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Poly(ethylene imine) functionalized mesoporous silica nanoparticle for biological applications
Zeolites and Related Materials: Trends, Targets and Challenges Proceedings of 4th International FEZA Conference A. Gédéon, P. Massiani and F. Babonneau (Editors) © 2008 Elsevier B.V. All rights reserved.
353
Poly(ethylene imine) functionalized mesoporous silica nanoparticle for biological applications Boris Ufer, Jessica M. Rosenholm, Alain Duchanoy, Lotta Bergman, Mika Lindén Center for Functional Materials, Department of Physical Chemistry, Åbo Akademi University, Porthansgatan 3-5, 20500 Turku, Finland
Abstract Functionalization of porous and non-porous silica nanoparticles by surface hyperbranching polymerization of aziridine on the outer surface building a poly(ethylene imine), PEI, layer is demonstrated. The surface concentration of amino groups on the silica particles is clearly higher for PEI functionalized particles as compared to particles functionalized by standard amino silanization. Further functionalization of this layer with a fluorescent dye makes the particles traceable in biological environment. The functionalized nanoparticles were investigated using SAXS, nitrogen adsorption, electrokinetic measurements, dynamic light scattering, SEM, and various optical spectroscopy methods, with special focus on the suspension stability. Keywords: mesoporous silica, poly(ethylene imine), fluorescein isothiocyanate
1. Introduction The utilization of silica nanoparticles as carriers for drugs has attracted a lot of recent interest [1]. The reason for this is on the one hand the tunable particle size and pore structure and on the other hand the possibility of functionalizing the surface using well documented methods [2]. This is an essential part of any state-of-the-art formulation, as the surface functions serve as anchoring points for further functionalization such as conjugation of biomolecules for targeting purposes or fluorescent dyes to introduce traceability. Importantly, these surface functions will also largely determine the extent of particle aggregation under given conditions. This is especially important under biological conditions where the salt concentration is normally high, which puts a limit to the possibility of utilizing electrostatic forces as the basis for dispersion stability. Poly(ethylene imine) (PEI) has become a versatile non-viral vector alternative for gene delivery [3] and amino-functionalized silica particles have also been used as DNA carriers for gene delivery [4]. Amino-functionalization has also been shown to be a promising route towards molecular gate-properties [5], which could be utilized for release-on-demand purposes. We could already show, that surface hyperbranching polymerization of aziridine is a highly efficient method for surface functionalization of non-porous silica nanoparticles [6] and mesoporous particles [7,8]. Here we report on ongoing results related to surface functionalization of mesoporous silica nanoparticles. An advantage of mesoporous particles as drug carrier matrixes is the possibility, due to their high surface area, for high drug uptake and to protect enclosed molecules against metabolism effects. Further functionalization of the particles by fluorophores allowing the particles to be traceable under biological conditions is also demonstrated, with special focus on optimizing the
354
B. Ufer et al.
linking chemistry so that high dispersion stability can be achieved under biologically relevant conditions.
2. Experimental Mesoporous silica nanoparticles (MSN) were synthesized according to literature procedures [9, 10] leading to highly monodisperse particles with a tunable particle size. Extraction was done according to [10] using ethanol and hydrochloric acid as solvent in an ultrasonic treatment. Aziridine was synthesized as described in [11]. Hyperbranching polymerization of monomeric aziridine on MSN was carried out in toluene at 70°C under argon atmosphere using catalytic amounts of acetic acid. Further functionalization of the surface layer with fluorescein isothiocyanate (FITC) as a fluorescent dye is done by adding an ethanolic dye solution to the MSN dispersed in buffered solution.
