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Fabrication and Characterization of Gd-DTPA-Loaded Chitosan–Poly(Acrylic Acid) Nanoparticles for Magnetic Resonance Imaging Arsalan Ahmed, Chao Zhang, Jian Guo, Yong Hu,* Xiqun Jiang* Gd-DTPA-loaded chitosan–poly(acrylic acid) nanoparticles (Gd-DTPA@CS–PAA NPs) were formulated based on the reaction system of water-soluble polymer–monomer pairs of acrylic acid in chitosan solution followed by sorption of Gd-DTPA. Morphological investigations revealed the spherical shape of these NPs with about 220 nm particle size. These NPs showed charge reversal characteristic in acidic solution. In vitro and in vivo magnetic characteristics of these NPs were explored to estimate their utilization in targeted enhanced magnetic resonance imaging. Relaxation studies showed that these NPs possessed pH susceptible relaxation properties, which could introduce in vivo-specific distribution of contrast agent. MRI experiment showed that these nanoparticles had better results in contrast enhancement, and the concentration of contrast agent increased in liver and brain with increment in time. Thus, these NPs could maintain in vivo long circulation and high relaxation rate and were suitable agents for magnetic resonance imaging.
Magnetic resonance imaging (MRI) is a widely employed technique for acquiring anatomical details of soft tissues[1] due to following benefits: non-ionization, harmlessness, and high resolution images with distinguished soft tissue
A. Ahmed, C. Zhang, Prof. Y. Hu National Laboratory of Solid State Microstructure, College of Engineering and Applied Sciences, Institute of Materials Engineering, Nanjing University, Nanjing, Jiangsu 210093, P. R. China E-mail: [email protected] J. Guo, Prof. X. Jiang Department of Polymer Science and Engineering, College of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, P. R. China E-mail: [email protected]
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contrast between different tissues.[2–4] The contrast between unlike tissues can be enhanced by utilizing paramagnetic compounds, for instance, gadolinium (III) complexes, as MRI contrast agents.[5,6] Gadolinium (Gd)based contrast agents can reduce the longitudinal relaxation time (T1) of surrounding water protons, and consequently improve the signal of MRI tomography.[7–9] The use of these contrast agents is common in medical imaging such as investigation of cancers and benign tumors, angiography scanning, determination of heart abnormalities, and detection of rupture of blood brain barrier (BBB).[10,11] Gadolinium-diethylenetriaminepentacetate (Gd-DTPA), owing to its enhanced stability and paramagnetic properties, is commonly used as T1 contrast agent in MRI. However, Gd-DTPA exhibits low relaxivity, short in vivo circulation time, and less targeting characteristics to tissues, which restrains its applications in MRI.[12] Therefore, high relaxivity, precise tissue detection, and minimum release of free
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1. Introduction
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Gd3þ during blood circulation are the preliminary requirement for better imaging. Recently, nanosized MRI probes have been considered an excellent tool for selective delivery of contrast agents to tissues due to their long blood circulation and accumulation in tissues.[13] Furthermore, because of decreased molecular tumbling rates, these MRI probes can possess enhanced relaxivity of loaded contrast agents.[14] In same manner, highly sensitive Gd-DTPAloaded liposomes, dendrimers, and NPs have been fabricated to enhance the MRI contrasts of soft tissues.[15–17] In order to acquire effective delivery of Gd-DTPA to tissues, nanocarriers, e.g., Gd-DTPA-loaded NPs need to cross many obstacles during their in vivo journey. While circulating in blood, they must prevent the adhesion to blood components and escape from reticuloendothelial system (RES).[18] Safe transport of contrast agent to specific tissue is affected by many factors, e.g., morphology of NPs, surface chemistry, and charges, and tissue specificity.[19–22] As an example, smaller NPs (less than 5 nm) can be removed from blood by glomerular filtration and larger NPs (larger than 250 nm) are captured by RES and eliminated from blood circulation in liver and spleen.[23] Therefore, aforementioned characteristics of NPs must be optimized to transport Gd-DTPA to tissues effectively. Biocompatible polymeric NPs offer the capabilities to escape from RES capture and transport drug to particular sites.[24] Several types of polymeric NPs have been designed for therapeutic and diagnostic purposes.[25–27] Among them, chitosan-based NPs are extensively utilized as carriers for drug and contrast agent delivery. Chitosan (CS) is a cationic polyelectrolyte which can form NPs with an anionic polyelectrolyte, e.g., alginate and plasmid.[28,29] In this report, chitosan–poly (acrylic acid) NPs (CS–PAA NPs) are formulated by polymerization of acrylic acid in chitosan solution.[30] Later, Gd-DTPA was adsorbed to CS–PAA NPs by placing NPs in Gd-DTPA solution (Scheme 1). These synthesized Gd-DTPA@CS–PAA NPs are supposed to show following characteristics. Firstly, the size of NPs could be controlled by altering the both constitute, i.e., chitosan and acrylic acid which could facilitate in formation of NPs with appropriate size. Secondly, the cationic amines of chitosan could be supportive for maximum incorporation of GdDTPA in CS–PAA NPs due to the interaction of anionic carboxylate group of Gd-DTPA with surface cationic amines of CS, which ensure them as promising contrast agents used
in the MRI measurements.[31] And thirdly, these NPs could demonstrate pH-stimulated degradable properties, resulting from the disruption of intermolecular interaction between NH3þ and COO– ions of chitosan and poly(acrylic acid), respectively, under strong acidic or basic environment.[32] The dissolution of these NPs at low pH could make them suitable candidate for targeting and transporting GdDTPA to tumor tissues since tumor sites are more acidic than normal tissue sites. For instance, pH of endosomes and lysosomes in tumors are approximately 5–6 and 4–5, respectively.[33] Thus, Gd-DTPA@CS–PAA NPs could be responsive to acidic condition.
2. Experimental Section 2.1. Materials Chitosan (CS) was obtained from Nantong Shuanglin Biological Product Inc. and was purified prior to use. Briefly, CS was dissolved in acetic acid solution (5% w/v) at 65 8C, filtered and precipitated in 5% NaOH solution, and dried under vacuum at 40 8C. The degree of deacetylation of CS was found to be 88% as measured by acid–base titration method,[34] and the average molecular weight of CS was about 80 kDa, which was determined by viscometric method.[35] Acrylic acid (AA) (Shanghai Guanghua Chemical Company) was distilled under reduced pressure before use. Potassium persulfate (K2S2O8) (Shanghai Chemical Reagent Co.) was re-crystallized in distilled water. Diethylene triamine pentaacetic acid (DTPA) and gadolinium oxide (Gd2O3) were also purchased from Shanghai Chemical Reagent Co. and utilized without further purification.
2.2. Synthesis of CS–PAA NPs CS–PAA NPs were fabricated by polymerization of acrylic acid in CS solution. Concisely, 0.125 mM chitosan solution was prepared by dissolving 0.5 g chitosan in 50 ml acrylic acidic under magnetic stirring. As the solution became clear, 0.01 g K2S2O8 was added to initiate the polymerization reaction. The reaction was carried out at 50 8C and about pH 4.0 under nitrogen atmosphere and magnetic stirring. After appearance of milky suspension, it was cooled, filtered to eliminate polymer aggregates, and dialyzed in phosphate buffer solution of pH 4.5 for 24 h (MWCO 10 000) to remove remaining monomers.
2.3. Synthesis of Gd-DTPA
Scheme 1. Formulation of Gd-DTPA@CS–PAA NPs.
3.7 g DTPA and 3.76 g Gd2O3 were dissolved in 40 ml deionized water and refluxed at 90 8C for 24 h. After that, free Gd3þ was determined by Arsenazo III using colorimetric method. Later, the solution was filtered and supernatant was precipitated in 200 ml ethanol, placed overnight, filtered, and dried under vacuum at room temperature for 8 h. Finally, the sample was pulverized under infrared lamp, vacuum-dried at room temperature for 24 h, and stored in desiccator.
