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Targeted Transfer of Polyethylenimine–Avidin–DNA Bioconjugates to Hematopoietic Cells Using Biotinylated Monoclonal Antibodies
Targeted Transfer of Polyethylenimine–Avidin–DNA Bioconjugates to Hematopoietic Cells Using Biotinylated Monoclonal Antibodies URSZULA WOJDA, JEFFERY L. MILLER Laboratory of Chemical Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892
Received 16 October 1999; revised 10 December 1999; accepted 15 December 1999
Here we examine whether attachment of biotinylated antibodies to proteins on the cell surface increases the transfection efficiency of polyethylenimine–avidin– DNA bioconjugate gene transfer. Preliminary experiments were performed to compare avidin endocytosis into cells incubated with biotinylated antibodies. Antibody biotinylation resulted in the endocytosis of avidin–FITC into nearly 100% of cells compared with no detectable binding or entry into unbiotinylated cells. Gene transfer was accomplished with avidin conjugated to polyethylenimine (PEI) at a molar ratio of 4:1 (PA4). Plasmid DNA encoding the green fluorescent protein (GFP) gene was condensed on the PA4, and transfection efficiencies were measured by flow cytometry as the percentage of cells that fluoresced at levels greater than two standard deviations above the negative control. Gene transfer efficiencies were compared among K562, HEL, and Jurkat leukemia cell lines. Control transfections with DNA alone or untargeted PEI–DNA resulted in ⱕ2% GFP positive cells. Targeting PEI–avidin–DNA to antibody biotinylated cells increased transfection efficiency several fold over untargeted PEI. For each cell type, the increase in transfection efficiency was not significantly different among four biotinylated antibodies tested (antiCD55, antiCD59, antiCD71, and antiCD98). These data suggest biotinylated antibodies may be useful for targeting polyethylenimine–avidin mediated gene transfer. © 2000 Wiley-Liss, Inc. and the American Pharmaceu-
ABSTRACT:
tical Association. J Pharm Sci 89: 674–681, 2000.
INTRODUCTION Bioconjugate vectors have been developed for the transfer of genes to improve the low transfection efficiency afforded by naked DNA. Several nonviral gene delivery vectors offer the advantages of safety, stability, and control of their physical Correspondence to: J. L. Miller (e-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 89, 674–681 (2000) © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association
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properties during synthesis and formulation. However, incremental improvements in the understanding of vector–cell interactions will be required to determine the true potential of these reagents. The development of cationic nonviral vectors over the last decade exemplifies the evolving nature of nonviral vector development.1 Cationic vectors are defined as a class of macromolecules that enhance the delivery of DNA by virtue of their positive charge. The cationic charge of several lipids causes an electrostatic affinity of
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derived liposomal preparations for both DNA and the cell membrane. Polylysine and other cationic peptides have also been developed in order to condense DNA, but their transfection efficiency relies on the release of the transferred DNA from endosomal compartments.2,3 Another polycationic vector, polyethylenimine (PEI), has recently gained favor due to its intrinsic endosomolytic properties. Every third backbone atom of PEI is an amino nitrogen providing exceptionally high pH buffering capacity. In the endosome, PEI acts as an efficient “proton sponge” probably triggering osmotic swelling and disruption of endosomal vesicles which promotes efficient gene transfer demonstrated in vitro and in vivo.4–6 Bioconjugation of polycations with celltargeting molecules has also been shown to increase their gene transfer efficiency.7 Adaptations of the polymeric vectors have been accomplished by conjugation to ligands for target cell receptors. The choice of cellular receptors is usually made on the basis of high expression levels on particular cell types. For instance, asialoglycoprotein receptors are expressed at high levels exclusively on hepatocytes and have been used to target gene delivery to those cells.8 Folate receptors are expressed at generally high levels on neoplastic cells and have been used for decades for the delivery of therapeutic folate analogues including gene transfer bioconjugates.9,10 The same concept of targeting highly expressed receptors has been successfully applied to the incorporation of transferrin into bioconjugates.11,12 In addition to natural ligands mentioned above, monoclonal antibodies directed against highly expressed surface receptors have been incorporated into the design of bioconjugates to improve their transfection efficiency.13 Such antibodies are selected due to their affinity for receptors having naturally high abundance on the targeted cell types.14 In this study, we investigate the use of biotinylated monoclonal antibodies as a means of delivering polyethylenimine–avidin (PA4) bioconjugates to cultured leukemia cells. Four separate biotinylated antibodies targeted to receptors expressed at high levels on hematopoietic cells (antiCD71B, antiCD98B, antiCD55B, and antiCD59B) were compared. We have previously demonstrated the use of direct biotinylation of the cell surface with biotin esters for this purpose.15 While the use of biotinylated antibodies resulted in a lower transfection efficiency than measured after direct membrane biotinylation,
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our results suggest that biotinylated antibody targeting significantly increases the efficiency of PEI-condensed plasmid DNA transfection.
