[CANCER RESEARCH 47, 4105-4108, August 1, 1987]

Preparation of Drug-Low Density Lipoprotein Complexes for Delivery ofAntitumoral Drugs via the Low Density Lipoprotein Pathway1

Bo Lundberg2

Department of Biochemistry and Pharmacy, ÀboAkademi, SF-20500Âbo, Finland

ABSTRACT

The receptor-mediated assimilation of low density lipoprotein (LDL)

by many cancer cells is much higher than that of normal cells. This factsuggests that lipoproteins with incorporated cytotoxic drugs may be usedas a carrier for chemotherapeutic agents to neoplastic cells. In this studya lipophilic cytotoxic compound is incorporated into reconstituted LDLby two different methods. Both the structure and cellular uptake werefound to be similar to those of native LDL. Tests of the cytotoxic activityon cultured cells demonstrated that the drug delivered to the cells via theLDL pathway was able to kill 100% of the cells. Heparin and a lowtemperature, which are known to inhibit uptake of LDL by the receptormechanism, abolished the cytotoxic activity of the drug-lipoprotein con

jugates. The results suggest that it may be possible to use reconstitutedLDL as a vehicle for lipophilic antineoplastic drugs in order to increasethe drug accumulation and selectivity in tumor cell populations with highLDL receptor activity.

INTRODUCTION

Cells acquire cholesterol for membrane synthesis primarilythrough the adsorptive endocytosis of plasma LDL3 (1). The

initial step in this uptake process is the high affinity binding ofLDL to a cell surface receptor followed by internalization andlysosomal degradation of the lipoprotein particle. The cholesterol released is used by the cell and the endogenous cholesterolsynthesis is inhibited (2). Cancer cells, which proliferate rapidly,need large amounts of cholesterol for synthesis of new membranes. A logical consequence of this is that cancer cells willhave higher LDL requirements and higher LDL receptor activities than normal cell types (3).

The LDL particle consists of an apolar core of neutral lipids(cholesteryl esters, triglycérides)surrounded by a polar shell ofphospholipid, unesterified cholesterol, and apo B that is recognized by the cell surface receptors. It is possible to replacethe core neutral lipids with exogenous neutral lipids or suitablehydrophobic compounds (4, 5). Such reconstituted m-LDL isessentially identical with native LDL as to the ability to bebound, incorporated, and degraded by cultured human fibro-blasts (5). Thus m-LDL reconstituted with an adequate toxiccompound may be used as a specific endogenous drug carrierfor targeting the drug to cancer cells with high LDL receptoractivity.

The criteria that must be met for an effective cancer therapyby use of the m-LDL system are: the cancer chemotherapeuticagent must be suitable for recombination with LDL, it must bespecific, i.e., must enter the cells by the LDL receptor pathway,and the amount of toxic m-LDL must be large enough to inhibitthe growth of or to kill the target cancer cells.

Received 12/1/86; revised 4/16/87; accepted 4/22/87.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' Supported by grants from The Cancer Society of Finland.2To whom requests for reprints should be addressed, at Department of

Biochemistry and Pharmacy, Porthansgatan 3, SF-20500 Àbo,Finland.3The abbreviations used are: LDL, low density lipoprotein; m-LDL, model

low density lipoprotein; PBS, phosphate buffered saline; SDOC, sodium deoxy-cholate; EPC, egg yolk phosphatidylcholine; compound 25, /V-|[(4-)3|3(oleo-yloxyjandrost- 5-en -17/3-yl]pentyl|oxy[carbonyl] - N, N -bis(2 -chloroethyl)amine;m-(25)LDL, complex of compound 25 with m-LDL; Apo B, apolipoprotein B.

MATERIALS AND METHODS

Materials. The cytotoxic mustard carbamate, Ar-(4-)-3^-(oleoyl-oxy)androst-5-en-17j3-yl(pentyl)oxy(carbonyl)-Ar,yV-bis(2-chloroethyl)a-mine, compound 25, was a gift from Dr. R. A. Firestone, Merck, Sharp,and Dohme Research Laboratories (Rahway, NJ) and synthesized asdescribed in (6). Sodium iodine-125 (16.3 mCi/Mg, carrier free, pH 7-11) was obtained from the Radiochemical Centre, Amersham, UK;heparin. ammonium salt (179 USP K-l units/mg) from Sigma Chemical Co., St. Louis, MO: sterol-ester acylhydrolase (EC 3.1.1.13) fromPseudomonas fluorescens from Boehringer Mannheim GmbH, GFR,and sodium deoxycholate (SDOC) from E. Merck AG, Darmstadt,GFR.