3. Results and Discussion The resulting mesoporous particles are highly monodispersed with adjustable particle sizes between 80 and 200 nm. They exhibited a wormlike mesopore structure as evidenced by small angle X-ray diffraction (not shown) and nitrogen sorption measurements (fig. 1a). Electrokinetic titration of pristine mesoporous silica material in water shows a typical isoelectric point (IEP) at pH 2 (fig 2). 1200 1100
3
Vol. Adsorbed [cm / g]
1000 900 800 700
a) pure silica
600 500 400
b) PEI functionalized
300 200 100 0 0,0
0,2
0,4
0,6
0,8
1,0
Rel. Pressure [p / p0]
Figure 1 Nitrogen physisorption isotherms for a) pure silica () and b) PEI functionalized silica nanoparticle (). 40
pure silica co-condensed post-funtionalized
30
Zeta Potential [mV]
20 10 0 -10 -20 -30 -40 -50 -60 2
4
6
8
10
pH
Figure 2. Electrokinetic titration curves for pure silica (), aminosilane co-condensed silica (), aminosilane post-functionalized material ().
355
Poly(ethylene imine) functionalized mesoporous silica…
Successful polymerization of poly(ethylene imine) is shown via thermogravimetric measurements which show a PEI content of 19 wt% up to 30 wt% and a change in the C-value obtained from nitrogen sorption data from 83 for pristine to 37 for functionalized material. Nitrogen sorption data also reveals polymerization on the outer surface of the particles leading to fully accessible pores without blocking and only a slight decrease in pore diameter (see fig. 1 and table 1). Table 1. Overview of the nitrogen physisorption data.
sample
AS (BET) [m2/g]
VP [cm3/g]
dP [Å]
Pure mesoporous silica Co-condensed Post functionalized PEI functionalized
987 742 838 715
1.52 0.49 1.06 1.08
42.5 25.8 37.8 35.4
Cvalue 76 166 41 32
IEP 2 6.7 8.1 9.2
The IEP for PEI functionalized material is shifted to higher pH values (fig. 3) compared to pristine silica particles also showing a successful functionalization of the outer particle surface. Compared to classic material functionalized due to co-condensation with aminosilane (fig. 2) or post-funtionalization with aminosilane (fig. 2), the material functionalized with PEI can reach higher IEP values and therefore electrostatic stabilization can be achieved also under biologically relevant pH conditions. This is not the case for co-condensed or post-funtionalized material as the IEP is too closed to the biologic relevant pH of 7.4 ± 0.5. This higher IEP value obtained for the PEIfunctionalized particles originates from the presence of amino groups on the surface, which are virtually fully protonated at pH values below 9 as the intrinsic pKa-value of PEI is 10.6 [12]. 60
PEI PEI + FITC
50
Zeta Potential [mV]
40 30 20 10 0 -10 -20 -30 6
8
10
12
pH
Figure 3. Electrokinetic titration curves for PEI functionallized material (*) and further FITC fluorophore labeled material ().
A further functionalization of the surface layer with FITC as a fluorescent dye, allows the observation of the silica particles in biological systems. As the fluorophore may have a great impact on suspension stability [6], it is important to choose a fluorophore and a linking chemistry which not influence the electrostatic stabilization. Here we
356
B. Ufer et al.
demonstrate that the outer layer of PEI can be performed without alteration of the isoelectric point (IEP) (see fig. 3).
4. Conclusion and Outlook Suspension stability is important in many bioapplications, including drug delivery and targeting of cells. As shown above, the suspension stability in a given electrostatically stabilized nanoparticle system is affected by the surface functionalization. PEIfunctionalized mesoporous silica nanoparticles are shown to be very promising candidates for biological applications due to their high positive effective surface charge. Furthermore the high surface area together with the high pore volume gives the chance to use this material as carrier for drug and to protect molecules from metabolism on their way into the cell [13].
5. Acknowledgments Financal support from the EU project NanoEar (Contract NMP4-CT-2006-026556; B.U., J.M.R., and A.D.) and the Academy of Finland project Biotarget (Contract 118196; L.B.) are gratefully acknowledged.
