Human Antibodies 21 (2012) 49–56 49DOI 10.3233/HAB-2012-0261IOS Press

Attachment of an anti-MUC1 monoclonalantibody to 5-FU loaded BSA nanoparticlesfor active targeting of breast cancer cells

Hasan Kouchakzadeha, Seyed Abbas Shojaosadatia,∗, Javad Mohammadnejadb, Malihe Paknejadc andMohammad Javad Rasaeed

aBiotechnology Group, Faculty of Chemical Engineering, Tarbiat Modares University, Tehran, IranbDepartment of Life Science Engineering, Faculty of New Sciences and Technologies, University of Tehran, Tehran,IrancDepartment of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, IrandMedical Biotechnology Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

Abstract. With PR81 as a murine monoclonal antibody (mAb) that was prepared against the human breast cancer, the MUC1receptor specific targeting is possible. In this study, PR81-conjugated bovine serum albumin (BSA) nanoparticles loaded withanticancer drug 5-fluorouracil (5-FU) were developed. Enzyme linked immunosorbant assay (ELISA) results showed highimmunoreactivity of PR81 mAb conjugated to nanoparticles towards MUC1 related peptide or native cancerous MUC1 andalmost no cross-reaction to non-specific proteins. In vitro experiments were performed to determine the ability of this new drugdelivery system on overcoming MCF-7 breast cancer cells in comparison with four other systems. The results revealed thatthese cell-type specific drug loaded nanoparticles could achieve more cell death as compared to when the 5-FU was used with nocarriers. Stability studies of produced drug delivery system proved high immunoreactivity of conjugated PR81 even after 11 daysof storage in room temperature.

Keywords: BSA nanoparticle, PR81 mAb, MUC1, MCF7 cell lines, 5-Fluorouracil

1. Introduction

The most important limitation of present cancerchemotherapy is the toxicity associated with the anti-cancer drugs to normal tissues, which results from thefact that anticancer drugs presently used in chemothera-py lack efficient selectivity towards tumor cells. There-fore, the selective controlling of drug concentration anddistribution within the tumor microenvironment is oneof the essential factors to achieve effective and safe can-cer chemotherapy [1–5]. One way to reach this aim is

∗Corresponding author: Seyed Abbas Shojaosadati, Biotechnolo-gy Group, Chemical Engineering Department, Faculty of Engineer-ing, Tarbiat Modares University, P.O. Box 14115-143, Tehran, Iran.Tel.: +98 21 82883341; Fax: +98 21 82883381; E-mail: shoja sa@modares.ac.ir.

the development of colloidal drug delivery systems [6–8]. In this regard, nanoparticles explain a high drugloading efficiency with trifling drug leakage as well asthe ability to avoid multidrug resistance period withgood storage stability [9,10]. Nanoparticles made ofbovine serum albumin (BSA) have gained much inter-est because of their inherent properties of biodegrad-ability, lack of toxicity, and nonantigenicity [11–13].Furthermore, the production of these particles is easyand a wide variety of drugs can be physically entrappedin them [9,10,14–17]. In addition, covalent linkage ofligands for targeted delivery of drugs to desired sitesof action is possible due to the presence of functionalgroups on the surface of these particles [9,18–21].

Targeted drug delivery can be achieved actively orpassively depending on the carrier system [22]. In theactive strategy, targeting ligand, such as antibodies, car-

