Surface Membrane Biotinylation Efficiently Mediates the Endocytosis

Surface Membrane Biotinylation Efficiently Mediates the. Endocytosis of Avidin Bioconjugates into Nucleated Cells. Urszula Wojda, Paul Goldsmith,† a...
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Bioconjugate Chem. 1999, 10, 1044−1050

Surface Membrane Biotinylation Efficiently Mediates the Endocytosis of Avidin Bioconjugates into Nucleated Cells Urszula Wojda, Paul Goldsmith,† and Jeffery L. Miller* Laboratory of Chemical Biology, Building 10, Room 9N308, National Institute of Diabetes and Digestive and Kidney Diseases and Metabolic Disease Branch, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892. Received May 21, 1999; Revised Manuscript Received August 3, 1999

Here we demonstrate that biotin covalently attached to cell surface obligates existing receptors to endocytose avidin bioconjugates into nucleated cells. Incubation of fluorescein-labeled avidin with biotinylated cell lines resulted in uniform and rapid surface attachment and endocytosis compared with no detectable association of the avidin-conjugated dye with unbiotinylated cells. Uptake was detected within minutes with efficiencies approaching 100% in cell lines and freshly obtained peripheral blood mononuclear cells. After 24 h, avidin was barely detectable on the surface of the nucleated cells. In marked contrast, fluorescent avidin remained exclusively on the external membrane of erythrocytes after 24 h. To investigate biotin-mediated endocytosis for the delivery of DNA, we prepared polyethylenimine-avidin (PEI-avidin) conjugates. Surface biotinylation significantly increased the transfection efficiencies of PEI-avidin condensed plasmid DNA coding green fluorescent protein (GFP) to the level of transferrin-receptor targeted gene delivery (15-20% GFP positive cells in culture after 48 h). The increase in transfection efficiency was blocked by the addition of free avidin or biotin to the culture medium. Biotin covalently bound to cell surface membrane proteins efficiently mediates the entry of avidin bioconjugates into nucleated cells.

INTRODUCTION

Endocytosis describes the processes whereby eukaryotic cells internalize a wide range of extracellular materials including nutrients and toxins (1). Receptor-mediated endocytosis involves the attachment of extracellular molecules (ligands) to specified molecules (receptors) present on cell membranes and transport of those receptor-ligand complexes to the cell interior. The ligands are either then recycled to the cell surface, degraded within lysosomal compartments, or escape degradation and enter the cytoplasm. The endocytic process is responsible for a vast array of beneficial processes ranging from surface ligand clearance to nutrient uptake. In addition, some toxins and viruses require receptor-mediated endocytosis in order to access to the interior of cells (2, 3). Several factors determine the specificity and efficiency with which ligands present within the extracellular space enter cells after binding to cell surface receptors. The absolute number of receptors expressed on the cell surface largely determines the quantity of specific ligands permitted to enter individual cells. For instance, iron uptake is regulated by the number of transferrin receptors available on the cell surface (4). When cells proliferate, more transferrin receptors are expressed on the cell surface to enhance iron uptake. In the case of proliferating erythroid cells, exceedingly high levels of transferrin receptors are present due to the requirement of iron for hemoglobin synthesis, but the number of receptors decreases as iron requirements decrease and the cells mature (4). The affinity with which ligands bind to their receptors may also regulate entry into cells. Although * To whom correspondence should be addressed, Laboratory of Chemical Biology. Phone: (301) 402-2373. Fax: (301) 4020101. E-mail: [email protected]. † Metabolic Disease Branch.

