Materials Science and Engineering C 52 (2015) 325–332

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Development of Gd(III) porphyrin-conjugated chitosan nanoparticles as contrast agents for magnetic resonance imaging Tania Jahanbin a, Hélène Sauriat-Dorizon b, Peter Spearman c, Soraya Benderbous a,⁎, Hafsa Korri-Youssoufi b,⁎ a b c

Université Paul Sabatier, Toulouse III, INSERM U825, CHU Purpan, 31059 Toulouse Cedex 9, France Institut de Chimie Moléculaire et des Matériaux d'Orsay, UMR CNRS 8182, ECBB, Université Paris-Sud, 91405 Orsay, France Faculty of Science, Engineering and Computing, University of Kingston, Penrhyn Road Kingston upon Thames Surrey KT1 2EE, London, UK

a r t i c l e

i n f o

Article history: Received 19 December 2014 Received in revised form 6 February 2015 Accepted 9 March 2015 Available online 10 April 2015 Keywords: Chitosan nanoparticles Dynamic light scattering Passive loading Gadolinium porphyrin Relaxivity

a b s t r a c t A novel magnetic resonance imaging (MRI) contrast agent based on gadolinium meso-tetrakis(4pyridyl)porphyrin [Gd(TPyP)] conjugated with chitosan nanoparticles has been developed. The chitosan nanoparticles were synthesized following an ionic gelation method and the conditions optimized to generate small nanoparticles (CNs) with a narrow size distribution of 35–65 nm. The gadolinium meso-tetrakis(4pyridyl)porphyrin [Gd(TPyP)] was loaded into chitosan nanoparticles by passive adsorption. The interaction of chitosan with Gd(TPyP) has been examined by UV–visible, Fourier transform infrared spectroscopies (FT-IR) and inductively coupled plasma mass spectrometry (ICP-MS), which indicate the successful association of Gd(TPyP) without any structural distortion throughout the chitosan nanoparticles. The potential of Gd(TPyP)CNs as MRI contrast agent has been investigated by magnetic resonance imaging (MRI) in-vitro. Relaxivities of Gd(TPyP)-CNs obtained from T1-weighted images, increased with Gd concentration and attained an optimum r1 of 38.35 mM−1 s−1, which is 12-fold higher compared to commercial Gd-DOTA (~4 mM−1 s−1 at 3T). The combination of such strong MRI contrast with the known properties of porphyrins in photodynamic therapy and biocompatibility of chitosan, presents a new perspective in using these compounds in cancer theranostics. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Since the first visualizing of the human body via magnetic resonance imaging (MRI) in 1977 [1,2], MRI has become the most widespread clinical diagnostic imaging technique in cancer therapy. MRI offers several significant advantages over other modalities such as high spatial resolution, noninvasiveness, absence of ionizing radiation, and capability to elicit both anatomic and physiologic information simultaneously [3]. Furthermore, MRI is also emerging as an advantageous technique in label detection, but suffers from relatively low sensitivity (insufficient contrast). In order to better distinguish targeted tissue from its surrounding, contrast of the images needs to be enhanced. One common approach to overcome the lack of MRI sensitivity is by way of contrast agents (CAs) to provide additional contrast. A tremendous effort has been devoted to designing contrast agents which exhibit high relaxivity, low toxicity, specificity, and suitably long intravascular duration and excretion time. Over recent decades, various macromolecular contrast agents have been developed [4], out of which porphyrin has attracted particular attention. It's an aromatic macrocyclic complex, exhibiting a great ⁎ Corresponding authors. E-mail addresses: [email protected] (S. Benderbous), hafsa.korri-youssoufi@u-psud.fr (H. Korri-Youssoufi).

http://dx.doi.org/10.1016/j.msec.2015.03.007 0928-4931/© 2015 Elsevier B.V. All rights reserved.

propensity for metallic cation chelation and highly stable. Porphyrin has attracted particular attention in cancer diagnosis and treatment due to its preferential uptake by tumor cells including sarcomas, carcinomas, and atheromatous plaque [5]. After the first report concerning the efficiency of water soluble Mn(II)-mesoporphyrin as a tumor targeting MRI contrast agent [6], numerous works have been devoted to study the potential of various water soluble metallo-porphyrins as MRI contrast agents. These porphyrins are substituted by anionic or cationic groups to undergo solubility in aqueous media [7]. The charged molecules could affect internalization and uptake in the cells. The main obstacle in the application of neutral porphyrin complexes in biomedical applications is their poor water solubility. However, it has been reported that by encapsulation or absorption of the therapeutic agent within hydrophilic polymeric nanoparticles, their solubility can be enhanced, which effectively decreases premature release and minimizes the administered dosage of therapeutic agents [8–11]. In the past few years, a number of polymeric nanoparticles have been synthesized to improve delivery efficiency and reduce toxicity of the drug [12–14]. Chitosan is one of the most promising polymers for pharmaceutical and biomedical applications [15,16]. Chitosan is a natural linear polysaccharide, composed of β-(1-4)-linked-2-amino-2-deoxy-β-Dglucopyranose and 2-acetamido-2-deoxy-β-D-glucopyranose, derived through partial deacetylation of chitin 52 [17]. Chitosan nanoparticles (CNs) have been intensively investigated for drug delivery administration

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Scheme 1. Schematic diagram depicting formation of chitosan nanoparticles (CNs) and Gd(TPyP)-CNs particles.

