Cancer has become a leading cause of death in industrialized countries [1
]. In light of the shortcomings of current treatment modalities for cancer, a critical thrust towards improving cancer therapy is to specifically target therapeutic agents to tumor cells while sparing healthy tissues from harm. This is one of the emerging interests in nanotechnology research. Nanoparticles (NPs) that are most used for cancer nanotechnology include polymers, dendrimers, liposomes, quantum dots, iron oxides, and gold nanoparticles [2
]. Among inorganic materials, gold NPs (AuNPs) exhibit unique optical properties, nontoxic nature, and straightforward surface functionalization, providing an attractive platform for cancer theranostics (therapy + diagnostic) development [3
At present, cancer therapy includes surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. AuNPs can destroy cancer cells by photothermal ablation, as exemplified by AuroShell [10
], through mechanical damage, or as drug delivery systems for anticancer agents, such as tumor necrosis factor [11
], doxorubicin [12
], or oxaliplatin [13
]. The boron-containing AuNPs represent a new class of therapeutics that can be used for boron neutron capture therapy (BNCT). BNCT is an experimental form of binary radiotherapy that selectively targets and damages tumor cells, even in the very challenging scenario, in which the malignancy is infiltrating into the surrounding normal tissue or is spreading in the whole organ [14
]. Clinical interest in BNCT has focused primarily on high-grade gliomas [16
]. However, boron-containing AuNPs may have limited clinical potential for brain tumors because they are unable to penetrate the blood–brain barrier (BBB) adequately, and they may be trapped in filtrating organs [20
]. Therefore, alternative or improved delivery agents are required to implement full clinical potential of BNCT. The delivery of pharmaceuticals through the brain capillary endothelial wall may be facilitated thru conjugation of therapeutics to brain drug delivery vectors. Since human serum albumin (HSA) is shown to undergo absorptive-mediated transcytosis through the BBB in vivo, HSA is a potential brain drug delivery vector in humans [21
Human serum albumin (HSA) is one of the most popular materials for designing nanoparticles [22
]. Since FDA approved Abraxane®
(paclitaxel-encapsulated albumin formulation), HSA has attracted increasing attention as a drug cargo. All the factors such as known structure of HSA obtained by X-ray analysis; the ability of the protein to bind and transport various drugs to certain organs of the body; the possibility to obtain recombinant HSA; its great stability at wide range of pH and temperature, as well as in different solvents; the possibility to store the protein in solution for many years make albumin an attractive tool for designing nanocomposite materials to diagnose and treat cancer diseases [22
]. The synergy between HSA and AuNPs results in interesting properties, such as increased nanoparticle stability, their reduced interaction with other plasma proteins, passive and active targeting of the conjugates to malignant cells, and hence increased selectivity [26
]. The transcytosis of albumin across the endothelium of blood vessels is achieved through the well-known gp60 pathway which promotes the albumin-bound drugs into tumor cells [29
]. Furthermore, active targeting by HSA-conjugated nanosystems involves interaction with several specific receptors, including Secreted Protein Acidic Rich in Cysteine (SPARC). SPARC, which is known as an extracellular matrix glycoprotein acid, is often seen in some neoplasms [30
]. It has been reported that SPARC is overexpressed in brain tumors, serving as a promoter to glioma progression and invasion, suggesting its potential therapeutic value [32
Nanoparticles with a suitable size to allow for accumulation through passive targeting, if combined with serum albumin, may demonstrate a dual ability to target the malignant cells through passive and active targeting, and therefore represent useful nanoplatforms for drug delivery with significant physiological stability. Authors’ study [28
] clearly suggests that stable conjugation of AuNP (core diameter 15 nm) with albumin prior to intravenous administration increases specificity and efficiency of AuNP in diseased target organs thus suggesting a potential role in nanomedicine and nanopharmacology. Therefore, the capping of AuNPs with boronated albumin can improve their biological properties, which may encourage the scientific community to investigate these nanosystems as BNCT drugs, as well as for photothermal therapy.
