Electrostatic Self-Assembly of PEG Copolymers onto Porous Silica

These results demonstrate that the electrostatic self-assembly of PEG copolymers onto silica nanoparticles used as drug nanocarriers is a robust and e...
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Langmuir 2008, 24, 8143-8150

8143

Electrostatic Self-Assembly of PEG Copolymers onto Porous Silica Nanoparticles Benjamin Thierry,*,† Lucie Zimmer,† Scott McNiven,‡ Kim Finnie,§ Christophe Barbe´,§ and Hans J. Griesser† Ian Wark Research Institute, UniVersity of South Australia, Mawson Lakes Campus, Mawson Lakes, Adelaide, South Australia 5095, Australia, Australian Nuclear Science and Technology Organisation, PriVate Mail Bag 1, Menai New South Wales 2234 Australia, and CeramiSphere Pty Ltd., New Illawarra Road, Menai, New South Wales 2234, Australia ReceiVed March 7, 2008. ReVised Manuscript ReceiVed May 7, 2008 A critical requirement toward the clinical use of nanocarriers in drug delivery applications is the development of optimal biointerfacial engineering procedures designed to resist biologically nonspecific adsorption events. Minimization of opsonization increases blood residence time and improves the ability to target solid tumors. We report the electrostatic self-assembly of polyethyleneimine-polyethylene glycol (PEI-PEG) copolymers onto porous silica nanoparticles. PEI-PEG copolymers were synthesized and their adsorption by self-assembly onto silica surfaces were investigated to achieve a better understanding of structure-activity relationships. Quartz-crystal microbalance (QCM) study confirmed the rapid and stable adsorption of the copolymers onto silica-coated QCM sensors driven by strong electrostatic interactions. XPS and FT-IR spectroscopy were used to analyze the coated surfaces, which indicated the presence of dense PEG layers on the silica nanoparticles. Dynamic light scattering was used to optimize the coating procedure. Monodisperse dispersions of the PEGylated nanoparticles were obtained in high yields and the thin PEG layers provided excellent colloidal stability. In vitro protein adsorption tests using 5% serum demonstrated the ability of the self-assembled copolymer layers to resist biologically nonspecific fouling and to prevent aggregation of the nanoparticles in physiological environments. These results demonstrate that the electrostatic self-assembly of PEG copolymers onto silica nanoparticles used as drug nanocarriers is a robust and efficient procedure, providing excellent control of their biointerfacial properties.

Introduction Efficient in vivo delivery and accumulation of therapeutic agents is the Holy Grail of pharmaceutical industries. Taking advantage of recent progress in control of matter at the nanoscale, “nanocarriers” are being developed with the promise of improved delivery and targeting of diagnostic and therapeutic agents.1,2 In vivo delivery of chemotherapeutics achieved through a nanoparticle approach aims at minimizing systemic toxicity while increasing therapeutic efficacy, for instance against solid tumors. Nanocarriers such as liposomes, micelles and solid polymeric nanoparticles are under active development and a few of these nanocarriers have already received approval for clinical use.2,3 Their small size, 1-100 nm, allows them to cross the abnormal fenestrated vasculature of a tumor and to accumulate at the tumor site, a process referred to as the enhanced permeation and retention (EPR) effect.4 One critical aspect of nanoparticulate drug delivery is the ability to carry high payloads of active therapeutic agents and to deliver these agents in a timely, efficient manner to the tissues to be treated. Silica particles present an interesting alternative to organic systems such as liposomes for drug delivery.5–11 * To whom correspondence may be addressed. E-mail: benjamin.thierry@ unisa.edu.au. Phone: +61 8 8302 3689. Fax: +61 8 8302 3683. † University of South Australia. ‡ Australian Nuclear Science and Technology Organisation. § CeramiSphere Pty Ltd.

(1) Jain, K. K. Technol. Cancer Res. Treat. 2005, 4, 407–416. (2) Vijayaraghavalu, S.; Raghavan, D.; Labhasetwar, V. Curr. Opin. InVestig. Drugs 2007, 8, 477–84. (3) Wang, M. D.; Shin, D. M.; Simons, J. W.; Nie, S. Expert ReV. Anticancer Ther. 2007, 7, 833–7. (4) Greish, K. J. Drug Target 2007, 15, 457–64. (5) Finnie, K. S.; Jacques, D. A.; McGann, M. J.; Blackford, M. G.; Barbe, C. J. J. Mater. Chem. 2006, 16, 4494–4498.

