International Journal of Pharmaceutics 458 (2013) 1–8

Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical Nanotechnology

HSA nanocapsules functionalized with monoclonal antibodies for targeted drug delivery Alexandra Rollett a , Tamara Reiter b , Anna Ohradanova-Repic c , Christian Machacek c , Artur Cavaco-Paulo d , Hannes Stockinger c , Georg M. Guebitz a,e,∗ a

University of Natural Resources and Life Sciences, Institute for Environmental Biotechnology, Konrad Lorenz Street 20, 3430 Tulln, Austria Graz University of Technology, Institute of Environmental Biotechnology, Petersgasse 12, 8010 Graz, Austria c Medical University of Vienna, Centre for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, 1090 Vienna, Austria d IBB – Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal e Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria b

a r t i c l e

i n f o

Article history: Received 19 August 2013 Received in revised form 1 October 2013 Accepted 5 October 2013 Available online 21 October 2013 Keywords: Targeted Drug delivery Albumin nanocapsules Cross linking Antibody

a b s t r a c t The chronic autoimmune disorder rheumatoid arthritis (RA) affects millions of adults and children every year. Chronically activated macrophages secreting enzymes and inflammatory cytokines play a key role in RA. Distinctive marker molecules on the macrophage surface could be used to design a targeted drug delivery device for the treatment of RA without affecting healthy cells and tissues. Here, different methods for covalent attachment of antibodies (mAb) recognizing MHC class II molecules found on macrophages onto human serum albumin (HSA) nanocapsules were compared. HSA nanocapsules were prepared with a hydrodynamic diameter of 500.7 ± 9.4 nm and a narrow size distribution as indicated by a polydispersity index (PDI) of 0.255 ± 0.024. This was achieved by using a sonochemical process avoiding toxic cross linking agents and emulsifiers. Covalent binding of mAb on the surface of HSA nanocapsules was realized using polyethyleneglycol (PEG)3000 as spacer molecule. The presence of mAb was confirmed by confocal laser scanning microscopy (CLSM) and enzyme-linked immunosorbent assay (ELISA). Specific binding of mAb-HSA nanocapsules to MHC class II molecules on antigen-presenting cells was demonstrated by flow cytometry analysis. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disorder characterized by inflammation of synovial joints, which affects yearly 0.5–1.0% of adults in industrialized countries (McInnes and Schett, 2011; Scott et al., 2010). Current treatments of RA are mainly based on non-steroidal anti-inflammatory drugs and cyclooxygenases (Rainsford, 2007). So called disease modifying anti-rheumatic drugs (DMARD) for example methotrexate, which reduce the rate of damage to bone and cartilage are also used for RA treatment. The major drawback of the currently used RA treatments is the cause of severe side effects, which can range from gastrointestinal over cardiovascular problems to the suppression of bone marrow (Schett, 2008).

∗ Corresponding author at: University of Natural Resources and Life Sciences, Institute for Environmental Biotechnology, Konrad Lorenz Street 20, 3430 Tulln, Austria. Tel.: +43 6645722600; fax: +43 227266280503. E-mail address: [email protected] (G.M. Guebitz). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.10.022

Consequently, alternative treatment strategies, which avoid non-specific interaction with healthy cells or tissues have emerged. It was found that chronically activated macrophages are one of the main contributors in RA whereas numbers and levels of macrophage activation correlate with the degree of joint inflammation and tissue degradation (Adamopoulos et al., 2006; Schett, 2008). Activated macrophages secrete hydrolytic enzymes and inflammatory cytokines. Depending on the type of activation, macrophages may display distinctive markers, which can be used to identify the state of activation. The markers CD163 and 25F9, e.g. indicate late phases of inflammation (Feghali and Wright, 1997; Zwadlo et al., 1987). Other markers such as MHC-II molecules, which are upregulated upon macrophage activation, could be well suited for the design of a targeted drug delivery device. Specific targeting of chronically activated macrophages by marker molecules found on macrophage surface could be an approach to deliver therapeutic agents without affecting healthy cells and tissues. Highly specific antibodies in combination with nanoparticles, carrying a concentrated amount of therapeutic molecules, could be an ideal targeted drug delivery system. Antibody–nanoparticle conjugates are currently used in two biomedical fields, namely