An important problem in anticancer therapy is to determine biodistribution of the drug carrier after administration. This problem is even more evident when nanosystems are used as boron carriers, as the optimal time window after the drug administration needs to be established for neutron irradiation. For this purpose, equipping the drug carrier with a tag for tracking its location can provide a solution. For example, for nanoparticle-based therapeutic strategies, incorporation of a positron or gamma emitter into the nanoparticle allows for tracking the drug location in a time-resolved fashion using positron emission tomography (PET) or single photon emission computerized tomography (SPECT) imaging [34
]. However, these methods have their limitations. While PET is useful for clinical applications due to its ability to provide information about spatial distribution of drugs in vivo, its spatial resolution of a millimeter scale makes it difficult to reveal precise drug location. Autoradiography enables subcellular spatial resolution for distribution of boron drugs, while it requires a neutron beam source for triggering nuclear reaction, which is not always available.
Among many detection modalities, 19
F MRI is advantageous for deep-tissue and noninvasive imaging in vivo [36
]. In vivo experiments using the C6 rat glioma model demonstrated that 19
F MRI in combination with 1
H MRI can selectively map the biodistribution of a 19
F-labelled borophenylalanine-fructose complex [39
]. The NMR sensitivity of 19
F is 0.83 relative to 1
H, has a 100% natural isotopic abundance ratio, and has a large chemical shift range (300 ppm). In addition, the human body itself provides a negligible endogenous 19
F MRI signal [36
], we have developed a novel anticancer albumin-trifluorothymidine theranostic conjugate PFT-Hcy-HSA-Cy7-pTFT. The in vivo antitumor effect of this albumin theranostic agent was evaluated on a U87 glioma-bearing mouse model [40
]. The results clearly demonstrated that the albumin theranostic can significantly suppress the tumor. Our initial results demonstrate the potential of PFT-Hcy-HSA-Cy7-pTFT conjugate to serve as an optical and 19
F MRI magnetic resonance imaging agent [38
]. Based on their detection sensitivity, the combination of 19
F MRI and fluorescence imaging is probably the most valuable one in multimodal molecular imaging.
Here, we report on the preparation and characterization of AuNPs stabilized with dual-labeled albumin, containing fluorophore and 19
F tags (bimodal serum albumin), which is functionalized with the chemotherapeutic agent (trifluorothymidine) or boron-rich anion (undecahydro-closo
-dodecaborate). The albumin theranostic conjugates allows for direct optical and 19
F magnetic resonance imaging [38
]. Fluorescence-based molecular imaging becomes increasingly important, mainly due to the development of highly sensitive cameras and very high spatial resolution. We used this modality in combination with computed tomography to generate anatomical details of an animal subject studied after albumin-AuNP conjugate to intravenous administration.
2. Experimental Section
Reagents and materials were purchased from Sigma-Aldrich (UK), unless otherwise indicated. Milli-Q water with conductivity greater than 18 MΩ/cm was used in all experiments. Gold (III) chloride trihydrate (HAuCl4 3H2O) and sodium citrate solution (Sigma–Aldrich Chem. Co., St. Louis, MO, USA) were used as received. HSA (A3782) was also obtained from Sigma–Aldrich Chem. Co. (St. Louis, MO, USA).
Dual-labeled albumin was synthesized according to the published procedure [37
] and kindly provided by Dr. Alexey S. Chubarov (Institute of Chemical Biology and Fundamental Medicine, SB RAS, Lavrentiev ave. 8, Novosibirsk, 630090, Russia).
) was synthesized according to the published procedure [41
] and kindly provided by Dr. Ludmila S. Koroleva (Institute of Chemical Biology and Fundamental Medicine, SB RAS, Lavrentiev ave. 8, Novosibirsk, 630090, Russia).
Maleimide functionalized of 5-trifluoromethyl-2’-deoxyuridine 5’-monophosphate was synthesized according to the published procedure [38
] and kindly provided by Dr. Vladimir A. Lisitskiy (Institute of Chemical Biology and Fundamental Medicine, SB RAS, Lavrentiev ave. 8, Novosibirsk, 630090, Russia).
The absorption spectra of colloidal solutions of AuNPs in quartz cells (1 cm path length) were registered on a Shimadzu UV-2100 spectrophotometer (Shimadzu, Japan). The concentrations of albumin solutions were determined by absorption at 292 nm, pH 13, using the molar extinction coefficient ε
= 4.44 × 104
] with a UV-1800 spectrometer (Shimadzu, Japan).