EXPERIMENTAL SECTION Cells, Antibodies, and Plasmid DNA K562 human chronic myelogenous leukemia, the human acute Jurkat T cell leukemia (clone E6-1) and HEL erythroleukemia cell lines were obtained from American Type Culture Collection and cultured in the recommended media supplemented with 10% fetal bovine serum (FBS; Biofluids, Rockville, MD) and with 25 g/ml gentamicin (Life Technologies, Gaithersburg, MD). Biotinylated monoclonal antibodies, antiCD55 (IgG2a), antiCD59 (IgG2a), antiCD71 (IgG2a), and antiCD98 (IgG1), were purchased from Pharmingen (San Diego, CA). IgG1 conjugated with FITC and IgG2a conjugated with Phycoerythrin (PE) were obtained from Coulter Corporation (Hialeah, FL). The plasmid pGT encoding for green fluorescence protein was prepared and purified as described elsewhere.16 Briefly, pGT was subcloned from the 780 base pairs DNA EcoRI/ NotI fragment encoding eGFP from pEGFP-N1 (Clontech, Palo Alto, CA) inserted into the pGreenLantern™-1 vector (Life Technologies, Gaithersburg, MD) digested with EcoRI and NotI. All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated in the text. Labeling Cells with Biotin, Biotinylated Antibodies, and Avidin–FITC Direct biotinylation of all cultured cells was performed as follows: 106 cultured cells were incubated in a final concentration of 0.5 ng sulfo-NHSbiotin per cell (1 mL of PBS final volume; 30 min at 4°C) and washed twice with PBS. For labeling with biotinylated antibodies, 106 cells were mixed with 20 L of the antibody in 100 L of PBS, incubated at 4°C for 30 min and washed twice with PBS. Isotypic control staining was performed with 10 L of IgG1 or IgG2a conjugated with FITC or PE. Cells labeled with biotin or with biotinylated antibodies were incubated with avidin conjugated with fluorescein isothiocyanate (Av–FITC, Pierce, Rockford, IL; 106 cells at a final concentration of 0.1 ng Av–FITC per cell, in 1 mL of PBS, for 30 min at 4°C), and washed twice with PBS. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 5, MAY 2000
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Preparation of Polyethylenimine–Avidin (PEI–Avidin) Conjugate Conjugation of PEI to avidin was performed as described previously.15 Briefly, branched polymer PEI (Fluka, Switzerland) of MW 800 kDa was prepared as a 5% w/v hydrochloride salt solution (800 L of commercial PEI, 7 mL of water, 200 L of 36% hydrochloric acid). As avidin is glycosylated, sodium periodate oxidation was used for the introduction of aldehyde residues onto the carbohydrate moieties. The aldehyde residues were then reacted with amine groups of PEI (Schiff base formation and reductive amination in the coupling buffer). A 218 L amount of 20 mg/ mL sodium periodate solution was added to 20 mg of lyophilized avidin dissolved in 2 mL of PBS (pH 7.4), and the sample was incubated for 60 min at 25°C. The reaction was quenched with glycerol followed by gel filtration on Sephadex G-25 superfine (PD10 Pharmacia) column. PEI was added at the molar ratio 1:4 (60 mg of PEI in 1.2 mL) to the fraction of avidin in PBS, and the sample was mixed vigorously for 1 h at 25°C. One milliliter of coupling buffer (20 mM Na3PO4, pH 7.5, 0.2 M NaCl, and 3 mg/mL NaCNBH3) was added to each sample followed by 1 h of incubation. The addition of the coupling buffer was repeated twice at 1-h intervals with a total of 3 mL of coupling buffer added to each sample prior to overnight incubation. Glycine in molar excess quenched the avidin for 1 h at 25°C. Finally, the conjugate was purified on the Macro-Prep High S cation-exchanger (Bio-Rad, Hercules, CA) with the 0.5–3 M NaCl gradient in 20 mM HEPES, pH 7.5, using a Gilson HPLC system. The main conjugate fraction (eluted between 1.3 and 3.0 M salt) was pooled, concentrated to 6 mL by ultrafiltration and dialyzed overnight against 3 × 1L of PBS, pH 7.4. The avidin content of each conjugate preparation was determined at 280 nm and PEI content by ninhydrin assay (NIN–SOL ninhydrin reagent from Pierce) at 570 nm. The reactions yielded 24.6 mg of PEI conjugated to 7.5 mg of avidin at the molar ratio of 1:3.7 (PA4). Transfection of Cells Transfection complexes of DNA with PEI or PEI– avidin (PA4) were prepared based on the optimization studies described earlier. 15 A 10-g amount of plasmid DNA was added and mixed gently with PEI or PA conjugates in PBS (total volume 0.5 mL). PEI–DNA or PA4–DNA comJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 5, MAY 2000
plexes were formed at molar ratios of PEI nitrogen to DNA phosphate of 6.4:1. After a 30-min incubation at 25°C, 0.5 mL of transfection mixture was added to the cells in 1.5 mL culture medium containing 10% FBS and gently mixed. Cells were biotinylated before transfections. All transfections were performed in 24-well plates (Costar, Cambridge, MA) with 5 × 105 cells per well. After a 4-h incubation with the transfection complexes, 1 mL of fresh culture media containing 15% FBS was added to each well. Transfection was assessed based on GFP expression by fluorescence microscopy and flow cytometry 48 h later. Flow Cytometry and Fluorescence Microscopy Flow cytometry analyses were performed using an EPICS ELITE ESP flow cytometer (Coulter, Hialeah, FL). In each experiment, 10,000 cells were analyzed using argon laser excitation and 525 nm (FITC; GFP) versus 575 nm (phycoerythrin (PE)) bandpass emission filters. Fluorescence microscopy was carried out using an Axiophot microscope with standard filter sets (Zeiss, Germany).
RESULTS Avidin Attached to Biotinylated Antibodies on the Cell Surface Is Efficiently Endocytosed in Nucleated Cells Preliminary experiments were performed to compare the endocytosis of fluorescein-conjugated avidin (Av–FITC) into directly biotinylated cells versus those coated with biotinylated antibodies. Av–FITC endocytosis into directly biotinylated cells was used as a positive control for these studies (Figure 1A,B). Biotinylated K562 cells displayed only surface fluorescence immediately after incubation with Av–FITC (Figure 1A). Over a 24-h period of observation, the fluorescence gradually shifted from the cell surface to the cell interior in nearly 100% of the cells. After 24 h, surface fluorescence was no longer observed, and the fluorescent label appeared as clusters on the interior of the cells (Figure 1B). Once endocytosed, the fluorescein remained inside the cells without evidence of recycling to the cell surface. Biotinylated antiCD59B (antiCD59B) antibodies were used due to their known ability to enter cells after crosslinking to CD59 on the cell surface.17 In a pattern similar to that observed after direct surface biotinylation,
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Figure 1. Fluorescence microscopy of cell immediately after the addition of avidin– FITC (A,C) and following 24 h of incubation at 37°C (B,D). Cells were fixed with 2% paraformaldehyde and observed under 100× objective. (A,B) Directly biotinylated K562 cells; (C,D) K562 cells labeled with biotinylated antiCD59 antibody (antiCD59B). Photographic exposure times varied with longer exposures required for cells labeled with antiCD59B. Each experiment was repeated several times with similar results.
