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CT-imaging guided drug delivery
Accepted Manuscript Title: Preparation and 68 Ga-radiolabeling of porous zirconia nanoparticle platform for PET/CT-imaging guided drug delivery operations Authors: Andras Polyak, L´ıvia Nasz´alyi Nagy, Judith Mihaly, Sebastian G¨orres, Alexander Wittneben, Ina Leiter, Jens P. Bankstahl, Laszlo Sajti, Mikl´os Kellermayer, Mikl´os Zr´ınyi, Tobias L. Ross PII: DOI: Reference:
S0731-7085(16)31014-7 http://dx.doi.org/doi:10.1016/j.jpba.2017.01.028 PBA 11035
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
7-11-2016 9-1-2017 12-1-2017
Please cite this article as: Andras Polyak, L´ıvia Nasz´alyi Nagy, Judith Mihaly, Sebastian G¨orres, Alexander Wittneben, Ina Leiter, Jens P.Bankstahl, Laszlo Sajti, Mikl´os Kellermayer, Mikl´os Zr´ınyi, Tobias L.Ross, Preparation and 68Garadiolabeling of porous zirconia nanoparticle platform for PET/CT-imaging guided drug delivery operations, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2017.01.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
Zirconia NPs as inorganic drug delivery systems have been examined previously. Aims: prospective PET imaging guided therapeutic operations with the NP system. DOTA bifunctional chelator has been attached onto the nanoparticles’ surface. DOTA-adsorbed zirconia nanoparticles were labeled with 68Ga PET-radioisotope. Preliminary biodistribution results were evaluated with microPET/CT.
1
Preparation and 68Ga-radiolabeling of porous zirconia nanoparticle platform for PET/CT-imaging guided drug delivery operations Andras Polyak1*, Lívia Naszályi Nagy2,3, Judith Mihaly3, Sebastian Görres1, Alexander Wittneben1, Ina Leiter1,4, Jens P. Bankstahl1, Laszlo Sajti5, Miklós Kellermayer2, Miklós Zrínyi2,6, Tobias L. Ross1 *Corresponding author 1
Department of Nuclear Medicine, Hannover Medical School Carl-Neuberg Str 1, 30627 Hannover, Germany 2
MTA-SE Molecular Biophysics Research Group, Semmelweis University, Budapest Tűzoltó Str 37-47, H-1094 Budapest, Hungary 3
Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar Tudósok Blvd 2, H-1117 Budapest, Hungary 4
Institute for Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine, Hannover Bünteweg 2, 30559 Hannover Germany 5
Nanotechnology Department, Laser Zentrum Hannover e.V., Hollerithallee 8, D-30419 Hannover, Germany 6
Nanochemistry Research Group, Semmelweis University, Budapest Nagyvárad Sqr 4, 1089 Budapest, Hungary
Abstract This paper describes the preparation of gallium-68 (68Ga) isotope labeled porous zirconia (ZrO2) nanoparticle (NP) platform of nearly 100 nm diameter and its first pharmacokinetic and biodistribution evaluation accomplished with a microPET/CT (µPet/CT) imaging system. Objectives of the investigations were to provide a nanoparticle platform which can be suitable for specific delivery of various therapeutic drugs using surface attached specific molecules as triggering agents, and at the same time, suitable for positron emission tomography (PET) tracing of the prospective drug delivery process. Radiolabeling was accomplished using DOTA bifunctional chelator. DOTA was successfully adsorbed onto the surface of nanoparticles, while the 68Ga-radiolabeling method proved to be simple and effective. In the course of biodistribution studies, the 68Ga-labeled DOTA-ZrNPs showed proper radiolabeling stability in their original suspension and in blood serum. µPet/CT imaging studies confirmed a RES-biodistribution profile indicating stable nano-sized labeled particles in vivo. Results proved that the new method offers the opportunity to examine further specifically targeted and drug payload carrier variants of zirconia NP systems using PET/CT imaging. Keywords:
68Ga,
nanoparticles,
DOTA,
PET,
2
theranostics,
imaging
guided
therapy
1. Introduction PET and SPECT molecular imaging methods can be of support for the development of new drug encapsulating and releasing micro- and nanoparticle (NP) systems, and at the same time, these pharmaceutical developments offer the opportunity to present new molecular imaging (diagnostic) and endoradiotherapeutic modalities, as well [1]. An effective theranostic drug delivery system shall be multifunctional: its characteristics shall make it suitable to transport and deliver a payload (drug molecule) with high drug loading capacity to the triggered tissue and cellular environment using surface attached targeting agents; and optionally, to carry contrast agent for tracing the whole process with non-invasive imaging method such as positron emission tomography [2]. Besides numerous organic based drug delivery systems (lipid membrane based compounds, solid biopolymer NPs, core-shell nanostructures) [3, 4], inorganic based drug carrier nanosystems have been also produced from different metals (Au, Fe, Ag) [5], metalloids (Si), transition-metals (Zr) or the latter’s oxides such as silica and zirconia [6, 7]. Biocompatible