Silica offers a biocompatible, stable matrix with no toxic degradation products and no significant immune response. Using sol-gel chemistry combined with emulsion technology, Barbe´ et al. have recently developed a novel controlled release system based on porous silica particles, with independent control over size and release rate.6,12 While the particle size is controlled by the emulsion chemistry, which provides an efficient “nanoreactor” for the sol-gel process, the release rate is controlled by the particles’ internal microstructure, the pore volume and size, the tortuosity, and the surface chemistry. Although many of these nanotechnology-based strategies have been successful in vitro, implementation in clinical practice has often proved inefficient. A major challenge toward the in vivo use of nanocarriers for drug delivery remains their rapid clearance from blood circulation and subsequent accumulation in the organs of the reticuloendothelial system (RES).13–15 The clearance process involves interaction with the mononuclear phagocyte system (MPS) and the complement system and is initiated through biologically nonspecific events (6) Barbe, C.; Bartlett, J.; Kong, L. AdV. Mater. 2004, 16, 1959–1961. (7) Trewyn, B. G.; Giri, S.; Slowing, I. I.; Lin, V. S. Chem. Commun. (Camb) 2007, 3236–45. (8) Giri, S.; Trewyn, B. G.; Lin, V. S. Nanomed 2007, 2, 99–111. (9) Kim, S.; Ohulchanskyy, T. Y.; Pudavar, H. E.; Pandey, R. K.; Prasad, P. N. J. Am. Chem. Soc. 2007, 129, 2669–75. (10) Ravi Kumar, M. N.; Sameti, M.; Mohapatra, S. S.; Kong, X.; Lockey, R. F.; Bakowsky, U.; Lindenblatt, G.; Schmidt, H.; Lehr, C. M. J. Nanosci. Nanotechnol. 2004, 4, 876–81. (11) Slowing, I. I.; Trewyn, B. G.; Lin, V. S. J. Am. Chem. Soc. 2007, 129, 8845–9. (12) Finnie, K. S.; Bartlett, J. R.; Barbe, C. J.; Kong, L. Langmuir 2007, 23, 3017–24. (13) Owens, D. E., 3rd; Peppas, N. A Int. J. Pharm. 2006, 307, 93–102. (14) Vonarbourg, A.; Passirani, C.; Saulnier, P.; Benoit, J. P. Biomaterials 2006, 27, 4356–73. (15) Romberg, B.; Hennink, W. E.; Storm, G. Pharm. Res. 2007,

10.1021/la8007206 CCC: $40.75  2008 American Chemical Society Published on Web 07/01/2008

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Figure 1. Schematic structure of the PEGylated mesoporous silica nanoparticles. A dense PEG shell is self-assembled onto the silica core to prevent biologically nonspecific adsorption.

such as binding with complement protein C3b, immunoglobulins G and M, fibronectin and apolipoproteins in the process of opsonisation or direct interaction with cells of the RES. Preliminary in vivo experiments showed that silica particles with a diameter larger than 40 nm were very rapidly filtered out by the RES; in contrast, a significant portion of 20 nm nanoparticles remained in blood circulation with a gradual accumulation in tumor tissues.6 The explanation for this surprising behavior may relate to the high curvature of the small particles, which influences protein adsorptions.16 To increase the drug loading and overall efficacy of silica nanoparticle-based drug delivery, however, larger particles (about 100 nm) are preferable. This has led us to investigate modification of the silica surface to improve the biointerfacial properties of our nanoparticles. Conjugation with polyethylene glycol brushes has been so far the most successful approach toward increasing circulation lifetime of colloidal nanostructures.14 PEGylated “stealth” liposomes have been a success story in the field of colloidal drug delivery. Although there is clear evidence that the PEG barriers on stealth liposomes are suboptimal and do not fully eliminate nonspecific biological adsorption events, they can nevertheless reduce blood clearance mechanisms, enabling blood residence times as long as 24 h.17–19 Less satisfactory results have been reported for PEGylated solid nanoparticulates, with frequent failure to achieve significantly improved in vivo bioavailability. Prevention of nonspecific events remains, however, a key goal in the design of long circulating solid nanoparticles. Procedures to achieve high density PEGylation of biomedical devices have been implemented and a good knowledge of structure-activity relationship is available.20,21 For instance, PEGylation under low solubility conditions has achieved highly fouling-resistant PEG layers on biomedical implants and tools.22 Nanoparticles are, however, far more challenging to surface-engineer and analyze. A major issue is the thermodynamically driven tendency to lower interfacial (16) Roach, P.; Farrar, D.; Perry, C. C. J. Am. Chem. Soc. 2006, 128, 3939–45. (17) Moghimi, S. M.; Szebeni, J. Prog. Lipid Res. 2003, 42, 463–78. (18) Yan, X.; Scherphof, G. L.; Kamps, J. A. J. Liposome Res. 2005, 15, 109–39. (19) Torchilin, V. P. Nat. ReV. Drug DiscoV. 2005, 4, 145–60. (20) Pasche, S.; Voros, J.; Griesser, H. J.; Spencer, N. D.; Textor, M. J. Phys. Chem. B 2005, 109, 17545–17552. (21) Pasche, S.; Textor, M.; Meagher, L.; Spencer, N. D.; Griesser, H. J. Langmuir 2005, 21, 6508–6520. (22) Kingshott, P.; Thissen, H.; Griesser, H. J. Biomaterials 2002, 23, 2043– 2056.

surface areas through irreversible aggregation. Nanoparticle PEGylation strategies therefore need to create an efficient steric/ entropic PEG barrier resistant to nonspecific adsorption events in a process compatible with maintaining the colloidal stability of the suspension. PEGylation of model polystyrene latex nanoparticles through hydrophobically driven self-assembly of Pluronic block copolymers such as poloxamer has been wellcharacterized and translated both in vitro and in vivo to reduced opsonization and increased blood circulation time.23,24 The ethylene glycol unit density per surface area and conformation of the PEG macromolecules has been shown to be the key to optimal resistance to protein adsorption and the subsequent improvement in bioavailability in vivo of the colloidal nanostructures.24,25 Electrostatic self-assembly of PEG-copolymers at solid-liquid interfaces has been demonstrated to be extremely efficient in preventing nonspecific adsorption events.20,21,26 The copolymer backbone is typically composed of a polycationic polymer such as Poly(L-lysine) (PLL) or polyethyleneimine (PEI), which can self-assemble electrostatically onto negatively charged surfaces such as metal oxides. The PEG macromolecule side chains extend into the liquid phase to form a solvated brush that provides efficient resistance to protein adsorption. The self-assembly process and efficiency of these PEG-copolymers have been investigated thoroughly on planar/macroscopic surfaces, but applications to colloidal systems have been few. Microspheres made of poly(lactic-co-glycolic acid) (PLGA) have been successfully coated with Poly(L-lysine)-g-poly(ethylene glycol) (PLL-PEG) and showed a decrease by 2 orders of magnitude in the amount of adsorbed proteins.27 PLGA nanoparticles have also been PEGylated using a PLL-PEG copolymer and further conjugated to folate, which resulted into improved uptake by target cells.28 The importance of customized and optimized procedures for the (23) Kabanov, A. V.; Alakhov, V. Y. Crit. ReV. Ther. Drug Carrier Syst. 2002, 19, 1–72. (24) Stolnik, S.; Daudali, B.; Arien, A.; Whetstone, J.; Heald, C. R.; Garnett, M. C.; Davis, S. S.; Illum, L. Biochim. Biophys. Acta 2001, 1514, 261–79. (25) Goppert, T. M.; Muller, R. H. Eur. J. Pharm. Biopharm. 2005, 60, 361– 72. (26) Malmsten, M.; Emoto, K.; Van Alstine, J. M. J. Colloid Interface Sci. 1998, 202, 507–517. (27) Muller, M.; Voros, J.; Csucs, G.; Walter, E.; Danuser, G.; Merkle, H. P.; Spencer, N. D.; Textor, M. J. Biomed. Mater. Res. A 2003, 66, 55–61. (28) Kim, S. H.; Jeong, J. H.; Chun, K. W.; Park, T. G. Langmuir 2005, 21, 8852–7.

