Peptide Derived from HIV-1 TAT Protein Destabilizes aMonolayer of Endothelial Cells in an in Vitro Model of theBlood-Brain Barrier and Allows Permeation of HighMolecular Weight Proteins*

Received for publication, June 26, 2012, and in revised form, November 8, 2012 Published, JBC Papers in Press, November 13, 2012, DOI 10.1074/jbc.M112.395384

Itzik Cooper‡§1, Keren Sasson¶, Vivian I. Teichberg‡†, Michal Schnaider-Beeri§, Mati Fridkin�2, and Yoram Shechter¶3

From the Departments of ‡Neurobiology, ¶Biological Chemistry, and Organic �Chemistry The Weizmann Institute of Science,Rehovot 76100, Israel and the §Sagol Neuroscience Center, Sheba Medical Center, Tel Hashomer, Ramat Gan 52621, Israel

Background: An in vitromodel for studying BBB opening and entry of impermeable substances was constructed.Results: A brain capillary endothelial cell monolayer was disrupted and opened to the penetration of impermeable therapeuticagents by C-TAT peptides.Conclusion: Experimental conditions enabling the blood to brain entry of impermeant therapeutic agents were established.Significance: Approaches toward overcoming states of major brain disorders were initiated.

Most chemotherapeutic agents are blood-brain barrier (BBB)impermeants. HIV-1-derived TAT protein variants contain atransmembrane domain, which may enable them to cross theBBB and reach the brain. Here we synthesized CAYGRK-KRRQRRR, a peptide containing a cysteine moiety attached totheN terminus of the transmembrane domain (C-TATpeptide),and studied its effects in an in vitro BBBmodel, which we foundto reflect penetration by a receptor-independent pathway. Incu-bation of the brain capillary endothelial cell monolayer with0.3–0.6 �mol/ml of this C-TAT peptide, for a period of 1–2 h,destabilizes brain capillary endothelial cell monolayer andintroduces the ability of impermeant therapeutic agents includ-ing highmolecular weight proteins to penetrate it substantially.The cysteinylmoiety at position1of theC-TATpeptide contrib-utes largely to the destabilizing potency and the penetrationefficacy of impermeant substances. The destabilizing effect wasreversed using heparin. In summary, experimental conditionsallowing a significant increase in entry of impermeant low andhigh molecular weight substances from the luminal (blood) tothe abluminal side (brain) were found in an in vitro BBB modelreflecting in vivo protein penetrability by a receptor-indepen-dent pathway.

A large variety of therapeutic agents with potential for treat-ing brain disorders are not in clinical use because they are

unable to cross the blood-brain barrier (BBB)4 followingperipheral administration (1). Even small bioactive peptidespenetrate the BBB only if highly lipophilic (2). Furthermore,therapeutically relevant proteins such as herceptin (transtu-zumab, a humanized IgG1 molecule) become ineffective, whenHER-2-positive metastases have reached the brain (3).The BBB is formed in part by tight junctions between adja-

cent endothelial cells that make up the cerebral capillaries.Tight junction proteins connect neighboring endothelial cellsto each other in a noncovalent fashion (4). Several pathologicalconditions are known to result inmarked disruption of the tightjunctions of the BBB (5–8). Such disruptions can lead to unde-sirable pathological consequences but might permit the pene-tration of therapeutic agents relevant for brain disorders. Clin-ically, all attempts so far to enhance penetration of therapeuticproteins via the BBB in quantities sufficient to treatmajor braindisorders were unsuccessful (9).A truncated form (71 amino acids) of the HIV-1 TAT (trans-

activator of transcription) proteinwas found to have the uniqueability to enter cells through the plasma membrane and accu-mulate intracellularly (10, 11). This is believed to take placethrough a plasmamembrane bilayer component, in a receptor-and transporter-independent fashion (12). This feature wasattributed to a nine-amino acid stretch of basic amino acids ofsequence RKKRRQRRRR, located in region IV of TAT protein(13). Peptide fragments containing this protein transductiondomain preserve this feature as well (14, 15). Studies in rodentsin vivo revealed that both TAT protein variants and peptides orfragments containing this protein transduction domain arecapable of penetrating the BBB and accumulate in brain tissue(16). This was the rationale to covalently link the peptide tobiologically active proteins for delivering them either intoperipheral tissues or to the CNS (13–15). Indeed such peptide-

* This work was supported by a joint Weizmann Institute of Science-ShebaMedical Center grant for Biomedical Research.