References [1]
C. Barbé, J. Bartlett, L. Kong, K. Finnie, H.Q. Lin, M. Larkin, S. Calleja, A. Bush, G. Calleja Adv. Mater., 16 (2004) 1959. [2] I.I. Slowing, B.G. Trewyn, S. Giri, V.S.-Y. Lin, Adv. Funct. Mater., 17 (2007) 1225. [3] W.T. Godbey, K.K. Wu, A.G. Mikos, J. Controlled Release, 60 (1999) 149. [4] M.N.V. Ravi Kumar, M. Sameti, S.S. Mohapatra, X. Kong, R.F. Lockey, U. Bakowsky, G. Lindenblatt, H. Schmidt, C.-M. Lehr, J. Nanosci. Nanotechnol., 4 (2004) 876. [5] R. Casasús, M.D. Marcos, R. Martínez-Máñez, J.V. Ros-Lis, J. Soto, L.A. Villaescusa, P. Amorós, D. Beltrán, C. Guillem, J. Latorre, J. Am. Chem. Soc., 124 (2004) 8612. [6] L. Bergman, J.M. Rosenholm, A.-B. Öst, A. Duchanoy, P. Kankaanpää, J. Heino, M. Lindén, J. Nanomater., 2008, submitted. [7] J.M. Rosenholm, A. Duchanoy, M. Lindén, Chem. Mater., 20 (2008) 1126. [8] J.M. Rosenholm, A. Penninkangas, M. Lindén, Chem. Comm., 2006, 3909. [9] J. Gu, W. Fan, A. Shimojima, T. Okubo, Small, 3 (2007) 1740. [10] K. Möller, J. Kobler, T. Bein, Adv. Funct. Mater., 17 (2007) 605. [11] W.A. Reeves, G.L. Drake Jr., C.L. Hoffpauir, J. Am. Chem. Soc., 73 (1951) 3522. [12] P.C. Griffiths, A. Paul, P. Stilbs, E. Petterson, Macromolecules, 38 (2005) 3539. [13] A. Vonarbourg, C. Passirani, P. Saulnier, J.-P. Benoit, Biomaterials, 27 (2006) 4356.
353
Poly(ethylene imine) functionalized mesoporous silica nanoparticle for biological applications Boris Ufer, Jessica M. Rosenholm, Alain Duchanoy, Lotta Bergman, Mika Lindén Center for Functional Materials, Department of Physical Chemistry, Åbo Akademi University, Porthansgatan 3-5, 20500 Turku, Finland
Abstract Functionalization of porous and non-porous silica nanoparticles by surface hyperbranching polymerization of aziridine on the outer surface building a poly(ethylene imine), PEI, layer is demonstrated. The surface concentration of amino groups on the silica particles is clearly higher for PEI functionalized particles as compared to particles functionalized by standard amino silanization. Further functionalization of this layer with a fluorescent dye makes the particles traceable in biological environment. The functionalized nanoparticles were investigated using SAXS, nitrogen adsorption, electrokinetic measurements, dynamic light scattering, SEM, and various optical spectroscopy methods, with special focus on the suspension stability. Keywords: mesoporous silica, poly(ethylene imine), fluorescein isothiocyanate
1. Introduction The utilization of silica nanoparticles as carriers for drugs has attracted a lot of recent interest [1]. The reason for this is on the one hand the tunable particle size and pore structure and on the other hand the possibility of functionalizing the surface using well documented methods [2]. This is an essential part of any state-of-the-art formulation, as the surface functions serve as anchoring points for further functionalization such as conjugation of biomolecules for targeting purposes or fluorescent dyes to introduce traceability. Importantly, these surface functions will also largely determine the extent of particle aggregation under given conditions. This is especially important under biological conditions where the salt concentration is normally high, which puts a limit to the possibility of utilizing electrostatic forces as the basis for dispersion stability. Poly(ethylene imine) (PEI) has become a versatile non-viral vector alternative for gene delivery [3] and amino-functionalized silica particles have also been used as DNA carriers for gene delivery [4]. Amino-functionalization has also been shown to be a promising route towards molecular gate-properties [5], which could be utilized for release-on-demand purposes. We could already show, that surface hyperbranching polymerization of aziridine is a highly efficient method for surface functionalization of non-porous silica nanoparticles [6] and mesoporous particles [7,8]. Here we report on ongoing results related to surface functionalization of mesoporous silica nanoparticles. An advantage of mesoporous particles as drug carrier matrixes is the possibility, due to their high surface area, for high drug uptake and to protect enclosed molecules against metabolism effects. Further functionalization of the particles by fluorophores allowing the particles to be traceable under biological conditions is also demonstrated, with special focus on optimizing the