receptors for interleukin-2 (IL-2R) exist in three forms: low, intermediate, and high affinity, only the high affinity form permits selective binding and uptake of the IL-2R targeted bioconjugates into cells (5). The entry of folic acid into cells is regulated both by the expression level of folate-binding receptors and the affinity of folate for those receptors (6). It is well-known that conjugation of a receptor-specific ligand with molecules normally restricted to the extracellular space leads to their endocytosis into cells expressing the corresponding ligand-specific receptor. Examples include folate and transferrin conjugated to fluorescent dyes (7, 8). Similarly, binding of specific monoclonal antibodies with affinities for individual cell surface receptors may result in their endocytosis (9). However, it remains unclear whether the modification of disparate receptors on the cell membrane by covalently bound molecules [defined here as receptor-adaptor(s)] is sufficient to mediate the endocytosis of adaptor-specific ligands. To test this hypothesis, biotin was covalently attached to proteins on the cell membranes to increase specifically the affinity of those biotin-modified proteins for avidin-containing molecules. Biotin was an ideal adaptor due to its small size, low toxicity in vivo (10, 11) and the well recognized affinity of biotin for avidin (12). Remarkably, surface biotinylation resulted in fluoresceintagged avidin entry into nearly 100% of cultured and primary cells tested compared with no detectable uptake by unbiotinylated cells. No evidence of exocytosis, surface recycling of the fluorescent probe, or growth inhibition was noted. A comparison of biotinylated-receptor and transferrin-receptor targeted polyethylenimine-DNA bioconjugates revealed equivalent transfection efficiencies. These data suggest that molecules covalently attached to proteins expressed on the cell membrane may be

10.1021/bc990059z Not subject to U.S. Copyright. Published 1999 by American Chemical Society Published on Web 09/21/1999

Biotin-Mediated Entry of Avidin Bioconjugates

generally useful for mediating the endocytosis of ligands normally confined to the extracellular space. EXPERIMENTAL PROCEDURES

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 µg/mL gentamicin (Life Technologies, Gaithesburg, MD). Primary hematopoietic cells were obtained from donated blood. Mononucleated cells and enucleated erythrocytes were isolated using Lymphocyte Separation Media (ICN Biomedicals, Aurora, OH) according to the manufacturer’s protocol. Primary cells expressing transferrin receptor at high levels were obtained by flow cytometry (13). Staining of 105 K562 cells with mouse anti-avidin-FITC monoclonal antibody (clone WC19.10, IgG1, Sigma, St. Louis, MO) and control IgG1-FITC (Immunotech, Westbrook, ME) was performed by incubation with 4 µL of antibody in 100 µL of PBS for 30 min at 4 °C. The plasmid pGT encoding for green fluorescence protein was prepared and purified as described elsewhere (14). 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. Biotinylation and Avidin-FITC Addition. Biotinylation of all cells was performed as follows: 106 cells were incubated in a final concentration of 0.5 ng of sulfoNHS-biotin/cell (Pierce, Rockford, IL) in PBS for 30 min at 4 °C and washed twice with PBS. Avidin or avidin conjugated with fluorescein isothiocyanate (Av-FITC, 0.1 ng/cell) was incubated with the cells in 1 mL of PBS. In the time-course studies, cells labeled with Av-FITC were incubated at 37 °C, and at each time-point, cells were collected, fixed with 2% p-formaldehyde, and analyzed by flow cytometry and fluorescence microscopy. Preparation of PEI-Avidin (PA) Conjugates. Polyethylenimine (PEI; Fluka, Switzerland) of MW 800 kDa was prepared as a 5% w/v hydrochloride salt solution (800 µL of commercial PEI, 7 mL of water, 200 µL of 36% hydrochloric acid). The pH of prepared PEI was 9.2, and the reagent was stored at 25 °C until used in conjugate preparation. Each avidin sample was prepared by dissolving 20 mg of lyophilized avidin in 2 mL of PBS, pH 7.4. To each sample of dissolved avidin was added 218 µL of 20 mg/mL sodium periodate solution, and the sample was wrapped with foil and incubated for 60 min at 25 °C. Since avidin is glycosylated, sodium periodate oxidation allows the introduction of aldehyde residues onto carbohydrates. Next the aldehyde residues can react with amine groups of PEI (Schiff base formation and reductive amination in the coupling buffer). Sodium periodate was dissolved in water (20 mg/mL) and stored at 25 °C protected from light. The reaction was quenched with glycerol followed by gel filtration on Sephadex G-25 superfine (PD10 Pharmacia) column. The resulting 3.5 mL fractions containing protein in PBS, pH 7.4, were used for conjugation with PEI. PEI was added to the avidin fractions at four different molar ratios: (A) 1: 2 (120 mg of PEI in 2.4 mL), (B) 1: 4 (60 mg of PEI in 1.2 mL), (C) 1: 8 (30 mg of PEI in 0.6 mL), and (D) 1: 16 (15