due to its various properties, such as biocompatibility, biodegradability, nontoxicity and mucoadhesive properties [18,19]. The core of CNs is capable of encapsulating different therapeutic agents [20], while the CNs' surface has a tendency to adhere to negatively charged cell surfaces which enable CNs to penetrate through cell membranes [21–23]. Chitosan has also shown the inhibitory growth effects on bladder tumor cells [24]. Thus, chitosan is largely favored for formulating drug delivery vehicles and contrast agent carriers for molecular imaging [25]. For this study, we demonstrate the potential of neutral metallated porphyrin Gd(III) meso-tetrakis(4-pyridyl)porphyrin [Gd(TPyP)], encapsulated in chitosan nanoparticles as MRI contrast enhancer. The synthesis of chitosan nanoparticles has been performed following ionic gelation and the conditions optimized to obtain small nanoparticles with narrow size distributions. Subsequently, Gd(TPyP) was conjugated with chitosan nanoparticles via a passive method (See Scheme 1). The loading of Gd(TPyP) to the nanoparticles has been studied by UV–visible spectroscopy and by inductively coupled plasma mass spectrometry (ICP-MS). Further characterization has been performed via scanning electron microscopy (SEM) equipped with energy-dispersive X-ray spectroscopy (EDX), dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FT-IR). The efficiency of Gd(TPyP)-CNs in aqueous solution as MRI contrast agents has been studied by MRI at 3T.

2. Experimental 2.1. Chemical materials Chitosan (from shrimp shells, medium molecular weight (MMv), deacetylation degree (DD) ≥85%), chitosan (high purity, low molecular weight (LMv = 60 kDa), deacetylation degree (DD) ≥ 93%), and pentasodium triphosphate (TPP) (purity ≥ 98%) were purchased from Sigma-Aldrich. TPyPH2 and Gd(TPyP) complexes were synthesized following the procedure described by Adler and Longo [26]. All the prepared solutions were filtered by syringe filter (pore size 0.22 μm, Milipore). All other reagents used were of analytical grade without further purification.

2.1.1. Synthesis of chitosan nanoparticles (CNs) The chitosan nanoparticles were prepared by ionic gelation following the reported method by Fan et al. [27] with some modifications. For this purpose, various concentrations (0.5, 0.7, 1, and 1.7 mg/mL) of two bulk chitosan polymers with MMv and DD = 85% and LMv and DD = 93% were dissolved in 70 mL of acid acetic (1%). The mixture was stirred vigorously overnight at room temperature. The pH of the solution was adjusted to 4.7 by adding dropwise the 20 wt.% NaOH aqueous solution under magnetic stirring followed by sonication for 20 min. In order to form the nanoparticles, 3 mL of the chitosan solution was heated at 60 °C for 10 min in a water bath and mixed with 1 mL of cooled TPP solution (1.25 mg/mL) under constant magnetic stirring in a cooled environment (6 °C). The transparent solution became opalescent after mixing of TPP and chitosan solution following by ultrasonication procedure [28], where the time of reaction and speed of agitation were optimized to obtain the smallest and mono-disperse CNs. The optimum time and speed of stirring were determined as 10 min and 700 rpm, respectively. After centrifugation at 12,500 rpm for 15 min (4 °C), the CNs were washed 3 times with distilled water and freeze-dried overnight at 2 °C (see SI, Fig. S1). 2.1.2. Preparation of Gd(TPyP) loaded chitosan nanoparticles Drugs can be loaded into nanoparticles either covalently and/or noncovalently which can be further divided into encapsulation and absorption techniques, loading during and after nanoparticle formation, respectively. Passive absorption has already been utilized in loading of the drugs in nanoparticles [29]. Herein, loading of Gd(TPyP) through CNs has been performed via passive absorption by which, various quantities of Gd(TPyP) (0.5, 1, 2, and 3 mg) were mixed with 1 mg of dry CNs. Afterwards, the mixture was dispersed in 1 mL of distilled water under magnetic stirring for 15 min at 4 °C, then ultrasonication for 5 min at 4 °C. The reactant solution was centrifuged at 12,500 rpm for 30 min at 4 °C and the supernatant was freeze-dried overnight at 2 °C. 2.2. Characterization of nanoparticles 2.2.1. Physicochemical characterization of CNs and Gd(TPyP)/CNs The hydrodynamic radius and polydispersity of CNs and Gd(TPyP)CNs were determined by dynamic light scattering using a photon

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correlation spectroscopy (PCS) assembly ZetaSizer-NanoZS (Malvern Instrument, UK) equipped with a standard 633 nm laser. A small quantity of CNs and Gd(TPyP)-CNs was dispersed in 1 mL of distilled water. The size and polydispersity of prepared colloids were calculated from an autocorrelation function of light scattered from particles. The measurements were carried out at 20 °C at a fixed scatter angle of 90°. Moreover, morphology of CNs and Gd(TPyP)-CNs was examined using scanning electron microscopy (SEM) (cambridgeS260, UK). A small drop of nanoparticle suspension was dropped on the gold surface and dried at room temperature. The quantitative analysis of Gd(TPyP)-CNs was examined by energy dispersive X-ray spectroscopy (EDX).

2.2.5. Determination of entrapment efficiency, loading capacity and yield of Gd(TPyP)/CNs In order to evaluate the entrapment efficiency and loading capacity of Gd(TPyP) into CNs, the prepared Gd(TPyP)-CNs were ultracentrifuged at 18,500 rpm for 20 min at 4 °C. The amount of free Gd(TPyP) in the supernatant after ultracentrifugation was measured by UV–visible spectrometry. The colorless (to the eye) solution was collected and the concentration of Gd(TPyP) was determined by monitoring the intensity of the characteristic porphyrin Soret band by UV–visible spectroscopy. The entrapment efficiency (EE), loading capacity (LC) and yield percentage of Gd(TPyP)/CNs were calculated as follows:

2.2.2. UV–visible spectroscopy Due to the characteristic UV–visible spectrum of the porphyrin complex, the ultraviolet–visible absorption spectroscopy has been used to study the concentration of the Gd(TPyP) loaded to CNs. The UV–vis spectra of prepared Gd(TPyP)-CNs was recorded by UVIKON XL spectrometer in the range of 350–700 nm at room temperature using quartz cuvettes (1 cm optical path).

