Selective Recognition of Rituximab-Functionalized Gold Nanoparticles

Oct 21, 2009 - Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia, V6T 1Z1 Canada, Department of ...
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Selective Recognition of Rituximab-Functionalized Gold Nanoparticles by Lymphoma Cells Studied with 3D Imaging Angelina Weiss,† Thomas C. Preston,† Jesse Popov,‡ Qifeng Li,† Sherry Wu,† Keng C. Chou,*,† Helen M. Burt,§ Marcel B. Bally,‡ and Ruth Signorell*,† Department of Chemistry, UniVersity of British Columbia, 2036 Main Mall, VancouVer, British Columbia, V6T 1Z1 Canada, Department of AdVanced Therapeutics, BC Cancer Agency, 675 West 10th AVenue, VancouVer, British Columbia, V5Z 1L3 Canada, and Faculty of Pharmaceutical Sciences, UniVersity of British Columbia, 2146 East Mall, VancouVer, British Columbia, V6T 1Z3 Canada ReceiVed: August 1, 2009; ReVised Manuscript ReceiVed: September 29, 2009

Several types of multivalent therapeutic antibody constructs have recently been described which exhibit an increased efficacy compared to free, bivalent antibody. For example, it has been shown that rituximab-coupled liposomes (devoid of encapsulated drug) show a stronger response than equal amounts of monomeric rituximab, supposedly due to the ability of the multivalent complex to hyper-cross-link its target in the plasma membrane. We sought to create a new type of multivalent antibody construct using gold nanoparticles, where rituximab is bound to the particle surface through a strong covalent bond. In the present study, rituximab-conjugated gold particles have been prepared with the aim of identifying suitable formulations for use in studies assessing the therapeutic potential of these novel formulations. Different types of rituximab-conjugated particles are prepared and characterized. The size of the particles, as well as the type of functionalization, is varied. In vitro studies with CD-20 positive human mantle cell lymphoma cells and CD-20 negative breast cancer cells combined with three-dimensional (3D) imaging allowed us to select an optimized rituximab-gold system for further studies. Selective recognition of these rituximab nanocarriers by lymphoma cells is demonstrated. 1. Introduction Rituximab (an anti-CD20 chimeric monoclonal antibody) is used for the treatment of B-cell non-Hodgkin’s lymphomas and B-cell leukemias (see refs 1–5 and references therein), as well as autoimmune disorders.1 Several mechanisms have been suggested to account for the antitumor activity of rituximab, including signaling-induced apoptosis, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity.6 Although the inclusion of rituximab in treatment regimens for lymphoma patients often leads to significant disease regression, a large percentage of the patients do not respond.7 In fact, in some distinct subclasses of lymphoma, such as mantle cell lymphoma, rituximab exhibits little therapeutic activity even though the diseased cells are known to overexpress CD-20. New formulations of rituximab have recently been described which increase the degree of apoptosis in target cell populations, supposedly as a result of hyper-cross-linking CD20.4,5 Such formulations consist of multimeric antibody constructs with increased valence compared to monomeric, bivalent rituximab. So far, rituximab-coated Dynabeads, dextran-rituximab polymers, and liposomes with grafted rituximab antibodies have been investigated (see ref 5 and references therein). In all cases, significant increases in the therapeutic activity of the antibody was observed for the multimeric carriers when compared with free rituximab. This is an interesting phenomenon that can be * To whom correspondence should be addressed. E-mail: signorell@ chem.ubc.ca (R.S.); [email protected] (K.C.C.). Fax: +1 604 822 2847 (R.S.); +1 604 822 2847 (K.C.C.). Phone: +1 604 822 9064 (R.S.); +1 604 822 5850 (K.C.C.). † Department of Chemistry, University of British Columbia. ‡ BC Cancer Agency. § Faculty of Pharmaceutical Sciences, University of British Columbia.

