363

Abstract

The water-soluble poly(methyl vinyl ether-co-maleic anhydride)

copolymer-bovine serum albumin bioconjugates were

synthesized in the presence of 1-ethyl-3-(3-dimetilamino-

propyl) carbodiimide hydrochloride as cross-linking agents via

microwave-assisted and conventional methods and characterized

by size-exclusion chromatography and high-performance liquid

chromatography. According to size-exclusion chromatography

and high-performance liquid chromatography results, the

bioconjugates synthesized in the microwave-assisted method

are more stable and effi cient than the conventional method.

The reaction time is shortened from 17 hours to 15 minutes by

means of the microwave-assisted method.

Keywords: Polyelectrolyte , protein , water soluble bioconjugate ,

microwave-assisted

Abbreviations: P(MVE-MA), Poly (methyl vinyl ether-co-maleic

anhydride) copolymer; BSA, Bovine serum albumin; WSPE,

Water-soluble polyelectrolyte; MA, Microbial antigen;

MWC, Microwave-assisted conjugates; CMC, Conventional

method conjugates; MWM, Microwave-assisted method; CM,

Conventional method; EDC, 1-ethyl-3-(3-dimetilaminopropyl)

karbodiimid hydrochloride; SEC, Size-exclusion chromatography;

HPLC, High-performance liquid chromatography

Artifi cial Cells, Blood Substitutes, and Biotechnology, 2012; 40: 363–368

Copyright © 2012 Informa Healthcare USA, Inc.

ISSN: 1073-1199 print / 1532-4184 online

DOI: 10.3109/10731199.2012.678942

Synthesis of microwave-assisted poly(methyl vinyl ether-co-maleic anhydride)-bovine serum albumin bioconjugates

Mesut Karahan 1 , Sevecen Tu ğ lu 2 & Zeynep Mustafaeva 2

1 Faculty of Engineering and Natural Sciences, Department of Bioengineering, Uskudar University, Uskudar, Istanbul,

Turkey and 2 Yildiz Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering,

Esenler-Istanbul, Turkey

Introduction

Water-soluble polyelectrolyte (WSPE)-microbial antigen

(MA) (proteins, peptide, polysaccharides, etc.) conjugates

have been the subject of many studies in the application areas

of biotechnology, medicine, and pharmacy. Th e WSPEs are

not immunogenic, possess carrier properties and adjuvant

activities simultaneously, and provide eff ective immuno

response. Th e use of this WSPE as the carriers of MA has

made it possible not only to increase the immune respon-

siveness of the organism by several orders of magnitude,

but it also provides eff ective immune protection (Mustafaev

1996, Karahan et al. 2010, Mansuro g lu et al. 2011, Karahan

2009, Petrov et al. 1992, Mustafaev and Sarac 1996, Dilgimen

et al. 2001, Mustafaev and Norimov 1990, Mustafaev et al.

1990, Mustafaev et al. 1996, Dincer et al. 1997, Shoukry et al.

2002, Akkili ç et al. 2007, Wu 2004, Karahan et al. 2007, Guney

et al. 1997, Peters 1985, Peters 1996, Mustafaev 2004, D ’ Souza

et al. 2004, cooper et al. 2005, Topuzogullari et al. 2007).

Maleic anhydride copolymers, which become polyelectro-

lytes in an aqueous medium due to the hydrolysis of anhydride

to the ring, are known to be eff ective in biomedical applica-

tions of drug transport systems and enzyme immobilization

(Ladaviere et al. 1999). Several characteristic properties have

been studied; namely, the two-step dissociation of carboxy-

lic groups (Minakata et al. 1980) and binding of counterions

(Kitano et al. 1987), pH-induced conformation transitions

(Kawaguchi et al. 1991), and a remarkable behavior of vis-

cosity that exhibits a maximum at the half-neutralization

point (Minakata et al. 1980, Kitano et al. 1987, Kawaguchi

et al. 1991, Ohno and Sugai 1990). Also, these copolymers

proved, so far, the most capable at binding molecules as

diff erent as nucleic acids and proteins such as BSA, which

is used as a model protein in many studies (Ladaviere et al.

