T m JOURNAL OF BIOUJCICAL C~~an1s-n~ 8 1994 by The American Society for Biochemistry and M l c l r Biology, h .

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Vol. 269, No. Issue of February

Delivery of Liposomes into Cultured Cells via Folate KB Receptor-mediated Endocytosis*

(Received for publication, June 25, 1993,and in revised form, September 1, 1993)

Robert J. Lee and Philip S LOWS .

From the Department of Chemistry, Purdue University, West Lafayette, Indiana 47907

Folic acid was covalently conjugated to 88-nm lipo- PEG coating is believed to inhibit nonspecific adsorption of somes via spacers of various lengths in an attempt to serum proteins (10) and to thereby prevent nonspecific recogtarget the liposomes KB cells expressing folate recepto nition of liposomes by macrophages (9-10). tors. Spacers of short and intermediate lengths were An equally serious limitation has the frequentinability been unable to mediate association of folate-conjugated lipo- to target the liposomes to the tissue of choice. In some cases, somes with receptor-bearing cells, however, use260 atissue-specific ligands have been difficult to of A polyethyleneglycol spacer (PEG, -3350) permitted where monoclonal antibodies to specific cellidentify, but even M, types have been avid uptake of the liposomes -2.6 x los sitedcell. The generated, a n incompatibility with PEG derivatization has at binding of folate-PEG liposomes to KB cells could be arisen, rendering the antibody-mediated targeting largely incompetitively inhibitedby excess free folate or by anti- effectual. Thus, although PEG coating appears to be necessary serum against the folate receptor, demonstrating the infor prolonged liposome survival in vivo, it simultaneously interaction is mediated by the cell surface folate-binding terferes with recognition of the target cell surface by the lipoprotein. Following binding, cell-associated folate-PEG some-linked antibody, forcing an apparentchoice between tarliposomeswereinternalizedbyfolate-receptor-medigeting and survivability (12). ated endocytosis at37 Cbut not at 4 C. These folateIn we PEG liposomes show potential for delivering large quan-this report, explore two modifications to current liposome technology to circumvent the apparent conflict between tities ofmolecular compounds low weight nontargetability and survivability. First, rather than derivatizea destructively into folate receptor-bearing cells.
Liposomes have recently received considerable scrutiny as possible vehicles for drug delivery largely because of desirable qualities not shared by other delivery strategies. Thus, liposomes can encapsulate large quantities of drug molecules eior ther within their aqueous interiorsdissolved into thehydrocarbonregions of their bilayers (1-3). Liposomes can also protect their contents from rapid filtration by the kidneys and degradation by proteases, esterases, and other destructive enzymes, thus enhancing the cargos residence time in the body (4). When attached to the proper antibody or other type of ligand, liposomes can sometimes be targeted to a tissue of choice, depending on the ability of the antibody or ligand to facilitate cell-specific docking (5-8). Once taken upby a target cell, liposomes may also facilitate the cytoplasmic delivery of encapsulated drug molecules by fusing with the membrane cell (2). Despite certain technical advantages, at least two obstacles currently impede the widespread implementation of liposomes as drug carriers in vivo. First, unmodified liposomes do not survive long in circulation, but instead are removed by macrophages of the reticuloendothelial system withina few hours of administration (10-12). Avoidance of this obstacle has been partially achieved by forming the liposomes from saturated lipids and cholesterol, and including gangliosides or polyethyleneglycol (PEG)-derivatized lipids within the bilayer (9-12). lipid headgroup directly with a cell-specific ligand, we attach the ligand to the distal end of a few lipid-conjugated PEG molecules on the liposome surface. In this manner, the ligand extends flexibly away from the liposome where it can randomly probe different regions of a cell surface and where it is not occluded by the attached forest of PEG molecules. Second, we employ a low molecular weight ligand (folic acid) as a targeting agent. Because high affinity receptorsfor folic acid are greatly enriched on certaincancer cells, it was reasoned that folic acid conjugation might allow targeting of the liposomes to neoplastic tissues. We demonstrate here thatfolate does indeed facilitate recognition of PEGderivatized liposomes by KB cells in culture, and thatfollowing cell surface binding, the liposomes are brought inside the targeted cells by folate receptor-mediated endocytosis.

