Polypeptide Point Modifications with Fatty Acid and Amphiphilic Block

Bioconjugate Chem. 2005, 16, 793−802

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Polypeptide Point Modifications with Fatty Acid and Amphiphilic Block Copolymers for Enhanced Brain Delivery Elena V. Batrakova,† Serguei V. Vinogradov,† Sandra M. Robinson,‡ Michael L. Niehoff,‡ William A. Banks,‡ and Alexander V. Kabanov*,† Department of Pharmaceutical Sciences and Center for Drug Delivery and Nanomedicine, College of Pharmacy, University of Nebraska Medical Center, 985830 Nebraska Medical Center, Omaha, Nebraska 68198, and GRECC Veterans Affairs Medical Center-St. Louis and Division of Geriatrics, Department of Internal Medicine, Saint Louis University School of Medicine, 915 North Grand Boulevard, St. Louis, MO 63104. Received November 9, 2004; Revised Manuscript Received April 29, 2005

There is a tremendous need to enhance delivery of therapeutic polypeptides to the brain to treat disorders of the central nervous system (CNS). The brain delivery of many polypeptides is severely restricted by the blood-brain barrier (BBB). The present study demonstrates that point modifications of a BBB-impermeable polypeptide, horseradish peroxidase (HRP), with lipophilic (stearoyl) or amphiphilic (Pluronic block copolymer) moieties considerably enhance the transport of this polypeptide across the BBB and accumulation of the polypeptide in the brain in vitro and in vivo. The enzymatic activity of the HRP was preserved after the transport. The modifications of the HRP with amphiphilic block copolymer moieties through degradable disulfide links resulted in the most effective transport of the HRP across in vitro brain microvessel endothelial cell monolayers and efficient delivery of HRP to the brain. Stearoyl modification of HRP improved its penetration by about 60% but also increased the clearance from blood. Pluronic modification using increased penetration of the BBB and had no significant effect on clearance so that uptake by brain was almost doubled. These results show that point modification can improve delivery of even highly impermeable polypeptides to the brain.

INTRODUCTION

Efficient delivery of therapeutic polypeptides across the blood-brain barrier (BBB) could result in improved diagnostics and treatment of diseases of central nervous system (CNS). Some examples include Parkinson’s and Alzheimer’s diseases (1-3), stroke (4, 5), lysosomal storage diseases (6-8), and human obesity (9, 10). The BBB is one of the most restrictive barriers in biology, which provides a formidable impediment for delivery of many polypeptides to the brain. Some peptides and regulatory proteins cross the BBB by saturable or nonsaturable mechanisms, whereas others cannot cross. Artificial hydrophobization of polypeptides with a small number of fatty acid residues (e.g., stearate or palmitate) was shown to enhance cellular uptake of these polypeptides in vitro (11). As a result of such modification, the polypeptide molecule remains water-soluble but also acquires lipophilic anchors that can target even very hydrophilic molecules to cell surfaces (12). The studies carried out in Russia over a decade ago have shown that modification of the Fab fragments of antibodies against gliofibrillar acid protein (GFAP) and brain-specific R-2glycoprotein (R-2GP) with stearate moieties led to an increased accumulation of the modified Fab fragments in the brains of rats (13, 14). Furthermore, a neuroleptic drug conjugated with stearoylated antibody Fab fragments was much more potent compared to the free drug. In comparison, fatty acylated Fab fragments of nonspecific antibodies did not accumulate in the brain but * To whom correspondence should be addressed. Tel: (402) 559-9364. Fax (402) 559-9365. [email protected]. † University of Nebraska Medical Center. ‡ Saint Louis University School of Medicine.

instead accumulated in the liver, while stearoylated Fab fragments of brain-specific antibodies displayed preferential accumulation in the brain. Subsequent studies using bovine brain microvessel endothelial cells (BBMEC) as an in vitro model of BBB demonstrated that stearoylation of ribonuclease A (approximately 13.6 kDa) increases the passage of this enzyme across the BBB by almost 9-fold (15). Based on this foundation the present work focused on the development of optimal modifications of polypeptides to enhance their delivery to the brain. The studies reported here show that chemical modification of polypeptides with lipophilic and amphiphilic groups can increase the permeability of a model polypeptide, horseradish peroxidase (HRP), 40 kDa, across the BBB in vitro and in vivo. More specifically, modification of HRP with (1) stearoyl chloride or (2) Pluronic block copolymers (poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide)) increased the rate of transport of HRP across the BBB and enhanced accumulation of HRP within the brain. The paper reports for the first time conjugation of a protein with amphiphilic block copolymer moieties via biodegradable disulfide links and demonstrates that such a modification is most successful for enhanced polypeptide delivery to the brain. EXPERIMENTAL PROCEDURES

Materials. Horseradish peroxidase type VI, MW 44 kDa (HRP), stearoyl chloride, sulfosuccinic acid bis(2ethylhexyl)ester (AOT), dithiobis(succinimidylpropionate) (DSP), disuccinimidylsuberate (DSS), 4-methoxytrityl chloride (MTr-CL), 1,1′-carbonyldiimidazole, ethylendiamine, 2,4,6-trinitrobenzenesulfonic acid (TNBS), trifluoroacetic acid, acetone, dichloromethane, dimethylform-

10.1021/bc049730c CCC: $30.25 © 2005 American Chemical Society Published on Web 06/24/2005

794 Bioconjugate Chem., Vol. 16, No. 4, 2005

Batrakova et al.