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mg of PEI in 0.3 mL). The samples were mixed vigorously for 1 h at 25 °C, and 1 mL 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 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. A 10 mm × 100 mm Macro-Prep High S support (Bio-Rad, Hercules, CA) cation-exchange column equilibrated at 20 mM Hepes, pH 7.5, containing 0.5 M NaCl was used to fractionate the samples with the 0.5-3 M NaCl gradient in 20 mM Hepes, pH 7.5, using Gilson HPLC system equipped with protein detection system at 280 and 214 nm. Some protein was eluted in the flow through. The main conjugate fraction of each sample was eluted between 1.3 and 3.0 M salt, pooled, concentrated to 6 mL by ultrafiltration, and dialyzed overnight against 3 × 1 L 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 conjugate reactions yielded the following products: (A) 31.2 mg of PEI conjugated to 5.2 mg avidin at the molar ratio of 1:2.0, (B) 12 mg of PEI conjugated to 4.36 mg avidin at the molar ratio of 1:4.36 (PA4), (C) 3.32 mg of PEI conjugated to 2.30 mg of avidin at the molar ratio of 1:8.31 (PA8), and (D) 1.05 mg of PEI conjugated to 1.35 mg of avidin at a molar ratio of 1:15.46 (PA16). The overall yield of these conjugates based on PEI was (A) 26.0%, (B) 20.0%, (C) 11.7%, and (D) 7.0%; based on avidin, the yield was (A) 26.0%, (B) 21.8%, (C) 11.5%, and (D) 6.8%. The conjugates were aliquoted and stored at -80 °C. Gel retardation assay of the PEI conjugates was performed in 1.2% agarose gels as previously described (15). Transfection of Cells. Transfection complexes of DNA with PEI or PEI-avidin (PA) conjugates were prepared as follows. Plasmid DNA was added and mixed gently with PEI or PA conjugates in PBS, in a total volume of 0.5 mL. PEI-DNA or PA-DNA complexes were formed at various molar ratios of PEI nitrogen to DNA phosphate (N:P) as indicated further in the text. Different N:P ratios were prepared by titrating DNA concentrations at a constant PEI concentration or by titrating PEI with the amount of DNA held constant. After 30 min of incubation in 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 transfection using standard procedure at 4 °C and washed with cold PBS. All transfections were performed in 24 well plates (Costar, Cambridge, MA) with 5 × 105 cells/well. After 4 h of 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. Transfection using Transferrin-PEI (TfPEI Kit, Bender MedSystems, Vienna, Austria) and with DMRIE-C Reagent (GIBCO BRL, Life Technologies, Grand Island, NY) was done according to manufacturer’s instructions. 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 versus 575 nm band-pass emission filters for fluorescein (FITC), green fluorescent protein (GFP), and phycoerythrin (PE) detection. Fluo-

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Figure 1. Addition of avidin-FITC to biotinylated K562 cells. Matching fluorescence (left panels) and phase (right panels) microscopy images of biotinylated cells incubated at 37 °C after addition of fluorescent avidin are shown (100× objective). Untreated biotinylated cells (A), and cells treated with avidinFITC and observed immediately (B), after 30 min (C), and after 24 h (D) were compared. Each experiment was repeated several times with similar results.

