In vivo diffusion of lactoferrin in brain extracellularspace is regulated by interactions withheparan sulfateRobert G. Thorne†, Aparna Lakkaraju‡, Enrique Rodriguez-Boulan‡§, and Charles Nicholson†¶

†Department of Physiology and Neuroscience, New York University School of Medicine, 550 First Avenue, New York, NY 10016; and ‡Margaret M. DysonVision Research Institute, Department of Ophthalmology and §Department of Cell and Developmental Biology, Weill Medical College of Cornell University,New York, NY 10021

Edited by Bertil Hille, University of Washington, Seattle, WA, and approved April 7, 2008 (received for review November 30, 2007)

The intercellular spaces between neurons and glia contain anamorphous, negatively charged extracellular matrix (ECM) withthe potential to shape and regulate the distribution of manydiffusing ions, proteins and drugs. However, little evidence existsfor direct regulation of extracellular diffusion by the ECM in livingtissue. Here, we demonstrate macromolecule sequestration by anECM component in vivo, using quantitative diffusion measure-ments from integrative optical imaging. Diffusion measurementsin free solution, supported by confocal imaging and binding assayswith cultured cells, were used to characterize the properties of afluorescently labeled protein, lactoferrin (Lf), and its associationwith heparin and heparan sulfate in vitro. In vivo diffusion mea-surements were then performed through an open cranial windowover rat somatosensory cortex to measure effective diffusioncoefficients (D*) under different conditions, revealing that D* forLf was reduced �60% by binding to heparan sulfate proteogly-cans, a prominent component of the ECM and cell surfaces in brain.Finally, we describe a method for quantifying heparan sulfatebinding site density from data for Lf and the structurally similarprotein transferrin, allowing us to predict a low micromolar con-centration of these binding sites in neocortex, the first estimate inliving tissue. Our results have significance for many tissues, be-cause heparan sulfate is synthesized by almost every type of cell inthe body. Quantifying ECM effects on diffusion will also aid in themodeling and design of drug delivery strategies for growth factorsand viral vectors, some of which are likely to interact with heparansulfate.

drug delivery � extracellular matrix � integrative optical imaging �somatosensory cortex � transferrin

The spread of diffusible signals in brain extracellular space(ECS) is influenced by the local environment, with clearance

mechanisms and the ECS volume fraction, tortuosity, and widthamong the best appreciated factors (1–3). The role that brainextracellular matrix (ECM) components play in modulatingdiffusion is less understood. Normal brain ECM is composedmostly of hyaluronic acid, a nonsulfated glycosaminoglycan, andproteoglycans carrying either chondroitin sulfate (CSPG) orheparan sulfate (HSPG) glycosaminoglycan side chains, alongwith the more recently identified reelin and tenascin glycopro-teins (4, 5). Although aging (6–8), pathological insults (9, 10), orgenetic modifications (11) resulting in altered brain ECM con-tent are often associated with changes in ECS volume fraction,the ability of ECM components to specifically bind and slow themigration of diffusing substances in the ECS remains an openquestion. A direct ECM effect on extracellular diffusion (e.g.,sequestration or slowing of a diffusing substance) has beenpostulated for proteins capable of binding HSPG (12, 13), butlittle evidence exists for this phenomenon in vivo.

HSPGs are thought to play essential roles in the physiology ofall organ systems (14). They comprise a large group that areeither cell-associated (e.g., the integral membrane syndecans and

the glycosyl-phosphatidylinositol-anchored glypicans) or se-creted (e.g., perlecan and agrin). Because secreted HSPG isoften bound to cells indirectly by �-dystroglycan, integrins, orother receptors at the cell surface (15), both forms presentessentially fixed sites of interaction for extracellular ligands. Invitro studies have shown many proteins are capable of binding tothe HS chains of cell-associated and secreted HSPGs (16); theseinteractions are suspected to underlie many phenomena, e.g.,shaping morphogen gradients during development (17, 18);trapping growth factors at the cell surface for subsequentreceptor activation (19); and sequestering pathogenic proteins(20, 21), in part by attenuating extracellular diffusion (22–25).Most protein-HS interactions are inferred from binding studies,using the closely related anticoagulant heparin (H), a highlysulfated glycosaminoglycan commonly isolated from mast cellsin the intestinal mucosa (25). Both H and HS are strongly anioniclinear polymers of uronic acid and glucosamine disaccharideunits, distinguished principally by higher degree of sulfation inH. Proteins that bind H typically bind HS, although the lowercharge density of HS results in interactions of lower affinity (26).Many proteins possess putative heparin-binding regions (27),clusters of positively charged amino acids capable of forming ionpairs with negatively charged glycosaminoglycan groups in aspecific manner (28). However, the capacity of a protein-HSinteraction to attenuate diffusion has not been quantified in vivo.