ISSN 1093-2607/12/$27.50 2012 – IOS Press and the authors. All rights reserved

50 H. Kouchakzadeh et al. / Attachment of an anti-MUC1 monoclonal antibody to 5-FU loaded BSA nanoparticles

bohydrates, aptamers, and small molecules, is conju-gated to carrier for increasing the delivery of a certaindrug to a specific target site [23–25]. One noteworthypoint is that when ligand is attached to the carrier, dueto the small size of the conjugate, it can only extravasateat the disease site but not normal vasculature, so, theligand cannot interact with normal tissues and produceside effects [26]. Many researchers have reported exactand well-organized cellular uptake of different carrierssuch as liposomes, micelles, and particles that had beentailored with a functional ligand with a high affinityfor target cells [27–30]. Monoclonal antibodies, whichcan specifically bind to preferentially overexpressedsurface receptors at target sites, may be coupled to thesurface of the long circulating nanocarriers [31–34].Long circulation time of nanocarriers will allow theireffective transport to the tumor site through the en-hanced permeability and retention (EPR) effect. Sur-face attachment of Poly ethyleneglycole (PEG) chainsprevent quick drug release and removal of nanocarriersby macrophages and therefore extend their circulationtime in the bloodstream [35–38]. In addition, mono-clonal antibodies can be coupled to the outer end of thePEG chains to achieve active targeting [39–43]. Local-ized diseases, such as cancers, not only have leaky vas-culature but also overexpress some epitopes or recep-tors that can be considered as targets [26]. The humanepithelial mucin, MUC1, is one of the transmembraneproteins that normally present on the luminal surfaceof the secretary glands [44]. This glycoprotein is un-usually overexpressed in different cancer tissues, par-ticularly in 80% of breast cancers [45–47]. In recentyears, different types of murine anti-MUC1 monoclon-al antibodies have been used for targeting of breast andovarian tumors and for breast tumor imaging [47–52],which confirms significant interest in MUC1 as a possi-ble target antigen for immunotherapy of breast cancer.

PR81 that was introduced by Paknejad et al. [53] isan anti-MUC1 murine monoclonal antibody of IgG1subclass against human breast carcinoma. The reactionof PR81 with the membrane extracts of the severalhuman breast cancerous tissues and the cell surface ofmany MUC1 positive cell lines has been proved [46,54]. Due to the high specific immunoreactivity, thePR81 seems to be a promising ligand for conjugationto nanoparticles for effective delivery of drugs in thehuman breast cancer chemotherapy.

In our previous studies, BSA was used as nanopar-ticles forming polymer because of its unique advan-tages [10]. 5-FU that is widely used in breast can-cer chemotherapy was considered as the model drug

in these studies. Preparation of 5-FU-loaded BSAnanoparticles was performed by an established phaseseparation method and drug-loading efficiency was op-timized. Afterward, linkage of activated PEG (PEGy-lation) to the surface of BSA nanopaticles was stud-ied and optimized in order to extend their circulationhalf-life in the bloodstream [35]. Consequently, the ob-jective of the present study is to develop and evalu-ate a new drug delivery system made of 5-FU load-ed BSA nanoparticles to which PR81 mAb be cou-pled through a heterobifunctional activated PEG chain.The immunoreactivity of PR81 mAb conjugated toBSA nanoparticleswith synthetic peptide (TSA-P1-24-BSA), native cancerous MUC1 purified from metastat-ic ascetic fluid and MUC1 related peptide were test-ed using ELISA procedure. The therapeutic effica-cy of PR81-modified 5-FU-loaded BSA nanopaticleswere evaluated by in vitro cytotoxicity and comparedwith four other systems including free 5-FU, bare BSAnanoparticles, PEGylated BSA nanoparticles and 5-FU-loaded PEGylated BSA nanopartcles by MTT as-say using MCF7 breast cancer cells. Additionally, sta-bility experimentswere organized to examine the PR81mAb immunoreactivity towards peptide related MUC1during 20 days in room temperature.

2. Materials and methods

2.1. Chemicals and reagents

BSA (fraction V, minimum 98%), glutaraldehyde8% aqueous solution, and 5-fluorouracil were obtainedfrom Sigma (Steinheim, Germany). 2-Iminoithiolane(Traut’s reagent) was purchased from Fluka (Seelze,Germany). The succinimidyl ester of methoxy poly(ethylene glycol) propionic acidwith an averagemolec-ular weight of 5.0 kDa (mPEG5000-SPA) was ob-tained from Sunbio (Orinda, USA). Cross-linker poly(ethylene glycol)-α-maleimide-ω-NHS ester with anaverage molecular weight of 7.5 kDa (NHS-PEG7500-Mal) was purchased from Jen Kem Technolo-gy (Texas, USA). Synthetic mucin peptide (TSA-P1-24 = TSAPDTRPAPGSTAPPAHGVTSAPDTR), cor-responding to the mucin core protein, which was chem-ically conjugated to BSA (TSA-P1-24-BSA) by reac-tion with glutaraldehyde,were purchased from Q-BIO-GENE Inc. (Kayserberg, France). The native cancer-ous MUC1 was purified from ascetic fluid of a patientwith aggressive small-cell lung carcinoma and metas-tasis to peritoneum, by an antibody-sepharose affinitycolumn [55]. All other reagents used in this study wereof analytical grade and were purchased from Merck(Darmstadt, Germany).