rescence microscopy of cells was carried out using an Axiophot microscope and standard filter sets (Zeiss, Germany). RESULTS

Entry of Fluorescein-Labeled Avidin into Cultured Cell Lines and Primary Hematopoietic Cells. Fluorescein-labeled avidin (Av-FITC) was used to examine kinetics of binding and possible internalization of avidin into biotinylated K562 leukemia cells. Untreated K562 cells showed very little autofluorescence or nonspecific binding of Av-FITC as assessed by flow cytometry and fluorescence microscopy (Figure 1A). When Av-FITC was added to biotinylated K562 cells, 100% of cells became fluorescently labeled. Av-FITC was well dispersed on the cell surfaces with several areas of increased fluorescence (Figure 1B). After 30 min, fluorescein was detected inside endosomal compartments in all the cells (Figure 1C). The fluorescence gradually shifted from the cell surface to the cell interior within the next 24 h as demonstrated by comparing the fluorescence and phase images of the same microscopic field (Figure 1D). Once endocytosed, the fluorescein remained inside the cells without evidence of recycling to the cell surface, and no exocytosis of fluorescent complexes was detected. Other biotinylated cell lines including HEL and Jurkat cells exhibited a nearly identical pattern and time-course of Av-FITC endocytosis under the same conditions, and no internalization was observed when the cells were incubated at 4 °C. In the absence of biotinylation, incubation of freshly isolated peripheral blood cells with Av-FITC did not

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increase their fluorescence to detectable levels (Figure 2A). We next biotinylated separate pools of erythrocytes and mononuclear cells and examined Av-FITC uptake in those cells. Immediately upon the addition of Av-FITC and after 24 h, 100% of the biotinylated erythrocytes exhibited intense fluorescence evenly distributed only on their surfaces (Figure 2B). Unlike erythrocytes, after 24 h, the pattern of fluorescence among the biotinylated mononuclear peripheral blood cells was similar to that observed in cell lines with nearly 100% of nucleated cells internalizing the fluorescent label (compare Figure 2C and Figure 1D). A comparison of brightfield and fluorescent views of individual cells (Figure 2, bottom panels) revealed a punctuate cytoplasmic distribution consistent with endosomal localization of the fluorescein. The flourescent label was also retained at a low level on the plasma membrane of several nucleated blood cells compared with no surface label retained on the K562 cells after 24 h (Figure 1D). Flow cytometry was used to examine whether avidin cross-linking affected the rate of avidin and biotin clearance from the cell surface or the growth of those cells. Untagged avidin was added to biotinylated cells at time ) 0 and fluorescent anti-avidin antibodies were used to measure the amount of avidin remaining on the surface over time. The surface level decreased by 20% within 15 min, and only 50% of avidin-bound biotin remained after 2 h (Figure 3A, dotted line). In contrast, biotin was cleared from the cell surface at a much slower rate in the absence of avidin cross-linking (Figure 3A, solid line). Biotin-mediated endocytosis of the avidin caused no significant decrease in cell growth as measured by cell counts (Figure 3B), and no change in cell viability was detected by dye exclusion. PEI-Avidin Conjugates Condense DNA and Transfect Biotinylated Hematopoietic Cells. We chose the model of DNA transfection to study the biotinmediated endocytosis of more complex avidin bioconjugates designed for the delivery of biologically active molecules to the nucleus. Polyethylenimine (PEI), a DNA condensing agent that may enhance endosomal escape, was conjugated with avidin. Green fluorescent protein (GFP) encoded plasmid DNA provided a specific marker of endosomal escape and trafficking of plasmid DNA to the nuclear compartment. Another advantage of this transfection model for our study was the low transfection efficiency of PEI-DNA among hematopoietic cells unless the PEI-DNA complexes were delivered by receptormediated endocytosis (15). Four PEI-avidin conjugates were prepared with increasing content of avidin as described in the Experimental Procedures. PA2, PA4, PA8, and PA16 define conjugates with avidin to PEI molar ratios of 2, 4, 8, and 16, respectively. The gel retardation assay showed a similar profile of cationic binding for PEI and the PEI-avidin conjugates as a function of PEI nitrogen:DNA phosphate (N:P) molar ratios. Consistent with the pK profile of PEI, approximately one of six nitrogen atoms in PEI is protonated at physiological pH (16). 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. Gene-transfer efficiencies of PEI-avidin conjugates were measured in K562 cells by flow cytometry. GFPexpressing cells were defined as those cells having a fluorescence at levels at least 2 standard deviations above the negative control. Optimization of transfecting conditions was performed based on avidin content in the