PEGylation of Porous Silica Nanoparticles

surface engineering of nanocarriers to improve in vivo bioavailability is becoming increasingly recognized.29,30 Electrostatic self-assembly of PEG copolymers on silica nanoparticles appears as a robust, environmentally friendly, versatile and easily up-scalable process (Figure 1). Toward improved bioavailability of porous silica nanoparticles used as a drug delivery system, we report in this paper the self-assembly of PEI-PEG copolymers, studied using quartz-crystal microbalance, X-ray photoelectron spectroscopy, FT-IR, transmission electron microscopy and dynamic light scattering. The resistance to nonspecific adsorption of proteins has been investigated in an in vitro model using model proteins and serum solutions.

Experimental Section Materials. Tergitol NP-9, cyclohexane, pentanol, tetramethoxysilane (TMOS), tetraethoxysilane (TEOS), branched polyethyleneimine (MW 25 000), methyl bromoacetate, N-hydroxysuccinimide (NHS), N,N′-dicyclohexylcarbodiimide (DCC), human serum albumin (HSA), lysozyme and KBr were purchased from Sigma and used as received. Methoxy polyethyleneglycol (mPEG) with average molecular weights of 3400 and 5000 were purchased from Carbomer. Water used in the experiments was purified with a Millipore water treatment system (organic content less than 5 ppb). Phosphate buffered saline (PBS) was obtained from Sigma and used at 0.15 M. Nanoparticle Synthesis. The particles were synthesized by combining sol-gel chemistry and emulsion polymerization as described previously.6,12 Typically, a water-in-oil microemulsion was prepared by mixing Tergitol NP-9, 1-pentanol, cyclohexane and an aqueous solution, which contained a catalyst for the sol-gel reaction (NH4OH). TEOS was added to the microemulsion and the resulting mixture was stirred for the appropriate time at room temperature. A polar solvent was then added to destabilize the emulsion, and the solid particles, about 100 nm in diameter (unless otherwise stated), were collected by centrifugation. The particles were then resuspended in water and washed several times using chloroform to remove residual surfactant. Copolymer Synthesis. The PEI-PEG copolymers were synthesized in a two-step procedure. First, methoxy polyethyleneglycols (mPEG) were converted into mPEG-acids based on a previously reported synthesis.31 Briefly, mPEGs (MW 3400 and 5000) were dried through azeotropic distillation with dry toluene and dissolved into dry THF. NaH was slowly added in a 1.5 molar excess and the mixture was stirred under N2 for 1 h. Methyl bromoacetate was added dropwise to the PEG solution and left to react for 20 h under N2. Solvent was removed and the residue was dissolved into 1 N NaOH for 2 h and acidified to pH 2 before being extracted with CHCl3. The organic phase was washed with H2O and dried with MgSO4. The solution was then filtered with Celite and then slowly precipitated into hexane. The yields of the reaction were 95% and 97% for PEG3400 and PEG5000, respectively (as determined by titration with NaOH). Next, the carboxylic acid groups introduced on the mPEGs were activated with DCC and NHS in dry DMSO before being reacted with primary amines of the polyethyleneimine in the presence of triethylamine. A percentage of primary amine groups in the PEI backbone of 25% was assumed for theoretical calculations. The solution was filtered off and then precipitated in diethyl ether to recover the PEI-PEG copolymer as a white solid. The copolymers were dialyzed against water for 5 days before being recovered by freeze-drying. Elemental analyses were used to determine the ratio of PEG to primary amines in the PEI backbone, which was calculated to be about 1:4.5 for both PEI-PEG3400 and PEI-PEG5000. The latter represents about 34 grafted PEG chains per PEI molecule. (29) Lee, H.; Lee, E.; Kim do, K.; Jang, N. K.; Jeong, Y. Y.; Jon, S. J. Am. Chem. Soc. 2006, 128, 7383–9. (30) Xie, J.; Xu, C.; Kohler, N.; Hou, Y.; Sun, S. AdV. Mater. 2007, 19, 3163–3166. (31) Sedlak, M.; Antonietti, M.; Colfen, H. Macromol. Chem. Phys. 1998, 199, 247–254.