To determine hydrodynamic diameter and zeta-potential of all NPs used in the study, we applied a Zetasizer Nano ZS Plus instrument (Malvern Instruments; Malvern, Worcestershire, UK). All measurements were made in triplicate and according to manufacturer instruction.
For electron microscopic characterization, suspensions of all studied AuNPs were adsorbed for 30 s on copper grids coated with formvar film, which was stabilized by carbon using a JEE-420 Vacuum Evaporator (Jeol, Tokyo, Japan). Then, excess liquid was removed and samples were negatively stained with phosphotungstic acid (pH 3.0) for 10 s. The grids were examined in a JEM 1400 (JEOL, Tokyo, Japan) electron microscope at an accelerating voltage of 80 kV, images were collected using a Veleta digital camera (EM SIS, Munster, Germany). The sizes of AuNPs were measured using the iTEM program, version 5.2 (EM SIS, Munster, Germany), on the camera screen.
1H and 19F NMR spectra were recorded on AV-300 NMR spectrometer (Bruker, Germany) at 300.13 and 282.7 MHz, (1H and 19F NMR spectra, respectively). The spectra were detected in 5 mm NMR sample tubes at 25 °C. The chemical shifts were expressed in parts per million (ppm, δ). Residual 1H NMR signal of the deuterated NMR solvent (D2O, δ 4.80 ppm) was used as a reference for all 1H chemical shifts. Perfluorobenzene (δ 0.00 ppm) was used as an external reference for chemical shifts in 19F NMR spectra. 31P NMR spectra were recorded on AV-400 and AV-300 NMR spectrometers (162 and 121 MHz, respectively). The 31P chemical shifts were calculated using an external standard of 85% H3PO4 (δ 0.00 ppm). The spin–spin coupling constants (J) are reported in hertz (Hz), and spin multiples are given as s (singlet), d (doublet), t (triplet), and m (multiplet). Broad peaks are indicated by the addition of br.
Low-molecular-weight materials (MW < 3 kDa) were removed from solutions of polymer conjugates by centrifugal filtration using Centricon concentrators with a MWCO of 3-kDa (Amicon Centriprep YM30, Millipore, Bedford, MA, USA).
2.3. Synthesis and Characterization of Theranostic Conjugate PFT-Hcy-HSA-Cy7-pTFT
The PFT-Hcy-HSA-Cy7-pTFT conjugate is a known HSA conjugate. The synthetic procedure of theranostic conjugates PFT-Hcy-HSA-Cy7-pTFT was performed out in the dark according to the method described elsewhere [38
The yield of PFT-Hcy-HSA-Cy7-pTFT was 50%. 19F NMR (PBS buffer, pH 7.4, δ, ppm): 108.0 (br m, CF3, perfluorotoluene residues), several overlapping signals at 100.0 ppm (m, CF3, 5-C). 31P NMR (D2O, δ, ppm): 8.89. UV–VIS (PBS buffer, pH 7.4): λmax nm (ε, M-1cm-1): 267 ((1.4 ± 0.1) × 105), 292 ((1.35 ± 0.1) × 105), and 762 ((1.8 ± 0.1) × 105). Fluorescence: the excitation and emission maximum wavelengths are 762 nm and 780 nm, respectively.
2.4. Synthesis and Characterization of Theranostic Conjugate HSA-Cy5-Hcy-TFAc-B12H12
The synthesis of HSA-Cy5-HcyTFAc conjugate was adapted from Lisitskiy et al. [38
]. Briefly, a solution (0.5 mL, 0.7 mM, 0.35 μmol) of dual-labeled albumin (HSA-Cy5-HcyTFAc) in PBS buffer (pH 7.4) was mixed with MID-B12
in DMSO (29.2 μL, 1.58 μmol, 0.166 M). The reaction mixture was incubated under constantly gently stirring at 37 °C in the dark for 17 h. The protein conjugates were purified by SEC utilizing a Millipore ultrafiltration tube and stored at 4 °C.