† Deceased April 26, 2011.1 To whom correspondence may be addressed: Dept. of Neurobiology, The

Weizmann Institute of Science, Rehovot 76100, Israel, and Sagol Neurosci-ence Center, Sheba Medical Center, Tel Hashomer, Ramat Gan, 52621,Israel. Tel.: 972-3-5303693; Fax: 972-3-5304752; E-mail: itzik.cooper@sheba.health.gov.il.

2 To whom correspondence may be addressed: Dept. of Biological Chemistry,The Weizmann Institute of Science, Rehovot 76100, Israel. Tel.: 972-8-9344530; Fax: 972-8-9344118; E-mail: yoram.shechter@weizmann.ac.il.

3 To whom correspondence may be addressed: Dept. of Organic Chemistry,The Weizmann Institute of Science, Rehovot 76100, Israel. Tel.: 972-8-9342505; Fax: 972-8-9344142; E-mail: mati.fridkin@weizmann.ac.il.

4 The abbreviations used are: BBB, blood-brain barrier; ES, electrospray; HSA,human serum albumin; TEER, transendothelial electrical resistance; Pe,permeability; PBEC, porcine brain endothelial cell; MAL, maleimide;PBEC-M, PBEC monolayer.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 53, pp. 44676 –44683, December 28, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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protein conjugates did reach the brain (17), but in quantities farbelow that required to treat amajor brain disorder such as braintumors (for instance glioblastomamultiforme (18)). Thus, con-ditions that allow the efficient penetration of therapeutic pro-teins from the periphery to the brain are still needed (19).In this study, we have synthesized cysteine-containing 13-a-

mino acid fragments, based on the protein transductiondomain of the TAT protein, postulating that the efficacy of thisprotein to cross the plasma membrane and to disrupt BBBtightness are interrelated. Efforts were then undertaken tomodify this peptide to a form that preserves its BBB disruptingproperty but with reduced penetrability into cell interiors. Wehave used an in vitro experimental system composed of tightjunction-linked primary porcine brain endothelial cells (PBEC)grown in culture (20, 21). This in vitro experimental systemreflects well BBB in vivo with regard to permeability (Pe) inagreement with findings made in rodents in vivo (22). Thetightness of this monolayer is determined by measuring thetransendothelial electrical resistance (TEER). As relevant forPe, this parameter is recognized as one of themost accurate andsensitive measures of BBB integrity (23). As for the BBB inmammals, major tight and adherence junction proteins areexpressed when endothelial cells contact each other (20, 21).This experimental system allowed us to determine the disrup-tion efficacy and the permeation potency of a large variety oftherapeutic agents, including high molecular weight proteins.Our efforts in this direction are described in this report.

EXPERIMENTAL PROCEDURES

Materials

C-TAT peptide (cysteine- and TAT-containing peptide,CAYGRKKRRQRRR) was synthesized by the manual conven-tional synthesis. An Fmoc (N-(9-fluorenyl)methoxycarbonyl)strategy was employed throughout the peptide chain assembly.The calculatedmass of the C-TATpeptide is 1734.07Da, foundby electrospray single quadropole mass spectroscopy analysis:ES (electrospray) � 1733.53 � 0.04 Da. HSA, 5,5�-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide, and fluresceine iso-thiocyanate were purchased from Sigma. PEG5-MAL (a 5-kDaPEG chain-containing maleimide moiety; CH3O-PEG5-C2H4-maleimide) was a product of Rapp Polymere (Tubingen, Ger-many). Na125I (carrier free) was obtained from New EnglandNuclear, and herceptin was purchased from Rosh (Basel, Swit-zerland). All othermaterials used in this studywere of analyticalgrade.

Chemical Synthesis

C-TAT-MAL (C-TAT peptide in which N-ethylmaleimidewas linked to the cysteine moiety) was prepared by reacting theC-TAT peptide withN-ethylmaleimide. The peptide (8.7 mg, 5�mol) dissolved in 1.0 ml of 0.1 M Hepes buffer, pH 7.4.N-Eth-ylmaleimide (20 �l) from a fresh solution of 0.5 M in N,N-di-methylformamidewas then added (2molar excess over the pep-tide). The reaction was continued for 5 min at 25 °C. Theproduct thus obtained was dialyzed for 10 h against H2O andlyophilized. C-TAT-MAL is 5,5�-dithiobis (2-nitrobenzoicacid)-negative, and the calculated mass for C-TAT-MAL is