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Figure 2. Examination of human peripheral blood cells. Cells were obtained from blood donations, biotinylated, and photographed 24 h after addition of avidin-FITC. Upper three panels show fluorescence microscopy of unbiotinylated, unseparated peripheral blood cells (A), biotinylated erythrocytes (B), and biotinylated mononuclear cells (C). (Lower panels) Enlargement of six individual mononuclear cells are shown in matching brightfield and fluorescent views for comparison.

conjugate, molar ratio of PEI nitrogen to DNA phosphate (N:P), cell number, and serum content. A comparison of gene-transfer efficiencies among the four conjugates was then performed. PA4 conjugates had the highest transfection efficiency when compared with PA2, PA8, and PA16 (Figure 4A). PA4 conjugates were most efficient at a N:P ratio of 6.4 (Figure 4B). At higher N:P ratios, increased cell toxicity was noted as a function of cation excess as reported elsewhere (15, 16). Reducing the amount of PA4-DNA added to cells resulted in a linear reduction in the transfection efficiency as measured by the percentage of cells expressing GFP after 48 h. To determine the relative efficiency of biotin-mediated endocytosis for DNA transfection among hematopoietic cells, PA4 transfection efficiency was compared with unconjugated PEI and transferrin-PEI bioconjugates, as well as a lipid-based reagent DMRIE-C (Figure 5). Transfection of the cell lines with PA4 and trasferrinPEI resulted in a significant increase in GFP expression when compared to PEI alone and a severalfold increase in transfection efficiency in comparison to lipid-based DMRIE-C. Incorporation of avidin into PEI significantly increased the transfection efficiency on biotinylated K562, Jurkat, and HEL cells to the level achieved with transferrin-PEI (15-20%). A significant increase in biotin-targeted transfection efficiency was detected among HEL cells (20 ( 1.9% PA4 versus 11.8 ( 1.0% transferrin-PEI). In contrast to the malignant cells, significant transfection of terminally differentiated or proliferating hematopoietic cells expressing the transferrin receptor at high levels was not demonstrable with any of the vectors (Figure 5, striped; dotted bars). PEI-Avidin Mediated Gene Transfer into Biotinylated Cells Is Blocked by Free Avidin or Biotin.

Free avidin or biotin was added to PA4 transfection mixtures in order to determine if PEI-avidin-mediated gene transfer required specific binding to the biotinmodified receptors. After formation of PA4-DNA complexes, 0.5 mg/mL biotin was added to the transfection mixture and incubated for 30 min. Alternatively, 10 µg/ mL free avidin was added to the biotinylated cells before the addition of PA4-DNA complexes. As shown in Figure 6, free biotin or avidin significantly blocked PA4-mediated gene transfer in all the cell lines examined. DISCUSSION