Lipoprotein. Human LDL (density 1.019-1.063 g/ml) was isolatedby differential density ultracentrifugation from fresh pooled humanserum using standard procedures (7). The centrifugations were performed in a Beckman SW-28 rotor at 27,000 rpm at 4'C for 24 h. The

purity of the lipoprotein preparations was checked by agarose gelelectrophoresis. LDL was labeled with iodine-125 to a specific activityof about 100 cpm/ng by the iodine monochloride method (8). LabeledLDL was passed through a Sephadex G-25 column and extensivelydialyzed against 0.15 M NaCl and 0.05% EDTA, pH 7.4, to removefree iodine. The lipoprotein preparations were filtered through a 0.22-¿imfilter and stored at 0°Cin sterile bottles.

Preparation of Cytotoxic m-LDL. The cytotoxic compound 25 wasincorporated into reconstituted m-LDL using the method describedbefore (5) replacing the core cholesteryl esters with the hydrophobiccompound 25. The structure of compound 25 is designed to be like thenative core lipid cholesteryl ester and it was found that the reconstitution properties were also very similar. In brief, LDL was delipidatedwith a 12-times weight excess of the detergent SDOC in 50 mM NaCl-50 mM sodium carbonate (pH 10). The solubilized apo B was separatedfrom the lipid components of LDL by gel filtration on a Sepharose CL-4B column. Reconstitution was then performed by addition of the apoB preparation to a sonicated microemulsion of compound 25 and EPC(2/1, w/w). The cytotoxic m-LDL complexes used for incubations withcells were then isolated by density gradient centrifugation using a 0-40% linear sucrose gradient.

An alternative reconstitution method, which involves delipidationwith an enzyme instead of detergent, was also developed. The cholesteryl ester core of 1 mg LDL was hydrolyzed with sterolester hydrolase(0.05 U) for 30 min at 37°C.The incubation mixture contained 0.25

mg EPC vesicles and 2 mg albumin in order to bind the reactionproducts; free cholesterol and free fatty acids. After completed incubation the reaction mixture was subjected to density gradient centrifugation using a 0-50% linear sucrose gradient. At the end of the centrifugation in a Beckman SW-60 Ti rotor (50,000 rpm, 24 h at 10°C)the

tubes were punctured and fractions were collected. The fractions wereanalyzed for protein and lipids as described under "Analytical Procedures." The protein-rich fractions were pooled, dialyzed against 0.01 MTris-HCl buffer, pH 7.4, and then incubated with a suspension ofcompound 25 for 30 min at 37°Cfollowed by a short sonication (5

min) in a bath sonifier at the same temperature. The compound 25suspension was prepared by the combined injection and sonicationprocedure described in (5) and the weight ratio between apo B andcompound 25 was 0.6:1. The resulting recombined cytotoxic m-LDLwas isolated by sucrose gradient centrifugation in the same way as forthe detergent method. After extensive dialyzation against PBS (0.01 M,pH 7.4) and filtration through a 0.45-^m Millipore filter (MilliporeCorp., Bedford, MA) the preparations were used for incubations withcells.

Cells. Diploid and nontransformed human lung fibroblasts weregrown in Dulbecco's modified Eagle medium supplemented with 2 mML-glutamine, 0.1 mM nonessential amino acid solution, 20 mM 4-(2-

4105

on April 4, 2020. © 1987 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

PREPARATION OF DRUG-LDL COMPLEXES

hydroxyethyl)-l-piperazineethanesulfonic acid, 0.08% (w/v) sodium bicarbonate, gentamicin (10 ¿ig/ml),and 10% (v/v) of foetal calf serum.In some experiments also cells of the SH-SY5Y subclone, derived froma human neuroblastoma of sympathetic adrenergic ganglion origin (9)were used. The SH-SY5Y cells were cultured in RPMI 1640 mediumsupplemented with 10% (v/v) foetal calf serum and streptomycin (100ng/ml). All cells were incubated with serum-free medium for 24 hbefore the experiments.

