Polyglycerol Dendrimers Bearing Rhodamine and Gd3+As Terminal Groups: Magneto-Optical

Polyglycerol dendrimers bearing rhodamine and Gd3+as terminal groups: Magneto-optical properties and phototoxicity against tumoral cells

Tessa Martins1, Marcelo G. Vivas2, Leonardo De Boni2, Cleber Renato Mendonça2, Alvaro A. Alencar de Queiroz1

1Centro de Estudos e Inovação em Materiais Biofuncionais Avançados/Universidade Federal de Itajubá (UNIFEI)

2Grupo de Fotônica, Instituto de Física de São Carlos/USP

AbstractToday, nanotechnologies provide technological power and tools that will enable for the development of new diagnostics and therapeutics for the cancer treatment. Dendrimers, a new category of hyper-structured macromolecules, provides multifunctional modularity at cancer-nanotechnology as receptors for malignant cells and fluorescent tracers. In this work, we have focused our attention for the synthesis of organic nanodots based on polyglycerol dendrimers (PGLD) containing rhodamine B (RhB) and gadolinium (Gd3+) for use as sensitive labels in two-photon fluorescence microscopy or tracers for tumoral cells and contrast agent in magnetic resonance image (RMI). The synthesis and optical characterization of PGLD(RhB):Gd3+ and their potential for use in diagnostic and therapeutic of cancer is explored.

1. Introduction

Optical molecular imaging in combination with nanobiotechnology is emerging as a powerful tool forstudying the temporal and spatial dynamics of tumor cells. Using exogenous targeted probes, researcherscan now perform non-invasive studies on living systems and visualize the imaging of receptor expressionand/or activity over time might have greater potential for treatment decisions based on early molecularresponse assessment. Optical imaging using fluorescent techniques represents a promising approach sinceit is inexpensive and involves no exposure to ionizing radiation, providing spatiotemporal resolution on thebasis of relatively small data sets compared with other conventional imaging methods [1].

Today, the Gd(III) complex of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) is commonly used in magnetic resonance imaging (MRI) as contrast agent (CA) in diagnostic medicine [2-3]. MRI is a diagnostic method producing anatomical images of organs and blood vessels with high contrast definition of normal and abnormal tissues.

Lanthanide metals such as Gd(III) shorten the relaxation time and presuppose a possibility of achieving higher sensitivities for the MRI examinations [4].The overall safety of gadolinium-containing contrast agents has been well established and the frequency ofadverse drug reactions with gadolinium-containing contrast agents is lower than with non-ionic iodinatedcontrast media. It may be beneficial in patients that cannot tolerate intravenous iodinated contrast materials. However, recent reports strongly correlated the development of nephrogenic systemic fibrosiswith exposure to gadolinium-containing MRI contrast agents in patientswith severe renal dysfunction and in post-liver transplantation patients [5-7].

Due to the severe adverse effects (limited sensitivity due to poor tissue selectivity) so that more efficient and selective agents for MRI are being actively sought.At the last decade a significantly interest have been devoted to the development of new contrast media for magnetic resonance imaging (MRI) based on poly(amidoamine) (PAMAM) dendrimers-gadolynium complexes [8-10].

Dendritic polymer nanoparticles have a great potential for applications in medicine. They are highly branched radial polymers that have specific and systematically variable size, shape and chemical structure. Their radial structure contains a high functionality density on the surface. Successive generations (G) increase the diameter and double the surface functional groups than the preceding generation. The systematically variable structuralarchitecture and the large internal free volume make dendrimers as attractive molecules for biomedical applications [11-14].

While development of dendrimer-based technologies has a tremendous potential for applications in medicine, these materials, particularly the cationic and higher generation amino-terminatedPAMAM dendrimers, have been shown to be toxic in vitro [15-18]. The results of the in vivo toxicity studies have shown that PAMAM dendrimers of high generations shows significantly toxicity to mammalian cells [19-22]. Additionally, PAMAM dendrimers (generation 2, 3 and 4) interact with erythrocyte membrane proteins causing changes in protein conformation. These changes increase with generation number and the concentrationof dendrimers.

