- Email: [email protected]
Surface modification of magnetic nanoparticles with oleylamine and gum Arabic
Available online at www.sciencedirect.com
Materials Science and Engineering C 28 (2008) 438 – 442 www.elsevier.com/locate/msec
Surface modification of magnetic nanoparticles with oleylamine and gum Arabic Otto C. Wilson Jr. a,⁎, Erin Blair a , Stephanie Kennedy a , Gloryvee Rivera a , Patrick Mehl b a
The Catholic University of America, Department of Biomedical Engineering, BONE/CRAB Lab, 620 Michigan Ave NE, Washington, DC 20064, United States b Biomagnetics Group, Vitreous State Lab, 620 Michigan Ave NE, Washington, DC 20064, United States Available online 11 April 2007
Abstract Magnetic nanoparticles (MNP) were synthesized under acidic conditions in the presence of oleylamine (OLA) and gum Arabic (GA) to study the influence of surface modification on MNP characteristics and cellular level bioactivity. Highly dispersed MNPs were formed from acidic solutions of ferric and ferrous chloride in the absence and presence of OLA. The MNP synthesized in the presence of GA formed large particle aggregates that exhibited rapid coagulation. The three types of MNP were characterized via thermogravimetric analysis (TGA), and dynamic and electrophoretic light scattering to determine particle size and zeta potential. Cytotoxicity and cell interactions were assessed for each of the three MNP samples using L929 fibroblast cells. OLA modified MNPs exhibited the highest level of cytotoxity. Approximately 27% of L929 fibroblast cells died after exposure to OLA modified MNP in comparison to 10% cell death for untreated, GA treated, and control cells. Cells that were exposed to MNPs could be translated in magnetic fields. The OLA and untreated MNPs could be detected in the cell cytoplasm while the GAmodified MNP clusters were located at the cell membrane based on light microscopy. © 2007 Elsevier B.V. All rights reserved. Keywords: Adsorption; Oleyl amine; Magnetic nanoparticles; Surface modification; Gum Arabic
1. Introduction Magnetic nanoparticles (MNPs) typically possess a magnetite (Fe3O4) or maghemite (γ-Fe2O3) core chemistry and range in size from 5–100 nm [1]. They are currently utilized in a wide variety of medical diagnostic and therapeutic uses such as hyperthermia treatments for cancer [2–4], cell sorting [5], and tissue engineering [6]. The chemical and physical characteristics of the MNPs play a critical role in determining successful interactions in each of these applications. Factors such as size, surface area, and magnetic properties are very important, but proper MNP surface chemistry is integral to enhance colloid stability and cell interactions. Biocompatible silica coated MNP (50 nm) were found to pass the blood–brain barrier without disturbing biological functions or showing any signs of toxicity [7]. Surface modification can also be used to minimize toxicity and/or undesired biological interactions such as removal by the reticuloendothelial system. Previous work with MNPs showed that gum Arabic (GA) is an effective agent to improve colloid stability [7] and enhanced ⁎ Corresponding author. Tel.: +1 202 319 5822; fax: +1 202 319 4287. E-mail address: [email protected] (O.C. Wilson). 0928-4931/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2007.04.008
cellular uptake by DU 145 prostate cancer cells [8]. However, phosphate preferentially adsorbs on MNPs and easily displaces GA, and decreases colloid stability [9]. One of the goals of this study is to modify the surface of MNPs with amine functional groups. This is for the purpose of developing a method to covalently link GA onto MNPs using amine based functionality. Oleylamine (OLA) consists of a long hydrophobic, monounsaturated fatty acid tail, with an amine group on one end. OLA has been used to stabilize solutions of a host of metal and metal oxide nanoparticles that are typically synthesized in nonaqueous solvents including gold [10] and cobalt [11] nanoparticles. The focus of this study is to investigate and report how surface modification of MNPs with GA and OLA influences particle size, colloid stability, and L929 fibroblast cell interactions. 2. Experimental procedures 2.1. MNP Synthesis Three types of MNPs (uncoated, OLA modified, GAmodified) were synthesized using a 2:1 molar ratio of ferric and ferrous chloride (Fisher Scientific, Fairlawn, NJ). The uncoated
O.C. Wilson Jr. et al. / Materials Science and Engineering C 28 (2008) 438–442
439
Table 1 Summary characterization data for the magnetic nanoparticles synthesized under acidic conditions MNP surface
TGA mass loss%
Size(nm)
Zeta potential (mV)
Uncoated OLA GA
5 15 35
25 90 1000
− 41 − 27 − 30
