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BEHAVIORAL PHARMACOLOGY
Institute for Pharmaceutical Technology, Biocenter of Johann Wolfgang Goethe-University, Frankfurt, Germany (K.M., S.D., E.H., J.K., K.L.); Institute for Medical Virology, University Hospital Medical School, Johann Wolfgang Goethe-Universität, Frankfurt, Germany (M.M.); Division of Clinical Chemistry, Department of Medicine, Albert-Ludwigs-University, Freiburg, Germany (M.M.H.); and Department of Pharmacology, Sechenov Medical Academy, Moscow, Russia (R.N.A.)
Received October 14, 2005; accepted March 17, 2006.
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). Nanoparticles have previously been shown to enable the transport of a number of drugs across the blood-brain barrier that normally cannot cross this barrier after i.v. injection (Kreuter et al., 1995; Kreuter, 2002; Steiniger et al., 2004). This blood-brain barrier transport was achieved by coating the particles with polysorbate 80 (Tween 80) and led to significant pharmacological and therapeutic effects in the brain (Kreuter et al., 1995, 2002; Alyautdin et al., 1997, 1998; Kreuter, 2002; Steiniger et al., 2004). The nanoparticle-bound drugs included peptides, such as the hexapeptide endorphin dalargin and the dipeptide kyotorphin, as well as other drugs, such as loperamide, tubocurarine, doxorubicin, and the N-methyl-D-aspartate receptor antagonists MRZ 2/576 and MRZ 2/596. Especially important was the finding that binding doxorubicin to nanoparticles greatly improved the survival of rats bearing the extremely aggressive glioblastoma 101/8 intracranially (Steiniger et al., 2004). In 20 to 40% of the rats, a total remission was observed histologically after 6 months, which is in sharp contrast with the normal life expectancy with this tumor of 12 to 18 days.
The mechanism of the enhanced blood-brain barrier transport with polysorbate 80-coated poly(butyl cyanoacrylate) nanoparticles has not yet been fully elucidated. Kreuter et al. (2002) postulated that the polysorbate coating led to the adsorption of apolipoprotein E and possibly apolipoprotein B from the blood after the i.v. injection. Thus, the nanoparticles could mimic lipoprotein particles, which would then interact with the lipoprotein receptors located on the brain capillary endothelial cells. A subsequent endocytosis of the nanoparticles together with bound drugs would then occur.
Apolipoprotein E plays an important role in the transport of lipoproteins to the brain (Nimpf and Schneider, 2000). Lipoproteins bind to and are internalized by the low-density lipoprotein receptor (LDL-R) and the LDL-R-related protein (Ribalta et al., 2003; Dergunov, 2004). The LDL-R is specifically up-regulated on the surface of the endothelium that forms the blood-brain barrier (Dehouck et al., 1997; Lucarelli et al., 2002). Apolipoprotein E-containing particles have been detected in human cerebrospinal fluid (Pitas et al., 1987).
In the present report, apolipoprotein E was coupled to human serum albumin nanoparticles (HSA-NP). HSA-NP are biodegradable (Weber et al., 2000a), easy to prepare in defined sizes (Langer et al., 2003), and carry reactive groups (thiol, amino, and carboxylic groups) on their surfaces that can be used for ligand binding and/or other surface modifications (Langer et al., 2000; Weber et al., 2000b; Wartlick et al., 2004). In contrast to the previously used polysorbate 80-coated poly(butyl cyanoacrylate) nanoparticles, HSA-NP offer the advantage that ligands can easily be attached by covalent linkage. Therefore, apolipoprotein E binding does not depend on adsorptive processes. Moreover, addition of surfactants is not necessary.
The opiate agonist loperamide was chosen as a model drug because loperamide does not exert antinociceptive effects after i.v. injection because it is not able to cross the blood-brain barrier. However, intracerebral administration of loperamide causes antinociceptive effects. Therefore, this drug was used as an indicator for successful transport across the blood-brain barrier (Alyautdin et al., 1997). Loperamide-loaded albumin nanoparticles with chemically bound apolipoprotein E were injected i.v. into ICR mice, and the resulting antinociceptive effects were examined by the tail-flick test (Isabel et al., 1981).