readily applied to gold nanoparticles, with the potential for valuable clinical applications. In this work, rituximab-conjugated gold nanoparticles are described as a novel multivalent antibody construct. Gold nanoparticles have been widely studied in the past few years for tumor imaging, detection, and cancer therapy including, for example, thermal therapy (see refs 8–17 and references therein). In some cases, gold nanoparticles have been functionalized with tumor-specific ligands or antibodies, resulting in active targeting of the nanoparticles to the tumor site.8–11 If therapeutic antibodies such as rituximab are employed in such a strategy, the antibody takes on a therapeutic role in addition to its targeting one. It is therefore important to assess the degree to which conjugation of the antibody to the assembly affects the activity of the antibody. In the case of rituximab, this is particularly important given that other multivalent rituximab complexes such as rituximab-coupled liposomes have displayed much higher efficacies than equal amounts of free rituximab.5 In the present study, we determine which type of antibody-gold particle system is the most suitable for serving as a multivalent antibody construct. For this purpose, we characterize and compare different types of nanosystems. The size of the particles (30 nm gold nanoparticles and 303 nm gold nanoshells) and the type of functionalization are varied. The latter includes nanocarriers in which rituximab is directly attached to the gold surface and two types of bioconjugates in which rituximab is linked by a polymer to the gold particle surface. The characterization includes in vitro tests with CD20-positive human lymphoid carcinoma cells, as well as breast adenocarcinoma cells, which do not express CD20. 3D imaging shows that rituximab-gold nanoparticles selectively bind at the cell membrane of CD-20 positive cells.

10.1021/jp907423z CCC: $40.75  2009 American Chemical Society Published on Web 10/21/2009

Recognition of Rituximab-Functionalized Gold Nanoparticles 2. Experimental Section Preparation of 30 nm Gold Nanoparticles (NPs). Gold NPs were prepared using standard methods.18,19 Briefly, an aqueous solution of tetrachloroauric acid (1%, 3.56 mL) was diluted to 100 mL and brought to a boil. Under rapid stirring, a sodium citrate solution (15.5 mM, 10 mL) was then quickly added. Over the course of ∼5 min, the appearance of the solution changed from colorless to burgundy. The heat source was then removed and stirring was continued for 1 h. The average diameter of the resulting gold NPs was subsequently determined by TEM to be 30 ( 5 nm (based on measurements of at least 100 particles). Ultraviolet-visible (UV-vis) absorption spectroscopy showed an absorption peak at 526 nm. The concentration of the gold NPs was 5.8 × 1011 particles/mL. The colloidal solution was stored in the dark at 4 °C. Preparation of 303 nm Gold Nanoshells (NSs). NSs were prepared using the method developed by Halas et al.20 Briefly, ammonium hydroxide (30% solution, 5.0 mL), ethanol (50 mL), tetraethyl orthosilicate (6.7 mmol, 1.5 mL), and (3-aminopropyl)trimethoxysilane (APTMS) (0.14 mmol, 25 µL) produced APTMS-functionalized silica particles with TEM measured diameters of 229 ( 11 nm (based on measurements of at least 100 particles). These were elaborated with gold seed particles (0.5 mL).21 The gold shell was grown using a stock tetrachloroauric acid/potassium carbonate solution (15 mL) (potassium carbonate (100 mg, 0.72 mmol), deionized water (94 mL), tetrachloroauric acid (1%, 6 mL)), deionized water (9 mL), the colloidal gold particle elaborated APTMS-functionalized silica particles (1.2 mL), and formaldehyde (60 µL). The solution was centrifuged (20 min, 2500 rpm), and the precipitate was redispersed in deionized water (30 mL). The average diameter of the resulting particles as determined by TEM was 303 ( 15 nm (based on measurements of at least 100 particles). This suggests an average gold coating thickness of about 37 nm. UV-vis absorption spectroscopy showed a broad peak, or more likely series of superimposed broad peaks, extending from 650 nm to beyond the scanning range of the spectrometer (>1100 nm). The concentration of the gold NSs was 2.6 × 109 particles/ mL. The colloidal solution was stored in the dark at 4 °C. Our interest in studying larger particles (303 nm) in spite of their tendency to aggregate arises from two effects: The larger particles can accommodate more antibodies on their surface and their smaller curvature could influence the clustering of these antibodies. Both effects could potentially influence the efficacy of rituximab. Preparation of Rituximab-Conjugated NPs (AB-NP) and NSs (AB-NS). Rituximab (AB, 200 µL, 10 mg/mL), obtained from the BC Cancer Agency, was mixed with either NP (50 µL) or NS (50 µL), and this mixture was allowed to react overnight at room temperature yielding the products AB-NP and AB-NS, respectively. The antibody was attached to gold particles through a gold-sulfur bond. These products were centrifuged twice (4000 rpm, 20 min) and redispersed in HBS buffer (100 µL, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 100 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid; pH 7.4)). Preparation of NPs ((AB-PEG)-NP) and NSs ((AB-PEG)NS) Conjugated with a Rituximab-PEG Complex. A rituximab-PEG-OPSS complex was formed via the nucleophilic reaction of one of the amino acid side chains of the antibody with the NHS group of orthopyridyl disulfide-PEGn-hydroxysuccinimide (MW 3400, OPSS-PEG-NHS, Laysan Bio, Inc.) under slightly basic conditions.22 OPSS-PEG-NHS (800 µL, 0.041 mg/mL in NaHCO3 buffer (100 mM sodium

