Bioconjugate Chem. 2003, 14, 1203−1208
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Influence of Targeting Ligand Flexibility on Receptor Binding of Particulate Drug Delivery Systems Verena Olivier,†,| Iris Meisen,‡ Barbara Meckelein,†,| Timothy R. Hirst,§,⊥ Jasna Peter-Katalinic,‡ M. Alexander Schmidt,† and Andreas Frey†,|,* Institut fu¨r Infektiologie, Zentrum fu¨r Molekularbiologie der Entzu¨ndung, von-Esmarch-Strasse 56, Westfa¨lische Wilhelms-Universita¨t, D-48149 Mu¨nster, Germany, Institut fu¨r Medizinische Physik und Biophysik, Robert-Koch-Strasse 31, Westfa¨lische Wilhelms-Universita¨t, D-48149 Mu¨nster, Germany, and Department of Pathology & Microbiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom . Received May 9, 2003; Revised Manuscript Received September 3, 2003
Receptor-mediated drug targeting via nanoengineered particulate delivery systems is an emerging field. However, little is known about how such magic bullets should be assembled to yield optimal targeting efficiency. Here we investigated the influence of targeting ligand flexibility on binding of ligand-coated microparticles to cell surface receptors. Using the ganglioside GM1-binding B subunit of cholera toxin as ligand and fluorescent microparticles as a model delivery system, conjugates with different numbers of linkages between ligand and particle were prepared and tested for their efficiency to bind to live fibroblast monolayers. Our results show that multiple bonds between ligand and particle reduce the targeting rate by up to 50% compared to constructs where ligands are attached via single aliphatic chains. Thus, for maximum performance, targeted particulate drug delivery systems should be assembled such that ligands are attached via single σ bonds only, allowing the ligand molecules to adopt an optimal binding conformation.
INTRODUCTION
Functional genomics, proteomics, and glycomics along with emerging technologies such as molecular breeding are expected to yield drugs with ever increasing potency and molecular specificity. Yet, except for antiinfectiva where foreign organisms are to be attacked, molecular specificity does not necessarily translate into cell typeor organ-specificity since the molecular target of the drug may be expressed at multiple locations throughout the body, exerting different functions at different sites. The predicament can be solved only when the drug is combined with a delivery technology that directs the compound exclusively to its desired site of action. This dilemma is most apparent in gene therapy and cancer medication, but also in areas less obvious such as oral vaccination where targeted delivery may be desirable. Orally administered proteinaceous antigens rapidly fall prey to the digestive enzymes of the gastrointestinal tract and give rise to oral immune tolerance, the default reaction of the mucosal immune system (1). To overcome this, it is believed that antigens must be delivered in intact form selectively to M cells, a special epithelial cell type overlying the organized mucosa-associated lymphoid tissue (2). To meet this requirement, we devised an M * Corresponding author. Laborgruppe Mukosaimmunologie, Abteilung Klinische Medizin, Forschungszentrum Borstel, Parkallee 22, D-23845 Borstel, Germany. Phone: +49-4537-188562. Fax: +49-4537-188-693. E-mail:
[email protected]. † Institut fu ¨ r Infektiologie. ‡ Institut fu ¨ r Medizinische Physik und Biophysik. § University of Bristol. | Present address: Laborgruppe Mukosaimmunologie, Abteilung Klinische Medizin, Forschungszentrum Borstel, Parkallee 22, D-23845 Borstel, Germany. ⊥ Present address: A14 - Main Quadrangle, The University of Sydney, NSW 2006, Australia.