EXPERIMENTAL.PROCEDURES LipMaterials-Egg phospholipids were obtained from Avanti Polar ids. Polyoxyethylene bis-amine (PEG bis-amine, M . -3350) wasobtained fromSigma.Calcein,Trauts reagent, and maleiimidocaproic acid were purchased from Fluka, and bicinchoninic acid reagent for protein assays was from Pierce Chemical Co. Other chemicals were purchased from major suppliers. KB cells, a human nasopharyngeal cancer cell line, was a gifl from the Purdue Cell Culture Laboratory. Antiserum against folate-binding protein was kindly provided by Chris Leamon in our laboratory. N-Maleiimidocaproylphosphatidylethanolamine (MC-PE) was pre(28.4mg in CH&U with an pared by activating maleiimidocaproic acid equimolar amount of dicyclohexylcarbodiimide (27.7 mg) and mixing (100 with egg phosphatidylethanolamine mg in 10 ml of CHsCl containing 20 p of triethylamine).Amino-PEG-SH was prepared by reacting l * The costs of publication of this article were defrayed in part by the Trauts reagent (8.2 in 200 pl of HaO) payment of page charges.This article must therefore hereby marked molar equivalentsof(200mg) in 1 II~M mg dissolved phosphate buffer be EDTA, 100 II~M aduertisementin accordance with 18 U.S.C. Section 1734 solely to and PEG bis-amine (1 ml, pH 8 0 .Lysine-SH was generated by the same procedure using .) indicate this fact. 10 mg of Trauts reagent and 13.2 mg of L-lysine. Because of the low $ To whom correspondence should be addressed. The abbreviations used are: PEG, polyethyleneglycol; MC-PE, N- reaction pH, we estimate that the vast majority of thiol groups were attached to the a-amino group of the lysine. N-Hydroxysuccinimide maleiimidocaproylphosphatidylethanolamine; PC, phosphatidylchomini- ester of folic acid (NHS-folate)was prepared by the following method: line; PE,phosphatidylethanolamine;FDMEM,folate-deficient mum essential media; NHS-folate, N-hydroxysuccinimide ester of folic folic acid (5g dissolved in 100 ml of dry dimethyl sulfoxide plus2.5ml of triethylamine)was reactedwith N-hydroxysuccinimide(2.6g) in the acid; PBS, phosphate-buffered saline.

3198

Folate-mediated Liposome Delivery into Cultured Cells

3199

MC

liposome-maleiimidc

liponomc-maleiimide

PEG-SH

FIG. Synthetic scheme for prepa1. ration o folate-PEG liposomes f

PEG-liposome

f d K Wid

MLS

Q
folatcPEG-lipoaome

presence of dicyclohexylcarbodiimide(4.7 g) overnight at room temperature. The by-product, dicyclohexylurea, was removed by filtration. The dimethyl sulfoxide solution was then concentrated under reduced pressure and heating, and NHS-folate was precipitated in diethylether. The

product, NHS-folate, was washed several times with anhydrous ether, dried under vacuum, and stored as a yellow powder. Cell Culture-Because most cell culture media contain folate at 103 times its normal serum level, KB cells were maintained in a modified