J. Phys. Chem. C, Vol. 113, No. 47, 2009 20253 bicarbonate, pH 8.5)) was mixed with rituximab (200 µL, 10 mg/mL) overnight at room temperature (1:1 molar ratio). The successful binding of rituximab to the OPSS-PEG-NHS could be verified by concentrating the sample with an Amicon filter device and examining the UV absorption spectrum of the filtrate. The absence of a peak at 280 nm (characteristic of the aromatic ring system of OPSS) indicated that a rituximab-PEG-OPSS complex had formed, as free OPSS-PEG-NHS would pass through the filter and be detected. Either NP (100 µL) or NS (100 µL) were mixed with this rituximab-PEG-OPSS complex overnight at room temperature yielding the products (AB-PEG)NP and (AB-PEG)-NS, respectively. These products were centrifuged twice (4000 rpm, 20 min) and redispersed in HBS buffer (100 µL). Preparation of NPs (AB-(PEG-NP)) and NSs (AB-(PEGNS)) Conjugated First with PEG then Rituximab. OPSSPEG-NHS (900 µL, 0.57 mg/mL in HBS buffer) was allowed to react with NP (100 µL) or NS (100 µL) overnight at room temperature. Both products were then centrifuged (10,000 rpm, 5 min), redispersed in NaHCO3 buffer (800 µL), and mixed with rituximab (200 µL, 10 mg/mL), at r.t. overnight yielding AB-(PEG-NP) and AB-(PEG-NS), respectively. These products were centrifuged twice (4,000 rpm, 20 min) and redispersed in HBS buffer (100 µL). Preparation of NPs (PEG-NP) and NSs (PEG-NS) Conjugated with PEG-SH. Poly(ethylene glycol) thiol (MW 2000, PEG-SH, Laysan Bio, Inc.) (900 µL, 0.50 mg/mL in HBS buffer) was allowed to react with NP (100 µL) or NS (100 µL) overnight at r.t. Both products were then centrifuged (10 000 rpm, 5 min), redispersed in HBS buffer (100 µL) yielding PEGNP and PEG-NS, respectively. Transmission Electron Microscopy (TEM). TEM was used to determine the average particle diameter. Due to the high visual contrast between the organic surface coatings and the metallic centers of the particles, both can be simultaneously characterized using TEM.23 Measurements were conducted on a Hitachi H7600 Transmission Electron Microscope with an operating voltage of 80-100 kV. Samples were prepared by placing 10 µL of the sample onto the surface of a Formvar/carbon 200 mesh, copper grid (Ted Pella, Inc.) and allowing the solvent to evaporate. Dynamic Light Scattering (DLS). DLS is a well-known technique for measuring the hydrodynamic diameter of particles dispersed in solution. It is especially useful if the particles are known to be spherical. All colloids prepared here were analyzed using this technique. Measurements were carried out with a Malvern Zetasizer 3000 HS instrument using disposable acrylic cuvettes (Sarstedt 67.755). Circular Dichroism (CD) Spectroscopy. To establish that the secondary structure of rituximab remained unmodified after the nucleophilic reaction with OPSS-PEG-NHS (vide supra), CD measurement were performed. In principle, CD spectroscopy can also be used to obtain information about the secondary structure of the antibody in any of the rituximab conjugated gold particles that were prepared.23,24 While the results were difficult to interpret in terms of secondary structure as the nearby gold interferes with the signal of the antibody, the spectra could provide evidence of conjugation of the particles in NP and NS with rituximab. CD spectra were recorded with a Jasco J810 CD spectropolarimeter, using a 0.2 mm rectangular quartz cuvette at room temperature. Compressed nitrogen (PP 4.9) was used as inert gas. Each spectrum was collected as an average of five scans in the range of 200-280 nm with a resolution of 0.5 nm.