Langmuir, Vol. 24, No. 15, 2008 8145 Quartz-Crystal Microbalance (QCM) Study of Copolymer Self-Assembly on Silica. QCM with dissipation measurements were performed on an E4 QCM (Q-Sense AB, Go¨teborg, Sweden). Silicacoated QCM AT-cut quartz crystal sensors with a fundamental frequency of 4.95 MHz were a gift from Scientific Solution (Australia). The sensors were plasma cleaned for 10 min prior to use. The copolymers were dissolved into Milli-Q water at a concentration of 1 mg/mL and the pH was adjusted to 8.5 with NaOH. The solution was then injected into the QCM at a flow rate of 100 µL/min and the 3rd, 5th, 7th, 9th and 11th overtone response in frequency and dissipation were continuously recorded. All measurements were performed at 24 ( 0.02 °C. PEGylation of Silica Nanoparticles. The PEI-PEG copolymers were dissolved into Milli-Q water at 2 mg/mL unless otherwise stated and the pH adjusted to 8.5. Typically, the nanoparticles were centrifuged at 13 400 rpm in a benchtop centrifuge, the supernatant discarded and the nanoparticles resuspended in H2O at 2 mg/mL. The nanoparticles suspension was then slowly added to the copolymer solution under strong agitation. The mixture was kept under sonication for 5 min and then left to stand for 55 min. The mixture was then distributed in 1.5 mL Eppendorf tubes and centrifuged 30 min at 13 400 rpm. The supernatants were removed and the nanoparticles resuspended in H2O. The washing procedure was repeated three times to remove unbound copolymer. Resistance to Nonspecific Protein Adsorption. To test the efficiency of the PEI-PEG self-assembled layers in resisting nonspecific protein adsorption, an in vitro assay was designed based on the procedure reported by Roach et al.16 Naked or PEGylated nanoparticles (2 mg/mL) were redispersed in PBS and sonicated for 5 min. Protein solutions consisted of (1) human serum albumin and lysozyme in PBS (1 mg of albumin and 1 mg of lysozyme per 1 mL of PBS) and (2) 10% fetal calf serum diluted into PBS. Protein solutions were mixed gently with the nanoparticles (1:1 volume) at room temperature. After 1 h, the nanoparticles were washed by centrifugation in PBS (×2), Milli-Q water (×2) and ethanol (×1) and characterized using XPS and FT-IR. Characterization. The nanoparticles were washed several times with water and ethanol prior to characterization by XPS. A concentrated nanoparticle solution in ethanol was drop-cast onto gold-coated silicon wafers and dried at room temperature. XPS analyses were conducted using a Kratos AXIS Ultra DLD X-ray photoelectron spectrometer with a monochromatic Al KR X-ray source and a hemispherical analyzer. The pass energy was 20 eV with a resolution of 0.3 eV for high-resolution spectra. Spectra were collected at a photoelectron takeoff angle of 90°. Binding energies were referenced to the C1s hydrocarbon carbon peak at 285.0 eV to compensate for surface charging effects. Component fitting of the high resolution spectra was performed using CasaXPS version 2.3.12 software. Shirley-type backgrounds were used and constrained to a full width at half-maximum between 0.9 and 1.5 eV. The peak fits used 70% Gaussian/30% Lorentzian peak shapes. FT-IR spectra were recorded on a Nicolet Magna-IR 750 spectrometer. Samples were measured as dispersions in KBr powder in diffuse reflection mode using a DRIFT setup. Nanoparticle size distribution measurements were carried out by dynamic light scattering (photon correlation spectroscopy) using a Zetasizer Nano ZS (Malvern Instruments) equipped with a 633 nm He-Ne laser. Size measurements were performed on dilute nanoparticle suspensions in 10-4 M NaCl and 0.15 M PBS. Fluctuations in the intensity of scattered light (at 90° to the incident) are analyzed through the use of first-order and second-order autocorrelation functions. The volume-weighted average diameter was obtained using the manufacturer’s software, which is based on a non-negative-least-squares regression analysis of the autocorrelation function. The instrument was calibrated using polystyrene latex standards from Duke Scientific. The Zetasizer instrument was used to measure the electrophoretic mobility of the nanoparticles and the Helmholtz-Smoluchowski equation was used to correlate electrophoretic mobilities to zeta potentials. Each sample was measured at 24 °C (1 min equilibration) three times while each measurement consisted of about 30 acquisitions. Silica nanoparticles were examined

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Figure 2. Typical QCM experiments for the PEI-PEG5000 copolymer on a silica-coated QCM sensor. (1) Injection of the copolymer; (2) rinse with PBS buffer; (3) rinse with PBS; (4) injection of the 5% serum solution in PBS; (5) rinse with PBS. Note the discontinuity in the time scale. Table 1. QCM-D Study of the Adsorption of the PEI-PEG Copolymers copolymer adsorption exposure to serum

PEI-PEG3400

PEI-PEG5000

-28 2.6 -1 0.6

-40 5.1 -1.5 0.3

∆F (Hz) ∆D (×106) ∆F (Hz) ∆D (×106)

using a JEOL 2010F scanning transmission electron microscope (TEM) equipped with a field emission gun electron source operated at 200 kV (JEOL, Japan).