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2.4. Fabrication of Gd-DTPA@CS–PAA NPs Gd-DTPA@CS–PAA NPs were prepared by sorption of Gd-DTPA on CS–PAA NPs. Briefly, Gd-DTPA was dissolved in 50 ml CS–PAA NPs solution, and the suspension was placed for 48 h in order to get maximum sorption of Gd-DTPA on CS–PAA NPs. Then, the suspension was centrifugated at10 000 rpm; the precipitates were collected and dialyzed against phosphate buffer solution (pH ¼ 4.5) in dialysis membrane bag of 10 000 MWCO for 24 h. The obtained NPs were freeze dried and stored at 4 8C.
2.5. Preparation of Gd-Poly(DTPA-Butanediamine) Copolymer
2.8. Characterization of Relaxivity The ability of contrast agents for MRI is elaborated by proton longitudinal relaxivity, which can be determined from the change in relaxation rate of water protons per concentration of paramagnetic cations and calculated by using expression.
1.52 g of 1,4-butanediamine (17.2 mmol) was dissolved in 40 ml DMSO, and 8.1 ml of triethylamine and 6.64 g DTPA (18.4 mmol) were added in it. This mixture was stirred at room temperature for 48 h to obtain a pale yellow solution. Then, the product was precipitated in 40 ml ethyl acetate, the supernatant was removed, precipitate was rinsed with ethyl acetate thrice, and the solvent was removed under vacuum at room temperature. The obtained product was dissolved in 40 ml water, and 6.6 g of gadolinium chloride (GdCl3) was added. Arsenazo III test was performed to check any free Gd3þ. The sample was then dialyzed against double distilled water using dialysis membrane of 10 000 MWCO, filtered, and the supernatant was lyophilized. Molecular weight was found to be 20 000 as measured by HPLC and the Gd content was 18% (w/w). The obtained product was termed as Gd-poly(DTPA-BTD) copolymer.
2.6. Gadolinium Loading Efficiency The content of Gd-DTPA inside the Gd-DTPA@CS–PAA NPs was measured by centrifugation method. In brief, the Gd-DTPA@CS– PAA NPs were centrifugated at 40 000 rpm for 30 min at 4 8C, and particles were allowed to settle down. The concentration of gadolinium in the supernatant was determined using ICP-AES technique. Later, the content of gadolinium adsorbed on NPs was calculated by subtracting the amount of gadolinium in the supernatant from the total amount of gadolinium. Finally, GdDTPA loading efficiency was obtained by following equation: Gadolinium loading efficiency ¼
vitro release rate of Gd-DTPA from NPs. 5 ml of Gd-DTPA@CS–PAA NPs (2.5 mg ml1) in different media were dialyzed at 37 8C using dialysis membrane with 10 000 MWCO. 0.1 ml solution from the exterior medium of dialysis bag was taken at defined time, and the concentration of Gd was measured by ICP-AES method.
weight of gadolinium in NPs weight of feeding gadolinium 100 ð1Þ
T11d r 1 ¼ 3þ Gd 1 T1
ð2Þ
where r1 is proton longitudinal relaxivity, 1/T1 and 1/T1d are the longitudinal relaxation rate contrast in the presence and absence of paramagnetic species respectively, and [Gd3þ] is the concentration of gadolinium cations.[36] The T1 values of Gd-DTPA and GdDTPA@CS–PAA NPs were measured in water at 37 8C on Bruker XP300 instrument using standard inversion-recovery pulse sequence.
2.9. In Vivo Relaxation Rate To measure T1 relaxation rate in blood, contrast agent was injected in rabbit. After certain time, blood was taken out from rabbit and the T1 relaxation rate was measured. Briefly, adult male rabbits with a body weight of approximately 2.0 0.5 kg were fixed in boxes and small amount (0.037 mmol kg1) of Gd-DTPA or Gd-DTPA@CS–PAA NPs were injected in rabbits, intravenously. After different time intervals, about 1 ml blood was taken out from the Rabbits and measured the T1 relaxation rate using Bruker XP300 instrument with standard inversion-recovery pulse sequence.
2.10. Biodistribution of Gd-DTPA@CS–PAA NPs Gd-DTPA or Gd-DTPA@CS–PAA NPs was intravenously injected into rabbits. After 4 h, the rabbits were anesthetized deeply to cause death and dissected. Heart, brain, kidney, liver, lung, and spleen were excised, and blood was collected from inferior vena cava. Organs were crushed using tissue pulverizer, dissolved in 90% HNO3, dried and ignited. The gadolinium content was then determined by ICP-AES.
where weight of feeding gadolinium was the total amount of gadolinium which had been added in the system.
2.11. In Vivo MRI of Gd-DTPA@CS–PAA NPs 2.7. Release Properties of Gd-DTPA from GdDTPA@CS-PAA NPs Dialysis method was used to evaluate in vitro release behavior of Gd-DTPA from Gd-DTPA@CS–PAA NPs. Distilled water, phosphate buffer solution (PBS) of pH ¼ 7.4 and pH ¼ 5.0, and bovine serum albumin (BSA) solution were utilized separately to measure the in
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MR imaging device (GE Company, 30 cm Helmoltz coil, 50 cm aperature, 0.2 T magnetic field) was utilized to obtain T1-weighted images of rabbit. The following parameters were adopted: spin-echo (SE) method, repetition time (TR) ¼ 500–400 ms, echo time (TE) ¼ 30– 25 ms. Rabbits were anesthetized and dosed with 0.1 mmol Gd-DTPA@CS–PAA NPs kg1 based on body weight of rabbit. The T1-weighted images were then taken in different time intervals.
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2.12. Characterization FT-IR spectra were measured on a Bruker IFS66V vacuum-type spectrometer in the range of 4 000–500 cm1. NPs were freeze dried and measured in KBr pellet. Dynamic light scattering (DLS) technique was employed to study particle size and its distribution. DLS experiments were performed on Malvern Zetasize 3000HS particle size analyzer at 25 8C with a Wavelength of 633.0 nm and scattering angle of 908. Nanoparticles suspensions with different pH values were prepared and their Zeta potential was measured on DPM-1-type electrophoresis apparatus (weight and measure standard agency of Shanghai). The morphology of NPs was studied by transmission electron microscope (TEM). The samples were diluted with water, placed on a nitrocellulose coated copper grid, stained with phosphotungstic acid (0.5% w/v), and observed under JEOL JEM-200CX TEM. Chemical composition of NPs surface was examined by X-ray photoelectron spectroscopy (XPS). GdDTPA@CS–PAA NPs were centrifuged at 50 000 rpm for 40 min using Du Pont Ultra Pro TM 80 ultracentrifuge. The isolated particles were then collected and freeze dried. Finally, the sample was finely ground and tested on VG ESCALAB MK-II photoelectron spectrometer.
