ART. CELLS, BLOOD SUBS., AND IMMOB. BIOTECH., 29(3), 203–212 (2001)

NONCOVALENT IMMOBILIZATIONOF BOVINE SERUM ALBUMIN ON

OXIDIZED CELLULOSE

Vijay Kumar1,* and Ganesh S. Deshpande2

1 Pharmaceutics Division, College of Pharmacy,The University of Iowa, Iowa City, IA 52242

2 SmithKline Beecham Consumer Healthcare, Parsippany,New Jersey

ABSTRACT

Immobilization of bovine serum albumin (BSA) on oxidized cel-lulose (OC) containing carboxylic groups, a biocompatible andbioresorbable polymer, was investigated in water and differentbuffer solutions (pH 2–7) at 5�C. The maximum amounts of BSAloaded on OC in pH 2, 3, 4, 6, and 7 buffer media were 9.82, 10.52,8.86, 9.16, 6.05, and 2.69% (w/w), respectively. In water, the cor-responding value was 9.82%. The release study was performed onpowder, pellet and suspension (in castor and sesame oils) dosageforms of OC-BSA immobilization products prepared in water andpH 2 buffer, in pH 7.4 phosphate buffer at 37�C. The results re-vealed the release of BSA to be the fastest from oil suspensions,intermediate from powders, and slowest from pellets. In conclu-sion, the results presented suggest that OC has the potential to beused as an immobilizing matrix for BSA and other proteins in wa-ter and pH 2– 4 buffer solutions.

203

Copyright � 2001 by Marcel Dekker, Inc. www.dekker.com

*Corresponding author. Fax: 1-319-335-9349; E-mail: vijay-kumar�uiowa.edu

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INTRODUCTION

In recent years, immobilization technology involving covalent and/or non-covalent fixation of drugs, enzymes, and proteins on soluble or insoluble poly-meric carriers has received considerable interest (1). It offers potential in drugdelivery, including drug targeting. Proteins immobilized on organic or inorganicpolymer supports frequently exhibit prolonged half-life, enhanced resistance todegradation, and decreased immunogenicity. Further, immobilized drugs/proteinscan be compressed into a pellet and used as an implant, or formulated as a suspen-sion and administered by injection. Cellulose and its derivatives such as celluloseacetates, cellulose nitrates, cellulose triacetate, and diethylaminoethylcellulose(DEAE-cellulose) have been extensively used as immobilizing matrices for a va-riety of agents, including proteins, whole cells, and enzymes (1). However, thesepolymers are not biodegradable and, hence, are not suitable for use as an injectableor implantable carrier system.

6-Carboxycellulose, commonly referred to as oxidized cellulose (OC,Fig. 1), is a biocompatible, bioresorbable material (2). It degrades in-vivo to pro-duce glucose and two or three carbon fragment units (3,4). The degradation rate,in general, increases with an increase in oxidation level and a decrease in degreeof polymerization (2). Currently, OC containing 16 –24% carboxylic content iscommercially available for use in humans to stop bleeding during surgery (5) andto prevent the formation and reformation of adhesions after surgery (6,7). OC hasalso been found to form a complex with a variety of substances containing aminegroups, including drugs (e.g., kanamycin sulfate (8), gentamycin (9,10), hydrox-ythiamine (11), methotrexate (11), and adrenaline (12)), enzymes (e.g., trypsin(13), proteinase (14,15), and cytokines (16)), and amino acids (17,18). These stud-ies show ionic interactions to be the driving force for complexation of these agentswith OC.

In this study, using bovine serum albumin (BSA) as a model protein, wehave found that the interaction of BSA on OC is pH dependent, increasing withdecreasing pH of the immobilization medium. This suggests that not only ionicinteractions but also hydrophobic forces appear to play an important role in theimmobilization of BSA on OC. Since the long-term goal of this research is the

204 KUMAR AND DESHPANDE

Figure 1. Structure of oxidized cellulose (OC).

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development of an OC-based biodegradable implantable or injectable deliverysystem for proteins, the release of BSA from powder, oil suspension, and tabletdosage forms of OC-BSA immobilization products was also studied, and the re-sults are included in this paper.

MATERIALS AND METHODS

Materials

Oxidized cellulose (carboxylic content 16%), sodium stearyl fumerate(PRUV�), and sesame oil were received from Eastman Chemical Co. (Kingsport,Tennessee, USA), Penwest Company (formerly Mendell Co. Inc., Patterson, NewYork, USA), and Croda Inc. (New York, NY, USA), respectively. BSA (A4503,Lot No. 129F0136) and castor oil were purchased from Sigma chemical Co. (St.Louis, Missouri, USA) and Ruger Chemicals (Hillside, New Jersey, USA), re-spectively. The Coomassie blue concentrate was obtained from Bio-Rad Labora-tories (Hercules, California, USA). All other chemicals were analytical grade.