3200

Folate-mediated Liposome Cultured Delivery Cells into

FDMEM and added to monolayers of KB cells (-5 x lo6) grown in 33-mm culture dishes. The cells were then incubated either at 4 or 37 C P for various lengths of time. Cell monolayers were washed four times with 2 ml of cold PBS and examined under an Olympus phase-contrast/ fluorescencemicroscope. To quantitatively determine the number of liposomes taken up by each cell, the cells were thoroughly washed in PBS, scraped off the culture dishes, and lysed in 1 ml of lysis buffer (PBS containing 1% Triton X-100). The cell-associated fluorescence was then measured in the lysis buffer extract in a Perkin-Elmer MPF-44 fluorescence spectrophotometer and converted to number of bound/ internalized liposomes based on standard curve obtained with known a calcein concentrations in the lysis buffer. The total number of cells was estimated by measuring total cellular protein using the bicinchoninic acid protein assay. To determine the fraction of cell-associated liposomes that were internalized, the cells were stripped of their surfacebound liposomes twowashes with acidic saline (pH 3) followed by one by wash with PBS. The remaining cell-associated liposomeswere considered internalized, as confirmed under Results. Role of Folate-binding Protein in Liposome Uptake-KB cells (-5 x lo5)grown in 33-mm culture dishes were treated with 60 n~ folate-PEG liposomes in PBS containing different concentrations of free folate. T Y ? * w T m e ~ ~ q q yDiminution in liposome binding was then assayed as outlined above. To fi determine whether folate binding activity was due to the folate-binding protein identified previously (lo), an anti-folate-bindingprotein antiseChannel Top (nm) rum known to block association of the receptor with its ligands was FIG. 2.Size distribution of folate-PEG liposomesprepared by incubated with KB cells monolayers for 30 min prior to addition of of PC and folate-PEG liposomes. After4 h of incubation at 37 C, the cells were rapid extrusion. Multilamelar vesicles composed egg PEG-PE were subjected to fivecycles of freezing and thawing and washed four times with cold PBS, and cell-associated fluorescence was subsequently extruded nine times through a 50-nm pore size polycarbonate membrane. Liposome size was determined by quasi-elastic light measured in the lysis buffer (see above). scattering on a Microtrac Ultrafine Particle Analyzer. RESULTS

I 1

f S s : g a a F F 3 % , , s

minimum essential medium from which folate was deleted (FDMEM). This medium was then supplemented with 10%heat-inactivated fetal calf serum which was the sole source of folate. For liposome uptake experiments, approximately lo5 cells were plated in 33-mm culture dishes and allowed to grow in FDMEM for 24 h before assays were commenced. Liposome Preparation and Folate Conjugation-The scheme we used to prepare folate-conjugated liposomes is illustrated in Fig. 1. Briefly, egg PC and MC-PE, an activated form of phosphatidylethanolamine, were dissolved at 4:lmolar ratio inCH,Cl and dried to a thin film in a round-bottom flask. The dried lipid washydrated in PBS containing 10 IILM calcein (a membrane-impermeant fluorescent dye), vortexed, and subjectedtofivecycles of freezing and thawing (14). The resulting liposomes were then extruded nine times through a 50-nm pore size polycarbonate membrane using a manual extruding device (Avestin). The resulting large unilamelar liposomes were separated from unencapsulated calcein by gel-filtration on a Sepharose CL-4B column (Pharmacia LKB Biotechnology Inc.). These liposomes (50 mg of lipid) containing an active maleiimide in the phosphatidylethanolamine headgroups were then reacted with excess ( 100 mg) amino-PEG-SH in PBS and separated from unreacted material on the same Sepharose CL4B column. The resulting PEG liposomes contained a free amino group a t the distal end of the PEG for subsequent use in folate conjugation. Based on the molecular weight of the PEGbis-amine given by the supplier and the dry weight of the PEG liposomes,we estimate that -3.5% of the total phospholipids were conjugatedto PEG. The method forconjugating liposomes tofolate was as follows: NHSfolate (20 mg) was dissolved in 50 pl of dimethyl sulfoxide and added slowly to the stimng liposome suspension (2 ml, pH adjusted to 1 using 1 1 M carbonatehicarbonate buffer, pH 11.5).After stimng for 15 min at room temperature, the reaction mixture was briefly centrifuged and the supernatant passed down a Sepharose CG4B column. The folate-PEG liposomes eluted in the void fraction. No significant leakage of calcein (<1%) observed during folate conjugation, as evidenced by the abwas sence of detectable fluorescence in the trailing fractions of the gel filtration column. The size distribution of the liposomes was determined by quasi-elastic light scattering in aMicrotrac Ultrafine Particle Analyzer (Leeds & Northrup). Phospholipid concentration was determined by a colorimetric assay previously described (18). Identical methods were used inthe preparation of folate-conjugatedliposomes with shorter spacers, except lysine-SH and cysteine were used instead of amino-PEG-SH.Folate-PEG liposomes contained on the average -365 covalently attached folates, representing derivatization of -1% of the total bilayer lipids. Cellular Uptake ofFolate-PEG Liposomes-Liposome samples encapsulating calcein (10rn internal concentration) were diluted in 1ml of