3. Results and Discussion 3.1. Formulation and Chemical Characterization of Gd-DTPA@CS–PAA NPs Polymer–monomer pairs polymerization is considered an attractive technique to fabricate CS–PAA NPs.[28] In this report, chitosan was dissolved in acrylic acid solution to form a clear solution and the polymerization of acrylic acid was initiated by adding K2S2O8. The static electronic interaction between carboxyl groups of PAA and cationic amino group of chitosan chain (Figure 1a) could cause the rolling up of macromolecular chains of chitosan. As the reaction proceeded, the solution color was shifted from clear solution to opalescent suspension, which showed the formation of CS–PAA NPs. The detailed reaction mechanism could found in our previous reports.[37,38] Furthermore, these types of NPs have been utilized in different applications of biomedical engineering, e.g., drug delivery and bioimaging.[38,39]
Figure 1b depicts the FTIR spectra of CS, PAA, and CS–PAA NPs. In the CS–PAA copolymer spectrum, absorption peak of amide band of I and amide band II at 1 662 and 1 586 cm1 related to pure chitosan diminished, while new absorption peaks at 1 731 and 1 628 cm1 emerged which could be attributed to carboxyl group of poly(acrylic acid) and NH3þ of chitosan. Similarly, broad peak at 2 500 cm1 described the existence of NH3þ too. Furthermore, peaks related to asymmetric and symmetric stretching vibrations of COO– at 1 532 and 1 414 cm1 were also observed, respectively. These evidences, obtained from FT-IR spectra, strengthened the concept that COO– of poly(acrylic acid) formed complex with chitosan. Gd-DTPA@CS–PAA NPs were prepared by absorption method. The NH3þ groups of chitosan interacted with COO– groups of Gd-DTPA, which could facilitate the sorption of high content of Gd-DTPA in CS–PAA NPs. The solution was placed for long time to obtain the maximum adsorption of Gd-DTPA in CS–PAA NPs. The small GdDTPA molecules, which were not adsorbed, were removed by dialysis. XPS characterization was performed to investigate the chemical composition of these Gd-DTPA@CS–PAA NPs, as shown in Figure 1c. The adsorbed content of gadolinium in Gd-DTPA@CS–PAA NPs was measured by analyzing the characteristic peak of Gd(4d) at a binding energy of about 185 eV. Figure 1c depicted the C(1s) peak at about 287 eV, which corresponded to hydrocarbons of chitosan, poly(acrylic acid) chains, and DTPA. N(1s), related to cationic amines of chitosan, has also showed its presence at about 402 eV. Moreover, O(1s) was obtained too, which could be attributed to carboxylate group of poly(acrylic acid), DTPA, and oxygen of chitosan.[40] In order to acquire detailed in-depth information of NPs surfaces, XPS analysis was performed at different angles of incidence, i.e., 308, 608, and 908. The measured content of each element is shown in Table 1. It was observed that molar percentage amount of gadolinium decreased with increasing incident angle, i.e., more of the gadolinium was adsorbed on the surface of NPs as compared to incorporation of Gd-DTPA inside the NPs.
Figure 1. Electrostatic interaction between CS and PAA which could lead the formation of NPs (a). FTIR spectra of CS, PAA, and CS–PAA NPs (b). XPS spectra of Gd-DTPA@CS–PAA NPs with the angle of incidence of 308, 608, and 908 (c).
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Table 1. Chemical composition of the surface of Gd-DTPA@CS– PAA NPs.
Angle of incidence [8] 30 60 90
C [mol%]
O [mol%]
N [mol%]
Gd [mol%]
64.40 64.02 64.88
30.37 30.38 29.59
5.01 5.41 5.36
0.22 0.18 0.18
Moreover, Gd-DTPA@CS–PAA NPs experience much reduction in zeta potential than CS–PAA NPs due to abundant number of carboxylate groups on the surfaces of Gd-DTPA@CS–PAA NPs. The pH responsiveness of these NPs could be utilized for long circulating and passive targeting of NPs. Figure 3a and b depicts the TEM images of CS-PAA NPs and Gd-DTPA@CS–PAA NPs, respectively. Both types of NPs have regular spherical shape in aqueous medium. After comparison of these two TEM images, it could be deduced that incorporation of GdDTPA in CS–PAA NPs did not disrupt the structure of NPs.
3.2. pH Responsiveness and Structural Description The size distribution and morphology of CS–PAA NPs and Gd-DTPA@CS–PAA NPs were studied by DLS and TEM, respectively. Figure 2a demonstrates the size distribution of these NPs. CS–PAA NPs had an average particle size of 220 nm and the particle size of Gd-DTPA@CS–PAA NPs was found to be 212 nm. The slight reduction in particle size could be associated with the interaction of anionic carboxylate group of Gd-DTPA and cationic amine group of chitosan. Luckily, the adsorption of Gd-DTPA on CS– PAA NPs did not affect the particle size significantly. The surface charges of CS-PAA and Gd-DTPA@CS–PAA NPs were determined by zeta potential measurements at different pH values. Figure 2b describes the trend of zeta potentials change in response to pH change. Zeta potential increases with the decrease in pH values. Negative-to-positive charge reversal property for both types of NPs was observed at pH greater than 6.4. The reported pKa value of CS is 6.5 and the carboxylic acid groups of PAA are ionized above pKa of 4.7.[41] It is reported that amines of CS and carboxylic acid groups of PAA and DTPA of these NPs are partially ionized at about pH 6.5.[42] However, when the pH is above 6.5, the ionization of carboxylic acid groups of PAA and DTPA become dominant over the ionization of amines of CS. Therefore, NPs acquire negative charges on the surfaces.
3.3. In Vitro Studies of Gd-DTPA@CS–PAA NPs Gd-DTPA@CS–PAA NPs were fabricated by utilizing sorption method and gadolinium was loaded in Gd-DTPA@CS– PAA NPs mainly through diffusion. Since the concentration of Gd-DTPA was higher outside the NPs than inside the NPs, Gd-DTPA was diffused from exterior solution (high concentration) to the interior of CS–PAA NPs (lower concentration). However, the presence of carboxylate groups in poly(acrylic acid) could retard the diffusion of Gd-DTPA inside the NPs. These two opposing processes could limit the absorption of Gd-DTPA inside the NPs and most of the Gd-DTPA was adsorbed on the surfaces of CS– PAA NPs. Table 2 explains the Gd-DTPA loading capacity of CS–PAA NPs. The amount of gadolinium before and after the dialysis was measured to get better understanding of incorporated amount of gadolinium in NPs. It was observed that majority of Gd-DTPA was loaded in NPs. However, increasing the concentration of Gd-DTPA did not enhance the gadolinium loading capability. It implied that absorption-releasing equilibrium was established due to the aforementioned two opposing processes, which prevented the further sorption of Gd-DTPA in NPs. In vitro release profile of Gd-DTPA from NPs was studied in different media to verify the stability and pH responsive release of Gd-DTPA from NPs, as shown in Figure 4. Since
Figure 2. Size distribution of CS–PAA NPs and Gd-DTPA@CS–PAA NPs (a). Zeta potential of CS–PAA NPs and Gd-DTPA@CS–PAA NPs at different pH (b).
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Figure 3. TEM images of CS–PAA NPs (a) and Gd-DTPA@CS–PAA NPs (b).
Table 2. Gadolinium incorporated efficiency of Gd-DTPA@CS–PAA NPs.
Gd-DTPA amount before dialysis [mg ml1]
No.
1 2 3
Gd-DTPA amount after dialysis [mg ml1]
Total
NPs
Solvent
Total
NPs
Solvent
6.0 4.0 3.0
3.74 2.53 1.90
2.26 1.47 1.10
4.14 2.59 2.04
3.52 2.39 1.79
0.62 0.20 0.25
water molecules can enter and exchange freely in NPs, owing to intermolecular interaction between water and ionic groups of NPs, similar release profiles were found in aqueous and or other release media such as PBS (pH 7.0) or BSA (pH 7.0) solutions. However, the hydrogen bonding between the carboxylate group of Gd-DTPA and cationic amine of chitosan could strengthen the possession of GdDTPA within NPs. Therefore, very small initial burst was observed, i.e., only about 15% was released in first 4 h (Figure 4a), and even only 40% Gd-DTPA was released in 2 d. Figure 4a suggests that these NPs exhibited a sustained
Gadolinium loading efficiency [%] 62.4 63.2 63.2
release of Gd-DTPA. Moreover, the effect of pH on the release of Gd-DTPA from NPs is shown in Figure 4b. Release profile of Gd-DTPA was drawn in PBS at pH 7.4 and pH 5.00. Similar sustained trend of Gd-DTPA release from NPs was observed. However, Gd-DTPA release at pH 5.00 was faster as compared to pH 7.4, which implied that strong acidic medium, owing to conversion of COO– to COOH, weakens the interaction between carboxylic acid groups of poly(acrylic acid) and Gd-DTPA and amine of chitosan, which accelerated the release of Gd-DTPA from NPs. These findings support the utilization of these NPs for MRI in biomedical fields.