Scheme 1. Synthesis of Pluronic-HRP Conjugatesa

a Step 1: Pluronic-amine is activated by bifunctional reagents (DSP or DSS) to obtain hydroxysuccinimide derivatives of Pluronic capable of reacting with the protein amino groups. Step 2: the excess of the reagents was removed by gel filtration, and the activated polymers were immediately reacted with HRP.

amide, anhydrous acetonitrile, and pyridine were obtained from Sigma-Aldrich Co. (St-Louis, MO). Pluronic P85 (lot no. WPOP-587A) and Pluronic L121 (lot no. WPAC-550B) were kindly provided by BASF Corp. (Parispany, NJ). All other reagents were of analytical grade. Chemical Modification of HRP with Stearic Acid Chloride in Reverse Micelles. Modification of amino groups of HRP by stearoyl chloride was performed in the reverse micelle system of AOT in octane as described earlier (12). Briefly, HRP solution (2.5 mg/mL) in 0.1 M sodium borate buffer (pH 9.5) was mixed with a 10-fold volume excess of 0.3 M AOT solution in octane. The emulsion was vigorously shaken for 10 min until an optically transparent solution was obtained. Following a 2-hour incubation at room temperature, the protein was precipitated in a 5-fold volume excess of cold acetone (-20 °C) with subsequent centrifugation at 2000 rpm. The precipitate was washed three times with cold acetone, dried, and dissolved in 0.1 M borate buffer (pH 9.5). The product was purified of low molecular residuals by gelpermeation chromatography on a Sephadex G25 column (Amersham, Piscataway, NJ). The protein recovery was determined by BCA Pierce method (16). The modification degree (the number of fatty residues introduced in a protein molecule) of HRP was determined by titration of free amino groups with TNBS as described below. Conjugation of HRP with Pluronic Block Copolymers. Preparation of Monoamino Derivatives of Pluronics P85 and L121. A Pluronic molecule contains two terminal hydroxyl groups, both of which can be used for conjugation with proteins. To obtain the monoamino derivative of Pluronic, one of the hydroxyl functions was protected with a 4-methoxytrityl (MTr) group. For this purpose, Pluronics (P85 or L121) were dried by coevaporation with anhydrous pyridine in vacuo at 50 °C. Then, the dried polymer was mixed with an equimolar amount of MTrCL in anhydrous pyridine, and the obtained mixture was incubated at room temperature for 3 h. The reaction was stopped by addition of methanol, and pyridine was

removed in vacuo by coevaporation with toluene. The monosubstituted product, MTr-Pluronic, was purified from nonmodified polymer and traces of bis-MTr-Pluronic by absorption chromatography on a Silicagel column (4.5 cm × 10 cm) in dichloromethane using stepwise elution with dichloromethane containing 2%, 5%, or 10% of methanol. Mono-MTr-Pluronic was dried by coevaporation with anhydrous acetonitrile in vacuo, and then incubated with 3-fold molar excess of 1,1′-carbonyldiimidazole in anhydrous acetonitrile for 4 h at 40 °C. Activated Pluronic was dissolved in ethanol and added dropwise to 10-fold excess of ethylenediamine in ethanol to obtain polymer with one side amino group as described elsewhere (17). The product was dialyzed against 10% ethanol to remove low molecular weight compounds. Removal of the MTr group from the polymer was achieved with 2% trifluoroacetic acid in dichloromethane. All synthetic steps have been controlled by thin-layer chromatography on Silicagel-coated plates using a dichloromethane/methanol (9:1) mixture as an eluent. Monoamino derivatives of Pluronics have been isolated by gelpermeation chromatography on the Sephadex LH-20 column (2.5 cm × 45 cm) in methanol as eluent. Colorimetric analysis of fractions with ninhydrine detected the presence of amino groups in the product. Amino-group content was analyzed by titration of the polymers with TNBS (18) and was equivalent to >45% substitution of all hydroxyl groups in Pluronics. Conjugation of HRP with Monoamino Derivatives of Pluronics P85 and L121 via a Biodegradable Link (DSP method). To obtain a biodegradable link, HRP was conjugated with Pluronics through a reducible disulfide bond (Scheme 1). Pluronic-amine is activated by a bifunctional reagent, DSP, to obtain a hydroxysuccinimide derivative of Pluronic capable of reacting with the protein amino groups (step 1). Excess of the reagent was removed by gel filtration, and the activated polymers were immediately reacted with HRP (step 2). Briefly, monoamino derivatives of Pluronics were first dissolved

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Polypeptide Modifications for Enhanced Brain Delivery