2

A. Rollett et al. / International Journal of Pharmaceutics 458 (2013) 1–8

therapy and diagnosis (Arruebo et al., 2009). In therapy the main focus is directed on cancer treatment. Several examples of mAbnanoparticles for cancer therapy were recently described in a review by Fay and Scott (2011). A variety of antibody–nanoparticles composed of different materials such as polylactic acid (Cirstoiu-Hapca et al., 2007; Kocbek et al., 2007), gelatin (Dinauer et al., 2005), lipid–polymer hybrids (Hu et al., 2010) or proteins such as human serum albumin (Steinhauser et al., 2006; Ulbrich et al., 2009; Wagner et al., 2010) were previously described. Human serum albumin based nanoparticles have several beneficial properties, such as biodegradability, biocompatibility, effective drug loading capacity due to drug-binding properties of natural albumin and water solubility (Müller et al., 1996). Within the amino acid sequence of HSA there are several functional groups which can be applied for a variety of specific or non-specific cross-linking reactions to link ligand molecules on the nanoparticle surface. We recently developed a method to produce HSA nanocapsules in a size range of ∼500 nm without using toxic cross-linking agents or emulsifiers (Rollett et al., 2012). In this study we covalently linked an antibody on HSA nanocapsules and showed the specific reaction toward MHC class II-positive THP-1 cells. 2. Materials and methods 2.1. Materials Human serum albumin (HSA), N-(3-dimethylaminopropyl)-Nethylcarbodiimide hydrochloride (EDAC), N-hydroxysuccinimide (NHS), n-dodecane, fluorescein 5(6)-isothiocyanate (FITC), 2iminothiolane hydrochloride (iminothiolane), sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), cysteine hydrochloride monohydrate, 5,5 -dithio-bis-(2-nitrobenacid), Celecoxib (4-[5-(4-Methylphenyl)-3-(trifluorozoic methyl)-1H-pyrazol-1-yl]benzenesulfonamide and O-(2-aminoethyl)-O 2-carboxyethyl)polyethylene glycol 3000 (NH2 -PEG3000 COOH) were purchased from Sigma-Aldrich (Steinheim, Germany). All other reagents were used in analytical grade. Mouse monoclonal antibody (mAb) to HLA − DR + DP (MEM136, IgG1) without fluorescent label and labeled with Alexa Fluor® 700 and mouse mAb to human serum albumin (anti-HSA mAb) (AL01 (IgG1)) were a kind gift from EXBIO Praha, a.s. (Vestec, Czech Republic). Anti-mouse IgG-HRP conjugate (anti-mouse mAb) from Amersham ECLTM Western Blotting System was purchased from GE-Healthcare (Vienna, Austria). Mouse mAb to alpha-fetoprotein that was used as an isotype control in flow cytometry experiments (AFP-12, IgG1) was a kind gift from Dr. Václav Hoˇrejˇsí (Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic). Allophycocyanin (APC)conjugated goat anti-mouse IgG + IgM antibody was purchased from Jackson ImmunoResearch, West Grove, PA, USA. All other reagents were used in analytical grade. 2.2. Methods 2.2.1. Synthesis of HSA nanocapsules Nanocapsules were prepared as previously described (Rollett et al., 2012). Briefly, for nanocapsule production, a mixture of 50% FITC-labeled and 50% unlabeled HSA was used. 10 mL HSA-solution (2 mg mL−1 in 100 mM potassium phosphate buffer, pH 8) were combined with 6.6 mL of dodecane. This mixture was sonicated using a 13 mm disrupter horn connected to Branson Sonifier 250 on ice using a total pulsation time of 2 min with 1 s pulsation followed by 2 s pause with amplitude setting of 10%. The horn amplitude is specified as 21.0 ␮m for an amplitude control setting

of 10%. A power of 70 W was required to maintain the amplitude for a given amplitude control setting of 50%. For phase separation the samples were stored at 4 ◦ C for 12 h. Afterwards the organic phase was removed and the nanocapsules were washed five times with 100 mM sodium-phosphate buffer, pH 7.4 by centrifuging (5 min, 5000 rpm) and pouring away the supernatant. To obtain nanocapsules with a homogeneous size distribution the solution was pre-filtered by pressure filtration (2 bar) using a cellulose acetate filter with a cut-off of 1 ␮m. A second filtration step using syringe filters with a cut-off of 0.8 ␮m was performed. Finally the capsule solution was re-concentrated using Vivaspin 30 kDa to a final concentration of 0.5 mg mL−1 . 2.2.2. Coupling of mAb onto the surface of HSA nanocapsules For the development of the methods unfiltered HSA capsules were used. For method optimization filtered HSA nanocapsules were used. 2.2.2.1. Method 1 – using EDAC as cross linker. The mAb was directly linked on HSA capsules using EDAC to cross link amino groups with carboxylic groups. Stock solutions of several reactants were prepared: NHS and EDAC 2.0 mg mL−1 in 0.1 M sodium phosphate buffer (pH 7.2); HSA capsules 1.4 mg mL−1 and labeled mAb 0.896 mg mL−1 in 100 mM sodium phosphate buffer (pH 7.2). HSA capsules (1.2 × 10−9 mol) were combined with EDAC (6.0 × 10−8 mol), NHS (6.0 × 10−8 mol) and mAb (1.0 × 10−10 mol) and reacted for 2 h at 25 ◦ C. Afterwards the reaction mixture was centrifuged 2 times for 10 min at 9000 × g to remove free mAb. The precipitate was investigated with ELISA and the wash solution was checked for antibody content by detecting the fluorescence signal of Alexa Fluor® 700. 2.2.2.2. Method 2 – sulfo-SMCC/iminothiolane activation. Amino groups of HSA capsules were modified with sulfo-SMCC. Stock solutions of several reactants were prepared: HSA capsules 1.4 mg mL−1 and labeled mAb 0.896 mg mL−1 in 100 mM sodium phosphate buffer (pH 7.2); sulfo-SMCC 2 mg mL−1 and iminothiolane 0.1 mg mL−1 in 100 mM sodium phosphate buffer (pH 7.2). HSA capsules (1.2 × 10−9 mol) were mixed with sulfo-SMCC (2.9 × 10−7 mol) at 25 ◦ C. After 1 h of incubation the modified capsules were washed with 100 mM sodium phosphate buffer (pH 7.2) by centrifuging at 9000 × g for 10 min. In a parallel reaction amino groups of mAb (1.0 × 10−10 mol) were modified with iminothiolane (5.0 × 10−9 mol) for 2 h at 25 ◦ C. The modified mAb were purified by SEC using a HiTrap desalting column (GE Healthcare Europe GmbH, Vienna, Austria) installed on an Äkta Purifier system (Amersham Pharmacia Biotech, Uppsala, Sweden), with 100 mM sodium phosphate buffer and 100 mM NaCl (pH 7.2) as eluent, at the flow rate of 1 mL min−1 . Fractions containing the mAb were collected and reconcentrated using Vivaspin 30 kDa. Activated HSA capsules were combined with modified mAb for 12 h at 25 ◦ C. To remove free mAb the mixture was treated like described above. 2.2.2.3. Method 3 – introduction of a PEG spacer. NH2 -PEG3000 COOH was covalently linked onto the surface of HSA capsules to work as a spacer. An additional stock solution of NH2 -PEG3000 COOH 2.0 mg mL−1 in 100 mM sodium phosphate buffer (pH 7.2) was prepared. HSA capsules (1.2 × 10−9 mol) were mixed with NH2 -PEG3000 -COOH (1.0 × 10−10 mol), EDAC (1.0 × 10−9 mol) and NHS (1.0 × 10−9 mol) and reacted at 25 ◦ C. After 2 h the PEG modified nanocapsules were separated from uncoupled NH2 PEG3000 -COOH or urea by washing with 100 mM sodium phosphate buffer (pH 7.2) using Vivacon 30 kDa at 5000 rpm for 2 min. In the next step amino groups of PEG were activated with sulfo-SMCC (2.9 × 10−7 mol) for 1 h at 25 ◦ C and washed like described before. In a parallel reaction amino groups of labeled mAb (1.0 × 10−10 mol)

