Journal of Pharmaceutical and Biomedical Analysis26 (2001) 219–224

Molecular recognition in a reconstituted tumor cellmembrane

Xin Hu a, Shaohua Wang a, Yufeng Zhang a, Xiandao Lu a, Xinpu Hou a,*,Angelica Ottova b, H. Ti Tien b

a Department of Physical Pharmacy, School of Pharmaceutical Science, Peking Uni�ersity,Beijing 100083, People’s Republic of China

b Physiology Department, Michigan State Uni�ersity, East Lansing, MI 48824, USA

Received 23 November 2000; received in revised form 22 January 2001; accepted 5 February 2001

Abstract

The design of an immunoliposome system for molecular recognition using reconstituted, hydrogel-supportedbilayer lipid membranes (sb-BLMs) is described. By monitoring the electrical properties, two kinds of recognition arefeasible: (i) the human bladder tumor cells, Ej and its antibody BDI-1, the lifetime of the reconstituted membrane is42 min; and (ii) the human rectum tumor cells, LOVO, the life of the reconstructed membrane is more than 40 min,the same as conventional BLM. Further, the anticancer drug, Adriamycin (Anticancer Res., 20 (2000) 1391), wasshown to be effective in such reconstituted systems, the life of which is less than 5 min. In these experiments, theactive ingredients of the Ej and LOVO cells were determined on reconstituted sb-BLMs. The key point is that thecomponent part being recognized on the BLM must be kept in its native state. © 2001 Elsevier Science B.V. All rightsreserved.

Keywords: Molecular recognition; Immunoliposomes; sb-BLM; Electrical properties; Anticancer drug; Adriamycin; Planar lipidbilayer; Bilayer lipid membrane

www.elsevier.com/locate/jpba

1. Introduction

As a drug carrier and in other applications,liposomes have been studied for many years [1–3]. The main problem of liposomes is that theyare mainly taken up by the reticuloendothelial

system when administrated in vivo and cannotget to the target sites, which greatly reduces thetherapeutic index of medicines, especially for an-ticancer drugs. One way to overcome thisdifficulty is by means of immunoliposomes,which are the liposomes linked with the con-cerned antibody or its fragments. In recentyears, several types of immunoliposomes havebeen developed and have shown promise in thisfield [4–7]. The difference between conventional

* Corresponding author.E-mail addresses: houxinpu@sun.bjmu.edu.cn (X. Hou), ot-

tova@pilot.msu.edu (A. Ottova).

0731-7085/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.

PII: S0731 -7085 (01 )00424 -1

X. Hu et al. / J. Pharm. Biomed. Anal. 26 (2001) 219–224220

liposomes and immunoliposomes lies in whetherthey can be recognized by the receptors of targetcells. The molecular recognition will result in aseries of physicochemical changes on the lipidmembrane, especially changes in electrical proper-ties. In this connection, it has been well estab-lished that the bilayer lipid membrane (planarBLM) possesses a similar structure as that of theliposomal membrane, e.g. double phosphatide bi-layer [3]. A modified BLM can be a simple modelfor investigating the molecular recognition occur-ring on liposomes in vitro. From another point ofview, the electrical change of the membrane issensitive to the specific interactions, such as anti-gen–antibody, receptor– ligand and enzyme–sub-strate [3]. This paper presents our attempt atestablishing a relationship between the host–guestreaction and its associated electrical change inreconstituted cell membranes for cancer research.

2. Experimental

2.1. Apparatus

The instrument used together with its softwaredata processing was a multifunctional analyzer(Model JI-100, Sino JinKer Electronic Co. Ltd,Tianjin, People’s Republic of China), which wasconnected to a personal computer (SAT, made inPeople’s Republic of China).

2.2. Reagents and materials

The phosphatidylcholine was extracted fromeggs and purified according to a well-known pro-cedure [7].The cholesterol was recrystallized (m.p.147.4–148.1°C) before use. The tumor cells ofhuman bladder, Ej, and rectum, LOVO, were giftsfrom Prof. Dinner, Alberta University, Canada.The antibody BDI-1 of Ej cells was supplied byProf. Xie Shusheng, School of Basic Medical Sci-ence, Peking University. The other reagents wereall of analytical grade and were used withoutfurther purification. The materials such as Teflonand plastic tubing and saturated calomel elec-trodes (SCE) were used as received.