Recently, we have focused our attention for the synthesis of organic nanodots based on polyglycerol dendrimers doped with rhodamines for use as sensitive labels in two-photon fluorescencemicroscopy or tracers for tumoral cells. The coupling of rhodamine as terminal groups of polyglycerol dendrimers may allow an enhancement of their sensing properties in biology, particularly, their fluorescence in acidic conditions

In this work the synthesis and characterization of the gadolinium−rhodamine polyglycerol dendrimers (RhB-PGLD-Gd3+) is reported. A major incentive for the use of PGLD dendrimers as frameworks for biological applications is that whenever they have been tested, they have been found to have good biocompatible properties [23-25]. The unique molecular features and properties of PGLD, like multiple reactive chain ends, their excellent water solubility and biocompatibilityrenders them as valuable compounds for RMI contrast agents.

1. Material and Methods

2.1. PGLD(RhB):Gd3+ synthesis and Characterization

PGLD (generation 4) was synthesized according to the modified process reported by Haaginvolving a two-step process based on allylation of glycerol and catalytic dihydroxylation [26]. In a three-neck flask previously purged with nitrogen gas (N2), PGLD G-3 (10.1 g, 5.98 mmol, OH groups: 143.5 mmol) was dissolved in 50 wt % of sodium hydroxide (57 mL) with heating. To the solution, TBAB(4.6 g, 14.3 mmol) was added and dispersed under vigorous stirring. Then, allyl chloride (75.7 mL, 930 mmol) was added over 30 h at 40 °C under vigorous stirring using the mechanical stirrer. After the addition of 143 mL of toluene to the mixture, the organic phase was separated and dried over MgSO4, filtered, and concentrated using a rotary evaporator. The crude was further purified by column chromatography (silica gel, petroleum ether/ethylacetate 10:1 to 1:1) to obtain a colorless oil. The purified PGLD G4 and was partially terminated with 2-(4-isothiocyanatobenzyl)-6-methyldiethylenetriaminepentaacetic acid (DPTA) chelating groups for gadolinium ion coordination.

Reactive aldehyde groups on PGLD were prepared by Schimdt reaction for the rhodamine B (RhB) coupling. The activated PGLD was coupled with RhB by reacting G4.0 PGLD with RhB isothiocyanate (Scheme 1). Thechemical structure was characterized by 1H-NMRspectroscopy using a 750 MHz FT-NMR spectrometer(Varian, Unity plus, CA).Yield: 63%,1H-NMR (750 MHz, DMF-d7): 4.79-4.58 (m, OH ofPGLD), 3.85-3.55 (m, CH and CH2 of PGLD), 3.47(s, C(CH2)2 of PGLD). MALDI-TOF MS calcd. for PGLD 3,92 kDa found 3,98 kDa.

Scheme 1. Preparation of RhB-conjugated PGLD (PGLD(RhB)).

The preparation ofgadolinium complex of dendrimer PGLD(RhB) was performed by adding the required equivalent of gadolinium salt (GdCl3.6H2O) to the functionalized dendrimer dissolved in deionized waterand by adjusting the pH between 6.0-6.5 using 0.1 M meglumine solution. After dyalisis the absence of free gadolinium ions was tested by using xylenol orange indicator at pH 5.8(acetate buffer).

2.2. Optical measurements

Two-photon absorption measurements were carried out employing the open aperture Z-scan technique [27] using 120-fs laser pulses from an optical parametric amplifier pumped by 150-fs pulses at 775 nm, delivered by a Ti:sapphire chirped pulse amplified system, operating at 1-kHz repetition rate. The Z-scan measurements were carried out with intensities ranging from 50 to 100 GW/cm2 (15 to 80 nJ/pulse), with beam waist size ranges from 16 to 19 m.The Z-scan setup is the same as that described in a previous publication [28]. The same laser system was used to excite the samples fluorescence, which was collected perpendicularly to excitation through an optical fiber attached to a spectrometer. The fluorescence intensity as a function of the excitation irradiance was measured at 700 nm.