EEð%Þ ¼

2.2.3. Fourier transmission infrared spectroscopy Fourier transmission infrared spectroscopy (FT-IR) of CNs, Gd(TPyP), and Gd(TPyP)-CNs was carried out with a Bruker IFS 66 spectrometer with ATR pike accessory over the range of 4000–800 cm− 1 using 10 scans at room temperature.

ðA−BÞ  100 A ðA−BÞ LCð%Þ ¼  100 C Yieldð%Þ ¼ ðactual mass=theoretical massÞ  100 where A is the initial added mass of Gd(TPyP), B is the mass of free Gd(TPyP) in the supernatant after centrifuge, and C is the mass of nanoparticles. Actual mass is the mass of Gd(TPyP)-CNs after freeze-drying while theoretical mass corresponds to initial total mass of CNs and Gd(TPyP). 3. Results and discussions 3.1. Size and polydispersity of CNs

2.2.4. Magnetic resonance imaging The magnetic resonance imaging (MRI) studies of Gd(TPyP)-CNs dispersed in water were performed with a 3T MRI apparatus (Philips ACHIEVA TX) at 25 °C. The longitudinal (r1) and transverse (r2) relaxivities of prepared colloids were evaluated from obtained T1 and T2 weighted images. T1-weighted images were acquired from spin echo sequences with an echo time (TE) of 8 ms and varying repetition time (TR) between 100–400 ms. T2-weighted images were obtained from the spin echo sequence with a long TR = 1500 ms and varying TE between 7 ms–40 ms. The other acquisition parameters were optimized as follows: field of view (FOV) = 154 × 69 × 5, slice thickness = 5 mm, flip angle = 90°, number of signal averages = 7, and voxel = 2 × 2 × 5 mm3 (resolution = 2.03 × 2.08 × 5). The presented high resolution T1 and T2 weighted images were obtained from spin echo sequence (TR = 400 ms and TE = 8 ms) and (TR = 1500 ms, TE = 40 ms) respectively, with voxel = 1 × 1 × 5 mm3 (1.01 × 0.98 × 5) and field of view (FOV) = 154 × 69 × 5, slice thickness = 5 mm, flip angle = 90°, and number of signal averages = 7. After image acquisition, the magnitudes of signal intensities within regions of interest (ROIs) were inferred manually by ImageJ software. The T1 and T2 MRI signal intensities have been fitted on a voxel basis with a     monoexponential function S1 ¼ S0 : 1− exp −TR T and S1 ¼ S0 : 1    to evaluate T1 and T2 relaxation times, respectively. exp −TE T 2

While the S0 in the first equation represents the signal intensity at very long TR (depends on the TE) and S0 in the second equation corresponds to the signal intensity at very small TE (depends on the TR). r1 and r2 relaxivities were obtained from the linear least square regression of the inverse of relaxation times 1/T1 and 1/T2 versus the concentration of paramagnetic ions as Eq. (1). 1 1 ¼ þ ri C TiðobsÞ TiðdiaÞ

i ¼ 1; 2

ð1Þ

where T 1 is the inverse of Ti (i = 1,2) of aqueous solution (s−1), T 1 is iðobsÞ

iðdiaÞ

the inverse of Ti (i = 1,2) of the solvent (s−1), C is the concentration of paramagnetic ion (mmol− 1) and ri is the paramagnetic relaxivity (mmol−1 s−1).

Chitosan is characterized by its degree of deacetylation (DD) and its viscosity in acidic solution (2%) which has an effect on shape and size of chitosan nanoparticles. Moreover, chitosan/TPP weight ratio plays an important role in controlling the mean diameter size of CNs, morphology and surface charge of CNs. In addition, molecular weight of chitosan as well as ionic strength, medium and pH affect the stability and physicochemical properties of CNs. In this study, bulk chitosan with medium (MMv) and low molecular weight (LMv) and deacetylation degree of 85% and 93%, respectively (see Experimental section), have been used to prepare CNs. CNs have been prepared with various chitosan concentration and the effect on their size and polydispersity have been studied via dynamic light scattering (DLS). The results obtained are listed in Table 1 and show that the size and polydispersity (PdI) of CNs depend strongly on the chitosan concentration employed during preparation. The smallest CNs particle size was obtained for the synthesis with concentration of 0.7 mg/mL of chitosan in which the mean diameter of CNs for MMv and LMv is 305 nm and 120 nm, respectively. At chitosan concentrations higher than 0.7 mg/mL, the average size of CNs obtained from MMv and LMv increased to 23 μm and 12 μm, respectively. The increase of particle size for high chitosan concentration could be related to the formation of aggregates in solution which has previously been reported [30]. In summary, the smallest particle with narrow size distribution was obtained at 0.7 mg/mL chitosan concentration for both MMv and LMv chitosan. Results in Table 1 also show that LMv chitosan leads to smallest CNs. Such effects of molecular weight on the size of chitosan nanoparticles have been also demonstrated within various methods of synthesis [31]. The occurrence of conformational change of chitosan Table 1 Mean particle size (nm) and PdI values obtained by dynamic light scattering from different chitosan concentration (mg/mL) with two different deacetylation degrees (85% and 93%). TPP = 1.25 mg/mL, chitosan–TPP mass ratio = 3.3/1, T = 20 °C in distilled water. Chitosan concentration (mg/mL)