1859 Da. Found by electrospray single quadropole mass spec-troscopy analysis: ES � 1858.49 � 0.21.

HO3S-C-TAT (a C-TAT peptide in which the cysteine moi-ety was converted to cysteic acid) was prepared by performicacid oxidation of the peptide. C-TATpeptide (12.2mg, 7�mol)was dissolved in 0.45 ml of formic acid. Hydrogen peroxide (50�l) was then added, and the reaction was carried out at 25 °Cover a period of 2 h. Performic acid was then removed by evap-oration. The product obtainedwas dissolved inH2O and lyoph-ilized. This procedure was repeated three times. HO3S-C-TATpeptide contains cysteic acid moiety (mol/mole) as verified byamino acid analysis following acid hydrolysis. The calculatedmass is 1782 Da, found by electrospray single quadropole massspectroscopy analysis: ES � 1781.52 � 0.06 Da.

Acetylated C-TAT peptide was prepared by dissolving 12.2mg of C-TAT in amixture of 1:1:0.5 ofH2O, pyridine and aceticanhydride. The reaction was carried out at 0 °C. The mediumwas evaporated, and the product was obtained, dissolved inH2O, and lyophilized. This procedurewas repeated three times.Acetylated C-TAT peptide was then dissolved in 0.1 MNa2CO3(pH 10.3, 1 h, 25 °C) for deacetylating the tyrosyl and the cys-teinyl moieties of this derivative. The pH was then dropped to6.0 with HCl, and the product was frozen until used.Fluresceine-labeled HSA was prepared by dissolving 67 mg

of HSA (1 �mol) in 0.5 ml of 0.1 M Na2CO3 (pH 10.3). Flures-ceine isothiocyanate 1.4 mg (3.6 molar excess over HSA) wasthen added, and the reaction was carried out for 1 h at 25 °C.The reaction mixture was then loaded on a Sephadex G-50column (1.7� 14 cm), equilibrated, and run in the same buffer.The tubes containing Fluresceine-labeled HSA were pooled,dialyzed against H2O, and lyophilized. Flures-HSA (HSA deriv-ative containing 1.7 mol of fluresceine/mol of HSA) preparedby this procedure contains 1.7 � 0.2 mol of fluresceine/mol ofHSA as determined by its absorbance at 500 nm using �500 �62,000.Cationized Flures-HSA was prepared by dissolving 20 mg of

Flures-HSA in 1.0 ml of 1 M 1,3-diaminopropane or in 1 ml of 1M arginine-amide. 1-Ethyl-3-(3-dimethyl aminopropyl) carbo-diimide (70 mg) was then added. The pH was adjusted to 6.0 �0.1. The reaction was carried out for 2 h. The derivatives thusobtained were dialyzed against H2O for 2 days with severalchanges of H2O and lyophilized. Flures-HSA that was cation-izedwith arginine-amide contains additional 64� 3moieties ofarginine-amide/mol of Flures-HSA as determined by aminoacid analysis following acid hydrolysis. The protein concentra-tion was calculated according to alanine (62 residues) and gly-cine (12 residues).

Biological and Chemophysical Procedures

Radiolabeling with Na125I—Radiolabeling of peptides andproteins with Na125I was carried out essentially by the proce-dure of Hunter and Greenwood (24) with slight modificationsthat were described in detail in Ref. 25.Primary Cultures of Brain Endothelial Cells—Primary cul-

tures of brain endothelial cells were isolated from freshly col-lected porcine brains as described previously (21, 26). ThesePBEC were validated as true BBB endothelial cells in previous

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studies (21, 26). The purity of the culture was confirmed byspecific staining for von Willebrand factor (20).In Vitro BBB Models and Transendothelial Electrical Resist-

ance Measurements—In a typical experiment, PBEC wereseeded at a density of 100,000 PBEC/cm2 on a microporousmembrane of a Transwell insert (Corning Costar, Acton, MA)placed into a 12-well plates. The cells were cultured in plat-ingmedium for up to 3 days until they reached confluency. Themedium was replaced with a serum-free medium (assaymedium) for an additional 24–48 h, and the integrity of thiscellular barrierwas determined bymeasuringTEER.AdecreaseinTEER reflects an increase in permeability and a loss of barrierfunction. TEER of the filter insert was recorded using anEndohm chamber connected to an EVOM resistance meter(World Precision Inst., Inc., Sarasota, FL). The TEER of eachfilter insert was calculated by subtracting the TEER of themicroporous membrane without PBEC and is reported as�cm2.When using a co-culture BBB in vitro system, Transwellinserts seededwith glial cells at the bottomwere used as blanks.The co-culture system was used in a “contact” configuration asdescribed in detail in Ref. 20. For testing the effects of the dif-ferent compounds onTEER, theywere diluted in assaymediumat the desired concentrations and added to the luminal (tomimic blood to brain passage) or abluminal (to mimic brain toblood passage) side of the inserts.Media—Platingmedium is composed of newborn calf serum