in methanol (70 µmol) and added dropwise to a stirred solution of DSP (0.2 mmol) in dimethylformamide at 25 °C. Following the 30-min reaction at room temperature, the DSP-modified Pluronic was purified by gel-filtration chromatography on a Sephadex LH 20 column in methanol. The product was concentrated in vacuo and dissolved in 20% ethanol. A solution of HRP (2 µmol) in 0.1 M borate (pH 9.5) was mixed with a DSP-modified Pluronic solution prepared earlier, and the homogeneous reaction mixture was incubated overnight at 4 °C. Modified HRP was precipitated in cold (-10 °C) ethanol, incubated at 4 °C for 30 min, and separated from the excess of DSPmodified Pluronic by centrifugation at 3000 rpm for 20 min at 4 °C. Brown precipitate was washed with cold ethanol and dried in vacuo. Pluronic-HRP conjugate was purified by cation-exchange chromatography on TSK CM650M resin with 10 mM sodium acetate (pH 4.4), 10 mM NaCl, and 5% ethanol as an eluent. First, elution of Pluronic-HRP was performed with this eluent, and then a gradient of NaCl (from 0.01 to 0.15 M) was used for elution of residual (unconjugated) HRP. Collected fractions were desalted in dialysis tubes with a molecular weight cutoff of 20 kDa (SpectroPor, Fisher Scientific, Pittsburg, PA) against water overnight at 4 °C and freezedried. The obtained conjugate was analyzed for protein and salt contents using the analytical procedures described below. Conjugation of HRP with Monoamino Derivatives of Pluronics P85 and L121 by Nonbiodegradable Bond (DSS method). To obtain a nonbiodegradable link, monoamino derivatives of Pluronics were conjugated with HRP similar to the method described above except using DSS, instead of DSP (Scheme 1). Analysis of HRP Conjugates. Protein Assay. The BCA Kit from Pierce (Rockford, IL) was used to measure protein content in the obtained HRP conjugates. The obtained conjugates (1.0 mg/mL) were dissolved in water and mixed with coloring solutions according to the manufacturer’s protocol. Mixtures were incubated for 30 min at 37 °C, and absorbance values were measured at 550 nm using a microplate reader Multiskan MCC/340 (Fisher Scientific, Pittsburgh, PA) as described elsewhere (16). HRP Enzymatic Activity. The enzymatic activity of HRP in the obtained conjugates was assessed with o-phenylenediamine as described earlier (12). Briefly, conjugate solutions (0.1 mg/mL) were mixed in a 96-well plate with the solution of o-phenylenediamine (5 mg/mL) in 0.1 M citrate buffer (pH 5) containing 1 mg/mL bovine serum albumin (BSA), 0.1% Triton, and hydrogen peroxide solution (0.02%). After incubating the mixture for 5 min at 37 °C, the reaction was stopped by the addition of 0.5% sodium sulfite in 2 M sulfuric acid, and absorbance was measured at 550 nm using the microplate reader Multiskan MCC/340. The activities were related to HRP concentration in the sample. Modification Degree. The modification degree of HRP (the number of fatty residues or Pluronic molecules introduced in a protein molecule) was determined by titration of the free amino groups of the protein with TNBS as described elsewhere (18). Briefly, solutions of native and modified protein (1 mg/mL) were mixed with 5% TNBS solution in 0.1 M sodium borate buffer (pH 9.5) in 96-well plate. Reaction mixtures were incubated for 1 h at 25 ° C, and absorbance was measured at 405 nm using the microplate reader. The degree of modification was calculated using following equation:

Si ) 4No(AoNo - Ai/Ni)/Ao

(1)

where No and Ni were protein content and Ao and Ai were amino group content in native and modified protein, respectively. Binding of HRP Conjugates to the Brain Endothelial Cells. BBMEC were used as an in vitro model of the BBB in these studies. BBMEC were isolated from cow brains using mechanical and enzymatic disruption of brain matter coupled with gradient separation as described earlier (19). Cells were seeded onto collagencoated, fibronectin-treated 24-well culture plates at a density of 50 000 cells/cm2 and grown using media consisting of 45% minimum essential medium (MEM), 45% Ham’s F-12 (F12), and 10% horse serum supplemented with antibiotics and heparin sulfate and used after reaching confluency (typically 10-12 days). All tissue culture media was obtained from Gibco Life Technologies, Inc. (Grand Island, NY). The BBMEC monolayers were incubated with unmodified HRP or the conjugates for various time points up to 120 min. Then, the cell monolayers were washed with 1% BSA in phosphate-buffered saline (PBS) and then washed in BSA-free PBS. The cells were then lysed in 1% Triton X100, and the net amount of the cell-bound enzyme was determined by colorimetric reaction with o-phenylenediamine in 0.1 M citrate buffer (pH 5) containing 0.1% Triton X100, 1 mg/mL BSA, and 0.02% hydrogen peroxide as described above. The amount of HRP was normalized by the amount of cell protein as determined by the Pierce BCA assay. Transport of HRP Conjugates across BBMEC Monolayers. Isolated BBMEC were seeded on fibronectin- and collagen-coated polycarbonate membrane inserts (Transwell, Costar Permeability Supports, Cole-Parmer Instruments Co, Vernon Hills, IL, Contd.; pore size 0.4 µm; diameter 24.0 mm) at a density of 250 000 cells per insert and allowed to grow and differentiate for up to complete maturation of the monolayers (typically within 14 days). The trans-epithelial electrical resistance (TEER) of the monolayers was recorded as an index of cell viability and monolayer integrity. The TEER values of the confluent BBMEC monolayers were no less than 130.0 Ω‚cm2. Polycarbonate membrane inserts with confluent BBMEC monolayers were placed in Side-Bi-Side diffusion cells from Crown Bio Scientific, Inc. (Somerville, NJ) maintained at 37 °C, and the transport of HRP conjugates across cell monolayers was determined as described earlier (20). Briefly, for luminal to abluminal transport studies, the assay buffer at the luminal (apical) side was removed and replaced with the assay buffer containing solutions of the modified or native HRP (40 µg/mL). The solutions in the donor chamber also contained 1 nM H3-mannitol as a paracellular marker (DuPont Corp., Boston, MA). At various time points, the solutions in the receiver chamber (at the basolateral side of the monolayers) and aliquots (20 µL) from the donor chamber (at the apical side of the monolayers) were removed for the determination of the HRP concentration. The enzyme activity of HRP was measured using the o-phenylenediamine method as described above. 3Hmannitol concentrations were determined using Beckman LS 6000 IC liquid scintillation counter (Beckman Counter, Inc., Fullerton, CA). All transport experiments were conducted in triplicate. Apparent permeability coefficients (Papp) of the polypletides were calculated using the following equation:

Papp )

1 dQ AC0 dt

(2)