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Figure 1. Representative TEM image of AB-NP. The dark regions are a consequence of the electron-dense gold while the light coronas are believed to represent the rituximab coating.

Fourier Transform Infrared (FT-IR) Spectroscopy. IR spectroscopy was also used as an indicator for the successful functionalization of gold nanoparticles with rituximab. IR spectra were recorded using a Bruker IFS 66 v/S system. During the experiment the measurement chamber was evacuated to a total pressure of 5 mbar to reduce the amount of interfering water. 100 scans were coadded at a resolution of 2 cm-1. The samples were dried on KBr windows in a vacuum chamber under gentle heating. UV-vis Spectroscopy. The surface plasmon resonances associated with gold nanostructures contain information about the particles structure (size and shape), the dielectric environment that the particle is embedded in (which can be strongly modified when particles are coated), and the dispersion of the nanostructures in solution (e.g., are particles in the colloid clustered together?).23,25 UV-vis spectroscopy was performed on a Varian Cary 50 spectrophotometer with 0.95 nm resolution at room temperature using different solvents. In Vitro Studies with Functionalized Gold Particles. A CD-20 positive human mantle cell lymphoma cell line, Z138,26 was cultured in RPMI 1640 media plus 10% fetal bovine serum and 2 mM L-glutamine at 37 °C under 5% CO2. For comparison a CD-20 negative breast adenocarcinoma cell line, SKBR3, was used. The SKBR3 cells were cultured in McCoy’s 5A growth media containing 10% fetal bovine serum at 37 °C under 5% CO2. Cell counts were performed using a hemocytometer and cell viability was assayed by a trypan blue dye exclusion assay. For each sample 400 000 cells were plated on a 12 mm round glass coverslip in 12-well tissue culture plates. Cells were established for 1 day prior to treatment. Poly-L-lysine coated coverslips were used for Z138 cells to promote cell attachment to the coverslips. The cells were washed with fresh medium before the concentrated gold nanoparticle conjugate solutions were added to the cells in 1.5 mL media. After incubation for 1, 24, or 48 h at 37 °C under 5% CO2, the cells were washed with medium and fixed for 15 min with 1.6% paraformaldehyde. Subsequently the cells were washed with PBS (137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM phosphate, pH 7.4) twice and mounted in 50% glycerol. Imaging of Gold Particles in Cell Cultures. Light scattering microscopy was employed to image the gold particles. Images were taken using a modified Olympus FV300 laser-scanning microscope with femtosecond laser pulses centered at 580 nm. The 580 nm laser beam was obtained by intracavity frequency doubling of a in-house-built KTP (KTiOPO4)-based optical parametric oscillator (OPO), which was pumped by a femtosecond Ti:Sapphire laser with wavelength of 805 nm and a repetition rate of 76 MHz. The input laser beam was linearly polarized, and only the cross-polarized scattered light from the

Figure 2. CD spectra for (a) AB and AB-PEG and (b) NP, AB-NP, (AB-PEG)-NP, and AB-(PEG-NP).