(10%), L-glutamine (2 mM), penicillin (100 units/ml), strepto-mycin (0.1mg/ml), and gentamicin (0.1mg/ml), all dissolved inEarl’s medium 199 (Sigma). The assay medium consists ofL-glutamine (2mM), penicillin (100 units/ml), streptomycin (0.1mg/ml), gentamicin (0.1 mg/ml), and hydrocortisone (550 nM)in DMEMdiluted 1:1 in Ham’s F-12medium (Biological Indus-tries, Beit Haemek, Israel).PermeabilityMeasurements—The PBEC permeability of this

in vitro BBB model to radiolabeled compounds ([14C]sucrose,[125I]HSA, and [125I]Herceptin) and fluorescently labeled com-pounds (doxorubicin or Fluores-HSA) was carried out asdescribed in detail in Refs. 20 and 27 with slight modifications.The tested compounds were added at the luminal side of theTranswell inserts. During the transport assay, the cells wereincubated at 37 °C. Samples (500 �l) were collected every 10min over a period of 40 min from the abluminal side andmeas-ured for their 14C or 125I content or for their fluorescent inten-sity. Pe values of filters having no PBEC seeded on them weresubtracted. Pe was obtained from the slope of the calculatedclearance curve as described in Ref. 27.

RESULTS

CationizedAlbumin Permeates through the PBECMonolayer—We prepared Flures-HSA and further cationized it by linkingeither 1,3-diaminopropane or arginine-amide, converting amajor part of the carboxylate moieties (�60%) into positivelycharged residues (“Experimental Procedures”). Both versions offluorescently labeled cationized HSA penetrated this PBECmonolayer efficiently (14–17 cm/s � 10�6; Fig. 1A). Bothderivatives had a marked effect in reducing TEER (Fig. 1B),suggesting that penetrability is secondary to destabilizing thetightness of this monolayer. Noncationized HSA neither pene-

trated nor destabilized this PBECmonolayer (Fig. 1). Thus, thisin vitro experimental system resembles BBB penetrability invivo, with regard to proteins that can enter this barrier byabsorptive mediated transendocytosis (1, 28).Synthesizing and Characterizing a Peptide That Opens BBB,

Based on TAT Protein Variants—Several TAT-containing pep-tide versions were synthesized: of these, we have selected as thefirst prototype a version containing a single cysteinyl moiety atposition 1 combined with fragment 46–57 of TAT protein,which contains the transduction domain (13). Table 1 summa-rizes several of the properties of this C-TAT peptide (CAY-GRKKRRQRRR). It migrates as a single peak on HPLC columnwith a retention time value of 4.605 min. It absorbs at 278 nmwith extinction coefficient (�278) of 2400 � 50, a value that is1.71 times higher than that expected from a peptide containinga single tyrosyl moiety (�278 � 1400). Mass spectrum analysisrevealed the corrected mass as calculated (1734.07 Da). Perfor-mic acid oxidation of this peptide yielded 1.0 � 0.1 mol/molcysteic acid, as determined by amino acid analysis following

FIGURE 1. Cationized derivatives of HSA decrease tightness and perme-ate across PBEC monolayer. A, Flures-HSA and Flures-HSA that was cation-ized with either arginine-amide or with 1,3-diaminopropane (9 mg/ml ofeach) were placed at the luminal side, and Pe values were measured over aperiod of 40 min. B, TEER measurements of PBEC 40 min after placing Flures-HSA or its cationized derivatives. The results are expressed as the means �S.E. (n � 3–7 inserts). ***, p � 0.001 versus Flures-HSA.