Here, we used integrative optical imaging (IOI) (3, 29) to studytwo fluorescently labeled proteins, lactoferrin (Lf) and trans-ferrin (Tf), to isolate the effect of a protein-HS interaction ondiffusion in vivo. Human Lf and Tf are iron-binding proteins verysimilar in size (�80,000 Mr), amino acid sequence homology(�60%) and overall bilobal structure (30). However, the aminoacid sequence of human Lf contains a region of basic amino acidsnear its N terminus that allows it to bind polyanions, such as H,HS, and DNA (31, 32); human Tf lacks such a region and doesnot bind H or other glycosaminoglycans under normal physio-logical conditions (33, 34). We first characterized the propertiesof Oregon green 514-labeled human Lf and Texas red-labeledhuman Tf, confirming their similar hydrodynamic sizes and theability of Lf, but not Tf, to bind H and cell-associated HSPG. Wenext determined D* values for each protein after injection intothe neocortex of anesthetized rats, showing that Lf diffusedsignificantly more slowly compared with Tf or Lf coinjected withH, allowing us to quantify the effect of Lf–HS binding on the

Author contributions: R.G.T., A.L., and C.N. designed research; R.G.T. and A.L. performedresearch; E.R.-B. and C.N. contributed new reagents/analytic tools; R.G.T. and A.L. analyzeddata; and R.G.T., A.L., and C.N. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

¶To whom correspondence should be addressed. E-mail: charles.nicholson@nyu.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0711345105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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diffusion of Lf through brain ECS. Our results directly demon-strate sequestration of a diffusing molecule by proteoglycans invivo.

ResultsFree Diffusion Coefficients (D) for Fluorescent Protein Conjugates. Dvalues were determined by IOI (3, 29) at 37°C, allowing estima-tion of apparent hydrodynamic diameters (dH) from the Stokes–Einstein equation [dH � (kT)/(3��D), where k is Boltzmann’sconstant, T is absolute temperature and � is the viscosity of water(6.9152 � 10�4 Pa�s at T � 310 K); see ref. 2.]. Fig. 1A showsrepresentative IOI image sequences (background fluorescencesubtracted) after pressure ejection of Lf or Tf solutions intodilute (0.3%) agarose, an essentially ‘‘free’’ medium. Fig. 1Bshows the Gaussian-shaped fluorescence intensity distributionsextracted from the images in Fig. 1 A, superimposed with theo-retical fits of the diffusion equation (3); the curves characteris-tically f latten and broaden with time as expected for diffusionfrom a point source. Fits to the data yielded the parameter �i

2/4at each time point, ti; regression of �i

2/4 upon ti in turn yieldeda straight line with a slope equal to D (Fig. 1C). Lf’s mean Dvalue was �30% lower with H (D � 5.0 � 0.5 � 10�7 cm2�s�1

with H (mean � SD), n � 16 measurements; D � 7.1 � 0.9 �10�7 cm2�s�1 without H, n � 14; P � 0.00011, ANOVA withStudent-Newman-Keuls post test; Fig. 1D), confirming forma-tion of an Lf–H complex with a significantly larger dH than Lfwithout H (Table 1); no significant differences in D wereobserved for Tf with H (D � 7.6 � 0.5 � 10�7 cm2�s�1, n � 18)

or without H (D � 7.5 � 0.5 � 10�7 cm2�s�1, n � 14) or betweenLf and Tf alone. The results verified H binding to Lf, but not Tf, andthe expected similarity in dH for Lf and Tf in solution (Table 1).