cell targeting strategy which exploits differences in cell surface receptor accessibility (3) and requires the antigen to be 30 nm in diameter and coated with a ligand directed against plasma membrane ganglioside GM1. Since we observed considerable differences in targeting efficiency in a cell culture model system depending on how the targeting ligand was attached to a particle surface (3, 4), we have chosen in this study to investigate the influence of rigidity of ligand attachment on the efficiency of ligand-mediated particle targeting. EXPERIMENTAL PROCEDURES
Production of Recombinant CTB. Recombinant cholera toxin B-subunit (rCTB) was prepared as described previously (5). Briefly, rCTB was expressed in secreted form in Vibrio sp. 60 containing the inducible vector pATA13 (6). rCTB was harvested from the culture supernatant by ultrafiltration and the concentrated retentate fraction subjected to hydrophobic interaction and cation exchange chromatography (5). The purified rCTB was desalted by gel permeation chromatography, equilibrated in Dulbecco’s phosphate-buffered saline (DPBS), pH 7.4, snap-frozen in liquid N2 and stored at -80 °C until required. Biotinylation of CTB. Mixtures of 5 µM rCTB and either 75, 25, or 8.35 µM NHS-LC-biotin (water soluble biotin (long arm) NHS, sulfosuccinimidyl-6-(biotinamido)hexanoate; Vector, Burlingame, CA) in a total volume of 200 µL of D-PBS were allowed to react for 30 min at RT on an end-to-end-mixer at 4 rpm before the reaction was quenched by adding 400 µL of 200 µM lysine in D-PBS to a final concentration of 133 µM. The solution was dialyzed extensively against D-PBS at 4 °C for 48 h using Slide-A-Lyzer Dialysis Cassettes (MWCO 10 000 Da; Pierce, Bonn, Germany), snap-frozen in liquid N2 and stored at -80 °C.
10.1021/bc034077z CCC: $25.00 © 2003 American Chemical Society Published on Web 10/28/2003
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Preparation of CTB-Coated Particles. CTB-coated microparticles (CTB-P) and biocytin-quenched control particles (Control-P) were prepared by coupling biotinylated CTB or biocytin (N--biotinyl-L-lysine) (Sigma, Taufkirchen, Germany) to avidin-coated, carboxy-modified, fluorescent latex particles (Molecular Probes, Eugene, OR) of 1 µm nominal size as described earlier (3). In 500 of µL of D-PBS, 5 × 108 or 1 × 109 red fluorescent particles were reacted under rocking at 1.5 rpm for 72 h at 4 °C in the dark with 21 µg (0.361 nmol) or 43 µg (0.738 nmol) of the differently biotinylated CTBs. Thus, regardless of its degree of biotinylation, CTB was applied in a 5.5-fold molar excess over the maximally achievable CTB particle load (approximately 80 000 CTB molecules per 1-µm particle, as calculated according to (3)). For the control particles, 4 × 109 green fluorescent particles in 1000 µL of D-PBS were reacted under the same conditions with 149 µg (400 nmol) biocytin, representing a 110fold molar excess of biocytin over biotin binding sites. Unreacted ligand was removed by centrifugation at 1100 × g and gently resuspending the particles in 1.8 mL of distilled water. Washes were repeated 10-12 times for all preparations before the particles were stored at concentrations of 2 × 108 to 3 × 109 particles/ml in 0.1 M HEPES-NaOH, pH 7.4, at 4 °C in the dark. GM1 Binding Assay. High-bind ELISA plates (Corning, Wiesbaden, Germany) coated overnight at 4 °C with 75 µL/well of 5 ng/mL ganglioside GM1 (Alexis, Gru¨nberg, Germany) in D-PBS were washed three times with 300 µL/well of D-PBS and blocked with 250 µL/well of 0.1% (w/v) ovalbumin (OVA) (Sigma) in D-PBS for 6-8 h at RT. After four washes with D-PBS, plates were incubated overnight at 4 °C with 75 µL/well of serially diluted biotinylated CTB in 0.1% (w/v) OVA/D-PBS, washed six times with PBST (D-PBS, 0.05% (v/v) Tween 20), and incubated for 90 min at RT with 75 µL/well of 1 µg/mL (8 pmol streptavidin/mL) peroxidase-labeled streptavidin (Vector) in 