MNPS were prepared by the dropwise addition of the mixed iron ion solution (20 ml) into an aqueous ammonium hydroxide solution that contained 5 ml of reagent ammonium hydroxide (Fisher Scientific, Fairlawn, NJ) and 75 ml of de-ionized water. The OLA modified particles were synthesized by ultrasonicating 2 ml of OLA and 73 ml of water to form an emulsion prior to adding five ml of reagent ammonium hydroxide. The mixed iron ion solution (20 ml) was added dropwise to the OLA emulsion while stirring. The GA-modified particles were synthesized by adding one g of GA (TIC Gums, Belcamp, MD) to 20 ml of the mixed iron ion solution prior to adding this solution dropwise to aqueous ammonium hydroxide solution (5 ml of ammonium hydroxide and 75 ml of water). In each case, the final concentration of Fe3+ was 0.5 M and the resulting solution had a pH of between 2 and 3, due to an excess of hydrochloric acid as a product of the reaction. The MNP samples were washed via centrifugation (10,000–25,000 rpm for 30–35 min) to remove counter ions and stored in de-ionized water. 2.2. MNP characterization The MNPs were characterized via thermogravimetric analysis (TGA) to determine mass loss from volatile components, dynamic light scattering to determine particle size and electrophoretic light scattering to determine zeta potential. TGA was performed on each of the 4 samples using a Shimadzu TGA-50 Thermogravimetric Analyzer (Kyoto, Japan). The particles were washed with de-ionized water and dried overnight, yielding an average of 25–30 mg of each sample. The dry solids were then ground into powder using a mortar and pestle. Each sample was placed in an alumina pan and heated to 800 °C at a rate of 10 °C/min in air. A Malvern Zetasizer 3000 was used to measure the particle size and zeta potential of the MNPs. Dynamic light scattering was used to measure the particle size of the MNP particles in dilute suspensions. Zeta potential measurements were collected for surface treated and untreated MNP samples at a particle concentration of 0.005 wt.% in 0.01 NaCl. The pH was adjusted to 7 to approximate physiological conditions.
Fig. 1. Optical micrograph of L929 mouse fibroblasts cells after overnight exposure to 5 μg/L untreated MNP (no external magnetic field).
2.3. MNP interactions with L929 mouse fibroblast cells 2.3.1. Live/dead cell assay MNPs (1–50 μg/L) were added to L929 fibroblast cultures containing about 10,000 cells/well. After a culture period of 1–2 days, a live/dead assay (Molecular Probes, Eugene, OR) was performed to determine the influence of MNP surface treatment on cell viability. In the assay, live cells are labeled with green fluorescent calcein dye (495 nm absorption), and dead cells with red fluorescent ethidium homodimer-1 (635 nm absorption). The results were quantified by calculating the number of green (or red) cells and dividing that by the total number of cells. 2.4. MNP cell uptake L929 mouse fibroblast cells were placed in a small Petri dish (50,000 cells/ml) and incubated with the surface treated MNP (5 μg/L) overnight. The cells were examined using a light microscope and subjected to a magnetic field using a small Nd– B–Fe magnet. A Cool-Snap Pro color CCD camera was used in conjunction with Image Pro Plus software for the image capture.
Table 2 Cytotoxicity data (% live cells) for the L929 mouse fibroblast cells after exposure to the surface modified MNP based on live/dead cell assay MNP particle exposure concentration
Uncoated OLA GA
50 μg/L
10 μg/L
5 μg/L
1 μg/L
98 – 96
94 75 97
81 85 90
85 59 70
Fig. 2. Optical micrograph of L929 mouse fibroblasts cells after overnight exposure to 5 μg/L untreated MNP (strong external magnetic field).
440
O.C. Wilson Jr. et al. / Materials Science and Engineering C 28 (2008) 438–442
Fig. 3. Higher magnification optical micrograph of untreated MNP exposed L929 mouse fibroblasts cells in the presence of a strong external magnetic field.
Images were captured before the application of the magnetic field, and then 1–2 min after the field was applied. 3. Results The MNPs that were synthesized displayed unique differences that reflected the influence of the surface modification agents. The OLA modified MNPs (OLA-MNP) were highly dispersed and remained so for extended periods of time. However, the GA-modified MNPs (GA-MNP) were highly agglomerated and rapidly sedimented. One of the more unusual findings of this study was that the untreated MNPs exhibited a higher degree of colloid stability than the OLA-MNP. Characterization data for the MNPs from TGA, DLS, and zeta potential are summarized in Table 1. The uncoated MNPs exhibited a mass loss of 5% on heating to 800 °C. This mass loss can be attributed to the volatilization of water, surface hydroxyl groups, and residual contaminants from the synthesis process. The OLA-coated nanoparticles demonstrated a higher mass loss of approximately 15% due to the thermal degradation of the organic molecule. The GA-modified MNPs exhibited the
Fig. 4. Optical micrograph of L929 cells after exposure to 5 μg/L untreated MNP. Contrast within cells indicates presence of MNP in the cell cytoplasm.