1997, Wang et al. 2008).

WSPE-MAs were synthesized via conventional meth-

ods such as complexes by physical mixture, complexes

in the presence of metal, conjugates via spacers or varied

cross-linkers, etc. However, conventional methods require

a long reaction time (Mustafaev 1996, Karahan et al. 2010,

Mansuro g lu et al. 2011, Karahan 2009, Dilgimen et al. 2001,

Mustafaev and Norimov 1990, Mustafaev et al. 1990, Akkili ç

et al. 2007, Karahan et al. 2007). For this purpose, we aimed

to show shorter reaction time using microwaves to synthe-

size WSPE-MA conjugates. Th e microwave-assisted method

involves the use of microwave radiations as an impact

on chemical synthesis. Microwave-assisted reactions are

provided by the thermal and/or non-thermal eff ect of this

radiation energy. Microwave energy is considered to be

due to dipole interactions providing a vibration feature to

the molecules so that they have a signifi cant impact for the

Correspondence: Mesut Karahan, Uskudar University, Faculty of Engineering and Natural Sciences, Department of Bioengineering, 34662 Uskudar, Turkey.

E-mail: mesut.karahan@gmail.com

(Received 16 December 2011; revised 9 February 2012; accepted 20 March 2012)

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Scheme 1. Microwave-assisted covalent conjugation reaction scheme of P(MVE-MA)-BSA.

Table I. Th e experimental ratios and concentrations of n BSA /n P(MVE-MA) [BSA Mw:66 kDa and P(MVE-MA) Mw:70 kDa] .

Ratios

Th e amount

of BSA (mg)

Th e amount of

P(MVE-MA) (mg)

Total PBS

solutions (ml)

0.25 0.94 1 3.90.5 1.88 1 3.91 3.76 1 3.93 11.28 1 3.95 18.80 1 3.9

conjugation reaction. Th is feature is called a non-thermal

feature of microwave energy (non-thermal eff ects) (Budarin

et al. 2011, Alesi et al. 2008).

In this study, to develop the synthetic polymeric

vaccine model systems, the poly(methyl vinyl ether-

co-maleic anhydride) copolymer-bovine serum albumin

(BSA) bioconjogates which were obtained by using 1-ethyl-3

-(3-dimetilaminopropyl) carbodiimide hydrochloride (EDC)

by conventional and microwave-assisted methods were per-

formed in varying rations at pH 7.0. All of the bioconjugates

were characterized by size-exclusion chromatography and

high-performance liquid chromatography methods.

Methods

Materials Poly(methyl vinyl ether-co-maleic anhydride) copolymers

(molecular weight: 41 kDa, Gantiez AN129BF) were supplied

from ICP Europe and used without further treatment. Th e

molecular weight of P(MVE-MA) (pK: 3.64 kJ.mol � 1 ) (Jens

et al. 2007) in phosphate buff er solution at pH 7.0 was found

to be Mw: 70 kDa, Mn: 63.388 kDa, Mz: 78.934 kDa by the

measurement technique of size exclusion chromatography.

Bovine serum albumin (Mw: 66 kDa, pI: 4.9) (Neurath and

Bailey 1953), 1-ethyl-3-(3-dimetilaminopropyl) carbodiim-

ide hydrochloride (EDC), and dimethyl sulfoxide (DMSO)

were purchased from Sigma Chemical Company (St. Louis,

USA); other chemicals such as NaOH (Fluka), sodium dihy-

drogen phosphate (NaH 2 PO 4 , Reiadel de Ha ë n), disodyum

hydrogen phosphate (Na 2 HPO 4 , Fluka), and NaN 3 (Appli-

chem) were used without further treatment. Ultra-pure water

was obtained from the Millipare Milli-Q gradient system. Th e

solutions of P(MVE-MA) used in this study were prepared in

DMSO solvent at room temperature with stirring over 12 h.

Th e solutions of BSA were prepared in phosphate-buff ered

saline PBS) at pH 7.0.