Results QCM Model Study. The in situ adsorption of the two PEIPEG copolymers on silica was studied using the QCM-D technique with silica coated QCM sensors. A typical QCM experiment for the PEI-PEG5000/silica system is presented in Figure 2 (5th overtones). A sharp decrease in frequency was observed upon injection of the copolymer in the QCM cell, indicating rapid adsorption of the copolymer, which results from the strong electrostatic interactions between the silica surface and the charged copolymer backbone. The negative frequency shift stabilized within a few minutes (∆F ) -28 Hz for PEIPEG3400 and ∆F ) -40 Hz for PEI-PEG5000). Simultaneously, a significant increase in the dissipation was observed (∆D ) 2.6 PEI-PEG3400 and ∆D ) 5.1 PEI-PEG5000), demonstrating the viscoelastic nature of the adsorbed films. The results of the individual polymer adsorption experiments are listed in Table 1. Only minimal changes in the frequency and dissipation were observed upon rinsing (t ) 3 h) for both copolymers. The adsorbed films were then equilibrated in PBS and the frequency and dissipation were monitored overnight. No significant desorption of the copolymers was observed. Next, the ability of the adsorbed copolymers to resist biologically nonspecific adsorption was investigated. A 5% serum solution in PBS was injected (t ) 18 h) in the QCM cell with a flow rate of 50 µL/min, and the proteins were left to adsorb for 2 h. The sensors were then flushed with PBS and the frequency shifts at equilibrium recorded as measurements of the amount of proteins adsorbed on the sensors. The measured frequency shifts were at the limit of the sensitivity of the technique (estimated about 1 Hz) for both copolymers ((∆F ) -1 Hz for PEI-PEG3400 and ∆F ) -1.5 Hz for PEI-PEG5000); in comparison, the frequency shift observed on a plain silica QCM sensor exposed to 5% serum was ∆F ) - 81 Hz.

Electrostatic Self-Assembly of the Copolymers on Silica Nanoparticles. The electrostatically driven self-assembly of the PEI-PEG copolymers onto silica nanoparticles was characterized using XPS and dynamic light scattering measurements. Characterization of the polymer coated silica nanoparticles using XPS revealed the presence of a peak at 400 eV which can be attributed to the N 1s signal (Figure 3). This peak, which was not present in the spectrum of the original particles, is indicative of polyethyleneimine and thus confirms the adsorption of the copolymer on the surface of the silica nanoparticles, at N ) 1.8% and 3.1% for PEI-PEG3400 and PEI-PEG5000, respectively (Table 2). High resolution examination of the 280-300 eV spectra (see Figure 3, right-hand panel) indicates the disappearance of the shakeup satellite peaks (due to the π-π* transitions in aromatic rings at 293 eV) characteristic of the Tergitol NP9 surfactant used in the silica nanoparticle synthesis (see Experimental Section). The C 1s high resolution spectra also showed a significant increase in the C-O component (at 286.5 eV), which can be attributed to PEG. As expected, the ratio of C-O (286.5 eV) to C-C/C-H (285 eV) carbon in the C 1s signal was noticeably higher for the PEI-PEG5000 coated sample in comparison to PEI-PEG3400 coated nanoparticles (4 vs 1.9). Dynamic light scattering measurements (Figure 4) indicated that the as-synthesized silica nanoparticles had a narrow size distribution with an average diameter of 126 ( 1 nm (volume distribution). Coating the nanoparticles with the PEI-PEG copolymers resulted in an increase in the average hydrodynamic diameter of the particles (134 ( 3 nm for PEI-PEG3400 and 139 ( 2 nm for PEI-PEG5000). Importantly, under optimized experimental conditions, aggregation of the nanoparticles could be completely prevented as shown in Figure 3. TEM analysis of the PEGylated nanoparticles confirmed the absence of aggregation (Figure 4). In contrast, direct mixing of the copolymer into the nanoparticle suspension produced irreversible aggregation and subsequent sedimentation of the nanoparticle aggregates within a few minutes. To get a further insight on the electrostatic adsorption process, a zeta-potential-titration experiment was performed. The nanoparticles were exposed to increasing amounts of the PEI-PEG3400 copolymer and the zeta potential of the PEGylated nanoparticles was measured in 10-4 M NaCl and 0.15 M PBS after washing off the unbound copolymers. Uncoated silica nanoparticles displayed a negative potential of -72 mV in 10-4 M NaCl. Rapid aggregation in PBS prevented reliable measurements of the zeta potential in PBS of the uncoated nanoparticles. At a copolymer/nanoparticle weight ratio of 0.1 and above, the negative charges on the silica nanoparticles were completely neutralized by the copolymer, resulting in a zeta potential around 0 mV in PBS (Figure 5). A net positive zeta potential of about 20 mV was, however, measured at low ionic strength. The latter can be assigned to the presence of the polyethyleneimine on the silica nanoparticle and consequent charge reversal. A zeta potential of +60 mV was measured in 10-4 M NaCl for PEI-coated nanoparticles. At lower copolymer/nanoparticle weight ratios, the adsorption of the copolymer only partially neutralized the silica, which resulted in zeta potentials of -58.6 and -11.3 mV for ratios of 0.001 and 0.01, respectively. Rapid aggregation of the nanoparticles was observed at a ratio of 0.01. Resistance to Biologically Nonspecific Adsorption Events. Changes in the percentage of N measured by X-ray photoelectron spectroscopy were used to quantify the amount of protein adsorbed on the silica nanoparticles. Nanoparticle suspensions with or without copolymer coatings were incubated in either a 1 mg/mL

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Figure 3. XPS survey and C 1s spectra of as-synthesized silica nanoparticles and PEGylated nanoparticles (PEI-PEG3400). The dashed lines in the C 1s spectra indicate the hydrocarbon (285 eV) and C-O/C-N components (286.5 eV). Table 2. Component Fitting of XPS C1s Peaks XPS elemental composition (%)