0.5 0.7 1.0 1.7

Chitosan(MMv) DD = 85%

Chitosan (LMv) DD = 93%

PdI

CNs (nm)

PdI

CNs (nm)

0.319 0.201 0.789 1

9158 305 980 23,860

0.289 0.139 0.687 1

8991 120 684 12,230

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due to its molecular weight could be attributed to differences in intramolecular hydrogen bonds and/or differences in charge distribution depending on chitosan chain length [31,34]. Chitosan with high molecular weight should have more intermolecular hydrogen bonding and their molecules can entangle easily which leads to the formation of large particles. While for the low molecular weight, the conformation of chitosan in solution is stiff and extended, leading to smaller particle sizes. The other parameter, which could affect the particle size of CNs, is deacetylation degree (DD) of chitosan as electrostatic, hydrophobic interactions, hydrogen bonding as well as van der Waals interaction could change with this parameter [32,33,35]. Hence, in our study, the deacetylation degree of bulk chitosan was studied with two polymers. As listed in Table 1, the average size of CNs obtained from bulk chitosan with DD of 93% is smaller than that obtained from 85% DD. This could be explained by an increase of chain rigidity at higher content of bulky acetyl groups causing more hydrophobic interaction by hydrogen bonding which is consistent with previously reported results [36]. The impact of stirring rate and time of reaction on the formation of CNs, was also studied (see Supporting information, Table S1). The smallest CNs are obtained after a 10 min reaction time and gentle stirring of 700 rpm. Longer times could lead to the coalition of nanoparticles and causing the formation of aggregates. Table 2 reviews the size and polydispersity of the CNs obtained in the present work compared to previously reported values in the literature. As seen from Table 2, the method developed in this work using ionic gelation with the optimization of chitosan concentration, time of reaction and the speed produces nanoparticles with small size with low polydispersity.

3.2. Gd(TPyP)-CNs formation and characterization 3.2.1. Loading amount The loading of Gd(TPyP) through the CNs was performed following passive absorption method by mixing the formed CNs with various concentrations of Gd(TPyP) as described in the Experimental section. Samples 1, 2, 3, and 4 are referred to 0.5, 1, 2, and 3 mg of Gd(TPyP)-loaded in 1 mg of CNs. The Gd(TPyP)-CNs complex was characterized by UV– visible spectroscopy following the Soret band of the metalloporphyrin in water. Fig. 1 shows the electronic absorption spectra of the Gd(TPyP)-loaded CNs compared to free Gd(TPyP) in ethanol. The same behavior was obtained for all the Gd(TPyP)-CNs complexes. The Gd(TPyP)-CNs spectra display an intense band at 422 nm designated as the Soret band and a band at 554 nm corresponding to a Q band which is typically observed for Gd(TPyP). This demonstrated that Gd(TPyP) was successfully loaded in chitosan nanoparticles. No modification in the Soret band was observed for the porphyrin–CNs complex suggesting the absence of structural modification in metalloporphyrin. The characteristic purple color of Gd(TPyP)-CNs intensified with increased loading of Gd(TPyP), and

Fig. 1. UV–visible spectra of Gd(TPyP) (in ethanol) and Gd(TPyP)-CNs after loading of increasing quantities of Gd(TPyP) to CNs in distilled water (samples 1–4).

all samples showed high solubility in water (inset of Fig. 1). UV–visible spectroscopy was used to determine the concentration of loaded Gd(TPyP) into CNs by monitoring the intensity of the Soret band (422 nm) of Gd(TPyP)-CNs taking into account the extinction coefficient of ε = 1.582 × 105 L·mol−1·cm−1. For comparison, quantitative measurements with inductively coupled plasma mass spectrometry (ICP-MS) were also performed on the same samples. This method measures the Gd(157) isotope to avoid interference from other lanthanides and gives the real value of Gd loaded in CNs. The two analytical methods show the increase of the amount of Gd(TPyP) loaded in chitosan nanoparticles from samples 1 to 4 and a linear correlation between the values obtained by UV-measurement and ICP-MS was observed (see Supporting information, Fig. S1). Entrapment efficiency (EE), loading capacity (LC) and yield percentage of prepared samples were evaluated as described in the Experimental section. The effect of different concentrations of Gd(TPyP) on EE, LC and yield (%) of Gd(TPyP) loaded CNs is presented in Fig. 2. It can be observed that EE did not change significantly and a maximum of (87%) was achieved at CNs:Gd(TPyP) ratio of 1:2, while LC and yield (%) reached a maximum at CNs:Gd(TPyP) ratio of 1:3. 100 90 80

LC (%) EE (%) Yield (%)

70 60

Table 2 Polydispersity and mean diameter of recently reported chitosan nanoparticles. Method of synthesis

Molecular weight (kDa)

Deacetylation degree (%)

Ionic gelation

60.0

93.0

150.0 620 24 200 350 LMv 43 44 208

– 90.0 95.0

Dry milling

90 93 92

diameter CNs (nm)

50

Polydispersity index (PdI)

Ref

120

0.139

200 321 153 180 210 186 735 849 2382

0.185 – –

This work [36] [37] [38]

40 30 20 10 0 1:0,5

0.38

[39] [40]

1:1

1:2

1:3

CNs:Gd(TPyP) Fig. 2. Entrapment efficiency (EE), loading capacity (LC) and yield percentage of Gd(TPyP)-CNs.