A. Rollett et al. / International Journal of Pharmaceutics 458 (2013) 1–8

were modified with iminothiolane (5.0 × 10−9 mol), purified and re-concentrated as described before. Activated PEG-HSA capsules were combined with modified mAb for 12 h at 25 ◦ C. To remove free mAb the mixture was treated like described above. 2.2.2.4. Method 4 – non-specific adsorption (Illum et al., 1983). mAb and HSA capsules were combined without any further cross-linker molecules to check if non-specific adsorption of antibody on capsules occurs. HSA capsules (1.2 × 10−9 mol) were combined with labeled mAb (1.0 × 10−10 mol) and reacted for 2 h at 25 ◦ C. Afterwards the reaction mixture was centrifuged 2 times for 10 min at 9000 × g to remove unlinked mAb. 2.2.2.5. Purification of particles. Method 3 was modified to obtain nano-sized mAb-HSA capsules. Filtered nanocapsules with a hydrodynamic diameter of ∼500 nm were used for this process. Stock solutions were prepared as described above. HSA nanocapsules (1.2 × 10−9 mol) were mixed with NH2 -PEG3000 -COOH (1.0 × 10−10 mol), EDAC (1.0 × 10−9 mol) and NHS (1.0 × 10−9 mol) in MES buffer (pH 6.0) and reacted at 25 ◦ C. After 2 h the PEG modified nanocapsules were separated from uncoupled NH2 PEG3000 -COOH or urea by washing 3 times with 100 mM sodium phosphate buffer (pH 7.2) using Vivacon 30 kDa at 5000 rpm for 2 min. In the next step, amino groups of PEG were activated with sulfo-SMCC (2.4 × 10−8 mol) for 1 h at 25 ◦ C and washed like described before. In a parallel reaction amino groups of mAb (1.0 × 10−10 mol) were modified with iminothiolane (5.0 × 10−9 mol), purified and re-concentrated as described before. Activated PEG-HSA capsules were combined with modified mAb for 12 h at 25 ◦ C and rotated at 60 rpm. To remove unlinked mAb the mixture was washed 2 times with 100 mM sodium phosphate buffer (pH 7.2) using Vivaspin 1000 kDa at 5000 rpm for 2 min. 2.2.3. Capsule characterization 2.2.3.1. HSA concentration. The HSA concentration was determined using Roti® -Nanoquant (Carl Roth GmbH+Co. KG, Karlsruhe, Germany) which is based on the method of Bradford. 2.2.3.2. Ellman’s assay for detection of thiol groups. After thiolation of the antibody the created thiol groups were detected by the Ellman’s assay, which is based on the formation of a yellow reaction product when DTNB reacts with free sulfhydryl groups (Ellman, 1959). Briefly a calibration curve with cysteine hydrochloride monohydrate (0.5–0.01 mM) was created by combining 190 ␮L cysteine with 10 ␮L of a DTNB stock solution (20 mM in 100 mM sodium phosphate buffer pH 7.2) and recording the absorbance at 412 nm after 10 min incubation. After the thiolation process the antibody was treated the same way and results were compared to the calibration curve to determine the exact amount of created sulfhydryl groups. 2.2.3.3. Dynamic light scattering (DLS). The mean size of HSA nanocapsules was determined by DLS using Zetasizer Nano ZS (Malvern Instruments GmbH, Herrenberg, Germany). Measurements were performed in triplicates at 25 ◦ C with angle detection at 173◦ . Size distribution was characterized by the PDI. The zeta potential was determined based on a combination of laser Doppler velocimetry and phase analysis light scattering using the Zetasizer Nano ZS. 2.2.3.4. Confocal laser scanning microscopy. Surface modified HSAcapsules in the micron range were analyzed as models by CLSM using a Leica TCS SPE Confocal microscope (Leica Microsystems GmbH, Mannheim, Germany). Laser light wavelengths of 635 nm and 488 nm were used for the excitation of Alexa Fluor® 700 and