Figure 4. Gadolinium release profile in different types of media (a) and effect of pH on release of Gd-DTPA from NPs (b).
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Figure 5. The water proton relaxation rate of Gd-DTPA@CS–PAA NPs with different Gd-DTPA loaded level after dialyzing against deionized water (a) and pH dependence of the longitudinal proton relaxivity rate for Gd-DTPA@CS–PAA NPs and Gd-DTPA (b).
In vitro relaxation properties of Gd-DTPA@CS–PAA NPs were examined. The relaxation rate was plotted versus different concentration of Gd-DTPA to calculate the proton longitudinal relaxivity r1. The value of r1 was similar to different loading content of Gd-DTPA in NPs, which was about 5.34 mM1 s1 (Figure 5a). This value is higher than the preloaded r1 value of Gd-DTPA (4.5 mM1 s1). It is consistent with the fact that r1 can be increased by attaching small molecular complex of gadolinium to macromolecules. For instance, r1 value of some gadolinium dendrimers could reach up to 5 800 mM1 s1, which is nearly 1 000 times greater than the original relaxation rate of small molecule.[43] However, slight increase in r1 of gadolinium was observed in our NPs system. It could be deduced that Gd-DTPA not only adsorbed on the surface of NPs but also distributed inside the NPs. The Gd-DTPA inside the NPS is relatively stable due to its interaction with chitosan, and water molecules exchange rate is very slow inside the NPs. Therefore, internal Gd-DTPA exhibited less enhancing impact on the outside water proton relaxation. We also observed the effect of pH change on longitudinal proton relaxation, as shown in Figure 5b. It was observed
that as the pH value increased, the r1 was reduced in both. However, a smooth trend was observed in Gd-DTPA@CS– PAA NPs which could be attributed to sustained release of Gd-DTPA from NPs. Moreover, the increase of r1 value in pure Gd-DTPA was greater than that of Gd-DTPA@CS–PAA NPs in response to low pH. This difference in increment of r1 in acidic environment might be related to electrostatic equilibrium between chitosan and poly(acrylic acid) polymeric chains. At low pH, some of the protons were substituted with NH3þ of chitosan, which had been associated with COO– of poly(acrylic acid) in NPs. Thus, the effect of H3Oþ was decreased. Consequently, slight change in r1 was observed with the reduction in pH.
3.4. In Vivo Properties of Gd-DTPA@CS–PAA NPs For studying the efficacy of Gd-DTPA@CS–PAA NPs during their circulation in blood, NPs were injected in rabbits intravenously and the in vivo relaxation characterization (0.037 mmol kg) was carried out. Simultaneously, we compared the relaxation properties of NPs with Gd-DTPA
Figure 6. Blood relaxation rate of Gd-DTPA@CS–PAA NPs, Gd-DTPA, and Gd-(DTPA-BTD) at different time intervals in rabbit (a), biodistribution of Gd3þ in rabbit (b), and organ relative distribution of Gd3þ in rabbit (c).
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and Gd-poly(DTPA-BTD). Figure 6a describes the graph of longitudinal proton relaxation in rabbit blood along with injection time. The relaxation enhancement of GdDTPA@CS–PAA NPs was found to be greatest, as compared to Gd-poly(DTPA-BTD) and Gd-DTPA. The r1 values of GdDTPA and Gd-poly(DTPA-BTD) copolymers were attenuated quickly and the relaxation effect was lost after 25 min. However, the relaxation attenuation of Gd-DTPA@CS–PAA
NPs was also fast in initial 25 min; later, it was gradually slowed down. Therefore, Gd-DTPA@CS–PAA NPs possessed enhanced blood relaxation properties. The biodistribution of Gd-DTPA@CS–PAA NPs in rabbit organs was also explored after 4 h injection. Figure 6b and c depicts the gadolinium content of Gd-DTPA@CS–PAA NPs and Gd-DTPA complex measured by ICP-AES distributed in different organs of rabbit, as well as the relative organ
Figure 7. Transverse T1-weighted MR images of rabbit brain (a) and liver (b) using SE technique: 1) no contrast agent after 146 min, and after administration 2) 1 min, 3) 8 min, 4) 29 min, 5) 62 min, 6) 146 min. TR/TE ¼ 450 ms/25ms (b) signal intensity of MRI (c).
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distribution of gadolinium in different organs, respectively. It was observed in Figure 6b that Gd-DTPA@CS–PAA NPs largely influenced the distribution of gadolinium in different organs. While injecting Gd-DTPA complex only, most of the gadolinium was accumulated in liver and kidney, and gadolinium did not exhibit its considerable presence in blood, which demonstrated that majority of Gd-DTPA complex was excreted from blood circulation within 4 h. Since Gd-DTPA cannot pass through the blood–brain barrier,[44] the distribution of Gd-DTPA in the brain was also very low. However, Gd-DTPA@CS–PAA NPs had less distribution in the kidney, which was significantly different from Gd-DTPA complex. These NPs were mainly concentrated in the liver and lungs.These results indicated that Gd-DTPA@CS–PAA NPs altered the distribution of gadolinium in the body organs and produced a specific targeting property for liver and lung. Therefore, these NPs could be utilized for the MR imaging of liver and lung cancer. The delivery to brain is difficult due to presence of BBB, which prevents the passage from blood to brain.[45] Luckily, chitosan-based NPs have shown their accumulation in brain.[46] It is noteworthy that Gd-DTPA@CS–PAA NPs could also cross the BBB and transported some of the gadolinium content to the brain. Since different organs vary in weight and size, it is significant to compare distribution of gadolinium on the basis of organ weights. Figure 6c exhibits the organ relative distribution of gadolinium content. It provides better understanding of gadolinium distribution in different organs, for instance, the distribution of Gadolinium in liver and kidney is more apparent in Figure 6c. Similarly, the significant distribution of gadolinium in spleen was only observed by Figure 6c, which described that these NPs could be used as spleen targeting MRI contrast agent.
3.5. Magnetic Resonance Imaging of Gd-DTPA@CS– PAA NPs Gd-DTPA@CS–PAA NPs were injected in rabbits to perform MRI experiment and examine the MRI signals in blood vessels, liver, and brain. Figure 7a and b is the T1-weighted MRI pictures of brain and liver of rabbit. These images results confirmed that these NPs contain the higher contrast ability than free Gd-DTPA complex, as the images of Gd-DTPA@CS–PAA NPs were much brighter than the images from free Gd-DTPA complex (Figure 7). The MRI signals in liver and brain were also significantly stronger than the signals without contrast agent, and the signal enhancement could be kept for long time. MRI images confirmed that these NPs could be delivered in brain and liver successfully and enhanced the MRI signal in these organs. Thus, Gd-DTPA@CS–PAA NPs could be a better carrier for MRI contrast agent. Figure 7c demonstrates the
4. Conclusion Polymeric CS–PAA nanoparticles were fabricated by utilizing intermolecular interaction between chitosan and acrylic acid, and Gd-DTPA was incorporated in them. These nanoparticles were sufficiently stable in normal aqueous solution with positive surface charges. They showed pH sensitivity as described by in vitro release and relaxation experiments. This novel quality makes them appropriate for safe journey of contrast agent at normal pH environment and targeted release in acidic environment. In vivo biodistribution and MRI studies have also demonstrated localization of contrast agent and enhanced contrast respectively.
Acknowledgements: This work is supported by the National Natural Science Foundation of China (no. 21474047 and 51173077) and Nature Science Foundation of Jiangsu Province (BK20141225).