Effect of Spacer Length on Liposome-CellInteractionsLarge unilamelar vesicles composed of egg PC and PEG-PE were prepared as described under Experimental Procedures and thensized by quasi-elastic light scattering. As seen in Fig. 2, the extruded liposomes exhibited a gaussian size distribution with mean diameter of 66 f 13 nm. When KB cell monolayers were incubated with liposomes containing the fluorescent probe calcein, uptake of the fluorescent unilamelar vesicles was found to depend critically on both the presence of attached folate and thelength of the spacer arm connecting the folate to the lipid head group. Thus, regardless of spacer chemistry, no uptake was observed in the absence of folate conjugation (Fig. 3). However,even with extensive folate conjugation, nocell association was seen unless a lengthy spacer separated folate from its bilayer anch:r (Fig. 4). Uptake of liposomesconstructed with the 250-APEG spacer was -37-fold greater than uptake of liposomes with the -23-A maleiimidocaproyl-lysine-SH spacer, which in turn did not measurably exceed nonspecific uptake of underivatized liposomes. Therefore, both folate and a long intervening spacer seem to be required for cellular recognition of the PEG-conjugated liposomes. Time Course of Folate-PEG Liposome Uptake K B Cells--In by order to study the kinetics of liposome uptake by receptorbearing cells, KB cell monolayers were incubated with 60 IIM calcein-containingfolate-PEG liposomes forvarious lengths of time, washed extensively, and evaluated forcell-associated fluorescence by fluorescence microscopy. Although fluorescence was confined to the cell periphery during early time points, as the incubation proceeded, increasing amounts of fluorescence were seen to enter the cell. By 4 h of incubation the entire cytoplasm fluoresced with well-defined punctuate intensities, probably representing the liposomes entrapped in endosomal compartments (Fig. 3). Only the nuclear region appeared somewhat dark, likely because the cytoplasm is thinner in those areas. Incontrast, PEG liposomescontaining the same concentration of encapsulated calcein but lacking folate remained dark throughout the incubation. Quantitative analysis of the intracellular fluorescence revealed that folate-PEG liposome uptake was linear over the first hour of incubation and gradually declined until no further

Folate-mediated Liposome Delivery into Cultured Cells

3201

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. .
I

:.

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FIG. Effectof folate derivatiza3.

tion on liposome uptake by cultured KB cells. Kl3 cell monolayers were incubatedwithcalcein-containing liposomes (60 IMO for 4 h a t 37 "C and viewed by either phase contrast (left panels) fluoor rescence microscopy (right panels). The upper two panels show the same field of cells following treatment with PEG liposomeslackingfolate(control),whereas the lower two panels display a field of cells treated with folate-PEG liposomes. The absence of cell-associated calcein fluorescence in thecontrol sample in contrast to the intense cell-associated fluorescence in the sample containing folate-derivatized liposomes reveals the sharp dependence of liposome recognition and uptake onfolate conjugation. Magnification is x 400.