sample was detected to reduce the background signal. A 60× objective lens (NA ) 1.2) was used to collect the scattered light from the samples. The power of the input laser beam was typically less than 0.5 mW. The scattered light was detected by a fiber-coupled photomultiplier tube (PMT), and the intensity was digitized by an analog-to-digital converter. The spatial resolution of the setup reaches about 250 nm. 3D images of the cells and the distribution of the gold nanoparticles were obtained by capturing a stack of 2D (x and y) images along the z-axis. The advantage of 3D-imaging is that it allows us to visualize whether the particles are attached to the cell membranes or have been incorporated into the cells by endocytosis, which is a crucial aspect for the present contribution. Note that fluorescence imaging of gold particles is not suitable here because of the low intensity.12,31 3. Results and Discussion 3.1. Rationale Behind the Designed Rituximab-Conjugated Particles. We have compared three different types of multivalent rituximab-coupled particles with the aim of assessing their suitability to selectively target CD-20 positive lymphoma cells in vitro. The criteria used in the present study included the stability of the functionalized gold particles in the cell culture medium as well as the functionality of the attached rituximab. More specifically, the latter concerned specific and selective binding to CD20-expressing lymphoma cells while the former assessed aggregation of the particles in tissue culture media. The NP and NS conjugates were prepared using three different strategies as described in the experimental section. AB-NP and AB-NS are conjugates in which the antibody is directly attached to the surface of the gold particles. For (AB-PEG)-NP and (ABPEG)-NS, the particles were conjugated to preformed rituximabPEG-OPSS complexes, which can be obtained by a nucleophilic reaction of one of the amino acid side chains with the NHS group of the OPSS-PEG-NHS molecule. The PEG molecule was introduced as a linker to reduce the steric hindrance and

Recognition of Rituximab-Functionalized Gold Nanoparticles

Figure 3. UV-vis spectra over time of (a) 50 µL of AB-NP in 950 µL of HBS buffer, (b) 50 µL of (AB-PEG)-NP in 950 µL of HBS buffer, (c) 50 µL of AB-NP in 950 µL of water, and (d) 50 µL of (AB-PEG)-NP in 950 µL of water.

the risk of conformational changes of the antibody, since the curvature of the particles in AB-NP and AB-NS may influence the conformation of the associated protein and this will, in turn, influence antibody avidity.27 Finally, in AB-(PEG-NP) and AB(PEG-NS) the OPSS-PEG-NHS was bound first to the surface of the particles dispersed in NP and NS formulations. Subsequently rituximab was coupled to the particles via a nucleophilic reaction with the particle associated NHS-groups. What differentiates these conjugates from (AB-PEG)-NP and (AB-PEG)NS is the manner in which the OPSS-PEG-rituximab was conjugated with NS or NP. As shown below, this greatly influences the binding avidity of the particle-antibody conjugates. It is important to recognize that in all three strategies the conjugate was attached to the gold particles through strong gold-sulfur bonds, a feature that differentiates this approach from those involving the use of lipid-based carriers where comparatively weak intermolecular interactions are used to couple rituximab.5 3.2. Characterization of the Rituximab-Gold Bioconjugates. TEM and DLS measurements confirm that for all three types of rituximab-conjugated gold particles, rituximab or the PEG-rituximab complex, respectively, are bound to the particle surface. A representative TEM image of AB-NP is provided in Figure 1. The darker regions are due to the presence of electron dense gold while the lighter corona is believed to be due to the surface associated rituximab (bare gold particles do not show the light corona). Similar results were found for AB-NS, (ABPEG)-NP, (AB-PEG)-NS, AB-(PEG-NP), and AB-(PEG-NS) (not shown). Although TEM clearly shows coated particles, it was not possible to distinguish between the different coating types with TEM. Differences between the particles with conjugated rituximab or PEG-rituximab were not apparent because the samples were dried prior to imaging and this drying step causes a collapse in the tertiary structure of the associated antibody. For this reason information on the original thickness of the coating was lost. Information on the hydrodynamic diameter of the particles in solution, however, can be extracted from DLS measurements. These provided information on the