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2,1

Gd(TPyP)-CNs

a

2100 2875

1322 1411 1203 1378 1641 1598

3300

892

Transmittance (%)

329

792 1027

Gd(TPyP)

1070 2094

1,4

3255

1197 890 1070 1334 850 985

1401

1594 1540

CNs

790 2107

0,7 1376 1538 1631 1203 1151

889 1022

2892

3243

3407

1066

1000

1500

2000

2500

3000

3500

4000

-1

Wavenumbers (cm )

b

Fig. 3. a) FT-IR spectra of CNs, Gd(TPyP) and Gd(TPyP)-CNs, b) EDX spectra of Gd(TPyP)-CNs.

3.2.2. Structural characterizations FT-IR spectroscopy of Gd(TPyP)-CNs compared to Gd(TPyP) and CNs was performed for structural characterization. Fig. 3a shows that Gd(TPyP) exhibits characteristic bands of the porphyrin units at 1594 cm−1 corresponding to stretching vibration (C_C), 1334 cm−1 (C_N stretch in pyrrole), 1401 (C–N), 1070, 890 (–C–H aromatic) and 790 cm− 1 (_C–H, aromatic ring vibration) [41–43]. Moreover, the observed band in the IR spectrum of Gd(TPyP) in the range of 1400 cm− 1 can be attributed to acetate stretching –C–O vibration ligand to metal which overlaps with the C_C stretching of porphyrin. The FT-IR spectrum of Gd(TPyP)-CNs is similar to the one of CNs with additional peaks referring to Gd(TPyP) with slight shifts. The peak at 1641 cm−1 in Gd(TPyP)-CNs spectrum is referred to carbonyl vibration of chitosan. This band shows a small shift from 1631 cm−1 for free chitosan to 1641 cm−1 after conjugation with Gd(TPyP). Moreover, the typical pyridyl vibration band of Gd(TPyP) at 1540 cm−1 disappears in the IR spectrum of the composite. This could be attributed to the interaction of chitosan with the pyridyl via hydrogen bonding of carbonyl band in chitosan which could be related to the coordination between chitosan and Gd(TPyP). The presence of Gd(III) in Gd(TPyP)-CNs has also been investigated using EDX-SEM. The EDX spectra were recorded at a number of spots, which confirmed the existence of Gd(III) in Gd(TPyP)-CNs (Fig. 3b).

3.2.3. Morphology characterizations In biomedical applications of nanoparticles, developing the nanostructures with well-defined uniform particle size and shape is highly demanding [44]. In imaging and drug delivery, the particles in the range of nanometers with uniform size and spherical shape enhance their ability to cross cell membranes and reduce the risk of rapid clearance from the body [44]. These criteria have been the basis of the optimization procedures that we have covered up to this point. The morphology of CNs and Gd(TPyP)-CNs has been studied via SEM and TEM respectively and results are presented in Fig. 4. From these images, CNs and Gd(TPyP)CNs appear uniform and spherical in shape and the absorption of Gd(TPyP) to CNs does not cause any change in the shape of nanoparticles (Fig. 4b). SEM and TEM images allow to measure the particle size of these nanoparticles which were determined around 35 to 50 nm. The typical size distribution by scattering intensity graphs of CNs and Gd(TPyP)-CNs is presented in Fig. 5. The size distributions of CNs and Gd(TPyP)-CNs in water solution show a single peak with maximum at 120 and 412 nm, respectively. The discrepancy in size measured with DLS and SEM and TEM can be attributed to the different measurement techniques. DLS measures the hydrodynamic radius of particles while SEM and TEM gives an actual diameter of nanoparticles in their dry state. The increase of the average size in DLS compared to the SEM and TEM image could be attributed to the enhancement of hydrodynamic diameter of particles due to their solvation sphere.

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a

b

Fig. 4. a) SEM image of chitosan nanoparticles CNs, b) TEM image of the composite Gd(TPyP)-CNs.

3.3. Magnetic resonance imaging The efficacy of suspension of Gd(TPyP)-loaded chitosan nanoparticles with different loading content in distilled water has been studied via magnetic resonance imaging at 3T and 25 °C. The high resolution T1 and T2 weighted images of Gd(TPyP)-CNs in water are presented in Fig. 6a. It is shown that the T1 signal intensity increases with the loading amount of Gd(TPyP) in CNs corresponding to an increase of Gd(III) concentration from 0.103 mM to 0.659 mM and vice versa for the T2 signal intensity. In order to approve the potential of this novel complex as MRI contrast agent, longitudinal and transverse relaxivities (r1 and r2) of Gd(TPyP)-CNs in aqueous solution have been evaluated and are presented in Fig. 6b. It's shown from these curves that the relaxation rate (1/T1 and 1/T2) of Gd(TPyP)-CNs increased linearly with Gd(III) concentration. The r1 of Gd(TPyP)-CNs in water was calculated at 38.35 mM−1 s−1 which is 56% higher than r1 of free Gd(TPyP) in ethanol (24.5 mM− 1 s−1) at 3T, while the evaluated r2 relaxivity of Gd(TPyP)-CNs in water is 33.43 mM−1 s−1 and decreased by 52% compared with free Gd(TPyP) in ethanol (69.97 mM−1 s−1) at 3T. Also a comparison of the measured r1 of Gd(TPyP)-CNs in water shows a

value 12 times higher than r1 of commercial Gd-DOTA at 3T which is a commercial product used generally as MRI contrast agent. Such a high relaxivity demonstrates the high potential application of this complex as MRI enhanced contrast agent. Due to the inner and outer sphere contributions on the paramagnetic relaxivity mechanism, relaxivity of contrast agents can be tuned via the number of coordinated water molecules to the paramagnetic ion, internal rotational motion, water exchange rate, water residency time and tumbling rate. One common strategy employed to enhance the relaxivity, is the conjugation of contrast agents with macromolecules which can efficiently delay the rotational motion of the complex, increasing rotational correlation time. Thus, the enhancement of r1 relaxivity with the conjugation of the Gd-porphyrin to CNs can be explained by a decrease in rotational correlation time. Moreover, this enhancement could be also related to an increase in exchange rate of nearby coordinated water molecules due to molecular structure of chitosan [48] and high Gd-loaded into nanoparticles. The enhancement of r1 relaxivity of Gd(TPyP) conjugated with chitosan nanoparticles is consistent with various reports. For example, the commercial contrast agent Gd-DTPA conjugated with CNs has shown higher r1 of 11.62 mM−1 s−1 compared to those of Gd-DTPA in aqueous solution