Fig. 5. Optical micrograph of L929 cells after exposure to 5 μg/L OLA treated MNP.
largest mass loss of 35%. The particle size of the MNPs was also influenced by the synthesis conditions. The smallest MNPs were obtained for the uncoated sample. The particle size was consistently below 50 nm and averaged around 25 nm. The OLA-MNPs had a particle size of 90 nm. The GA-MNPs were much larger due to extensive agglomeration and had a particle size of approximately 1000 nm. The zeta potential (measured at pH 7) of all MNPs was negative and −41 mV, − 27 mV, and − 30 mV for MNP, OLA-MNP, and GA-MNP, respectively. The results of the live dead cell assay (Table 2) indicated that the L929 fibroblast cells that were exposed to OLA modified MNPs displayed a higher percentage of dead cells. The data shown in Table 2 is a compilation of four samples that were analyzed at MNP loadings of 50, 10, 5, and 1 μg/L. The amount of live cells varied from 59–85% for OLA-MNP. The unmodified MNPs and GA-modified MNPS had cell survival rates of 81–98% and 71–96%. The control had a 90% cell survival rate. It is interesting to note that according to our experimental results, the lower MNP concentrations seemed to
Fig. 6. Optical micrograph of L929 cells after exposure to 5 μg/L GA treated MNP. GA-modified MNP agglomerates appear located at the outer surface of the cell membrane.
O.C. Wilson Jr. et al. / Materials Science and Engineering C 28 (2008) 438–442
result in higher cell mortality. More experiments will be conducted to study this effect further. The cells were placed in a magnetic field to verify interactions between the MNPs and L929 fibroblast cells. The fibroblast cells were incubated overnight with the surface modified MNPs samples at a concentration of 5 μg/L. L929 fibroblast cells that were exposed to untreated, OLA treated and GA treated MNPs showed movement and migration along the direction of the magnetic field. The most notable interactions with the magnetic field occurred for the L929 cells exposed to the untreated MNPs. Optical micrographs for L929 cells exposed to untreated MNPs both in the absence and presence of the magnetic field are shown in Figs. 1–3. Fig. 1 shows the fibroblast cells exposed to untreated MNPs in the absence of a strong external magnetic field. Figs. 2 and 3 show the same L929 fibroblast cell culture in the presence of a strong external magnetic field. The fibroblast cells aged with untreated MNPs formed chains and could be manipulated with magnetic fields more easily than cells exposed to OLA or GA treated MNPs. Light microscopy was also used to visualize the location of the magnetic nanoparticles within the cells. Higher magnification optical micrograph photos (Figs. 4–6) of the surface treated MNPs showed differences in contrast that may indicate the location of the MNPs with respect to the cell membrane. The uncoated MNPs (Fig. 4) and OLA-coated MNPs (Fig. 5) seem to be concentrated in the cytoplasm while the GA-coated particles (Fig. 6) are concentrated on the surface of the cells. 4. Discussion One of the more interesting results of this study was that the synthesis of MNPs using acidic conditions (i.e. the final pH was approximately 2–3) greatly improved the colloid stability of unmodified nanoparticles. In each synthesis case, the resulting MNP suspension was approximately pH 2–3, due to an excess of hydrochloric acid as a reaction product. Typical MNP synthesis reactions such as the Massart method use excess base to maintain alkaline pH conditions and the resulting MNPs exhibit a high degree of clumping. Post synthesis treatments are required to improve the degree of particle dispersion such as the use of surface modification agents or peptization by lowering the pH or adding ferric nitrate [12,13]. Both the unmodified and OLA modified MNPs exhibited a high degree of colloid stability. The colloid stability of the unmodified MNPs resulted from synthesis conditions that gave rise to a post synthesis peptization reaction. The high level of agglomeration that was exhibited by the GAmodified MNP most likely occurred during the synthesis of the MNP. The addition of GA to preformed MNP resulted in a well dispersed particle suspension [7,8] but the presence of GA during MNP synthesis resulted in agglomerated particles. The extreme agglomeration that was exhibited by the GA-modified MNPs may be attributed to complex bridging interactions. During coprecipitation, a bridge may form when part of the large GA molecule is adsorbed onto the surface of two or more particles. This may explain why the co-precipitated particles form larger agglomerates [8]. The zeta potential measurements indicated