H. Kouchakzadeh et al. / Attachment of an anti-MUC1 monoclonal antibody to 5-FU loaded BSA nanoparticles 51

2.2. Monoclonal antibody

The PR81 is a murine anti-MUC1mAb of IgG1 classand subclass, containing kappa light chain. This mAbexhibited good and similar affinities (2.19× 108 M−1)towards the TSA-P1-24 and A-P1-15, the two synthet-ic 27- and 16-amino acid peptides that included thecore tandem repeat sequence of MUC1 [53]. The an-tibody PR81 has been obtained from the original sup-plier (Medical Biotechnology Department, Faculty ofMedical Sciences, Tarbiat Modares University, Tehran,Iran).

2.3. Preparation and separation of 5-FU-loaded BSAnanoparticles

5-FU-loaded BSA nanoparticles were prepared by anoptimized desolvation process [10]. Briefly, 0.2 g BSAin 2.0 mL of 2 mg/ml 5-FU aqueous solution titrated topH 8.4, was converted to nanoparticles by continuousaddition of 3.7 mL ethanol by A syringe pump (WPI,Switzerland) at the rate of 1.0 mL/min under stirring(550 rpm) at room temperature. 176 μL of 8% glu-taraldehyde aqueous solution was added subsequentlyto induce particle cross-linking. After performing thecross-linking process under stirring of the suspensionover night, possible microparticles were precipitatedby centrifugation (15,000 × g, 2 min) and discarded.Nanoparticleswere separated by two cycles of centrifu-gation (25,000 × g, 20 min) and redispersion of thepellet to the original volume in 10 mM NaCl pH 9.This leads to production of nanoparticles with averagesize of 210 nm and zeta potential of −31.7 mV. Then,BSA nanoparticle solution was freeze-dried to yieldnanoparticle powder.

2.4. PR81 mAb thiolation

A crucial step in attachment of PR81 to nanoparti-cles is thiolation of the monoclonal antibody used astargeting ligand. Sulfhydryl groups were introducedonto the PR81 molecules to provide a reactive site forthe conjugation with BSA-based nanoparticles. Prima-ry amino groups of the monoclonal antibody can reactwith Traut’s reagent, leading to creation of sulfhydrylgroups. In this step, PR81 was dissolved in phosphatebuffer pH 8.0 at the concentration of 1.0 mg/mL. Forthe introduction of thiol groups, PR81 mAb was incu-bated with a 50-fold molar excess of 2-iminothiolanesolution for 2 h at 20◦C as previously described [40].The antibody was purified by dialysis for 48 h. Theresulting solution contained thiolated PR81 mAb at aconcentration of about 1 mg/mL.

2.5. Surface modification of drug-loadednanoparticles

PEGylation of BSA nanoparticles was performedunder optimum condition [35]. A solution of 6.5 mMNHS-PEG7500-Mal and 5 mg/mL BSA nanopaticle inphosphate buffer pH 7.0 was incubated for 10 min at27◦C under constant shaking (Eppendorf thermomix-er, 550 rpm). The nanoparticles were purified by cen-trifugation and redispersion as mentioned above. Forthe coupling reaction, 1.0 ml of the sulfhydryl-reactivenanoparticle suspension was incubated with 0.5 ml ofthe thiolated PR81, to achieve a covalent linkage be-tween antibody and the nanoparticle system. Thisamount of antibody is molar equivalent with 60% ofmaleimide groups. The sample was incubated for 12 hat 20◦C under constant shaking (600 rpm). The samplewas purified from unreacted PR81 mAb by centrifu-gation and redispersion as described before. To de-termine the amount of unbounded antibody, the result-ing supernatant was collected and analyzed by Brad-ford method. A schematic representation of this drugdelivery system for targeting of MUC1 positive cellsis shown in Fig. 1. The PR81-modified drug-loadedBSA nanoparticle solution was freeze-dried to form thepowder of nanoparticles. In addition, mPEG5000-SPAwas attached to drug-loaded BSA nanoparticle and thesolution was freeze-dried to produce powder for use inin vitro experiments.

2.6. PR81 immunoreactivity measurement

The immunoreactivity of thiolated and conjugatedPR81 mAb with native MUC1 and synthetic mucinpeptide was investigated using ELISA method. NativeMUC1 and synthetic mucin peptide were used as PR81receptors in ELISA plate.