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Materials
Human serum albumin (fraction V), verapamil, and glutaraldehyde 25% aqueous solution were purchased from Sigma (Steinheim, Germany), and 2-iminothiolane/HCl (Traut's reagent), D-Salt Dextran Desalting columns, and NeutrAvidin were obtained from Pierce (Rockford, IL). Anti-apolipoprotein E antibody, alkaline phosphatase-conjugated anti-goat IgG antibody (rabbit), and apolipoprotein E (mixture of all the human apolipoprotein E isoforms) were purchased from Calbiochem (Schwalbach, Germany). Biotin-4-fluorescein was obtained from Molecular Probes Europe BV (Leiden, The Netherlands). Mouse serum from male ICR (CD-1) mice was obtained from Harlan-Winkelmann. Recombinant human apolipoprotein E3, apolipoprotein E2 Arg142Cys, and apolipoprotein E2 Sendai were expressed in the baculovirus system and purified as described before (Hoffmann et al., 2001).
Nanoparticle Preparation
The HSA-NP were prepared by an established desolvation method and were characterized with regard to particle size as described previously (Weber et al., 2000a). The resulting nanoparticles were purified by 3-fold centrifugation (16,100g, 8 min) and redispersion in water. Redispersion was performed in an ultrasonication bath.
The particle sizes of modified and unmodified nanoparticles were measured using a Zetasizer 3000 HSa (Malvern Instruments, Malvern, Worcestershire, UK).
Preparation of NeutrAvidin-Modified Nanoparticles
NeutrAvidin Binding to Nanoparticles. Purified nanoparticles were activated using the cross-linker NHS-PEG3400-Mal (Nektar, Huntsville, AL) to achieve a sulfhydryl-reactive particle system. A volume of 500 µl of cross-linker solution [NHS-PEG3400-Mal, 60 mg/ml in phosphate-buffered saline (PBS), pH 8.0] was added to 2.0 ml of nanoparticle dispersion (20 mg/ml in PBS buffer, pH 8.0). The mixture was incubated under shaking for 1 h at room temperature. Afterward, the activated nanoparticles were purified by centrifugation and redispersion as described above. Thereafter, NeutrAvidin was conjugated to the activated HSA-NP by heterobi-functional cross-linking as described previously (Langer et al., 2000). An aliquot (10.0 mg) of NeutrAvidin was dissolved in 1.0 ml of TEA buffer (pH 8.0), and 1.2 mg of 2-iminothiolane (Traut's reagent) in 1.0 ml of TEA buffer (pH 8.0) was added. After 12-h incubation at room temperature, the thiolated protein was purified by size exclusion chromatography (D-Salt Desalting column). The number of introduced sulfhydryl groups in the NeutrAvidin molecule was measured with 5,5'-dithio-bis-(2-nitrobenzoic acid) (Ellman's reagent) as described previously (Langer et al., 2000; Weber et al., 2000b). For the conjugation, 1 ml of thiolated and purified NeutrAvidin solution was added to 1 ml of sulfhydryl-reactive HSA-NP. The mixture was incubated under shaking for 12 h at room temperature. The nonreacted thiolated NeutrAvidin was removed by nanoparticle centrifugation and redispersion in water. The supernatants of the centrifugation steps were assayed spectrophotometrically at 280 nm to determine uncoupled NeutrAvidin.