J. Phys. Chem. C, Vol. 113, No. 47, 2009 20255 average size of particles in the liquid phase and therefore allowed us to distinguish between thinner (rituximab only) and thicker (PEG-rituximab) coatings. For NP, NS, and the pure monomeric antibody in solution we found an average diameter of 33, 255, and 10 nm, respectively. AB-NPs were found to have diameters around 50 nm, while the AB-(PEG-NP) and (AB-PEG)-NP formulations exhibited diameters between 70 and 80 nm. The diameters of these particles were greater than those of the original NPs. These results provide evidence that rituximab and PEG-rituximab were conjugated to the particles. The DLS particle size data were found to be consistent with what would be expected if the surface of the gold particles were coated with rituximab and PEG-rituximab: the PEG-linker leads to a larger diameter. DLS results obtained for antibody coupled to NS particles were equivocal because of highly variable light scattering data that could be traced back to the aggregation of the nanoshells (vide infra). Additional evidence supporting the fact that rituximab and PEG-rituximab were bound to the surface of the particles comes from FT-IR and CD spectroscopic data. In both these cases, however, it should be noted that small amounts of unbound antibody in the sample solution may contribute to the spectral data obtained and this may influence the interpretations of the results. The IR spectra (not shown here) exhibit the characteristic absorption peaks of the antibody for all coated particles, indicating that rituximab is attached to the particles in all the prepared conjugates. Unfortunately, the rather poor quality of the IR spectra prevents us from drawing any further conclusions on the structure or concentration of the bound antibody.28,29 Information on the secondary structure of bound antibody can be gained from CD spectroscopy. Figure 2a shows the CD spectrum of both rituximab alone (AB) and the OPSS-PEGrituximab complex (PEG-AB). Similarities in these spectra confirm that conjugation of rituximab to OPSS-PEG-NHS did not change the general secondary structure of the antibody.23,24 The retention of this secondary structure is crucial for the selectivity and efficacy of the antibody. Information about the secondary structure of rituximab following its conjugation to particles is more difficult to assess from the CD spectra because of the interference with the gold. CD spectra from the different conjugated NPs, as shown in Figure 2b, suggest contributions from both rituximab and the gold nanoparticles and are consistent with other evidence indicating that the antibody is bound to or near the gold particle surface. These CD spectra also imply that the change in the secondary structure of the rituximab in AB-NP is more pronounced than in AB-(PEGNP) and (AB-PEG)-NP. UV-vis spectroscopy data also indicated that rituximab and PEG-rituximab are conjugated to the surface of the particles in NP. The coating of particles by rituximab and PEG-rituximab can be probed by measuring a small redshift of the surface plasmon resonance, as well as by changes in the UV-vis spectrum of gold particles prior to and following conjugation.14,15 This can be explained by a change in the overall particle size, as well as a change in the refractive index near the gold particle surface.30 For NP, the plasmon peak was red-shifted by 5 nm after conjugation. For NS, it was difficult to assess the redshifts that occurred after conjugation because the plasmon peaks were quite broad. TEM, DLS, IR, CD, and UV-vis spectroscopy combined clearly demonstrate that rituximab and PEG-rituximab are bound to the surface of the particles after conjugation. It is, however, important to note here that none of these methods provide any information on the type of the bond that has formed

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Figure 4. Representative light scattering images of Z138 cells (left) and SKBR3 cells (right)

Figure 5. Representative light scattering images of Z138 cells after incubation for 1 h with PEG-NP. Only a very small fraction of the gold particles were incorporated.