2.7. Cell line and cell culture

A MUC1-positive cell line, MCF 7, was purchasedfrom Pasteur Institute (Tehran, Iran). The MCF 7 cellline is a kind of human breast carcinoma that expressesthe MUC1 antigen on the plasma membrane in thecell culture. The cell line was grown in RPMI 1640with 10% FCS, 100 Unit/mL penicillin, 100 μg/mLstreptomycin, 2 mM glutamine and 5 μg/L insulin.

2.8. In vitro cell viability

The cytotoxicity of BSA targeted nanoparticles wasevaluated and compared with four other systems, in-

52 H. Kouchakzadeh et al. / Attachment of an anti-MUC1 monoclonal antibody to 5-FU loaded BSA nanoparticles

Fig. 1. Schematic representation of the produced drug delivery system for MUC1 targeting.

cluding free 5-FU, bare BSA nanoparticles, PEGylatedBSA nanopaticles, and 5-FU-loaded PEGylated BSAnanoparticles, using MCF 7 cancer cell lines in an MTTassay. The MCF 7 cells (3 × 104 cells/well) wereplaced on the sterile 96-well plate and were grown toconfluence. Every experiment was done in triplicateand the percentage of cell growth inhibition was as-sayed during 5 days with incubation at 37◦C. In ad-dition, similar wells containing untreated MCF 7 cellswere used as control. 5-FU concentration in all of theformulations was adjusted to be the same as that of freedrug.

2.9. Storage stability

The immunoreactivity of PR81 mAb in the produceddrug delivery system with peptide related MUC1 wasdetermined by ELISA method during 20 days using anon-specific protein as a reference. The aim of thismeasurement was to find the number of days duringwhich the immunoreactivity of PR81 mAb exhibited ahigh level.

3. Results and discussion

3.1. PR81 mAb thiolation

Free sulfhydryl groups bear the risk of oxidativedisulfide bridge formation leading to dimmers or even

higher oligomers of PR81. These by-products couldaffect the biological activity of PR81 and thus wereundesirable. For preventing the formation of disul-fide bonds EDTA with the concentration of 0.1 M wasadded to the buffers used in all steps including thiolationand dialysis [56]. The amount of thiol groups withinthe antibody was quantified through disulfide buildingwith Ellmans’s reagent. Within this reaction 2-nitro-5-thiobenzoic acid (TNB) was released stoichiometrical-ly, which can be quantified at 412 nm. The free thiolgroups were quantified in triplicate. The molar ratio offree thiol groups per

PR81 mAb after purification was calculated to be0.77 ± 0.18 by the use of a standard curve. Follow-ing thiolation experiment an ELISA method was usedfor assessing the immunoreactivity of thiolated PR81mAb. As it is presented in Fig. 2, thiolated PR81 mAbdemonstrated high immunoreactivity towards MUC1related peptide and native cancerous MUC1 and almostno cross-reaction towards non-specific proteins used inthis study (BSA). These results verifies high and suit-able reactivity of PR81 after modification with Traut’sreagent.

3.2. Immunoreactivity of PR81 mAb conjugated tonanoparticles

The molar ratio of thiolated PR81 mAb to activatedmaleimide groups of PEG chain on BSA nanoparticles

H. Kouchakzadeh et al. / Attachment of an anti-MUC1 monoclonal antibody to 5-FU loaded BSA nanoparticles 53

Fig. 2. Reactivity of thiolated PR81 (5 μg/ml) towards TSA-P1-24-BSA (0.5 μg/ml), cancerous MUC1 (0.2 μg/ml) and BSA (0.5 μg/ml). Notethe concentration of TSA-P1-24-BSA (0.5 μg/ml) is higher than MUC1 (0.2 μg/ml).

