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NANOTECHNOLOGIES Targeted Delivery of Cisplatin by Сonnexin 43 Vector Nanogels to the Focus of Experimental Glioma C6 N. V. Nukolova1,2,3, V. P. Baklaushev1,2, T. O. Abakumova1,3, P. A. Mel’nikov2, M. A. Abakumov1,3, G. M. Yusubalieva1, D. A. Bychkov4, A. V. Kabanov3,5, and V. P. Chekhonin1,2 Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 157, No. 4, pp. 527-533, April, 2014 Original article submitted October 25, 2013 The aim of this study was to create a nanocontainer conjugated with monoclonal antibodies to connexin 43 (Cx43) that is actively expressed at the periphery of C6 glioma and in the astroglia roll zone. Stable vector nanogels with high (up to 35%) cisplatin load were synthesized. The antitumor effects of Cx43-modified cisplatin-loaded nanogels, free cisplatin, and nonspecific drugs were carried out on C6 glioma model. Vector nanogels reduced systemic toxicity of cisplatin, effectively inhibited tumor growth, and significantly prolonged the lifespan of animals with experimental tumors. Key Words: cisplatin; nanogels; targeted delivery; monoclonal antibodies to connexin-43; brain tumors Many drugs are poorly dissolved in water, are unstable, rapidly captured by the lymph system, and eliminated [4,12]. In addition, they are often characterized by high systemic toxicity (which is particularly important for alkylating platinum-containing cytostatics) and their selective delivery to the tumor is of crucial importance. The priority task of targeted delivery is the development of a drug delivery systems (DDS) with drugs packed in nanocontainers [12,13]. This packing prolongs the lifetime of the drug in circula1 Department of Basic and Applied Neurobiology, V. P. Serbsky State Research Center of Social and Forensic Psychiatry, Ministry of Health of the Russian Federation; 2Department of Medical Nanobiotechnologies, Biomedical Faculty, N. I. Pirogov Russian National Research Medical University, Ministry of Health of the Russian Federation; 3 Department of Chemical Enzymology, Chemical Faculty, 4Geological Faculty, M. V. Lomonosov Moscow State University; 5Department of Molecular Pharmacology, Pharmaceutical Division, University of North Carolina, Chapel-Hill, USA. Address for correspondence:
[email protected]. N. V. Nukolova
tion, reduces its toxicity, and improves biodistribution [4,12,13]. Various DDS (liposomes, micelles, nanotubules, nanogels, etc.) has their advantages and flaws and the choice of a nanocontainer for a certain drug is an important problem, because this choice is determined by the nature of vehicle and morphology and functions of the target organ. Polymeric nanogels is a new class of nanocontainers [6,11]. Due to regulated physicochemical properties of nanogels, it is possible to stimulate drug release from the nanocontainers only under certain conditions or in specific cells/organs [11]. Specific structure of the vascular network in brain tumors (high vascularization, high vascular permeability, impaired blood-brain barrier [5]) prompt the use of nanocontainer systems with drugs [2]. The development of controlled targeted DDS by conjugation of the nanocontainers with vector groups [7,8] has been in the focus of attention during the recent years. We have used monoclonal antibodies to connexin 43 (Cx43) as vector groups, because Cx43
0007-4888/14/15740524 © 2014 Springer Science+Business Media New York
N. V. Nukolova, V. P. Baklaushev, et al.
is actively expressed in brain tumor and/or peritumoral space [1,2]. We studied the therapeutic efficiency of cisplatinloaded nanogels conjugated with monoclonal antibodies to Cx43 associated with C6 glioma.