HPLC gel fi ltration BSA, polyelectrolyte (PE), and water-soluble PE-BSA bio-

conjugates were separated using HPLC. Th e molecular

masses of proteins and the fraction compositions of the

polymer – protein mixtures or conjugates were estimated

by gel fi ltration chromatography, using column Shim-Pack

Diol-300 (7.9 mm ID�50 cm) with Shim-Pack Pre-column

Diol (4.0 mm ID�5 cm) at room temperature. A Shimadzu

model LC-6AD pump was run in diff erent buff ers at a fl ow

rate of 1.0 ml/min. All solutions were fi ltered with 0.45 μ m

Sartorius RC-membrane fi lters before injection. A 20 μ l

sample volume was injected for analysis.

Th e eluate was monitored at 280 nm with Shimadzu

SPD-10AV VP Model UV – vis Detector. PE-BSA bioconjugates

were fractioned using a Shimadzu FRC-10A model fraction

collector. Th e standards used to calibrate the column were

thyroglobulin (670 kDa), immunoglobulin (155 kDa), BSA

(66 kDa), ovalbumin (44 kDa), and myoglobin (16.9 kDa).

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Microwave-assisted P(MVE-MA) 365

Figure 1. Conventional Method Conjugates (CMC): HPLC chromatograms of BSA, P(MVE-MA), and BSA-P(MVE-MA) conjugates prepared at ratio on n BSA /n P(MVE-MA) : 0.25(1), 0.5(2), 1(3), 3(4), 5(5).

PBS was prepared from the Millipore MilliQ Gradient

system, and consisted of 50 mM phosphate and 150 mM

sodium chloride for pH 7.0 studies. Mobile phase solutions

were fi ltered through a 0.45 μ m cellulose nitrate fi lter and

were degassed before use.

SEC measurements A Viscotek TDA 302 detector system with refractive index

(660 nm), right angle light scattering (670 nm), and

four-capillary diff erential viscometer detectors was used for

on-line SEC signal detection. A separate UV detector obtained

from Viscotek was connected to this detector system and

the detectors were in the following order: UV – LS – RI – VIS.

0.2 μ m nylon pre-fi lter that was used between the column

and detectors; HPLC pump, degasser, and autosampler with

100 μ l injection loop with built-in Viscotek GPCmax VE 2001

pump system, which is connected to the detectors. Omni

SEC 4.1 software was used for the acquisition and analysis

of SEC data.

A Viscotek quadruple detector array was calibrated

using a BSA monomer peak in a mobile phase of phosphate-

buff ered saline (PBS) at 1.0 ml/min fl ow rate 0.185 (Kendrick

et al. 2001) and 0.66 (Wen et al. 1996) were used as dn/dc value

and extinction coeffi cient of BSA, respectively. A Shim-Pack

Diol 300 column in the dimensions of 500 mm length and

7.9 mm inlet diameter was used for separating BSA, PE, and

PE-BSA conjugates. Elution was isocratic and at a fl ow rate of

1.0 ml/min. PBS was prepared using ultra-pure water from a

Millipore MilliQ Gradient system, and consisted of 50 mM

phosphate and 150 mM sodium chloride; pH 6.0 and 7.0.

buff er solutions were fi ltered through a 0.45 μ m Millipore

cellulose nitrate fi lter and were degassed before use.

Microwave (multi-mode) Milestone ’ s MicroSYNTH (Sorisole, Italy) labstation, com-

bined with the microwave technology, satisfi es the needs

of the various chemical research laboratories. In fact, the

diff erent accessories that can be fi tted inside the multimode

microwave cavity cover a large ramp of volume, from 2 ml

to 500 ml, diff erent values of pressure from 1 to 50 bar, and

give the possibility of reaching temperatures up to 250 o C.

Researchers can also choose to perform one reaction at a

time or to perform parallel synthesis with Milestone ’ s rotors

for up to 24 reactions at the same time (Favretto 2003).