Av–FITC endocytosis was observed following incubation with antiCD59B antibody. Immediately after the addition of Av–FITC (Figure 1C), all cells displayed well-dispersed surface fluorescence with small areas of increased intensity. Within 24 h, notable changes in the pattern of fluorescence were recorded. The fluorescence shifted from well-dispersed clusters and dots of high intensity on the cell surfaces (capping) to a fluorescence within more localized endosomal compartments in the cell interior (Figure 1D). The internalization of Av–FITC was observed in nearly 100% of the cells both after direct biotinylation as well as after antibody biotinylation, as demonstrated by comparing the fluorescence and phase images of the same microscopic field (not shown). It was also noted that cells formed clustered groups directly after avidin labeling, probably as a result of some avidin bridging among neighbor cells (Figure 1A,C). After endocytosis of the surface avidin, the cells no longer showed the tendency to cluster (Figure 1B,D). Neither direct biotinylation or indirect biotinylation with antiCD59B followed by Av–FITC resulted in the internalization of fluorescent complexes when cells were incubated at 4°C over the 24-h period (not shown). Untreated cells, cells incubated with isotypic control antibodies, and cells incubated with Av–FITC alone showed no increases in fluorescence as assessed by flow cytometry or fluorescence microscopy. In addition, other
leukemic cell lines including HEL and Jurkat cells exhibited a nearly identical pattern and time course of Av–FITC endocytosis following incubation with biotinylated antiCD59 antibody. K562 Cells Labeled With Biotin as Well as Biotinylated AntiCD59 Antibody Are Efficiently Transfected by Avidin–PEI (PA4) Conjugates On the basis of the remarkable efficiency of biotin-targeted endocytosis in these cells, we hypothesized that biotinylated antiCD59 antibody may be useful to target gene delivery using the vector constructed by conjugation of avidin with a DNA condensing agent polyethylenimine (PEI–avidin). PEI–avidin conjugates were prepared at the molar ratio of avidin to PEI of 4:1 (PA4).15 The gel retardation assay showed a similar profile of cationic DNA binding for PEI and the PEI–avidin (PA4) conjugate as a function of PEI nitrogen:DNA phosphate (N:P) molar ratios. Accordingly to the pK profile of PEI, approximately one of six nitrogen atoms in PEI is protonated at physiological pH.4 Our gel retardation assay demonstrated that cationic binding of PEI or PEI– avidin completely neutralized the anionic charge of plasmid DNA and prevented electrophoretic mobility at N:P ratios above 3. For the transfection experiments, complexes of DNA with untargeted PEI or PA4 were formed at an N:P ratio of 6.4:1. This ratio provided the most efficient PA4 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 5, MAY 2000
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gate in cells directly labeled with biotin (positive control) resulted in the 14.5 ± 2.2% GFP expressing cells (11-fold increase over untargeted PEI). In the cells biotinylated with antiCD59B antibody, PA4 also provided a significant increase in transfection efficiency (6.8 ± 0.6%; 5.2-fold increase over untargeted PEI).
Figure 2. GFP expression in transfected K562 cells. The percentage of cells expressing GFP 48 h after transfections performed in triplicate are shown. Bars denote untransfected K562 cells (control), cells transfected with PEI (PEI), cells labeled directly with NHS– biotin (Biotin), and cells labeled with biotinylated antibody (antiCD59B).
transfection within the N:P range of 3:1 to 11:1.15 In contrast to PA4, untargeted PEI provided very limited gene transfer, independently of the N:P ratio. Green fluorescent protein (GFP) encoded plasmid DNA was chosen as a specific marker of successful transfection since endosomal escape, trafficking of plasmid DNA to the nuclear compartment, and high-level protein expression of transferred gene are required for the cells to fluoresce at detectable levels. GFP-expressing cells were defined as those cells having a fluorescence at levels at least two standard deviations above the negative control. Gene transfer efficiencies were measured in K562 cells by flow cytometry after 48 h and are shown on Figure 2. Untargeted PEI on unbiotinylated and biotinylated cells resulted in only 1.3 ± 0.5% of cells expressing GFP, while transfection using PEI–avidin (PA4) conju-
Different Biotinylated Antibodies Targeted With PEI–Avidin (PA4) Provide Similar Gene Transfer Efficiency and Expression Pattern in K562 Cells Due to the lower level of antiCD59B-targeted transfection compared to direct surface biotinylation, we next studied the transfection efficiency of PA4-mediated gene transfer in K562 cells after labeling with four separate biotinylated antibodies: antiCD59B, antiCD55B, antiCD71B, and antiCD98B. The four antigens (CD59, CD55, CD71, and CD98) were chosen due to their structural differences despite consistently high-level expression among hematopoietic cells.18 While CD59 and CD55 represent GPI-anchored proteins, CD71 and CD98 have transmembrane domains. In addition, CD71 (the transferrin receptor) has been shown to mediate high-level transfection of PEI into K562 cells.11 Figure 3 demonstrates the transfection efficiency and GFP expression levels among cells incubated with the four individual antibodies. Each dot represents a cell with the corresponding GFP expression intensity shown on the y axis. As shown in Figure 3, the transfection efficiency was similar for all biotinylated antibodies tested, with somewhat higher transfection efficiency provided by antiCD71 antibody directed against transferrin receptor. The distribution of GFP expressing
Figure 3. Flow cytometry analysis of control versus transfected K562 cells expressing GFP after PA4-mediated transfection. The cells were directly biotinylated or incubated with the biotinylated antibodies (antiCD59B, antiCD55B, antiCD71B, and antiCD98B) prior to transfection. GFP expression is shown as the percentage of cells expressing GFP at low, medium and high levels (values are indicated at the right of each box). The range of fluorescence intensity for each level was defined by the log scale with expression defined by fluorescence ⱖ2 SD above the negative control. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 5, MAY 2000
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cells among three logarithmic levels was also similar with the four antibodies. Interestingly, the pattern included a population of very highlevel expressing clones in all cases. PEI–Avidin (PA4) Gene Transfer Efficiency Correlates With the Amount of Biotin Available on the Cell Surface and Is Cell-Type Specific To determine whether the lower transfection efficiency associated with biotinylated antibody targeted cells was due to a lower avidin binding capacity among those cells, we next measured the relative level of biotin on their cell surfaces. Figure 4 shows a comparison of the avidin–FITC mean fluorescence measured on K562 cells before and after labeling with biotin or biotinylated antiCD59 antibody. While biotinylated antiCD59 provides a 9-fold increase in the avidin–FITC fluorescence above the control level (mean fluorescence 11.4 versus control 1.27), a 90-fold increase in avidin–FITC fluorescence was observed in directly biotinylated cells (mean fluorescence 110.2). Therefore, the higher transfection efficiency observed after direct biotinylation of the cell surface correlates with a higher level of biotin on the cell surfaces available for PA4 binding. We also compared PA4 transfection efficiency between three different leukemic cell lines: K562 cells, HEL cells, and Jurkat cells (Figure 5). In all cell types, the transfection efficiencies measured after antibody biotinylation were significantly higher than the untargeted PEI controls. The increase in transfection efficiency measured after targeting with the four antibodies was similar for each cell line. In erythroleukemic K562 and HEL cells, antiCD71 antibody directed against transferrin receptor proved to be slightly more efficient than the other antibodies. Jurkat cells had the lowest transfection efficiency with all four biotinylated antibodies.
