-treated loaded virosomes in DPBS served as appropriate controls. After 2 days post-injection, parenchymal cell types (hepatocytes) were isolated as described in an earlier report by a member of our group (10Ramani K. Hassan Q. Venkaiah B. Hasnain S.E. Sarkar D.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11886-11890Crossref PubMed Scopus (53) Google Scholar) and plated on 12-well plastic plates (Falcon®, BD Biosciences) coated with VITROGEN® as per their protocol in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Following incubation in CO2 for 24 h, plates were examined for EGFP expression as described above. The genomic DNA and total RNA were extracted by standard procedures (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) and subjected to PCR amplification/RT-PCR of the EGFP gene (transcripts). (i) PCR Amplification of EGFP Gene—Total genomic DNA isolation from hepatocytes was done using TRIzol. The primer set designed for EGFP amplification included the following: sense, 5′-TGACCCTGAAGTTCATCTGCACCA-3′; antisense, 5′-TTGATGCCGTTCTTCTGCTTGTCG-3′. PCR amplification was performed using Taq DNA polymerase (Invitrogen) with a cycling profile of 94 °C for 45 s, 55 °C for 45 s, 72 °C for 45 s, for 30 cycles, and a final extension of 72 °C for 10 min (same in the case of β-actin primers, Stratagene). A specific amplified product of 361 bp of the EGFP gene was visualized by ethidium bromide staining on a 1.2% agarose gel and transferred to nylon membrane. As a control, the β-actin gene was also amplified and a product of 650 bp was obtained. Southern hybridization was performed to further confirm the specificity of the PCR products using a 720-bp NotI/HindIII EGFP gene fragment derived from the plasmid pEGFP-N1, labeled with [α-32P]dCTP (by the random primer-labeling technique). (ii) RT-PCR Amplification of EGFP Gene-specific Transcript—Total hepatic RNA from various mice or from HepG2 cells was isolated using TRIzol reagent (Invitrogen). DNase I (Invitrogen)-treated RNA was reverse transcribed using Superscript RNase H- RT (Invitrogen) and gene-specific antisense primer as per the manufacturer's protocol. PCR amplification (30 cycles) of the RT product was performed using high-fidelity Platinum Taq DNA polymerase (Invitrogen) with a cycling profile as mentioned before. The amplified product of 361 bp of the EGFP gene was visualized by ethidium bromide staining on a 1.2% agarose gel and transferred to nylon membrane and subsequently Southern hybridized. As a control, β-actin mRNA was also amplified by RT-PCR for each sample and a product of 650 bp was obtained. Following intravenous (tail vein) injection (0.8 mg of F protein containing 4 μg of plasmid DNA) of various pBVluc (containing the firefly luciferase gene under control of the cytomegalovirus promoter as described earlier by Ramani et al. (10Ramani K. Hassan Q. Venkaiah B. Hasnain S.E. Sarkar D.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11886-11890Crossref PubMed Scopus (53) Google Scholar))-loaded virosomes (FV, LCFV, LHFV, and LHFV(H)), parenchymal cells were isolated as above. The cells were washed twice with Tricine-buffered saline and suspended in the same. For luciferase assay, the Triton X-100-solubilized cell lysate (from 1 × 107 cells) was mixed with luciferase assay buffer containing 1 mm luciferin (Roche Molecular Biochemicals). Luciferase activity (in mV) was measured in a 1250 Luminometer (BIO-Orbit, Finland) as described earlier (10Ramani K. Hassan Q. Venkaiah B. Hasnain S.E. Sarkar D.