3

FITC, respectively. The emitted light was detected in the range of 641–771 nm for Alexa Fluor® 700 and 506–542 nm for FITC. For each channel and each field of view, laser intensity, photomultiplier gain and offset were individually modified to optimize the signal/noise ratio. Confocal stacks were acquired with an ACS APO 63.0 × 1.30 oil objective by applying a Z-step of 6.38 ␮m. Volume rendering and three dimensional models were created with the software Imaris7.0 (Bitplane, Zurich, Switzerland). 2.2.3.5. Determination of antibody in wash fraction. All wash fractions were analyzed for the content of antibody by measuring the absorbance of Alexa Fluor® 700 at 700 nm with a NanoDropTM 2000c Spectrophotometer (Thermo Fisher Scientific, Wilmington, USA). 2.2.3.6. Enzyme-linked immunosorbent assay (ELISA). To analyze the presence of mAb on the surface of HSA nanocapsules an ELISA was performed. Wells of ELISA plates (Greiner high and medium binding 96 well plates, Sigma-Aldrich, Steinheim, Germany) were coated with 50 ␮L of mAb-HSA nanocapsules solution (ca 5.0 ␮g mL−1 ), non-surface modified HSA nanocapsules (0.5 and 5.0 ␮g mL−1 ) and buffer only for 12 h. After washing with buffer (0.05% detergent Tween 20 in PBS, pH 7.4) and blocking for 1 h (1% skimmed dry milk in washing buffer) the samples were incubated for 1 h at 23 ◦ C with a horseradish peroxidaselabeled anti-mouse mAb (anti-mouse mAb). A control incubation to detect HSA capsules was performed where the samples were incubated with an anti-HSA antibody for 1 h followed by incubation with the secondary horseradish peroxidase-labeled antibody (antimouse mAb) for 1 h. The immune-reaction was visualized using o-phenylenediamine as a substrate (Sigma-Aldrich). The reaction was stopped by the addition of 3 M HCl and absorbance was read in a microplate reader at 492 nm. 2.2.4. Antibody function 2.2.4.1. Cells. The Jurkat T cell line E6.1 and the monocytic line THP1 were purchased from the American Type Culture Collection. Cells were maintained in RPMI 1640 medium supplemented with 2 mM l-glutamine, 50 ␮g mL−1 streptomycin, and 50 U mL−1 penicillin and 10% heat inactivated FCS (all from Invitrogen) in a humidified atmosphere with 5% CO2 at 37 ◦ C. To upregulate MHC class II molecules on the cell surface, THP-1 cells were incubated with 25 ng mL−1 (500 U mL−1 ) recombinant human interferon-gamma (IFN-␥, Peprotech, Rocky Hill, NJ) for 24 h before flow cytometry. 2.2.4.2. Flow cytometry. Cells were washed with staining buffer (PBS containing 1% BSA and 0.02% NaN3 and incubated for 30 min with 4.8 ␮g mL−1 human IgG on ice to prevent nonspecific binding of the mAbs to Fc receptors. Then, the mAb-HSA nanocapsules, the free primary mAb, the non-modified HSA nanocapsules or the isotype control mAb AFP-12 (all 20 ␮g mL−1 was added and the cells were incubated for 30 min on ice. Cells were washed with staining buffer and incubated with APC-conjugated secondary antibody or 30 min on ice. After a final wash, cells were analyzed on an LSRII flow cytometer (BD Biosciences) and the data further processed with the FloJo software (Treestar). Live cells were gated according to their forward- and side-scatter characteristics dead cells were excluded using DAPI. 2.2.5. Loading with celecoxib HSA capsules were loaded with celecoxib as follows. 10 mg celecoxib were dissolved in 400 ␮L cyclohexane and added to 10 mL of the above described HSA-solution (2 mg mL−1 in 100 mM potassium phosphate buffer, pH 8). The mixture was sonicated, stored and washed as described above.

4

A. Rollett et al. / International Journal of Pharmaceutics 458 (2013) 1–8

Fig. 1. Comparison of different strategies for the coupling of mAb onto HSA-capsules for the purpose of targeted drug delivery: left axis: ELISA to detect mAb on HSA-capsule surface. HSA-capsules modified with mAb were analyzed with a secondary antibody in comparison to non-modified HSA-capsules and blocking buffer alone. Surface bound mAb was detected by incubation with a horseradish peroxidase-labeled secondary antibody. Control incubation to detect HSA was performed with an anti-HSA antibody followed by incubation with the secondary antibody. Right axis: wash solutions of coupling reactions were analyzed photometrically to detect unlinked Alexa Fluor® 700 labeled mAb.