Measurement of Free Biotin Binding Sites. NeutrAvidin-modified HSA-NP were analyzed for free biotin binding sites by a noncumulative titration with the fluorescent probe biotin-4-fluorescein at pH 7.5 (Balthasar et al., 2005). A 96-well plate was preincubated with 250 µl of human serum albumin solution (2 mg/ml) for 5 days. After washing, the wells were filled with 150 µl of a suspension of NeutrAvidin-modified HSA-NP adjusted to approximately 20 nM NeutrAvidin concentration. A 400 nM biotin-4-fluorescein solution was added in increasing amounts to the nanoparticle suspension (0–40 µl). After incubation for 3 h at room temperature, the fluorescence was measured by a BMG Fluostar Galaxy microplate reader (Offenburg, Germany).
Loperamide Loading of Nanoparticles
An aliquot (20 mg) of the purified HSA-NP was incubated with 6.6 and 3.3 mg of loperamide in an ethanol/water solution, respectively. After an incubation time of 2 h, the unbound loperamide was removed by centrifugation and redispersion of the nanoparticles in water. For the desorption experiments, aliquots of the drug-loaded nanoparticles were diluted 1:10 with purified water, 10% (v/v) mouse serum, 90% (v/v) mouse serum, and 1% (m/v) polysorbate 80, respectively. After an incubation period of 15 min, the HSA-NP were separated from unbound loperamide by ultrafiltration over a membrane with 30-kDa molecular mass cutoff (Amicon Ultrafree-MC centrifugal filter devices; Millipore, Bedford, MA).
In the collected supernatants and ultrafiltrates, the amount of unbound loperamide was determined by a high-performance liquid chromatography (HPLC) method (see below). The drug loading of the nanoparticles was calculated as the difference between the added initial amount of loperamide and the drug detected in the supernatants.
Apolipoprotein E Surface Modification of NeutrAvidin-Modified Nanoparticles
Apolipoprotein E Biotinylation. To enable the attachment of apolipoprotein E to NeutrAvidin-modified nanoparticles, apolipoprotein E was biotinylated according to a standard protein modification protocol with PFP-Biotin (Pierce). Apolipoprotein E was dissolved in PBS (pH 7.0) at a concentration of 167 µg/ml. The biotinylated protein was separated from low molecular weight compounds by a dextran desalting column. The efficiency of the biotinylation process was determined by Western blot analysis as described below.
Binding of Biotinylated Apolipoprotein E to NeutrAvidin-Modified Nanoparticles. The loperamide-loaded NeutrAvidin-modified nanoparticles were redispersed in water to a particle concentration of 20 mg/ml. Thereafter, 167 µg of biotinylated apolipoprotein E was added, resulting in a final concentration of 10 mg/ml nanoparticles and 80 µg/ml apolipoprotein E. After 12-h incubation, the nanoparticle supernatant was analyzed for unbound apolipoprotein E by immunoblotting as described below.
Determination of Biotin-Apolipoprotein E Binding and Biotinylation Efficiency by Immunoblotting (Western Blot). Polyacrylamide gels (15 g of polyacrylamide/100 ml) were loaded with 15.0-µl aliquots of biotin-apolipoprotein E and the supernatants of the apolipoprotein E-modified nanoparticles. After electrophoresis, gels were transferred to a nitrocellulose membrane, and unspecific binding was blocked with a 5% bovine serum albumin solution. For the detection of unbound antibody, one membrane was incubated with an anti-apolipoprotein E (goat) antibody. In a second step, an anti-goat (rabbit) antibody conjugated with alkaline phosphatase was added. The other membrane was incubated with a streptavidin-alkaline phosphatase conjugate. The alkaline phosphatase on both membranes was reacted with a bromochloroindolyl phosphate and a nitro tetrazolium substrate to a visible dark purple band.