DISCUSSION Immense effort has recently been directed toward developing targeted bioconjugates for gene delivery in order to increase transfection efficiency.7 Targeting is achieved by coupling DNA bearing molecules with ligands for cell surface receptors or with antibodies directed to those receptors. This approach has led to cell transfection with folate conjugates,9 transferrin conjugates,11 galactose conjugates,8 and conjugates incorporating integrin-binding motifs.19 Biotin and avidin have been used extensively in the design of gene trans-
Figure 4. Fluorescence of avidin–FITC added to (A) unbiotinylated cells; (B) cells directly biotinylated with sulfo-NHS-biotin; (C) cells indirectly biotinylated with antiCD59 antibody (see Methods for details). Mean fluorescence values (fluorescence units) measured by flow cytometry on 10,000 cells are shown.
fer bioconjugates because of the remarkable affinity of avidin for biotin (Ka ⳱ 1015 M−1), and the availability of these reagents.20 Hematopoietic progenitor cells bearing the c-kit receptor have been successfully transfected with plasmid DNA condensed polylysine–streptavidin after linking to biotinylated steel factor (c-kit receptor ligand).21 Among viral systems, avidin–biotin designs have been used to expand their tropism.14,22 In addition, oligonucleotides as well as pharmaceutical peptides have been delivered to the brain after biotin–avidin coupling with the antibodies directed at CD71.23,24 The use of biotin to modify the target cell surface has more recently been used in vector designs. Direct biotinylation of surface proteins15 or synthetic sugars on the cell surJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 5, MAY 2000
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Figure 5. Comparison of PA4 transfection in K562, HEL, and Jurkat cells. Prior to transfection, the cells were directly biotinylated with NHS-biotin (striped bars) or antibody biotinylated with one out of four biotinylated antibodies: antiCD59B (open bars), antiCD55B (filled bars), antiCD71B (dotted bars), and antiCD98B (gray bars). Flow cytometry was used to determine the percentage of cells expressing GFP 48 hours after transfection. Values are shown as a means ± standard deviation of duplicate experiments. PEI transfection of unbiotinylated cells is included as a control (dashed bars).
face25 has been reported for this purpose. Biotinylation of the cell surface has also been used for red blood cell survival studies26 and immunomodulation.27 Our gene transfer vector design is simple and consists of DNA condensed on avidin-conjugated PEI. Avidin itself behaves as a ligand for targeting rather than a bridge between biotin molecules covering both vector and target cell. Cell surface biotinylation provides the needed receptors for avidin binding. The unique feature of this targeting strategy is the possibility of using the same vector to target disparate cellular types after direct biotinylation versus similar cellular subsets using antibody biotinylation. This study demonstrates the usefulness of biotinylated antibodies for targeting PEI–avidin bioconjugates. The rapid and almost complete internalization of avidin bound to both directly biotinylated cell surfaces as well as those cells covered with biotinylated antibody suggests that mechanisms which control the clearance of ligand molecules from the cell surface may be involved in both targeting approaches.28 Consistent with the rapid endocytosis of avidin–FITC after attachment to biotinylated antibodies, we were also able to demonstrate a significant increase in transfection efficiency and high-level expression of the transfected DNA. Surprisingly, the use of separate biotinylated antibodies resulted in only minor differences in the transfection efficiency of the PA4–DNA complexes. Transmembrane proteins like CD71 and JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 5, MAY 2000
CD98 are endocytosed by a clathrin-dependent mechanism,29,30 while the GPI–proteins (CD55 and CD59) are thought to undergo caveolaeassociated endocytosis.31,32 While transferrin receptor antibodies (CD71) proved to be slightly more efficient in the erythroleukemic cell lines (K562 and HEL cells), the use of CD59-, CD55-, and CD98-targeted antibodies resulted in equivalent transfection efficiency. This result suggests that PEI-mediated endosomal lysis may be effective in both endosomal pathways, or that both pathways have shared features. Direct membrane biotinylation resulted in the highest transfection efficiency of the PA4–DNA complexes in all the cell lines. This high level is consistent with the high amount of biotin able to bind avidin on the cell surface after direct biotinylation. A similar correlation of receptor numbers and transfection efficiency has been demonstrated elsewhere.11 We conclude that biotinylated antibodies are useful for increasing the transfection efficiency of PEI bioconjugates in leukemic hematopoietic cells, but this increase is less than that achieved after the covalent addition of biotin directly to the cell surface.