Synthesis of WSPE-BSA bioconjugates Cross-linking agents (cross-binders) are chemical reagents

that ensure the formation of covalent bonds of molecules

and have many diff erent varieties, according to the appli-

cation purposes in processes. Th ey are known as EDC

or EDAC 1-ethyl-3-(3-dimetilaminopropil) carbodiimide

hydrochloride derivatives of the most commonly used con-

jugation reaction of biological molecules. Conjugation of the

molecule that contains primary amine and carboxyl groups

can be realized using EDC. According to the conjugate for-

mation process, primarily the N-substituted carbodiimides

react with carboxylic acids and they form the highly reactive

O-acylisourea intermediate product. Th en, the active inter-

mediate product reacts with nucleophilic groups such as the

primary amines to perform the conjugates via amide bond

formation. According to the literature, to ensure the highest

activity of carbodiimides the optimum pH range is 4.7 to 6.0

(Hermanson 1996).

P(MVE-MA)-BSA bioconjugates were synthesized via two

methods: conventional and microwave-assisted. A sche-

matic representation of the reaction procedure is shown in

Scheme 1.

In order to produce P(MVE-MA)-BSA bioconjugates

with a conventional method, the solution of poly(methyl

vinyl ether-co-maleic anhydride) (1 mg) dissolved in DMSO

(0.1 ml), stirred at room temperature and 1.9 ml PBS was

added to the solution to ensure the amount of organic phase

was at a maximum 5% rate of total solvent, but to prevent any

possible formation of micelles, PBS was added under rapid

mixing. EDC was added more than four times the amount

of the copolymer (4 mg), and pH of solution was adjusted

to 4.7 in order to provide the highest reactivity of the EDC.

After 3 h stirring, BSA solution in diff erent concentrations

(n BSA /n P(MVE-MA) : 0.25, 0.5, 1.0, 3.0 and 5.0) (Table I) was

added to the reaction mixture and stirred for 12 h. Th e total

volume of the obtained solution was completed to 4 ml

and was stirred 2 h more than the pH of solution, which

was adjusted to 7.0 using 1 N NaOH solution prepared in

ultra-pure water. Th e total reaction time was 17 h.

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Figure 3. SEC [UV (a), LS (b)] chromatograms of the conjugates by conventional method of BSA-P(MVE-MA) bioconjugates prepared at the ratios of n BSA /n P(MVE-MA) : 0.25(1), 0.5(2), 1(3), 3(4), 5(5).

Figure 4. SEC [UV (a), LS (b)] chromatograms of the conjugates by means of the microwave-assisted method of BSA-P(MVE-MA) bioconjugates prepared at the ratios of n BSA /n P(MVE-MA) : 0.25(1), 0.5(2), 1(3), 3(4), 5(5).

Figure 2. Microwave Assisted Conjugates (MWC): HPLC chromatograms of BSA, P(MVE-MA), and BSA-P(MVE-MA) conjugates prepared at ratio on n BSA /n P(MVE-MA) : 0.25(1), 0.5(2), 1(3), 3(4), 5(5).

In order to obtain P(MVE-MA)-BSA bioconjugates via

the microwave-assisted method, the solution of poly(methyl

vinyl ether-co-maleic anhydride) (1 mg) was dissolved in

0.1 ml DMSO, stirred at room temperature, 1.9 ml PBS was

added to the solution, and EDC was added in more than four

times the amount of the copolymer (pH 4.7). After 3 min stir-

ring of solution under microwave [75 Watt (W), Microsynth,

Milestone (R) , BG, Italy], to compare the results with the clas-

sical method, BSA solution in diff erent concentrations (n BSA /

n P(MVE-MA) : 0.25, 0.5, 1.0, 3.0 and 5.0) (Table I) was added

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Microwave-assisted P(MVE-MA) 367

0 1 2 3 4 5

0

10

20

30

40 1 2

Are

a of

UV

(Arb

itrar

y U

nits

)

nBSA/nP[MVE-MA]

Figure 5. Th e area of the UV of the conjugates (MWC) (1) and conjugates (CMC) (2) prepared at the ratios of n BSA /n P(MVE-MA) : 0.25, 0.5, 1, 3, 5.

0 1 2 3 4 5

0

10

20

30

40

50

60

70 1 2

Are

a of

LS

(Arb

itrar

y U

nits

)

nBSA/nP[MVE-MA]

Figure 6. Th e area of the LS of the conjugates (MWC) (1) and conjugates (CMC) (2) prepared at the ratios of n BSA /n P(MVE-MA) : 0.25, 0.5, 1, 3, 5.

peaks increase, as were obtained by UV and LS detectors

(Topuzogullari et al. 2007). According to these fi gures, the

highest peak intensities were obtained in n BSA /n P(MVE-MA) : 5.