ceramic material zirconia is used for several biomedical applications such as dental and chirurgical implants [8, 9], because it shows advantageous bioactivity for osteoporosis. Some research groups have overcome synthetic difficulties and have prepared different drug carrier systems using zirconia [10, 11]. In our previous studies, zirconia based NP systems as potential prospective inorganic drug delivery systems have been examined [6, 7]. Affinity of the studied porous zirconia NPs for adsorbing significant quantity of carboxyl and/or amine bearing drug molecules (in one single synthetic step) was studied by means of three anticancer agents: D,L-a-difluoromethylornithine (DFMO, eflornithine), doxorubicin and ursolic acid. Resulted anticancer drug loaded particles were radiolabeled with a Technetium-99m (99mTc) isotope and studied in SPECT/CT normal biodistribution studies and they proved to be stable in physiological medium [7]. Native zirconia surface itself was suitable for adsorbing the radiolanthanide lutetium-177 without application of complexing molecule parts and the 177Lu-labeled compound was tested as a beta-emitter therapeutic radiopharmaceutical candidate for local, specific radiotherapeutic applications [12]. The present paper describes briefly the preparation of DOTA (tetraazacyclododecane-1,4,7,10tetraacetic acid) bifunctional chelator adsorbed and gallium-68 (68Ga) isotope labeled porous zirconia (ZrO2) nanoparticle (NP) variant and its first in vivo µPET/CT pharmacokinetic evaluation. Posttransition metal radioisotope 68Ga (T½=67.6 min, 1.89 MeV, 89%) is positron emitter nuclide and ideal for shorter tracing period PET investigations. Besides,
68
Ga has the advantage that as daughter of
Germanium-68 (T½=270.8 d) and it can be produced from locally installed 68Ge/68Ga generators. 3
The aims of investigations were to provide a nanoparticle platform which can be suitable for prospective specific delivery of various therapeutic drugs using surface attached specific molecules as triggering agents, and at the same time, suitable for PET tracing of the drug delivery process [13]. By proving the successful DOTA adsorption, we also aimed at providing an easily applicable (switchable) method for radiolabeling with several further radioisotopes including
67
Ga and
111
In for SPECT
imaging, 64Cu for extended tracing time PET studies, 166Ho, 90Y for radionuclide therapy and the 44Sc47
Sc isotope pair in order to achieve radiotheranostic (combined PET and endoradiotherapeutic)
modalities [1].
2. Materials and methods Zirconia nanoparticles have been synthesized in ethanol. The synthesis was based on a method previously described [14]. (See details of preparation in supplementary data.) Briefly, 0.4 ml of 0.1 M CsCl solution was added to 100 ml of abs. ethanol and heated to 60°C while stirring at 300 rpm. The reaction was carried out under argon gas. After homogenization, 3.2 ml of TBOZ was quickly added to it. 4 hours later the reaction mixture was centrifuged at 4000 rpm for 10 min and the sediment was discarded. The solid content of the stable white supernatant was 2.5±0.1 mg/ml. The product was kept at 4°C. DOTA bifunctional chelator was adsorbed on the surface of zirconia NPs in ethanol (0.5 mg DOTA/mg ZrO2). 24 hours later the product was dialyzed first against MilliQ water, and then three times against 0.25 M ammonium acetate buffer pH 4.1 (solid content 2.08±0.1 mg/ml). In parallel, the same experimental setup was used to obtain ZrO2-DOTA in HEPES buffer pH 8.0 (solid content 2.5±0.1 mg/ml), as well as native ZrO2 in both buffers. Size distribution evaluation of the samples was performed by dynamic light scattering (DLS) on an AVID Nano w310i instrument and by scanning electron microscopy (SEM). In order to evaluate the strength of DOTA adsorption, zeta potential values of the samples were determined at 20°C using a Malvern Zetasizer Nano ZS (Malvern, Worcs, UK) equipped with a He–Ne laser (l = 633 nm) and a backscatter detector at a fixed angle of 173°. SEM images were recorded on a Quanta 400F (Fei company, The Netherlands). For sample preparation, a drop of the solution was placed on a carboncoated sample disc and dried at room temperature. The diameters of 200 particles were measured with Image J software and classified to obtain an average Feret particle size distribution. The surface structure of ZrO2 and ZrO2-DOTA samples was investigated with attenuated total reflection infrared (ATR-FTIR) spectroscopy using a Varian Scimitar 2000 FTIR spectrometer (Varian Inc.) equipped with an MCT (mercury–cadmium–telluride) detector and a single reflection ATR unit (‘Golden Gate’, SPECAC Ltd, UK) with a diamond ATR element. Scans were performed in the wavenumber region 4000–500 cm-1. In general, 4 cm-1 resolution and records of 128 scans were applied. 4
68
Ga isotope was obtained from
68
Ge/68Ga isotope generator (Cyclotron Co. Obninsk, Russia).