ACKNOWLEDGMENTS We thank J. Muthoni Njoroge for assistance with the flow cytometry studies and Alexander Gubin for the subcloning and preparation of plasmid DNA used in these studies.
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Received 16 October 1999; revised 10 December 1999; accepted 15 December 1999
Here we examine whether attachment of biotinylated antibodies to proteins on the cell surface increases the transfection efficiency of polyethylenimine–avidin– DNA bioconjugate gene transfer. Preliminary experiments were performed to compare avidin endocytosis into cells incubated with biotinylated antibodies. Antibody biotinylation resulted in the endocytosis of avidin–FITC into nearly 100% of cells compared with no detectable binding or entry into unbiotinylated cells. Gene transfer was accomplished with avidin conjugated to polyethylenimine (PEI) at a molar ratio of 4:1 (PA4). Plasmid DNA encoding the green fluorescent protein (GFP) gene was condensed on the PA4, and transfection efficiencies were measured by flow cytometry as the percentage of cells that fluoresced at levels greater than two standard deviations above the negative control. Gene transfer efficiencies were compared among K562, HEL, and Jurkat leukemia cell lines. Control transfections with DNA alone or untargeted PEI–DNA resulted in ⱕ2% GFP positive cells. Targeting PEI–avidin–DNA to antibody biotinylated cells increased transfection efficiency several fold over untargeted PEI. For each cell type, the increase in transfection efficiency was not significantly different among four biotinylated antibodies tested (antiCD55, antiCD59, antiCD71, and antiCD98). These data suggest biotinylated antibodies may be useful for targeting polyethylenimine–avidin mediated gene transfer. © 2000 Wiley-Liss, Inc. and the American Pharmaceu-
ABSTRACT:
tical Association. J Pharm Sci 89: 674–681, 2000.
INTRODUCTION Bioconjugate vectors have been developed for the transfer of genes to improve the low transfection efficiency afforded by naked DNA. Several nonviral gene delivery vectors offer the advantages of safety, stability, and control of their physical Correspondence to: J. L. Miller (e-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 89, 674–681 (2000) © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association
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properties during synthesis and formulation. However, incremental improvements in the understanding of vector–cell interactions will be required to determine the true potential of these reagents. The development of cationic nonviral vectors over the last decade exemplifies the evolving nature of nonviral vector development.1 Cationic vectors are defined as a class of macromolecules that enhance the delivery of DNA by virtue of their positive charge. The cationic charge of several lipids causes an electrostatic affinity of
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derived liposomal preparations for both DNA and the cell membrane. Polylysine and other cationic peptides have also been developed in order to condense DNA, but their transfection efficiency relies on the release of the transferred DNA from endosomal compartments.2,3 Another polycationic vector, polyethylenimine (PEI), has recently gained favor due to its intrinsic endosomolytic properties. Every third backbone atom of PEI is an amino nitrogen providing exceptionally high pH buffering capacity. In the endosome, PEI acts as an efficient “proton sponge” probably triggering osmotic swelling and disruption of endosomal vesicles which promotes efficient gene transfer demonstrated in vitro and in vivo.4–6 Bioconjugation of polycations with celltargeting molecules has also been shown to increase their gene transfer efficiency.7 Adaptations of the polymeric vectors have been accomplished by conjugation to ligands for target cell receptors. The choice of cellular receptors is usually made on the basis of high expression levels on particular cell types. For instance, asialoglycoprotein receptors are expressed at high levels exclusively on hepatocytes and have been used to target gene delivery to those cells.8 Folate receptors are expressed at generally high levels on neoplastic cells and have been used for decades for the delivery of therapeutic folate analogues including gene transfer bioconjugates.9,10 The same concept of targeting highly expressed receptors has been successfully applied to the incorporation of transferrin into bioconjugates.11,12 In addition to natural ligands mentioned above, monoclonal antibodies directed against highly expressed surface receptors have been incorporated into the design of bioconjugates to improve their transfection efficiency.13 Such antibodies are selected due to their affinity for receptors having naturally high abundance on the targeted cell types.14 In this study, we investigate the use of biotinylated monoclonal antibodies as a means of delivering polyethylenimine–avidin (PA4) bioconjugates to cultured leukemia cells. Four separate biotinylated antibodies targeted to receptors expressed at high levels on hematopoietic cells (antiCD71B, antiCD98B, antiCD55B, and antiCD59B) were compared. We have previously demonstrated the use of direct biotinylation of the cell surface with biotin esters for this purpose.15 While the use of biotinylated antibodies resulted in a lower transfection efficiency than measured after direct membrane biotinylation,
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our results suggest that biotinylated antibody targeting significantly increases the efficiency of PEI-condensed plasmid DNA transfection.