Covalent Binding of Apolipoprotein E to Nanoparticles via a Polyethylene Glycol Cross-Linker
HSA-NP were activated using the cross-linker NHS-PEG3400-Mal to achieve a sulfhydryl-reactive particle system as described above. Thereafter, apolipoprotein E was conjugated to the activated HSA-NP by heterobifunctional cross-linking as shown previously (Langer et al., 2000). Aliquots (500 µg) of different apolipoprotein E derivatives (apolipoprotein E3, apolipoprotein E2 Arg142Cys, and apolipoprotein E Sendai) were dissolved in 1.0 ml of TEA buffer (pH 8.0), and 2-iminothiolane (Traut's reagent) was added in a 50-fold molar excess concentration. After a 12-h incubation period at room temperature, the thiolated protein was purified by size exclusion chromatography (D-Salt column). For the conjugation, 500 µg of thiolated and purified apolipoprotein E was added to 25 mg of sulfhydryl-reactive HSA-NP. The mixture was incubated under shaking for 12 h at room temperature. The unreacted thiolated apolipoprotein E was removed by centrifugation and redispersion of the particles in ethanol/water (2.6% ethanol v/v). Determination of apolipoprotein E binding was performed by immunoblotting as described above.
Approximately 20 mg of the purified apolipoprotein E-modified HSA-NP was incubated with 6.6 mg of loperamide in an ethanol/water solution. After an incubation time of 2 h, the unbound loperamide was removed by centrifugation and determined by HPLC. The loperamide-loaded apolipoprotein E-PEG-NP were redispersed in water.
Preparation of Polysorbate 80-Coated HSA-NP
Nanoparticles without apolipoprotein E but coated with polysorbate 80 were prepared to evaluate whether surfactant coating causes similar effects on loperamide-loaded HSA-NP as previously shown for poly(butyl cyanoacrylate) nanoparticles (Alyautdin et al., 1997). Loperamide was adsorbed to NeutrAvidin-modified nanoparticles as described above. Then, the loperamide-loaded nanoparticles were incubated with polysorbate 80 (1% m/v) solution for 30 min.
Preparation of Loperamide Solution
Loperamide was dissolved in ethanol/water (2.6% ethanol v/v) at a concentration of 0.7 mg/ml.
Determination of Loperamide Loading
The unbound loperamide in the supernatants and ultrafiltrates of the nanoparticles was detected by an HPLC method (Chen et al., 2000). The chromatographic separations were carried out using aliquots (20 µl) of the supernatants collected during particle purification. The aliquots were injected into a Luna 250 x 4.6 mm, 5-µm particle C18 (2) column (Phenomenex, Aschaffenburg, Germany). The flow rate was set to 1.0 ml/min during the separation, with the mobile phase composed of acetonitrile/sodium phosphate solution (pH 2.3, 20 mM)/diethylamine (40:60:0.08, v/v/v) resulting in an elution time of 22 min.
Assessment of P-Glycoprotein Activity
The neuroblastoma cell line UKF-NB-3rVCR10 that shows high P-glycoprotein (P-gp) expression (Kotchetkov et al., 2005; Michaelis et al., 2006) was used for experiments. Functional status of P-gp in UKF-NB-3rVCR10 cells was assessed by a rhodamine-123 dye efflux assay as described previously (Kotchetkov et al., 2003). In brief, 2 x 106 cells were incubated with rhodamine-123 0.4 µM for 60 min, washed twice, and incubated for an additional hour without rhodamine-123 to allow efflux of rhodamine by P-gp. Fluorescence was determined using Becton Dickinson FACScan, CellQuest software (Becton Dickinson) and expressed and quantified in terms of relative fluorescence units.
Animal Testing
The animal experiments were performed in accordance with the German Tierschutzgesetz and the Allgemeine Verwaltungsvorschrift zur Durchführung des Tierschutzgesetzes and were authorized by the Regierungspräsidium Darmstadt (VI 63-19c 20/15-F 116/11). In the study with the NeutrAvidin-modified nanoparticles, mice were divided into six groups with nine animals per group (Table 1). Each mouse in groups 1a (loperamide-loaded HSA-NP linked to apolipoprotein E via biotin-NeutrAvidin complex), 2 (loperamide-loaded, polysorbate 80-coated HSA-NP), 3 (loperamide solution), and 4 (loperamide-loaded HSA-NP) received a dose of 7.0 mg/kg loperamide into the tail vein. The animals of group 1b (loperamide-loaded HSA-NP linked to apolipoprotein E via biotin-NeutrAvidin complex) received a dose of 4.0 mg/kg loperamide into the tail vein. The mice of group 5 received 100 mg/kg empty nanoparticles into the tail vein, equivalent to the nanoparticle concentration that was administered to mice of groups 1a, 1b, 2, and 4.