For the determination of celecoxib the capsules were disintegrated by freeze drying and re-dissolving the powder in 130 mM NaHCO3 buffer (pH 7.0). The solution was then analyzed by HPLC analysis using an established method by Chow et al. (2004). Briefly reversed phase HPLC separation was carried out using an Eclipse XDB-C8 column (5 ␮m, 4.6 mm × 150 mm, Agilent) at 25 ◦ C. The mobile phase consisted of acetonitrile, tetrahydrofuran, and 0.02 M sodium acetate buffer, pH 5 in the ratio of 30:8:62. The flow rate of the mobile phase was 1.5 mL min−1 and a ramp from 30% to 40% acetonitrile in 30 min was used. 3. Results and discussion 3.1. mAb-coupling – method development We recently developed a method for the sonochemical production of HSA capsules in micron- and nano-range functionalized with folic acid for targeted drug delivery to chronically activated macrophages (Rollett et al., 2012). This method is characterized by the complete avoidance of toxic cross-linking chemicals. However, to enhance specificity of targeting to activated macrophages we have linked mAb to HLA-DR and HSA-DP in this study. To do this we tested different methods to link antibodies onto HSA capsules, with a main focus on keeping the reaction fast and simple. Pre-experiments using HSA capsules in micron-range were performed to elaborate the most suitable method for further studies. After the coupling reactions, samples were washed and wash solutions were analyzed for antibody content by measuring the absorbance of Alexa Fluor® 700. Furthermore, ELISA experiments were performed to detect mAb on HSA capsules using secondary antibodies for the detection of mAb. The results are summarized in Fig. 1. The most commonly used method for coupling of antibodies on various kinds of NPs involves thiolation of mAb with iminothiolane followed by coupling to NPs using a heterobifunctional

cross-linker eventually including a spacer e.g. NHS-PEG5000-Mal (Steinhauser et al., 2006; Ulbrich et al., 2009; Wagner et al., 2010). Other approaches are based on heterobifunctional cross-linkers without spacer function e.g. SMCC or EDAC (Kocbek et al., 2007; Shuvaev et al., 2011). In method 1 we used the water soluble, heterobifunctional cross-linker EDAC to link primary amino groups with carboxylic groups. Usually this cross-linker activates carboxylic groups of reactant 1 to form a stable intermediate which in a further step reacts with the amino groups of reactant 2 (Hermanson, 2008). To avoid intermolecular cross-linking of the reactants, the antibody and the HSA capsules were incubated at once with the crosslinker molecule. In contrast to the other methods investigated here, with this simple method no activation of reactants is necessary. In method 2, the antibody was thiolated with iminothiolane to create thiol groups, which can be further linked to amino groups of HSA capsules by the heterobifunctional cross-linker, sulfo-SMCC. The thiolation of mAb enhances the specificity of the reaction since in that way, thiol groups are only found on the mAb but not on HSA capsules. This thiolation process was previously optimized, regarding the amount of thiol groups created, by Steinhauser et al. (2006). According to the results of the Ellman’s assay for detection of free sulfhydryl groups 8.09 ± 0.91 thiol groups per antibody were created in contrast to untreated mAb where no free thiol groups could be detected. The 3rd method used in this study was the most complex. Here additionally to thiolation of mAb, a NH2 -PEG3000 -COOH was introduced as spacer. Furthermore a method without the use of cross-linker molecules was tested, to check if non-specific adsorption of antibody on HSA capsules was occurring (method 4). When looking at the results of the antibody content in the wash solutions, surprisingly only method 3 showed a lower absorbance of Alexa Fluor® 700 compared to the other methods (Fig. 1 right axis). Hence, we assumed that in this experiment the antibody must be found on the surface of the capsules. However, the results of ELISA

A. Rollett et al. / International Journal of Pharmaceutics 458 (2013) 1–8

5

Fig. 2. Model and 3-D reconstruction of CLSM image of FITC-labeled HSA capsule carrying Alexa Fluor® 700 labeled antibody on the capsule surface. The capsules were prepared with method 3. Green: HSA, red: antibody. (For interpretation of the references to color in this text, the reader is referred to the web version of the article.)

detecting antibodies on HSA capsules also showed that only with method 3 a significant amount of mAb was found on capsule surface (Fig. 1 left axis). When looking at the results of method 4 we can exclude non-specific adsorption of mAb on HSA-capsules, which is an important fact since HSA is well known for its extraordinary binding capacity (Fasano et al., 2005). With method 1 and method 2 obviously no covalent binding of mAb on HSA-capsules had occurred, neither the detection of mAb in wash solutions nor the ELISA experiment gave an indication for mAb binding on HSA-capsules. EDAC and sulfo-SMCC without spacer are therefore not suitable for this purpose. Regarding the fact that only with method 3 mAb was detected on HSA-capsules we assumed that a spacer molecule is essential for the covalent binding of mAb on HSA-capsules. As a comprehensive method for the determination of antibody presence on HSA capsules we used confocal laser scanning microscopy. With this method we could visualize the particles and not only determine the presence of antibody on HSA capsules but also characterize the three dimensional structure of the capsules. This method was used previously for the successful characterization of modified HSA nanocapsules (Rollett et al., 2012). With the three dimensional reconstruction of a single particle the shape of a HSA capsule surrounding a cavity where drugs could be loaded was confirmed (see Fig. 2). Additionally we could see a similar trend as described above where method 3 was the most successful. With this method a layer of fluorescent labeled antibody surrounding the HSA capsule can be clearly seen in the reconstruction image. Interpreting these results we assumed that a spacer is necessary for the successful cross-linking of an antibody on HSA nanocapsules. Based on these results, method 3, which is characterized by the introduction of a NH2 -PEG3000 -COOH spacer, was used for subsequent experiments. Additionally PEG is known to work as stealthing agent to prevent non-specific uptake of nanoparticles by macrophages (Cavaco-Paulo et al., 2013).