EXPERIMENTAL SECTION Cells, Antibodies, and Plasmid DNA K562 human chronic myelogenous leukemia, the human acute Jurkat T cell leukemia (clone E6-1) and HEL erythroleukemia cell lines were obtained from American Type Culture Collection and cultured in the recommended media supplemented with 10% fetal bovine serum (FBS; Biofluids, Rockville, MD) and with 25 g/ml gentamicin (Life Technologies, Gaithersburg, MD). Biotinylated monoclonal antibodies, antiCD55 (IgG2a), antiCD59 (IgG2a), antiCD71 (IgG2a), and antiCD98 (IgG1), were purchased from Pharmingen (San Diego, CA). IgG1 conjugated with FITC and IgG2a conjugated with Phycoerythrin (PE) were obtained from Coulter Corporation (Hialeah, FL). The plasmid pGT encoding for green fluorescence protein was prepared and purified as described elsewhere.16 Briefly, pGT was subcloned from the 780 base pairs DNA EcoRI/ NotI fragment encoding eGFP from pEGFP-N1 (Clontech, Palo Alto, CA) inserted into the pGreenLantern™-1 vector (Life Technologies, Gaithersburg, MD) digested with EcoRI and NotI. All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated in the text. Labeling Cells with Biotin, Biotinylated Antibodies, and Avidin–FITC Direct biotinylation of all cultured cells was performed as follows: 106 cultured cells were incubated in a final concentration of 0.5 ng sulfo-NHSbiotin per cell (1 mL of PBS final volume; 30 min at 4°C) and washed twice with PBS. For labeling with biotinylated antibodies, 106 cells were mixed with 20 L of the antibody in 100 L of PBS, incubated at 4°C for 30 min and washed twice with PBS. Isotypic control staining was performed with 10 L of IgG1 or IgG2a conjugated with FITC or PE. Cells labeled with biotin or with biotinylated antibodies were incubated with avidin conjugated with fluorescein isothiocyanate (Av–FITC, Pierce, Rockford, IL; 106 cells at a final concentration of 0.1 ng Av–FITC per cell, in 1 mL of PBS, for 30 min at 4°C), and washed twice with PBS. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 5, MAY 2000
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Preparation of Polyethylenimine–Avidin (PEI–Avidin) Conjugate Conjugation of PEI to avidin was performed as described previously.15 Briefly, branched polymer PEI (Fluka, Switzerland) of MW 800 kDa was prepared as a 5% w/v hydrochloride salt solution (800 L of commercial PEI, 7 mL of water, 200 L of 36% hydrochloric acid). As avidin is glycosylated, sodium periodate oxidation was used for the introduction of aldehyde residues onto the carbohydrate moieties. The aldehyde residues were then reacted with amine groups of PEI (Schiff base formation and reductive amination in the coupling buffer). A 218 L amount of 20 mg/ mL sodium periodate solution was added to 20 mg of lyophilized avidin dissolved in 2 mL of PBS (pH 7.4), and the sample was incubated for 60 min at 25°C. The reaction was quenched with glycerol followed by gel filtration on Sephadex G-25 superfine (PD10 Pharmacia) column. PEI was added at the molar ratio 1:4 (60 mg of PEI in 1.2 mL) to the fraction of avidin in PBS, and the sample was mixed vigorously for 1 h at 25°C. One milliliter of coupling buffer (20 mM Na3PO4, pH 7.5, 0.2 M NaCl, and 3 mg/mL NaCNBH3) was added to each sample followed by 1 h of incubation. The addition of the coupling buffer was repeated twice at 1-h intervals with a total of 3 mL of coupling buffer added to each sample prior to overnight incubation. Glycine in molar excess quenched the avidin for 1 h at 25°C. Finally, the conjugate was purified on the Macro-Prep High S cation-exchanger (Bio-Rad, Hercules, CA) with the 0.5–3 M NaCl gradient in 20 mM HEPES, pH 7.5, using a Gilson HPLC system. The main conjugate fraction (eluted between 1.3 and 3.0 M salt) was pooled, concentrated to 6 mL by ultrafiltration and dialyzed overnight against 3 × 1L of PBS, pH 7.4. The avidin content of each conjugate preparation was determined at 280 nm and PEI content by ninhydrin assay (NIN–SOL ninhydrin reagent from Pierce) at 570 nm. The reactions yielded 24.6 mg of PEI conjugated to 7.5 mg of avidin at the molar ratio of 1:3.7 (PA4). Transfection of Cells Transfection complexes of DNA with PEI or PEI– avidin (PA4) were prepared based on the optimization studies described earlier. 15 A 10-g amount of plasmid DNA was added and mixed gently with PEI or PA conjugates in PBS (total volume 0.5 mL). PEI–DNA or PA4–DNA comJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 5, MAY 2000
plexes were formed at molar ratios of PEI nitrogen to DNA phosphate of 6.4:1. After a 30-min incubation at 25°C, 0.5 mL of transfection mixture was added to the cells in 1.5 mL culture medium containing 10% FBS and gently mixed. Cells were biotinylated before transfections. All transfections were performed in 24-well plates (Costar, Cambridge, MA) with 5 × 105 cells per well. After a 4-h incubation with the transfection complexes, 1 mL of fresh culture media containing 15% FBS was added to each well. Transfection was assessed based on GFP expression by fluorescence microscopy and flow cytometry 48 h later. Flow Cytometry and Fluorescence Microscopy Flow cytometry analyses were performed using an EPICS ELITE ESP flow cytometer (Coulter, Hialeah, FL). In each experiment, 10,000 cells were analyzed using argon laser excitation and 525 nm (FITC; GFP) versus 575 nm (phycoerythrin (PE)) bandpass emission filters. Fluorescence microscopy was carried out using an Axiophot microscope with standard filter sets (Zeiss, Germany).
RESULTS Avidin Attached to Biotinylated Antibodies on the Cell Surface Is Efficiently Endocytosed in Nucleated Cells Preliminary experiments were performed to compare the endocytosis of fluorescein-conjugated avidin (Av–FITC) into directly biotinylated cells versus those coated with biotinylated antibodies. Av–FITC endocytosis into directly biotinylated cells was used as a positive control for these studies (Figure 1A,B). Biotinylated K562 cells displayed only surface fluorescence immediately after incubation with Av–FITC (Figure 1A). Over a 24-h period of observation, the fluorescence gradually shifted from the cell surface to the cell interior in nearly 100% of the cells. After 24 h, surface fluorescence was no longer observed, and the fluorescent label appeared as clusters on the interior of the cells (Figure 1B). Once endocytosed, the fluorescein remained inside the cells without evidence of recycling to the cell surface. Biotinylated antiCD59B (antiCD59B) antibodies were used due to their known ability to enter cells after crosslinking to CD59 on the cell surface.17 In a pattern similar to that observed after direct surface biotinylation,
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Figure 1. Fluorescence microscopy of cell immediately after the addition of avidin– FITC (A,C) and following 24 h of incubation at 37°C (B,D). Cells were fixed with 2% paraformaldehyde and observed under 100× objective. (A,B) Directly biotinylated K562 cells; (C,D) K562 cells labeled with biotinylated antiCD59 antibody (antiCD59B). Photographic exposure times varied with longer exposures required for cells labeled with antiCD59B. Each experiment was repeated several times with similar results.