% contribution in the C 1s simulation

sample

C

N

O

Si

CC/CH

CN/CO

O-C-O/CdO/ N-CdO

O-CdO

silica PEI-PEG3400 PEI-PEG5000

13.5 27.8 28.2

1.8 3.1

61 49.6 53.4

25.5 20.6 15.2

44.7 31.1 19

29.6 58.2 76.2

7.7 9.1 4.3

1.4 1.6 -

human serum albumin/lysozyme solution or 5% fetal calf serum in PBS. After washing off loosely bound proteins using three rounds of centrifugation, the suspensions were redispersed in ethanol and concentrated solutions were slowly evaporated onto gold coated silicon wafers for XPS analyses. The XPS N signals measured on nanoparticles before and after exposure to protein solutions are presented in Figure 6. The nitrogen content of the naked silica nanoparticles increased significantly after incubation in protein solutions, from 0% to 9.2% and 7% for HSA/Lysozyme and 5% serum, respectively. Adsorption of proteins on uncoated silica particles also led to a significant increase in the XPS C 1s component at 288.2 eV (data not shown), which can be attributed to the amide C of proteins. In contrast, the nitrogen percentage remained constant within experimental uncertainty with ∆N ) 0.1% for both HSA/lysozyme and 5% serum solutions after protein adsorption with nanoparticles PEGylated with PEI-PEG3400, indicating absence of protein adsorption within the limit of sensitivity of XPS (estimated at ∆N ) 0.2%). Nanoparticles coated with PEI-PEG5000 copolymer showed a slight increase in N when exposed to the 5% serum solution (∆N ) 0.3%) and

a constant nitrogen signal in the model protein solution (∆N ) 0.1%). No significant changes in the amide peak at 288.2 eV were observed for any of these samples. The XPS results were further confirmed by FT-IR analyses (Figure 6). As-synthesized silica nanoparticles showed characteristic features at 950 and 1090 cm-1 due to Si-OH and Si-O-Si bands. The broad absorption band observed between 4000 and 3000 cm-1 corresponds to the fundamental stretching vibrations of different types of hydroxyl groups. Absorption bands at 2886 and 2960 cm-1 could also be detected and are assigned to the presence of residual surfactant molecules on the nanoparticles (-CH2CH2 stretching). IR analysis of nanoparticles exposed to the protein solutions revealed significantly different spectra, with the appearance of two strong absorption bands characteristics of protein amides at 1650 and 1540 cm-1. The

Figure 4. Volume distribution of as synthesized silica nanoparticles and PEGylated nanoparticles. The calculated hydrodynamic diameters are the average of three independent measurements. Inset: TEM image of PEGylated silica nanoparticles (PEI-PEG3400).

Figure 5. Zeta potential values in PBS (0.15 M) of PEGylated nanoparticles (PEI-PEG3400) as a function of the polymer-to-nanoparticles weight ratio. The inset shows the values measured at low ionic strength (10-4 M NaCl).

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Figure 6. Resistance to nonspecific adsorption onto PEGylated silica nanoparticles. Left panel: XPS N% of as synthesized and PEGylated nanoparticles before and after incubation with protein solutions. Right panel: FTIR spectra of (a) as synthesized nanoparticles, (b) as synthesized nanoparticles after incubation in 5% serum solution, (c) PEGylated nanoparticles, and (d) PEGylated particles after incubation in 5% serum solution. The dashed lines indicate the characteristic amide I and II bands.

overlapping of the absorption bands of silica, Tergitol surfactant and PEG prevented detailed IR analysis of the copolymer selfassembly. The absence of the characteristic amide I and II bands in the IR spectra of the PEGylated nanoparticles after exposure to the protein solutions for both PEI-PEG3400 and PEI-PEG5000, however, confirmed absence of biological fouling (within the sensitivity of the technique).

Discussion Our results show that PEGylation of the porous silica nanoparticles markedly changes their interfacial properties, which provides the basis for in vivo studies that are currently underway and will be reported elsewhere. The in vitro results presented above shed light on three key aspects: the self-assembly process, the colloidal stability, and the resistance to nonspecific adsorption events. Copolymer Self-Assembly on Silica. The electrostatic selfassembly on macroscopic surfaces of PEGylated polyamine copolymers such as PLL-PEG and PEI-PEG has been one of few methodologies capable of imparting total resistance to biologically nonspecific adsorption events under physiological conditions. It is also a facile process. Importantly, the biointerfacial properties of the self-assembled layers can be optimized through chemical design, for instance by controlling the PEG to backbone grafting ratio and/or the molecular weight of the PEG moieties. In this work, two PEI-PEG copolymers were synthesized with two different PEG molecular weights (PEI-PEG3000 and PEIPEG5000) while keeping the grafting ratio constant at 1:4.5 PEG molecules per primary amino group available for coupling on the polyethyleneimine backbone. Cationic copolymers such as PLL-PEG have been shown to self-assemble at the surface of metal oxides such as niobiua, titania, silica and conductive tin oxide, with the PEG macromolecules extending into the aqueous liquid phase. In the self-assembly process, the adsorbed amount of copolymer and its conformation is determined by the strength of the electrostatic copolymer-surface interaction as well as the intra- and intermolecular copolymer repulsion. To characterize the copolymer self-assembly process on silica, the quartz crystal microbalance technique has been selected, as it provides insights on both the mass and the structure of the adsorbed layers. Changes in the frequency and dissipation for several harmonics were recorded in real time and can be directly related to properties of the adsorbed layers on the sensors, such as mass and viscoelasticity.