Furthermore, when comparing the fate of micro and nanoparticles in intestinal tissue, it was found that nanoparticles showed a better performance since they diffuse throughout the sub-mucosal layers while microparticles were predominantly localized in the epithelial lining (Desai et al., 1996). For this reason we focused on the optimization of the production of mAb-HSA particles in order to obtain nanocapsules with an average diameter below 1 ␮m and a narrow size distribution. By filtration of HSA capsules we obtained nanoparticles with a diameter of 500.7 ± 9.4 nm and a narrow size distribution indicated by a PDI of 0.255 ± 0.024. Method 3 was used to link the antibody on the surface of these nanocapsules. The filtration steps for re-concentration and removal of unlinked antibody were optimized to receive nanocapsules with a narrow size distribution. The final antibody modified nanocapsules had a diameter of 815.1 ± 27.51 nm and a PDI of 0.329 ± 0.063 nm. The increased diameter is an evidence for the successful linking of mAb on the capsules. No particles in the size range of an antibody (∼12 nm) (Shire et al., 2010) were found in the preparation, which let us conclude that unlinked antibody molecules were removed thoroughly. 3.3. Antibody presence and binding characteristics Previous studies have shown that macrophage activation plays a significant role in the pathogenesis of RA (Schett, 2008; Xia et al.,

3.2. Purification of particles The optimum size of nanoparticles for cell uptake is dependent on multiple factors including the target cell and specific properties of the particle formulation, therefore it is very difficult to predict the optimum size for the cell uptake (Kelly et al., 2011). Although macrophages are able to take up particles greater than 1 ␮m by phagocytosis (Hasegawa et al., 2007), other research groups have found that the cell uptake of particles in the nanorange is more efficient when compared to microparticles (Panyam and Labhasetwar, 2003; Suri et al., 2007). However, to prevent unspecific phagocytic uptake of drug delivery particles by the whole macrophage population of the body uncontrolled phagocytosis of micro particles should be suppressed.

Fig. 3. ELISA to determine the presence of mAb on HSA nanocapsules. Modified HSA nanocapsules and non-modified nanocapsules were analyzed by incubation with an anti-HSA antibody followed by incubation with a horseradish peroxidase-labeled anti-mouse mAb. Presence of mAb was detected by incubation with horseradish peroxidase-labeled anti-mouse mAb only.

6

A. Rollett et al. / International Journal of Pharmaceutics 458 (2013) 1–8

Fig. 4. Flow cytometry analysis of MHC class II-positive THP-1 cells and MHC class II-negative Jurkat cells to compare the binding properties of mAb modified HSA nanocapsules and unconjugated anti-MHC class II mAb MEM-136. The binding was visualized using APC-labeled secondary antibody. The specific staining is shown in black histograms, overlaid with the staining using isotype control mAb AFP-12, shown in filled gray histograms. Numbers represent percentage of cells within the positive gate that was set to a cut-off of 0.5% using the isotype control mAb.

2009). Therefore, a device targeting marker molecules found on macrophages can be an ideal targeted drug delivery system. The mAb, which was chosen for this experiment, recognizes a common epitope on beta-chain of human HLA-DR and HLA-DP (Koch et al., 1999). Antigen-presenting cells like macrophages, dendritic cells

or B cells are expressing MHC class II molecules, where DR and DP are the isotopes (Janeway et al., 2001). For the analysis of the nanocapsules regarding the presence of the antibody on the surface of the capsules and the binding characteristics of the cross-linked antibody to MHC II positive

Fig. 5. Reversed phase HPLC analysis to detect celecoxib loaded into HSA-capsules after disintegration was performed using an Eclipse C8 column. Dotted line: blank (celecoxib (1 mg mL−1 )); solid line: disintegrated HSA-capsules loaded with celecoxib. The mobile phase consisted of acetonitrile, tetrahydrofuran and 0.02 M sodium acetate buffer, pH 5 in a ratio of 30:8:62. The flow rate of the mobile phase was 1.5 mL min−1 and a ramp from 30% to 40% acetonitrile in 30 min was used. Celecoxib was found after a retention time of 16.20 min.

A. Rollett et al. / International Journal of Pharmaceutics 458 (2013) 1–8

or negative cells another charge of mAb-HSA nanocapsules was prepared. An ELISA was performed to detect on the one hand the presence of HSA nanocapsules and on the other hand the presence of mAb on the surface of the HSA nanocapsules. Preliminary experiments confirmed that HSA nanocapsules were binding on ELISA plates and were thus detectable by this method. As shown in Fig. 3, the presence of HSA nanocapsules can be determined by detection with an anti-HSA antibody. With this experiment we could also exclude unspecific binding of the anti-mouse mAb to the capsules, since no signal could be detected when incubating unmodified HSA nanocapsules with anti-mouse mAb only. When looking at the mAb-HSA nanocapsules, the anti-mouse mAb is giving an enhanced signal, which proves the presence of the antibody on the capsules. Knowing that the antibody is present on the capsules, in a next step we tested the binding ability of the cross linked antibody to MHC II molecules on living cells. For this flow cytometry analysis was carried out where MHC class II-positive THP-1 cells and MHC class II-negative Jurkat cells were employed. When comparing the binding pattern of cross-linked mAb and free mAb (Fig. 4) it can be seen that the patterns are similar, indicating that the cross-linked mAb recognized its antigen very well. Furthermore, we conclude from this experiment that no unspecific binding to MHC class IInegative cells is occurring. 3.4. Loading with celecoxib HSA capsules were loaded with celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor approved for relief of the signs and symptoms of inflammatory diseases like RA (Tive, 2000). Loading with the hydrophobic drug was achieved during the ultrasound based preparation and approved by HPLC analysis. Therefore the capsules were disintegrated and the free celecoxib was determined. In Fig. 5 it can be clearly seen that free celecoxib is present in the disintegrated HSA capsule solution (see peak at 16.20 min retention time). With this experiment we could show that the prepared HSA capsules can be easily loaded during preparation with a hydrophobic antirheumatic drug such as celecoxib. 4. Conclusion Biodegradable protein nanocapsules in a size range of 500 nm were prepared out of human serum albumin and subsequently modified on the surface with an antibody for the purpose of targeted drug delivery. Three different methods for covalent binding of mAb on HSA-capsules were compared. ELISA and CLSM analysis showed that NH2 -PEG3000 -COOH is necessary as spacer for the successful cross linking of mAb on the surface of HSA-capsules. Optimizing filtration and washing steps we prepared mAb modified HSA nanocapsules with a diameter of ∼800 nm and a narrow size distribution indicated by a PDI of 0.329. Furthermore FACS analysis demonstrated that the antibody attached to the capsule surface still recognizes its antigen without any unspecific binding to MHC class II-negative cells. The prepared mAb-HSA nanocapsules would thus be well suited for the purpose of targeted drug delivery of therapeutics for the treatment of rheumatoid arthritis. Antibodies targeting other marker molecules could also be linked on HSA nanocapsules using this developed method. Acknowledgements This work has received funding from the European Union Seventh Framework Program (FP7/2007–2013) under grant agreement