increase incell fluorescence was observed by the 4-h time point (Fig. 5). This gradual decline in the rate of internalization appears to derive from saturation of the uptakesystem rather than depletion of liposomes, since liposome concentration in the medium did not measurably decline over the course of the incubation. We, therefore, conclude that folate-PEG liposomes are taken up KB cells via a saturable mechanism followed by by internalization into intracellular compartments. Comparison of the kinetics of liposome uptake with internalization of protein-folate conjugates (14) shows that theformer proceeds much slower than the latter. In the of the previcase ously characterized uptake of folate-conjugated serum albumin (14), saturation occurred within 30 min of incubation at 37 "C; in the present situation, saturation wasnot achieved for 4 h. We suggest this kinetic difference is due the larger to size of the liposomes which may result in slower penetration through the viscous carbohydrate layer coating the cell surface. We also observed that only -2.5 x lo5 liposomes are bound or taken up by each KB cell at saturation. This quantity also muchlower is

than the number serum albumin conjugates internalized by of the samecells (-2 x lo7) (14). Because folate-binding proteins exist in clusters on the cell membrane, it is conceivable that binding of a folate-PEG liposome to one receptor could occlude the binding of liposomes to adjacent receptors in the same cluster. Alternatively, one liposome might occupy many receptors due to the multivalency of the folate-PEG liposome construct. Quantitation of Surface-bound and Internalized LiposomesT distinguish surface bound from internalized liposomes, KJ3 o cells were incubated for 4 h with various concentrations of calcein-containing liposomes and thenwashed with either cold PBS to remove unattached liposomes or with acidic saline to strip bound but not internalized liposomes. The cells were then lysed and assayed for residual cell fluorescence using the protocol described under "Experimental Procedures." As shown in Fig. 6a, incubation of cells a t 4 "C with increasing concentrations of folate-PEG liposomes led to increased binding of the M liposomes to thecell surface. By 60 n liposome concentration,

Folate-mediated Liposome Delivery into Cultured Cells

Ib further demonstratethe role of the folate-binding protein (folate receptor) in folate-PEG liposome uptake, KEi cells were preincubated with antiserum to the folate receptor and then assayed for liposome uptake. As shown in Fig. 8, cell association of folate-PEG liposomes was inhibited by anti-folate-binding protein antiserum butnot by preimmune serum. Therefore, the uptakeof folate-PEG liposomes is probably mediated by the same folate-binding protein that catalyzes folate internalization (16, 19).