To measure the fluorescence lifetime of the samples, we used the second harmonic of Q-switched and mode-locked Nd:YAG laser with 70 ps of pulse duration. The second harmonic is focused in a BBO (beta barium borate) crystal to generate 266 nm. The 266 nm beam was focused into the sample with a lens of focal length f = 12 cm. The sample was placed in a 2 mm-thick fused silica cuvete. The fluorescence signal was collected perpendicularly to the excitation beam by an optical fiber positioned close to the fluorescent spot. The signal is acquired by a silicon photodetector with a rise time of approximately 0.5 ns, and subsequently averaged and recorded with a digital oscilloscope (5 GS/s). The fluorescence lifetime is determined by deconvolution of the decay curves and subsequent fitting using exponential functions. With this system we are limited to measure lifetimes higher than 0.5 ns with a typical accuracy of approximately 0.2 ns, obtained by the standard deviation of the mean lifetime, determined by repeating the experiment several times.

2.3. Relaxation Measurements

Solutions of PGLD(RhB):Gd3+ were prepared in water at 5 concentrations (0.9-5 mM Gd). The longitudinal relaxation time (T1) for each concentration was measured using an inversion recovery pulse sequence on a Bruker 400MHz spectrometer at a frequency of 400.137 MHz at 299K and 310K. Each inversion recovery experiment (25/polymer) yielded 2D spectra which were processed using NUTS software and fit to a three-parameter model. Relaxivities (R1) were obtained from linear least squares determination of the slopes of 1/T1 vs [Gd] plots.

2.4. Biological assays

The human breast cancer cells (MCF-7 cell lines) obtained from National Cell Bank of EUA (ATCC) were cultured at a seeding density of 4.0×104 cells per cm2 onto the designated culture plates using RPMI 1640 (Gibco, UK) supplemented with 10% FBS (Gibco, UK), 100 /mL streptomycin (Invitrogen, Paisley, UK) and 100 units/mL penicillin G (Invitrogen, Paisley, UK).

For both, cellular uptake and cytotoxicity assay, the 40-50% confluent cells were washed twice with serum free media (SFM). The cytotoxicity of PGLD(RhB):Gd3+ nanoparticles was evaluated using methylthiazo..letetrazolium(MTT) assay (Sigma Aldrich Co. Germany) in T47D cells. Briefly, cultured cells in 96-well plates were exposed to various ratios of the PGLD(RhB):Gd3+ for 4 hr. They were then washed once withphosphate buffered saline (PBS), replenished with normal culture medium and incubated at 37 oC for 24 hr. The normal culture medium was replaced with 200 mL fresh media and then 50 mL MTT reagent (2.5 mg/mL in PBS) was added to each well. Following a 4 hr incubation period at 37 oC, medium was removed and cells were exposed to 200 mL DMSO and 50 mL of Sorenson buffer (pH 7.4). Cultures were incubated for 30 min. at 37 oC and then UV absorbance was measured at 550 nm using a spectrophotometric plate reader (SUNRISE TECAN, Austria).

For cellular uptake assay, the 40-50% confluent cells were washed twice with serum free media (SFM), and then exposed to the PGLD(RhB):Gd3+for 4 hr at 37 oC incubation. After that, the cells were washed with SFM, replenished with normal culture medium and incubated at 37oC for further 24 hr. The cells were then washed 3 times with sterile PBS prior to fixation. Fixation involved washing the cells 3 times with PBS, followed by 10 min. incubation with 2% formaldehyde in PBS at room temperature. Cells were then washed a further 3 times with PBS and mounted on slides using Vectashield Hardset mounting medium with or without DAPI nuclear stain (Vector Labs, Peterborough, UK). Cellular internalization of the PGLD(Rh):Gd3+ by MCF-7 cells was examined by fluorescence microscopy utilizing an Olympus BX51 compoundfluorescence microscope equipped with a BX-RFA fluorescence illuminator and catadioptric UMPlanFL-BD objectives. Intermediate magnifications were obtained using a U-CA magnifying device (Olympus optical Co., Ltd. Tokyo, Japan) which was inserted between objective and camera. To optimize fluorescence excitation, U-MWU2 cube at 330-385 nm, U-MWB2 cube at 460-490 nm and U-MF2 cube at 546-563 nm were used for DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI)).