The yield of HSA-Cy5-HcyTFAc-B12H12 conjugate was ~65%. UV–VIS (PBS buffer, pH 7.4): λmax 278 nm (ε = (3.88 ± 0.1) × 104) and λmax 646 nm (ε = (2.7 ± 0.1) × 105). Inductively coupled plasma atomic emission spectroscopy: 2.6 B12H12 residues per albumin.
2.5. Synthesis of Gold Nanoparticles
Gold nanoparticles (AuNPs) were prepared using citrate method [43
]. In brief, 5 mL (38.8 mM) of sodium citrate solution was added to 45 mL (1.1 mM) of HAuCl4
O boiling water solution under intense stirring. The mixture was stirred for 20 min and then kept for 24 h at room temperature, after that, the suspension was passed through a 0.45 µm filter (Millipore, USA) and stored at 4 °C. According to TEM data, the obtained AuNPs had a spherical shape and a diameter of 12 ± 0.1 nm. Hydrodynamic diameter of AuNPs was 17.40 ± 0.40 nm, zeta potential was −33.60 ± 2.04 mV, and the maximum absorption spectrum was 520 nm.
2.6. Synthesis and Characterization of Multifunctional Human Serum Albumin-Therapeutic Nucleotide Conjugates alb-TFT-AuNP and alb-boron-AuNP
To obtain albumin-modified AuNPs, citrate-AuNPs (0.9 mL of 1.16 nM) were incubated with conjugates PFT-Hcy-HSA-Cy7-pTFT and HSA-Cy5-Hcy-TFAc-B12H12 (0.1 mL of 0.1 mM) in PBS for 24 h at room temperature. The reaction mixtures were centrifuged for 15 min (13,000 rpm) to remove unbound albumin conjugates. The pellets were resuspended in PBS and kept at 4 °C. The alb-TFT-AuNP had a zeta potential of −32.05 ± 4.70 mV and a hydrodynamic diameter of 25.93 ± 0.60 nm; the diameter measured by TEM was 17.55 ± 1.14 nm. The alb-boron-AuNP had a zeta potential of −22.43 ± 1.12 mV and a hydrodynamic diameter of 28.93 ± 0.08 nm; the diameter measured by TEM was 17.25 ± 1.58 nm.
The nanoparticle suspensions in PBS were kept at 4 °C and application was done in the in vitro and in vivo studies within 3–10 days. Prior to tail vein injection, zeta-potential and hydrodynamic diameter of albumin-AuNP conjugates were determined by dynamic light scattering measurements. Noteworthy, the physicochemical parameters of the used AuNPs conjugates remained unaltered during storage.
2.7. Cell Viability Assay (MTT Test)
Human mammary adenocarcinoma cell line MCF-7 was cultured in IMDM medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), penicillin (100 units/mL), and streptomycin (100 μg/mL) at 37 °C and 5% CO2 in humid atmosphere.
The inhibition of cell growth was assessed by MTT test, based on the reduction in MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide into formazan by mitochondrial NAD(P)H-dependent oxidoreductase enzymes [44
]. Cells in exponential growth phase were seeded in 96-well plates (2000 cells per well). The cells were allowed to attach for 24 h and were treated with HSA, alb-boron-AuNP, alb-TFT-AuNP, HSA-Cy5-Hcy-TFAc-B12
, PFT-Hcy-HSA-Cy7-TFT, or pTFT. The solutions of albumin conjugates were applied in medium with HSA-equivalent concentrations ranging from 0.06 to 60 µM for 72 h at 37 °C. MTT solution was added to each well at final concentration of 0.5 mg/mL, and the plates were incubated at 37 °C for 2 h. The medium was removed and the dark blue crystals of formazan were dissolved in 100 μL of isopropanol. The absorbance at 570 nm (peak) and 620 nm (baseline) was determined using a microplate reader Multiscan FC (Thermo Fisher Scientific Corporation). All concentrations were performed in triplicate. The values are given as mean ± standard deviation (S.D.) values, and all measurements were repeated three times.