DISCUSSION Previous work from this laboratory has demonstrated that functionally active proteins can be nondestructively delivered mrpvcr m e Iysinc-SH unino-PEG-SH i n into the cytoplasm of folate receptor-bearing cells if the proType of Spacer teins are first conjugated to folic acid (14-16). In thisreport, we FIG. 4. Effect of spacer length on the uptake of folate-conjufurther document that liposome-encapsulatedmolecules can be gated liposomesby KB cells. Folate-conjugated liposomes were presimilarly targeted to KB cells by tethering the folic acid to the pared as described under Experimental Procedures. spacer refers lipid bilayer via a long spacer. Because cells expressing large No to liposomes in which folate was directly conjugated to amino group the of egg PE in liposomes composed of egg PC/egg PE (8:2). Cysteine spacer numbers of folate receptors are primarily malignant in nature refers to liposomes composed of egg PC/MC-PE (8:2) that were conju- (19-211, entrapment of antineoplastic agents in folate-PEG ligated to folate via the amino group of cysteine. The thiol group of the posomes could conceivably prove useful in the treatment of cysteine was in turn linked to egg PE in the bilayer via reaction with certain types of cancer. maleiimidocaproicacid, as described under Experimental Procedures. The use of a polyethyleneglycol spacer to attach folate to a The length of this spacer is estimated to be -15 A. Lysine-SH and amino-PEG-SHrefer to liposomes in which lysine or PEG bis-amine targeted liposome may have advantages beyond its capacity to derivatized with Trauts reagent was attached lipid bilayer in the enable bulky liposome docking at a cell surface. Thus, PEG to the same way as the cysteine s acer The lengths of these two spacers are derivatization is now commonlyused to prevent liposome phagapproximately 22 and 250 respectively. ocytosis by the reticuloendothelial system. Such stealth lipothis association had reached roughly 1.3 x 105/cell.That these somes are reported to survive more than 24 h in circulation fluorescent liposomes are only surface bound and not endocy- compared with only -2 h observed fortheir unprotected countosed was demonstrated by the ability of an acid saline wash to terparts (12). Further, as pointed out earlier, the length and quantitatively elute them from the cell (Fig. 6a). In contrast, flexibility of the PEG spacer may permit formation of multiple when liposomeincubation was conducted at 37 C and followed tethers between a liposome and a folate receptor cluster. Beby a similar acid saline wash, only half of the liposomes could cause the affinity of such multivalent attachments can be orberemoved (Fig. 6b). The residual liposomes, roughly 1.2 x ders of magnitude higher than the association of monovalent 105/cell, must be sequestered and protected from acid strip- folate with a single receptor, competition from endogenousfoping during the 37 C incubation. Since folate receptor binding lates for receptor occupancy in vivo should be minimized. Thus, proceeds normally at both 4 and 37 C,but endocytosis is per- as shown in Fig. 7, although 0.2 mra free folate may be able to mitted only at the latter temperature (14), we interpret these reduce liposome binding to KB cells to half its normal value, data to suggest that the folate-conjugated liposomes are also folate concentrations in the serum seldom exceed 20 m (131, endocytosed onlyat the higher incubation temperature. These and should, therefore, not compromiseliposome binding in internalized liposomes are probably responsible for the punc- vivo. Although virtually any molecule can be deliveredinto folatetate fluorescence that dominates the cytoplasm in cells incubated at 37 C (Fig. 3). At 4 C, no cytoplasmic fluorescenceis receptor bearing cells by direct conjugation to folic acid (14-161, encapsulation of the therapeutic agent within a folate-PEG seen (data not shown). preferred for many reasons. First, direct modiInhibition of Folate-PEG Liposome Uptake by Free Folate or liposome may be is Antiserum against Folate-binding Protein-In order to evalu- fication of the targeted drug not required, since it can simply ate therole of folate in the cellular uptake of folate-PEG lipo- be entrapped within a folate-conjugatedliposome. Whereas desomes, KB cells were treated with 60 m folate-PEG liposomes livery of large proteins may be relatively unaffected by this in PBS containing increasing concentrations of free folate. As distinction, for low molecular weight drugs with few or no poscritical. Even shown in Fig. 7, uptake of the folate-PEG liposomes was com- sible sites of modification, the advantage could be petitively inhibited by excess free folate. Curiously, although in cases where derivatization is chemically feasible, biological free folate has aKd of 0.01-1 m for the folate receptor (131, our activity might still be lost, rendering the folate targeting caparesults show that 0.2 mra folate is required to reduce cellular bility useless. Second, encapsulation within a liposome can amliposome uptake by 50%. Therefore, folate-PEGliposomes must plify the number of drugs delivered at each receptor. Although exhibit a much higher affinity for the cells than free folate. We calculations reveal that only 2.5 x lo5 receptordcell may be believe the reason for this enhanced avidity is that thesize of occupied at saturation (see Fig. 4), if each liposome contains the liposome and length of the PEG spacers allow more than -lo4 drug molecules (-0.16 M), the net delivery may still be one folateAiposome bind a cell surface receptor. The fact that enhanced by a factor of 100. Third, liposome-encapsulateddrug to folate receptors normally exist in large clusters (13) and that molecules may be shielded from enzymetic degradation that folate-linked lipid molecules can freely diffuse over the lipo- might inactivate otherwise unprotected drug molecules. some surface allows for multivalent interactions. Theoretically, Fourth, because of the liposomes slower filtration through the the dissociation constant of a multivalent interaction is roughly kidneys and due to the possibility of enshrouding the liposome the product of the dissociation constants of the individual in- in a forest of PEG, the circulating lifespan of a folate-PEG teractions. However, because in this case the competition is liposome may be greatly elevated over that of its unencapsubetween folate on a liposome and free folic acid, relative diffu- lated cargo. Thus, foreign macromoleculesare commonly scavenged by the reticuloendothelial system, while small molecular sion rates and off-rate constants may complicate this mathweight drugs should be quickly filtered by the kidneys. HOWematical description.

