Membrane fusion mediated cytosolic drug delivery through sc
Membrane fusion mediated cytosolic drug delivery through sc. Fv targeted Sendai viral envelopes Mukesh Kumar Research Associate 1. National Brain Research Centre, Manesar, Haryana 2. All India Institute of Medical Sciences, New Delhi
Human Placental Alkaline Phosphatase (PAP) » PAP is an isozyme of alkaline phosphatase » 513 amino acid peripheral membrane glycoprotein anchored to the outer surface of plasma membrane via a phosphatidylinositol linkage. PAP Phosphatidyl Inositol Anchor » Dimer of two identical subunits with molecular weight of 64 k. Da each. 2
» Expression » is restricted to the second and third-trimester of human placenta. It is a marker for placental cells as well as for tumors of reproductive organs like ovarian tumor, seminomas, and cervical tumors. Nearly universally seen in germ cell tumors. Characteristics: - Increased heat stability, differential inhibition by various uncompetitive inhibitors like L-Phenylalanine, L-Leucine, peptides like L-Phe. Gly-Gly. 3
Features that make it an attractive immunotherapeuic target • Cell surface localization • Clathrin mediated endocytosis • Low shedding into circulation • Ectopic expression in various malignancies viz. choriocarcinoma, germ cells(>80%), seminomas, uterus and ovary, breast, colon, head and neck 4
Human Alkaline Phosphatases Subunit mol. wt. Tissue non. Specific (TNAP) 69 k. Da Subunit association Dimer dimer Heat lability Labile 70 o. C; 5 min Homology with PAP 60% 90% Urea inhibition 2 mol/L 70% 40% Levamisole inhibition 10 µmol/L 50% insensitive Amino – acid / peptide inhibiton L-Homoarginine L-Phe, L-Leu, L-Phe-gly Intestinal (IAP) 68 k. Da Placental ( PAP) 64 k. Da dimer 70 o. C; 30 min 25%
Alkaline Phosphatase Isozymes PAP • L-Phe-gly BONE INTESTINAL • L-Homo THESE INHIBITORS ARE : (i) Isozyme specific (ii) Bind to defined sites on the molecule • L-Phe
Phagemid Filamentous Phage
Bio-panning from Human Phage Display Antibody Library Tomlinson’s human antibody library was used for selection. Bio-panning » TG 1 (E. coli) - “Suppressor strain”- recognizes stop codon (TAG) as glutamate- surface display of sc. Fv » HB 2151(E. coli) - “Non-suppressor” strain- recognize amber stop codon-for soluble expression of sc. Fv.
Selection of PAP binding monoclonal phage sc. Fv PEG precipitated monoclonal phage ELISA. Cross- reactivity profile of P 4 C 8 in presence of varying amounts of PAP/IAP/BAP. P 4 C 8 is showing good binding to PAP consistently. P 4 C 8 shows maximum inhibition in binding to PAP in the presence of 1. 5µg of free PAP
Soluble expression and binding of sc. Fv SDS-PAGE of soluble expression of clones showing 29 k. D protein Binding of dialyzed soluble sc. Fvs of monoclonals on PAP coated plate Binding of soluble sc. Fv of P 4 C 8 to PAP is maximum
Cellular Internalization Three strategic categories to enhance endosomal escape : - (i) Molecular ferries (ii) Leakage-inducing molecules (iii)Physico-chemical techniques
Enveloped viral fusion The hemifusion model for bilayer fusion.
SENDAI, A PARAMYXOVIRIDAE ( HEMOLYTIC VIRUS FAMILY ) Enveloped animal virus, containing single stranded RNA as genetic material. Ø Discovered in early fifties in “Sendai” Japan, also known as “HVJ” Ø It requires physiological p. H and temperature to enter/infect host cells. Ø Mechanisms of entry fairly resolved. However, the exact role of HN help to F-protein in membrane fusion is yet to be deciphered. Ø It is highly expected to be non-pathogenic for human. Ø
F-protein ü Its a glycoprotein which mediates p. H-independent fusion of viral envelope with the plasma membrane of host cell. ü Synthesized as inactive precursor F 0 (60 k. D)– proteolytic cleavage – F-protein - (disulfide-linked F 1 (45 k. D) and F 2 (15 k. D) poly peptide) ü Fusion peptide – Hydrophobic stretch of 20 -25 amino acid at N-terminus of F 1 –essential for biological activity of the protein 15
Preparation of Drug loaded Virosomes Ramani et. al. Proc. Natl. Acad. Sci. USA Vol. 95, pp. 11886 -11890, September 1998 16
Cloning Strategy of sc. Fv with F-protein fragment Chimeric sc. Fv has been used with F-protein for virosome reconstitution.