Fig. 5. Size distribution of CNs and Gd(TPyP)-CNs dissolved in water at 20 °C via DLS.

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a

b

331

Relaxivity of Gd(TPyP)-CNs at 3T

1/Ti(i=1,2)

b)

Gd(III) concentration (mM) Fig. 6. a) High resolution T1 (TR = 400 ms, TE = 8 ms) and T2 (TR = 1500 ms, TE = 40 ms) weighted spin echo MR images of different concentrations of Gd(TPyP)-CNs and b) longitudinal and transverse relaxivities of Gd(TPyP)-CNs in water at 3T and 25 °C.

Relaxivity (mM-1.s-1)

(3.62 mM−1 s−1) at 0.5T [45]. Mn-DTPA-CNs in aqueous solution has shown a high r1 of 7.21 mM− 1 s−1 at 0.5T and 32 °C compared with those of Mn-DTPA (3.5 mM−1 s−1) [46]. Gd-phostriamine (MS-325) chelated with chitosan-β-cyclodextrins also showed high r1 relaxivity (27.5 mM−1·s−1) compared with that of free macromolecule [47]. The higher r2 relaxivity of free Gd(TPyP) (69.97 mM−1·s-1) compared to the encapsulated one (33.43 mM−1 s−1), see Fig. 7, can be related to the poor solubility of free Gd(TPyP) in organic solvent forming aggregates that influence the r2 relaxivity, as it is known that the r2 relaxivity increases with the aggregates [49]. By contrast, Gd(TPyP)CNs show great dispersity in water, which might lead to obtain the lower r2 relaxivity compared to the free compound. While, MRI is a powerful tool for noninvasively mapping biological structure and function, it is important to keep in mind that the method has ubiquitous limitation such as signal-to-noise ratio, which could be improved by employing the exogenous contrast agents. By administration of exogenous contrast agents to enhance T1 differences, a relatively large agent concentration must be delivered. Typically, for conventional agents, concentrations in excess of approximately 50 mM are required. Whereas in our study, the high in-vitro relaxivity at low concentration (less than 1 mM) with r1/r2 ratio nearby unity in moderate field (3T) are the advantages of this new composite (Gd(TPyP)-CNs) applied as MR imaging contrast agent at high magnetic fields. 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0

r1 r2

4. Conclusion Chitosan nanoparticles have been synthesized following an ionic gelation method and loaded with Gd(TPyP) to obtain contrast agents for MRI. The optimization approach investigated for chitosan nanoparticle formulation and loading process leads to nanoparticles with a diameter in the range of 45–65 nm and hydrodynamic radius of 412 nm. UV–visible and FT-IR studies demonstrated that Gd(TPyP) successfully conjugated with the CNs and furthermore, direct attachment of the porphyrin complex to the chitosan polymer network was demonstrated following the hydrogen interaction between pyridyl in the meso position of the macrocycle and the carbonyl of chitosan. No evident distortion of the macrocycle was observed based on the similarity of the absorption spectra of Gd(TPyP)-CNs complex and free Gd(TPyP). We demonstrated that the encapsulation efficiency and loading capacity of Gd(TPyP)-CNs were attained at 87% and 47% respectively. MR imaging of Gd(TPyP)-CNs invokes an enhancement of the contrast of MR images at low concentration as compared to isolated Gd(TPyP). Gd(TPyP)-CNs demonstrate relatively high relaxivities (almost 12 times) in moderate field (3T) compared to conventional Gd-based MR contrast agents such as Gd-Dota. Referring to MRI studies, we can conclude that the Gd(TPyP)-CNs nanoparticles offer great potential to be considered as novel MRI contrast agents. As some porphyrins are already used in drugs for photodynamic therapy agents, such complexes could be investigated for their association with the chitosan nanoparticles developed in this work metallo- and applied in theranostics investigation of some cancers. Acknowledgments The authors gratefully acknowledge Mr. François Brisset and Dr. J. Swinden for their kind technical cooperations and valuable contributions for this work. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2015.03.007.

Gd(TPyP)-CNS Gd(TPyP) Gd-DOTA in water in water in ethanol at 3T and 25°C at 3T and 25°C at 3T and 25°C Fig. 7. r1 and r2 relaxivities of Gd(TPyP), Gd(TPyP)-CNs, Gd-DOTA in aqueous solution at 3T and 25 °C.

References [1] R. Damadian, M. Goldsmith, L. Minkoff, NMR in cancer: XVI. FONAR image of the live human body, Physiol. Chem. Phys. 9 (1) (1977) 97–100 (108).