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Apolipoprotein E3, apolipoprotein E2 Sendai, and apolipoprotein E2 Arg142Cys were directly bound to the HSA-NP via a PEG cross-linker as outlined above. Animals were divided into three groups, each consisting of nine animals per group (Table 2). Each mouse received a dose of 7.0 mg/kg loperamide.
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The nociceptive threshold of the mice was tested after the injection of the different preparations using the tail-flick test (Tail-Flick Analgesia Meter; Ugo Basile, Comerio, Italy). The tail-flick analgesia meter measures pain sensitivity in mice as they respond to the application of heat to a small area of their tails.
To prevent tail tissue damage, the experiments were terminated after 10 s if no response was evoked (cutoff time). This time point was considered to indicate complete analgesia. For each animal, the tail-flick latency was determined before dosing of any preparation (= predrug latency). Tail-flick latencies were measured at 15, 30, 45, and 60 min after dosing. In groups with analgesic effects, the tail-flick test was performed over a period up to 180 min.
The response latencies were converted to percent maximal possible effect (%MPE) using the following term (eq. 1):
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), the covalent attachment of the avidin derivative to the particle surface was performed by the use of a poly(ethylene glycol)-based cross-linker (Fig. 1). Thiolation of NeutrAvidin using 2-iminothiolane resulted in 1.93 ± 0.93 mol of sulfhydryl groups per mole of NeutrAvidin. After conjugation of PEG cross-linker activated HSA-NP to the sulfhydryl groups of NeutrAvidin, monodisperse nanoparticles with a particle diameter of 340 ± 8.6 nm were obtained. The photometric analysis of the collected supernatants of the purification steps showed that 16 to 21% of the used Neutr-Avidin was covalently attached to the nanoparticle surfaces. Control experiments with nonactivated nanoparticles revealed no nonspecific NeutrAvidin adsorption. Approximately 3.69 x 10–10 mol of NeutrAvidin was bound per milligram of nanoparticles. A mathematical calculation indicated that approximately 6000 NeutrAvidin molecules were attached to one nanoparticle.
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Measurement of Free Biotin Binding Sites. The capacity of the coupled NeutrAvidin to bind biotin was assessed by a biotin-4-fluorescein assay (Fig. 2). The titration experiment resulted in a hump region reflecting progressive quenching of specifically bound biotin-4-fluorescein. A steep linear increase in fluorescence occurred after saturation of the biotin binding sites. The saturation corresponds to the breakpoint of the two resulting curves. The breakpoint of the curves in Fig. 2 showed a saturation of the biotin binding sites at a 43.9 nM biotin-4-fluorescein concentration. Therefore, 2.92 ± 0.3 (n = 6) biotin binding sites out of 4 per avidin molecule on the particle surface were detected for NeutrAvidin-modified HSA-NP.
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Similar results were obtained in Western blots for the direct covalent coupling of apolipoprotein E3, apolipoprotein E2 Sendai, and apolipoprotein E2 Arg142Cys to NHS-PEG3400-Mal-activated HSA-NP: coupling of apolipoprotein E3 and apolipoprotein E2 Sendai resulted in 19 ± 1 µg of apolipoprotein per milligram of nanoparticles, and apolipoprotein E2 Arg142Cys coupling resulted in 11 ± 2 µg of apolipoprotein per milligram.