441
that all three of the samples are within the range of electrostatic colloid stability. However, the GA-modified MNPs were so large, that gravitational forces predominated over electrostatic repulsive forces measured by the zeta potential. The GA-modified MNP particles settled out of solution within 30 min, while the OLAcoated and uncoated particles remained in suspension for weeks after synthesis. The cytoplasm of the cells exposed to unmodified and OLA modified MNPs appeared darker due to the internalization of the MNPs. This result is most likely a size effect rather than a surface chemistry effect because smaller (100 nm) GA-modified MNPs were noted to traverse the outer cell membrane of DU 145 prostate cancer cells and localize within the cytoplasm [8]. In this study, the GA-modified MNP with an agglomerate size in the range of 1 um were too large to pass by the cell membrane, but they did appear to adhere to the cell membrane. In addition, the rapid sedimentation of the larger GA-modified MNP agglomerates would cause them to settle out of solution and be less available for interaction with the fibroblast cells. Some fibroblasts that were exposed to untreated MNPs did exhibit both a darkened cytoplasm and nucleus, which indicates that the untreated MNPs may be able to traverse the nuclear membrane. Further studies on the untreated MNPs will be conducted to ascertain whether the MNPs can pass the nuclear membrane. This study shows that size and surface chemistry effects may be useful to target MNPs to specific regions of the cell and within specific tissues. A number of studies have demonstrated that insulin [14] and lactoferrin and ceruloplasmin [15] surface modified MNPs can be localized at the cell membrane. Results of the insulin surface modification study showed that uncoated MNPs were endocytosed by the cells while the insulin-coated MNPs attached to the cell membrane. Insulin was also noted to cause an increase in cell proliferation and viability. 5. Conclusions OLA influenced the colloidal stability, size, and bioactivity of MNPs. Oleylamine improved the colloidal stability of MNPs on a level comparable to MNPs synthesized under acidic conditions. Cytotoxicity results indicated that the OLA-MNPs were more toxic to cells than the untreated MNPs or GA-MNP. Particle uptake by fibroblasts was demonstrated by application of magnetic field and the alignment of the MNP containing cells along the magnetic field. Light microscopy images showed that the GA-coated particles remained on the surface of the fibroblast cells due to their large size. The uncoated and OLA-MNP were able to cross the cell membrane and enter the cytoplasm. References [1] [2] [3] [4]
C.C. Berry, J. Mater. Chem. 15 (2005) 543. S. Mornet, S. Vasseur, F. Grasset, E. Duguet, J. Mater. Chem. 14 (2004) 2161. O.V. Salata, J. Nanobiotechnology 2 (2004) 1. Q.A. Pankhurst, J. Connolly, S.K. Jones, J. Dobson, J. Phys. D. Appl. Phys. 36 (2003) R167. [5] J.H. Clement, M. Schwalbe, N. Buske, K. Wagner, M. Schnabelrauch, P. Gornert, K.O. Kliche, K. Pachmann, W. Weitschies, K. Hoffken, J. Cancer. Res. Clin. Oncol. 132 (2006) 287.
442
O.C. Wilson Jr. et al. / Materials Science and Engineering C 28 (2008) 438–442
[6] K. Shimizu, A. Ito, H. Honda, J. Biomed. Mater. Res. B. Appl. Biomater. 77 (2006) 265. [7] D.N. Williams, K.A. Gold, T.R. Pulliam-Holoman, S.H. Ehrman, O.C. Wilson Jr., J. Nanopart. Res. 8 (2006) 749. [8] D.N. Williams. Ph.D. Thesis, University of Maryland, 2003. [9] C. Roque, O.C. Wilson Jr., submitted to Mat Sci Eng C, 2006. [10] M. Aslam, L. Fu, M. Su, K. Vijayamohanan, V.P. Dravid, J. Mater. Chem. 14 (2004) 1795.
[11] N. Wu, L. Fu, M. Su, M. Aslam, K.C. Wong, V.P. Dravid, Nano Lett. 4 (2004) 383. [12] F.A. Tourinho, R. Franck, R. Massart, J. Mater. Sci. 25 (1990) 3249. [13] F.A. Tourinho, J. Depeyrot, G.J. da Silva, M.C.L. Lara, Brazil. J. Phys. 28 (1998) 413. [14] A.K. Gupta, C. Berry, M. Gupta, A. Curtis, IEEE Trans. Nanobioscience 2 (2003) 255. [15] A.K. Gupta, A.S. Curtis, Biomaterials 25 (2004) 3029.