(covalent bonds or unspecific weak interactions). In particular for AB-(PEG-NP), the cell culture studies described below indicate that a large portion of the antibodies may be attached to the PEG-NP system through weak interactions instead of covalent bonds. UV-vis spectroscopy was also a useful technique in determining the stability of the prepared colloids over time. Particle aggregation leads to the emergence of new plasmon modes so that the colloidal stability of these systems can readily be monitored over time.30 Furthermore, larger aggregates that form in solution typically precipitate quickly, which decreases the overall absorbance of the spectrum. More importantly, aggregation in cell culture media may influence in vitro results. Using UV-vis, the agglomeration behavior of the particles was shown to be dependent on gold particle size, conjugation, and solvent. The colloids NS, AB-NS, AB-(PEG-NS), and (AB-PEG)-NS were all found to be unstable in solution and, in particular, in cell culture media. Therefore, the nanoshells were not considered to be suitable nanocarriers of rituximab. For AB-NP and (ABPEG)-NP mixed with HBS buffer, the UV-vis spectra at various points in time are shown in panels a and b of Figure 3, respectively. Both AB-NP and (AB-PEG)-NP showed good stability in HBS, in contrast to NP, which aggregated immediately and irreversibly when added to HBS (i.e., 50 µL NP in 950 µL HBS, spectra not shown). Figure 3a and b also reveals that AB-NP was less stable than (AB-PEG)-NP, when mixed with HBS. The comparatively poor stability of AB-NP could be improved by adding PEG-SH to the colloid (data not shown), while the addition of PEG-SH to (AB-PEG)-NP had no observable effect. This behavior suggests that the aggregation of the AB-NP colloid is caused by a nonuniform distribution

of AB over the particle surface, providing less steric stabilization to the antibody than (AB-PEG)-NP or simply leaving patches of gold more accessible to the solvent. In addition to studying colloidal stability in HBS, the stability of the different particles was determined in low ionic strength solutions (distilled water). In contrast to what was observed with HBS, NP was stable in an aqueous solution but AB-NP (Figure 3c) and (AB-PEG)NP (Figure 3d) showed a clear tendency to aggregate. We attribute this to a reduced stability of the antibody in solution under conditions of low ionic strength. Results for AB-(PEGNP) are not shown here, as they were similar to those of (ABPEG)-NP. 3.3. Gold Particle Binding to CD-20 Positive Cells in Vitro. Light scattering images were obtained in order to gain information about the ability of rituximab-conjugated particles to selectively bind to CD20 on the surface of target cells. For these studies two cell lines were usedsZ138 mantle cell lymphoma cells which express CD20 and SKBR3 breast adenocarcinoma cells which do not. Light scattering images of Z138 and SKBR3 cells are shown in panels a and b of Figure 4, respectively. While both cell lines appeared bluish and scattered the light, a different cell shape and size may be seen. Z138 cells were spherical with a diameter of about 10 µm, whereas SKBR3 cells appeared irregular with a larger size of about 20 µm. To ensure that binding of AB-functionalized nanocarriers to the cell membrane was not a result of nonspecific binding, both cell lines were incubated with NP and PEG-NP; i.e., particles without antibody. The cells were incubated as described above for 1, 24, and 48 h at 37 °C. Cell growth and viability was not changed relative to an untreated control cell population under these conditions. Neither of the cell lines showed binding of the particles to the cell membrane regardless of incubation times, while uptake into the cytoplasm was observed. After 1 h incubation time, the Z138 cells still had a very low level of incorporated particles (Figure 5). Over the same incubation time the CD-20 negative SKBR3 cells had taken up a large number of particles, a much more efficient uptake than noted for Z138.12,13 We observed that Z138 cells incorporated about the same amount of particles from PEG-NP in 48 h as SKBR3 cells did in only 1 h. The 3D scan images of single cells (similar to Figure 6b) let us take a look into the cell interior revealing that the particles were located in the cytoplasm and not attached to the membrane. To show the selectivity of rituximab-conjugated nanoparticles to CD20 at the membrane of Z138 cells, the cell lines (Z138 and SKBR3) were incubated for 1 h with AB-NP, AB-(PEG-

Recognition of Rituximab-Functionalized Gold Nanoparticles

Figure 6. Light scattering images of SKBR3 cells that have been incubated with (AB-PEG)-NP. (a) Several cells. The gold particles are the bright dots inside the cells. (b) Image of a single cell (left) and 3D image of this cell (right). The 3D image shows that the particles are in the cytoplasm and not attached to the cell membrane.