Confocal Imaging of Lf and Tf Binding to Cultured Cells and the Effectof H. Before measuring diffusion in vivo, we studied the bindingcharacteristics of each fluorescent Lf and Tf conjugate in vitro.Lf binding to cells is mediated mainly by high capacity, lowaffinity binding to cell surface HSPG, whereas uptake dependson low capacity, high affinity interactions with specific receptorssuch as the low density lipoprotein receptor-related protein(LRP) (35, 36). In contrast, Tf binding and internalization aremediated by low capacity, high affinity interactions with the Tfreceptor (37, 38). We first evaluated binding of Oregon green514-labeled human Lf to cultured Madin–Darby canine kidney(MDCK) type II cells, because cell surface HSPG content isparticularly well characterized in this cell line (39). As expected,Lf binding to polarized MDCK cells was robust and nearlyabolished in the presence of H at 4°C (Fig. 2A); Lf binding tonormal rat kidney (NRK) fibroblasts was similarly robust andheparin-sensitive (Fig. 2B). Pharmacological inhibition of LRPin MDCK cells attenuated uptake but did not affect surfacebinding [supporting information (SI) Fig. S1], suggesting thatinitial binding was primarily due to cell surface HSPG and notLRP. In contrast, binding of Texas red-labeled human Tf tohuman d407 cells was heparin-insensitive (Fig. 2C), whereas nodiscernible binding was observed for Tf, with or without H, toNRK cells (Fig. 2D). The inability of Tf to bind NRK cells wasnot anticipated, because these cells display Tf receptors on theirsurface (40); Tf species differences or the presence of thefluorophore likely provide the explanation. The results indicatethe fluorescent human Tf conjugate lacks affinity for the rat Tfreceptor and, importantly, confirm heparin-sensitive binding ofLf, but not Tf, to cell surface HS sites.

In Vivo Diffusion Measurements in Brain. D* values were measuredat a depth of 200 �m in the somatosensory cortex of anesthetizedrats by IOI (Fig. 3A), as described in ref. 3. Representative imagesequences and extracted fluorescence intensity distributionsafter pressure ejection of Lf (Fig. 3 B and C), Lf � H (Fig. 3 Dand E), and Tf (Fig. 3 F and G) showed hindered diffusion inbrain relative to agarose. Direct comparison of matched fluo-

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Fig. 1. Free diffusion measurements. (A) Representative images after ejection of Lf or Tf, with or without H, into dilute agarose. (Scale bars: 200 �m.) (B)Fluorescence intensity (I) profiles were extracted from each image (data in blue) and fit to the diffusion equation (fits in red) along a single axis (depicted in Aat the far left). (C) Linear regression of data in A and B; �i

2 � 4D(ti � t0), so regression of �i2/4 on ti returns a slope of D (data transformed to a zero y-intercept).

Fitting yielded the following D values: Lf, 6.5 � 10�7 cm2�s�1; Lf � H, 4.6 � 10�7 cm2�s�1; Tf, 7.2 � 10�7 cm2�s�1; and Tf � H, 7.9 � 10�7 cm2�s�1. (D) Summary data(mean � SD; *, P � 0.0002, ANOVA).

Table 1. Stokes–Einstein diameters and in vivo tortuosity (�) forfluorescently labeled lactoferrin and transferrin

Molecule dH, nm�experimental �

(D/D*)1/2

�predicted†

ECSpl ECScyl

Lf 9.29 � 0.32 (n�14) 3.50 � 0.19 2.27 2.29Lf � H 13.1 � 0.30 (n�16) 2.54 � 0.09 2.60 2.69Tf 8.81 � 0.17 (n�14) 2.29 � 0.11 2.24 2.24Tf � H 8.68 � 0.13 (n�18) 2.28 � 0.07 2.23 2.23

Experimental values (mean � SEM) were determined at 37 � 0.5°C.†� predicted for inert substance of specified dH from restricted diffusionmodels of planar (ECSpl) or cylindrical (ECScyl) ECS pores (3).