Received: February 4, 2015; Revised: March 11, 2015; Published online:DOI: 10.1002/mabi.201500034 Keywords: chitosan; Gd-DTPA; magnetic resonance imaging; nanoparticles; poly(acrylic acid)
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Fabrication and Characterization of Gd-DTPA-Loaded Chitosan–Poly(Acrylic Acid) Nanoparticles for Magnetic Resonance Imaging Arsalan Ahmed, Chao Zhang, Jian Guo, Yong Hu,* Xiqun Jiang* Gd-DTPA-loaded chitosan–poly(acrylic acid) nanoparticles (Gd-DTPA@CS–PAA NPs) were formulated based on the reaction system of water-soluble polymer–monomer pairs of acrylic acid in chitosan solution followed by sorption of Gd-DTPA. Morphological investigations revealed the spherical shape of these NPs with about 220 nm particle size. These NPs showed charge reversal characteristic in acidic solution. In vitro and in vivo magnetic characteristics of these NPs were explored to estimate their utilization in targeted enhanced magnetic resonance imaging. Relaxation studies showed that these NPs possessed pH susceptible relaxation properties, which could introduce in vivo-specific distribution of contrast agent. MRI experiment showed that these nanoparticles had better results in contrast enhancement, and the concentration of contrast agent increased in liver and brain with increment in time. Thus, these NPs could maintain in vivo long circulation and high relaxation rate and were suitable agents for magnetic resonance imaging.
Magnetic resonance imaging (MRI) is a widely employed technique for acquiring anatomical details of soft tissues[1] due to following benefits: non-ionization, harmlessness, and high resolution images with distinguished soft tissue
A. Ahmed, C. Zhang, Prof. Y. Hu National Laboratory of Solid State Microstructure, College of Engineering and Applied Sciences, Institute of Materials Engineering, Nanjing University, Nanjing, Jiangsu 210093, P. R. China E-mail: [email protected] J. Guo, Prof. X. Jiang Department of Polymer Science and Engineering, College of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, P. R. China E-mail: [email protected]
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contrast between different tissues.[2–4] The contrast between unlike tissues can be enhanced by utilizing paramagnetic compounds, for instance, gadolinium (III) complexes, as MRI contrast agents.[5,6] Gadolinium (Gd)based contrast agents can reduce the longitudinal relaxation time (T1) of surrounding water protons, and consequently improve the signal of MRI tomography.[7–9] The use of these contrast agents is common in medical imaging such as investigation of cancers and benign tumors, angiography scanning, determination of heart abnormalities, and detection of rupture of blood brain barrier (BBB).[10,11] Gadolinium-diethylenetriaminepentacetate (Gd-DTPA), owing to its enhanced stability and paramagnetic properties, is commonly used as T1 contrast agent in MRI. However, Gd-DTPA exhibits low relaxivity, short in vivo circulation time, and less targeting characteristics to tissues, which restrains its applications in MRI.[12] Therefore, high relaxivity, precise tissue detection, and minimum release of free
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1. Introduction
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Gd3þ during blood circulation are the preliminary requirement for better imaging. Recently, nanosized MRI probes have been considered an excellent tool for selective delivery of contrast agents to tissues due to their long blood circulation and accumulation in tissues.[13] Furthermore, because of decreased molecular tumbling rates, these MRI probes can possess enhanced relaxivity of loaded contrast agents.[14] In same manner, highly sensitive Gd-DTPAloaded liposomes, dendrimers, and NPs have been fabricated to enhance the MRI contrasts of soft tissues.[15–17] In order to acquire effective delivery of Gd-DTPA to tissues, nanocarriers, e.g., Gd-DTPA-loaded NPs need to cross many obstacles during their in vivo journey. While circulating in blood, they must prevent the adhesion to blood components and escape from reticuloendothelial system (RES).[18] Safe transport of contrast agent to specific tissue is affected by many factors, e.g., morphology of NPs, surface chemistry, and charges, and tissue specificity.[19–22] As an example, smaller NPs (less than 5 nm) can be removed from blood by glomerular filtration and larger NPs (larger than 250 nm) are captured by RES and eliminated from blood circulation in liver and spleen.[23] Therefore, aforementioned characteristics of NPs must be optimized to transport Gd-DTPA to tissues effectively. Biocompatible polymeric NPs offer the capabilities to escape from RES capture and transport drug to particular sites.[24] Several types of polymeric NPs have been designed for therapeutic and diagnostic purposes.[25–27] Among them, chitosan-based NPs are extensively utilized as carriers for drug and contrast agent delivery. Chitosan (CS) is a cationic polyelectrolyte which can form NPs with an anionic polyelectrolyte, e.g., alginate and plasmid.[28,29] In this report, chitosan–poly (acrylic acid) NPs (CS–PAA NPs) are formulated by polymerization of acrylic acid in chitosan solution.[30] Later, Gd-DTPA was adsorbed to CS–PAA NPs by placing NPs in Gd-DTPA solution (Scheme 1). These synthesized Gd-DTPA@CS–PAA NPs are supposed to show following characteristics. Firstly, the size of NPs could be controlled by altering the both constitute, i.e., chitosan and acrylic acid which could facilitate in formation of NPs with appropriate size. Secondly, the cationic amines of chitosan could be supportive for maximum incorporation of GdDTPA in CS–PAA NPs due to the interaction of anionic carboxylate group of Gd-DTPA with surface cationic amines of CS, which ensure them as promising contrast agents used
in the MRI measurements.[31] And thirdly, these NPs could demonstrate pH-stimulated degradable properties, resulting from the disruption of intermolecular interaction between NH3þ and COO– ions of chitosan and poly(acrylic acid), respectively, under strong acidic or basic environment.[32] The dissolution of these NPs at low pH could make them suitable candidate for targeting and transporting GdDTPA to tumor tissues since tumor sites are more acidic than normal tissue sites. For instance, pH of endosomes and lysosomes in tumors are approximately 5–6 and 4–5, respectively.[33] Thus, Gd-DTPA@CS–PAA NPs could be responsive to acidic condition.
2. Experimental Section 2.1. Materials Chitosan (CS) was obtained from Nantong Shuanglin Biological Product Inc. and was purified prior to use. Briefly, CS was dissolved in acetic acid solution (5% w/v) at 65 8C, filtered and precipitated in 5% NaOH solution, and dried under vacuum at 40 8C. The degree of deacetylation of CS was found to be 88% as measured by acid–base titration method,[34] and the average molecular weight of CS was about 80 kDa, which was determined by viscometric method.[35] Acrylic acid (AA) (Shanghai Guanghua Chemical Company) was distilled under reduced pressure before use. Potassium persulfate (K2S2O8) (Shanghai Chemical Reagent Co.) was re-crystallized in distilled water. Diethylene triamine pentaacetic acid (DTPA) and gadolinium oxide (Gd2O3) were also purchased from Shanghai Chemical Reagent Co. and utilized without further purification.
2.2. Synthesis of CS–PAA NPs CS–PAA NPs were fabricated by polymerization of acrylic acid in CS solution. Concisely, 0.125 mM chitosan solution was prepared by dissolving 0.5 g chitosan in 50 ml acrylic acidic under magnetic stirring. As the solution became clear, 0.01 g K2S2O8 was added to initiate the polymerization reaction. The reaction was carried out at 50 8C and about pH 4.0 under nitrogen atmosphere and magnetic stirring. After appearance of milky suspension, it was cooled, filtered to eliminate polymer aggregates, and dialyzed in phosphate buffer solution of pH 4.5 for 24 h (MWCO 10 000) to remove remaining monomers.
2.3. Synthesis of Gd-DTPA
Scheme 1. Formulation of Gd-DTPA@CS–PAA NPs.
3.7 g DTPA and 3.76 g Gd2O3 were dissolved in 40 ml deionized water and refluxed at 90 8C for 24 h. After that, free Gd3þ was determined by Arsenazo III using colorimetric method. Later, the solution was filtered and supernatant was precipitated in 200 ml ethanol, placed overnight, filtered, and dried under vacuum at room temperature for 8 h. Finally, the sample was pulverized under infrared lamp, vacuum-dried at room temperature for 24 h, and stored in desiccator.