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acid hydrolysis. C-TAT peptide is highly soluble in H2O (20mg/ml).C-TAT Peptide Decreases TEER in a Concentration- and

Time-dependent Fashion—Fig. 2A shows the effect of severalconcentrations of C-TATpeptide placed at theTranswell lumi-nal side, which resembles the blood side of the BBB, caused bythe asymmetry alignment of the cells grown on collagen-coatedinserts (26), on TEER over a period of 2 h. At a C-TAT peptideconcentration of 0.6�mol/ml, TEER values were decreased in atime-dependent fashion amounted to 47, 13, and 2% of initialvalue following 0.5, 1.0, and 2 h of incubation, respectively. At0.3 �mol/ml C-TAT peptide, TEER decreased to 48, 27, and16% of initial value following 0.5, 1.0, and 2 h of incubation,respectively. A lower concentration of the C-TATpeptide (0.06�mol/ml) decreased TEER by 26%, following 1 h of incubationwith no further decrease at 2 h (Fig. 2A).We also studied the destabilizing effect of theC-TATpeptide

placed at the abluminal side (“brain” side). TEER value hasdecreased as well with time, although at a lower rate, as com-pared with the same peptide concentration placed at the lumi-nal side. However, in both cases TEER values decreased to�2–7% of initial value following 2 h of incubation.Lastly, we have compared the destabilizing effect of the

C-TAT peptide, side by side on a monoculture of PBEC asdescribed above and a contact co-culture made of endothelialcells on one side of the insert and glial cells seeded on the bot-tom of the Transwell inserts (20). TEER was �1.5 times higherin the co-culture (707 � 16 versus 458 � 30 �*cm2, respec-tively). C-TAT peptide, however, was nearly equipotent indecreasing TEER with time (t1⁄2 � 0.4 � 0.02 h; Fig. 2B). Thus,the added glial cells enhance significantly BBB tightness butnot the efficacy of the C-TAT peptide to disrupt it, suggestingthat the effect of C-TAT on the barrier properties is primarilyon the endothelial cells.Two Patterns of BBB Opening by C-TAT Peptide—Heparin

was documented to associate with TAT protein transductiondomain (29). Fig. 3 shows that heparin associates with theC-TAT peptide as well. Heparin, at a 2:1molar ratio suppressesthe peptide efficacy to reduce TEER over a period of 2 h (Fig. 3).We therefore added heparin at 0, 1, 5, and 30min following the

addition of the C-TAT peptide to the PBECmonolayer (PBEC-M), and TEER values were monitored over a period of 2 h.Exposure of PBEC-M to the C-TAT peptide for a period of 1min prior to neutralizing it with heparin reduced TEER by�60%, and this was followed by nearly full recovery of TEERwithin a period of 2 h. Nearly the same pattern is seen following5 min of exposure of PBEC-M to the peptide prior to neutral-izing it. More prolonged exposure (30 min) of PBEC-M to thepeptide prior to neutralizing it, yielded large decrease in TEERwith little restoring efficacy in the 2-h time frame shown in Fig.3, although after 24 h, PBEC-M has restored to large extent itsTEER values (data not shown). Thus, C-TAT peptide appearsto yield two distinct patterns of BBB opening. The first onetakes place within a short (1 min) transitory exposure and isnearly fully reversible, where the second pattern takes place

TABLE 1Chemical features of C-TAT peptide

Characteristic

Migration on analytical HPLCretention timea

Single peak, 4.605 min

Absorbance at 278 nmb �278 � 2400 � 50Mass spectrac Calculated 1734.07 daltons, found

(ES) 1733.53 � 0.04 daltonsCysteic acid content following

performic acid oxidationd1.0 � 0.1 mol/mol

Solubility in H2O 20 mg/mla Conducted with a linear gradient from 0 to 100% of solution A (0.1% TFA) tosolution B (acetonitrile H2O 75:25 in 0.1% TFA) using a chromolith Rp 18e(100 � 4 mm) column.

b Determined by UV spectroscopy. Peptide concentration was determined by acidhydrolysis of 20-�l aliquot followed by amino acid analysis and calculated ac-cording to alanine (one residue) and glycine (one residue).

c Mass spectroscopy was determined by the electrospray ionization technique.d Cysteic acid content was determined by acid hydrolysis and amino acid analysisof an aliquot following performic acid oxidation (“Experimental Procedures”).Peptide concentration was calculated according to alanine and glycine (one resi-due of each).

FIGURE 2. Time- and dose-dependent effect of the C-TAT peptide ondestabilizing BBB in vitro systems. A, the indicated concentrations of theC-TAT peptide were added to the luminal or abluminal side of the Transwellswith PBEC cultured in monolayers, and TEER values were monitored over aperiod of 2 h. The data are presented as the means � S.E. (n � 4 –9 inserts). B,side by side comparison of TEER and Pe for monoculture versus co-culture BBBin vitro model. C-TAT at 0.6 mM was applied to the luminal side of either amonoculture or co-culture (with the addition of glial cells at the bottom of theTranswell insert (20)). The TEER was then monitored for 2 h. The results areexpressed as the means � S.E. for three inserts each. The data used in thisfigure were driven only from a side by side comparison from cells (PBEC andglial) prepared in the same preparation.