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rescence intensity distributions (Fig. 4A) and linear regressionsto obtain D* (Fig. 4B) showed pronounced differences in thediffusion behavior of Lf, Lf � H, and Tf in brain. Lf diffusion inbrain was remarkably slow, resulting in a mean D* value almost60% lower than Tf (D* � 0.58 � 0.1 � 10�7 cm2�s�1 for Lf, N �4 animals, n � 12; D* � 1.4 � 0.3 � 10�7 cm2�s�1 for Tf, N �4, n � 8; P � 0.00013) (Fig. 4C), despite their similar sizes (dH� 9 nm). Lf also diffused significantly slower alone than whencomplexed with H (D* � 0.78 � 0.1 � 10�7 cm2�s�1 with H, N �4, n � 10; P � 0.018; Fig. 4C), despite the complex’s larger size(dH � 13 nm). Control experiments showed that H had no effecton D* for Tf (D* � 1.5 � 0.2 � 10�7 cm2�s�1 with H, N � 4, n �9) or Texas red-labeled 3,000 Mr dextran (dex3), a relativelyneutral, inert probe (D* � 4.7 � 1.0 � 10�7 cm2�s�1 with H, N �3, n � 10; D* � 5.0 � 1.0 � 10�7 cm2�s�1 without H, N � 5, n �6). Our in vivo results were, therefore, characterized by Lf’s slowdiffusion, its enhancement when complexed to H, and theabsence of any effect of H on the diffusion of Tf or dex3.

Prediction of Heparan Sulfate (HS) Binding Site Density from Exper-imental Data. Values of the experimental tortuosity [� � (D/D*)1/2] for Lf � H and Tf � H, determined from mean D andD*, are listed in Table 1. The dimensionless parameter � is usefulfor characterizing hindrances to diffusion in complex mediabecause the dependence of free diffusion on dH is alreadyaccounted for by the inclusion of D in the definition of �. Thestudy in ref. 3 provided relationships that predict � in vivo forinert substances of a specified size, based on a model for � wherenormal neocortical ECS was envisioned as an isoporous envi-ronment of fluid-filled planar or cylindrical pores having a width,dECS, of 38 or 64 nm, respectively. This model separated � intotwo components, referred to here as �1 and �2:

� � �DD*

� �D�

D*DD�

� �1�DD�

� �1�2, [1]

where D� is an interstitial diffusion coefficient. The first com-ponent, �1, represents the hindrance expected for a vanishinglysmall inert molecule arising from obstructions (e.g., cells and theECM) and possibly other factors (e.g., cell cavities or deadspaces), whereas the second component, �2, represents theincreased hindrance expected from restricted diffusion due tosteric hindrance and drag as dH approaches dECS (see ref. 3).

Experimental data and modeling suggest that �1 � 1.6, whereas�2 may be estimated from analytical expressions that depend ondH, dECS, and pore geometry (3). Table 1 lists predicted � valuesfrom this two-component model, based on the measured val-ues of dH for Lf and Tf, with and without H. Experimental �values for Tf � H and the Lf � H complex agreed well withmodel predictions. However, Lf’s experimental � was signifi-cantly larger than predicted, suggesting the existence of a sourceof hindrance for Lf in addition to �1 and �2.

We hypothesized Lf binding to fixed HS sites could provide anexplanation for Lf’s high � in neocortical ECS (Fig. S2). It is wellappreciated that a chemical reaction, such as binding, cansignificantly affect the diffusion process (41). The simplestprocess would be where some portion of a diffusing substancebecomes immobilized by reversibly binding to fixed elements(e.g., HS chains within brain ECS); if a linear equilibriumrelationship exists between the immobilized and diffusing sub-stance (i.e., cA�B � cA � R, where cA�B and cA are concentrationsof the immobilized and free substance, respectively, and R is aconstant) and the binding process is sufficiently rapid so as to beconsidered instantaneous, solutions to the diffusion equationtake the following form (41):

DR � 1

2 cA �cA

t. [2]

If we further stipulate that the concentration of binding sites inthe ECS, cB, is constant and define an equilibrium dissociationconstant for binding, KD � (cA�cB)/cA�B, Eq. 2 becomes thefollowing (42):

DcB

KD� 1

2cA �cA

t. [3]

Eq. 3 is identical to the solution for simple diffusion withoutbinding, except that a modified diffusion coefficient, D � D/(cB/KD� 1), is used in place of D. Following this treatment, we can add tothe previous model (Eq. 1) a third component, �3, to account forrapid, reversible binding to fixed sites within brain ECS:

� � �DD*

� �D� D DD*D�D

� �1�2�DD

� �1�2�3, [4]

Fig. 2. Binding of fluorescently labeled Lf and Tf to cultured cells in the presence or absence of H. Representative confocal images of Lf binding to MDCK cells(A), Lf binding to NRK cells (B), Tf binding to d407 cells (C), and Tf binding to NRK cells (D) at 4°C. (Scale bars: 10 �m.)