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2.4. Fabrication of Gd-DTPA@CS–PAA NPs Gd-DTPA@CS–PAA NPs were prepared by sorption of Gd-DTPA on CS–PAA NPs. Briefly, Gd-DTPA was dissolved in 50 ml CS–PAA NPs solution, and the suspension was placed for 48 h in order to get maximum sorption of Gd-DTPA on CS–PAA NPs. Then, the suspension was centrifugated at10 000 rpm; the precipitates were collected and dialyzed against phosphate buffer solution (pH ¼ 4.5) in dialysis membrane bag of 10 000 MWCO for 24 h. The obtained NPs were freeze dried and stored at 4 8C.
2.5. Preparation of Gd-Poly(DTPA-Butanediamine) Copolymer
2.8. Characterization of Relaxivity The ability of contrast agents for MRI is elaborated by proton longitudinal relaxivity, which can be determined from the change in relaxation rate of water protons per concentration of paramagnetic cations and calculated by using expression.
1.52 g of 1,4-butanediamine (17.2 mmol) was dissolved in 40 ml DMSO, and 8.1 ml of triethylamine and 6.64 g DTPA (18.4 mmol) were added in it. This mixture was stirred at room temperature for 48 h to obtain a pale yellow solution. Then, the product was precipitated in 40 ml ethyl acetate, the supernatant was removed, precipitate was rinsed with ethyl acetate thrice, and the solvent was removed under vacuum at room temperature. The obtained product was dissolved in 40 ml water, and 6.6 g of gadolinium chloride (GdCl3) was added. Arsenazo III test was performed to check any free Gd3þ. The sample was then dialyzed against double distilled water using dialysis membrane of 10 000 MWCO, filtered, and the supernatant was lyophilized. Molecular weight was found to be 20 000 as measured by HPLC and the Gd content was 18% (w/w). The obtained product was termed as Gd-poly(DTPA-BTD) copolymer.
2.6. Gadolinium Loading Efficiency The content of Gd-DTPA inside the Gd-DTPA@CS–PAA NPs was measured by centrifugation method. In brief, the Gd-DTPA@CS– PAA NPs were centrifugated at 40 000 rpm for 30 min at 4 8C, and particles were allowed to settle down. The concentration of gadolinium in the supernatant was determined using ICP-AES technique. Later, the content of gadolinium adsorbed on NPs was calculated by subtracting the amount of gadolinium in the supernatant from the total amount of gadolinium. Finally, GdDTPA loading efficiency was obtained by following equation: Gadolinium loading efficiency ¼
vitro release rate of Gd-DTPA from NPs. 5 ml of Gd-DTPA@CS–PAA NPs (2.5 mg ml1) in different media were dialyzed at 37 8C using dialysis membrane with 10 000 MWCO. 0.1 ml solution from the exterior medium of dialysis bag was taken at defined time, and the concentration of Gd was measured by ICP-AES method.
weight of gadolinium in NPs weight of feeding gadolinium 100 ð1Þ
T11d r 1 ¼ 3þ Gd 1 T1
ð2Þ
where r1 is proton longitudinal relaxivity, 1/T1 and 1/T1d are the longitudinal relaxation rate contrast in the presence and absence of paramagnetic species respectively, and [Gd3þ] is the concentration of gadolinium cations.[36] The T1 values of Gd-DTPA and GdDTPA@CS–PAA NPs were measured in water at 37 8C on Bruker XP300 instrument using standard inversion-recovery pulse sequence.
2.9. In Vivo Relaxation Rate To measure T1 relaxation rate in blood, contrast agent was injected in rabbit. After certain time, blood was taken out from rabbit and the T1 relaxation rate was measured. Briefly, adult male rabbits with a body weight of approximately 2.0 0.5 kg were fixed in boxes and small amount (0.037 mmol kg1) of Gd-DTPA or Gd-DTPA@CS–PAA NPs were injected in rabbits, intravenously. After different time intervals, about 1 ml blood was taken out from the Rabbits and measured the T1 relaxation rate using Bruker XP300 instrument with standard inversion-recovery pulse sequence.
2.10. Biodistribution of Gd-DTPA@CS–PAA NPs Gd-DTPA or Gd-DTPA@CS–PAA NPs was intravenously injected into rabbits. After 4 h, the rabbits were anesthetized deeply to cause death and dissected. Heart, brain, kidney, liver, lung, and spleen were excised, and blood was collected from inferior vena cava. Organs were crushed using tissue pulverizer, dissolved in 90% HNO3, dried and ignited. The gadolinium content was then determined by ICP-AES.
where weight of feeding gadolinium was the total amount of gadolinium which had been added in the system.
2.11. In Vivo MRI of Gd-DTPA@CS–PAA NPs 2.7. Release Properties of Gd-DTPA from GdDTPA@CS-PAA NPs Dialysis method was used to evaluate in vitro release behavior of Gd-DTPA from Gd-DTPA@CS–PAA NPs. Distilled water, phosphate buffer solution (PBS) of pH ¼ 7.4 and pH ¼ 5.0, and bovine serum albumin (BSA) solution were utilized separately to measure the in
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MR imaging device (GE Company, 30 cm Helmoltz coil, 50 cm aperature, 0.2 T magnetic field) was utilized to obtain T1-weighted images of rabbit. The following parameters were adopted: spin-echo (SE) method, repetition time (TR) ¼ 500–400 ms, echo time (TE) ¼ 30– 25 ms. Rabbits were anesthetized and dosed with 0.1 mmol Gd-DTPA@CS–PAA NPs kg1 based on body weight of rabbit. The T1-weighted images were then taken in different time intervals.
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2.12. Characterization FT-IR spectra were measured on a Bruker IFS66V vacuum-type spectrometer in the range of 4 000–500 cm1. NPs were freeze dried and measured in KBr pellet. Dynamic light scattering (DLS) technique was employed to study particle size and its distribution. DLS experiments were performed on Malvern Zetasize 3000HS particle size analyzer at 25 8C with a Wavelength of 633.0 nm and scattering angle of 908. Nanoparticles suspensions with different pH values were prepared and their Zeta potential was measured on DPM-1-type electrophoresis apparatus (weight and measure standard agency of Shanghai). The morphology of NPs was studied by transmission electron microscope (TEM). The samples were diluted with water, placed on a nitrocellulose coated copper grid, stained with phosphotungstic acid (0.5% w/v), and observed under JEOL JEM-200CX TEM. Chemical composition of NPs surface was examined by X-ray photoelectron spectroscopy (XPS). GdDTPA@CS–PAA NPs were centrifuged at 50 000 rpm for 40 min using Du Pont Ultra Pro TM 80 ultracentrifuge. The isolated particles were then collected and freeze dried. Finally, the sample was finely ground and tested on VG ESCALAB MK-II photoelectron spectrometer.