68
Ga
was available in aqueous solution after preconditioning by AG 1-X8 Resin (Bio-Rad) columns. For radiolabeling, 0.2 mg of ZrO2-DOTA-NPs were incubated in 68Ga eluate for 20 mins at 95°C in HEPES buffer (pH: 4). Radiolabeling efficiency was checked in pH 4.0 HEPES buffered original suspension and in blood serum up to 180 minutes postlabeling by thin layer chromatography (TLC). In course of PET/CT biodistribution studies, Mus Musculus C57BL/6N mice were injected with 12 MBq activity of the 68Ga-labeled nanosystem. Animals were scanned by Siemens Inveon µPET/CT imaging system, in vivo results were processed by PMOD software.
3. Results and discussion The stable supernatant of ZrO2-DOTA-NP synthesis has a main particle population of 94±19 nm diameter with a low amount of larger secondary particles according to DLS measurement (see Fig 1, part A). SEM investigations revealed very similar size distribution with an average of 112±18 nm showing only insignificant amount of larger aggregates (see Fig 1, part B and low magnification images in Fig. S-2 and S-3 in E. Supplementary Information). Solid content was 2.45 mg/ml. Zeta potential of ZrO2-DOTA-NPs in pH 8.0 HEPES buffer was found to be -17±1,7 mV while in pH 4.1 acetate buffer it was 25.2±1.0 mV. In the case of acetate buffer we can suggest that acetate ions are adsorbing to the surface of zirconia (see comparison to non-buffered zirconia zeta potential-pH values shown by dotted arrow in Fig 2 A). The zeta potential values of DOTA-modified samples are shifted towards lower charge (solid arrows in Fig 2 A), as expected due to the zwitterionic nature of DOTA (-5.44±0.3 mV in HEPES, 20.7±1.2 mV in acetate buffer). This indicates a strong interaction between zirconia and DOTA chelator, and the resulting product is expected to be stable at pH 4 due to its relatively high surface charge.
5
Figure 1. Part A: Mass averaged size distribution function of zirconia NPs evaluated by DLS. Part B: representative SEM image of zirconia NPs
For ZrO2-DOTA-NP samples, at both pH values, some low-intensity IR bands prove the attachment of the DOTA chelator (marked by dotted lines in Fig 1, Part B). The bands at 1558 and at 1482, 1453 and 1386 cm-1 are assigned to the antisymmetric and symmetric stretching modes, respectively, of COO -, supporting the surface adsorption of DOTA. The band at 1661 cm-1 may be assigned to the un-ionized COOH groups of DOTA [15]. The presence of hydrogen carbonate (HCO3- at 1625 and 1457 cm-1) and of monodentate carbonate (m-CO32- at 1540 and 1385 cm-1) species on native porous ZrO2 nanoparticle surface at neutral pH was already demonstrated by ATR-FTIR spectroscopy [7]. Using buffers with different pH, the character of surface species is markedly changed. At acidic pH (pH=4) using ammonium-acetate buffer the amount of the surface adsorbed hydrogen carbonate is suppressed and the IR spectrum is dominated by characteristic stretching bands of protonated carboxylate (1708 cm-1) and those of ionic carboxylate (asCOO- at 1552 cm-1 and sCOO- at 1413 and 1278 cm-1). It seems plausible that also the buffer acetate adsorbs to the zirconia surface and this result is confirmed by zeta potential experiments. As to the zirconia surface at pH=8 (HEPES buffer), again, the bands belonging to hydrogen carbonate surface species are suppressed (the shifted band towards 1664 cm-1 belongs to ionic HCO3-), while new bands around 1552 and 1417 cm-1 suggest the preferable formation of surface carboxylate species. (The broad, strong band at 1183 cm -1 belongs to the -SO2 stretching of the HEPES molecules.)
6
Figure 2. Part A: Zeta-potential values of native surface ( ZrO2) and DOTA-bearing buffered zirconia ( ZrO2-DOTA) samples compared to native surface non-buffered zirconia (, the pH was set using HCl and NaOH solutions). Acetate adsorption is shown by dotted arrow, DOTA adsorption is shown by solid arrows. Part B: IR spectra of adsorbed carbonate/carboxylate species on ZrO2 and ZrO2-DOTA samples at pH=4 and pH=8, compared with those of native porous zirconia (ZrO2).
68
The Ga-radiolabeling showed high efficiency and proper stability in original buffered NP suspension as well as in blood serum. In pH 4.0 HEPES buffer, the labeling efficiency was between 90.5% and 97.5% up to 180 minutes while 80.2% to 94.7% in human blood serum. Control DLS examinations of 68
Ga-DOTA-ZrNP system confirmed proper particle size stability after the 68Ga-labeling process.
7
Figure 3. Parts A and B: Representative µPET/CT slice series of 68Ga-DOTA-ZrNP system in two C57BL/6N mice (A and B series) 60 minutes after i.v. application. Part C: time-activity curve recorded by µPET/CT up to 60 minutes. Massive and permanent RES organ (liver, spleen) uptake, slight lung activity, extended blood retention (concluded by heart activities) and slow excretion via kidneys could be observed. In PET layers (in A and B), the brighter tone represents the higher activity concentration.