Materials Science and Engineering C 28 (2008) 438 – 442 www.elsevier.com/locate/msec
Surface modification of magnetic nanoparticles with oleylamine and gum Arabic Otto C. Wilson Jr. a,⁎, Erin Blair a , Stephanie Kennedy a , Gloryvee Rivera a , Patrick Mehl b a
The Catholic University of America, Department of Biomedical Engineering, BONE/CRAB Lab, 620 Michigan Ave NE, Washington, DC 20064, United States b Biomagnetics Group, Vitreous State Lab, 620 Michigan Ave NE, Washington, DC 20064, United States Available online 11 April 2007
Abstract Magnetic nanoparticles (MNP) were synthesized under acidic conditions in the presence of oleylamine (OLA) and gum Arabic (GA) to study the influence of surface modification on MNP characteristics and cellular level bioactivity. Highly dispersed MNPs were formed from acidic solutions of ferric and ferrous chloride in the absence and presence of OLA. The MNP synthesized in the presence of GA formed large particle aggregates that exhibited rapid coagulation. The three types of MNP were characterized via thermogravimetric analysis (TGA), and dynamic and electrophoretic light scattering to determine particle size and zeta potential. Cytotoxicity and cell interactions were assessed for each of the three MNP samples using L929 fibroblast cells. OLA modified MNPs exhibited the highest level of cytotoxity. Approximately 27% of L929 fibroblast cells died after exposure to OLA modified MNP in comparison to 10% cell death for untreated, GA treated, and control cells. Cells that were exposed to MNPs could be translated in magnetic fields. The OLA and untreated MNPs could be detected in the cell cytoplasm while the GAmodified MNP clusters were located at the cell membrane based on light microscopy. © 2007 Elsevier B.V. All rights reserved. Keywords: Adsorption; Oleyl amine; Magnetic nanoparticles; Surface modification; Gum Arabic
1. Introduction Magnetic nanoparticles (MNPs) typically possess a magnetite (Fe3O4) or maghemite (γ-Fe2O3) core chemistry and range in size from 5–100 nm [1]. They are currently utilized in a wide variety of medical diagnostic and therapeutic uses such as hyperthermia treatments for cancer [2–4], cell sorting [5], and tissue engineering [6]. The chemical and physical characteristics of the MNPs play a critical role in determining successful interactions in each of these applications. Factors such as size, surface area, and magnetic properties are very important, but proper MNP surface chemistry is integral to enhance colloid stability and cell interactions. Biocompatible silica coated MNP (50 nm) were found to pass the blood–brain barrier without disturbing biological functions or showing any signs of toxicity [7]. Surface modification can also be used to minimize toxicity and/or undesired biological interactions such as removal by the reticuloendothelial system. Previous work with MNPs showed that gum Arabic (GA) is an effective agent to improve colloid stability [7] and enhanced ⁎ Corresponding author. Tel.: +1 202 319 5822; fax: +1 202 319 4287. E-mail address: [email protected] (O.C. Wilson). 0928-4931/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2007.04.008
cellular uptake by DU 145 prostate cancer cells [8]. However, phosphate preferentially adsorbs on MNPs and easily displaces GA, and decreases colloid stability [9]. One of the goals of this study is to modify the surface of MNPs with amine functional groups. This is for the purpose of developing a method to covalently link GA onto MNPs using amine based functionality. Oleylamine (OLA) consists of a long hydrophobic, monounsaturated fatty acid tail, with an amine group on one end. OLA has been used to stabilize solutions of a host of metal and metal oxide nanoparticles that are typically synthesized in nonaqueous solvents including gold [10] and cobalt [11] nanoparticles. The focus of this study is to investigate and report how surface modification of MNPs with GA and OLA influences particle size, colloid stability, and L929 fibroblast cell interactions. 2. Experimental procedures 2.1. MNP Synthesis Three types of MNPs (uncoated, OLA modified, GAmodified) were synthesized using a 2:1 molar ratio of ferric and ferrous chloride (Fisher Scientific, Fairlawn, NJ). The uncoated
O.C. Wilson Jr. et al. / Materials Science and Engineering C 28 (2008) 438–442
439
Table 1 Summary characterization data for the magnetic nanoparticles synthesized under acidic conditions MNP surface
TGA mass loss%
Size(nm)
Zeta potential (mV)
Uncoated OLA GA
5 15 35
25 90 1000
− 41 − 27 − 30