Choice of Cell Lines and Controls He. La cells - have PAP; Controls – Heat inactivated Csc. Fv-F-virosome alone Csc. Fv-F-virosome at 10°C Sa. Os cells - BAP only, no PAP Sa. Os transfected with PAP - have PAP with BAP CHO - no PAP Octadecyl rhodamine B chloride (R 18) Detection of Membrane Fusion Pictorial representation of a lipid-mixing assay based on fluorescence self-quenching. 18
Fold expression PAP Expression Analysis of Transfected Sa. Os-2 Real-Time analysis of PAP expression in transiently transfected Sa. Os-2 cell line. ~42 fold increase in the PAP RNA level was observed. Flow-cytometer analysis of the PAP transfected Sa. Os cells. PAP expressed on the surface of the cells were detected with anti-PAP monoclonal as a primary Ab and anti-mouse alexa 596 as secondary Ab. 14% PAP transfected cells were positively stained for PAP.
Fusion Kinetics of sc. Fv Engineered Virosome with Cell Lines He. La cells Sa. Os-2 Sa. Os (T) CHO - Natural expression of PAP on surface - No PAP but BAP expression on surface - PAP transfected Sa. Os - No expression of PAP on surface Kinetics of sc. Fv-F-virosome fusion with different cell lines. Dequenched fluorescence of R-18 shows that the initial membrane fusion is sc. Fv dependent.
FITC-lysozyme delivery by sc. Fv-F-virosome HC = Heat treatment to virosomes abrogates fusion mediated delivery of the cargo without affecting sc. Fv mediated endocytosis. C = Virosome without sc. Fv. Fusion mediated cytosolic delivery of FITC-Lysozyme and fluorescence, Quantification by Image. J, CTCF = Corrected Total Cell Fluorescence
Doxorubicin delivery by sc. Fv-F-virosome HC = Heat treatment to virosomes abrogates fusion mediated delivery of the cargo without affecting sc. Fv mediated endocytosis. Fusion mediated cytosolic delivery of doxorubicin and fluorescence quantification by Image. J, CTCF = Corrected Total Cell Fluorescence
Cell Survival Analysis after Virosome Mediated Doxorubicin Treatment He. LA (HC) - Heat Control He. La (control)- He. La with F-virosome, no sc. Fv Sa. Os(UT) - Untransfected Sa. Os(T) - PAP Transfected Sa. Os CHO – Negative control Trypan blue exclusion assay for cell survival after Doxorubicin packaged virosomal delivery. (significance level of P-value is <0. 05) Significant reduction in He. La cells and PLAP transfected Sa. Os cells viability
Effect of Endocytotic Inhibitor on Cellular Internalization Delivery of FITC-lysozyme to PAP expressing He. La cells by immunovirosome with or without cytochalasin B (10µM). In presence of cytochalasin. B (cyto(+)), the cargo is delivered by membrane fusion mediated pathway.
Localization of Intracellular RITC-lysozyme Delivered by Immuno-virosome Yellow fluorescence indicates endocytotic route of internalization. Reduced yellow fluorescence in panel A in comparison to Panel B but no such change in red fluorescence confirms membrane fusion mediated cytosolic delivery by Immuno-virosome. Live cell confocal microscopy for intracellular localization of RITC-lysozyme in He. La cells in presence and absence of cytochalasin. B (10µM). Lysotracker dye was used for tracking endocytotic route of internalization. HC – Heat control in terms of endocytotic route of entry.
Efficacy of sc. Fv-F-virosome Efficacy of Csc. Fv-F-virosome for RITC-lysozyme delivery to He. La cells. Cells without primary antibody against PAP (H 17 E 2) were negative control. App. 68% PAP positive cells were also positive for RITC-lysozyme.
Graphical presentation of the modality sc. Fv targeted Sendai viral membrane efficiently delivers the payload in cytosol via membrane fusion mediated process. Chimeric sc. Fv- sc. Fv fused with trans-membrane and cytosolic region of F-protein with a linker in between. PAP- Placental Alkaline Phosphatase
Conclusions v sc. Fv anchored on the virosome surface helps in binding of the virosome specifically to PAP expressing cells and subsequent membrane fusion is mediated by F-protein. v Most of the cargo (FITC-lysozyme/ doxorubicin) is delivered directly to the cytosol. v Enhancement to endosomal escape may result in less immunogenicity of the developed modality. The data is under review for publication.
Acknowledgement Dr. Subrata Sinha, Director, National Brain Research Centre, Manesar, Haryana Dr. P. Chattopadhyay, Professor, Department of Biochemistry, AIIMS, N. Delhi Dr. D. P. Sarkar, Professor, Department of Biochemistry, South Campus, University of Delhi Dr. Kunzang chosdol, Additional Professor, Department of Biochemistry, AIIMS, N. Delhi Prashant Mani, Department of Biochemistry, South Campus, University of Delhi Lab members and staff of the Department of Biochemistry, AIIMS, New Delhi Department of Biotechnology (DBT) and ICMR for funding UGC for fellowship
Thanks
Deglycosylated immunovirosome did not fuse with hepatic cells but retained its fusion ability with He. La cells expressing PAP with greater intensity than the virosomes without PNGase treatment
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