1 ,

Folate-mediated Liposome Delivery into Cultured Cells

2.5

3203

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2.5
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2.0

3.0

4.0

5.0

Free folate Concentration (M) FIG. Inhibition of folate-PEG liposome uptake by 7. free folic FIG. Time dependence of liposome uptake by iIB cells. KB acid. KB cells were incubated for 4 h with calcein-containingfolate5. cells were incubated with calcein-containingliposomes (60 m)at 37 "C PEG liposomes(60 m)in thepresence of various concentrations of free for different lengths of time. The total amount of cell-associated lipo- folic acid at 37 "C. m e r thorough washing, the cells were lysed in 1% somes was determined by fluorescence spectroscopy following cell lysis Triton X-100, and the number of cell-associated liposomes was deterin 1% Tritm X-100.Open circles (0) liposomes; closed circles (0) mined by fluorescence spectroscopy. PEG folate-PEG liposomes.Error bars represent one standard deviation (n = Incubation time (hour.)

3).

a)

3.0

P "1
2.0

0.0

2
20
40

60

0.0

0.1

0.2

0.3

Q.4

0.5

Liposome Concentration (nM)

Added serum

(mi)

FIG. Inhibition of folate-PEG liposome uptake by antiserum 8. to the folate-binding protein. cells were preincubated for 30 min KB at 37 "C with different amounts of either nonspecificrabbit serum (0) or rabbit antiserum raised against the cell surface folate receptor (0). Calcein containing folate-PEG liposomes I ) were then added.After (60 " 4 h of further incubation at 37 "C, the cells were washed with cold PBS, lysed in 1% Triton X-100, the quantity of cell-associated liposomes and was determined by fluorescence spectroscopy.

liposomes avoids most concerns over competition with endogenous folate for common cellsurface receptors. Where adjacent folate receptors are found unoccupied, the affinity of a multivalent liposome attachment willfavorliposome association over that of free folate. Although the value of antibody-mediated liposome targeting should not be minimized, for specific delivery to cancer cells, the use of folate as a targeting agent may be preferred. Thus, folate is nonallergenic whereas antibodies may elicit an im0 20 40 00 Llposome Concentration (nM) mune response (24). Folate mediates liposome endocytosis into non-lysosomal compartments (141, whereas antibodies freFIG. 6. Evaluation of the fraction of cell-associated liposomes that are internalized as a function of liposome concentration quently promote uptake into lysosomes. Folate is compatible and temperature.KB cells were incubated with different concentra- with PEG-derivatized lipids in contradistinction to antibodytions of calcein-containingliposomes for h at 4 "C (a) 37 "C (b).The conjugated liposomes, which lose their binding affinity upon 4 or cells were then washed four times in PBS to remove unattached liposomes and either stripped of surface-bound liposomes at low pH or le& incorporation of PEG lipids (12).And the folate ligand is inexn unmodified. Filled circles (O), cells treated with folate-PEG liposomes pensive, stable duringstorage and i vivo circulation, intrinsiand washed with PBS. Filled triangles (A),cells treated with folate- cally nontoxic to cells, and easy to conjugate to the desired and PEG liposomes and washed with pH 3 saline. Open circles (0) open liposomes. Taken together, these advantages suggest that fotriangles (A) are cells treated with PEG liposomes and washed with late-PEG liposomes warrant scrutiny as a possible delivery PBS or pH 3 saline, respectively. system for chemotherapeutic agents. Finally, the observations that both excess free folate and ever, in the guise of a folate-labeled stealth liposome, the tar- antiserum to the cell surface folate binding protein inhibit fogeted contents should survive the above hazards for many late-PEG liposome endocytosissuggest that uptake occurs via hours. Finally, as mentioned previously, the use of folate-PEG the classical folate endocytosis pathway. documented by othAs