7

NMP4-LA-2009-228827 NANOFOL. We thank Exbio from Czech Republic for providing antibodies. References Adamopoulos, I.E., Sabokbar, A., Wordsworth, B.P., Carr, A., Ferguson, D.J., Athanasou, N.A., 2006. Synovial fluid macrophages are capable of osteoclast formation and resorption. J. Pathol. 208, 35–43. Arruebo, M., Valladares, M., González-Fernández, Á., 2009. Antibody-conjugated nanoparticles for biomedical applications. J. Nanomater., 24. Cavaco-Paulo, A., Nogueira, E., Loureiro, A., Nogueira, P., Freitas, J., Almeida, C.R., Harmark, J., Hebert, H., Moreira, A., Carmo, A.M., Preto, A., Gomes, A., 2013. Liposome and protein based stealth nanoparticles. Faraday Discuss., http://dx.doi.org/10.1039/C3FD00057E. Chow, H.-.S., Anavy, N., Salazar, D., Frank, D.H., Alberts, D.S., 2004. Determination of celecoxib in human plasma using solid-phase extraction and high-performance liquid chromatography. J. Pharm. Biomed. Anal. 34, 167–174, http://dx.doi.org/10.1016/j.japna.2003.08.018. Cirstoiu-Hapca, A., Bossy-Nobs, L., Buchegger, F., Gurny, R., Delie, F., 2007. Differential tumor cell targeting of anti-HER2 (Herceptin® ) and antiCD20 (Mabthera® ) coupled nanoparticles. Int. J. Pharm. 331, 190–196, http://dx.doi.org/10.1016/j.ijpharm.2006.12.002. Desai, M.P., Labhasetwar, V., Amidon, G.L., Levy, R.J., 1996. Gastrointestinal uptake of biodegradable microparticles: effect of particle size. Pharm. Res. 13 (12), 1838–1845. Dinauer, N., Balthasar, S., Weber, C., Kreuter, J., Langer, K., von Briesen, H., 2005. Selective targeting of antibody-conjugated nanoparticles to leukemic cells and primary T-lymphocytes. Biomaterials 26, 5898–5906, http://dx.doi.org/10.1016/j.biomaterials.2005.02.038. Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77, http://dx.doi.org/10.1016/0003-9861(59)90090-6. Fasano, M., Curry, S., Terreno, E., Galliano, M., Fanali, G., Narciso, P., Notari, S., Ascenzi, P., 2005. The extraordinary ligand binding properties of human serum albumin. IUBMB Life 57, 787–796, http://dx.doi.org/10.1080/15216540500404093. Fay, F., Scott, C.J., 2011. Antibody-targeted nanoparticles for cancer therapy. Immunotherapy 3, 381–394, http://dx.doi.org/10.2217/imt.11.5. Feghali, C.A., Wright, T.M., 1997. Cytokines in acute and chronic inflammation. Front. Biosci. 2, d12–d26. Hasegawa, T., Hirota, K., Tomoda, K., Ito, F., Inagawa, H., Kochi, C., Soma, G., Makino, K., Terada, H., 2007. Phagocytic activity of alveolar macrophages toward polystyrene latex microspheres and PLGA microspheres loaded with anti-tuberculosis agent. Colloids Surf., B 60, 221–228, http://dx.doi.org/10.1016/j.colsurfb.2007.06.017. Hermanson, G., 2008. Bioconjugate Techniques, second ed. Academic Press, Inc, San Diego, USA. Hu, C.J., Kaushal, S., Cao, H.S.T., Aryal, S., Sartor, M., Esener, S., Bouvet, M., Zhang, L., 2010. Half-antibody functionalized lipid–polymer hybrid nanoparticles for targeted drug delivery to carcinoembryonic antigen presenting pancreatic cancer cells. Mol. Pharm. 7, 914–920, http://dx.doi.org/10.1021/mp900316a. Illum, L., Jones, P.D.E., Kreuter, J., Baldwin, R.W., Davis, S.S., 1983. Adsorption of monoclonal antibodies to polyhexylcyanoacrylate nanoparticles and subsequent immunospecific binding to tumour cells in vitro. Int. J. Pharm. 17, 65–76, http://dx.doi.org/10.1016/0378-5173(83)90019-4. Janeway, C.A.J., Travers, P., Walport, M., Shlomchik, M.J., 2001. Immunobiology: The Immune System in Health and Disease, fifth ed. Garland Science, New York. Kelly, C., Jefferies, C., Cryan, S., 2011. Targeted liposomal drug delivery to monocytes and macrophages. J. Drug Deliv.. Kocbek, P., Obermajer, N., Cegnar, M., Kos, J., Kristl, J., 2007. Targeting cancer cells using PLGA nanoparticles surface modified with monoclonal antibody. J. Controlled Release 120, 18–26, http://dx.doi.org/10.1016/j.jconrel.2007.03.012. ˇ ´ J., Steinlein, y, Koch, C., Staffler, G., Hüttinger, R., Hilgert, I., Prager, E., Cern P., Majdic, O., Hoˇrejˇsí, V., Stockinger, H., 1999. T cell activation-associated epitopes of CD147 in regulation of the T cell response, and their definition by antibody affinity and antigen density. Int. Immunol. 11, 777–786, http://dx.doi.org/10.1093/intimm/11.5.777. McInnes, I.B., Schett, G., 2011. The pathogenesis of rheumatoid arthritis. N. Engl. J. Med. 365, 2205–2219, http://dx.doi.org/10.1056/NEJMra1004965. Müller, B.G., Leuenberger, H., Kissel, T., 1996. Albumin nanospheres as carriers for passive drug targeting: an optimized manufacturing technique. Pharm. Res. 13, 32–37. Panyam, J., Labhasetwar, V., 2003. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv. Drug Deliv. Rev. 55, 329–347. Rainsford, K.D., 2007. Anti-inflammatory drugs in the 21st century. Subcell Biochem. 42, 3–27. Rollett, A., Reiter, T., Nogueira, P., Cardinale, M., Loureiro, A., Gomes, A., Cavaco-Paulo, A., Moreira, A., Carmo, A.M., Guebitz, G.M., 2012. Folic acid-functionalized human serum albumin nanocapsules for targeted drug delivery to chronically activated macrophages. Int. J. Pharm. 427, 460–466, http://dx.doi.org/10.1016/j.ijpharm.2012.02.028. Schett, G., 2008. Review: immune cells and mediators of inflammatory arthritis. Autoimmunity 41, 224–229. Scott, D.L., Wolfe, F., Huizinga, T.W., 2010. Rheumatoid arthritis. Lancet 376, 1094–1108, http://dx.doi.org/10.1016/S0140-6736(10)60826-4. Shire, S.J., Gombotz, W., Bechtold-Peters, K., Andya, J., 2010. Current Trends in Monoclonal Antibody Development and Manufacturing. Springer, New York, USA.