354
B. Ufer et al.
linking chemistry so that high dispersion stability can be achieved under biologically relevant conditions.
2. Experimental Mesoporous silica nanoparticles (MSN) were synthesized according to literature procedures [9, 10] leading to highly monodisperse particles with a tunable particle size. Extraction was done according to [10] using ethanol and hydrochloric acid as solvent in an ultrasonic treatment. Aziridine was synthesized as described in [11]. Hyperbranching polymerization of monomeric aziridine on MSN was carried out in toluene at 70°C under argon atmosphere using catalytic amounts of acetic acid. Further functionalization of the surface layer with fluorescein isothiocyanate (FITC) as a fluorescent dye is done by adding an ethanolic dye solution to the MSN dispersed in buffered solution.
3. Results and Discussion The resulting mesoporous particles are highly monodispersed with adjustable particle sizes between 80 and 200 nm. They exhibited a wormlike mesopore structure as evidenced by small angle X-ray diffraction (not shown) and nitrogen sorption measurements (fig. 1a). Electrokinetic titration of pristine mesoporous silica material in water shows a typical isoelectric point (IEP) at pH 2 (fig 2). 1200 1100
3
Vol. Adsorbed [cm / g]
1000 900 800 700
a) pure silica
600 500 400
b) PEI functionalized
300 200 100 0 0,0
0,2
0,4
0,6
0,8
1,0
Rel. Pressure [p / p0]
Figure 1 Nitrogen physisorption isotherms for a) pure silica () and b) PEI functionalized silica nanoparticle (). 40
pure silica co-condensed post-funtionalized
30
Zeta Potential [mV]
20 10 0 -10 -20 -30 -40 -50 -60 2
4
6
8
10
pH
Figure 2. Electrokinetic titration curves for pure silica (), aminosilane co-condensed silica (), aminosilane post-functionalized material ().
355
Poly(ethylene imine) functionalized mesoporous silica…
Successful polymerization of poly(ethylene imine) is shown via thermogravimetric measurements which show a PEI content of 19 wt% up to 30 wt% and a change in the C-value obtained from nitrogen sorption data from 83 for pristine to 37 for functionalized material. Nitrogen sorption data also reveals polymerization on the outer surface of the particles leading to fully accessible pores without blocking and only a slight decrease in pore diameter (see fig. 1 and table 1). Table 1. Overview of the nitrogen physisorption data.
sample
AS (BET) [m2/g]
VP [cm3/g]
dP [Å]
Pure mesoporous silica Co-condensed Post functionalized PEI functionalized
987 742 838 715
1.52 0.49 1.06 1.08
42.5 25.8 37.8 35.4
Cvalue 76 166 41 32
IEP 2 6.7 8.1 9.2
The IEP for PEI functionalized material is shifted to higher pH values (fig. 3) compared to pristine silica particles also showing a successful functionalization of the outer particle surface. Compared to classic material functionalized due to co-condensation with aminosilane (fig. 2) or post-funtionalization with aminosilane (fig. 2), the material functionalized with PEI can reach higher IEP values and therefore electrostatic stabilization can be achieved also under biologically relevant pH conditions. This is not the case for co-condensed or post-funtionalized material as the IEP is too closed to the biologic relevant pH of 7.4 ± 0.5. This higher IEP value obtained for the PEIfunctionalized particles originates from the presence of amino groups on the surface, which are virtually fully protonated at pH values below 9 as the intrinsic pKa-value of PEI is 10.6 [12]. 60
PEI PEI + FITC
50
Zeta Potential [mV]
40 30 20 10 0 -10 -20 -30 6
8
10
12
pH
Figure 3. Electrokinetic titration curves for PEI functionallized material (*) and further FITC fluorophore labeled material ().