Cellular Uptake and Metabolism of |"SI|LDL and |'25I]m-LDL. Determinations of binding, incorporation, and degradation of [12SI]LDLand ['"Ijm-LDL were done essentially as described by Brown and

Goldstein (10). The degradation was determined from the amount oftrichloroacetic acid-soluble radioactivity in the incubation medium afterextraction of free iodine with chloroform. Values for bound lipoproteinwere obtained by incubation of the cells with a solution containingheparin (5 mg/ml) in PBS. The flasks were then washed three timeswith cold PBS and the cells were detached by gentle scraping. Aliquotswere taken for determination of protein and radioactivity (incorporatedlipoprotein).

Determination of Toxicity. The toxicity of m-LDL with incorporatedcompound 25 [m-(2S)LDL] was measured by its ability to kill humanfibroblasts or neuroblastoma cells in culture. The cell death was checkedby Trypan blue exclusion. Selectivity was measured by comparing theeffects of m-(25)LDL on cell viability in the presence or absence ofheparin (3 mg/ml) in the medium. Heparin prevents the binding ofLDL to the receptor (11). In some experiments the incubations wereperformed at 4'C, which does not prevent the binding but the internal-

ization of the lipoprotein particle (11).Analytical Procedures. Protein was measured by the modified Lowry

method (12) using albumin as standard. Lipids were extracted withchloroform/methanol (2/1, v/v). Quantitative thin layer chromatogra-phy was performed with precoated silica gel 60 plates (Merck). Theplates were developed first to half the width in chloroform/methanol/acetic acid/water (50/25/8/4), and then to the top in petroleum ether/benzene/acetic acid (30/8/1). Densitometric quantification was madeafter detection by spraying with an aqueous solution containing sulphuric acid 20% (v/v), ammonium sulphate 20%, and K2CrO3 0.6%,and heating at 160"C for 30 min. Cholesterol was also determined by

an enzymatic method using the kit from Boehringer Mannheim GmbH(catalogue no. 310328). Compound 25 was determined as free cholesterol after hydrolysis with 0.6 N ethanolic KOH.

RESULTS

Characterization of the Cytotoxic m-LDL. The delipidation ofLDL with SDOC gave an apo B product, which was virtuallyfree of lipids in agreement with earlier results (5). The apo B-lipid complex obtained after incubation with sterol-ester hydro-lase and separation by sucrose gradient centrifugation had thefollowing composition: protein 33.2%, phospholipid 30.2%,cholesterol 14.6%, cholesteryl ester 3.3%, triglycéride15.6%,and free fatty acid 3.1%. After recombination of the SDOCdelipidated apo B with a compound 25-EPC (2:1) microemul-sion, or alternatively the sterol-ester hydrolase delipidatedLDL, with a compound 25 suspension, the resulting complexeswere isolated by sucrose gradient centrifugation (Fig. IA). Them-(25)LDL particles appeared as peaks at densities about 1.07g/ml. The weight ratios between cytotoxic drug and apo B werequite constant in the major part of the peaks. Fractions in thedensity interval 1.020-1.070 g/ml were pooled for further characterization and incubation with cells. The recovery of compound 25 in the final preparations was about 65 and 60% forthe SDOC and hydrolase reconstitution method, respectively.The modified Krieger method (13) was also tested in this respectand the yield obtained was 8%.

The final m-(25)LDL particles were subjected to analyticalgel filtration on a Sepharose CL-2B column. They appeared asa symmetrical peak near that of native LDL (Fig. \B). Theinserted electron micrograph in Fig. \B demonstrates that them-(25)LDL particles have a spherical form. The retention ofthe m-(25)LDL preparations in a Heparin-Sepharose column

Fract ¡onnumber

¿812

300

- 200£

Q- 100

10

^O)3-6

JL

1.12

1.09

1.06

1.03

1.00

10 20 30 ¿0

Fraction number50

Fig. 1. Characterization of compound 25: m-LDL complexes prepared by thedetergent (O) or by the hydrolase (A) method, respectively (see "Materials andMethods"). I. density gradient centrifugation in a 0-40% sucrose gradient. Bar,

fractions taken for incubations with cells and further characterization; insert,ratios between compound 25 and protein (apo B) in the fractions; dotted line, theposition of native LDL in the density gradient. B, Sepharose CL-4B columnchromatography. Dotted line, elution pattern of native LDL shown as a comparison; arrow, void volume (I,,) of the column; insert, electron micrograph ofcytotoxic m-LDL particles; bar, 30 nm.