Loperamide Adsorption. After incubation of 20 mg of HSA-NP with 6.6 and 3.3 mg of loperamide, a loading efficiency of 92.7 or 84.6 µg of loperamide per milligram of HSA-NP was obtained, respectively (Fig. 4). To prove the drug-loading stability in the presence of serum or polysorbate 80, the preparations were diluted 1:10 with water, 10% mouse serum, 90% mouse serum, or 1% polysorbate 80. Under all the conditions, no significant reduction of the loperamide loading efficiency was observed. This shows that because of its lipophilic nature, the adsorption of loperamide to nanoparticle surfaces led to a stable drug loading.
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Assessment of P-gp Activity in the Presence of Nanoparticles. The neuroblastoma cells UKF-NB-3rVCR10 were treated with 200 µg/ml nanoparticles during and after rhodamine-123 incubation. Verapamil inhibits P-gp activity. Therefore, verapamil (5 µg/ml) treatment served as positive control for P-gp inhibition. Nanoparticles may induce a fluorescence shift by autofluorescence. Incubation of unstained cells with nanoparticles did not result in a significant fluorescence signal (data not shown). Verapamil (5 µg/ml) caused a more than 30-fold increase in rhodamine-123 intracellular accumulation (Fig. 5). In contrast, treatment with the nanoparticle preparations did not result in a significant increase of rhodamine-123 accumulation. This shows that nanoparticles do not inhibit P-gp function.
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Animal Testing. Loperamide-loaded HSA-NP linked via biotin-NeutrAvidin complexes to apolipoprotein E induced analgesic effects in a dose-dependent manner (Fig. 6). Treatment of animals with loperamide-loaded apolipoprotein E HSA-NP (7 mg/kg loperamide) (group 1a) resulted in 100% MPE as early as 15 min after administration. After 120 min, no statistically significant analgesic effects were detectable. Treatment of animals with loperamide-loaded apolipoprotein E HSA-NP (4 mg/kg loperamide) (group 1b) resulted in 92% MPE. The use of loperamide-loaded polysorbate 80-coated HSA-NP (group 2) resulted in 100% MPE within 15 min after administration but, compared with group 1a, started to decline earlier. Treatment of animals in group 3 (7 mg/kg loperamide solution), group 4 (loperamide-loaded HSA-NP, 7 mg/kg loperamide), or group 5 (unloaded apolipoprotein E nanoparticles) did not exhibit statistically significant analgesic effects. Evaluation of noneffective controls was finished after 60 min to avoid unnecessary harm to the animals.
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). In accordance with previous results, directly after desolvation HSA-NP could be prepared in a defined size of 200 nm (Langer et al., 2003). Avidin and biotin form the most stable naturally occurring complex (Ka = 1015 M–1) (Green, 1990). The use of avidin-biotin complexes for the coupling of molecules to HSA-NP has been described before by our group (Langer et al., 2000; Wartlick et al., 2004; Balthasar et al., 2005). Avidin was coupled to the amino groups by the heterobifunctional cross-linker NHS-PEG3400-Mal (Wartlick et al., 2004). The conjugation of functional proteins by protein chemistry bears the risk of losing protein functionality. Therefore, a quantification of the biotin binding sites was performed by the biotin-4-fluorescein assay modified after Kada et al. (Balthasar et al., 2005). Our results revealed 2.9 biotin binding sites per coupled NeutrAvidin molecule. This shows that not all the theoretically available biotin binding sites (four per NeutrAvidin molecule) were available for biotin binding. Steric hindrance and binding of the molecules at the active binding sites are likely to be responsible for this result.