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where dQ/dt is the flux across the cell monolayers, A is the surface area of the membrane, and C0 is the initial concentration of the drug. Localization of HRP Conjugates in BBMEC Monolayers. For this study, HRP conjugated with Pluronic P85 via the biodegradable DSP bond (Scheme 1) was used. The native and modified proteins were labeled with Alexa Fluor 594 protein labeling kit (Molecular Probes Inc., Eugene, OR) according to the manufacture’s protocol. BBMEC grown on chamber slides (Fisher, St. Louis, MO) were incubated with Alexa-labeled native or P85modified HRP (1 mg/mL) in assay buffer for 2 h at 37 °C. Following incubation, the loading solutions were removed, and cell monolayers were washed three times with ice-cold PBS containing 1% BSA and examined with a Zeiss LSM 410 confocal laser scanning microscope, CLSM (Goettinger, Germany). All pictures were taken at the same brightness and contrast. In Vivo Transport of St-HRP and Pluronic-HRP Conjugates to the Brain. Radioactive Labeling of HRP. Native, Pluronic P85-modified, or stearoyl-modified HRP were labeled by the chloramine-T method (i.e., excluding the sodium metabisulfite treatment) (21). Briefly, 1 mCi of 131I (New England Nuclear, Boston, MA), 10 µg of chloramine T, and 5.0 µg of protein were incubated together for 60 s. The reaction was halted by adding 10 µg of sodium metabisulfite. The iodinated materials were purified by filtration on Sephadex G10 (Sigma Chemical Co., St. Louis, MO) columns. Incorporation of the radioactive 131I, as determined by TCA/brine precipitation, was greater than 90% for all materials, and the specific activities were estimated to be between 50 and 150 Ci/g of protein. Albumin was labeled with 99mTc with stannous tartrate (22) and purified on a column of Sephadex G-10. Measurement of Unidirectional Influx Rates. Male CD-1 mice (Charles River Labs, Wilmington, MA) weighing 20-30 g were anesthetized with an ip injection of 40% ethyl carbamate. The left jugular vein and right carotid artery were isolated. A volume of 0.2 mL of 1% BSA in lactated Ringer’s solution was injected into the jugular vein. The injection contained 1 × 105 cpm of a radioactively iodinated native or Pluronic- or stearoylmodified HRP. Between 2 and 180 min after injection, blood was obtained from the carotid artery, and the mouse was decapitated. The brain was removed, the pineal and pituitaryglands were discarded, the remainder of the brain was weighed, and the levels of radioactivity were determined in a γ counter that could simultaneously count 131I. The whole blood was centrifuged at 5000g for 10 min at 4 °C, and the level of radioactivity was determined in the resulting serum. The brain/serum ratio was calculated with the equation

brain/serum ratio (µL/g) ) (cpm/g of brain)/ (cpm/µL of serum) (3) To adjust for clearance of the protein from the blood, exposure time (expt) was calculated from the equation

expt ) [I0tCp(J)dJ]/Cpt

(4)

where Cpt is the level of radioactivity in serum at time t, and J is the dummy variable for time (23, 24). Brain/ serum ratios of the protein content were plotted against expt, and the regression line for the linear portion of the relation was calculated. The slope of this relation measures Ki and the intercept measures the distribution volume within brain at t ) 0 (Vi in units of µL/g). Lack of a statistically significant relation between the brain/

serum ratios and expt indicates either that the protein does not cross the BBB or that a steady state between the rate of influx and efflux has already been reached. Calculation of the Percent of Injected Proteins Taken up by Brain. The clearance of the radioactively labeled native, Pluronic-modified, or stearoyl-modified HRP from blood was first characterized. The amount of radioactivity injected intravenously was divided by the level of radioactivity in serum to yield the percent of injected radioactivity present in 1 mL of serum (% inj/mL):

% inj/mL ) 100(cpm of injected radioactivity)/ (cpm/mL of serum) (5) To calculate the percent of the injected dose of the protein entering a gram of brain (% inj/g), % inj/mL was divided by 1000 to convert it to units of % inj/µL. The brain/serum ratios for the HRPs were corrected for vascular contamination by subtracting10 µL/g. The resulting brain/serum ratio was then multiplied by % inj/ µL to yield % inj/g. The area under the curve (AUC) was calculated with Prism 4.0 software, which uses the trapezoidal method. Capillary Depletion. To distinguish whether the native, stearoyl-modified, or Pluronic-modified HRP taken up by brain tissue was sequestered by the brain endothelial cells, which comprise the BBB or was transported fully across them into brain tissue, we performed capillary depletion with the protocol adapted to mice (25) from rats (26). CD-1 male mice anesthetized with ip ethyl carbamate received an iv injection of 0.2 mL of lactated Ringer’s solution containing 1% BSA, 1 × 106 cpm of a radioactively labeled native, stearoyl-modified, or Pluronicmodified HRP, and 1 × 106 of 99mTc-labeled albumin. Blood from the abdominal aorta was collected. The thorax was then opened to expose the heart, both jugular veins were severed, the descending thoracic aorta was clamped, and 20 mL of lactated Ringer’s solution was injected into the left ventricle of the heart within about 1 min. The cerebral cortex was removed 30 or 120 min after iv injection. The cerebral cortex was weighed and emulsified with a glass homogenizer (10 strokes) in 0.8 mL of physiological buffer (10 mM HEPES, 141 mM NaCl, 4 mM KCl, 2.8 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, and 10 mM D-glucose adjusted to pH 7.4). Dextran solution (1.6 mL of a 26% solution) was added to the homogenate, and the suspension was vigorously mixed and homogenized again (3 strokes). Homogenization was performed at 4 °C in less than 1 min. An aliquot of the homogenate was centrifuged at 5400g for 15 min at 4 °C in a Beckman TL-100 ultracentrifuge with a TLS-55 swinging bucket rotor. The pellet containing the brain vasculature and the supernatant containing the brain parenchyma were carefully separated. The levels of radioactivity in these fractions and in the aortic serum were determined in a γ counter. The fractions were expressed as volumes of distribution in µL/g, and the parenchymal fraction for HRP was corrected for vascular contamination by subtracting the value for the parenchymal fraction for albumin (Pv). The amount of HRP retained by brain capillaries (5) was measured by the volume of distribution for the vascular fraction from this experiment. Statistical Analysis. All statistical tests were performed by Microsoft Excel XP program using the twotailed heteroscedastic t-tests. A minimum p value of 0.05 was estimated as the significance level for all tests. SEM values for modified and native HRP accumulation and transfer levels were less than 10% of the mean.