(LKB, Sweden) was identical with that of native LDL (data notshown). More than 99% of the m-(25)LDL preparations wasprecipitated with dextran sulphate-MgCh (14) which is knownto precipitate native LDL. It can be concluded that the reconstituted cytotoxic m-LDL particles are structurally similar tonative LDL. The stability of the preparations was good and noaggregation was noted after 4 weeks at 4°C.

Experiments were also performed to study the stability of m-LDL particles in human serum and a possible transfer of drugto other lipoprotein groups. Preparations of m-(25)LDL witha hydrogen-3 label in the steroid moiety (270 cpm/^ig) wereincubated at 37°Cfor 4 h in human serum. After completed

incubation the lipoprotein fractions were separated by densitygradient centrifugation and the distribution of radioactivity wasmeasured. The stability of the LDL-drug complexes was foundto be very good and no significant amount of the lipophilicsteroid mustard could be detected in other lipoprotein fractionsthan the LDL one.

Metabolism of |125I]LDLand [125I]m-(25)LDL. As a test of thebiological activity of the m-(25)LDL preparations the binding,incorporation, and degradation were studied in cultured human

4106

on April 4, 2020. © 1987 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

PREPARATION OF DRUG-LDL COMPLEXES

fibroblasts. The data in Fig. 2 show that the m-(25)LDL particles are taken up and metabolized by the cells at a rate similarto that of native LDL.

Cytotoxic Activity of m-(25)LDL. The ability of the m-(25)LDL preparations to kill cells was studied with humanfibroblasts and neuroblastoma cells in culture. The results aresummarized in Table 1. A consistent feature was that at prele-thal concentrations of cytotoxic m-LDL the cells showed adeleterious response but recovered after a few hours. Dead cellsbegan to appear at a concentration of 5 fig/ml during 5 h ofincubation with detergent m-(25)LDL. If the incubation timewas extended to 20 h an appreciable part of the cells was killedand especially prone to the treatment were the neuroblastomacells. When the concentration of cytotoxic m-LDL was increased further the toxic effect was enhanced and at concentrations above 20 ng/ml all cells were killed already after 5 h. Thecytotoxic m-LDL prepared with apo B, delipidated by the sterolester hydrolase method, showed the same properties, but theeffective concentration was somewhat higher than that forSDOC m-(25)LDL. However, a treatment of both cell typeswith a concentration of 20 Mg/ml during 20 h killed all cells.

If heparin (3 mg/ml) was added to the incubation mediumthe toxic effect of the m-(25)LDL preparations was greatlyreduced and even at a concentration of 30 Mg/ml the cells wereviable after 48 h of incubation. A lowering of the incubationtemperature to 4°Cabolished the toxic effect, but if the temperature then was raised to 37°Cthe cytotoxic activity was

restored.

200-

100-5500--C•g"

400-010^300-0>uen^200-Q£

100-(NIÈoí

c 500-•oCtu

D400-a"gì

300-

200-100-BindiIncoriDegraigTlorat

Tdati

TononTïITTT

Fig. 2. Cellular binding, incorporation, and degradation of [l25I]m-(25)LDLcomplexes prepared by the detergent (.•()or the hydrolase method (B) comparedwith those of native ['"I]LDL (C). Confluent human flbroblasts in culture werepreincubatecl for 24 h in serum-free growth medium before the addition of thelabeled lipoproteins (5 ^g/ml). After completed incubation (4 h at 37'C) the cellsand medium were assayed for binding, incorporation, and degradation of drug-LDL and native LDL as described under "Materials and Methods."

Table 1 Cytoloxicity of the reconstituted m-LDL preparations containingcompound 25 tested on cultured cells grown in serum-free medium

The cells used, human fibroblasts and human neuroblastoma cells (SH-SY5Y),were preincubated in serum-free medium for 24 h prior to the addition of thecytotoxic m-LDL preparation. The toxicity of the drug-LDL complexes wasmeasured by counting the dead cells after completed incubation. In some experiments the receptor-mediated uptake of cytotoxic LDL was inhibited by additionof heparin (3 mg/ml) or by incubation at 4*C.