Immobilization of BSA on OC

Stock solutions of BSA in water and aqueous buffer solutions (pH 2 and 3:0.01M citrate buffer; pH 4 and 6: 0.01M acetate buffer; and pH 7: 0.01M phos-phate buffer), corresponding to BSA concentration of 1 mg/ml, were prepared. Toa 10 ml BSA stock solution, 100 mg of OC was added and the mixture was stirredat 5�C for different time periods using a magnetic stirrer. The reaction suspensionwas then centrifuged for 10 minutes at 2000 r.p.m. Hundred microliters of thesupernatant was removed and analyzed immediately for BSA by the Bradfordtest (vide infra). The residue was collected, freeze-dried, and stored in a freezer(� 5�C) until used. All immobilization reactions were conducted in triplicate.Control solutions containing BSA only were prepared and subjected to identicalconditions prior to analysis.

Determination of BSA Loading

The amount of BSA bound to OC was determined by the Bradford method(19) using Coomassie blue as a reagent. The latter was prepared by diluting onepart of the Coomassie blue concentrate with four parts of distilled water. ForBSA analysis, 2.5 ml of the Coomassie reagent and 100 ml of the BSA solutionwere taken in a stoppered quartz cell (1 cm width) and mixed. At the end of oneminute, absorbance measurements were made at 595 nm using an ultraviolet-

IMMOBILIZATION OF BSA ON OC 205

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visible spectrophotometer (Shimadzu UV 2100U, Shimadzu Corp., Kyoto, Japan).The amount of BSA bound to OC was determined from the difference in BSAconcentrations before and after treating the solution with OC. Control BSA solu-tions (i.e., without OC), containing the same amount of BSA as was used in thereaction sample, were also prepared and analyzed as described above. All assayswere performed in triplicate.

Preparation of OC-BSA Pellets

OC-BSA immobilization products prepared in water and 0.01M citratebuffer (pH 2.0) were used in the study. They were compressed, with and without0.5% PRUV�, on a Carver press using a 1/3 inch die and flat-faced punches. Eachpellet weighed 30 mg, and showed a hardness value of 6 –7 kp as measured on aSchlenniger 2E /106 hardness tester.

Release Studies

The release of BSA was studied from powder, pellet (with and without 0.5%PRUV�), and oil suspension dosage forms of OC-BSA prepared in water and pH2 buffer solution. Oils used in the study were castor oil and sesame oil. An accu-rately weighed amount of the test sample was suspended in a 10 ml phosphatebuffer solution (pH 7.4) containing 10% (v/v) ethanol using a shaker at 37�C.Hundred microliters of the supernatant were withdrawn at 4, 10, and 24 hours andthen every 24 hours for 6 days, and analyzed immediately for BSA by the Brad-ford method (19) described above. The control used in the study was a pH 7.4phosphate buffer solution containing the same amount of BSA as was present inthe test sample and 10% (v/v) ethanol. This solution was stored at 37�C and ana-lyzed at the same time intervals as was used for the test samples.

RESULTS AND DISCUSSION

Immobilization of BSA on OC

The binding of BSA to OC as a function of time in water and different pHbuffer solutions is shown in Fig. 2. As is evident, the immobilization of BSA onOC was complete in about two hours in water, about four hours in pH 2 and 3buffer solutions, and about six hours in pH 4, 6, and 7 buffer media. No loss ofBSA was observed in control solutions, suggesting that BSA is stable under ex-perimental conditions used. The maximum loading of BSA on OC achieved inwater and different pH buffer solutions are presented in Table 1.

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As is evident from Figure 3, the immobilization of BSA on OC increaseslinearly with a decrease in the pH of the immobilization medium (R 2 � 0.9147).The high loading of BSA seen in water can attributed to the pH effect because OCbeing a weak polyacid (pKa 3.5 and 4.0) (22) partially dissociates in water andproduces a suspension with a pH of about 2.4.

IMMOBILIZATION OF BSA ON OC 207

Figure 2. Effect of reaction duration on the binding of BSA to OC (n�3).

Table I. Amounts of BSA Loaded on OC

Reaction Medium pH Percent Binding (Std. Dev.)

pH 2 10.52 � 0.15pH 3 8.96 � 0.22pH 4 9.16 � 0.23pH 6 6.05 � 0.08pH 7 2.69 � 0.07Water (pH 2.4a) 9.82 � 0.30

a pH of the OC dispersion.