0.1% (w/v) OVA/D-PBS. After six washes with PBST, plates were developed using the 3,3′,5,5′-tetramethylbenzidine substrate system of Frey et al. (7). Nano-Electrospray Ionization Mass Spectrometric Determination of Biotinylation. Biotinylated CTB (5 µg) was dialyzed extensively against 20% (v/v) acetic acid for 48 h at 4 °C in QuixSep microdialysis chambers (Roth, Karlsruhe, Germany) using Spectra/Por7 dialysis membranes (MWCO 3500; Spectrum, Rancho Dominguez, CA), lyophilized, and redissolved in 8.6 µL of 25% (v/ v) acetic acid (final concentration 10 µM CTB) of which 5 µL (50 pmol) was used for mass spectrometry. Measurements were carried out on a nano-electrospray Q-TOF instrument fitted with a Z-spray nano-ESI source (Micromass, Manchester, UK) using omega nanospray glass capillaries prepared from borosilicate glass (Hilgenberg, Malsfeld, Germany) on a Kopf vertical pipet puller, model 720 (David Kopf Instruments, Tujunga, CA). Electrospray potential (900-1100 V) was applied via a stainless steel wire electrode inside the nanospray source (8). Biochemical Determination of Biotinylation. The differently biotinylated CTBs were serially diluted (2-fold, 6.7 µg/mL-3.3 ng/mL) in 0.1% (w/v) BSA/PBS and 3 µL of each dilution was pipetted onto nitrocellulose membranes (4.5 × 13 cm, 0.2 µm pore size; Schleicher & Schuell, Dassel, Germany). After being dried and washed twice with 20 mL of D-PBS and once with PBST, membranes were blocked for 5 h in 20 mL of blocking solution (1% (w/v) casein (Hammarsten grade; BDH, Poole, UK), 0.02% (w/v) thimerosal, 0.05% (v/v) Tween 20 in D-PBS) at RT. Dotted CTB labeled with a 15-, 5-, or 1.67-fold molar excess of biotinylation reagent was
Olivier et al.
then incubated in 20 mL of blocking solution for 24 h at 4 °C with 6834 ng (54.4 pmol), 2278 ng (18.1 pmol), or 759 ng (6.0 pmol) peroxidase-labeled streptavidin, each representing a 20-fold molar excess of streptavidin over the maximum amount of biotin theoretically present on the membrane. After four washes in PBST and two in D-PBS, 800 µL of LumiLightPLUS Western blotting substrate (Roche, Mannheim, Germany) was applied, and chemiluminescence was quantitated on a Lumi-ImagerF1 (Roche). Serially diluted OVA (2-fold, 6.7 µg/mL-3.3 ng/mL) biotinylated with a 15-fold molar excess of NHSLC-biotin served as standard. Binding of Microparticles to BALB/c 3T3 Fibroblasts. BALB/c 3T3 fibroblasts (clone A31; ATCC, Rockville, MD) were grown in high glucose (25 mM) Dulbecco’s modified Eagle’s medium (DMEM) (PAA Laboratories, Linz, Austria) supplemented with 10% (v/v) newborn calf serum (defined grade; Hyclone, Bonn, Germany), 2 mM glutamine, 100 U/mL penicillin/100 µg/mL streptomycin, and 25 mM HEPES (PAA) at 37 °C in a humidified atmosphere containing 10% (v/v) CO2. For particle binding, cells (passages 70-86) were seeded onto round (13 mm diameter) glass coverslips (Bellco, Vineland, NJ) in 24-well tissue culture plates and used at 14-17 days post confluence. Monolayers were washed gently five times with 1 mL of prewarmed (37 °C) D-PBS containing 0.9 mM CaCl2 and 0.5 mM MgCl2 (CM-PBS) before 500 µL of a suspension containing 2.5 × 107 particles/mL each of CTB-P and Control-P in DMEM, 4 mM glutamine, and 25 mM HEPES were applied. For competition assays, coverslips were preincubated for 15 min with 250 µL of 20 µg/mL CTB in DMEM, 4 mM glutamine, and 25 mM HEPES before 250 µL of 5 × 107 particles/mL each of CTB-P and Control-P in the same buffer were added. After 60 min at 37 °C, coverslips were washed gently five times with 1 mL of prewarmed CM-PBS, fixed in 1 mL of 3% (w/v) paraformaldehyde in D-PBS for 90-120 min at RT in the dark, and washed three times in D-PBS and once in distilled water and mounted on glass slides with Mowiol 4-88 (Calbiochem-Novabiochem, Bad Soden, Germany) containing 2.5% (w/v) DABCO (1,4-diazabicyclo[2.2.2]octane; Sigma). Cells were examined en face with a Zeiss Axiophot microscope equipped for