2.8. Animal Care, Maintenance of Tumors, and Experimental Procedures
The trials with the mice of the SCID line were performed at the Center for Genetic Resources of Laboratory Animals at the Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences (RFMEFI62119X0023). All animal experiments described in this paper were carried out in compliance with the principles of humanity according to directive 86/609/EEC of the European Community. All efforts were made to minimize the number of animals used and their suffering.
The study was conducted on 8 male mice of the SCID (SHO-PrkdcscidHrhr) line of the SPF status at the age 6–7 weeks. The animals were kept in sn SPF-vivarium of the Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences (SB RAS) in unisexual family groups of 4 individuals in individually ventilated cages (IVC) of the OptiMice system (Animal Care Systems) under the controlled conditions (at a temperature of 22–26 °C, relative humidity of 30–60%, and light mode light/dark 14/10 h with dawn at 1:00 a.m.). The Sniff feed (Germany) and deionized water enriched by Severyanka mineral mixture (St. Petersburg) were given to the animals ad libitum.
U87 line of glioma cells were used in the experiments [45
]. About 2–3 weeks before the beginning of the experiment, the U87 glioma cell culture, which is stored in the cryobank of the Center of Collective Use SPF-vivarium of the Institute of Cytology and Genetics SB RAS in liquid nitrogen, is defrosted and cultivated for 5–7 passages on a DMEM/F12 medium (1:1) with a 10% fetal serum (FBS) produced by Invitrogen. Before the injection, the tumor cells were removed from the substrate by a tripsin/versene solution, and the precipitate was thoroughly resuspended in the medium without a serum after centrifuging for 5 min at 1000 revolutions per min (bringing to the concentration 100,000 cells in 1 μL).
Orthotopic xenotransplantation of U87 cells is done into mice of the SCID line. Before the operation, the animal was placed in the chamber with the air inflow of 450–500 mL/min and isoflurane concentration in the air mixture of 1.5%. After 3 min, the animal was transferred onto a heated operating table with a surface temperature of 37 °C and placed under an anesthetic mask with an isoflurane in oxygen mix (1.5%, flow rate 200 mL/min) for anesthesia. The mice (n = 8) received incranial injection of inocula of 300 × 103 tumor cells at left hemisphere near the hippocampus (-4 mm Bregma). The cell suspension was introduced in the subcortical brain structures through a hole in the animal’s cranium. For this, a skin incision 3–4 mm in length was made on the head in the caudal–cranial direction near the bregma, and 5 μL of the cell suspension was introduced through the hole in the cranium.
Before FLECT/CT scanning, animals were immobilized using 75 µL/10 g body weight of Dorbene (Laboratories SYVA S.A., Leon, Spain) and 5 min later, 80 µL/10 g body weight of Zoletil (Virbac Sante Animale, Carros, France).
Mice were imaged in InSyTe FLECT/CTTM system (TriFoil Imaging, Chatsworth, CA, USA). CT was performed using next parameters: tube voltage 35 kV, tube current 950 µA, and exposure time 180 ms. The entire object was scanning, according to the principle of continuous helical radiation of 720 projections with 360-degree coverage. Image resolution reaches 25 × 25 × 25 microns. Total time of study was 15 min. FLECT was performed using the 730 nm laser, and the fluorescence signal was filtered with 813 nm filter emission. Exposure time was 16 msec. The entire object was scanning, according to the principle of continuous helical radiation of 116 projections with 360-degree coverage. Image resolution reaches 1 mm × 1 mm × 1 mm. Total time of study was 40 min. The images were reconstructed using the TriFoil Imaging software (TriFoil Imaging, Chatsworth, CA, USA). CT and FLECT reconstructions were combined using VivoQuant v2.5 (Invicro, Boston, MA, USA).
The investigation of PFT-Hcy-HSA-Cy7-pTFT and alb-TFT-AuNP distributions in SCID mice with the brain tumor caused by the intracranial injection of a human glioblastoma cell line U87 were held 4 weeks after the injection of tumor cells. Before FLECT/CT scanning, 0.1 mL of 0.1 mM solution of albumin conjugates was intravenously injected in the mouse. The mice were imaged by InSyTe FLECT/CTTM system in vivo over time after injection of albumin conjugates (at 1 and 72 h after intravenous injection).