8418 � www.pnas.org�cgi�doi�10.1073�pnas.0711345105 Thorne et al.

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where

�3 � �cB

KD� 1 �

�

�1�2. [5]

Eq. 5 can be used to extract information about HS binding sitesfrom the experimental � for Lf, provided that (i) the bindingprocess described by the treatment is justified; (ii) either cB or KDare known; and (iii) the product �1�2 can be estimated, either byusing Lf’s dH (with the analytical expressions in ref. 3), experi-mental � measurements with Lf where HS binding is prevented,or experimental � measurements with a closely related surrogatethat does not bind HS.

The validity of Eq. 5 necessitates that the binding events takeplace much faster than diffusion. Based on the rate constant forLf diffusion in brain (i.e., D*/L2, where L is on the order of a fewhundred micrometers) and the binding kinetics estimated for asimilar protein-HS interaction in vitro (22), Eq. 5 appearsjustified.

Neither cB for HS binding sites nor KD for Lf–HS binding inbrain are available. Because our diffusion measurements usedOregon green 514-labeled human Lf in a rat tissue, we estimatedKD by quantifying binding of this f luorescent conjugate to NRKcells. A low affinity class of putative HSPG binding sites werecharacterized by a KD of 2.6 �M (Fig. S3), in line with priormeasurements using Lf and cell lines from other species (SIResults and Discussion).

Finally, �/�1�2 (Eq. 5) for Lf was estimated by using either Lf’sdH with analytical expressions (3) or the experimental � for Tfas a surrogate for nonbinding Lf, with �3 � 1.53 in both cases.Using this value for �3 and the experimental KD for low affinitybinding of Lf to NRK cells allowed us to estimate cB � 3.5 �M[i.e., �0.7 �M based on total tissue volume, given an ECSvolume fraction of 0.2 (7)]. Our in vivo diffusion measurementsin rat somatosensory cortex are, therefore, consistent with theexistence of HSPG at the site of injection, resulting in a densityof fixed HS binding sites for Lf in the low �M range.

DiscussionHere, we report in vivo D* values for two biologically relevantproteins in the CNS. We have shown that Lf diffusion is

open cranial window BF FL pre-inj FL post-inj

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Fig. 3. Diffusion in brain versus free solution. (A) For brain measurements,Lf or Tf solutions were pressure ejected from a micropipette at a depth of 200�m in normoxic neocortex. Left to Right: Low-power view of open cranialwindow; brightfield (BF) view of rectangle indicated in the leftmost imageafter insertion of micropipette; micropipette and background fluorescence(FL) before pressure injection (pre-inj); and fluorescent cloud of molecules justafter injection (post-inj), showing the six axes used for diffusion analysis. (B)Representative images after Lf ejection into free solution (dilute agarose) orbrain (cortex). (C) Profiles and theoretical fits along the 0° axis for images in B,yielding D � 7.0 � 10�7 cm2�s�1 and D* � 6.1 � 10�8 cm2�s�1. (D) Representativeimages after ejection of Lf � H solution. (E) Profiles and theoretical fits alongthe 0° axis for images in D, yielding D � 5.1 � 10�7 cm2�s�1 and D* � 8.4 � 10�8

cm2�s�1. (F) Representative images after ejection of Tf solution. (G) Profiles andtheoretical fits along the 0° axis for images in F, yielding D � 8.0 � 10�7 cm2�s�1

and D* � 1.5 � 10�7 cm2�s�1. (Scale bars: 200 �m.)