epifluorescence (Zeiss, Jena, Germany) and were either photographed or recorded with a video camera. Statistics. For quantitation of microparticle binding, digital images of five randomly selected nonoverlapping regions (top, bottom, center, left, and right) of each coverslip were printed and counted manually. Statistical analysis was performed using the StatView 4.51 software (Abacus Concepts, Berkeley, CA). Mixed probes of testand control particles were considered paired samples. Results of independent experiments were treated as unpaired samples. Statistical analyses were considered significant only if p < 0.05. RESULTS AND DISCUSSION
When particle surfaces are to be derivatized with biomolecules, coupling is often achieved in an ill-defined manner, either by physisorption via van der Waals and electrostatic forces or by chemisorption after preactivation of the surface or in a one-pot reaction with a condensation reagent. Although macromolecules bound this way may generally retain their biological activity, it is extremely difficult to assess the degree of their functionality once the bioconjugate is formed. To tackle this problem we devised a strategy to bind a ligand to a polyvalent microparticle via a defined number of linkers.
Influence of Ligand Flexibility on Receptor Binding
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Figure 1. Analysis of CTB biotinylation. A: Representative nano-ESI MS analysis in positive ion mode of CTB biotinylated with 1.67-, 5-, and 15-fold molar excess (1.67×-me, 5×-me, 15×-me) of NHS-LC-biotin. Species A: unmodified CTB; species B: phosphoric acid adduct of the CTB monomer; species C: monobiotinylated CTB; species D: CTB carrying two biotin labels. B: Regression analysis of mass spectrometric versus biochemical analysis of CTB biotinylation. Degrees of biotinylation deduced from the nanoESI MS data closely correlate (simple regression R2: 0.997; one-way ANOVA, p < 0.05) with background-corrected signal intensities (mean ( standard error) obtained from a dot-blot biotin detection assay where biotinylated ovalbumin served as standard (100%).
This enables us to deduce the rigidity of the conjugate and investigate its influence on the functionality of the probe. As a ligand the ganglioside GM1-binding B subunit of cholera toxin (CTB) was chosen. CTB (SWISS-PROT: P01556) is a torus- or donut-shaped homopentameric protein of 58222 Da bearing five identical receptor binding sites arranged in 5-fold symmetry at the bottom of the torus (9) and 10 potentially derivatizable amino functions on each subunit. While one of them (Lys-91) tolerates derivatization (10, 11), although it is directly involved in GM1-binding (9), succinylation of a second lysine (Lys-34) does not impair the biological activity of its own but that of a neighboring subunit (10). Besides that, integrity of the lone tryptophan (Trp-88) is crucial for GM1-binding (10). To link CTB to the particle surface we decided to biotinylate the ligand to various degrees at its amino functions thus creating single or multiple bonds when coupling it to avidin-coated microparticles. Differently Biotinylated CTBs. Recombinant CTB was produced in functional pentameric form in a Vibrio species and biotinylated with NHS-LC-biotin (sulfosuccinimidyl-6-(biotinamido)hexanoate), an active ester derivative of biotin, which carries a flexible 2.2 nm spacer and reacts with R- and -amino functions of proteins. Since preservation of at least about 50% of its GM1binding activity requires that no more than 30% of the amino functions of CTB be converted into amide bonds (12), a maximum of a 15-fold molar excess of NHS-LCbiotin should be used for derivatization when assuming full conversion of the active ester. We therefore carried out three different biotinylation reactions with 1.67-, 5-, and 15-fold molar excess of NHS-LC-biotin. The actual degree of biotinylation was determined using nano-electrospray