Polypeptide Modifications for Enhanced Brain Delivery RESULTS

Chemical Modification of HRP with Stearic Acid Chloride in Reverse Micelles. The HRP contains six amino groups available for chemical modification. To modify these groups with stearic acid residues, the enzyme was reacted with stearoyl chloride in the reverse micelle system of AOT in octane (12). Based on the titration of the remaining free amino groups of the protein with TNBS (see Experimental Procedures section), an average of 1.1 stearic acid residues per protein molecule was introduced onto HRP under these conditions. The solubility of the stearoylated HRP (St-HRP) was approximately 1 mg/mL, its RZ (absorbance ratio A403/A280) was 2.7, and the catalytic activity was 50% of the native HRP activity (RZ of native HRP was 3.0). Conjugation of HRP with Pluronic Block Copolymers. Pluronics have a three-block ABA structure, where the A block is hydrophilic poly(ethylene oxide) (PEO) and the B block is a lipophilic poly(propylene oxide) (PPO) chain. Variations in the length of the blocks result in copolymers with different molecular masses and hydrophilic-lipophilic balances (HLB). We synthesized conjugates of HRP with Pluronic P85, a copolymer of intermediate lipophilicity, and Pluronic L121, a highly lipophilic copolymer. Initially monoamino derivatives of P85 and L121 were prepared as described in the Experimental Procedures section. These derivatives, “Pluronic-amine” were then conjugated to HRP using a two-step procedure shown in Scheme 1. First, Pluronic-amines were activated with DSP or DSS reagents. Second, the products of these reactions were linked to the amino groups of HRP. The DSP reagent was used to introduce a degradable disulfide linkage between the HRP and Pluronic molecules, while the DSS linkage was not degradable. To vary the number of copolymer chains per protein molecule, different excesses of activated Pluronic were used. The conjugation steps are described in greater detail in the Experimental Procedures section. The modified proteins were isolated by precipitation in cold ethanol and then purified by ion-exchange chromatography (Figure 1). TNBS analysis demonstrated that the products contained mainly mono- and bis-substituted protein. The fractions of unmodified HRP did not exceed 25%. The unmodified HRP was discarded, while the fractions of modified protein (peaks 1 and 2) were combined for each conjugate and used in subsequent studies. Overall, four conjugates with stable or degradable linkages and different modification degrees were synthesized as shown in Table 1. Residual activity of modified proteins was about 90-100%. Binding and Transport of Stearoyl-Modified HRP across BBMEC Monolayers. The stearic acid was conjugated to the HRP molecule via a nonbiodegradable bond. The effect of this modification on the binding of HRP with brain endothelial cells was studied using BBMEC monolayers. The results are presented in Figure 2. Modification of HRP resulted in about a 3-fold increase of its binding with the BBMEC. Figure 3 shows the effect of stearoyl modification on HRP transport across BBMEC monolayers from the apical to the basolateral side. As is seen in Figure 3, HRP modification increased the apparent permeability coefficient (Papp) about 1.6 times (3.8 × 10-7 cm/s) when compared with the nonmodified HRP (2.4 × 10-7 cm/s). Equally important, there were no changes in TEER or permeability of a paracellular marker, 3H-mannitol, demonstrating that the integrity of the monolayers was not altered.

Figure 1. Chromatographic isolation of the HRP-Pluronic conjugates using TSK CM-650 column (2 cm × 15 cm) in NaCl salt gradient (0.01-0.2 M) (pH 4.5, elution rate 2 mL/min, detection at 280 nm). The peaks correspond to (1) bis-substituted HRP, (2) monosubstituted HRP, and (3) unmodified HRP. The substitution degree in each fraction was confirmed by the TNBS assay. Table 1. Characteristics of HRP-Pluronic Conjugates conjugate HRP-P85 (1) HRP-P85 (2) HRP-P85 (3) HRP-L121

residual Pluronic modification activity, %b excess type of link degreea 50 50 70 70

degradable stable degradable degradable

1.4 1.6 2.0 2.1

97 100 89 95

a Average number of Pluronic chains per HRP molecule determined using TNBS titration assay. b Compared to the native HRP, o-phenylenediamine reaction.

Figure 2. Binding of unmodified HRP and St-HRP with BBMEC monolayers. Data are mean ( SEM (n ) 4). Asterisk (/) shows significant difference between HRP and St-HRP groups. Modification of HRP resulted in about a 3-fold increase of its binding with the cells.

Binding and Transport of Pluronic-HRP Conjugates across BBMEC Monolayers. Figure 4 presents the results of the study of binding of various PluronicHRP conjugates with BBMEC monolayers. The cells were


Figure 3. Apical to basolateral permeability of HRP and StHRP in BBMEC monolayers. Data are mean ( SEM (n ) 4). Asterisk (/) shows a significant difference between HRP and St-HRP. Stearoyl modification of the protein results in about a 1.6-fold increase in its transport across the BBMEC.

Figure 4. Binding of unmodified HRP and Pluronic-HRP conjugates (described in Table 1) with BBMEC monolayers at 60 min incubation. Results are mean ( SEM (n ) 4). Asterisk (/) shows a significant difference compared to HRP. Modification of HRP with Pluronic increases the binding (about 6 times for HRP-L121) of this protein with BBMEC.