8
Experimental animals tolerated i.v. application of NPs well, no side effects could be recorded during the entire in vivo imaging study and in the post-anaesthesia period. µPET/CT imaging studies confirmed a typical RES-biodistribution profile specified to nanocolloid based products [16], visually (see Fig 3, A-B) and quantitatively (Fig 3, section C) reinforcing the stable particle size. In the two animals most of the activity (more than 70% of ID) has been accumulated in the main RES organs liver (avg. 4.7391 ±0.4619 MBq/cm3 and 4.9358 ±0.2129 MBq/cm3 activity concentrations after 10 and 60 minutes, respectively) and spleen (1.9510 ±0.3633 MBq/cm3 activity concentration 35 min p.i.) [16]. Compared to the RES organ uptakes, relative low lung uptake and slowly decreasing could be detected during the entire tracing period (max. act. conc. was 0.6008 ±0.1709 MBq/cm 3 at 2.75 min p.i.), proving the lack of in vivo aggregated colloid fraction of NPs trapped by pulmonary lung capillaries [17], indicating that the very low amount of aggregates present in the sample is not supposed to interfere with the biological use. On the other hand, relative high and elongated traceable heart activities (max. act. conc. was 1.4857 ±0.0607 MBq/cm 3 at 2.75 min p.i.) and negligible kidney uptakes paired to slowly increasing urinary bladder values have been also suggested extended blood retention of NPs whereas slow excretion via kidneys and urinary tract.
4. Conclusions In conclusion, in vitro characterizations have confirmed that using our method, DOTA bifunctional chelator was successfully adsorbed onto the surface of ZrO2 nanoparticles while the following characterizations confirmed strong DOTA-adsorption, then after the
68
Ga-radiolabeling method in
68
pH4 environment at 95°C proved to be simple and effective. The new Ga-DOTA-ZrNP complex had durable radiochemical and colloidal stability after p.i., as well. Furthermore, in vivo µPET/CT biodistribution trials have confirmed the product’s in vivo radioisotope-adsorbing and size stability; and therefore demonstrated the possibility of achieving prospective PET/CT-imaging guided drug delivery operations using targeted and drug loaded variants of
68
Ga-DOTA-labeled porous zirconia
nanoparticle platforms. Further research with active targeted and radiolabeled ZrO2 nanoparticles is still needed to explore the potential of these inorganic based nanocarrier systems.
Acknowledgements This study was supported by the project K 115259 of the Hungarian Scientific Research Fund (OTKA, Hungary). The authors also received financial support from the Deutsche Forschungsgemeinschaft within the excellence cluster REBRITH (Exc62/1), and from the Volkswagenstiftung within the project of Biofabrication for NIFE.
9
References [1] K. Stockhofe, J.M. Postema, H. Schieferstein, T.L. Ross, Radiolabeling of nanoparticles and polymers for PET imaging. Pharmaceuticals, 7 (2014) 392-418. [2] R. Rossin, D. Pan, K. Qi, J.L. Turner, X. Sun, K.L. Wooley, M.J. Welch, 64Cu-labeled folateconjugated shell cross-linked nanoparticles for tumor imaging and radiotherapy: Synthesis, radiolabeling, and biologic evaluation. Journal of Nuclear Medicine, 46 (2005) 1210-1218. [3] V.P. Torchilin, Immunoliposomes and PEGylated immunoliposomes: possible use for targeted delivery of imaging agents. ImmunoMethods, 4 (1994) 244-258. [4] A. Polyak, I. Hajdu, M. Bodnar, G. Trencsenyi, Z. Postenyi, V. Haasz, G. Janoki, G.A. Janoki, L. Balogh, J. Borbely, (99m)Tc-labelled nanosystem as tumour imaging agent for SPECT and SPECT/CT modalities. International Journal of Pharmaceutics, 449 (2013) 10-17. [5] M. Liong, J. Lu, M. Kovochich, T. Xia, S.G. Ruehm, A.E. Nel, F. Tamanoi, J.I. Zink, Multifunctional inorganic nanoparticles for imaging, targeting, and drug delivery. ACS Nano, 2 (2008) 889-896. [6] L. Naszalyi