The first nanoparticle system that was shown to transport molecules across the blood-brain barrier was polysorbate 80-coated poly(butyl cyanoacrylate) nanoparticles (Alyautdin et al., 1997, 1998; Kreuter, 2002). There are different possibilities how this transport of substances across the blood-brain barrier may be facilitated by polysorbate 80-coated nanoparticles: 1) NP may adsorb at the endothelium forming the blood-brain barrier and therefore yield a concentration gradient leading to an enhanced brain uptake of the delivered drug; 2) the surfactant may affect the blood-brain barrier integrity by solubilization of membrane lipids; 3) nanoparticles may induce opening of the tight junctions; 4) the surfactant may inhibit the blood-brain barrier multidrug efflux system; 5) nanoparticles may be endocytosed into the brain endothelial cells and release their transported substances that are further delivered to the brain (Kreuter, 2004); or 6) nanoparticles may be transcytosed through the endothelium forming the blood-brain barrier. Possibly, various mechanisms contribute to the brain uptake of drugs mediated by polysorbate 80-coated poly(butyl cyanoacrylate) nanoparticles. However, there are some indications that favor the idea that nanoparticle binding to a lipoprotein receptor like LDL-R or low-density lipoprotein receptor-related protein (LRP) may play a critical role during brain drug uptake mediated by polysorbate 80-coated poly(butyl cyanoacrylate) nanoparticles. Apolipoprotein E binds to lipoprotein receptors including LDL-R (De Loof et al., 1986; Kreuter, 2004) and LRP (Croy et al., 2004; Dergunov, 2004), and apolipoprotein E was found to be specifically adsorbed on the surface of polysorbate 80-coated poly(butyl cyanoacrylate) nanoparticles from human citrate-stabilized plasma (Kreuter, 2004). Moreover, the LDL-R is up-regulated on the brain endothelium compared with peripheral vessels (Vasile et al., 1983; Meresse et al., 1989; Dehouck et al., 1997; Lucarelli et al., 2002), and lipoprotein particles seem to be transported across the blood-brain barrier by transcytosis (Dehouck et al., 1997). Coating of poly(butyl cyanoacrylate) nanoparticles with apolipoprotein E and apolipoprotein B in the absence of surfactant was sufficient to enable drug transport to the brain (Kreuter et al., 2002). Taken together, these findings suggest that adsorption of apolipoprotein E to polysorbate 80-coated poly(butyl cyanoacrylate) nanoparticles and subsequent apolipoprotein E binding to a receptor of the lipoprotein receptor family followed by endocytosis and/or transcytosis may be a central mechanism during polysorbate 80-coated poly(butyl cyanoacrylate)-NP-mediated drug trans-port to the brain.
Here, we investigated whether HSA-NP with covalently linked apolipoprotein E also was able to transport loperamide to the brain. Loperamide does not cross the blood-brain barrier but exerts antinociceptive effects when administered directly to the brain (DeHaven-Hudkins et al., 1999). Investigation of loperamide-loaded, apolipoprotein E-coupled HSA-NP revealed strong antinociceptive effects, whereas nonmodified HSA-NP were unable to transport loperamide across the blood-brain barrier. This further underlines that binding of apolipoprotein E to the nanoparticles can mediate drug transport to the brain. Moreover, the use of a well defined system that does not depend on adsorption processes but carries apolipoprotein E covalently fixed to the nanoparticle surface has large advantages. Such a system avoids intravasal application of surfactants, which may lead to significant adverse effects (Varma et al., 1985; Gelderblom et al., 2001). Although loperamide was simply adsorbed to the surface of the nanoparticles, loading and desorption experiments revealed a stable drug attachment even in the presence of different concentrations of serum or surfactants such as polysorbate 80. Therefore, compared with poly(butyl cyanoacrylate)-NP, the apolipoprotein E-coupled HSA-NP fulfills the requirements for a surfactant-free and well defined system with stable drug and drug targeting ligand modifications.