ionization mass spectrometry (nano-ESI MS). In Figure 1A the resulting spectra are shown, depicting a representative analysis of CTB, biotinylated with 1.67-, 5-, or 15-fold molar excess of NHS-LC-biotin. Several species named A, B, C, D were detected in each spectrum. The different charge states, +8 to +12, obtained for each species allowed the determination of the average molecular masses which are given in each spectrum. Species A represents the unmodified CTB-monomer (calcd average mass 11 645.36). Species B is generated by a noncovalent phosphoric acid adduct of the unmodified CTB-monomer (calcd average mass 11 743.36). Species C is assigned to a monoderivatized monomer (calcd average mass 11 984.82) and species D corresponds to a CTB monomer derivatized with 2 biotin moieties (calcd average mass 12 324.28). On the
Table 1. Degree of Biotinylation of CTB Depending on the Amount of Biotinylation Reagent Used derivatization as determined by molar excess of biotinylation reagent 15-fold 5-fold 1.67-fold
nano-ESI-MS: no. of biotins per pentamera
dot-blot: biotinylation as % of standardb
2.75 ( 0.10 0.82 ( 0.06 0.24 ( 0.00
122.79 ( 11.61 43.39 ( 2.20 11.37 ( 0.85
a Data are derived from three different m/z ratios. b Data are derived from five different dot blots of each sample. Results are given as mean ( standard error.
basis of these data the average degree of biotinylation of a CTB pentamer can be deduced by comparing the background-corrected relative signal intensities of the unmodified and differently biotinylated CTB monomers using eq 1,
biotins per CTB pentamer )
c + 2d × 5 (1) 100 + c + d
where c is the relative signal intensity of CTB monomers carrying 1 biotin label, d the relative signal intensity of CTB monomers carrying two biotin labels, and the signal intensity of nonbiotinylated CTB monomer is set 100%. The respective degrees of biotinylation obtained with 1.67-, 5-, or 15-fold excess of NHS-LC-biotin are summarized in Table 1. They were confirmed in a quantitative dot-blot assay, which correlated strongly with the mass spectrometric data (Figure 1B). The relative differences in signal intensity between the differently biotinylated CTBs were normalized on a standard and are included in Table 1. According to these data, the actual derivatization yield for CTB is directly correlated with the molar excess of NHS-LC-biotin applied (simple regression R2: 1.0, oneway ANOVA, p < 0.025) while the reaction yields for biotinylation reagent increased slightly from 14.3% for a 1.67-fold molar excess over 16.5% for a 5-fold excess to 18.3% for a 15-fold molar excess. Thus, no signs of saturation in the derivatization reactions are visible, which is to be expected when less than five amino functions are derivatized on a homopentameric protein molecule. Biological Activity of the Differently Biotinylated CTBs. Due to the five equal receptor binding sites, CTB is markedly tolerant toward derivatization of its primary amino functions. Hybrid CTB pentamers renatured from equal amounts of monomers which were either formylated at Trp-88 or succinylated at Lys-34 have only 1.25
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Figure 2. Biological activity of biotinylated CTB. ELISA-type assay with serial dilutions of CTBs derivatized with 1.67- (2), 5- (b), and 15- (9) fold molar excess of NHS-LC-biotin. Conjugates were captured on ganglioside GM1-coated microtiter plates and visualized via streptavidin-horseradish peroxidase and a colorogenic substrate. Data represent mean ( standard error of three independent experiments. Inset: Regression analysis of signal intensities of streptavidin-binding to GM1-captured biotinylated CTB versus degree of biotinylation. Backgroundcorrected absorbances extrapolated for saturated GM1-binding (mean ( standard error) closely correlate (simple regression R2: 1.0; one-way ANOVA, p < 0.005) with degrees of biotinylation determined by mass spectrometry.