3. Results and Discussion:

3.1. PGLD(Rh):Gd3+ Synthesis and Characterization

The formation of dendritic PGLD structure was well demonstrated by the NMR spectroscopic data (Figure 1). Inthe 1H-NMR spectra, the characteristic peaks of PGLD appeared in the regions of  2.0–2.3,  4.1–4.4, and 5.0–5.7 ppm, while the characteristic protons of the Gd3+ chelatingportions showed broad absorption around  3.5 ppm.

(A) /
(B)

Figure 1- 1H-NMR (400 MHz, D2O) (A) and Maldi Tof(B) spectroscopy of PGLD G4.

The PGLD(RhB):Gd3+structure was further established by GPC analyses evaluating their relativemolecular sizes. Figure 2 shows GPC traces for the PGLD conjugated with RhB amd Gd3+, where intense peaks appearing atthe retention time of 12.4, 12.8 and 14.2 min corresponded to PGLD and their conjugates PGLD(RhB) and PGLD(RhB):Gd3+, respectively.

Figure 2- Overlay of GPC traces for PGLD (A), PGLD(RhB) and PGLD(RhB):Gd3+. Efluent:

dimethylformamide (DMF); flow rate: 0.6 mL/min; detector: reflactive index.

3.2. Optical properties of PGLD(Rh):Gd3+

The linear absorption spectra of PGLD G4 (dashed line) and PGLD(RhB):Gd3+ (solid line) in ethanol solutions are showed in Figure 3. The PGLD G4 is fully transparent at wavelengths longer than 350 nm, while the PGLD(RhB):Gd3+ present a lowest energy absorption band between 470-570 nm related to -* transitions from the ground to first excited state (S0→S1), characteristic of the RhB [29]. In Figure3, the squares represent the photoluminescence (PL) spectra excited at 530 nm, which present a strong emission band between 530 to 650 nm with maxima peaks at 563 nm.

Figure 3 –The solid lines, squares and circles show, respectively, the linear absorption, fluorescence and two-photon absorption spectra for PGLD(RhB):Gd3+. The dashed lines represent the linear absorption spectrum of PGLD G4.

Figure 3 also illustrates the two-photon absorption (empty circles) spectrum of the PGLD(RhB):Gd3+ obtained by open-aperture Z-scan measurements, similar to the ones presented in Fig. 2 (see symbols). The decrease observed in the normalized transmittance as a function of the z position indicates a 2PA process, since excitation takes place in nonresonant conditions. In Figure4, the solid lines represent the fitting obtained employing the theory described in literature[30].

Figure 4 – Open-aperture Z-scan curves for PGLD(RhB):Gd3+ in ethanol at 700 (squares) and 750 nm (circles). The solid lines represent the fitting employing the theory described in ref [30].

The nonlinear spectra (circles in Fig. 3) present a 2PA allowed band centered at approximately 700 nm, with 2PA cross-section value around 20 GM. This band corresponds to a transition to a higher energy state located at the UV region (S0→Sn / 350 nm). The 2PA cross-sections, reported in the literature for RhB [31], present values between 20-200 GM for wavelengths between 600-900 nm. The decrease in the 2PA cross-section magnitude of PGLD(RhB):Gd3+ with respect to RhB is because of the high molecular weight of PGLD G4, which does not contribute to the two-photon process.