Th e area of UV and LS of bioconjugates is plotted in

Figures 5 and 6. UV areas MWC were four times bigger

than CMC, and LS areas of MWC were three times larger than

CMC. Th at is, in direct proportion to the CMC and MWC, the

peak area seems to have increased when n BSA /n P(MVE-MA)

ratio increased. Th e molecular weights of the CMC and MWC

increase depending on molecule sizes.

As a result of the measurements, as long as the greater

amount of BSA was banded to P(MVE-MA), the larger peak

was observed. In addition, the reaction time of P(MVE-MA)-

BSA bioconjugates under microwave-assisted conditions

was shortened from 17 h to 15 min because of the non-

thermal eff ect of the microwave energy ’ s dipolar polarization

mechanism on the molecules.

Conclusion

Water-soluble and stable P(MVE-MA)-BSA bioconjugates

have been synthesized successfully via a microwave-assisted

method with shortened reaction time. Th e best ratio of

bioconjugates was determined to be n BSA /n P(MVE-MA) : 5. It is

suggested that microwave-assistance is a useful technique to

develop a synthetic vaccine model system by means of short

reaction time and synthesized stable bioconjugates without

protein denaturation.

Acknowledgements

Th e authors dedicate this report to the memory of the

Founder Head of Yıldız Technical University Bioengineer-

ing Department, the precious man of science, Prof. Dr. M.I.

Mustafaev. Th is research was supported by a grant from the

T.R. Prime Ministry State Planning Organization (Project

Number 25-DPT-07 - 04-01).

Declaration of interest

Th e authors report no confl icts of interest. Th e authors alone

are responsible for the content and writing of the paper.

to the reaction mixture and stirred for (75 W) 10 min. Th e

total volume of the obtained solution was completed to

4 ml and was stirred (75 W) 2 min more, then the pH value

of the mixture was adjusted to 7.0 with 1N NaOH. Th e total

reaction time was 15 min. Th e BSA/poly(methyl vinyl ether-

co-maleic anhydride) copolymers ratios, n BSA /n P(MVE-MA) ,

were calculated using the equation n � CN A /M, where n

is the number of the molecules in 1 ml, M is the molecular

weight of components, N A is the avagadro ’ s number, and

C represents concentration in g/ml. Results are given in

Table I.

Results and discussion

To compare the CM and MWM by means of reaction time, the

reaction time was shortened from 17 h to 15 min via MWM.

HPLC and SEC were used for the P(MVE-MA)-BSA bio-

conjugates formation. HPLC chromatograms belonging to

CMC and MWC are given in Figures 1 and 2, respectively.

According to these chromatograms, the trimer peak of BSA

proteins was seen between 13.5 and 19 min. P(MVE-MA)

copolymer didn ’ t show any peak-like baseline, but as a result

of the BSA and P(MVE-MA) interactions, the peaks of the

conjugates have been observed between 9 and 11.5 minutes

because of the occurrence of BSA-copolymer conjugates,

which have larger molecular size and molecular weight

than free-form P(MVE-MA). When obtained conjugates are

compared among themselves, if the added amount of BSA

to a fi xed amount of polymer rises, intensity increases of

the conjugate peaks are observed. When comparing the CM

and MWM, larger peak areas were observed in MWM due to

the bigger molecular size and weight of P(MVE-MA)-BSA

bioconjugates than CM.

Th e UV and LS chromatograms of synthesized P(MVE-

MA)-BSA bioconjugates using CM and MWM are illustrated

in Figures 3a,3b and 4a,4b, respectively. Th ese fi gures show

the chromatograms of P(MVE-MA)-BSA bioconjugates

at diff erent ratios of molecular concentrations of compo-

nents (n BSA /n P(MVE-MA) ) at pH 7.0, and it can be seen that,

with an increase in the number of BSA molecules in the

bioconjugate [the concentration of P(MVE-MA) is kept

constant], the areas of bioconjugates ’ chromatographic

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