functional receptor binding sites left but still retain 40% of the original affinity for GM1 (10). Consequently, CTB should tolerate the up to 2.75 biotins which we introduced even if labeling occurred exclusively at amino groups essential for receptor interaction. However, affinity may decrease with progressive labeling under these circumstances. Moreover, selective biotinylation of these sites will attach the CTB molecule to the avidin-coated microparticle the wrong way around with its receptor binding interface pointing toward the particle core. We therefore needed to know whether biotin was present at locations (top and/or side of the torus) which would allow CTB to conjugate to avidin-coated particles in the desired
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way and whether this biotinylation pattern persists for the different degrees of biotinylation. To address this question, soluble biotinylated CTB was first allowed to bind to a GM1-coated microtiter plate before the biotin moieties were detected with a streptavidin-horseradish peroxidase conjugate. In this setup, biotinylation of amino functions located at the bottom of the torus, i.e., the GM1-binding interface of CTB, cannot be detected because they are buried beneath the CTB molecule inaccessible for streptavidin-horseradish peroxidase. Since signals were not only detectable but also increased linearly with the number of biotins per CTB pentamer (Figure 2), all three preparations did indeed carry labels at the desired part of the torus and exhibited a similar affinity for GM1, although the result does not imply that some kind of site-selective labeling has occurred. Nevertheless, a substantial proportion of biotinylated CTB in each preparation can link to avidincoated particles such that their GM1-binding site points away from the particle core and all preparations are comparable in their biological activity. Targeting Efficiency of Microparticles Conjugated with CTB in Mono- or Polyvalent Manner. Since molecules biotinylated at an unfavorable position cannot be eliminated from a preparation we decided to circumvent this predicament by using rather large particles of 1 µm diameter as an experimental delivery system. They offer a parking area for approximately 80 000 CTB molecules (3, 4), enough to generate a surface coat that is representative of the original distribution of top/side- and bottom-labeled CTB. Since the proportion of bottom-labeled CTB was shown to be constant in all preparations, its effect will then be canceled out when comparing the biological activity of different CTB-coated particles (CTB-P). As internal control for binding assays, inert microparticles of different color (Control-P) were prepared by quenching their biotin binding sites in a charge neutral manner using excess biocytin. BALB/c 3T3 fibroblasts were chosen as an appropriate target cell since they express high levels of GM1 receptor (13) which is readily accessible to the 1 µm particles used here (3, 4). To simulate a real targeting situation, cells were grown as confluent monolayers and then exposed
Figure 3. CTB-mediated binding of microparticles to BALB/c 3T3 fibroblasts. Cell monolayers (133 mm2) were exposed to 2.5 × 107 particles each of CTB-P (A-C) and Control-P (D-F) for 1 h at 37 °C. Particles had been coated with CTB derivatized with 1.67-fold (A), 5-fold (B), or 15-fold (C) molar excess of biotinylation reagent. Particle binding was visualized by fluorescence microscopy and quantitated. Binding of CTB-P was significantly higher than binding of Control-P for all CTB-P preparations (two-tailed, paired t-tests, p < 0.0005), scale bar, 200 µm. G: In the presence of 10 µg/mL free CTB binding of CTB-P prepared from CTB derivatized with 1.67-fold molar excess of biotinylation reagent was significantly lower (*) than in the absence of free CTB (two-tailed, unpaired t-test, p < 0.0001) and indistinguishable from control levels (Control-P) (two-tailed, paired t-test, p ≈ 0.9). Data given are means ( standard error.