Nagy, A. Polyak, J. Mihaly, A. Szecsenyi, I.C. Szigyarto, Z. Czegeny, E. Jakab, P. Nemeth, B. Magda, P. Szabo, Z. Veres, K. Jemnitz, I. Bertoti, R.P. Joba, G. Trencsenyi, L. Balogh, A. Bota, Silica@zirconia@poly(malic acid) nanoparticles: promising nanocarriers for theranostic applications. Journal of Materials Chemistry B, 4 (2016) 4420-4429. [7] L.N. Nagy, J. Mihály, A. Polyák, B. Debreczeni, B. Császár, I.C. Szigyártó, A. Wacha, Z. Czégény, E. Jakab, S. Klébert, Inherently fluorescent and porous zirconia colloids: preparation, characterization and drug adsorption studies. Journal of Materials Chemistry B, 3 (2015) 7529-7537. [8] M. Hisbergues, S. Vendeville, P. Vendeville, Zirconia: Established facts and perspectives for a biomaterial in dental implantology. Journal of biomedical materials research. Part B, Applied Biomaterials, 88 (2009) 519-529. [9] R.J. Kohal, M. Bachle, W. Att, S. Chaar, B. Altmann, A. Renz, F. Butz, Osteoblast and bone tissue response to surface modified zirconia and titanium implant materials. Dental materials : Official Publication of the Academy of Dental Materials, 29 (2013) 763-776. [10] D. Manoharan, A. Loganathan, V. Kurapati, V.J. Nesamony, Unique sharp photoluminescence of size-controlled sonochemically synthesized zirconia nanoparticles. Ultrasonics Sonochemistry, 23 (2015) 174-184. [11] F. Masoodiyeh, J. Karimi-Sabet, A.R. Khanchi, M.R. Mozdianfard, Zirconia nanoparticle synthesis in sub and supercritical water — particle morphology and chemical equilibria. Powder Technology, 269 (2015) 461-469. [12] A. Polyak, L.N. Nagy, E. Drotár, G. Dabasi, R.P. Jóba, Z. Pöstényi, R. Mikolajczak, A. Bóta, L. Balogh, Lu-177-labeled zirconia particles for radiation synovectomy. Cancer Biotherapy and Radiopharmaceuticals, 30 (2015) 433-438. [13] T. Lammers, F. Kiessling, W.E. Hennink, G. Storm, Nanotheranostics and image-guided drug delivery: current concepts and future directions. Molecular Pharmaceutics, 7 (2010) 1899-1912. [14] J. Widoniak, S. Eiden-Assmann, G. Maret, Synthesis and Characterisation of Monodisperse Zirconia Particles. European Journal of Inorganic Chemistry, 2005 (2005) 3149-3155. [15] J.H. Lee, D.-Y. Jung, Immobilization of Gd (III)-DOTA Complexes in Layered Double Hydroxides Thin Film. Bull Korean Chem. Soc. 34 (2013) 3488-3490. [16] N. Bertrand, J.C. Leroux, The journey of a drug-carrier in the body: An anatomo-physiological perspective. Journal of Controlled Release, 161 (2012) 152-163. [17] H.L. Kutscher, P. Chao, M. Deshmukh, Y. Singh, P. Hu, L.B. Joseph, D.C. Reimer, S. Stein, D.L. Laskin, P.J. Sinko, Threshold size for optimal passive pulmonary targeting and retention of rigid microparticles in rats. Journal of controlled release : Official Journal of the Controlled Release Society, 143 (2010) 31-37.
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S0731-7085(16)31014-7 http://dx.doi.org/doi:10.1016/j.jpba.2017.01.028 PBA 11035
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
7-11-2016 9-1-2017 12-1-2017
Please cite this article as: Andras Polyak, L´ıvia Nasz´alyi Nagy, Judith Mihaly, Sebastian G¨orres, Alexander Wittneben, Ina Leiter, Jens P.Bankstahl, Laszlo Sajti, Mikl´os Kellermayer, Mikl´os Zr´ınyi, Tobias L.Ross, Preparation and 68Garadiolabeling of porous zirconia nanoparticle platform for PET/CT-imaging guided drug delivery operations, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2017.01.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
Zirconia NPs as inorganic drug delivery systems have been examined previously. Aims: prospective PET imaging guided therapeutic operations with the NP system. DOTA bifunctional chelator has been attached onto the nanoparticles’ surface. DOTA-adsorbed zirconia nanoparticles were labeled with 68Ga PET-radioisotope. Preliminary biodistribution results were evaluated with microPET/CT.
