Development of fluorescent- and radio-traceable T1307-polymeric micelles as biomedical agents for cancer diagnosis: biodistribution on 4T1 tumor-bearing mice

In recent years, nanocarriers have been studied as promising pharmaceutical tools for controlled drug-delivery, treatment-efficacy follow-up and disease imaging. Among them, X-shaped amphiphilic polymeric micelles (Tetronic®, poloxamines) display great potential due to their biocompatibility and non-toxic effects, among others. In the present work, polymeric micelles based on the T1307 copolymer were initially decorated with a 4,4-difluoro-4-bora-3a,4a-diaza-s -indacene (BODIPY)-fluorophore in order to determinate its in vivo biodistribution on 4T1 tumor-bearing mice. However, unfavorable results with this probe led to two different strategies. On the one hand, the BODIPY-loaded micelles, L-T1307-BODIPY , and on the other hand, the 99m Tc -radiolabeled micelles, L-T1307- 99m Tc , were analyzed separately in vivo . The results indicated that T1307 accumulates mainly in the stomach, the kidneys, the lungs and the tumor, reaching the maximum organ-accumulation 2 hours after intravenous injection. Additionally, and according to the results obtained for L-T1307- 99m Tc , the capture of the polymeric micelles in organs could be observed up to 24 hours after injection. The results obtained in this work were promising towards the development of new radiotracer agents for breast cancer based on X-shaped polymeric micelles.


INTRODUCTION
Cancer is the second leading cause of death in the world. Among the different types of cancer are the solid tumors, i.e., carcinomas, sarcomas and lymphomas. In this sense, nanotechnology has been growing very fast in terms of developing new strategies against solid tumors, by optimizing diagnostic efficiency, developing novel anticancer treatments, and reducing the toxicity and improving the aqueous solubility of the drug candidates (Glisoni, Sosnik, 2014a;Lv et al., 2013;Glisoni et al., 2013;Glisoni et al., 2012). The use of nanotechnology is purportedly related to the enhanced permeability and retention effect (EPR), which takes advantage of the properties of solid tumors that may promote angiogenesis and ensure high blood supply to the growing mass, causing imperfect vascular structures and a significant Development of fluorescent-and radiotraceable T1307-polymeric micelles as biomedical agents for cancer diagnosis: biodistribution on 4T1 tumor-bearing mice Nicole Lecot, Gonzalo Rodríguez, Valentina Stancov, Marcelo Fernández, Mercedes González, Romina J. Glisoni, Pablo Cabral, Hugo Cerecetto, lack of lymphatic drainage. The EPR could allow the extravasation of nanomaterials and their accumulation inside the pathological site (Eawsakul et al., 2017;Maeda et al., 2000).
Nanomedicines allow early tumor diagnosis, which is the most important fact to increase patient survival (Oda et al., 2017). Several types of nanosystems for diagnostic imaging have been described; among them, quantum dots, gold nanoparticles, carbon nanotubes, silica nanoparticles, liposomes, nano micelles and dendrimers can be highlighted (Acharya, Mitra, Cholkar, 2017). These nanosystems have the following advantages, among others: i) their nanometrical size, ii) the possibility of surface functionalization for active drug-delivery, iii) their passive targeting, iv) the solubilization of poorly soluble molecules in aqueous milieu, v) the protection of encapsulated substances from degradation and metabolism, vi) improved pharmacokinetic effects. These nanodevices particularly used in diagnostic imaging have been described coupled with magnetic resonance, optical, nuclear, computed tomography and ultrasound imaging (Acharya, Mitra, Cholkar, 2017;Marques Grallert et al., 2012). Advanced thermosensitive nanomaterials are promising "smart materials" for diagnosis when stimulated at a particular temperature range (Nardecchia et al., 2019;Cohn, Sosnik, Levy, 2003).
Considering this background, the purpose of our study was the development of T1307 PMs as nanocarriers for forthcoming in vivo studies as diagnostic agents. To this end, we studied the biodistribution of tumor-bearing mice, using two T1307-probes: (i) fluorescent-and (ii) radioactive-labeled.