Based on preliminary experiments with silica nanoparticles, a 1 mg/mL concentration and slightly alkaline (pH 8.5-9) conditions were used for the copolymer adsorption step. As the isoelectric point of silica is about 2 and the pKa of PEI around 9-10, strong electrostatic interactions are expected to occur at this pH. Another consideration was to minimize the adsorption of the copolymer through nonelectrostatic interactions between silica and PEO chains, which can occur at acidic and neutral pH.32,33 The changes in both QCM frequency and dissipation occurring upon injection of the copolymers into the flow cell confirmed the adsorption of the PEI-PEGs onto the silica-coated QCM sensor. The kinetics of the adsorption were rapid, with equilibrium reached after a few minutes. The frequency shift and dissipation increase observed for PEI-PEG5000 were greater than those obtained for PEI-PEG3400, which indicates that, in first approximation, a higher coverage of the silica surface by the copolymer is achieved when using a higher molecular weight PEG. Assuming that the adsorbed mass (∆m) is evenly distributed, rigidly attached and small compared to the mass of the crystal,34 the frequency shift ∆F can be related to the adsorbed mass per surface unit using the Sauerbrey equation:

∆m )

C∆F n

(1)

where n is the overtone number and C the constant that describes the sensitivity of the instrument to changes in mass (C ) 17.7 ng cm-2 Hz-1). Using the Sauerbrey approximation, an adsorbed mass of 495 and 708 ng cm-2 can be calculated for PEI-PEG3400 and PEI-PEG5000, respectively. While the copolymer is strongly bound to the silica surface through electrostatic interactions, the PEG side-chains extend into the aqueous liquid phase and can therefore couple with bulk water. The QCM measured mass is thus the mass of the adsorbed copolymer plus the mass of the coupled water and is often referred to as the wet mass. A QCM-D study investigating the self-assembly on silica of a PLL-PEG copolymer with PEG 5000 and a grafting ratio of 3.5 to the PLL backbone (MW ) 20 000) reported a total adsorbed mass of 1240 ng cm-2, and optical waveguide lightmode spectroscopy (32) Iruthayaraj, J.; Poptoshev, E.; Vareikis, A. V.; Makuska, R.; van der Wal, A.; Claesson, P. M. Macromolecules 2005, 38, 6152–6160. (33) Muller, D.; Malmsten, M.; Tanodekaew, S.; Booth, C. J. Colloid Interface Sci. 2000, 228, 317–325. (34) Hook, F.; Kasemo, B.; Nylander, T.; Fant, C.; Sott, K.; Elwing, H. Anal. Chem. 2001, 73, 5796–804.

PEGylation of Porous Silica Nanoparticles

was used to differentiate the copolymer dry mass from the coupled solvent mass, which was determined to be 80% of the total wet mass.35 A major feature of the QCM-D technique is its ability to provide insights on the viscoelastic nature of the adsorbed layers. When the voltage driving the QCM sensor is turned off, a damping of the oscillations occurs resulting from energy dissipation within the adsorbed layer and coupling with the liquid phase. The decay rate of the amplitude thus reflects the energy dissipation and therefore the viscoelastic properties of the adsorbed layer. As expected, the PEI-PEG5000 adsorbed layer was more dissipative than the PEI-PEG3400 layer. The QCM results were further confirmed by XPS analyses of silica nanoparticles after copolymer coating, showing an increased ratio of carbon-to-silicon signal for the PEI-PEG5000 coated nanoparticles (1.85 for PEI-PEG5000 vs 1.35 for PEI-PEG3400). Analogous results were obtained on glass substrates (data not shown). An interesting feature was revealed by XPS high resolution analyses of the PEGylated nanoparticles. Residual Tergitol surfactant could be detected on the as-synthesized, “naked” nanoparticles by the shakeup satellite component in the C 1s signal at 293 eV. The disappearance of this satellite signal after PEGylation demonstrates that the PEI-PEG copolymers displaced the residual surfactant on the silica surface, indicative of strong interactions between the polyamino backbone of the copolymer and the negatively charged silica nanoparticles. The stability of the self-assembled coating in physiological conditions is critical for the intended applications. The PEGylated silica QCM sensors were incubated for up to 16 h in PBS and the signal was continuously monitored. No desorption of the copolymers was observed. Similarly, the PEGylated nanoparticles remained stable in PBS for days with no aggregation, as demonstrated by dynamic light scattering measurements. Colloidal Stability and Steric Considerations. Along with their biointerfacial properties and ability to resist biologically nonspecific adsorption events, the size distribution of drug-loaded nanocarriers dramatically influences their fate in biological environments.36–38 A recent study suggested that the excellent circulation time observed with PEGylated “stealth” liposomes is at least partially related to the protective effect of the PEG layer against liposome-liposome aggregation.39 In addition, although the exact particle size threshold is still unclear, tumor accumulation through the EPR effect is limited by anatomical considerations such as the defect size of abnormal vasculatures.4 Thus, measurement and control of particle size and aggregation is critical. PEGylation strategies therefore need (1) to avoid irreversible aggregation of the nanoparticles during the coating procedure and (2) to provide colloidal stability of the suspension in complex biological environments. The electrostatic adsorption of polyelectrolytes onto solid nanoparticles has been the subject of theoretical and experimental studies.40–42 A major issue is to avoid aggregation of the nanoparticles during the adsorption of the polyelectrolytes. Aggregation can occur via two main mechanisms: direct (35) Muller, M. T.; Yan, X. P.; Lee, S. W.; Perry, S. S.; Spencer, N. D. Macromolecules 2005, 38, 5706–5713. (36) Gaumet, M.; Vargas, A.; Gurny, R.; Delie, F. Eur. J. Pharm. Biopharm. 2007, (37) Chithrani, B. D.; Chan, W. C. W. Nano Lett. 2007, 7, 1542–1550. (38) Lai, S. K.; O’Hanlon, D. E.; Harrold, S.; Man, S. T.; Wang, Y. Y.; Cone, R.; Hanes, J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 1482–7. (39) Dos Santos, N.; Allen, C.; Doppen, A. M.; Anantha, M.; Cox, K. A. K.; Gallagher, R. C.; Karlsson, G.; Edwards, K.; Kenner, G.; Samuels, L.; Webb, M. S.; Bally, M. B. Biochim. Biophys. Acta-Biomembranes 2007, 1768, 1367– 1377. (40) Winkler, R. G.; Cherstvy, A. G. J. Phys. Chem. B 2007, 111, 8486–8493. (41) Gittins, D. I.; Caruso, F. J. Phys. Chem. B 2001, 105, 6846–6852. (42) Schneider, G.; Decher, G. Nano Lett. 2004, 4, 1833–1839.