incubated with unmodified HRP or the conjugates for 60 min, and the net amount of the cell-bound enzyme was determined as described above. There was practically no difference in binding between the unmodified HRP and the HRP-P85 (2) containing a nondegradable link. However, binding of conjugates containing the cleavable disulfide link, HRP-P85 (3) and HRP-L121, was increased significantly. The most pronounced effect (over 6-fold increase in binding) was observed in the case of the lipophilic Pluronic L121 conjugate. This result was consistent with our earlier report suggesting strong binding of this lipophilic block copolymer with the BBMEC membranes (27). HRP-P85 (3) displayed a only a 2-fold increase in cell binding compared to HRP. The study of the permeability of various Pluronic-HRP conjugates across BBMEC was carried out in two steps. The initial experiment compared Pluronic-HRP conjugates with nondegradable and degradable links and unmodified HRP (data not shown). Modification of HRP with P85 via nondegradable link, HRP-P85 (2) resulted in about a 2-fold increase in the HRP transport. The conjugate containing a cleavable link, HRP-P85 (3), was transported almost 6 times faster than the unmodified HRP. Finally, the conjugate with the lipophilic Pluronic L121, also containing a cleavable linkage, displayed the highest rates of transport across the BBMEC monolayers. The rate of its transport was over 10-times higher than that of the unmodified HRP. Noteworthy, the amount of HRP transported across the BBMEC monolayers was determined using enzymatic reaction with the HRP

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Figure 5. Apical to basolateral permeability of HRP and Pluronic-HRP in BBMEC monolayers. Data are mean ( SEM (n ) 4). Asterisk (/) shows significant difference between HRP and Pluronic-HRP groups. Conjugation of HRP with Pluronics via the biodegradable linkage resulted in the greatest increase (over 10-fold for HRP-L121) in permeability across the BBMEC.

substrate, o-phenylenediamine, suggesting that enzymatic activity of the protein was preserved during the transport across the cellular monolayers. Based on this experiment, Figure 5 compared conjugates of HRP with degradable links, HRP-L121 and HRP-P85 (3), as well as St-HRP and unmodified HRP. There were no changes in 3H-mannitol permeability (not shown). At the same time, there were substantial differences in the permeability of different HRP samples increasing in the order HRP < HRP-St < HRP-P85 (3) < HRP-L121. Overall, conjugation of the enzyme with the highly hydrophobic Pluronic L121 via the biodegradable link resulted in the greatest increase in its transport across BBMEC monolayers. The Papp values were as follows: HRP 1 × 10-7 cm/s; St-HRP 3 × 10-7 cm/s; HRP-P85 (3) 7.8 × 10-7 cm/s; HRP-L121 10.4 × 10-7 cm/s. A separate experiment using HRP-P85 (3) labeled with Alexa FLUOR 594 determined that the amount of HRP associated with BBMEC monolayers was approximately 11.8% ( 0.3% at 60 min and 12.1% ( 0.4% at 120 min, while the amount of transferred enzyme was increasing in time reaching 3.6% ( 0.4% at 120 min. Localization of HRP Conjugates in BBMEC Monolayers. Figure 6 shows confocal microscopy images of intracellular localization of a nonmodified protein, HRP (A, B), and HRP conjugated with Pluronic block copolymer P85 (C, D) in BBMEC monolayers. Pictures B and D represent fluorescent images; A and C represent differential interference contrast (DIC) and fluorescence images combined. HRP was linked to Pluronic P85 by a biodegradable disulfide linkage (conjugate HRP-P85 (3)). As is seen in the figure, unmodified HRP does not enter the barrier cells (A, B). Modification of the protein with Pluronic block copolymer drastically enhanced transport of HRP into the cells with accumulation in the cytoplasm, nuclei, and other cellular organelles (C, D). Transport of St-HRP into the Brains of Mice. Figure 7 shows that linear relations existed between brain/serum ratios and exposure times for St-HRP (r ) 0.979, n ) 7, p , 0.001) to about 100 min and for HRP (r ) 0.959, n ) 10, p , 0.001) for over 250 min. This indicates passage across the BBB for both compounds and allows computation of Ki for those time periods. The Ki for St-HRP was 0.192 ( 0.018 µL/(g‚min) and for HRP



Figure 6. The confocal microscopy images showing the intracellular localization of Alexa-HRP (A, B), and Alexa-P85-HRP (C, D) in BBMEC monolayers. Pictures B and D represent fluorescent images; A and C represent differential interference contrast (DIC) and fluorescence images combined. HRP was linked to Pluronic P85 via a biodegradable disulfide linkage. The cells were exposed to Alexa-labeled native and modified HRP solutions for 2 h and washed with PBS. Modification of the protein with Pluronic L121 drastically enhanced transport of HRP into the cells with accumulation in the cytoplasm, nuclei, and other cellular organelles.

was 0.079 ( 0.008 µL/(g‚min), a 2.4-fold difference. The two values differed from each other at the p < 0.001 level. The rate for HRP is only a little faster than the upper range usually measured for the entry of albumin. Figure 8A shows the clearance of St-HRP and HRP from the blood. The half-time clearance (calculated with the first time point excluded for each compound) was 89.5 min for St-HRP and 79.6 min for HRP. The volume of distribution for St-HRP was 7.67 mL and for HRP was 5.93 mL. Statistical analysis showed that the slopes (halftime clearance rates) of these lines were not different, but that the intercepts (volume of distribution) were different: F(1,1) ) 7.65, p < 0.05. The percent of the injected dose taken up by brain (%inj/g) depends on two factors: the rate of entry into brain and the peripheral pharmacokinetics (such as halftime clearance rate and volume of distribution). Figure 9A shows the %inj/g for the first 180 min after injection of these compounds. St-HRP peaked higher and sooner. The ratio of the AUC for stearoyl-modified protein vs native protein (St-HRP/HRP) was 1.6 for the first 90 min of the experiment, but only 1.13 for the entire 180 min. Figure 10 A,B shows results for capillary depletion after vascular washout and correction for albumin space with Tc-albumin for St-HRP and HRP. As is seen in