3204

Folate-mediated Liposome Delivery into Cultured Cells

Martin, F. J. (1991) m . Natl. h dSei. U.S. A. 88, 11460-11464 P a. 1 . Allen, T M., Hansen, C., Martin, F., Redemann. C., and Martin, F. J. (1991) 1 . Biochirn. Biophys. Acta 1066,29-36 12. Klibanov, A. L., Maruyama, K , Beckerleg, A. M., Ibrchilin,V. P., and Huang, L.(1991) Biochirn. Biophys. Acta 1062, 142-148 13. Kamen, B. A,, Wang, M.-T., Streckfuss, A. J., Peryea, X.,and Anderson, R. 0. W. (1988) Biol. Chern. 2 8 13602-13609 J. 8, 14. Leamon, C. P., and Low, P.S. (1991) Proc. Natl. Acad. Sci. U.S. A. 88,55725576 J. 15. Leamon, C. P., and Low, P. S. (1992) Biol. Chern. 267,2496624971 . Biochern. J. 291,855-860 16. Leamon, C. P ,and Low, P. S. (1993) 17. MacDonald, R.C., MacDonalds,R. I., Menco, B. P. M., Takeshita, K , Subbarao, N. K., and Hu, L. (1991) Biochirn. Biophys. Acta 1061,297403 18. Stewart, J. C. (1980)hl.Biochern. 104,486469 19. Anderson, R. G. W., h e n , B. A,, Rothberg, K. G., Lacey, S.W (1992) . Science 255,410-411 20. Weitman, S.D., Lark, R. H., Conery, L. R., Fost, D. W., Frasca, V., Zurawsky, V. R., Jr., and Kamen, B. A. (1992) Cancer Res. 62,33964401 21. Kane. M. A., Portillo, R. M., Elwood, P. C., Antony, A. C., and Kolhouse, J. F. (1986) Biol. Chern. 261,4449 J. 22. Canevari, S., Mezzanzanica, D., Menard, S., Femni, S., Moretta, L., and Int. Colnaghi, M.I. (1992) J. Cancer 7,4244 23. Bolhuis, R. H., Lamers, C. H.J., Goey, S. H., Eggermont, A. M. M., Trimlms, L. J. B. M. Z., Stoter, G., Lanzavecchia,A,, D Re, E., Miotti, S., Raspagliesi, F., i Int. Rivoltini, L., and Colnaghi, M. I. (1992) J. Cancer 7, 76-61 24. Phillips, N.C., and Emili, A. (1991) Immunol. Lett. 30,291-296 25. Rothberg, K. G., Ying,Y., Kolhouse, J. F., Kamen, B. A., and Anderson, R.G . W . (1990) . Cell Biol. 110, 637-649 J 26. Turek, J. J., Leamon, C. P., and Low,P. S. (1993) Cell Sci. 106,423430 J.
10. Papaha4jopoulos, D., Allen, T. M., Gabizon, A,, Mayhew, E., Matthay, K, Huang, S. K , Lee, K-D., Woodle, M. C., Lasic, D. D., Redemann, C.. and

ers (13,19, 25), this pathway begins with folate binding to a cluster of folate receptors located in uncoated pits termed caveolae. Previous studies from our laboratory have shown that proteins as large as ferritin (Mr -440,000) can be co-endocytosed as covalent conjugates of folate at these uncoated pits (26). The present investigation extends this size limit considerably by demonstrating that liposomes with a mean diameter of 66 nm can enter cells by the same route. Preliminary data indicate that -200-nm folate-PEG liposomes are not internalized by the same cells. It will be interesting to learn whether the size limit on endocytosed folate conjugates corresponds to the -50 nm diameter of the caveolae (19).
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