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11886-11890Crossref PubMed Scopus (53) Google Scholar). Four groups of mice (three mouse per group) were injected (0.8 mg of F protein containing 4 μg of plasmid DNA) with various pEGFP-N1-loaded virosomes (FV, LCFV, LHFV, and LHFV(H)). After 6 months a portion of each liver from the injected mice was immediately fixed in Bouins fixative and dehydrated through graded alcohol and xylene followed by embedding in paraffin wax. Such embedded tissues were cut into 5-μm sections, deparaffinized, and rehydrated. For immunostaining, sections were washed in PBS for 10 min and blocked with 3% bovine serum albumin in PBS for 1 h at 37°C. Subsequently, sections were incubated with EGFP-specific monoclonal antibody (JL-8) for 1 h at 37 °C. After three washes with PBS, sections were incubated with fluorescein isothiocyanate-labeled goat anti-mouse IgG at 37 °C for 1 h. All antibody dilutions were made in 1.5% bovine serum albumin in PBS. Following extensive washes with PBS, the slides were counterstained with Mayer hematoxylin, washed in distilled water, and mounted in glycerol. The specificity of immunostaining was verified by the use of PBS in place of primary antibody (data not shown). The sections were examined using fluorescence microscopy (Nikon fluorescein isothiocyanate filter set) and photographed (magnification, ×20). The CD spectra were recorded on a Jasco (J-715) spectropolarimeter fitted with a Peltier-based temperature controller (PTC-43). The instrument was calibrated with (+)-10-camphorsulfonic acid. Subsequently, the spectral measurements of the virosome samples (0.04 mg/ml F protein in LHFV and LCFV samples) were made using a 0.2-cm path-length cell at room temperature with constant purging of nitrogen. Each protein sample was subjected to 30 scans to obtain the final spectrum. The respective buffer baseline was subtracted from the spectra of the protein samples and data were presented as ellipticity expressed in millidegree. Fusion of F-virosomes with Target Cell Membrane Is Enhanced by Histidylated Cationic Lipid—To evaluate the particle size, virosomal preparations were subjected to photon correlation spectrometry measurements. It revealed the mean diameter of FVs to be 181 nm, which conforms well to the average size reported earlier by transmission electron microscopy (17Bagai S. Sarkar D.P. Biochim. Biophys. Acta. 1993; 1152: 15-25Crossref PubMed Scopus (23) Google Scholar), on the other hand, LHFV and LCFV preparations were found to be 298 and 232 nm, respectively (data not shown). ξ potential measurements of these virosomes reflected net negative charge for FV and net positive charge for both LHFV and LCFV. The membrane fusion activity of F protein in FV is well correlated with its ability to hemolyse various RBCs upon WGA-mediated binding of F protein to cell surface sugar molecules (7Bagai S. Puri A. Blumenthal R. Sarkar D.P. J. Virol. 1993; 67: 3312-3318Crossref PubMed Google Scholar). Therefore, to begin with mouse, RBC was chosen as appropriate target cells with a view to check and compare the efficiency of membrane fusion by various virosomal preparations in the presence of WGA. As shown in Fig. 2a, various doses of LH molecules significantly enhanced the hemolytic activity of LHFVs. With 2 mg, LH incorporated into 50 mg of total Sendai virus protein resulted in a 4-fold increase in hemolytic activity. The specificity of the effect of LH was established by parallel control with LC (having the same amount of positively charged head group but lacks histidine moiety, Fig. 1), which did not affect hemolysis over FVs alone. Strikingly, same amount of LH in LH/CHOL liposomes mixed with FVs did not affect hemolysis over FVs alone. HNFVs, containing HN that markedly enhances its lytic activity (7Bagai S. Puri A. Blumenthal R. Sarkar D.P. J. Virol. 