Figure 10A (performed at 30 min), little or no St-HRP or HRP taken up by brain had been retained by capillaries but had all entered the brain parenchymal space. Two-way ANOVA showed significant effects for compound (p < 0.001), region (p < 0.001), and interaction (p < 0.001). Figure 10B shows parenchymal levels at 30 and 120 min. More HRP and St-HRP was found in the parenchymal compartment at 120 than 30 min, but at both times, entry favored St-HRP. A two-way ANOVA was significant for compound (p < 0.05) and time (p < 0.05) but not interaction. Transport of HRP-P85 Conjugate into the Brain of Mice. Pluronic block copolymer with intermediate hydrophilic-lipophilic properties, P85, conjugated with HRP by biodegradable linkage (conjugate HRP-P85 (3), Table 1) was used for these studies. The unidirectional influx rate (Ki) for HRP-P85 was 0.0800 ( 0.0173 µL/ (g‚min) (regression line based on n ) 10, r ) 0.853, p < 0.005), and the Ki for HRP was 0.0206 ( 0.0081 µL/(g‚min) (n ) 10, r ) 0.669, p < 0.05), a 3.9-fold increase, Figure 7B. The significant p values show that there was a statistically significant correlation between brain/serum ratios and exposure time and so indicates that both compounds crossed the BBB. The line for HRPP85 was linear for about 180 min and for HRP for about


Batrakova et al.

Figure 7. Comparison of blood-to-brain transport for St-HRP (A) and HRP-P85 (B) vs HRP. Modification of HRP increased Ki by 2.4-fold for St-HRP and by 3.9-fold for HRP-P85. Figure 9. Modification of the protein with stearoyl or Pluronic moiety increased the AUC for brain uptake by 13% (St-HRP) and 82% (HRP-P85) during the 180 min of the experiment.

of HRP. The ratio of the area under the curve (HRPP85/HRP) was 1.82. Figure 10 shows results for capillary depletion after vascular washout and correction for albumin space with Tc-albumin. Figure 10C shows that at 30 min little or no HRP-P85 or HRP taken up by brain had been retained by capillaries but had all entered the brain parenchymal space. Two-way ANOVA test showed significant effects for region (p < 0.05) but not for compound or interaction. The lack of difference between these compounds is consistent with the %inj/g results, which showed HRP-P85 and HRP similar at that time point. Figure 10D shows parenchymal levels at 30 and 120 min. More HRP and HRP-P85 was found in the parenchymal compartment at 120 than 30 min, but at 120 min, entry favored HRP-P85. A two-way ANOVA test was significant for compound, time, and interaction all at the p < 0.001 level. DISCUSSION Figure 8. The percent of injected radioactivity present in 1 mL of serum (%inj/g) following injection of St-HRP and HRP (A) or HRP-P85 and HRP (B).

360 min. The Ki for HRP-P85 was about 3.9 times higher than the Ki for HRP, and this was significantly different: F(1,16) ) 9.27, p < 0.01. The half-time clearance from blood did not differ between the two compounds (Figure 8B), being 131 min for HPR-P85 and 94.5 min for HRP (2 min time points excluded). The volume of distribution after iv injection also did not differ between HPR-P85 (5.17 mL) and native HRP (3.82 mL). Figure 9B compares the values for %inj/g for HRPP85 and HRP. The curves differed little for the first 30 min, but thereafter, uptake of HRP-P85 exceeded that

Strategies for improving transport of therapeutic polypeptides to the brain are crucial for the successful applications of polypeptides for diagnostics and treatment of CNS diseases. The present in vitro and in vivo study demonstrates that point modifications of a model polypeptide, HRP, with lipophilic or amphiphilic groups greatly enhance the transport and accumulation of HRP in the brain. The key for such modifications is to achieve a delicate balance in hydrophilic-lipophilic properties of the modified polypeptides. On one hand, the modified polypeptide should be lipophilic to interact with and incorporate into the membranes of microvessel endothelial cells, but on the other hand, the protein should retain its high solubility in the blood stream. To accomplish this goal for stearoylated polypeptide, we used reverse micelles of a surfactant, AOT, producing microreactors and allowing a point conjugation of each polypeptide molecule



Figure 10. Capillary depletion compared for HRP and St-HRP (A,B) or HRP and HRP-P85 (C,D) 30 min after injection. Results show that all studied proteins crossed the BBB to enter the parenchymal compartment of the brain. The amount of native HRP (white bars, A-D), St-HRP (black bars, A and B), or HRP-P85 (black bars, C and D) retained in the brain is shown for capillaries and parenchyma (30 min after iv injection, A and C) or for parenchyma only (30 and 120 min after iv injection, B and D). Modification of HRP with stearoyl or Pluronic moiety significantly increased protein accumulation in the brain.

with 1-2 strearic acid residuals of the aliphatic chain. Based on the earlier reports (11, 15, 28), a stearic derivative was chosen as the most appropriate for this purpose. Consequently, total binding and transport of the stearoyl-modified HRP was increased about 2-3 times in the brain microvessel endothelial cells in vitro. A similar principle was used in the case of a protein modification with Pluronic block copolymers; the designed conjugates had different balances between hydrophilic and lipophilic properties. Modification of HRP with Pluronic L121 was the most promising for the CNS delivery of the conjugate. The total binding and transport of Pluronic P85 and L121 modified HRP were increased up to 6 and 10 times, respectively, compared with the native HRP. A method of conjugation using biodegradable and nonbiodegradable links was also varied. As expected, the modification of the protein via a biodegradable link resulted in the most effective transport of the protein across the brain microvessel endothelial cell monolayers. We speculate that being highly hydrophilic the HRP molecule requires a highly lipophilic moiety to obtain the optimal hydrophilic-lipophilic balance of the conjugate. Such a lipophilic group has a tendency to remain associated with the cellular membrane as an anchor. Therefore, a biodegradable link allowed the polypeptide molecule to separate from its lipophilic moiety and enter the brain after the conjugate passed membranes of the barrier cells. Data obtained by confocal microscopy visualizes this effect. Modification of the protein with Pluronic P85 via biodegradable link drastically enhanced transport of HRP into the cells and its accumulation in the cytoplasm, nuclei, and other cellular organelles. We suggest that the protein conjugate was initially bound to the cellular membrane through the Pluronic moiety incorporated into the lipid bilayer followed by cleavage of the HRP molecule from the Pluronic anchors. Another crucial point is to preserve the function of the modified protein (1) after the modification and (2) after the transport across the BBB. From this prospective, modification of HRP with Pluronic block copolymers was more preferable than conjugation with stearoyl moiety;