One mechanism cells use to determine which molecules are endocytosed is the expression of high-affinity receptors on their external membranes. In this way, nutrients such as iron, folic acid, and lipoproteins enter cells to meet metabolic needs while other molecules not useful to the cell are excluded. When a single receptor binds one or more ligands, the number of binding sites per receptor and the number of receptors per cell is limited in order to regulated ligand entry (17, 18). Receptorbinding specificity also plays a large role in restricting the tropism of several viruses (19). Here we have demonstrated that covalently added adaptor molecules can modify the cell surface in order to specifically mediate the endocytosis of extracellular elements. We used biotin as a receptor-adaptor due to the well-recognized interaction of biotin with avidin and the lack of avidin-specific receptors on human cells. The covalent addition of a sulfoN-hydroxysuccinimide (sulfo-NHS) group to biotin permits conjugation to free amine groups, primarily the epsilon amine group of lysine residue in proteins. The receptor-mediated nature of avidin endocytosis was confirmed by the ability of free avidin or biotin to block

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Figure 3. Kinetics of biotin and avidin clearance and cell growth before and after avidin addition. (A) Flow cytometry was used to measure the clearance of avidin and biotin from the cell surface. (solid line) K562 cells were biotinylated and the amount of biotin remaining on the cell surface was measured by relative fluorescence after the addition of avidin-FITC. (dotted line) Unlabeled avidin was added to biotinylated K562 cells at time ) 0 and the clearance of avidin was measured using anti-avidin antibodies. The data points show the mean fluorescence values ( standard deviation of triplicate experiments with time ) 0 representing 100%. (B) Following biotinylation and incubation with avidin-FITC, cell growth was measured on 3 successive days. Numbers of cells are means of triplicate experiments with standard deviation bars. (solid bars) control K562 cells, (open bars) biotinylated K562 cells, (hashed bars) biotinylated K562 cells incubated with avidin.

the process. The nonspecificity of the biotinylation reaction for free amines on the cell surface additionally suggests that membrane elements not classically associated with endocytosis may be adapted for this purpose. Avidin and biotin have been used extensively in the design of bioconjugates due to their remarkable affinity (Ka ) 1015 M-1), low cost, and availability (20). Surface biotinylation of human red blood cells has been used in humans as an alternative to 51Cr in labeling studies (21). Biotinylation has little effect on the stability of red blood cells in vivo, and surface biotinylation rarely modifies their antigenicity as measured by serological assays (22). However, internalization of the biotin molecules is prevented by the inability of erythrocytes to endocytose their membrane (23) (Figure 2B). The addition of avidin to biotinylated red blood cells also increases their immunogenicity due to their inability to clear bound avidin from the surface (24). In marked contrast with erythrocytes, nucleated cells rapidly endocytose the surface avidin as demonstrated here.

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Figure 4. Transfection efficiency of PEI-avidin conjugate variants. (A) Transfection efficiency of PEI-avidin (PA) conjugates at increasing molar ratios of avidin: PA2, PA4, PA8, and PA16. (B) efficiency of PA4 in function of N:P ratio. PA4 was tested on biotinylated K562 cells (triangles) and compared to experiment with PEI alone on K562 cells (squares). The same experiment was performed for all PA conjugates. PEI or PA conjugates were mixed with 10 µg of DNA (pGT) in 0.5 mL of PBS at a growing N:P ratios as indicated on the figure and added to 5 × 105 cells/well in 1.5 mL culture medium (DMEM containing 10% serum), in a 24 well plate. After 4 h, 1 mL of DMEM containing 15% FBS was added to the cells. Flow cytometry was used to determine the percentage of cells expressing GFP 48 h after transfection. Values are shown as means ( standard deviation of triplicate transfections.

The dissociation constant of avidin-biotin is several powers of magnitude higher than that of naturally occurring receptor ligand pairs even at very low pH (25). Due to the high affinity and stability of the avidin-biotin bond, we expected a significant portion of the endocytosed avidin to be continuously recycled to the cell surface. However, within the limits of detection, the surface staining pattern of fluorescent avidin suggests most of the avidin was removed from the cell surface within 24 h (Figures 1 and 2).These findings probably reflect the ability of nucleated cells to redirect cross-linked receptors from the recycling pathway to the lysosomal compartment. This redirection away from recycling pathways and lysosomal retention of multivalent ligands has been demonstrated in the case of homodimerric cross-linking of Fc receptors (26) as well as oligomerization of transferrin receptors (27). The tetravalency of avidin for biotin