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Fig. 4. Diffusion in brain: Comparison of profiles and summary data. (A)Representative profiles and theoretical fits for Lf, Lf � H, and Tf. (B) Linearregression of data in A; regression of �i

2/4 on ti returns a slope of D* (datatransformed to a zero y-intercept). Fitting yielded the following D* values: Lf,5.5 � 10�8 cm2�s�1; Lf � H, 8.5 � 10�8 cm2�s�1; and Tf, 1.6 � 10�7 cm2�s�1. (C)Summary data (mean � SD; *, P � 0.02; **, P � 0.0002, ANOVA).

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dramatically hindered in the adult rat neocortex whereas Tf, aprotein very similar to Lf in size and structure, diffuses asexpected for an inert molecule. The different in vivo diffusionproperties of Lf and Tf provide evidence that HSPGs can playan important role in modulating extracellular diffusion in vivo,because Lf’s potential to bind HS is among the principal featuresdistinguishing the two proteins. HS chains can extend remark-able lengths, e.g., 50 nm or more for a 30,000 Mr chain (43), sowe hypothesize that both secreted and cell-associated forms ofHSPG slowed diffusion of Lf by binding Lf as it migrated throughbrain ECS.

An important aspect of our study is the close agreement observedbetween predicted values of � from a described theoretical model(3) and the experimental � values for Tf � H and the Lf–H complex.Indeed, we arrived at a prediction of neocortical HS binding sitedensity in the ECS, cB � 3.5 �M, by extending this previous modelto account for the effect of rapid, reversible binding on themeasured value of � for Lf. Although a similar strategy has beenapplied to describe the diffusion and binding of a protein to HS inisolated corneal basement membranes (22), our study does so usingin vivo measurements. Our estimate of cB in the rat somatosensorycortex appears reasonable when compared against extrapolationsfrom postmortem HS purification (SI Results and Discussion). Werecognize that HSPG forms and concentrations may vary acrossdifferent CNS areas; these variations will only become apparentafter further study.

Suitability of Lf and Tf as in Vivo Diffusion Probes. Lf and Tf havebeen particularly well characterized in terms of their structuralsimilarities (30), stable monomeric solution properties (44, 45) andin vitro binding behavior (31–34). Our D values for each proteinwere consistent with those obtained by other methods [e.g., Dvalues of 7.3–7.9 � 10�7 cm2�s�1 and 8.1–8.3 � 10�7 cm2�s�1 (37°C)are obtained for Lf and Tf, respectively, from small angle x-ray andneutron scattering data (46, 47)]. The reduction in D observed forLf in the presence of H was, therefore, consistent with Lf–Hcomplex formation and not Lf aggregation.

We used Lf as an HS-binding probe and Tf as a nonbindingsurrogate for our diffusion measurements in brain. Normal braindoes not contain H, so Lf binding in brain ECS was assumedpredominantly because of cell- and ECM-associated HSPGs. Lfbinding to other negatively charged ECM components in brain, e.g.,CSPG or hyaluronic acid, although possible, was not consideredimportant because these interactions are much weaker than thosewith HS (32, 48). Importantly, we expected to compete off Lf–HSbinding in brain with H, which has both a higher charge density (Hcontains an average of 2.7 negative charges per disaccharide versus�2 for HS) and stronger affinity for Lf compared with HS (26, 32).

High affinity receptors for Lf (e.g., LRP) and Tf are present inbrain (37, 49); however, their concentration and KD are reportedlyin the low nanomolar range (36, 37), multiple orders of magnitudebelow the likely tissue concentration profiles used to determine D*(both Lf and Tf were used at concentrations of �60 �M in theinjecting micropipette) and beyond the expected limits of detectionby our present system. Low capacity, high affinity binding, ifpresent, would have been saturated over the entire course of ourmeasurements, contributing negligibly to the imaged concentrationprofiles. Finally, experimental � for Tf � H and the Lf–H complexagreed well with the predicted � for diffusing inert substances ofequivalent size, suggesting that neither the Tf nor Lf–H measure-ments were appreciably affected by binding to HS or other sites.