Pluronic-based conjugates retained 90-100% of the native enzyme activity, whereas St-HRP retained only 50%. A considerable decrease in the enzymatic activity of St-HRP could be a result of the last step of the synthesis, when obtained conjugate was precipitated by ice-cold acetone. Nevertheless, all obtained conjugates retain their enzymatic activity during their transport across the BBMEC monolayers in vitro, as was detected by an HRP substrate, o-phenylenediamine. In vivo studies found that (1) hydrophobization with stearoyl group or (2) amphiphilic modification with a Pluronic block copolymer had increased the rate of HRP penetration across the BBB and increased accumulation of HRP by the brain. In vivo studies of BBB permeability can differ from in vitro studies because clearance, serum protein binding, and tissue sequestration are factors that can influence in vivo results. Lipophilic modifications are especially notorious for increasing the ability of compounds to cross the BBB while decreasing the amount of compound accumulated by brain. This paradoxical effect occurs because lipophilic modification increases uptake by all tissues, not just brain, and so reduces the amount of the compound available for transport across the BBB. If the decrease in the amount of compound available for transport exceeds the increase in transport rate, the net effect is to decrease the amount of compound taken up by brain. Here we found that, in comparison to HRP, StHRP had an increase in transport rate of 2.4-fold. However, volume of distribution as measured in Figure 8A was increased, indicating that there was increased uptake by other tissues. These two factors resulted in a net uptake by brain of St-HRP as measured by %inj/g of about 60% for the first 90 min after injection and an increase of about 13% over the entire study period. Another problem with lipid modification as discussed above is that compounds can partition into cell membranes and become trapped there. For in vivo BBB, this results in material being retained by the capillaries and not being able to enter the brain parenchymal space. Here, the capillary depletion method was used to assess this possibility. It was found that although more St-HRP


than HRP was retained by capillaries, the vast majority of St-HRP entered brain parenchyma. Overall modification with Pluronic block copolymers appeared to be more promising for HRP delivery to the brain. Permeation of the BBB in vivo was increased almost 4-fold with no statistically significant effects on peripheral pharmacokinetics or entry into the parenchymal space. The %inj/g was almost twice as great for HRP-P85 as for HRP. CONCLUSIONS

This study indicates that simple and straightforward modification of polypeptides with lipophilic or amphiphilic groups can considerably increase the transport of HRP into the brain, while preserving the enymatic activity of HRP. A delicate balance between lipophilic and hydrophilic properties of the conjugate should be achieved to accomplish its efficient delivery into the brain. To achieve this balance, each unique polypeptide may require tailoring of the lipophilic moiety to match the hydrophilic-lipophilic characteristics of the native polypeptide. ACKNOWLEDGMENT

This work was supported by National Institutes of Health Grants R01 NS36229, R01 NS41863, and R01 AA12743 and VA Merit Review. LITERATURE CITED (1) Brinton, R. D. (1999) A women’s health issue: Alzheimer’s disease and strategies for maintaining cognitive health. Int. J. Fertil. Women’s Med. 44, 174-185. (2) Gozes, I. (2001) Neuroprotective peptide drug delivery and development: potential new therapeutics. Trends Neurosci. 24, 700-705. (3) Kroll, R. A., and Neuwelt, E. A. (1998) Outwitting the bloodbrain barrier for therapeutic purposes: osmotic opening and other means. Neurosurgery 42, 1083-1099; discussion 10991100. (4) Koliatsos, V. E., Clatterbuck, R. E., Nauta, H. J., Knusel, B., Burton, L. E., Hefti, F. F., Mobley, W. C., and Price, D. L. (1991) Human nerve growth factor prevents degeneration of basal forebrain cholinergic neurons in primates. Ann. Neurol. 30, 831-840. (5) Dogrukol-Ak, D., Banks, W. A., Tuncel, N., and Tuncel, M. (2003) Passage of vasoactive intestinal peptide across the blood-brain barrier. Peptides 24, 437-444. (6) Desnick, R. J., and Schuchman, E. H. (2002) Enzyme replacement and enhancement therapies: lessons from lysosomal disorders. Nat. Rev. Genet. 3, 954-966. (7) Downs-Kelly, E., Jones, M., Alroy, J., Cavanagh, K., King, B., Lucas, R., Baker, J., Kraemer, S., and Hopwood, J. (2000) Caprine mucopolysaccharidosis IIID: a preliminary trial of enzyme replacement therapy. J. Mol. Neurosci. 15, 251-262. (8) Urayama, A., Grubb, J. H., Sly, W. S., and Banks, W. A. (2004) Developmentally regulated mannose 6-phosphate receptor-mediated transport of a lysosomal enzyme across the blood-brain barrier. Proc. Natl. Acad. Sci. U.S.A. 101, 1265812663. (9) Banks, W. A., and Farrell, C. L. (2003) Impaired transport of leptin across the blood-brain barrier in obesity is acquired and reversible. Am. J. Physiol. Endocrinol. Metab. 285, E1015. (10) Banks, W., and Lebel, C. (2002) Strategies for the delivery of leptin to the CNS. J. Drug Target 10, 297-308. (11) Kabanov, A., Levashov, A., and Alakhov, V. (1989) Lipid modification of proteins and their membrane transport. Protein Eng. 3, 39-42. (12) Slepnev, V., Phalente, L., Labrousse, H., Melik-Nubarov, N., Mayau, V., Goud, B., Buttin, G., and Kabanov, A. (1995) Fatty acid acylated peroxidase as a model for the study of

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BC049730C

Polypeptide Point Modifications with Fatty Acid and Amphiphilic Block

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