332

T. Jahanbin et al. / Materials Science and Engineering C 52 (2015) 325–332

[2] W.S. Hinshaw, P.A. Bottomley, G.N. Holland, Radiographic thin-section image of the human wrist by nuclear magnetic resonance, Nature 270 (5639) (Dec. 1977) 722–723. [3] G.M. Lanza, P.M. Winter, S.D. Caruthers, A.M. Morawski, A.H. Schmieder, K.C. Crowder, S.A. Wickline, Magnetic resonance molecular imaging with nanoparticles, J. Nucl. Cardiol. 11 (6) (2004) 733–743. [4] R. Mewis, S. Archibald, Biomedical applications of macrocyclic ligand complexes, Coord. Chem. Rev. 254 (15–16) (2010) 1686–1712. [5] D.N. Reinhoudt, Supramolecular Materials and Technologies, Wiley, 2008. [6] C. Chen, J.S.J. Cohen, C.C.E. Myers, M. Sohn, Paramagnetic metalloporphyrins as potential contrast agents in NMR imaging, FEBS Lett. 168 (1984) 70–74. [7] T. Jahanbin, H. Sauriat-Dorizon, S. Benderbous, H. Korri-Youssoufi, Gadolinium meso-tetrakis(4–pyridyl)porphyrin as a MRI contrast agent: comparison with anionic and cationic metallo-porphyrins, J. Coll. Sci. Biotechnol. (2015) (in press). [8] S.K. Sahoo, S. Parveen, J.J. Panda, The present and future of nanotechnology in human health care, Nanomed. Nanotechnol. Biol. Med. 3 (1) (2007) 20–31. [9] S.K. Sahoo, V. Labhasetwar, Nanotech approaches to drug delivery and imaging, Drug Discov. Today 8 (24) (2003) 1112–1120. [10] S. Parveen, S. Sahoo, Polymeric nanoparticles for cancer therapy, J. Drug Target. 16 (2) (2008) 108–123. [11] M. Das, C. Mohanty, S. Sahoo, Ligand-based targeted therapy for cancer tissue, Expert Opin. Drug Deliv. 6 (3) (2009) 285–304. [12] K.S. Soppimath, T.M. Aminabhavi, A.R. Kulkarni, W.E. Rudzinski, Biodegradable polymeric nanoparticles as drug delivery devices, J. Control. Release 70 (1–2) (2001) 1–20. [13] K.E. Uhrich, S.M. Cannizzaro, R.S. Langer, K.M. Shakesheff, Polymeric systems for controlled drug release, Chem. Rev. 99 (11) (1999) 3181–3198. [14] D.F. Ranney, Biomimetic transport and rational drug delivery, Biochem. Pharmacol. 59 (2) (2000) 105–114. [15] P. Giunchedi, I. Genta, B. Conti, R.A.A. Muzzarelli, U. Conte, Preparation and characterization of ampicillin loaded methylpyrrolidinone chitosan and chitosan microspheres, Biomaterials 19 (1–3) (1998) 157–161. [16] P. Calvo, C. Remuñán-López, J.L. Vila-Jato, M.J. Alonso, Novel hydrophilic chitosanpolyethylene oxide nanoparticles as protein carriers, J. Appl. Polym. Sci. 63 (1) (Jan. 1997) 125–132. [17] L. Ilium, Chitosan and its Use as a pharmaceutical excipient, Pharm. Res. 15 (9) (1998). [18] K. Bowman, K.W. Leong, Chitosan nanoparticles for oral drug and gene delivery, Int. J. Nanomedicine 2 (1) (2006) 117–128. [19] N.G.M. Schipper, K.M. Vårum, P. Stenberg, G. Ocklind, H. Lennernäs, P. Artursson, Chitosans as absorption enhancers of poorly absorbable drugs: 3: influence of mucus on absorption enhancement, Eur. J. Pharm. Sci. 8 (4) (1999) 335–343. [20] A. Trapani, J. Sitterberg, U. Bakowsky, T. Kissel, The potential of glycol chitosan nanoparticles as carrier for low water soluble drugs, Int. J. Pharm. 375 (1–2) (2009) 97–106. [21] P. Artursson, T. Lindmark, S.S. Davis, L. Illum, Effect of chitosan on the permeability of monolayers of intestinal epithelial cells (Caco-2), Pharm. Res. 11 (9) (1994). [22] G. Borchard, H.L. Lueβen, A.G. de Boer, J.C. Verhoef, C.-M. Lehr, H.E. Junginger, The potential of mucoadhesive polymers in enhancing intestinal peptide drug absorption. III: effects of chitosan-glutamate and carbomer on epithelial tight junctions in vitro, J. Control. Release 39 (2–3) (1996) 131–138. [23] K.A. Janes, P. Calvo, M.J. Alonso, Polysaccharide colloidal particles as delivery systems for macromolecules, Adv. Drug Deliv. Rev. 47 (1) (2001) 83–97. [24] M. Hasegawa, K. Yagi, S. Iwakawa, M. Hirai, Chitosan induces apoptosis via caspase3 activation in bladder tumor cells, Cancer Sci. 4 (2001) 459–466. [25] S. Hein, K. Wang, Chitosan composites for biomedical applications: status, challenges and perspectives, Mater. Sci. Technol. 24 (2008) 1053–1061. [26] A.D. Adler, F.R. Longo, J.D. Finarelli, J. Goldmacher, J. Assour, L. Korsakoff, A simplified synthesis for meso-tetraphenylporphine, J. Org. Chem. 32 (2) (Feb. 1967) 476. [27] W. Fan, W. Yan, Z. Xu, H. Ni, Formation mechanism of monodisperse, low molecular weight chitosan nanoparticles by ionic gelation technique, Colloids Surf. B Biointerfaces 90 (2012) 21–27.