MATERIALS AND METHODS Synthesis of vector CDDP-loaded nanogels. Nanogels based on polyethylene glycol and polymethacrylic acid block copolymer PEG-b-PMAA (Polymer Source Inc.) were synthesized as described previously using 1,2-ethylene diamine for cross-linking [8]. The nanogels were loaded with cisplatin (CDDP, SigmaAldrich) in water at 37oC (pH 9.0). Free cisplatin was removed using a NAP-10 desalinating column (Sephadex G-25). The concentration of Pt(II) in nanogels was measured by X-ray fluorescent analysis on a ReSPECT substance composition analyzer (Tolokonnikov Company) using the “dry drop” method with molybdenum as the inner standard. Nanogels were conjugated with monoclonal antibodies to Cx43 (MAbCx43) via flexible PEG bridges. Antibodies were isolated from ascitic fluid of BALB/c mouse by affinity chromatography on protein-A agarose and dialyzed against PBS. Immunoglobulins G served as control. The procedure was as follows: 10fold molar excess of 2-iminathiolane (Thermo Scientific) was added to antibody solution in 0.1 M borate buffer with 5 mM EDTA (pH 8) and mixed for 45 min at ambient temperature. Activated antibodies were then incubated with 10-fold molar excess of maleimide-PEG-NH2 (NH2-PEG-MAL, Creative PEGWorks) at 4oC. The resultant pegylated antibodies (MAb-PEG-NH2) were purified by repeated centrifugation using Amicon YM-30 filters (PBS, pH 7.4). The nanogel carboxyl groups were then activated (10 eq. N-(3-dimethyl-aminopropyl)-N-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (NHS; pH 5.5-6.0; 10 min), purified on a NAP-10 column (PBS, pH 7.4), and mixed with MAb-PEG-NH2 (PBS, pH 7.4, 4oC, 8 h). Nanogel-antibody conjugates were purified by gel filtration on a 2×50 cm column packed with Sephadex CL-6B (PSB pH 7.4, flow velocity 0.5 ml/min) with an UV detector (280 nm). The appropriate fractions of eluted samples were concentrated on Amicon YM-30 filters and the content of conjugated antibodies was measured using microBCA kits (Thermo Scientific) according to manufacturer’s instruction. The concentration of conjugated antibodies was calculated as the proportion of antibody weight (μg) to nanogel weight (mg). Analysis of vector nanogels. The size, polydispersion, and charge (ζ-potential) of nanogels were
525 measured by dynamic light scatter on a Zetasizer Nano ZS analyzer (Malvern Instruments Ltd.). Specific activities of antibodies conjugated with nanogels were verified by ELISA with immobilized recombinant antigen (Cx43 extracellular fragment) [1]. Peroxidase-labeled polyclonal antibodies A3682 (Sigma-Aldrich) served as the second antibodies. The immunoperoxidase reaction was developed with TMB ready-to-use solution (Invitrogen). Enzyme activity of specific immune complexes formed on the solid phase were detected on a spectrophotometer (Bio-tec ELx800) at λ=450 nm. Accumulation of vector nanogels in C6 glioma cells. Rat C6 glioma cells (ATCC No. CCL107) were cultured in basic DMEM with 1% 100 mM sodium pyruvate, 1% 200 mM L-glutamine, 1% antibioticantimycotic (10,000 U/ml penicillin, 10,000 μg/ml streptomycin, and 25 μg/ml amphotericin), and 10% newborn calf serum (Invitrogen). Dissociation was carried out with 0.05% trypsin-EDTA (Invitrogen). Glioma C6 cells were cultured in Petri dishes (Coverglas Bottom Dish; Ted Pella) to 80% confluence, after which LysoTracker Red lysosomal probe (Invitrogen) was added (0.2 μl/100 μl medium) and after 30-min incubation, fluorescein-labeled nanogels (0.5 mg/ml) were added. The samples were incubated for 1 h at ambient temperature and 5% CO2, then washed thoroughly with PBS, and fluorescent analysis was carried out on A1R MP laser confocal microscope (Nikon). Antitumor activity of CDDP-loaded nanogels. The study was carried out on 3-month-old adult female Wistar rats (230-280 g; n=31). Intracranial C6 glioma was induced by stereotaxic implantation of 4×105 C6 glioma cells as described previously [3]. Magnetic resonance tomography of the brain was carried in all rats on day 5 after implantation. The animals with verified glioma growth were distributed into groups, 5-7 per group, by the mean volume of the tumor. The drugs were injected intravenously (200 μl; 5 mg/kg cisplatin) on days 7, 14, 21 after glioma implantation. Group 1 rats received free cisplatin, group 2 ones were injected with Cx43 nanogel/cisplatin, group 3 with IgG nanogel/cisplatin, and group 4 rats received nanogel/cisplatin. Group 5 animals (control) received 5% glucose solution (solvent for all drugs). Magnetic imaging was carried out on a ClinScan 7T magnetic resonance tomograph (Bruker). For obtaining T2-weighed images in the coronary plane, frequency separation of fat signals was used with the following Turbo Spin Echo (TSE) sequence parameters: TR/TE=4000/43 msec, 0.5 mm section thickness, 288×320 matrix, FOV=40 mm. Parameters for transverse plane T2-weighed images were as follows: TR/ TE=5110/57 msec, section thickness 0.5 mm, 288×320 matrix, FOV=40 mm. Dynamic morphometric analysis
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of glioma volume on T2 weighed images was carried out using ImageJ software (National Institute of Mental Health). Morphometric data were statistically processed by the nonparametric Mann–Whitney test for two unrelated groups at p≤0.05 level of significance.