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following prolonged (30 min) exposure of the PBEC-M to thepeptide, yielding a more extensive and less reversible BBBopening pattern.Cysteine 1 of the C-TAT Peptide Contributes Significantly to

the PBEC-M Destabilizing Potency of the Peptide—Initially weelongated the C-TAT peptide by adding additional argininemoiety at the C-terminal position. This 14-amino acid peptide(CAYGRKKRRQRRRR) was as effective as the shorter C-TATversion (not shown), indicating that additional positive chargeat the C-terminal end is not required for enhancing its destabi-lizing efficacy. Subsequently we prepared two additional ver-sions of this longer C-TAT peptide. The first one lacks its cys-teinylmoiety at position 1 (AYGRKKRRQRRRR;TATpeptide),and in the second version the cysteinyl moiety was replacedwith valine (VAYGRKKRRQRRRR; V-TAT peptide). Valinehas rather similar atomic volume and degree of hydrophobicityas cysteine but lacks the weakly acidic and the reducing efficacyof cysteine. As shown in Fig. 4, the C-TAT peptide is approxi-mately twice as potent as the TAT peptide in disruptingPBEC-M, as well as capable of fully reducing TEER, 2 h afteraddition. V-TAT peptide was nearly as potent as the C-TATpeptide in this respect (Fig. 4). Thus, the destabilizing efficacy ofTAT containing peptides is significantly enhanced if either cys-teine or valine are present at theN-terminal end. It appears thata hydrophobic side chain moiety, e.g., -CH2-SH (cysteine) or-CH(CH3)2 (valine), is responsible for this increase in potency.Further derivatization of the sulfhydryl moiety of the C-TATpeptide yielded inactive derivatives.Structure-Function Relationships—Table 2 shows the poten-

cies of several C-TAT peptide derivatives to reduce TEER valueof PBEC monolayer following 1 h of incubation with 0.6�mol/ml of each derivative. Derivatization of the free cysteinylmoiety with NEM, linking to it PEG5-MAL, or oxidizing it tocysteic acid, yielded derivatives having reduced BBB disruptingpotency. Interestingly, acetylation of the C-TAT peptide underconditions that preserve the cysteinyl-moiety in its reduced

form yielded a C-TAT peptide derivative that preserved �47%of its destabilizing potency (Table 2). Thus, the positive chargesof the two lysine moieties in this peptide can be replaced bynoncharged amino acid moieties.C-TAT Peptide Turns PBEC Monolayer Penetrable to Low

MolecularWeight Substances—We incubated the PBECmono-layer with 0.6 �mol/ml C-TAT peptide for 2 h at 37 °C. [14C]-Sucrose (molecular weight � 342) or doxorubicin HCl (molec-

FIGURE 3. PBEC monolayer recovers after neutralization of C-TAT withheparin. C-TAT was added to the luminal side of the in vitro BBB model at 0.6mM, and heparin was added at 2:1 molar ratio in several time points afterward.TEER values were then monitored. The data are expressed as percentages ofinitial TEER values normalized to control inserts and presented as themeans � S.E. values (n � 3 inserts).

FIGURE 4. Cysteine 1 of the C-TAT peptide contributes significantly to thepeptide PBEC-M destabilizing potency. Two versions of the longer C-TATpeptide were synthesized. The first one lacks its cysteinyl moiety at position 1(AYGRKKRRQRRRR; TAT), and in the second version the cysteinyl moiety wasreplaced with valine (VAYGRKKRRQRRRR; V-TAT). The peptides were added tothe luminal side of the Transwells with PBEC-cultured in monolayers, andTEER values were monitored over a period of 2 h. The data are presented asthe means � S.E. (n � 3 inserts).

TABLE 2Structure-function studies: effect of modified derivatives of C-TATpeptide on destabilizing PBEC-MC-TATpeptide and the indicated derivatives of the C-TAT peptide (0.6�mol/ml ofeach) were placed at the luminal side of the Transwell, and TEER values weredetermined following 1 h of incubation at 37 °C. The data are presented as themeans � S.E. (n � 5).