1993; 67: 3312-3318Crossref PubMed Google Scholar), served as a positive control, and incorporation of LH and LC molecules in HNFVs did not enhance the hemolytic activity any further. Similar results were also obtained with RBCs from human and rabbit (data not shown). The high performance liquid chromatography analysis of the sucrose density gradient-purified virosomes showed 85% efficiency of incorporation using 2 mg of LH against 50 mg of Sendai virus (Fig. 2a, inset). The gradient purification of virosomes (12Hsu M. Scheid A. Choppin P.W. J. Biol. Chem. 1981; 256: 3557-3563Abstract Full Text PDF PubMed Google Scholar) ensured the co-grafting of LH and LC molecules with F protein in the viral membrane. Similar profile of incorporation (and size) was also obtained with HA containing virosomes and GV (data not shown). The incorporation profile of LC in FVs followed the same trend (data not shown). Calculations based on the protein/lipid composition of virosomes, molecular weights of F (HA and G) and cationic lipids, about 60 molecules LH (and LC), were estimated to be associated with one molecule of F (HA and G) protein in the respective virosomal membranes (having 2 mg of LH with 50 mg of Sendai, influenza, and vesicular stomatitis virus). All further experiments were performed at this stoichiometry. NBD-PE-labeled FVs, LHFVs, and LCFVs, containing equivalent amounts of F protein of identical electrophoretic mobility (Fig. 2b), had equal affinity for mouse RBCs and HepG2 cells but showed negligible attachments to HeLa cells at 4 °C (Fig. 2, c and d). Because our main goal is to demonstrate targeted gene delivery in liver cells using LH-modified FVs, HepG2 cells are included at this point as the only target cells of choice (7Bagai S. Puri A. Blumenthal R. Sarkar D.P. J. Virol. 1993; 67: 3312-3318Crossref PubMed Google Scholar) to establish the role of LH molecules in enhancing membrane fusion induced by F protein in culture conditions. HeLa cells are taken as negative controls to reveal the binding specificity of the terminal β-galactose moieties of F protein to the asialoglycoprotein receptors on the hepatocyte cell surface. As shown in Fig. 2e, the presence of LH in the viral membrane markedly enhanced the kinetics (∼8-fold increase) as well as the extent of hemolysis. LH/cholesterol liposomes mixed with FVs failed to exert such an effect. Fluorescent micrographs showing escalated movement of NBD-PE (dequenching of fluorescence) from LHFVs to mouse RBCs indicated membrane fusion (compare panels A and B with C in Fig. 3) and the transfer of RITC-lysozyme from loaded virosomes to an increased number (about 2-fold) of HepG2 cells (compare panel 1 and 2 with 3 in Fig. 4a) indicated core mixing. This is also in" @default.
• W1982619130 created "2016-06-24" @default.
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• W1982619130 creator A5020779635 @default.
• W1982619130 creator A5029919048 @default.
• W1982619130 creator A5037583910 @default.
• W1982619130 creator A5047254599 @default.
• W1982619130 creator A5078491857 @default.
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• W1982619130 date "2005-10-01" @default.
• W1982619130 modified "2023-09-26" @default.
• W1982619130 title "Histidylated Lipid-modified Sendai Viral Envelopes Mediate Enhanced Membrane Fusion and Potentiate Targeted Gene Delivery" @default.
• W1982619130 cites W1413998355 @default.
• W1982619130 cites W1480671701 @default.
• W1982619130 cites W1600024407 @default.
• W1982619130 cites W1914528802 @default.
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• W1982619130 cites W1992566630 @default.
• W1982619130 cites W2010814343 @default.
• W1982619130 cites W2011659579 @default.
• W1982619130 cites W2013270869 @default.
• W1982619130 cites W2015159733 @default.
• W1982619130 cites W2044194952 @default.
• W1982619130 cites W2044486273 @default.
• W1982619130 cites W2045472151 @default.
• W1982619130 cites W2045526448 @default.
• W1982619130 cites W2048484727 @default.
• W1982619130 cites W2061510929 @default.
• W1982619130 cites W2062495546 @default.
• W1982619130 cites W2069031909 @default.
• W1982619130 cites W2077850392 @default.
• W1982619130 cites W2078968390 @default.
• W1982619130 cites W2081577524 @default.
• W1982619130 cites W2082268572 @default.
• W1982619130 cites W2085516892 @default.
• W1982619130 cites W2105582248 @default.
• W1982619130 cites W2121325844 @default.
• W1982619130 cites W2142613671 @default.
• W1982619130 cites W2159953901 @default.
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