A further functionalization of the surface layer with FITC as a fluorescent dye, allows the observation of the silica particles in biological systems. As the fluorophore may have a great impact on suspension stability [6], it is important to choose a fluorophore and a linking chemistry which not influence the electrostatic stabilization. Here we
356
B. Ufer et al.
demonstrate that the outer layer of PEI can be performed without alteration of the isoelectric point (IEP) (see fig. 3).
4. Conclusion and Outlook Suspension stability is important in many bioapplications, including drug delivery and targeting of cells. As shown above, the suspension stability in a given electrostatically stabilized nanoparticle system is affected by the surface functionalization. PEIfunctionalized mesoporous silica nanoparticles are shown to be very promising candidates for biological applications due to their high positive effective surface charge. Furthermore the high surface area together with the high pore volume gives the chance to use this material as carrier for drug and to protect molecules from metabolism on their way into the cell [13].
5. Acknowledgments Financal support from the EU project NanoEar (Contract NMP4-CT-2006-026556; B.U., J.M.R., and A.D.) and the Academy of Finland project Biotarget (Contract 118196; L.B.) are gratefully acknowledged.
References [1]
C. Barbé, J. Bartlett, L. Kong, K. Finnie, H.Q. Lin, M. Larkin, S. Calleja, A. Bush, G. Calleja Adv. Mater., 16 (2004) 1959. [2] I.I. Slowing, B.G. Trewyn, S. Giri, V.S.-Y. Lin, Adv. Funct. Mater., 17 (2007) 1225. [3] W.T. Godbey, K.K. Wu, A.G. Mikos, J. Controlled Release, 60 (1999) 149. [4] M.N.V. Ravi Kumar, M. Sameti, S.S. Mohapatra, X. Kong, R.F. Lockey, U. Bakowsky, G. Lindenblatt, H. Schmidt, C.-M. Lehr, J. Nanosci. Nanotechnol., 4 (2004) 876. [5] R. Casasús, M.D. Marcos, R. Martínez-Máñez, J.V. Ros-Lis, J. Soto, L.A. Villaescusa, P. Amorós, D. Beltrán, C. Guillem, J. Latorre, J. Am. Chem. Soc., 124 (2004) 8612. [6] L. Bergman, J.M. Rosenholm, A.-B. Öst, A. Duchanoy, P. Kankaanpää, J. Heino, M. Lindén, J. Nanomater., 2008, submitted. [7] J.M. Rosenholm, A. Duchanoy, M. Lindén, Chem. Mater., 20 (2008) 1126. [8] J.M. Rosenholm, A. Penninkangas, M. Lindén, Chem. Comm., 2006, 3909. [9] J. Gu, W. Fan, A. Shimojima, T. Okubo, Small, 3 (2007) 1740. [10] K. Möller, J. Kobler, T. Bein, Adv. Funct. Mater., 17 (2007) 605. [11] W.A. Reeves, G.L. Drake Jr., C.L. Hoffpauir, J. Am. Chem. Soc., 73 (1951) 3522. [12] P.C. Griffiths, A. Paul, P. Stilbs, E. Petterson, Macromolecules, 38 (2005) 3539. [13] A. Vonarbourg, C. Passirani, P. Saulnier, J.-P. Benoit, Biomaterials, 27 (2006) 4356.