2.3. Procedure

2.3.1. Preparation of agar electrode andsalt-bridge-supported BLM (sb-BLM)

The freshly prepared lecithin and cholesterol(4:1, mg/mg lecithin concentration: 20 mg/ml)were dissolved in n-decane. The Teflon tube wasselected, the diameter of which was 0.5 mm andthe length of which was about 2.0–4.0 cm, thensealed at the tip of a thick plastic tube. One endof the tube was about 1 cm in diameter and theother about 1 mm; it was 10 cm long. The Teflontube was immersed into hot agar solution (�80,2.5%), and the air in two tubes was excludedthrough the upper (thick) end, so the surface ofhot liquid agar rose slowly. After the thin Teflontube was filled with agar completely, it was takenout and cooled, then KCl (0.1 M) solution waspoured into the plastic tube and one SCE was putin it as an inner reference electrode. According toRef. [3], the agar-filled tubing was cut at its endwith a surgeon’s knife and immersed immediatelyinto the BLM-forming solution for 30 s, thentaken out to be inserted into 0.1 mol/l KCl, whereanother SCE was placed as outer reference elec-trode. The two SCEs were connected to the mea-suring instrument described above to monitor thechange of electrical signals on sb-BLM.

2.3.2. The construction of tumor cell membraneson sb-BLM

The Ej and LOVO cells were each ground, afterbeing cultured to reproduce sufficient amount forexperimental need, and mixed with PBS (pH 7.0).The sediment was collected and washed with PBSagain after centrifugation at high speed (13 000g,for 30 min) and lyophilized (�20, for 48 h). Themembrane powder in lipid solution (1 mg/ml) wassonicated briefly and used for reconstituted tumorcell membrane experiments (e.g. Ej–sb-BLM andLOVO–sb-BLM) similar to the conventional sb-BLMs mentioned above.

2.3.3. The antigen–antibody interactionIn the outer KCl solution of the Ej–sb-BLM

system, 25, 50 and 100 �l BDI-1 were added,respectively, under the same conditions of addi-tion of 100 �l BDI-1 as control in the LOVO–sb-

X. Hu et al. / J. Pharm. Biomed. Anal. 26 (2001) 219–224 221

BLM system; soon afterwards, the cyclic voltam-mogram and electrical parameters were recorded.

2.3.4. The interaction of sb-BLM and anticancerdrug

To study the influence of the anticancer drugAdriamycin (ADM), 1 mg/ml solution was addeddropwise and the change in the BLM was moni-tored from the screen in the reconstitutedLOVO–sb-BLM system. A conventional sb-BLMwas used as a control in this experiment.

3. Results and discussion

In the majority of literature available, thepreparation of immunoliposomes is based on spe-cific interactions in water solutions (e.g. antigen–antibody, etc.), from which the conclusion isdrawn that the same interaction must occur in thelipid surrounding, especially for the aggregationof a quantity of phospholipids (e.g. liposomes). Inmany cases this is correct, but in many experi-ments another situation is often seen, in which therecognition between host and guest, which ishighly active in water, does not occur or is veryweak in liposomes. This may be due to the loss ofmacromolecular activity during preparation. Onthe other hand, it is very possible that the reactiveability may change in a different polar medium.In previous experiments, we have only focusedattention on the appropriate conditions of prepa-ration; when the immunoliposomes prepared weretested in vivo, the outcome was not ideal. By thistime, much manpower and many material re-sources had been used. How can specific recogni-tion be detected in lipid surrounding beforepreparing immunoliposomes? The BLM is a verygood model, the determination of its elec-troparameters is direct and straightforward, andthe sensitivity of the method is excellent (theconcentration of Ag or Ab usually being less than10−9 M). At the same time, the method can becoupled with computer technology, so that onlinedetection may be realized, as will be reported indue course.

3.1. Optimization of sb-BLM

The conventional BLM is formed in a smallhole, the boundary of the hole supports the BLM.The c-BLM separates an electrolyte solution intoan inner and an outer section and two SCEsinserted in the two sections are linked to theexternal circuits. The main disadvantage of c-BLMs is their limitation in satisfying the experi-mental need. First, the forming of a BLM isdifficult and time-consuming. Secondly, the BLMis very fragile on account of the disturbance fromthe bathing solution on both sides of it. Usuallythe lifetime of a c-BLM is less than 30 min (atbest a few hours). The sb-BLM has overcome theinstability of the c-BLM, because one side is theinterface of aqueous solution/BLM, while theother is that of agar gel/BLM. The sb-BLM cantransmit not only electrons but also ions, whichare more suitable for biomembrane experiments,because the majority of charged particles con-ducted are cations and anions, which play animportant role in biological processes. The previ-ous method of preparation of sb-BLMs was com-plex and liable to rupture. In preparing the agarelectrode, we have optimized the method reportedpreviously [3]. Compared with the former method,the optimizations are: (a) Ag–AgCl was replacedby SCE because SCE is more commonly used inelectrochemistry, since it contains its own salt-bridge; and (b) the inner reference electrode wasin solution, not inserted in the agar gel. By reasonof these optimizations, the new reference electrodeis steadier, and the inner reference electrode neednot be glued to the outer layer of the electrode,which is the key point that influences the stabilityand fidelity in BLM experiments. With these sb-BLMs, typical cyclic voltammograms were ob-tained, from which the resistance (Rm) andcapacitance (Cm) of the membrane were easilycalculated. However, unlike the c-BLM, the sb-BLM could not be observed directly through amicroscope. The completion of BLM formationwas measured using Cm, which reflects the thick-ness of the membrane, by considering the BLM tobe a parallel plate capacitor [8]. The Rm wasanother principal parameter that depicted theability of the BLM to conduct electrical charges.