Dead cells (%)

Preparation"SDOCm-(25)LDL+Heparin4'CHydrolasem-(25)LDL-t-Heparin4'CConcentration(Mg/ml)'55101020,30303010102020,303030Time(h)520520520205205202020Humanli

broblasts520SO801000010506010000SH-SYSY1080301001002015908010040

' SDOC m-(25)LDL and hydrolase m-(25)LDL = eytotoxie m-LDL preparedby the detergent or sterol ester acylhydrolase method, respectively (see "Materialsand Methods").

* Concentration of cytotoxic compound. Values, means of at least three exper

iments.

DISCUSSION

A major problem in cancer chemotherapy with cytotoxicagents is the lack of selectivity, which leads to severe toxiceffects on normal cells. Consequently, attempts are being madeto discover ways to deliver the cytotoxic agents specifically tothe neoplastic cells. Such studies have been performed with thechemotherapeutic agents linked to DNA (15), antibodies (16),hormones (17), and liposomes (18). However, the results of invivo administration have not been satisfactory because of rapidclearance from the circulation by reticuloendothelial cells orstability problems with the drug-carrier vehicles.

The use of the LDL receptor pathway for delivery of cytotoxicagents to cancer cells has been proposed by several authors (19,20). A recent in vivo study supports the hypothesis that LDLwith incorporated cytotoxic drugs may be valuable as a carriersystem (13). The present report describes the successful incorporation of a lipophilic cytotoxic agent, a steroid mustardcarbamate, into the core of reconstituted LDL and the evaluation of the biological activity of the resulting complex. Both thestructure and the cellular uptake of the drug-bearing particleare similar to those of native LDL. The critical step in thereconstitution procedures is the delipidation of apo B, becauseit is known that even minor changes in the structure of apo Bcan result in a fast clearance in the circulation of the reconstituted LDL by the reticuloendothelial cells. It was thus considered important to develop a mild reconstitution method as analternative to that presented before, which uses a detergent forthe delipidation of apo B (5). This new procedure does notinvolve organic solvents or detergent, but enzymatic hydrolysisfor the delipidation of LDL. Recent structural studies of apo Bhave confirmed that it retains its native conformation by recombination with lipid after a mild delipidation (21). Both recombination procedures gave m-LDL particles with similar structure and cellular uptake, but the cytotoxic activity of the hydrolase m-(25)LDL was somewhat lower than that of the SDOCm-(25)LDL. The explanation is obviously that the former conjugate has a lower ratio of cytotoxic compound per apo B,which leads to a lower incorporation of the drug via the receptorpathway.

The specificity of the preparations used in this study seems4107

on April 4, 2020. © 1987 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

PREPARATION OF DRUG-LDL COMPLEXES

to be good. The values for binding, uptake, and degradationwere similar to those obtained for native LDL and those obtained by Goldstein and Brown (1). The incorporation of m-(25)LDL by cultured cells was blocked by heparin, which isknown to selectively release LDL bound to the cell-surfacereceptor (10). The cytotoxic activity was also prevented bylowering of the temperature to 4°C,which inhibits the incor

poration of the lipoprotein particle (10). Compared to otherreconstitution methods, those described here give a much higheryield. The simple incubation of a drug with LDL gave onlyincorporation of 0.5% of the drug added (22). The modifiedKrieger method (13), which otherwise seems to be favorable,gave a yield of 8% in our hands. These values can be comparedto the yield of about 60% obtained in this study. Anotherimportant factor from the practical point of view is the highstability of the preparations during storage. It can also be notedthat LDL can be stored frozen with preserved clearance rate inthe circulation (13).

Large efforts have been made to adopt liposomes (lipidvesicles) to the use for administration of drugs (23). However,the rapid receptor-mediated endocytosis of cytotoxic m-LDLmight provide distinct advantages over the trapping of antineo-plastic drugs in liposomes. The main reasons for this statementare that liposomes are subjected to destruction by blood components, primarily by lipoproteins (24) and to a fast clearancefrom the circulation (25). In the m-(25)LDL particles the lipo-philic drug is incorporated into a physically stable core and theclearance of an adequately reconstituted m-LDL is similar tothat of native LDL (13). The cytotoxic m-LDL complexesdescribed in the present study were also shown to be stable inserum, which is of essential importance for a successful in vivouse of the preparation. On the other hand, drawbacks comparedwith the liposome vehicle are that the drug must be lipophilicand in a liquid or liquid crystalline state at body temperature(5). However, compared to hitherto clinically used administration vehicles for lipophilic drugs, which cause toxic reactionsthemselves (26), the m-LDL particle seems to provide majoradvantages.