3. Results and Discussion 3.1. Formulation and Chemical Characterization of Gd-DTPA@CS–PAA NPs Polymer–monomer pairs polymerization is considered an attractive technique to fabricate CS–PAA NPs.[28] In this report, chitosan was dissolved in acrylic acid solution to form a clear solution and the polymerization of acrylic acid was initiated by adding K2S2O8. The static electronic interaction between carboxyl groups of PAA and cationic amino group of chitosan chain (Figure 1a) could cause the rolling up of macromolecular chains of chitosan. As the reaction proceeded, the solution color was shifted from clear solution to opalescent suspension, which showed the formation of CS–PAA NPs. The detailed reaction mechanism could found in our previous reports.[37,38] Furthermore, these types of NPs have been utilized in different applications of biomedical engineering, e.g., drug delivery and bioimaging.[38,39]
Figure 1b depicts the FTIR spectra of CS, PAA, and CS–PAA NPs. In the CS–PAA copolymer spectrum, absorption peak of amide band of I and amide band II at 1 662 and 1 586 cm1 related to pure chitosan diminished, while new absorption peaks at 1 731 and 1 628 cm1 emerged which could be attributed to carboxyl group of poly(acrylic acid) and NH3þ of chitosan. Similarly, broad peak at 2 500 cm1 described the existence of NH3þ too. Furthermore, peaks related to asymmetric and symmetric stretching vibrations of COO– at 1 532 and 1 414 cm1 were also observed, respectively. These evidences, obtained from FT-IR spectra, strengthened the concept that COO– of poly(acrylic acid) formed complex with chitosan. Gd-DTPA@CS–PAA NPs were prepared by absorption method. The NH3þ groups of chitosan interacted with COO– groups of Gd-DTPA, which could facilitate the sorption of high content of Gd-DTPA in CS–PAA NPs. The solution was placed for long time to obtain the maximum adsorption of Gd-DTPA in CS–PAA NPs. The small GdDTPA molecules, which were not adsorbed, were removed by dialysis. XPS characterization was performed to investigate the chemical composition of these Gd-DTPA@CS–PAA NPs, as shown in Figure 1c. The adsorbed content of gadolinium in Gd-DTPA@CS–PAA NPs was measured by analyzing the characteristic peak of Gd(4d) at a binding energy of about 185 eV. Figure 1c depicted the C(1s) peak at about 287 eV, which corresponded to hydrocarbons of chitosan, poly(acrylic acid) chains, and DTPA. N(1s), related to cationic amines of chitosan, has also showed its presence at about 402 eV. Moreover, O(1s) was obtained too, which could be attributed to carboxylate group of poly(acrylic acid), DTPA, and oxygen of chitosan.[40] In order to acquire detailed in-depth information of NPs surfaces, XPS analysis was performed at different angles of incidence, i.e., 308, 608, and 908. The measured content of each element is shown in Table 1. It was observed that molar percentage amount of gadolinium decreased with increasing incident angle, i.e., more of the gadolinium was adsorbed on the surface of NPs as compared to incorporation of Gd-DTPA inside the NPs.
Figure 1. Electrostatic interaction between CS and PAA which could lead the formation of NPs (a). FTIR spectra of CS, PAA, and CS–PAA NPs (b). XPS spectra of Gd-DTPA@CS–PAA NPs with the angle of incidence of 308, 608, and 908 (c).
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Table 1. Chemical composition of the surface of Gd-DTPA@CS– PAA NPs.
Angle of incidence [8] 30 60 90
C [mol%]
O [mol%]
N [mol%]
Gd [mol%]
64.40 64.02 64.88
30.37 30.38 29.59
5.01 5.41 5.36
0.22 0.18 0.18
Moreover, Gd-DTPA@CS–PAA NPs experience much reduction in zeta potential than CS–PAA NPs due to abundant number of carboxylate groups on the surfaces of Gd-DTPA@CS–PAA NPs. The pH responsiveness of these NPs could be utilized for long circulating and passive targeting of NPs. Figure 3a and b depicts the TEM images of CS-PAA NPs and Gd-DTPA@CS–PAA NPs, respectively. Both types of NPs have regular spherical shape in aqueous medium. After comparison of these two TEM images, it could be deduced that incorporation of GdDTPA in CS–PAA NPs did not disrupt the structure of NPs.
3.2. pH Responsiveness and Structural Description The size distribution and morphology of CS–PAA NPs and Gd-DTPA@CS–PAA NPs were studied by DLS and TEM, respectively. Figure 2a demonstrates the size distribution of these NPs. CS–PAA NPs had an average particle size of 220 nm and the particle size of Gd-DTPA@CS–PAA NPs was found to be 212 nm. The slight reduction in particle size could be associated with the interaction of anionic carboxylate group of Gd-DTPA and cationic amine group of chitosan. Luckily, the adsorption of Gd-DTPA on CS– PAA NPs did not affect the particle size significantly. The surface charges of CS-PAA and Gd-DTPA@CS–PAA NPs were determined by zeta potential measurements at different pH values. Figure 2b describes the trend of zeta potentials change in response to pH change. Zeta potential increases with the decrease in pH values. Negative-to-positive charge reversal property for both types of NPs was observed at pH greater than 6.4. The reported pKa value of CS is 6.5 and the carboxylic acid groups of PAA are ionized above pKa of 4.7.[41] It is reported that amines of CS and carboxylic acid groups of PAA and DTPA of these NPs are partially ionized at about pH 6.5.[42] However, when the pH is above 6.5, the ionization of carboxylic acid groups of PAA and DTPA become dominant over the ionization of amines of CS. Therefore, NPs acquire negative charges on the surfaces.
3.3. In Vitro Studies of Gd-DTPA@CS–PAA NPs Gd-DTPA@CS–PAA NPs were fabricated by utilizing sorption method and gadolinium was loaded in Gd-DTPA@CS– PAA NPs mainly through diffusion. Since the concentration of Gd-DTPA was higher outside the NPs than inside the NPs, Gd-DTPA was diffused from exterior solution (high concentration) to the interior of CS–PAA NPs (lower concentration). However, the presence of carboxylate groups in poly(acrylic acid) could retard the diffusion of Gd-DTPA inside the NPs. These two opposing processes could limit the absorption of Gd-DTPA inside the NPs and most of the Gd-DTPA was adsorbed on the surfaces of CS– PAA NPs. Table 2 explains the Gd-DTPA loading capacity of CS–PAA NPs. The amount of gadolinium before and after the dialysis was measured to get better understanding of incorporated amount of gadolinium in NPs. It was observed that majority of Gd-DTPA was loaded in NPs. However, increasing the concentration of Gd-DTPA did not enhance the gadolinium loading capability. It implied that absorption-releasing equilibrium was established due to the aforementioned two opposing processes, which prevented the further sorption of Gd-DTPA in NPs. In vitro release profile of Gd-DTPA from NPs was studied in different media to verify the stability and pH responsive release of Gd-DTPA from NPs, as shown in Figure 4. Since
Figure 2. Size distribution of CS–PAA NPs and Gd-DTPA@CS–PAA NPs (a). Zeta potential of CS–PAA NPs and Gd-DTPA@CS–PAA NPs at different pH (b).
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Figure 3. TEM images of CS–PAA NPs (a) and Gd-DTPA@CS–PAA NPs (b).
Table 2. Gadolinium incorporated efficiency of Gd-DTPA@CS–PAA NPs.
Gd-DTPA amount before dialysis [mg ml1]
No.
1 2 3
Gd-DTPA amount after dialysis [mg ml1]
Total
NPs
Solvent
Total
NPs
Solvent
6.0 4.0 3.0
3.74 2.53 1.90
2.26 1.47 1.10
4.14 2.59 2.04
3.52 2.39 1.79
0.62 0.20 0.25
water molecules can enter and exchange freely in NPs, owing to intermolecular interaction between water and ionic groups of NPs, similar release profiles were found in aqueous and or other release media such as PBS (pH 7.0) or BSA (pH 7.0) solutions. However, the hydrogen bonding between the carboxylate group of Gd-DTPA and cationic amine of chitosan could strengthen the possession of GdDTPA within NPs. Therefore, very small initial burst was observed, i.e., only about 15% was released in first 4 h (Figure 4a), and even only 40% Gd-DTPA was released in 2 d. Figure 4a suggests that these NPs exhibited a sustained
Gadolinium loading efficiency [%] 62.4 63.2 63.2
release of Gd-DTPA. Moreover, the effect of pH on the release of Gd-DTPA from NPs is shown in Figure 4b. Release profile of Gd-DTPA was drawn in PBS at pH 7.4 and pH 5.00. Similar sustained trend of Gd-DTPA release from NPs was observed. However, Gd-DTPA release at pH 5.00 was faster as compared to pH 7.4, which implied that strong acidic medium, owing to conversion of COO– to COOH, weakens the interaction between carboxylic acid groups of poly(acrylic acid) and Gd-DTPA and amine of chitosan, which accelerated the release of Gd-DTPA from NPs. These findings support the utilization of these NPs for MRI in biomedical fields.