1
Preparation and 68Ga-radiolabeling of porous zirconia nanoparticle platform for PET/CT-imaging guided drug delivery operations Andras Polyak1*, Lívia Naszályi Nagy2,3, Judith Mihaly3, Sebastian Görres1, Alexander Wittneben1, Ina Leiter1,4, Jens P. Bankstahl1, Laszlo Sajti5, Miklós Kellermayer2, Miklós Zrínyi2,6, Tobias L. Ross1 *Corresponding author 1
Department of Nuclear Medicine, Hannover Medical School Carl-Neuberg Str 1, 30627 Hannover, Germany 2
MTA-SE Molecular Biophysics Research Group, Semmelweis University, Budapest Tűzoltó Str 37-47, H-1094 Budapest, Hungary 3
Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar Tudósok Blvd 2, H-1117 Budapest, Hungary 4
Institute for Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine, Hannover Bünteweg 2, 30559 Hannover Germany 5
Nanotechnology Department, Laser Zentrum Hannover e.V., Hollerithallee 8, D-30419 Hannover, Germany 6
Nanochemistry Research Group, Semmelweis University, Budapest Nagyvárad Sqr 4, 1089 Budapest, Hungary
Abstract This paper describes the preparation of gallium-68 (68Ga) isotope labeled porous zirconia (ZrO2) nanoparticle (NP) platform of nearly 100 nm diameter and its first pharmacokinetic and biodistribution evaluation accomplished with a microPET/CT (µPet/CT) imaging system. Objectives of the investigations were to provide a nanoparticle platform which can be suitable for specific delivery of various therapeutic drugs using surface attached specific molecules as triggering agents, and at the same time, suitable for positron emission tomography (PET) tracing of the prospective drug delivery process. Radiolabeling was accomplished using DOTA bifunctional chelator. DOTA was successfully adsorbed onto the surface of nanoparticles, while the 68Ga-radiolabeling method proved to be simple and effective. In the course of biodistribution studies, the 68Ga-labeled DOTA-ZrNPs showed proper radiolabeling stability in their original suspension and in blood serum. µPet/CT imaging studies confirmed a RES-biodistribution profile indicating stable nano-sized labeled particles in vivo. Results proved that the new method offers the opportunity to examine further specifically targeted and drug payload carrier variants of zirconia NP systems using PET/CT imaging. Keywords:
68Ga,
nanoparticles,
DOTA,
PET,
2
theranostics,
imaging
guided
therapy
1. Introduction PET and SPECT molecular imaging methods can be of support for the development of new drug encapsulating and releasing micro- and nanoparticle (NP) systems, and at the same time, these pharmaceutical developments offer the opportunity to present new molecular imaging (diagnostic) and endoradiotherapeutic modalities, as well [1]. An effective theranostic drug delivery system shall be multifunctional: its characteristics shall make it suitable to transport and deliver a payload (drug molecule) with high drug loading capacity to the triggered tissue and cellular environment using surface attached targeting agents; and optionally, to carry contrast agent for tracing the whole process with non-invasive imaging method such as positron emission tomography [2]. Besides numerous organic based drug delivery systems (lipid membrane based compounds, solid biopolymer NPs, core-shell nanostructures) [3, 4], inorganic based drug carrier nanosystems have been also produced from different metals (Au, Fe, Ag) [5], metalloids (Si), transition-metals (Zr) or the latter’s oxides such as silica and zirconia [6, 7]. Biocompatible ceramic material zirconia is used for several biomedical applications such as dental and chirurgical implants [8, 9], because it shows advantageous bioactivity for osteoporosis. Some research groups have overcome synthetic difficulties and have prepared different drug carrier systems using zirconia [10, 11]. In our previous studies, zirconia based NP systems as potential prospective inorganic drug delivery systems have been examined [6, 7]. Affinity of the studied porous zirconia NPs for adsorbing significant quantity of carboxyl and/or amine bearing drug molecules (in one single synthetic step) was studied by means of three anticancer agents: D,L-a-difluoromethylornithine (DFMO, eflornithine), doxorubicin and ursolic acid. Resulted anticancer drug loaded particles were radiolabeled with a Technetium-99m (99mTc) isotope and studied in SPECT/CT normal biodistribution studies and they proved to be stable in physiological medium [7]. Native zirconia surface itself was suitable for adsorbing the radiolanthanide lutetium-177 without application of complexing molecule parts and the 177Lu-labeled compound was tested as a beta-emitter therapeutic radiopharmaceutical candidate for local, specific radiotherapeutic applications [12]. The present paper describes briefly the preparation of DOTA (tetraazacyclododecane-1,4,7,10tetraacetic acid) bifunctional chelator adsorbed and gallium-68 (68Ga) isotope labeled porous zirconia (ZrO2) nanoparticle (NP) variant and its first in vivo µPET/CT pharmacokinetic evaluation. Posttransition metal radioisotope 68Ga (T½=67.6 min, 1.89 MeV, 89%) is positron emitter nuclide and ideal for shorter tracing period PET investigations. Besides,
68
Ga has the advantage that as daughter of
Germanium-68 (T½=270.8 d) and it can be produced from locally installed 68Ge/68Ga generators. 3
The aims of investigations were to provide a nanoparticle platform which can be suitable for prospective specific delivery of various therapeutic drugs using surface attached specific molecules as triggering agents, and at the same time, suitable for PET tracing of the drug delivery process [13]. By proving the successful DOTA adsorption, we also aimed at providing an easily applicable (switchable) method for radiolabeling with several further radioisotopes including
67
Ga and
111
In for SPECT
imaging, 64Cu for extended tracing time PET studies, 166Ho, 90Y for radionuclide therapy and the 44Sc47
Sc isotope pair in order to achieve radiotheranostic (combined PET and endoradiotherapeutic)
modalities [1].