MNPS were prepared by the dropwise addition of the mixed iron ion solution (20 ml) into an aqueous ammonium hydroxide solution that contained 5 ml of reagent ammonium hydroxide (Fisher Scientific, Fairlawn, NJ) and 75 ml of de-ionized water. The OLA modified particles were synthesized by ultrasonicating 2 ml of OLA and 73 ml of water to form an emulsion prior to adding five ml of reagent ammonium hydroxide. The mixed iron ion solution (20 ml) was added dropwise to the OLA emulsion while stirring. The GA-modified particles were synthesized by adding one g of GA (TIC Gums, Belcamp, MD) to 20 ml of the mixed iron ion solution prior to adding this solution dropwise to aqueous ammonium hydroxide solution (5 ml of ammonium hydroxide and 75 ml of water). In each case, the final concentration of Fe3+ was 0.5 M and the resulting solution had a pH of between 2 and 3, due to an excess of hydrochloric acid as a product of the reaction. The MNP samples were washed via centrifugation (10,000–25,000 rpm for 30–35 min) to remove counter ions and stored in de-ionized water. 2.2. MNP characterization The MNPs were characterized via thermogravimetric analysis (TGA) to determine mass loss from volatile components, dynamic light scattering to determine particle size and electrophoretic light scattering to determine zeta potential. TGA was performed on each of the 4 samples using a Shimadzu TGA-50 Thermogravimetric Analyzer (Kyoto, Japan). The particles were washed with de-ionized water and dried overnight, yielding an average of 25–30 mg of each sample. The dry solids were then ground into powder using a mortar and pestle. Each sample was placed in an alumina pan and heated to 800 °C at a rate of 10 °C/min in air. A Malvern Zetasizer 3000 was used to measure the particle size and zeta potential of the MNPs. Dynamic light scattering was used to measure the particle size of the MNP particles in dilute suspensions. Zeta potential measurements were collected for surface treated and untreated MNP samples at a particle concentration of 0.005 wt.% in 0.01 NaCl. The pH was adjusted to 7 to approximate physiological conditions.
Fig. 1. Optical micrograph of L929 mouse fibroblasts cells after overnight exposure to 5 μg/L untreated MNP (no external magnetic field).
2.3. MNP interactions with L929 mouse fibroblast cells 2.3.1. Live/dead cell assay MNPs (1–50 μg/L) were added to L929 fibroblast cultures containing about 10,000 cells/well. After a culture period of 1–2 days, a live/dead assay (Molecular Probes, Eugene, OR) was performed to determine the influence of MNP surface treatment on cell viability. In the assay, live cells are labeled with green fluorescent calcein dye (495 nm absorption), and dead cells with red fluorescent ethidium homodimer-1 (635 nm absorption). The results were quantified by calculating the number of green (or red) cells and dividing that by the total number of cells. 2.4. MNP cell uptake L929 mouse fibroblast cells were placed in a small Petri dish (50,000 cells/ml) and incubated with the surface treated MNP (5 μg/L) overnight. The cells were examined using a light microscope and subjected to a magnetic field using a small Nd– B–Fe magnet. A Cool-Snap Pro color CCD camera was used in conjunction with Image Pro Plus software for the image capture.
Table 2 Cytotoxicity data (% live cells) for the L929 mouse fibroblast cells after exposure to the surface modified MNP based on live/dead cell assay MNP particle exposure concentration
Uncoated OLA GA
50 μg/L
10 μg/L
5 μg/L
1 μg/L
98 – 96
94 75 97
81 85 90
85 59 70
Fig. 2. Optical micrograph of L929 mouse fibroblasts cells after overnight exposure to 5 μg/L untreated MNP (strong external magnetic field).
440
O.C. Wilson Jr. et al. / Materials Science and Engineering C 28 (2008) 438–442
Fig. 3. Higher magnification optical micrograph of untreated MNP exposed L929 mouse fibroblasts cells in the presence of a strong external magnetic field.
Images were captured before the application of the magnetic field, and then 1–2 min after the field was applied. 3. Results The MNPs that were synthesized displayed unique differences that reflected the influence of the surface modification agents. The OLA modified MNPs (OLA-MNP) were highly dispersed and remained so for extended periods of time. However, the GA-modified MNPs (GA-MNP) were highly agglomerated and rapidly sedimented. One of the more unusual findings of this study was that the untreated MNPs exhibited a higher degree of colloid stability than the OLA-MNP. Characterization data for the MNPs from TGA, DLS, and zeta potential are summarized in Table 1. The uncoated MNPs exhibited a mass loss of 5% on heating to 800 °C. This mass loss can be attributed to the volatilization of water, surface hydroxyl groups, and residual contaminants from the synthesis process. The OLA-coated nanoparticles demonstrated a higher mass loss of approximately 15% due to the thermal degradation of the organic molecule. The GA-modified MNPs exhibited the
Fig. 4. Optical micrograph of L929 cells after exposure to 5 μg/L untreated MNP. Contrast within cells indicates presence of MNP in the cell cytoplasm.