Figure 4. Gadolinium release profile in different types of media (a) and effect of pH on release of Gd-DTPA from NPs (b).
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Figure 5. The water proton relaxation rate of Gd-DTPA@CS–PAA NPs with different Gd-DTPA loaded level after dialyzing against deionized water (a) and pH dependence of the longitudinal proton relaxivity rate for Gd-DTPA@CS–PAA NPs and Gd-DTPA (b).
In vitro relaxation properties of Gd-DTPA@CS–PAA NPs were examined. The relaxation rate was plotted versus different concentration of Gd-DTPA to calculate the proton longitudinal relaxivity r1. The value of r1 was similar to different loading content of Gd-DTPA in NPs, which was about 5.34 mM1 s1 (Figure 5a). This value is higher than the preloaded r1 value of Gd-DTPA (4.5 mM1 s1). It is consistent with the fact that r1 can be increased by attaching small molecular complex of gadolinium to macromolecules. For instance, r1 value of some gadolinium dendrimers could reach up to 5 800 mM1 s1, which is nearly 1 000 times greater than the original relaxation rate of small molecule.[43] However, slight increase in r1 of gadolinium was observed in our NPs system. It could be deduced that Gd-DTPA not only adsorbed on the surface of NPs but also distributed inside the NPs. The Gd-DTPA inside the NPS is relatively stable due to its interaction with chitosan, and water molecules exchange rate is very slow inside the NPs. Therefore, internal Gd-DTPA exhibited less enhancing impact on the outside water proton relaxation. We also observed the effect of pH change on longitudinal proton relaxation, as shown in Figure 5b. It was observed
that as the pH value increased, the r1 was reduced in both. However, a smooth trend was observed in Gd-DTPA@CS– PAA NPs which could be attributed to sustained release of Gd-DTPA from NPs. Moreover, the increase of r1 value in pure Gd-DTPA was greater than that of Gd-DTPA@CS–PAA NPs in response to low pH. This difference in increment of r1 in acidic environment might be related to electrostatic equilibrium between chitosan and poly(acrylic acid) polymeric chains. At low pH, some of the protons were substituted with NH3þ of chitosan, which had been associated with COO– of poly(acrylic acid) in NPs. Thus, the effect of H3Oþ was decreased. Consequently, slight change in r1 was observed with the reduction in pH.
3.4. In Vivo Properties of Gd-DTPA@CS–PAA NPs For studying the efficacy of Gd-DTPA@CS–PAA NPs during their circulation in blood, NPs were injected in rabbits intravenously and the in vivo relaxation characterization (0.037 mmol kg) was carried out. Simultaneously, we compared the relaxation properties of NPs with Gd-DTPA
Figure 6. Blood relaxation rate of Gd-DTPA@CS–PAA NPs, Gd-DTPA, and Gd-(DTPA-BTD) at different time intervals in rabbit (a), biodistribution of Gd3þ in rabbit (b), and organ relative distribution of Gd3þ in rabbit (c).
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and Gd-poly(DTPA-BTD). Figure 6a describes the graph of longitudinal proton relaxation in rabbit blood along with injection time. The relaxation enhancement of GdDTPA@CS–PAA NPs was found to be greatest, as compared to Gd-poly(DTPA-BTD) and Gd-DTPA. The r1 values of GdDTPA and Gd-poly(DTPA-BTD) copolymers were attenuated quickly and the relaxation effect was lost after 25 min. However, the relaxation attenuation of Gd-DTPA@CS–PAA
NPs was also fast in initial 25 min; later, it was gradually slowed down. Therefore, Gd-DTPA@CS–PAA NPs possessed enhanced blood relaxation properties. The biodistribution of Gd-DTPA@CS–PAA NPs in rabbit organs was also explored after 4 h injection. Figure 6b and c depicts the gadolinium content of Gd-DTPA@CS–PAA NPs and Gd-DTPA complex measured by ICP-AES distributed in different organs of rabbit, as well as the relative organ
Figure 7. Transverse T1-weighted MR images of rabbit brain (a) and liver (b) using SE technique: 1) no contrast agent after 146 min, and after administration 2) 1 min, 3) 8 min, 4) 29 min, 5) 62 min, 6) 146 min. TR/TE ¼ 450 ms/25ms (b) signal intensity of MRI (c).
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distribution of gadolinium in different organs, respectively. It was observed in Figure 6b that Gd-DTPA@CS–PAA NPs largely influenced the distribution of gadolinium in different organs. While injecting Gd-DTPA complex only, most of the gadolinium was accumulated in liver and kidney, and gadolinium did not exhibit its considerable presence in blood, which demonstrated that majority of Gd-DTPA complex was excreted from blood circulation within 4 h. Since Gd-DTPA cannot pass through the blood–brain barrier,[44] the distribution of Gd-DTPA in the brain was also very low. However, Gd-DTPA@CS–PAA NPs had less distribution in the kidney, which was significantly different from Gd-DTPA complex. These NPs were mainly concentrated in the liver and lungs.These results indicated that Gd-DTPA@CS–PAA NPs altered the distribution of gadolinium in the body organs and produced a specific targeting property for liver and lung. Therefore, these NPs could be utilized for the MR imaging of liver and lung cancer. The delivery to brain is difficult due to presence of BBB, which prevents the passage from blood to brain.[45] Luckily, chitosan-based NPs have shown their accumulation in brain.[46] It is noteworthy that Gd-DTPA@CS–PAA NPs could also cross the BBB and transported some of the gadolinium content to the brain. Since different organs vary in weight and size, it is significant to compare distribution of gadolinium on the basis of organ weights. Figure 6c exhibits the organ relative distribution of gadolinium content. It provides better understanding of gadolinium distribution in different organs, for instance, the distribution of Gadolinium in liver and kidney is more apparent in Figure 6c. Similarly, the significant distribution of gadolinium in spleen was only observed by Figure 6c, which described that these NPs could be used as spleen targeting MRI contrast agent.
3.5. Magnetic Resonance Imaging of Gd-DTPA@CS– PAA NPs Gd-DTPA@CS–PAA NPs were injected in rabbits to perform MRI experiment and examine the MRI signals in blood vessels, liver, and brain. Figure 7a and b is the T1-weighted MRI pictures of brain and liver of rabbit. These images results confirmed that these NPs contain the higher contrast ability than free Gd-DTPA complex, as the images of Gd-DTPA@CS–PAA NPs were much brighter than the images from free Gd-DTPA complex (Figure 7). The MRI signals in liver and brain were also significantly stronger than the signals without contrast agent, and the signal enhancement could be kept for long time. MRI images confirmed that these NPs could be delivered in brain and liver successfully and enhanced the MRI signal in these organs. Thus, Gd-DTPA@CS–PAA NPs could be a better carrier for MRI contrast agent. Figure 7c demonstrates the
4. Conclusion Polymeric CS–PAA nanoparticles were fabricated by utilizing intermolecular interaction between chitosan and acrylic acid, and Gd-DTPA was incorporated in them. These nanoparticles were sufficiently stable in normal aqueous solution with positive surface charges. They showed pH sensitivity as described by in vitro release and relaxation experiments. This novel quality makes them appropriate for safe journey of contrast agent at normal pH environment and targeted release in acidic environment. In vivo biodistribution and MRI studies have also demonstrated localization of contrast agent and enhanced contrast respectively.
Acknowledgements: This work is supported by the National Natural Science Foundation of China (no. 21474047 and 51173077) and Nature Science Foundation of Jiangsu Province (BK20141225).
Received: February 4, 2015; Revised: March 11, 2015; Published online:DOI: 10.1002/mabi.201500034 Keywords: chitosan; Gd-DTPA; magnetic resonance imaging; nanoparticles; poly(acrylic acid)
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