2. Materials and methods Zirconia nanoparticles have been synthesized in ethanol. The synthesis was based on a method previously described [14]. (See details of preparation in supplementary data.) Briefly, 0.4 ml of 0.1 M CsCl solution was added to 100 ml of abs. ethanol and heated to 60°C while stirring at 300 rpm. The reaction was carried out under argon gas. After homogenization, 3.2 ml of TBOZ was quickly added to it. 4 hours later the reaction mixture was centrifuged at 4000 rpm for 10 min and the sediment was discarded. The solid content of the stable white supernatant was 2.5±0.1 mg/ml. The product was kept at 4°C. DOTA bifunctional chelator was adsorbed on the surface of zirconia NPs in ethanol (0.5 mg DOTA/mg ZrO2). 24 hours later the product was dialyzed first against MilliQ water, and then three times against 0.25 M ammonium acetate buffer pH 4.1 (solid content 2.08±0.1 mg/ml). In parallel, the same experimental setup was used to obtain ZrO2-DOTA in HEPES buffer pH 8.0 (solid content 2.5±0.1 mg/ml), as well as native ZrO2 in both buffers. Size distribution evaluation of the samples was performed by dynamic light scattering (DLS) on an AVID Nano w310i instrument and by scanning electron microscopy (SEM). In order to evaluate the strength of DOTA adsorption, zeta potential values of the samples were determined at 20°C using a Malvern Zetasizer Nano ZS (Malvern, Worcs, UK) equipped with a He–Ne laser (l = 633 nm) and a backscatter detector at a fixed angle of 173°. SEM images were recorded on a Quanta 400F (Fei company, The Netherlands). For sample preparation, a drop of the solution was placed on a carboncoated sample disc and dried at room temperature. The diameters of 200 particles were measured with Image J software and classified to obtain an average Feret particle size distribution. The surface structure of ZrO2 and ZrO2-DOTA samples was investigated with attenuated total reflection infrared (ATR-FTIR) spectroscopy using a Varian Scimitar 2000 FTIR spectrometer (Varian Inc.) equipped with an MCT (mercury–cadmium–telluride) detector and a single reflection ATR unit (‘Golden Gate’, SPECAC Ltd, UK) with a diamond ATR element. Scans were performed in the wavenumber region 4000–500 cm-1. In general, 4 cm-1 resolution and records of 128 scans were applied. 4
68
Ga isotope was obtained from
68
Ge/68Ga isotope generator (Cyclotron Co. Obninsk, Russia).
68
Ga
was available in aqueous solution after preconditioning by AG 1-X8 Resin (Bio-Rad) columns. For radiolabeling, 0.2 mg of ZrO2-DOTA-NPs were incubated in 68Ga eluate for 20 mins at 95°C in HEPES buffer (pH: 4). Radiolabeling efficiency was checked in pH 4.0 HEPES buffered original suspension and in blood serum up to 180 minutes postlabeling by thin layer chromatography (TLC). In course of PET/CT biodistribution studies, Mus Musculus C57BL/6N mice were injected with 12 MBq activity of the 68Ga-labeled nanosystem. Animals were scanned by Siemens Inveon µPET/CT imaging system, in vivo results were processed by PMOD software.
3. Results and discussion The stable supernatant of ZrO2-DOTA-NP synthesis has a main particle population of 94±19 nm diameter with a low amount of larger secondary particles according to DLS measurement (see Fig 1, part A). SEM investigations revealed very similar size distribution with an average of 112±18 nm showing only insignificant amount of larger aggregates (see Fig 1, part B and low magnification images in Fig. S-2 and S-3 in E. Supplementary Information). Solid content was 2.45 mg/ml. Zeta potential of ZrO2-DOTA-NPs in pH 8.0 HEPES buffer was found to be -17±1,7 mV while in pH 4.1 acetate buffer it was 25.2±1.0 mV. In the case of acetate buffer we can suggest that acetate ions are adsorbing to the surface of zirconia (see comparison to non-buffered zirconia zeta potential-pH values shown by dotted arrow in Fig 2 A). The zeta potential values of DOTA-modified samples are shifted towards lower charge (solid arrows in Fig 2 A), as expected due to the zwitterionic nature of DOTA (-5.44±0.3 mV in HEPES, 20.7±1.2 mV in acetate buffer). This indicates a strong interaction between zirconia and DOTA chelator, and the resulting product is expected to be stable at pH 4 due to its relatively high surface charge.
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Figure 1. Part A: Mass averaged size distribution function of zirconia NPs evaluated by DLS. Part B: representative SEM image of zirconia NPs
For ZrO2-DOTA-NP samples, at both pH values, some low-intensity IR bands prove the attachment of the DOTA chelator (marked by dotted lines in Fig 1, Part B). The bands at 1558 and at 1482, 1453 and 1386 cm-1 are assigned to the antisymmetric and symmetric stretching modes, respectively, of COO -, supporting the surface adsorption of DOTA. The band at 1661 cm-1 may be assigned to the un-ionized COOH groups of DOTA [15]. The presence of hydrogen carbonate (HCO3- at 1625 and 1457 cm-1) and of monodentate carbonate (m-CO32- at 1540 and 1385 cm-1) species on native porous ZrO2 nanoparticle surface at neutral pH was already demonstrated by ATR-FTIR spectroscopy [7]. Using buffers with different pH, the character of surface species is markedly changed. At acidic pH (pH=4) using ammonium-acetate buffer the amount of the surface adsorbed hydrogen carbonate is suppressed and the IR spectrum is dominated by characteristic stretching bands of protonated carboxylate (1708 cm-1) and those of ionic carboxylate (asCOO- at 1552 cm-1 and sCOO- at 1413 and 1278 cm-1). It seems plausible that also the buffer acetate adsorbs to the zirconia surface and this result is confirmed by zeta potential experiments. As to the zirconia surface at pH=8 (HEPES buffer), again, the bands belonging to hydrogen carbonate surface species are suppressed (the shifted band towards 1664 cm-1 belongs to ionic HCO3-), while new bands around 1552 and 1417 cm-1 suggest the preferable formation of surface carboxylate species. (The broad, strong band at 1183 cm -1 belongs to the -SO2 stretching of the HEPES molecules.)