Fig. 5. Optical micrograph of L929 cells after exposure to 5 μg/L OLA treated MNP.
largest mass loss of 35%. The particle size of the MNPs was also influenced by the synthesis conditions. The smallest MNPs were obtained for the uncoated sample. The particle size was consistently below 50 nm and averaged around 25 nm. The OLA-MNPs had a particle size of 90 nm. The GA-MNPs were much larger due to extensive agglomeration and had a particle size of approximately 1000 nm. The zeta potential (measured at pH 7) of all MNPs was negative and −41 mV, − 27 mV, and − 30 mV for MNP, OLA-MNP, and GA-MNP, respectively. The results of the live dead cell assay (Table 2) indicated that the L929 fibroblast cells that were exposed to OLA modified MNPs displayed a higher percentage of dead cells. The data shown in Table 2 is a compilation of four samples that were analyzed at MNP loadings of 50, 10, 5, and 1 μg/L. The amount of live cells varied from 59–85% for OLA-MNP. The unmodified MNPs and GA-modified MNPS had cell survival rates of 81–98% and 71–96%. The control had a 90% cell survival rate. It is interesting to note that according to our experimental results, the lower MNP concentrations seemed to
Fig. 6. Optical micrograph of L929 cells after exposure to 5 μg/L GA treated MNP. GA-modified MNP agglomerates appear located at the outer surface of the cell membrane.
O.C. Wilson Jr. et al. / Materials Science and Engineering C 28 (2008) 438–442
result in higher cell mortality. More experiments will be conducted to study this effect further. The cells were placed in a magnetic field to verify interactions between the MNPs and L929 fibroblast cells. The fibroblast cells were incubated overnight with the surface modified MNPs samples at a concentration of 5 μg/L. L929 fibroblast cells that were exposed to untreated, OLA treated and GA treated MNPs showed movement and migration along the direction of the magnetic field. The most notable interactions with the magnetic field occurred for the L929 cells exposed to the untreated MNPs. Optical micrographs for L929 cells exposed to untreated MNPs both in the absence and presence of the magnetic field are shown in Figs. 1–3. Fig. 1 shows the fibroblast cells exposed to untreated MNPs in the absence of a strong external magnetic field. Figs. 2 and 3 show the same L929 fibroblast cell culture in the presence of a strong external magnetic field. The fibroblast cells aged with untreated MNPs formed chains and could be manipulated with magnetic fields more easily than cells exposed to OLA or GA treated MNPs. Light microscopy was also used to visualize the location of the magnetic nanoparticles within the cells. Higher magnification optical micrograph photos (Figs. 4–6) of the surface treated MNPs showed differences in contrast that may indicate the location of the MNPs with respect to the cell membrane. The uncoated MNPs (Fig. 4) and OLA-coated MNPs (Fig. 5) seem to be concentrated in the cytoplasm while the GA-coated particles (Fig. 6) are concentrated on the surface of the cells. 4. Discussion One of the more interesting results of this study was that the synthesis of MNPs using acidic conditions (i.e. the final pH was approximately 2–3) greatly improved the colloid stability of unmodified nanoparticles. In each synthesis case, the resulting MNP suspension was approximately pH 2–3, due to an excess of hydrochloric acid as a reaction product. Typical MNP synthesis reactions such as the Massart method use excess base to maintain alkaline pH conditions and the resulting MNPs exhibit a high degree of clumping. Post synthesis treatments are required to improve the degree of particle dispersion such as the use of surface modification agents or peptization by lowering the pH or adding ferric nitrate [12,13]. Both the unmodified and OLA modified MNPs exhibited a high degree of colloid stability. The colloid stability of the unmodified MNPs resulted from synthesis conditions that gave rise to a post synthesis peptization reaction. The high level of agglomeration that was exhibited by the GAmodified MNP most likely occurred during the synthesis of the MNP. The addition of GA to preformed MNP resulted in a well dispersed particle suspension [7,8] but the presence of GA during MNP synthesis resulted in agglomerated particles. The extreme agglomeration that was exhibited by the GA-modified MNPs may be attributed to complex bridging interactions. During coprecipitation, a bridge may form when part of the large GA molecule is adsorbed onto the surface of two or more particles. This may explain why the co-precipitated particles form larger agglomerates [8]. The zeta potential measurements indicated