Influence of Ligand Flexibility on Receptor Binding
Figure 4. Influence of degree of biotinylation on the binding of CTB-particles to BALB/c 3T3 fibroblasts. A: Degree of biotinylation of CTB derivatized with 1.67-, 5- and 15-fold molar excess (1.67×-me, 5×-me, 15×-me) of biotinylation reagent. The number of biotin labels per CTB pentamer (mean ( standard error) represents the maximum number of ligand-particle bonds. B: Efficiency of CTB-mediated particle binding to apical surfaces of cultured BALB/c 3T3 fibroblasts. Data from seven independent experiments are displayed as n-fold better binding of CTB-P than Control-P (geometric mean */÷ standard error). Particles coated with CTBs that can form no more than one single bond with avidin particles (1.67×-me and 5×-me) showed significantly higher binding (*) to cell surfaces than particles coated with CTB that can form multiple bonds with a particle (15×-me) (one-way ANOVA, Fisher’s PLSD, p < 0.05).
to a suspension of 2 × 105 CTB-P per mm2 along with the same number of Control-P as internal standard in physiological buffer. All three CTB-P preparations showed binding to the apical plasma membranes of 3T3 fibroblasts (Figure 3A-C) which was significantly higher than that of Control-P (Figure 3D-F). Binding was exclusively mediated via the ligand since competition with soluble CTB reduced binding of CTB-P to control levels as exemplified in Figure 3G for CTB-P prepared with 1.67fold molar excess of biotinylation reagent. However, when comparing the targeting efficiency of the different CTB-P preparations significantly lower binding was observed for particles derived from CTB which was derivatized with a 15-fold than for particles coated with CTB derivatized with 1.67- or 5-fold molar excess of biotinylation reagent (Figure 4). Binding of the former CTB-P which may contain a mean of 2.75 bonds between ligand and particle (Figure 4A) was reduced significantly by up to 50% (one-way ANOVA, Fisher’s PLSD, p < 0.05) compared to preparations in which CTB is linked via no more than a single bond (calculated average values: 0.82 and 0.24 bonds, respectively) to the experimental particulate delivery system (Figure 4B). As chemical bonds occur in integers only, both a 0.82- and a 0.24-fold biotinylated CTB should of course behave the same in that only the biotinylated matter reacts with the particle while the nonbiotinylated CTB is lost during CTB-P preparation. As 0.24-fold biotinylated ligand still harbors less double-labels than 0.82-fold labeled CTB it should perform even better than the latter on particulate delivery systems which was indeed the case (Figure 4B). However, differences between these two CTB-P preparations were not statistically significant. In conclusion, our results demonstrate that conjugating targeting ligands via multiple bonds to particulate delivery systems impairs the performance of the final conjugate to a significant extent. A possible reason for this adverse behavior is proposed in Figure 5. It shows that a monovalent aliphatic linker gives a ligand considerably more freedom to rotate and bend than bi- or polyvalent linkages which will tether it in a rotational
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Figure 5. Proposed effect of ligand flexibility on targeting efficiency. Schematic diagram of ligand-particle conjugates binding to cell surfaces. Ligands conjugated to a particle surface via a single σ bond (A) are able to adopt an optimal position for receptor interaction. Ligands conjugated via multiple bonds to a particle surface (B) are sterically constrained which prevents them to adopt an optimal binding position.