Av–FITC endocytosis was observed following incubation with antiCD59B antibody. Immediately after the addition of Av–FITC (Figure 1C), all cells displayed well-dispersed surface fluorescence with small areas of increased intensity. Within 24 h, notable changes in the pattern of fluorescence were recorded. The fluorescence shifted from well-dispersed clusters and dots of high intensity on the cell surfaces (capping) to a fluorescence within more localized endosomal compartments in the cell interior (Figure 1D). The internalization of Av–FITC was observed in nearly 100% of the cells both after direct biotinylation as well as after antibody biotinylation, as demonstrated by comparing the fluorescence and phase images of the same microscopic field (not shown). It was also noted that cells formed clustered groups directly after avidin labeling, probably as a result of some avidin bridging among neighbor cells (Figure 1A,C). After endocytosis of the surface avidin, the cells no longer showed the tendency to cluster (Figure 1B,D). Neither direct biotinylation or indirect biotinylation with antiCD59B followed by Av–FITC resulted in the internalization of fluorescent complexes when cells were incubated at 4°C over the 24-h period (not shown). Untreated cells, cells incubated with isotypic control antibodies, and cells incubated with Av–FITC alone showed no increases in fluorescence as assessed by flow cytometry or fluorescence microscopy. In addition, other
leukemic cell lines including HEL and Jurkat cells exhibited a nearly identical pattern and time course of Av–FITC endocytosis following incubation with biotinylated antiCD59 antibody. K562 Cells Labeled With Biotin as Well as Biotinylated AntiCD59 Antibody Are Efficiently Transfected by Avidin–PEI (PA4) Conjugates On the basis of the remarkable efficiency of biotin-targeted endocytosis in these cells, we hypothesized that biotinylated antiCD59 antibody may be useful to target gene delivery using the vector constructed by conjugation of avidin with a DNA condensing agent polyethylenimine (PEI–avidin). PEI–avidin conjugates were prepared at the molar ratio of avidin to PEI of 4:1 (PA4).15 The gel retardation assay showed a similar profile of cationic DNA binding for PEI and the PEI–avidin (PA4) conjugate as a function of PEI nitrogen:DNA phosphate (N:P) molar ratios. Accordingly to the pK profile of PEI, approximately one of six nitrogen atoms in PEI is protonated at physiological pH.4 Our gel retardation assay demonstrated that cationic binding of PEI or PEI– avidin completely neutralized the anionic charge of plasmid DNA and prevented electrophoretic mobility at N:P ratios above 3. For the transfection experiments, complexes of DNA with untargeted PEI or PA4 were formed at an N:P ratio of 6.4:1. This ratio provided the most efficient PA4 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 5, MAY 2000
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gate in cells directly labeled with biotin (positive control) resulted in the 14.5 ± 2.2% GFP expressing cells (11-fold increase over untargeted PEI). In the cells biotinylated with antiCD59B antibody, PA4 also provided a significant increase in transfection efficiency (6.8 ± 0.6%; 5.2-fold increase over untargeted PEI).
Figure 2. GFP expression in transfected K562 cells. The percentage of cells expressing GFP 48 h after transfections performed in triplicate are shown. Bars denote untransfected K562 cells (control), cells transfected with PEI (PEI), cells labeled directly with NHS– biotin (Biotin), and cells labeled with biotinylated antibody (antiCD59B).
transfection within the N:P range of 3:1 to 11:1.15 In contrast to PA4, untargeted PEI provided very limited gene transfer, independently of the N:P ratio. Green fluorescent protein (GFP) encoded plasmid DNA was chosen as a specific marker of successful transfection since endosomal escape, trafficking of plasmid DNA to the nuclear compartment, and high-level protein expression of transferred gene are required for the cells to fluoresce at detectable levels. GFP-expressing cells were defined as those cells having a fluorescence at levels at least two standard deviations above the negative control. Gene transfer efficiencies were measured in K562 cells by flow cytometry after 48 h and are shown on Figure 2. Untargeted PEI on unbiotinylated and biotinylated cells resulted in only 1.3 ± 0.5% of cells expressing GFP, while transfection using PEI–avidin (PA4) conju-
Different Biotinylated Antibodies Targeted With PEI–Avidin (PA4) Provide Similar Gene Transfer Efficiency and Expression Pattern in K562 Cells Due to the lower level of antiCD59B-targeted transfection compared to direct surface biotinylation, we next studied the transfection efficiency of PA4-mediated gene transfer in K562 cells after labeling with four separate biotinylated antibodies: antiCD59B, antiCD55B, antiCD71B, and antiCD98B. The four antigens (CD59, CD55, CD71, and CD98) were chosen due to their structural differences despite consistently high-level expression among hematopoietic cells.18 While CD59 and CD55 represent GPI-anchored proteins, CD71 and CD98 have transmembrane domains. In addition, CD71 (the transferrin receptor) has been shown to mediate high-level transfection of PEI into K562 cells.11 Figure 3 demonstrates the transfection efficiency and GFP expression levels among cells incubated with the four individual antibodies. Each dot represents a cell with the corresponding GFP expression intensity shown on the y axis. As shown in Figure 3, the transfection efficiency was similar for all biotinylated antibodies tested, with somewhat higher transfection efficiency provided by antiCD71 antibody directed against transferrin receptor. The distribution of GFP expressing
Figure 3. Flow cytometry analysis of control versus transfected K562 cells expressing GFP after PA4-mediated transfection. The cells were directly biotinylated or incubated with the biotinylated antibodies (antiCD59B, antiCD55B, antiCD71B, and antiCD98B) prior to transfection. GFP expression is shown as the percentage of cells expressing GFP at low, medium and high levels (values are indicated at the right of each box). The range of fluorescence intensity for each level was defined by the log scale with expression defined by fluorescence ⱖ2 SD above the negative control. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 5, MAY 2000
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cells among three logarithmic levels was also similar with the four antibodies. Interestingly, the pattern included a population of very highlevel expressing clones in all cases. PEI–Avidin (PA4) Gene Transfer Efficiency Correlates With the Amount of Biotin Available on the Cell Surface and Is Cell-Type Specific To determine whether the lower transfection efficiency associated with biotinylated antibody targeted cells was due to a lower avidin binding capacity among those cells, we next measured the relative level of biotin on their cell surfaces. Figure 4 shows a comparison of the avidin–FITC mean fluorescence measured on K562 cells before and after labeling with biotin or biotinylated antiCD59 antibody. While biotinylated antiCD59 provides a 9-fold increase in the avidin–FITC fluorescence above the control level (mean fluorescence 11.4 versus control 1.27), a 90-fold increase in avidin–FITC fluorescence was observed in directly biotinylated cells (mean fluorescence 110.2). Therefore, the higher transfection efficiency observed after direct biotinylation of the cell surface correlates with a higher level of biotin on the cell surfaces available for PA4 binding. We also compared PA4 transfection efficiency between three different leukemic cell lines: K562 cells, HEL cells, and Jurkat cells (Figure 5). In all cell types, the transfection efficiencies measured after antibody biotinylation were significantly higher than the untargeted PEI controls. The increase in transfection efficiency measured after targeting with the four antibodies was similar for each cell line. In erythroleukemic K562 and HEL cells, antiCD71 antibody directed against transferrin receptor proved to be slightly more efficient than the other antibodies. Jurkat cells had the lowest transfection efficiency with all four biotinylated antibodies.