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nanoparticle-nanoparticle interactions consequent upon charge neutralization and bridging of the nanoparticles by adsorbing polyelectrolytes. To minimize polyelectrolyte-bridging aggregation, a relatively short PEI backbone (average MW ) 25 kDa) for the PEI-PEG copolymers has been used in this study. In addition, strong agitation was maintained throughout the coating procedure to reduce interparticle aggregation. Charge neutralization resulting from the adsorption of PEI-PEGs has been investigated by zeta potential measurements. For polymer-tonanoparticle weight ratios above 0.1, complete screening of the native charges on the silica nanoparticles in PBS could be observed. For polymer to nanoparticle weight ratios below 0.1, the adsorbed copolymer layers were unable to screen completely the native charges. In addition, for a polymer-to-nanoparticle ratio of 0.01, the reduced charges on the nanoparticles combined with insufficient steric repulsion from a low density PEG layer resulted in rapid aggregation and sedimentation of the nanoparticles. Under optimal self-assembly experimental conditions, no aggregation was observed and the PEGylated nanoparticles were readily recovered in an excellent yield. The PEGylated nanoparticles resuspended into PBS were stable at room temperature for days with no observable sedimentation. Dynamic light scattering measurements demonstrated the colloidal stability of the PEGylated nanoparticles (PBS) up to 50 °C. TEM confirmed the absence of aggregation. The difference in average diameters as measured with dynamic light scattering and TEM can be explained by swelling of the mesoporous silica nanoparticles.43 PEGylation of 50 and 20 nm silica nanoparticles has also been successfully achieved (data not shown), further demonstrating the robustness of the self-assembly process used here. Significant aggregation was however observed by dynamic light scattering for the 20 nm nanoparticles. A small but significant increase in the average hydrodynamic diameter of the nanoparticles, obtained by careful dynamic light scattering measurements confirmed the presence of the PEG shell on the nanoparticles. As shown in Figure 4, the uncoated silica particles had an average diameter of 126 ( 1 nm, which increase upon PEGylation to 134 ( 3 (PEI-PEG3400) and 139 ( 2 nm (PEI-PEG5000). Using the radius of gyration Rg of free PEG in solution, one can estimate the conformation of the molecules: If the hydrodynamic layer thickness is more than twice the radius of gyration, the most likely conformation is that of the PEG chains extending in the liquid phase solution and forming brushes. Rg can be empirically calculated by44

Rg[nm] ) 0.181N0.58

(2)

where N is the number of EG per PEG chain. The hydrodynamic thicknesses of the PEG shells can be estimated from the dynamic light scattering experiments, although more advanced measurements are required to take into consideration the core-shell nature of the PEGylated nanoparticles. To a first approximation, the data suggest that the PEI-PEG5000 copolymer adsorbed onto the 100 nm silica nanoparticles is in the regime of overlapping PEG chains (measured thickness ) 6.5 nm, which is >2Rg ) 5.6 for PEG 5000). For the PEI-PEG3400 copolymer, however, the data indicate that the PEG chains are only weakly overlapping (measured thickness ) 4 nm, which is 95% which may be required for clinical applications; our procedures appear sufficiently efficient to merit in vivo assessment. Preliminary in vivo experiments have indicated promise of the present strategy for equipping drug-loaded porous silica nanoparticles with a protective PEG layer, by showing a significant increase in blood circulation time between non-PEGylated and PEGylated silica particles. Work is underway to document the biodistribution of PEGylated nanoparticles and to deliver drugs to tumor sites.

Conclusions PEI-PEG copolymers were synthesized with two different PEG molecular weights and their self-assembly onto porous silica nanoparticles was characterized using QCM, XPS, FT-IR, TEM and dynamic light scattering. The copolymers readily adsorbed onto silica due to strong electrostatic interactions and remained stable in physiological environments such as diluted serum. The efficiency of the PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption events was investigated in vitro, using protein solutions and diluted serum. Protein fouling was at the limit of sensitivity of both QCM and XPS for nanoparticles coated with the two PEI-PEG copolymers investigated, and no aggregation was observed for coated nanoparticles, demonstrating the efficiency of the self-assembled PEG layers to improve the biointerfacial properties of porous silica nanoparticles. The self-assembly of PEI-PEG copolymer onto silica nanoparticles is a promising strategy toward their in vivo use as drug nanocarriers. Acknowledgements We thank Mark Blackford at ANSTO for help with the TEM analysis and Dr. Linggen Kong from Ceramisphere Pty Ltd. for helpful discussion regarding the synthesis of the nanoparticles. This work was supported by the International Science Linkages program established under the Australian Government’s innovation statement Backing Australia’s Ability and by the South Australia Cancer Council. LA8007206 (47) Wagner, M. S.; McArthur, S. L.; Shen, M. C.; Horbett, T. A.; Castner, D. G. J. Biomater. Sci.-Polym. Ed. 2002, 13, 407–428.