441
that all three of the samples are within the range of electrostatic colloid stability. However, the GA-modified MNPs were so large, that gravitational forces predominated over electrostatic repulsive forces measured by the zeta potential. The GA-modified MNP particles settled out of solution within 30 min, while the OLAcoated and uncoated particles remained in suspension for weeks after synthesis. The cytoplasm of the cells exposed to unmodified and OLA modified MNPs appeared darker due to the internalization of the MNPs. This result is most likely a size effect rather than a surface chemistry effect because smaller (100 nm) GA-modified MNPs were noted to traverse the outer cell membrane of DU 145 prostate cancer cells and localize within the cytoplasm [8]. In this study, the GA-modified MNP with an agglomerate size in the range of 1 um were too large to pass by the cell membrane, but they did appear to adhere to the cell membrane. In addition, the rapid sedimentation of the larger GA-modified MNP agglomerates would cause them to settle out of solution and be less available for interaction with the fibroblast cells. Some fibroblasts that were exposed to untreated MNPs did exhibit both a darkened cytoplasm and nucleus, which indicates that the untreated MNPs may be able to traverse the nuclear membrane. Further studies on the untreated MNPs will be conducted to ascertain whether the MNPs can pass the nuclear membrane. This study shows that size and surface chemistry effects may be useful to target MNPs to specific regions of the cell and within specific tissues. A number of studies have demonstrated that insulin [14] and lactoferrin and ceruloplasmin [15] surface modified MNPs can be localized at the cell membrane. Results of the insulin surface modification study showed that uncoated MNPs were endocytosed by the cells while the insulin-coated MNPs attached to the cell membrane. Insulin was also noted to cause an increase in cell proliferation and viability. 5. Conclusions OLA influenced the colloidal stability, size, and bioactivity of MNPs. Oleylamine improved the colloidal stability of MNPs on a level comparable to MNPs synthesized under acidic conditions. Cytotoxicity results indicated that the OLA-MNPs were more toxic to cells than the untreated MNPs or GA-MNP. Particle uptake by fibroblasts was demonstrated by application of magnetic field and the alignment of the MNP containing cells along the magnetic field. Light microscopy images showed that the GA-coated particles remained on the surface of the fibroblast cells due to their large size. The uncoated and OLA-MNP were able to cross the cell membrane and enter the cytoplasm. References [1] [2] [3] [4]
C.C. Berry, J. Mater. Chem. 15 (2005) 543. S. Mornet, S. Vasseur, F. Grasset, E. Duguet, J. Mater. Chem. 14 (2004) 2161. O.V. Salata, J. Nanobiotechnology 2 (2004) 1. Q.A. Pankhurst, J. Connolly, S.K. Jones, J. Dobson, J. Phys. D. Appl. Phys. 36 (2003) R167. [5] J.H. Clement, M. Schwalbe, N. Buske, K. Wagner, M. Schnabelrauch, P. Gornert, K.O. Kliche, K. Pachmann, W. Weitschies, K. Hoffken, J. Cancer. Res. Clin. Oncol. 132 (2006) 287.
442
O.C. Wilson Jr. et al. / Materials Science and Engineering C 28 (2008) 438–442
[6] K. Shimizu, A. Ito, H. Honda, J. Biomed. Mater. Res. B. Appl. Biomater. 77 (2006) 265. [7] D.N. Williams, K.A. Gold, T.R. Pulliam-Holoman, S.H. Ehrman, O.C. Wilson Jr., J. Nanopart. Res. 8 (2006) 749. [8] D.N. Williams. Ph.D. Thesis, University of Maryland, 2003. [9] C. Roque, O.C. Wilson Jr., submitted to Mat Sci Eng C, 2006. [10] M. Aslam, L. Fu, M. Su, K. Vijayamohanan, V.P. Dravid, J. Mater. Chem. 14 (2004) 1795.
[11] N. Wu, L. Fu, M. Su, M. Aslam, K.C. Wong, V.P. Dravid, Nano Lett. 4 (2004) 383. [12] F.A. Tourinho, R. Franck, R. Massart, J. Mater. Sci. 25 (1990) 3249. [13] F.A. Tourinho, J. Depeyrot, G.J. da Silva, M.C.L. Lara, Brazil. J. Phys. 28 (1998) 413. [14] A.K. Gupta, C. Berry, M. Gupta, A. Curtis, IEEE Trans. Nanobioscience 2 (2003) 255. [15] A.K. Gupta, A.S. Curtis, Biomaterials 25 (2004) 3029.