8

A. Rollett et al. / International Journal of Pharmaceutics 458 (2013) 1–8

Shuvaev, V.V., Tliba, S., Pick, J., Arguiri, E., Christofidou-Solomidou, M., Albelda, S.M., Muzykantov, V.R., 2011. Modulation of endothelial targeting by size of antibody–antioxidant enzyme conjugates. J. Controlled Release 149, 236–241, http://dx.doi.org/10.1016/j.jconrel.2010.10.026. Steinhauser, I., Spänkuch, B., Strebhardt, K., Langer, K., 2006. Trastuzumab-modified nanoparticles: optimisation of preparation and uptake in cancer cells. Biomaterials 27, 4975–4983, http://dx.doi.org/10.1016/j.biomaterials.2006.05.016. Suri, S., Fenniri, H., Singh, B., 2007. Nanotechnology-based drug delivery systems. J. Occup. Med. Toxicol. 2, 16. Tive, L., 2000. Celecoxib clinical profile. Rheumatology 39, 21–28, http://dx.doi.org/10.1093/rheumatology/39.suppl 2.21. Ulbrich, K., Hekmatara, T., Herbert, E., Kreuter, J., 2009. Transferrin- and transferrin-receptor-antibody-modified nanoparticles enable drug delivery across the blood-brain barrier (BBB). Eur. J. Pharm. Biopharm. 71, 251–256, http://dx.doi.org/10.1016/j.ejpb.2008.08.021.

Wagner, S., Rothweiler, F., Anhorn, M.G., Sauer, D., Riemann, I., Weiss, E.C., KatsenGloba, A., Michaelis, M., Cinatl Jr., J., Schwartz, D., Kreuter, J., von Briesen, H., Langer, K., 2010. Enhanced drug targeting by attachment of an anti ␣v integrin antibody to doxorubicin loaded human serum albumin nanoparticles. Biomaterials 31, 2388–2398, http://dx.doi.org/10.1016/j.biomaterials.2009.11. 093. Xia, W., Hilgenbrink, A.R., Matteson, E.L., Lockwood, M.B., Cheng, J.X., Low, P.S., 2009. A functional folate receptor is induced during macrophage activation and can be used to target drugs to activated macrophages. Blood 113, 438–446. Zwadlo, G., Voegeli, R., Schulze Osthoff, K., Sorg, C., 1987. A monoclonal antibody to a novel differentiation antigen on human macrophages associated with the down-regulatory phase of the inflammatory process. Exp. Cell. Biol. 55 (6), 295–304.