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Figure 2. Part A: Zeta-potential values of native surface ( ZrO2) and DOTA-bearing buffered zirconia ( ZrO2-DOTA) samples compared to native surface non-buffered zirconia (, the pH was set using HCl and NaOH solutions). Acetate adsorption is shown by dotted arrow, DOTA adsorption is shown by solid arrows. Part B: IR spectra of adsorbed carbonate/carboxylate species on ZrO2 and ZrO2-DOTA samples at pH=4 and pH=8, compared with those of native porous zirconia (ZrO2).
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The Ga-radiolabeling showed high efficiency and proper stability in original buffered NP suspension as well as in blood serum. In pH 4.0 HEPES buffer, the labeling efficiency was between 90.5% and 97.5% up to 180 minutes while 80.2% to 94.7% in human blood serum. Control DLS examinations of 68
Ga-DOTA-ZrNP system confirmed proper particle size stability after the 68Ga-labeling process.
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Figure 3. Parts A and B: Representative µPET/CT slice series of 68Ga-DOTA-ZrNP system in two C57BL/6N mice (A and B series) 60 minutes after i.v. application. Part C: time-activity curve recorded by µPET/CT up to 60 minutes. Massive and permanent RES organ (liver, spleen) uptake, slight lung activity, extended blood retention (concluded by heart activities) and slow excretion via kidneys could be observed. In PET layers (in A and B), the brighter tone represents the higher activity concentration.
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Experimental animals tolerated i.v. application of NPs well, no side effects could be recorded during the entire in vivo imaging study and in the post-anaesthesia period. µPET/CT imaging studies confirmed a typical RES-biodistribution profile specified to nanocolloid based products [16], visually (see Fig 3, A-B) and quantitatively (Fig 3, section C) reinforcing the stable particle size. In the two animals most of the activity (more than 70% of ID) has been accumulated in the main RES organs liver (avg. 4.7391 ±0.4619 MBq/cm3 and 4.9358 ±0.2129 MBq/cm3 activity concentrations after 10 and 60 minutes, respectively) and spleen (1.9510 ±0.3633 MBq/cm3 activity concentration 35 min p.i.) [16]. Compared to the RES organ uptakes, relative low lung uptake and slowly decreasing could be detected during the entire tracing period (max. act. conc. was 0.6008 ±0.1709 MBq/cm 3 at 2.75 min p.i.), proving the lack of in vivo aggregated colloid fraction of NPs trapped by pulmonary lung capillaries [17], indicating that the very low amount of aggregates present in the sample is not supposed to interfere with the biological use. On the other hand, relative high and elongated traceable heart activities (max. act. conc. was 1.4857 ±0.0607 MBq/cm 3 at 2.75 min p.i.) and negligible kidney uptakes paired to slowly increasing urinary bladder values have been also suggested extended blood retention of NPs whereas slow excretion via kidneys and urinary tract.
4. Conclusions In conclusion, in vitro characterizations have confirmed that using our method, DOTA bifunctional chelator was successfully adsorbed onto the surface of ZrO2 nanoparticles while the following characterizations confirmed strong DOTA-adsorption, then after the
68
Ga-radiolabeling method in
68
pH4 environment at 95°C proved to be simple and effective. The new Ga-DOTA-ZrNP complex had durable radiochemical and colloidal stability after p.i., as well. Furthermore, in vivo µPET/CT biodistribution trials have confirmed the product’s in vivo radioisotope-adsorbing and size stability; and therefore demonstrated the possibility of achieving prospective PET/CT-imaging guided drug delivery operations using targeted and drug loaded variants of
68
Ga-DOTA-labeled porous zirconia
nanoparticle platforms. Further research with active targeted and radiolabeled ZrO2 nanoparticles is still needed to explore the potential of these inorganic based nanocarrier systems.
Acknowledgements This study was supported by the project K 115259 of the Hungarian Scientific Research Fund (OTKA, Hungary). The authors also received financial support from the Deutsche Forschungsgemeinschaft within the excellence cluster REBRITH (Exc62/1), and from the Volkswagenstiftung within the project of Biofabrication for NIFE.
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