RESULTS Nanogels are ultrasoft nanomaterials consisting of polymeric hydrophilic or amphiphilic chains [8]. Negatively charged nanogels (140-150 nm, -25±5 mV) synthesized by us did not aggregate and remained stable at pH 4.5-11.0 and concentrations of up to 7 mg/ml. In order to render vector characteristics to the nanogels, they were conjugated with monoclonal antibodies to Cx43 (MAbCx43) via PEG bridges. At stage 1, the antibodies were thiolated using Trout reagent (10-fold molar excess). Increasing the amount of thiolating agent (to 30 eq.) led to inactivation of antibod-
ies, which was confirmed by ELISA. After pegylated antibodies were prepared, they were conjugated with NHS-activated nanogels. In addition to protection of activity of conjugated monoclonal antibodies (MAb), it was essential to make them available for binding with specific antigen, and hence, flexible PEG bridges consisting of 170 monomer moieties were used. The final product was purified by gel filtration chromatography removing free antibodies not fixed to the nanogel (Fig. 1, b, fraction 2), whereas MAb-modified and nonmodified nanogels remained in the mixture (Fig. 1, b, fraction 1). As the nanogels were modified using a 10-fold molar excess of pegylated antibodies, the content of nonmodified nanogels in the final mixture was most likely extremely low, if any. Protein concentration in vector nanogel preparations was 85±5 μg/mg nanogel. Analysis of MAbCx43 nanogels by ELISA showed that 60% initial immunocytochemical activity of conjugated antibodies was preserved. Similar conju-
Fig. 1. Gel filtration chromatography on Sepharose CL-6B. a) Calibration curves with dextrane blue (I; 200 kDa) and yeast alcoholdehydrogenase (II; 150 kDa); b) separation of reaction mixture containing MAb-nanogel; fractions (1) and (2) correspond to MAb-nanogel and free MAb; c) fluorescence intensity of nonmodified nanogels (1) and FITC-IgG-nanogels (2).
N. V. Nukolova, V. P. Baklaushev, et al.
gation of nanogels with fluorescence-labeled antibodies (FITC-IgG) was carried out. Fluorescence intensity of purified conjugates was studied by spectrophotometry (492 nm). The difference in the fluorescence intensities between the vector and nonvector (negative control) nanogels indicated effective modification of nanogels with FITC-labeled antibodies (Fig. 1, c). The hydrodynamic diameter of the nanogels virtually did not change after conjugation with antibodies (120-130 nm vs. 140-150 nm). However, ζ-potential of MAb nanogel increased significantly (from -25±2 to -17±3 mV). This could be attributed to partial shielding of the charge by conjugated antibodies and by lesser mobility of negatively charged PMAA segments. Dispersion of MAb nanogels remained stable in saline (0.15 M NaCl; pH 7.4). Immunochemical activity of conjugated antibodies was preserved for at least 15 days (4oC and -20oC). Antitumor drug cisplatin (CDDP) was added to the nanogel at optimal pH of the medium and drug/ nanogel molar proportion [CDDP]/[COOH]=0.5 [10]. Cisplatin binds to PMAA blocks in the nanogel via the formation of a coordination complex with Pt(II). The size of nanogels loaded with the drug decreased
527 and ζ-potential increased, which indicated neutralization of the nanogel carboxyl groups and effective incorporation of the drug in the nanogels (110±5 nm, -12±3 mV). The nanogel capacity (drug weight/loaded nanogel weight ratio) reached 35±3%. Importantly, CDDP was slowly released from the nanogels and the velocity of this process depended on medium ionic strength and pH [8,9]. The release was more rapid at pH