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ular weight � 580) was then added to the Transwell luminalside, and the extent of permeability after a period of 2 h wasquantitated. Both substances penetrated the PBEC monolayer(Fig. 5). Pe values were 17.8 � 1.8 � 10�6 and 3.8 � 1.8 � 10�6

cm/s for [14C]sucrose and doxorubicin, respectively, exceedingthe Pe values obtained in untreated PBECmonolayer by 38- and14-fold, respectively (Fig. 5). Thus, incubation with the C-TATpeptide alters PBEC in a way that allows the entrance of lowmolecular weight substances from the luminal to the abluminalside to a considerable extent.Permeation of High Molecular Weight Proteins Following

PBEC Monolayer Treatment with C-TAT Peptide—Next, wehave studied the permeability of [125I]HSA (molecular mass �66 kDa) and of [125I]herceptin (molecular mass � 145 kDa)following the incubation of the PBEC monolayer withC-TAT peptide under the same conditions used in Fig. 5.Both proteins penetrated the treated PBEC monolayer to asignificant extent (Fig. 6). Penetrability amounted to 1.9–3.2 � 10�6 cm/s, exceeding 15–18 times that found in con-trol PBEC monolayer. Thus, the destabilizing effect of theC-TAT peptide enabled the entrance of high molecularweight proteins via this PBECmonolayer from the luminal tothe abluminal side.As shown in the co-culture experimental system, the addi-

tion of glial cells largely enhanced the tightness of the endothe-lial cell monolayer as judged by increased TEER (Fig. 2B). Toassess whether this increase in BBB integrity imposes a pro-found decrease in protein penetrability following disruptionwith theC-TATpeptide, we compared [125I]herceptin penetra-bility with both systems under identical experimental condi-tions. Pe of [125I]herceptin in the co-culture system amountedto 43 � 3% of that obtained in the monoculture system. How-ever, the same extent of penetration was reached by increasingthe incubation time with the C-TAT peptide to 3 h or doublingits concentration (data not shown).

DISCUSSION

In this study we were interested in finding conditions thatpermit significant permeations of impermeant substancesincluding therapeutic proteins from the periphery to the brain.Major attempts have been undertaken worldwide in this direc-tion. A common approach is to link or fuse BBB-impermeablepeptide or protein to a “shuttle protein” that enters the BBB byreceptor-mediated transendocytosis (30). Such approachesmay deliver low (pmol) amounts of a peptide or a protein fromthe periphery to the CNS. From a clinical standpoint, however,this approach appears inadequate. Other directionswere there-fore attempted. Brown et al. (31) have demonstrated opening ofthe BBB in patients with malignant brain tumors using hyper-osmolar mannitol. This procedure increased to some extentBBB penetrability of 86Rb and of [14C]sucrose but had little orno effect in enhancing penetrability of high molecular weightproteins.In this study we have used an in vitro model of the BBB,

composed of PBECs grown in culture (20, 21). Initially we haveanalyzed whether this experimental system reflects BBB pene-trability of high molecular weight proteins as well. As validatedin rodents in vivo, we found that HSA (molecular mass � 66kDa), an impermeant molecule, becomes permeable followingcationization (Fig. 1). This opened to us the options of findingconditions that weakened BBB tightness and allowed penetra-tion of therapeutic proteins, two parameters that are extremelyproblematic for evaluation in vivo in a quantitative fashion.Several mechanisms have been proposed by which TAT pro-

tein disrupts BBB, including the decrease of tight junction pro-teins expression and relocalization (32–34). It is reasonable toassume that the C-TAT peptide may have effects similar tothose of the TAT protein on the expression of tight junctionproteins. This would suggest a paracellular passage afterC-TAT peptide treatment. In designing our BBB-disruptingpeptide prototype, we hypothesized that in addition to thetransduction domain, additional site(s) located in other regions

FIGURE 5. Effect of C-TAT peptide treatment on penetration of low molec-ular weight substances. PBEC-M in Transwells were incubated with mediumalone or with medium containing C-TAT peptide (0.6 �mol/ml) for 2 h at 37 °C.[14C]Sucrose or doxorubicin HCl were then added at the luminal side, andtheir permeability across PBEC-M to the abluminal side was determined bymeasuring Pe values every 10 min over a period of 40 min. The results areexpressed as the means � S.E. of the independent assays (n � 3– 4 inserts).*, p � 0.05; ***, p � 0.001 versus untreated.