Fig. 3. Reactivity of PR81 conjugated to 5-FU loaded BSA nanoparticles (2.5 μg/ml) towards TSA-P1-24-BSA (0.5 μg/ml), cancerous MUC1(0.2 μg/ml) and BSA (0.5 μg/ml). Note the concentration of TSA-P1-24-BSA (0.5 μg/ml) is higher than MUC1 (0.2 μg/ml).

was 0.6. After conjugation of antibody to nanoparticlesand separation, as described before, the supernatantwascollected for the quantification of PR81 mAb. Brad-ford assay results showed that the protein concentrationin the supernatant was negligible and so nearly all ofthe thiolated PR81 molecules were conjugated to BSAnanoparticles. The immunoreactivity of PR81 mAb inthe produced drug delivery system was evaluated byELISA method. As Fig. 3 reveals, the immunoreac-tivity of conjugated PR81 mAb towards MUC1 relatedpeptide and native cancerous MUC1 is much higherthan non-specific proteins, but it is lower than the thi-olated PR81 mAb. These results demonstrate that thebiological activity of PR81 mAb after attachment haddiminished rather than thiolated PR81 mAb but it islikewise in high level.

3.3. In vitro evaluation

At in vitro experiments, cytotoxic efficiency offree 5-FU, bare BSA nanoparticles, PEGylated BSA

nanoparticles and PEGylated BSA nanoparticles load-ed by 5-FU was compared with the new produceddrug delivery system PR81-modified 5-FU loadedBSAnanoparticles. MTT method was conducted for theanalysis of MCF 7 cells. In addition, MCF 7 wells wereused as control. The average of lively cells after 5 daysin encounteringwith these different systems is reportedin Fig. 4. As this figure demonstrates, the cytotoxic ef-ficiency of PR81-modified 5-FU loaded BSA nanopar-ticles is more than other systems and so this new drugdelivery system can be considered as the best systemfor overcoming the MUC1 positive MCF 7 breast can-cer cell lines. This drug delivery system may produceeven better results in in vivo tests.

3.4. Storage stability

The produced drug delivery system was freeze-driedand the immunoreactivity of PR81 mAb was measuredduring 20 days by ELISA method. The samples were

54 H. Kouchakzadeh et al. / Attachment of an anti-MUC1 monoclonal antibody to 5-FU loaded BSA nanoparticles

Fig. 4. In vitro results for five drug delivery systems against MCF 7 cell line: 5-FU = the drug 5-Fluorouracil was added directly, NP = onlynanoparticles were added, PEG-NP = PEGylated nanoparticles were added, PEG-NP-5-FU = PEGylated nanoparticles loaded by 5-Fluorouracilwere added, PR81-PEG-NP-5-FU = nanoparticles loaded by 5-Fluorouracil and conjugated to PR81 mAb were added. Untreated MCF7 cellswere considered as control. The results indicated highest cell death (lowest viability) in case of PR81-PEG-NP-5-FU. All assays were performedin triplicate (n = 3, mean ± SD).

Fig. 5. Storage stability results of PR81-modified 5-FU-loaded BSA nanoparticles. Reactivity of PR81 conjugated to nanoparticles toward MUC1(0.2 μg/well). All assays were performed in triplicate (n = 3, mean ± SD).

stored in room temperature. As is shown in Fig. 5, Af-ter 4 days of storage, immunoreactivity of PR81 mAbdecreased but it is in appropriate range due to high-er immunoreactivity towards peptide related MUC1 incomparisonwith referenceBSA. The results reveal thatthe immunoreactivetity of PR81 mAb towards peptiderelated MUC1 after 11 days of storage in room tem-perature is in an acceptable level and this drug deliverysystem can be stored for about 11 days for further usagein room temperature.

4. Conclusion

In this study a novel specific drug carrier system,which was enable a specific transport of 5-FU to MUC1

overexperessing breast cancer cells, was developed.The cytotoxicity of this system was compared with oth-er systems including free 5-FU, bare BSA nanoparti-cles, PEGylated BSA nanoparticles, and 5-FU loadedPEGylated BSA nanoparticles. MTT assay of MCF 7breast cancer cell lines after 5 days showed that the cy-totoxicity efficiency of this novel drug delivery systemis higher than other investigated systems and thus thissystem is more efficient and can be a promising systemfor future use in breast cancer chemotherapy. Storagestability of this drug delivery system at room tempera-ture demonstrated that the PR81 mAb biological activ-ity is in high level within 11 days without adding anyretentive materials. In vitro results persuade us for per-forming in vivo and clinical experiments for accessing

H. Kouchakzadeh et al. / Attachment of an anti-MUC1 monoclonal antibody to 5-FU loaded BSA nanoparticles 55

progress in breast cancer chemotherapy by the use ofthis drug delivery system.

Acknowledgment

We appreciate partial support of this research by Ira-nian National Science Foundation (INSF).

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