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The isoelectric point of BSA is 4.8–5.0 (23). Thus, in pH 2 buffer and water,it appears that the immobilization of BSA on OC takes place via both physicaladsorption (i.e., hydrogen bonding, hydrophobic interactions, and/or van der Waalforces) and ionic interaction (1,20), the former being the predominant mechanism.At higher pH conditions, however, OC-COOH progressively ionizes to OC-COO�, while BSA converts from a fully protonated form, (NH3

�-BSA-COOH),to a zwitterion (NH3

�-BSA-COO�) and subsequently to NH3�-BSA-COO�.

Thus, as the pH increases, more and more ionic interaction between OC and BSAdominates, causing a progressive increase in the formation the water-soluble OC-BSA complex, and consequently, a decrease in the yield of the insoluble OC-BSAimmobilization product. At or above pH 7.0, OC and BSA predominantly exist asOC-COO� and NH3-BSA-COO�, respectively, and hence, the yield of OC-BSAis significantly decreased.

Release Studies

The release profiles of BSA from powder, pellet and suspension dosageforms of OC-BSA, prepared in water, are shown in Fig 4. All samples, except forthe pellets that had no PRUV�, exhibited an initial fast release (‘‘burst effect’’).In the first four hours, the amounts of BSA released from OC-BSA suspensions in

208 KUMAR AND DESHPANDE

Figure 3. Relationship between immobilization of BSA on OC and pH of the medium.

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sesame oil and castor oil were about 69% and 55%, respectively, from OC-BSApowder about 37%, and from OC-BSA pellet containing 0.05% PRUV� about28%. After four hours, all samples released BSA slowly. After six days, the 100%of BSA were released from castor and sesame oil suspensions of OC-BSA. Thepowder and pellet, with and without PRUV�, forms, in contrast, released only90%, 64%, and 72% of BSA, respectively, over the same time period.

Fig 5 shows the release profiles of BSA from OC-BSA prepared in pH 2.0buffer solution. As is evident, both castor oil and sesame oil suspensions of OC-BSA exhibited similar release profiles. OC-BSA pellets with and without PRUV�also showed no significant difference in their release patterns. After four hours,the amounts of BSA released from OC-BSA suspensions, powder, and pelletswere 49%, 28%, and 11%, and After six days, about 84%, 69%, and 59%,respectively.

The above results show that (i) irrespective of whether OC-BSA was pre-pared in water or pH 2.0 buffer, the release of BSA was the fastest from oil sus-pensions, intermediate from the powder forms, and the slowest from the pelletformulations, and (ii) OC-BSA prepared in water released BSA at a faster ratethan that made in pH 2.0 buffer solution. The faster release of BSA from thesesame oil and castor oil suspensions compared to that from the respective OC-

IMMOBILIZATION OF BSA ON OC 209

Figure 4. Release profiles of BSA from powder, pellet, and oil suspension dosage formsof OC-BSA prepared in water (n�3).

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BSA powders may be due to the plasticization effect of oils on OC. The differencein the release properties of OC-BSA prepared in water versus that made in pH 2buffer could be due to different proportions of interaction forces present betweenOC and BSA.

CONCLUSION

The results presented above show that the immobilization of BSA on OC isfavored at low pH conditions and in water. The release of BSA was the fastest(84 –100%) from OC-BSA suspension in oils (castor and sesame oils), interme-diate (69–90%) from OC-BSA powders, and the slowest (59–72%) from OC-BSA pellets, over six days, suggesting that the pellet form is useful as a sustained-release delivery system whereas powder and suspension forms may serve as apulse-release delivery system. The presence of PRUV� in the pellets did notchange the release profile of BSA.

Further work to (i) characterize the secondary structure of BSA in the insol-uble fraction of the immobilization product and (ii) isolate and identify the solublefraction of OC-BSA complex by freeze drying, is in progress. Immobilization ofother proteins, such as lysozyme, on OC is also being investigated. Lysozyme (23)has an isoelectric point of ca. 11. Therefore, unlike BSA, it is expected to show

210 KUMAR AND DESHPANDE

Figure 5. Release profiles of BSA from powder, pellet, and oil suspension dosage formsof OC-BSA prepared in pH 2.0 buffer solution (n�3).

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higher affinity to OC at a physiological pH. The successful demonstration of bind-ing of various proteins on OC may allow its use not only as a carrier for proteinsin the design of a biodegradable delivery system but also as an adjuvant in thedevelopment a solid vaccine.

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