Biotin-Mediated Entry of Avidin Bioconjugates

Figure 5. Comparison of PEI-avidin transfection efficiency in hematopoietic cells with other transfection reagents. Transfection efficiency using PEI, Transferrin-PEI, PEI-avidin (PA4), or DMRI-C with pGT plasmid was compared under similar conditions in K562 cells (open bars), Jurkat cells (solid bars), HEL cells (dashed bars), primary mononucleated cells from buffy coat (striped bars) and in proliferating primary erythroid cells demonstrating extremely high level transferrin receptor expression (dotted bars). Efficiency is represented as percentages of GFP expressing cells assessed by flow cytometry from three independent experiments done in duplicate.

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conjugates (30), transferrin conjugates (15), galactose conjugates (31, 32), conjugates with integrin binding motifs (33), and antibody-coupled conjugates (34) have been developed for this purpose. Despite recent advances in targeting strategies, transfection of targeted nonviral vectors generally does not result in high-level gene expression in a majority of cells. Our data suggest the generally low transfection efficiency in this setting may be due to cell type specific barriers that are postendocytotic and not specifically related to the cell cycle. Additional explanations include the low sensitivity of GFP as a marker of gene transfection (35), and the physical state of the DNA-bioconjugate complexes (32). In the peripheral blood mononuclear cells studied here, little is known about the catabolism of transferred plasmid DNA, cytoplasmic trafficking after endosomal release, or nuclear localization. Despite the low level of effective gene transfer in these cells, the extremely high efficiency of biotin-mediated endocytosis in primary cells suggests receptor adaptation may prove useful for the entry of other therapeutic bioconjugates or the targeted clearance of extracellular toxins. Conceptually, receptoradaptation should provide a generic means for delivering bioconjugates across tissue and species boundaries. ACKNOWLEDGMENT

We thank Dr. A. Schechter for critical reading of the manuscript, Dr. A. Gubin for his assistance in the preparation of plasmid DNA, and J. Muthoni Njoroge for technical assistance with flow cytometry. LITERATURE CITED

Figure 6. Transfection efficiency of PEI-avidin in biotinylated cells in the presence of excess free biotin or avidin. A total of 8 µg of PEI alone or in PEI-avidin (PA4) complexed with 10 µg of pGT (N:P ratio ) 6.4) with and without addition of free biotin or avidin was used to transfect biotinylated K562 cells (open bars), Jurkat cells (solid bars) and HEL cells (dashed bars). Additional biotin was added to 0.5 mg/mL final concentration to preformed PEI-DNA or PA4-DNA complexes, the mixture was incubated for 30 min at 25 °C and used to transfect cells in the medium supplemented with 0.5 mg/mL biotin. Avidin was added to the 10 µg/mL concentration to cells in media and incubated for 30 min at 25 °C before transfection with PEIDNA or PA4-DNA containing 10 µg/mL avidin. Forty-eight hours later, the percentage of GFP expressing cells was assessed by flow cytometry and is shown as means of triplicate studies ( standard deviation.

results in the clustering of biotinylated proteins on the cell surface (28). Therefore, trafficking of avidin-biotin protein complexes to lysosomal compartments may serve as a default pathway for the clearance of irreversibly clustered proteins from the cell surface. Endocytosis triggered by cross-linking of membrane domains containing glycosylphosphatidyinisitol-anchored receptors may also be involved in this process (29). Our study has not determined whether specific endocytic elements, such as clathrin, or specific signaling cascades are required for biotin-mediated endocytosis to occur. In addition to the study of endocytosis itself, receptor adaptation on nucleated cells may be useful for targeting bioconjugates into cells. Immense efforts have recently been directed toward increasing the efficiency of gene transfer and other technologies by targeting membrane components classically associated with receptor-mediated endocytosis. Among nonviral vectors, folate-targeted

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