Figure 5 shows the quadratic dependence observed for the fluorescence intensity as a function of the excitation laser irradiance at 700 nm, which confirms the two-photon nature of the nonlinear optical process. The inset (Fig. 5) shows the fluorescence spectra excited via two-photon absorption of 700 nm.

Figure 5 – Two-photon induced fluorescence signal as function of the laser power (log-log scale) for PGLD(RhB):Gd3+ at 700 nm. The solid line represents a linear fitting with a slope of approximately 2.0. This result confirms the two-photon nature of the nonlinear process. The inset shows the fluorescence spectra excited via two-photon absorption at 700 nm.

In order to measure the fluorescence lifetime for RhB and PGLD(RhB):Gd3+, we use 70 ps laser pulses at 532 nm. Figure 6 shows the deconvoluted fluorescence decay signal for RhB (squares) and PGLD(RhB):Gd3+ (circles) as well as the lifetime values obtained by fitting the data with a mono-exponential function. The fluorescence lifetime for RhB/ethanol is about 2.5 ns, while for the PGLD(RhB):Gd3+/ethanol, we obtained the lifetime of 3.7 ns. The lifetime value obtained for RhB is in good agreement with those already reported in the literature, using different techniques [32-33]. The fluorescence lifetime of PGLD(RhB):Gd3+ is higher than the one observed for RhB alone, which indicates that, in fact, there is a chemical interaction between them, which corroborates the results of Maldi-Tof spectroscopy.

Figure 6– Time-resolved fluorescence for RhB (squares) and PGLD/RhB (circles) obtained using 70 picoseconds laser excitation at 532 nm. The fluorescence lifetime was determined through the deconvolution method and a mono-exponential function.

3.3. Relaxivity properties of PGLD(Rh):Gd3+

Contrast enhancement in MRI is due to the ability of the paramagnetic Gd3+ cation to shorten the longitudinal (T1) and transverse (T2) relaxation times of water protons in the surrounding tissues. The effectiveness of gadolinium chelates as MRI contrast agents is usually assessed in vitro by measuring the corresponding relaxivities r1 and r2, defined as the longitudinal and transverse relaxation rates, respectively, for a millimolar solution of Gd complex. The commercial CAs routinely used for clinical diagnosis have longitudinal relaxivities (r1) ranging from 3.5 to 5 mM-1 s-1 [34].

Contrast agents improve image enhancement by increasing the relaxation rate ofwater protons in surrounding tissue through dipole-dipole interactions between the protonnuclear spins and the local magnetic field from the unpaired electron spins of theparamagnetic agent. The role of Gd complexes as T1 agents limits our concerns to thelongitudinal relaxation rate (1/T1). The overall relaxivity, 1/T1, can be expressed as thetotal contribution of the diamagnetic contribution from the solvent (1/T1d) and the paramagnetic contribution (1/T1p), which is proportional to the concentration of the paramagnetic species:

The overall proton relaxivity can be separated into two main contributions, inner sphere

(IS) and outer sphere (OS) relaxivity (Eq. 2 and 3):

Inner sphere relaxivity results from the chemical exchange of the water protons (coordinated to Gd+3) with the bulk water, while outer sphere relaxivity results from interaction between the Gd+3 electrons and the bulk water protons diffusing around the paramagnetic center. In addition to the inner sphere water coordinated to the Gd+3, additional water molecules can be hydrogen-bonded to the ligand (usually to the carboxylate groups) or to the inner sphere water molecule. These water molecules also contribute to the overall relaxivity of the agent, and are referred to as “second sphere” relaxation. Due to the difficulty in separating and evaluating this additional contribution, it is usually neglected or taken into account as part of the outer sphere contribution even though changing the second sphere contribution can potentially increase the overall proton relaxivity of Gd+3 contrast agents.

For low molecular weight complexes, the inner and outer sphere mechanisms have been found to contribute to the overall relaxation to roughly the same degree. Since the outer sphere contribution will remain relatively unchanged, increased relaxivities are achieved by optimizing the parameters that influence the inner sphere contribution.