X. Hu et al. / J. Pharm. Biomed. Anal. 26 (2001) 219–224222

Fig. 1. The lifetime of sb-BLM as a function of added anti-body BDI-1. As for LOVO–sb-BLM the BDI-1 added did notaffect the membrane.

maintaining time of the BLM can be measured. Inour laboratory, the maintaining time of the sb-BLM is often more than 2.5 h, which is muchsteadier than that of the c-BLM. When the mem-brane compositions of the Ej and LOVO cellswere changed, the lifetime of the reconstitutedsb-BLM usually would decrease slightly; however,the value of Rm and Cm did not changesignificantly.

3.2. Specific interactions

In the Ej–sb-BLM system, the membrane wasvery fragile and was broken soon after its anti-body BDI-1 was added. This may be clearly seenin Fig. 1. As for LOVO–sb-BLM the BDI-1added did not affect the membrane; the longevitywas the same as that of LOVO–sb-BLM withoutthe antibody being added (usually �2500 s). This

The change of Rm is related to the interaction ledby the ingredient on the BLM and the substancefrom the bathing solution. When the BLMbreaks, Rm reduces exponentially, from which the

Fig. 2. The longevity of sb-BLM monitored in terms of membrane resistance (Rm). This is illustrated in (a) upper: LOVO-sb-BLMwith added antibody (usually�2500s), (b) lower: Ej-sb-BLM with antibody added. The arrows denote the time when the antibodywas added.

X. Hu et al. / J. Pharm. Biomed. Anal. 26 (2001) 219–224 223

Fig. 3. The longevity of sb-BLM monitored in terms of membrane resistance (Rm) in the presence of ADM to the common sb-BLM:(a) Upper: the membrane did not break until the concentration of ADM reached 0.5%. In contrast, (b) lower: the membrane ofLOVO–sb-BLM ruptured when as little as 0.0032% ADM was added, and the maintaining times were all less than 200 s. The arrowsdenote the time when the antibody or drug was added.

is illustrated in Fig. 2a and b. The arrows de-note the time when the antibody or drug wasadded.

While adding ADM to the common sb-BLM,the membrane did not break until the concen-tration of ADM reached 0.5%. In contrast, themembrane of LOVO–sb-BLM ruptured when aslittle as 0.0032% ADM was added, and themaintaining times were all less than 200 s. Thisis illustrated in Fig. 3a and b. The arrows de-note the time when the antibody or drug wasadded.

These results fully suggested the relationshipbetween the molecular recognition on BLM andthe electrical properties (e.g. Rm). For strong in-teraction of Ej and BDI-1, the life of sb-BLMreduced with the increase in the amount of anti-body. In the LOVO–sb-BLM system, there wasno such recognition and the membrane couldsurvive for a long time.

The test on ADM and BLM shows that themolecular recognition must have taken place onthe reconstituted BLM, but the accurate func-tional sites are not clear at present. As an effec-tive anticancer drug, ADM generally is deemedto act on the DNA of tumor cells [11]; thisexperiment revealed another potential mecha-nism of ADM, namely that the BLM may playa partial role in the antitumor process. In thisconnection, it has been reported that standardanticancer drugs, ADM (Doxorubicin) andTaxol have been assessed by apoptosis, DNAsynthesis, growth rate by MIT assay, uptake ofamino acid, and by morphological changes [8–10].

4. Conclusions

This study is aimed at the design of an im-

X. Hu et al. / J. Pharm. Biomed. Anal. 26 (2001) 219–224224

munoliposome system for molecular recognitionusing reconstituted, hydrogel-supported bilayerlipid membranes (sb-BLMs). We have shown that,by monitoring the electrical properties, two kindsof recognition are feasible: (i) the human bladdertumor cells, Ej and its antibody BDI-1, the life-time of the reconstituted membrane is 1000 s; and(ii) the human rectum tumor cells, LOVO, the lifeof the reconstructed membrane is more than 2500s, the same as for conventional BLM. Further, theanticancer drug, ADM [11], was shown to beeffective in such reconstituted systems, the life ofwhich is less than 240 s.

The specific recognition that occurred on theBLM can be monitored and measured online byits electrical change, which is a possible way ofdesigning and screening effective immunolipo-somes in vitro; thus the preparation of immuno-liposomes is no longer random. The key point isthat the component part being recognized onBLM must be kept in native state. In these exper-iments, the active ingredients of Ej and LOVOcells were determined on reconstituted sb-BLM inthe light of the previous investigation [12]; unlikethe phenomena that occurred on c-BLM, the re-sults showed that there was a decrease in Rm andalso that the membrane could not be maintainedany more; yet, the sb-BLM was more stable thanc-BLM, both in theory and practice, whichdemonstrates the recognition and interaction onBLMs.

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