[28] E.S.K. Tang, M. Huang, L.Y. Lim, Ultrasonication of chitosan and chitosan nanoparticles, Int. J. Pharm. 265 (1–2) (Oct. 2003) 103–114. [29] M. Athar, A. Das, Therapeutic nanoparticles: state-of-the-art of nanomedicine, Adv. Mater. Rev. 1 (2014) 25–37. [30] Q. Gan, T. Wang, C. Cochrane, P. McCarron, Modulation of surface charge, particle size and morphological properties of chitosan–TPP nanoparticles intended for gene delivery, Colloids Surf. B Biointerfaces 44 (2–3) (2005) 65–73. [31] M.L. Tsaih, R.H. Chen, Effect of molecular weight and urea on the conformation of chitosan molecules in dilute solutions, Int. J. Biol. Macromol. 20 (3) (1997) 233–240. [32] P. Sorlier, C. Viton, A. Domard, Relation between solution properties and degree of acetylation of chitosan: role of aging, Biomacromolecules 3 (6) (2002) 1336–1342. [33] C. Schatz, C. Viton, T. Delair, C. Pichot, A. Domard, Typical physicochemical behaviors of chitosan in aqueous solution, Biomacromolecules 4 (3) (2003) 641–648. [34] C. Schatz, C. Pichot, T. Delair, C. Viton, A. Domard, Static light scattering studies on chitosan solutions: from macromolecular chains to colloidal dispersions, Langmuir 19 (23) (2003) 9896–9903. [35] C.J. Van Oss, M.K. Chaudhury, R.J. Good, Interfacial Lifshitz-van der Waals and polar interactions in macroscopic systems, Chem. Rev. 88 (6) (1988) 927–941. [36] A. Rampino, M. Borgogna, P. Blasi, Chitosan nanoparticles: preparation, size evolution and stability, Int. J. Pharm. 455 (2013) 219–228. [37] H. Liu, C. Gao, Preparation and properties of ionically cross‐linked chitosan nanoparticles, Polym. Adv. Technol. 20 (2009) 613–619. [38] Y. Wu, W. Yang, C. Wang, J. Hu, S. Fu, Chitosan nanoparticles as a novel delivery system for ammonium glycyrrhizinate, Int. J. Pharm. 295 (2005) 235–245. [39] A. Floris, M. Meloni, F. Lai, Cavitation effect on chitosan nanoparticle size: a possible approach to protect drugs from ultrasonic stress, Carbohydr. Polym. 94 (2013) 619–625. [40] H. Kwak, S. Kim, Physicochemical and biofunctional properties of crab chitosan nanoparticles, J. Nanosci. Nanotechnol. 13 (2013) 5296–5304. [41] Z.-C. Sun, Y.-B. She, Y. Zhou, X.-F. Song, K. Li, Synthesis, characterization and spectral properties of substituted tetraphenylporphyrin iron chloride complexes, Molecules 16 (4) (2011) 2960–2970. [42] N.E. Shi, G. Yin, M. Han, L. Jiang, Z. Xu, Self-assembly of two different hierarchical nanostructures on either side of an organic supramolecular film in one step. Chemistry 14 (20) (Jan. 2008) 6255–6259. [43] M.S. Saeedi, S. Tangestaninejad, M. Moghadam, V. Mirkhani, I. MohammadpoorBaltork, A.R. Khosropour, Magnetic nanoparticles supported manganese(III) tetrapyridylporphyrin catalyst via covalent interaction: A highly efficient and reusable catalyst for the oxidation of hydrocarbons, Polyhedron 49 (1) (25 January 2013) 158–166. [44] S. Svenson, D.A. Tomalia, Dendrimers in biomedical applications—reflections on the field, Adv. Drug Deliv. Rev. 57 (15) (2005) 2106–2129. [45] Y. Huang, B. Cao, X. Yang, Q. Zhang, X. Han, Z. Guo, Gd complexes of diethylenetriaminepentaacetic acid conjugates of low-molecular-weight chitosan oligosaccharide as a new liver-specific MRI contrast agent. Magn. Reson. Imaging 31 (4) (May 2013) 604–609. [46] Y. Huang, X. Zhang, Q. Zhang, X. Dai, J. Wu, Evaluation of diethylenetriaminepentaacetic acid–manganese(II) complexes modified by narrow molecular weight distribution of chitosan oligosaccharides as potential magnetic resonance imaging contrast agents, Magn. Reson. Imaging 29 (4) (2011) 554–560. [47] S. Aime, E. Gianolio, F. Uggeri, S. Tagliapietra, A. Barge, G. Cravotto, New paramagnetic supramolecular adducts for MRI applications based on non-covalent interactions between Gd(III)-complexes and beta- or gamma-cyclodextrin units anchored to chitosan. J. Inorg. Biochem. 100 (5–6) (May 2006) 931–938. [48] A. Szpak, G. Kania, T. Skórka, W. Tokarz, S. Zapotoczny, M. Nowakowska, Stable aqueous dispersion of superparamagnetic iron oxide nanoparticles protected by charged chitosan derivatives, J. Nanoparticle Res. 15 (1) (Jan. 2013) 1372. [49] L. Faucher, Y. Gossuin, A. Hocq, Marc-AndreFortin, Impact of agglomeration on the relaxometric properties of paramagnetic ultra-small gadolinium oxide nanoparticles, Nanotechnology 22 (29) (2011) 10, http://dx.doi.org/10.1088/0957-4484/22/ 29/295103.

Development of Gd(III) porphyrin-conjugated chitosan nanoparticles as contrast agents for magnetic resonance imaging.

A novel magnetic resonance imaging (MRI) contrast agent based on gadolinium meso-tetrakis(4-pyridyl)porphyrin [Gd(TPyP)] conjugated with chitosan nano...
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