Fatty Acid Binding Domain Mediated Conjugation of Ultrafine Magnetic Nanoparticles with Albumin Protein
© to the authors 2008
Received: 4 September 2008
Accepted: 11 November 2008
Published: 22 November 2008
A novel bioconjugate of stearic acid capped maghemite nanoparticle (γ-Fe2O3) with bovine serum albumin (BSA) was developed by taking recourse to the fatty acid binding property of the protein. From FT-IR study, it was found that conjugation took place covalently between the amine group of protein molecule and carboxyl group of stearic acid capped maghemite nanoparticle. TEM study further signified the morphology of the proposed nanobioconjuagte. The binding constant of nanoparticle with protein molecule was evaluated from the optical property studies. Also, magnetic measurement (M–H) showed retaining of magnetic property by significant values of saturation magnetization and other hysteretic parameters.
Bioconjugation of magnetic nanoparticles is basically done to make it compatible for numerous biomedical applications such as MRI contrast enhancement, drug delivery, detoxification of biological fluids, immunoassay, tissue repair, hyperthermia etc. [1–6]. Besides these, bioconjugate systems are also being applied in various large-scale bioprocesses such as nucleic acid detachment, protein separation, magnetic biosensor etc. [7–9]. All these biorelated applications require the use of magnetic nanoparticles that should have size smaller than 10 nm with overall narrow particle size distribution, so that the particles have uniform and unique properties . This is mainly because of the fact that particles at this size range have the advantage of showing well-established magnetic properties which reduces the possibility of particle aggregation upon magnetic attraction in a magnetic dispersion . For fabricating a bioconjugate, it generally involves lots of surface chemistry work . Normally, these are synthesized by modifying the nanoparticle surface with some chemical linker molecule, so that it can further interact with next incoming bio molecular entity with the help of free functional group of linker molecule. This procedure was already well established but supposed to be having some difficulty in the sense of stability of linker molecule due to various biochemical events. This might occur mostly under in vivo condition when applying the bioconjugate system in the targeted delivery of neoplastic compounds to tumor cell.
To overcome this difficulty, we proposed some biologically evolved linker moiety (fatty acid binding domain) to fabricate a novel bioconjugate in covalent fashion by utilizing the molecular recognition property of bio molecular system, such as bovine serum albumin (BSA) protein. It is supposed to be an important substitute over the synthetic linker in designing the bioconjugate covalently so that it can be applied gently under preceding condition. This can also be called as natural anchor molecule that is functional in several of its biological activities. Here, BSA is chosen as the material of interest for the bioconjugation purpose because of the fatty acid binding domain of BSA, which helps in the conjugation of stearic acid capped nanoparticle with the protein moiety . Also, serum albumin is the major vehicle for transport of nonesterified fatty acids in the circulation . Magnetic bioconjugate of stearic acid capped ultrafine maghemite nanoparticle with BSA molecule is quite advantageous in case of stability because of the molecular recognition ability of the BSA molecule towards fatty acid itself and its well stability under physiological condition (pH = 7.4).
High purity (>99%) iron (III) nitrate [Fe(NO3)3 · 9H2O], stearic acid [C18H36O2], and tetrahydrofuran (THF) were used for the synthesis of maghemite nanoparticle. The albumin protein required for the bioconjugation purpose was also of high purity from Spectrochem India Pvt. Ltd.
Ultrafine maghemite nanoparticles (γ-Fe2O3) were synthesized through a gentle chemistry route . High purity iron (III) nitrate [Fe(NO3)3 · 9H2O] and stearic acid [C18H36O2 in the ratio of 1:2 were used as initial ingredients. The homogeneous solution of molten mixture was then heated at 125 °C for 11/2 h to form a reddish brown viscous mass which then subsequently treated with THF. The powdery precipitates were collected through centrifugation and dried completely in an air oven at 70 °C. The dried precipitates were further subjected to heat treatment at 250 °C for holding time of 30 min inside an electrically heating furnace to get the nanoparticles. The synthesis of the conjugate of bovine serum albumin with maghemite nanoparticles (γ-Fe2O3) was carried out by transferring 3 mmol of BSA into PBS buffer. The mixture was then kept at 4 °C and allowed to stand for 12 h, so that protein sample gets completely soluble in the PBS buffer. After this, 1 mmol of maghemite nanoparticles (γ-Fe2O3) were mixed with the BSA containing buffer and the mixture was vortexed for 1 h at room temperature. The vortexed mixture is again stored at 4 °C for 2 h for the stabilization purpose. This was basically done to control the covalent interaction taking place between stearic acid coated maghemite nanoparticles with that of protein molecule. Further, the mixture was centrifuged at high speed for 15 min to get the magnetic nanobioconjugate consisting of maghemite nanoparticles (γ-Fe2O3) and BSA. The pellet portion is collected and allowed to vacuum dried and the supernatant being kept for further characterization.
The formation of maghemite–BSA nanobioconjugate had been studied with the help of FT-IR spectrometer. The spectrum was recorded in the transmission mode on a Nicollet Impact 410-spectrometer. The dried samples of maghemite, BSA as well as the magnetic nanobioconjugate were grounded with KBr and the mixture was compressed into a pellet for characterization. Transmission electron microscopy (TEM) study for the conjugate of bovine serum albumin with maghemite nanoparticle (MNP/BSA) as well as MNP itself was carried out using a JEM-100CX model operated at 100 KV. The photoluminescence spectrum for all samples was taken with Perkin Elmer LS-55 fluorescence spectrometer. The magnetic properties of the resulting bioconjugate as well as maghemite nanoparticles had been studied with vibrating sample magnetometer (VSM, Lakeshore, 7410) for confirming the retaining of magnetic properties by the nanoparticles after the formation of nanobioconjugate.
Results and Discussion
In summary, we developed a simple technique for the synthesis of bioconjugate of maghemite nanoparticles with BSA molecule by using the covalent interaction between the fatty acid binding domains of BSA molecule with stearic acid capped nanoparticles. This will lead to the development of non-toxic iron oxide nanoparticles using BSA as a biocompatible passivating agent. We confirmed the formation of the same from the FT-IR spectra as well as TEM micrograph. We also established the well retaining of magnetic property of nanoparticles after the formation of bioconjugate from M–H study. It is worth mentioning here that, this is the first report on conjugation of nanoparticles with biomolecules by utilizing biologically evolved linker moiety in covalent fashion. The designed magnetic bioconjugate seems to be applicable for targeted delivery purpose to a neoplastic cell due to the receptor action of the BSA molecule by binding to a wide variety of lipophilic compounds such as steroid present over cancer cell.
One of the author, PD, gratefully acknowledge the financial support by DAE-BRNS, Govt. of India (vide project no. 2007/20/34/04-BRNS/1865) under DAE Young Scientist Research Award. The authors would like to extend sincere thanks to CIF, IIT Guwhati, India and RSIC–NEHU for VSM and TEM facility.
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