DISCUSSION Immense effort has recently been directed toward developing targeted bioconjugates for gene delivery in order to increase transfection efficiency.7 Targeting is achieved by coupling DNA bearing molecules with ligands for cell surface receptors or with antibodies directed to those receptors. This approach has led to cell transfection with folate conjugates,9 transferrin conjugates,11 galactose conjugates,8 and conjugates incorporating integrin-binding motifs.19 Biotin and avidin have been used extensively in the design of gene trans-
Figure 4. Fluorescence of avidin–FITC added to (A) unbiotinylated cells; (B) cells directly biotinylated with sulfo-NHS-biotin; (C) cells indirectly biotinylated with antiCD59 antibody (see Methods for details). Mean fluorescence values (fluorescence units) measured by flow cytometry on 10,000 cells are shown.
fer bioconjugates because of the remarkable affinity of avidin for biotin (Ka ⳱ 1015 M−1), and the availability of these reagents.20 Hematopoietic progenitor cells bearing the c-kit receptor have been successfully transfected with plasmid DNA condensed polylysine–streptavidin after linking to biotinylated steel factor (c-kit receptor ligand).21 Among viral systems, avidin–biotin designs have been used to expand their tropism.14,22 In addition, oligonucleotides as well as pharmaceutical peptides have been delivered to the brain after biotin–avidin coupling with the antibodies directed at CD71.23,24 The use of biotin to modify the target cell surface has more recently been used in vector designs. Direct biotinylation of surface proteins15 or synthetic sugars on the cell surJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 5, MAY 2000
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Figure 5. Comparison of PA4 transfection in K562, HEL, and Jurkat cells. Prior to transfection, the cells were directly biotinylated with NHS-biotin (striped bars) or antibody biotinylated with one out of four biotinylated antibodies: antiCD59B (open bars), antiCD55B (filled bars), antiCD71B (dotted bars), and antiCD98B (gray bars). Flow cytometry was used to determine the percentage of cells expressing GFP 48 hours after transfection. Values are shown as a means ± standard deviation of duplicate experiments. PEI transfection of unbiotinylated cells is included as a control (dashed bars).
face25 has been reported for this purpose. Biotinylation of the cell surface has also been used for red blood cell survival studies26 and immunomodulation.27 Our gene transfer vector design is simple and consists of DNA condensed on avidin-conjugated PEI. Avidin itself behaves as a ligand for targeting rather than a bridge between biotin molecules covering both vector and target cell. Cell surface biotinylation provides the needed receptors for avidin binding. The unique feature of this targeting strategy is the possibility of using the same vector to target disparate cellular types after direct biotinylation versus similar cellular subsets using antibody biotinylation. This study demonstrates the usefulness of biotinylated antibodies for targeting PEI–avidin bioconjugates. The rapid and almost complete internalization of avidin bound to both directly biotinylated cell surfaces as well as those cells covered with biotinylated antibody suggests that mechanisms which control the clearance of ligand molecules from the cell surface may be involved in both targeting approaches.28 Consistent with the rapid endocytosis of avidin–FITC after attachment to biotinylated antibodies, we were also able to demonstrate a significant increase in transfection efficiency and high-level expression of the transfected DNA. Surprisingly, the use of separate biotinylated antibodies resulted in only minor differences in the transfection efficiency of the PA4–DNA complexes. Transmembrane proteins like CD71 and JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 5, MAY 2000
CD98 are endocytosed by a clathrin-dependent mechanism,29,30 while the GPI–proteins (CD55 and CD59) are thought to undergo caveolaeassociated endocytosis.31,32 While transferrin receptor antibodies (CD71) proved to be slightly more efficient in the erythroleukemic cell lines (K562 and HEL cells), the use of CD59-, CD55-, and CD98-targeted antibodies resulted in equivalent transfection efficiency. This result suggests that PEI-mediated endosomal lysis may be effective in both endosomal pathways, or that both pathways have shared features. Direct membrane biotinylation resulted in the highest transfection efficiency of the PA4–DNA complexes in all the cell lines. This high level is consistent with the high amount of biotin able to bind avidin on the cell surface after direct biotinylation. A similar correlation of receptor numbers and transfection efficiency has been demonstrated elsewhere.11 We conclude that biotinylated antibodies are useful for increasing the transfection efficiency of PEI bioconjugates in leukemic hematopoietic cells, but this increase is less than that achieved after the covalent addition of biotin directly to the cell surface.
ACKNOWLEDGMENTS We thank J. Muthoni Njoroge for assistance with the flow cytometry studies and Alexander Gubin for the subcloning and preparation of plasmid DNA used in these studies.
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