NP), and (AB-PEG)-NP. The uptake of (AB-PEG)-NP by the CD-20 negative SKBR3 cells is illustrated by representative scattering images in Figure 6. It was found comparable to that observed when using antibody-free PEG-NP, i.e., the particles are incorporated into the cell by nonspecific endocytosis. 3D images of single cells taken after 1 h (right panel of Figure 6b) showed a large number of particles inside the cytoplasm without any indication of selective recognition at the membrane of these cells. A different pattern was noted for Z138 cells as illustrated in Figure 7. The particles from (AB-PEG)-NP are clearly bound to the membrane of Z138 cells, which express CD20. The particles have not entered the cytoplasm, but are attached to the membrane (see the 3D image in Figure 7b). The specific binding of antibody-conjugated particles correlates well with the results found for similar systems which target oral or breast cancer.13,14 Contrary to those studies, however, we do not observe any significant nonspecific binding. The 3D imaging data allowed as to select an optimized nanosystem for further development. As mentioned above, NS and their conjugates were problematic due to the strong instability of the colloid when added to the cell medium. This aggregation suggests that the particles will precipitate, making recognition unlikely, as these precipitated clusters cannot diffuse in solution and encounter cells. Indeed, this was verified by light scattering images that showed large aggregates and the absence of recognition by cells. The other conjugates of NP, AB-NP and AB-(PEG-NP), showed distinctly less recognition compared to (AB-PEG)-NP. In the case of AB-NP this might be because of a change in the structure of rituximab or steric hindrance. The CD spectra discussed above give some indication that the secondary structure of rituximab bound to the gold surface might have been changed significantly compared with the structure of the unbound, monomeric antibody. For AB-(PEG-NP), an unsuccessful/nonspecific binding of rituximab to the OPSS-PEGNHS/gold nanoparticle complex may possibly explain the lack of activity. It is also possible that the nucleophilic reaction between OPSS-PEG-NHS and rituximab was unsuccessful or

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Figure 7. Light scattering images of Z138 cells that have been incubated with (AB-PEG)-NP. (a) Several cells. The gold particles on the cell membrane lead to strong scattering. (b) Image of a single cell (left) and 3D image of this cell (right). The 3D image indicates that the particles are attached to the cell membrane by selective recognition of rituximab by CD20.

only partially successful due to the steric hindrance caused by the OPSS-PEG-NHS being restrained to a monolayer on the gold surface. Due to this, instead of forming covalently bound OPSS-PEG-rituximab complexes it is possible that only weakly interacting complexes formed. Such a complex would likely be unstable in the cell medium and would dissociate prior to recognition. 4. Conclusions This study demonstrates that rituximabsa therapeutic antibody used in the treatment of lymphomas and leukemiasscan be successfully attached to the surface of gold nanoparticles, creating multivalent antibody complexes. Characterization techniques such as TEM, DLS, and UV-vis, CD, and IR spectroscopy provided strong evidence for the successful conjugation of rituximab with either gold nanoparticles or nanoshells. Light scattering images were used to demonstrate the selective recognition of the antibody-conjugated particles at the membrane of CD20-positive Z138 lymphoma cells. No recognition was observed for nanocarriers without conjugated rituximab, as well as for SKBR3 cells, which do not express CD20. Different types of rituximab-functionalized particles were compared and our systematic investigations reveal that only one type is suitable for further studies: gold nanoparticles (30 nm) conjugated with a preformed rituximab-PEG-OPSS complex (the colloid referred to as (AB-PEG)-NP in this work). These formed stable conjugates with high antibody activity and were easily transported in cell culture media. Acknowledgment. This project was financially supported by the Natural Sciences and Engineering Research Council of Canada, the Canada Foundation for Innovation through LASIR and an individual grant of KCC, and by the A. P. Sloan Foundation (RS). T.C.P. acknowledges NSERC for the provision of a fellowship. M.B.B. is supported by a grant from the Canadian Institutes of Health Research (CIHR), and J.P. is a recipient of a CIHR and Michael Smith Foundation for Health Research Graduate Fellowships.

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