axis tangential to the particle surface or even totally immobilize it in a position that is dictated by the location of the cross-linker on the ligand and therefore by the relative reactivities of its surface functional groups. For optimal targeting efficiency drug targeting systems should therefore be designed such that the targeting ligand is attached via a single σ bond only. A powerful technology to achieve such tailored linkers on proteins may arise from current efforts to expand the genetic code in order to incorporate unnatural amino acids (14) which eventually will provide a solitary handle for attaching the ligand to the particulate drug delivery system. Alternatively, protein ligands may be substituted by chemically less complex molecules such as peptides where a single attachment handle can readily be introduced (15). ACKNOWLEDGMENT
This work was supported by the Deutsche Forschungsgemeinschaft (grant no. FR 958/2-2&3). B.M. is a recipient of a personal stipend from the state of NordrheinWestfalen. LITERATURE CITED (1) Garside, P., and Mowat, A. M. (2001) Oral tolerance. Semin. Immunol. 13, 177-185. (2) Neutra, M. R., Frey, A., and Kraehenbuhl, J. P. (1996) Epithelial M cells: gateways for mucosal infection and immunization. Cell 86, 345-348. (3) Frey, A., Giannasca, K. T., Weltzin, R., Giannasca, P. J., Reggio, H., Lencer, W. I., and Neutra, M. R. (1996) Role of the glycocalyx in regulating access of microparticles to apical plasma membranes of intestinal epithelial cells: implications for microbial attachment and oral vaccine targeting. J. Exp. Med. 184, 1045-1059. (4) Frey, A., Meckelein, B., and Schmidt, M. A. (1999) Grafting protein ligand monolayers onto the surface of microparticles for probing the accessibility of cell surface receptors. Bioconjugate Chem. 10, 562-571. (5) Richards, C. M., Aman, A. T., Hirst, T. R., Hill, T. J., and Williams, N. A. (2001) Protective mucosal immunity to ocular herpes simplex virus type 1 infection in mice by using Escherichia coli heat-labile enterotoxin B subunit as an adjuvant. J. Virol. 75, 1664-1671. (6) Aman, A. T., Fraser, S., Merritt, E. A., Rodigherio, C., Kenny, M., Ahn, M., Hol, W. G., Williams, N. A., Lencer, W. I., and Hirst, T. R. (2001) A mutant cholera toxin B subunit
1208 Bioconjugate Chem., Vol. 14, No. 6, 2003 that binds GM1- ganglioside but lacks immunomodulatory or toxic activity. Proc. Natl. Acad. Sci. U.S.A. 98, 8536-8541. (7) Frey, A., Meckelein, B., Externest, D., and Schmidt, M. A. (2000) A stable and highly sensitive 3,3′,5,5′-tetramethylbenzidine-based substrate reagent for enzyme-linked immunosorbent assays. J. Immunol. Methods 233, 47-56. (8) Alving, K., Paulsen, H., and Peter-Katalinic, J. (1999) Characterization of O-glycosylation sites in MUC2 glycopeptides by nanoelectrospray QTOF mass spectrometry. J. Mass Spectrom. 34, 395-407. (9) Merritt, E. A., Sarfaty, S., van den Akker, F., L’Hoir, C., Martial, J. A., and Hol, W. G. (1994) Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide. Protein Sci. 3, 166-175. (10) De Wolf, M. J., and Dierick, W. S. (1994) Regeneration of active receptor recognition domains on the B subunit of cholera toxin by formation of hybrids from chemically inactivated derivatives. Biochim. Biophys. Acta 1223, 285-295. (11) McCann, J. A., Mertz, J. A., Czworkowski, J., and Picking, W. D. (1997) Conformational changes in cholera toxin B
Olivier et al. subunit-ganglioside GM1 complexes are elicited by environmental pH and evoke changes in membrane structure. Biochemistry 36, 9169-9178. (12) Ludwig, D. S., Holmes, R. K., and Schoolnik, G. K. (1985) Chemical and immunochemical studies on the receptor binding domain of cholera toxin B subunit. J. Biol. Chem. 260, 12528-12534. (13) Critchley, D. R., Streuli, C. H., Kellie, S., Ansell, S., and Patel, B. (1982) Characterization of the cholera toxin receptor on Balb/c 3T3 cells as a ganglioside similar to, or identical with, ganglioside GM1. No evidence for galactoproteins with receptor activity. Biochem. J. 204, 209-219. (14) Wang, L., and Schultz, P. G. (2002) Expanding the genetic code. Chem. Commun. (Cambridge, U.K.) 1-11. (15) Akerman, M. E., Chan, W. C., Laakkonen, P., Bhatia, S. N., and Ruoslahti, E. (2002) Nanocrystal targeting in vivo. Proc. Natl. Acad. Sci. U.S.A. 99, 12617-12621.
BC034077Z