In humans, three common variants of apolipoprotein E exist. Apolipoprotein E3 is the most frequent one, followed by apolipoprotein E4 and apolipoprotein E2 in Caucasian populations (Davignon et al., 1988; Corder et al., 1993; Weisgraber, 1994). For our initial experiments with NeutrAvidin-modified HSA-NP, a mixture of all three apolipoprotein E molecules was used. However, within the apolipoprotein E protein family, apolipoprotein E3 and apolipoprotein E4 show a higher binding affinity to the LDL-R compared with apolipoprotein E2 (Hoffmann et al., 2001; Ribalta et al., 2003). Therefore, in following experiments, different apolipoprotein E molecules were used to shed further light on the uptake mechanism of apolipoprotein E-coupled nanoparticles to the brain. Apolipoprotein E2 is defective in binding to lipoprotein receptors (Schneider et al., 1981; Dong et al., 1996). In accordance with our hypothesis, apolipoprotein E2 Sendai, which is characterized by <5% LDL-R binding compared with apolipoprotein E3 (Hoffmann et al., 2001), did not enable a loperamide transport to the brain when coupled to loperamide-loaded HSA-NP. In contrast, apolipoprotein E3-modified loperamide-loaded HSA-NP exerted antinociceptive effects, indicating that apolipoprotein E3 alone is sufficient to mediate loperamide transport to the brain. Investigation of loperamide-loaded HSA-NP coupled to apolipoprotein E2 Arg142Cys, an apolipoprotein E3 analog associated with the dominant form of type III hyperlipidemia with reduced LDL-R binding similar to apolipoprotein E2 Sendai (M. M. Hoffmann, unpublished) and with strongly diminished heparin binding (Ji et al., 1994), yielded no antinociceptive effects. These data suggest that apolipoprotein E3 mediates drug transport to the brain by a specific process and not by nonspecific events. Based on the results of the P-gp activity assay, a simple inhibition of the P-gp function by apolipoprotein E3-modified nanoparticles can be excluded. Apolipoprotein E2 Sendai LDL-R binding is substantially decreased, whereas heparin binding is only slightly affected (Hoffmann et al., 2001), supporting the hypothesis that apolipoprotein E-coupled nanoparticles may mimic lipoprotein particles. These lipoprotein particles may interact with receptors present on the blood-brain barrier (e.g., LDL-R, LRP), which mediate drug transport into the brain.
In this scenario, apolipoprotein E-coated nanoparticles would act as "Trojan Horses" for bound drugs. Bound drugs then may further be transported into the brain by diffusion after release within the endothelial cells or, alternatively, by transcytosis, which was observed in vitro for LDL by Dehouck et al. (1997). Therefore, apolipoprotein E or polysorbate-coated nanoparticles hold great promise as carriers for brain and central nervous system delivery of a variety of essential drugs, including cytostatics for brain tumors and peptides (Kreuter et al., 1995; Kreuter, 2002), for which the blood-brain barrier is normally an impermeable barrier.
In conclusion, we show in this report that HSA-NP coupled to apolipoprotein E are sufficient to mediate loperamide transport to the brain. Our data suggest that only apolipoprotein E proteins that have a high binding affinity to lipoprotein receptor are able to mediate loperamide transport into the brain. Therefore, apolipoprotein E-coupled nanoparticles may mimic lipoprotein particles that are endocytosed into the blood-brain barrier endothelium and transcytosed through the blood-brain barrier endothelium into the brain.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: LDL-R, low-density lipoprotein receptor; HSA-NP, nanoparticle(s) made of human serum albumin; PBS, phosphate-buffered saline; TEA, triethanolamine; HPLC, high-performance liquid chromatography; PEG, polyethylene glycol; P-gp, P-glycoprotein; %MPE, percentage maximal possible effect; LRP, low-density lipoprotein receptor-related protein.
Address correspondence to: Klaus Langer, Institute for Pharmaceutical Technology, Biocenter of Johann Wolfgang Goethe-University, Marie-Curie-Strasse 9, D-60439 Frankfurt, Germany. E-mail: k.langer{at}em.uni-frankfurt.de
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apolipoprotein E HSA-NP with 7.0 mg/kg loperamide; 
apolipoprotein E HSA-NP with 4.0 mg/kg loperamide; 
HSA-NP with loperamide (no apolipoprotein E attached); 
loperamide solution; 
apolipoprotein E HSA-NP without loperamide; and 
polysorbate 80-coated HSA-NP with 7.0 mg/kg loperamide.