Equipments
Fourier-transform infrared (FTIR) spectra were acquired, in solid state (KBr), using an IR-Prestige21 FTIR-ATR infrared spectrophotometer (Shimadzu, Kyoto, Japan) with Happ-Genzel apodization. The analyzed region was in the range between 4000-400 cm -1 (10 scans, spectral resolution of 4 cm -1 ). The solid lyophilized samples (T1307-BODIPY, L-T1307-BODIPY and lyophilized pristine T1307 were mounted on the ATR metal-glass plate and the spectra were obtained with the IR SOLUTION spectrum software, which were subsequently processed using Origin 8. Nuclear Magnetic Resonance (NMR) spectra were performed in a Bruker DPX-400 spectrometer, operating at a frequency of 400.13 MHz for 1 H and 100.77 for 13 C. The NMR spectra were analyzed in 15 % w/v CDCl 3 solutions for BODIPY-ester and T1307-BODIPY (n=2), and in 15 % w/v DMSO-d 6 solutions for L-T1307-BODIPY (n=3). The spectra were obtained using the MestReNova 8.0 software.
Fluorescence spectra were performed in a microplate reader (Thermo Scientific™ Varioskan™ LUX multimode microplate reader, USA).
Particle size and zeta potential of L-T1307-BODIPY micelles were obtained from five repeated measurements by a dynamic laser-diffraction particle-size detector and a Malvern Zeta analyzer (Nano-ZS, Malvern Instruments, Malvern, UK), respectively. The measuring process was kept at 25 °C.
Radioactivity was counted in a CRC7 Capintec dose calibrator and in a solid scintillation counter detector with 3"×3" NaI(Tl) crystal associated with a single channel analyzer (ORTEC, Oak Ridge, TN).

Animals
Balb/c female mice weighing 18-20 g were produced and provided by Unidad de Reactivos para Biomodelos de Experimentacion (URBE), Facultad de Medicina, Universidad de la República, Uruguay. The authors state that they followed the principles outlined in the Declaration of Helsinki for all animal experimental investigations. Animals were housed in wire mesh cages at 20 ± 2 o C with 12 h artificial light-dark cycles. The animals were fed ad libitum to standard pellet diet and water and were used after a minimum of 3 days acclimation to the housing conditions.
All protocols for animal experimentation were carried out in accordance with procedures authorized by the Ethical Committee for Animal Experimentation, Uruguay, by whom this project was previously approved (CHEA-UdelaR Protocol number 240011-001547-16).

Data analysis
The statistical analysis was performed using the Student's t-test (in a comparison between two groups); p level less or equal to 0.05 was defined to determine statistically significant differences.

Synthesis of the T1307-BODIPY
The synthesis of T1307-BODIPY followed a twostep reaction (Figure 1): synthesis of BODIPY-ester and conjugation of T1307 with BODIPY-ester to form T1307-BODIPY.

Synthesis of the BODIPY-ester
A mixture of formyl-BODIPY (80 mg, 0.28 mmol), phosphonium ylide (192 mg, 0.55 mmol) and triethylamine (69 μL) was stirred at room temperature during three days under a nitrogen atmosphere. The mixture was evaporated in vacuo and the product was isolated using a preparative thin layer chromatography (SiO 2 , n-hexane:methylenedichloride (9:1)). Red solid, Synthesis of the T1307-BODIPY (Glisoni, Sosnik, 2014b)  a Microcon® centrifugal filter (cut off 10 kDa) with the low-binding membrane of the mixture at 12,000 rpm for 10 minutes. Micelles were lyophilized and stored at 4 °C. The L-T1307-BODIPY was resuspended in MilliQ water and filtered by 0.22 µM for further studies (pH = 6.2). The encapsulation efficiency (%EE) was 82.7 %, particle size was 243 nm, polydispersity index was 0.32, and zeta potential was -2.9 ± 0.32 mV. Loading of T1307 with the radioactive probe (L-T1307-99mTc) (García et al., 2018;Fernández et al., 2015;Giglio et al., 2008) In order to label T1307 directly by stannous reduction of 99m TcO 4 -, SnF 2 . 2H 2 O (0.2 mL of stock ethanolic solution, 1 mg/mL) was added to a solution of T1307 (0.5 mL of Milli-Q water, 0.15 g/mL) and Na 99m TcO 4 (14 mCi). The pH was then adjusted to 6.5. The mixture was incubated at room temperature for 20 minutes, then transferred to a Microcon® centrifugal filter (cut off 10 kDa) with the low-binding Ultracel® membrane, and centrifuged at 12,000 rpm for 10 minutes. The collected aqueous supernatant was used to determine the labeling yield, radiochemical purity (RP) and for further studies in animals (pH = 6.9). The labeling yield and RP were estimated by i) an ascending instant thin layer chromatography (ITLC) using the chromatographic systems: a) saline; b) pyridine:acetic acid:water (3:5:1.5 v/v), and ii) RP-HPLC using the conditions indicated above with UV and gamma detections. RP: 91.9 %.