FIGURE 6. Effect of C-TAT peptide treatment on penetration of highmolecular weight substances. PBEC-M in Transwells were incubated withmedium alone or with medium containing C-TAT peptide (0.6 �mol/ml) for2 h at 37 °C. [125I]HSA or [125I]herceptin was then added at the luminal side,and their permeability across PBEC-M to the abluminal side was determinedby measuring Pe values every 10 min over a period of 40 min. The results areexpressed as the means � S.E. of the independent assays (n � 4 – 6 inserts).**, p � 0.01; ***, p � 0.001 versus untreated.

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of the TAT protein may be required to extend the BBB disrup-tion potency of a transduction domain-containing fragment.Postulating that such an additional site must be a conservedentity amongTATprotein variants, we have selected a cysteinylmoiety as a representative of the seven very conserved cysteinesof region II. NMR studies suggested that the flexibility of theC-TATprotein allows regions II and IV to be in close proximityto each other (35, 36). For that reason we have placed the cys-teinylmoiety at position 1 for allowing its approach to the vicin-ity of the transduction domain if the appropriate structuralalterations do take place at aqueous physiological media.Indeed the inclusion of a cysteinylmoiety at position 1 yielded aTAT-containing peptide that is considerable more potent indestabilizing the PBECmonolayer (Fig. 4). A free cysteinyl sidechain moiety (-CH2-SH) appears to be required. Its derivatiza-tion yields less potent derivatives (Table 2). A low concentra-tion of the C-TAT peptide (0.3–0.6 �mol/ml) destabilizesPBEC monolayer within a period of 1.5–2 h (Fig. 2). Short (1min) exposure of PBEC-M to the peptide followed by its neu-tralization with heparin yielded 60% decrease in TEER within30 min followed by nearly full TEER recovery within the next2 h (Fig. 3). This desirable transitory opening feature is impor-tant for clinical implications.Although not definitive yet, our efforts to obtain a peptide

derivative capable of disrupting BBB but lacking its ability topenetrate into cell interiors were not successful so far. Forexample, PEG5-MAL-C-TAT peptide (a conjugate of PEG5-MAL linked to the cysteine moiety of the C-TAT peptide) isimpermeable into cell interiors5 and ineffective as well in desta-bilizing the PBEC monolayer (Table 2). It therefore seems thatthese two functions may be interrelated and cannot be dissoci-ated. From a clinical point of view, this implies that in vivoexperimental conditions need to be found to bring the C-TATpeptide into close proximity to the BBB, to minimize its uptakeby peripheral tissue. Considering the usage of cationized pro-teins as BBB-opening agents, the efficiency is dependent on thedosage and the type of cationization. Although difficult to com-pare, we found on a molar basis that 1,3-diaminopropane-cat-ionized HSA is �3–5 times more potent than the C-TAT pep-tide in disrupting and opening PBEC-M to impermeantsubstances (data not shown).Finally, we stress that acetylation of the C-TAT peptide

under conditions that leave the cysteinyl moiety nonmodifiedyielded a derivative that preserved �47% of its BBB-disruptingpotency (Table 2). Thus, these two lysyl moieties can bereplaced, derivatized, or modified upon developing a secondgeneration of BBB opening peptides with improved therapeuticefficacies.6In summary, we have used here an in vitro BBB model that

appears to reflect BBB in vivo by at least the following five cri-teria: very low penetrating capacity of [14C]sucrose; high TEERvalue (400�*cm) (20, 21); similar expression pattern ofmajortight and adherence junction proteins (20); negligible penetra-

bility of doxorubicin, [125I]HSA, and [125I]herceptin (Figs. 5 and6); and the conversion of fluorescein-HSA into a penetrablespecies following cationization (Fig. 1). This model assisted usin developing, experimental conditions that destabilizes BBBand allows the delivery of therapeutic agents including highmolecular weight proteins, from the periphery to the brain inquantities thatmight be sufficient to treatmajor CNS disordersand malignant brain tumors.

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C-TAT Peptide Opens Blood-Brain Barrier to Impermeant Agents

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and Yoram ShechterItzik Cooper, Keren Sasson, Vivian I. Teichberg, Michal Schnaider-Beeri, Mati Fridkin

High Molecular Weight Proteins Model of the Blood-Brain Barrier and Allows Permeation ofin VitroCells in an

Peptide Derived from HIV-1 TAT Protein Destabilizes a Monolayer of Endothelial

doi: 10.1074/jbc.M112.395384 originally published online November 13, 20122012, 287:44676-44683.J. Biol. Chem. 

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