Under /// vivo conditions the liver and adrenal gland areparticularly active in assimilating LDL by the receptor pathway(27). Therefore, it is likely that both liver and adrenal wouldsuffer during therapy. However, it may be possible to downregulate the receptor-mediated LDL uptake in these organs bypretreatment with steroids and bile acids (28). Another objection which can be raised against the possible use of LDL as adrug carrier is the competition due to the presence of endogenous LDL in the blood. It might be advantageous to reduce thelevel of LDL in the patient, which reduces the competition forthe cytotoxic m-LDL by native LDL. Especially favorable inthis respect is the situation in the cerebrospinal fluid, whichdoes not contain LDL (29). Malignant glioma might thereforebe treated with toxic m-LDL without competition with nativeLDL. The use of apo B as a carrier for targeting of drugs tocancer cells will never reach absolute selectivity, because therewill always be an uptake by normal cells as well. Nevertheless,since most drugs in use today are totally untargeted, even partialsuccess would be an appreciable advantage, especially in cancertreatment where the side effects are a severe problem.

REFERENCES

1. Goldstein, J. L., and Brown, M. S. The low-density lipoprotein pathway andits relation to atherosclerosis. Annu. Rev. Biochem., 46: 897-930, 1977.

2. Brown, M. S.. Dana, S. E., and Goldstein, J. L. Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reducíaseactivity in cultured human fibroblasts.J. Biol. Chem., 249: 789-796, 1974.

3. Vitols, S., Garthon, G., Ost, À.,and Peterson, C. Elevated low-densitylipoprotein receptor activity in leukemic cells with monocytic differentiation.Blood, 63: 1186-1193, 1984.

4. Krieger, M., Brown, M. S., Faust, J. R., and Goldstein, J. L. Replacementof endogenous cholesteryl esters of low density lipoprotein with exogenouscholesteryl linoleate. J. Biol. Chem., 253: 4093-4101, 1978.

5. Lundberg, B., and Suominen, L. Preparation of biologically active analogsof serum low density lipoprotein. J. Lipid Res., 25: 550-558, 1984.

6. Firestone, R. A., Pisano, J. M., Falck. J. R., McPhaul, M. M., and Krieger,M. Selective delivery of cytotoxic compounds to cells by the LDL pathway.J. Med. Chem., 27:1037-1043, 1984.

7. Lindgren, F. T., Jensen, L. C., and Hatch, F. T. The isolation and quantitativeanalysis of serum lipoproteins. in: G. J. Nelson (ed.). Blood Lipids andLipoproteins: Quantitation, Composition, and Metabolism, pp. 181-275.New York: Wiley-Interscience, 1972.

8. Bilheimer, D. W., Eisenberg, S., and Levy, R. I. The metabolism of very-lowdensity lipoprotein proteins. I. Preliminary in vitro and in vivo observations.Biodi ini Biophys. Acta. 260: 212-221. 1972.

9. Biedler, J. L., Helson, L., and Spengler, B. A. Morphology and growth,tumorigenicity, and cytogenetics of human neuroblastoma cells in continuousculture. Cancer Res., 33: 2643-2652, 1973.

10. Brown, M. S., and Goldstein, J. L. Analysis of a mutant strain of humanfibroblasts with a defect in the internalization of receptor bound low densitylipoprotein. Cell, 9: 663-674, 1976.

11. Goldstein, J. L., Basu, S. K., Brunschede, G. Y., and Brown, M. S. Releaseof low density lipoprotein from its cell surface receptor by sulfated glucosa-minoglycans. Cell, 7: 85-95, 1976.

12. Markwell, M. A. K.. Hass, S. M., Bieber, L. L., and Tolbert, N. E. ModifiedLowry procedure to simplify protein determination in membranous andlipoprotein samples. Anal. Biochem., 87: 206-210, 1978.

13. Masquelier, M., Vitols, S., and Peterson, C. Low density lipoprotein (LDL)as a carrier of antitumoral drugs: in vivo fate of drug-LDL complexes inmice. Cancer Res., 46: 3842-3847, 1986.