Loading of T1307 with the fluorescent probe (L-T1307-BODIPY)
The typical encapsulation process was developed as follows: pristine T1307 (50 mg) was hydrated in PBS (0.4 mL) and kept at 4 o C for 30 minutes, after which the PBS was adjusted to a final volume of 0.5 mL and BODIPY-ester (2.9 mg) was added, followed by 1 h of stirring at room temperature. To remove the unencapsulated fluorescent probe, it was centrifuged using Nicole Lecot, Gonzalo Rodríguez, Valentina Stancov, Marcelo Fernández, Mercedes González, Romina J. Glisoni, Pablo Cabral, Hugo Cerecetto, grown grown in a humidified incubator containing 5 % CO 2 and maintained at 37 o C. The cells were centrifuged at 1,000 rpm for 5 minutes. The supernatant was removed and the pellet was resuspended in RPMI.
In vivo studies Tumoral model (Dávila et al., 2019;Gao et al., 2016) Cell suspensions were prepared at 7 × 10 6 cells/mL in RPMI milieu. Afterwards, five-six week old Balb/c female mice were inoculated subcutaneously (after the preparation of cell suspension) into the fourth inguinal mammary fat pad (100 μL/mouse). Animals were palpated daily in order to record the presence, location, and volume of all tumors. Tumor diameters were measured daily with a sterile caliper, calculated using the ellipsoidal method volume. Palpable tumors (̴ 100 mm 3 ) developed 5 days after the cell inoculation. Tumor diameters were measured daily with a vernier caliber (Ostrand-Rosenberg). The two diameters of the tumor, long (L) and short (C), were perpendicular to each other and covered the largest portion of the tumor in each direction. Tumor volume (V) was calculated using the following equation: V = (C 2 × L)/2 (Chiang et al., 2014;Dávila et al., 2019).
In vivo and ex vivo fluorescence imaging of L-T1307-BODIPY on Balb/c mice with primary mammary tumors induced with 4T1 cells (Calzada et al., 2017) At day 14 after the cell inoculation, fluorescence imaging study was conducted on Balb/c mice with induced 4T1 tumors by the intravenous injection (IV) of 50 mg of L-T1307-BODIPY per kg of body weight, via the animals' tails. For these experiments, the animals were anesthetized with isoflurane immediately before the injections. After being introduced into an optical imaging platform (In-Vivo MS FX PRO instrument, Bruker, Billerica, USA), the animals were measured using the X-ray and fluorescence modes (10 seconds acquisition, at excitation and emission wavelengths of 480 nm and 535 nm), 1 and 24 hours after the IV injections, n=3 for each time point. After each imaging time point, mice were sacrificed for organ dissection, macroscopic examination, biodistribution and ex vivo imaging was carried out separately using the aforementioned imaging equipment. Animals without injection were used as negative control.
Biodistribution assay of L-T1307-99mTc on Balb/c mice with primary mammary tumors induced with 4T1 cells Biodistribution study of the radioactive probe was performed by IV injections, via tail, of 856 μCi of the L-T1307-99m Tc on Balb/c mice with and without 4T1induced tumors (day 14 after cell inoculation). The animals, n=3 for each time point, were sacrificed by cervical dislocation 1, 2, 4 and 24 hours after the IV injections. The radioactivity in organs and tissues was measured in the solid scintillation counter detector described above. Organ weight correction was applied. The results are expressed as the percentage uptake of injected dose per tissue weight (%Act/g).