Implications for Drug Delivery. Our findings may be especiallyrelevant for methods of drug delivery that involve the injectionor infusion of drugs or gene therapy vectors directly into CNStissue, e.g., convection enhanced delivery. Studies have shownthat H can increase the gross distribution volume or effect sizeof certain protein growth factors (50) or the adeno-associatedvirus serotype 2 gene therapy vector (51) after administrationinto the mammalian CNS in vivo, but the mechanism of Henhancement was uncertain. Our findings with Lf provide amechanistic explanation that can be extrapolated to other pro-teins possessing heparin-binding regions and may even be usedto predict � for such proteins, provided KD is known. Finally, wenote that although H complexation of Lf enhanced transportcompared with Lf alone, the Lf–H D* did not increase to thelevel of Tf’s D*, because the Lf–H complex was nearly 4 nmlarger than Tf. Increased size will be accompanied by increaseddiffusional hindrance, becoming more pronounced as the size ofthe diffusing substance approaches the brain ECS width limit (3).

Materials and MethodsFluorescent Conjugates. Oregon green 514-labeled human Lf (OG514-Lf; iron-saturated; 2.9 mol of OG514/mol Lf; Molecular Probes) and Texas red-labeledhuman Tf (TR-Tf; iron-saturated; 2.0 mol of TR/mol Tf; Molecular Probes) wereused at a concentration of 5 mg/ml in PBS (pH 7.2) unless indicated otherwise.TR-dex3 (0.26 mol of TR/mol dex3; Molecular Probes) was used at a concen-tration of 1 mM in a solution of 154 mM NaCl. Some solutions were made upwith heparin sodium (50 mg/ml; Grade I-A from porcine intestinal mucosa,with H polymer chains mostly in the range 17,000–19,000 Mr; Sigma). Allsolutions were vortexed briefly and centrifuged at 12,000 � g before use.

Animal Preparation for Diffusion Measurements. Experiments with femaleSprague–Dawley rats (160–260 g) were carried out at the New York UniversitySchool of Medicine in accordance with National Institutes of Health guidelinesand local Institutional Animal Care and Use Committee regulations as de-scribed in ref. 3.

Diffusion Measurements. We used the IOI method (2, 29), as described for in vivomeasurements in ref. 3, with slight modifications. The diffusion equation (equa-tion 4 in ref. 3) was fitted to the upper 90% of fluorescence intensity curves, usinga nonlinear simplex algorithm, yielding estimates for a parameter, �i, at a suc-cession of times, ti. Linear regression of �i

2/4 upon ti yielded a slope of D* (or D).Curves were extracted from the data along each of six axes (Fig. 3A) and the finalvalueofD*(orD) foreachmeasurementwastakenfromtheaverageoffouraxes,excluding the two axes giving highest and lowest values. Transforming to a zeroy-intercept (subtracting D* � t0 from �i

2/4 and plotting versus ti) was used tofacilitate comparison. Typically, Lf and Tf diffusion were imaged over timesranging from 20 to 50 s for D and 200 to 300 s for D*.

In Vitro Lf Binding Measurements. Madin–Darby canine kidney (MDCK) type IIcells were cultured on Transwell filters (BD Biosciences) in DMEM plus 5% FBSfor 4 days. Normal rat kidney (NRK) 49F cells and human retinal pigmentepithelial cells (d407) (a gift of R. C. Hunt, University of South Carolina,Columbia) were each cultured on coverslips in DMEM plus 5% FBS or DMEMplus 3% FBS, respectively, for 48 h. All cells were incubated at 4°C for 20 minin recording medium (20 mM Hepes, 4.5 g/liter glucose, 1% nonessentialamino acids, 1% FBS in HBSS) after which OG514-Lf (50 �g/ml) or TR-Tf (100�g/ml) were added for 1 h in the presence or absence of H (100 �g/ml). Cellswere then rinsed twice with cold recording medium and immediately fixed.Images were acquired with a Leica TCS-SP2 laser scanning confocal microscopeand processed identically for all conditions. Additional experiments on Lfinternalization and binding were conducted with MDCK and NRK cells (SIExperimental Procedures).

ACKNOWLEDGMENTS. Thisworkwas supportedbyNational InstitutesofHealthGrants R01-NS28642 (to C.N.) and R01-EY08538 (to E.R.B.), a Dyson FoundationGrant (to E.R.B.), a Research to Prevent Blindness Foundation Grant (to E.R.B.),and an American Health Assistance Foundation Grant (to E.R.B.).

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