14. Warnick, G. R., and Albers, J. J. A comprehensive evaluation of the heparin-manganese precipitation procedure for estimating high density lipoproteincholesterol. J. Lipid Res., 19: 65-76, 1978.

15. Trouet, A., Deprez-de Campeneere, D., and de Duve, C. Chemotherapythrough lysosomes with DNA-daunorubicin complexes. Nature (Lond.), 239:110-112, 1972.

16. Levy, R.. Jurwitz, E., Marón, R., and Sela, M. The specific cytotoxic effectsof daunomycin conjugated to antitumor antibodies. Cancer Res., 35: 1182-1186, 1975.

17. Wall, M. E., Abernathy, G. S., Carrol, F. I., and Taylor, D. J. The effects ofsome steroidal alkylating agents on experimental animal mammary tumorand leukemia systems. J. Med. Chem., 12: 810-818, 1969.

18. Gregoriades, G., Wills, E. J., Swain, C. P., and Tavill, A. S. Drugcarrierpotential of liposomes in cancer chemotherapy. Lancet, /: 1313-1316, 1974.

19. Gal, D., Ohashi, M., MacDonald, P. C., Buchsbaum, H. J.. and Simpson, E.R. LDL as a potential vehicle for chemotherapeutic agents and radionucleo-tides in the management of gynecologic neoplasms. Am. J. Obstet. Gynecol.,139: 877-885, 1981.

20. Norata, G., Canti, G., Ricci, L., Nicolin, A., Trezzi, E., and Catapano, A. L.In vivo assimilation of low density lipoproteins by a fibrosarcoma tumourline in mice. Cancer Lett., 25: 203-208, 1984.

21. Singh, S., and Lee, D. M. Conformational studies of lipoprotein B andapolipoprotein B: effects of disulfide reducing agents sulfhydryl blockingagent, denaturing agents, pH and storage. Biochim. Biophys. Acta, 876:460-468, 1986.

22. Iwanik, M. J., Shaw, K. V., Ledwith, B. J., Yanovich, S., and Shaw, J. M.Preparation and interaction of a low-density lipoprotein:daunomycin complex with P388 leukemic cells. Cancer Res., 44: 1206-1215, 1984.

23. Gregoriades, G., and Allison, A. C. (éd.),Liposomes in Biological Systems,New York: John Wiley and Sons, 1980.

24. Kirby, C., Clarke, J., and Gregoriades, G. The effect of the cholesterolcontent of small unilamellar liposomes on their stability in vivo and in vitro.Biochem. 3., 186: 591-598,1980.

25. Abra, R. M., and Hunt, C. A. Liposome disposition in vivo III. Dose andvesicle-size effects. Biochim. Biophys. Acta, 666: 493-503, 1981.

26. Blum, R. H., Garnick, M. B., Israel, M., Canellos, G. P., Henderson, I. C.,and Frei, E. Initial clinical evaluation of Ar-trifluoroacetyladriamycin-14-valerate (AD-32), an adriamycin analog. Cancer Treat. Rep., 63: 919-923,1979.

27. Pittman, R. C., Attie, A. D.. Carew, T. E., and Steinberg, D. Tissue sites ofdegradation of low density lipoprotein: application of a method for determining the fate of plasma proteins. Proc. Nati. Acad. Sci. USA, 76: 5345-5349,1979.

28. Hynds, S. A., Welsh, J., Stewart, J. M., Jack, A., Soukop, M., McArdle, C.S., Caiman, K. C., Packard, C. J., and Shepherd, J. Low-density lipoproteinmetabolism in mice with soft tissue tumours. Biochim. Biophys. Acta, 795:589-595. 1984.

29. Roheim, P. S., Carey, M., Forte, T., and Vega, G. L. Apolipoproteins inhuman cerebrospinal fluid. Proc. Nati. Acad. Sci. USA, 76: 4646-4649,1979.

4108

on April 4, 2020. © 1987 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1987;47:4105-4108. Cancer Res   Bo Lundberg  PathwayDelivery of Antitumoral Drugs via the Low Density Lipoprotein Preparation of Drug-Low Density Lipoprotein Complexes for

  Updated version

  http://cancerres.aacrjournals.org/content/47/15/4105

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/47/15/4105To request permission to re-use all or part of this article, use this link

on April 4, 2020. © 1987 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from