Preparation and physicochemical characterization of fluorescent T1307-BODIPY and L-T1307-BODIPY probes
In order to generate a physiologically stable T1307probe, we initially proposed the covalent conjugation between this copolymer and an adequate BODIPYderivative (Rodríguez et al., 2017). For that reason, we planned to use an ester containing BODIPY, i.e., BODIPY-ester (Figure 1), that could be able to react with the free-hydroxyl groups of the T1307 copolymer, via a conventional transesterification process in the presence of Sn(Oct) 2 as catalyst (Glisoni, Sosnik, 2014b). In this sense, a T1307 decorated with BODIPY moieties (T1307-BODIPY, Figure 1) was successfully prepared, through an assisted-microwave procedure, but 1 H NMR, 13 C NMR, and IR spectroscopies suggested that the final product was the result of hydroxyl-1,4-addition followed by esterhydrolysis, incorporating four units of BODIPY for each unit of T1307 ( Figure 2B and 3). The T1307-BODIPY fluorescent probe had the same emission spectrum as the BODIPY-ester but did not show the same fluorescence intensity (Figure 4). Moreover, it displayed very poor aqueous solubility (in PBS and PBS with up to 10 % of DMSO) and the aqueous solution had acidic pH (5.8). For this reason, all our attempts to carry out in vivo studies were unsuccessful.
Studies with fluorescent probe-loaded T1307 PMs (L-T1307-BODIPY) BODIPY-ester loaded within T1307 PMs was successfully performed in a short time, producing L-T1307-BODIPY with a %EE of 82.7 and nanometric size. The NMR and IR spectra of L-T1307-BODIPY confirmed a correct encapsulation process without structure modification of the BODIPY-ester fluorophore (compare Figures 2A and 2C, and see Figure 3).
After the IV injection of L-T1307-BODIPY, mice did not reveal toxicity effects on during in vivo studies, according to the Irwin test (Dávila et al., 2019). This test allowed us to consider the qualitative effects of L-T1307-BODIPY on behavior and physiological function, in the first dose that has observable effects as well as in doses that not induce behavioral toxicity.
It was possible to observe an accumulation of BODIPY-fluorophore in the region of the tumor after 1 hour of biodistribution ( Figure 5A, left). After 24 hours of biodistribution, fluorescence was diffuse ( Figure 5A, right), not only evident in the tumor region but also in other areas that could indicate biodistribution by the circulation of this type of nanomaterial (PMs). In order to know the real fluorescence-contribution of each tissue and organ, the ex vivo analyses were done in each time point (Figures 5B and 5C). In these studies, we could observe the typical intestinal and stomach fluorescence, due to the presence of chlorophyll contained in the feedpellets, although the signal was significantly different to the control 1 hour after injection. Additionally, significant fluorescence accumulation in tumor and kidney tissue (not significantly different) by L-T1307-BODIPY was observed. Ex vivo studies, 24 hours after L-T1307-BODIPY injection, revealed no fluorescence differences in comparison with untreated animals (controls) ( Figure 5C). Studies with radioactive probe-loaded T1307 PMs (L-T1307-99m Tc) 99m Tc loaded within T1307 PMs was successfully performed in a short time, producing good-yield L-T1307- 99m Tc. Afterwards, Microcon® centrifugation yielded the desired probe with acceptable amounts of free 99m TcO 4 and 99m TcO 2 , according to the chromatographic studies, being the RP of 91.9 %, and without any negative effect on the properties of these PMs (aqueous solubilization, adequate pH and nanometric size).