Aptamer-Modified Temperature-Sensitive ... - ACS Publications

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Aptamer-modified Temperature-sensitive Liposomal Contrast Agent for MR Imaging Kunchi Zhang, Min Liu, Xiaoyan Tong, Na Sun, Lu Zhou, Yi Cao, Jine Wang, Hailu Zhang*, Renjun Pei*

Key Laboratory of Nano-Bio Interface, Division of Nanobiomedicine and Nanobionics, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou, 215123, China. E-mail: [email protected]

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ABSTRACT: A novel aptamer modified thermosensitive liposome was designed as an efficient MR imaging probe. In this paper, Gd-DTPA was encapsulated into an optimized TSL formulation, followed by conjugating with AS1411 for specific targeting against tumor cells which overexpress nucleolin receptors. The resulting liposomes were extensively characterized in vitro as a contrast agent. As-prepared TSLs-AS1411 had a diameter about 136.1 nm. No obvious cytotoxicity was observed from MTT assay, which illustrated that the liposomes exhibited excellent biocompatibility. Compared to the control incubation at 37 oC, the liposomes modified with AS1411 exhibited much higher T1 relaxivity in MCF-7 cells incubated at 42 oC. These data indicate that the Gd-encapsulated TSLs-AS1411 may be a promising tool in early cancer diagnosis.

Keywords: Aptamer, targeted, themosensitive liposome, MR imaging.


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1. INTRODUCTION Magnetic resonance imaging (MRI) contrast agents have been extensively used to enhance image contrast between pathological and normal tissues, because of its noninvasiveness, superior tissue resolution, and precise three-dimensional positioning ability. But in order to improve the diagnostic sensitivity and accuracy, contrast agents are indispensable.1-3 Various paramagnetic compounds have been evaluated as MR contrast agents, but chelated gadolinium (Gd) compounds continue to be the most widely used. Currently, clinically used contrast agents are mainly small molecule gadolinium chelates such as Gd-DTPA (Magnevist), Gd-DTPA-BMA (Omniscan), Gd-BOPTA (MultiHance) and so on. However, these small molecule contrast agents suffer some drawbacks including lack of specificity, short circulation time and insufficient relaxivity.4-6 Recent efforts for designing new contrast agents have focused on macromolecular contrast agents, which can efficiently enhance the relaxivity and prolong the circulation time.7-9 Liposomes, with a bilayer structure, have been paid much attention during the past 30 years as pharmaceutical carriers of great potential.10 Liposomes have been developed as contrast agents carriers by encapsulating chelated Gd within the aqueous lumen of liposomes with high loading of Gd 33-40. However, the relaxivity of this kind of liposome-based MRI agents would be significantly reduced due to the slow flux of water across the membrane bilayer.11 The immobilization of chelated Gd on the surface of liposomes could increase the exchange of bulk water protons with


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the contrast agents.12 But this method did not take the advantage of high loading of Gd of encapsulating agents within the lumen of liposomes. Thermosensitive liposomes (TSLs), one of the unique liposomes with tunable release properties, are commonly used to deliver drugs to the target site in a controlled manner. TSLs are composed of lipid bilayers that undergo temperature-dependent phase transitions from gel to liquid phase and permeable at elevated temperatures, resulting in rapid release of encapsulating cargoes at the gel-to-liquid crystalline phase-transition temperature (Tm).13-15 TSLs have the potential of high loading of Gd and permeability to water or fast releasing of the encapsulated Gd.16-18 In comparison to other ligands, DNA aptamers are stable in harsh environment and have been demonstrated to be nonimmunogenic.1 AS1411, A 28-nucleotide guanosine-rich (G-rich) DNA sequence, is commonly known as anti-nucleolin aptamer. AS1411 can bind to the nucleolin receptors which are over expressed on the tumor cells20. The combination of aptamers with nanomaterials as a MR imaging probe therefore represents a powerful diagnostic tool for the detection of cancer in early stage21. However, there are the limited reports using the aptamer-targeted MR imaging probes to show their potential for cancer cell and tissue imaging.22-24 Especially, the aptamer-targeted liposomes have not yet been developed for cancer cell MR imaging. With the purpose of investigating targeted thermosensitive liposomes contrast agents, herein, we report the preparation of thermosensitive liposome encapsulated with Gd-DTPA, followed by modifying with AS1411. The PEG chains on the surface of liposomes will improve the stability of liposomes and


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have a reduced tendency to leak contrast agents while in circulation. Our results showed that the AS1411 modified TSLs showed enhanced imaging effect on MCF-7 cells in response to a mild hyperthermic treatment.

2. EXPERIMENTAL SECTION 2.1

Materials.

Dipalmitoyl

phosphatidylcholine

(DPPC),

monostearoyl

phosphatidylcholine (MSPC) and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine -N-[carboxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000-carboxylic acid) were purchased from Avanti (Avanti Polar Lipids, Inc. USA). Fatty acid methyl ester sulfonate (MES), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. Roswell Park Memorial Institute Medium (RPMI-1640), Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were obtained from HyClone (Logan, Utah, USA). Gadopentetate dimeglumine (Gd-DTPA) were obtained from Consun Pharmaceutical Co., Ltd. (Guangzhou, China). All the other reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and used without further purification.

2.2 Liposome preparation. TSLs encapsulating Gd-DTPA were prepared by lipid film hydration method. Phospholipids [DPPC: MSPC: DSPE-PEG2000-COOH (86 : 10 : 4)] were dissolved in chloroform and the organic solvent was evaporated under vacuum for 60 min at 40 oC using a rotary evaporator. The dried lipid film was


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hydrated with Gd-DTPA solution to achieve a final lipid with the concentration of 5 mM. Then, the TSLs were prepared by extruding the above solution through 100 nm polycarbonate filter membrane 10 times at 60 oC using an Extruder25. The free Gd-DTPA was removed through a Sephadex G-75 column and the carboxylated liposomes (TSLs-COOH) were obtained. AS1411 aptamer was conjugated onto TSL-COOH through EDC chemistry. Briefly, TSL-COOH was dispersed in MES buffer (pH 6.0) with continuous stirring. Then, EDC (10 mg/mL) and sulfo-NHS (10 mg/mL) were added to the above solution respectively. After stirring for 15 min, the AS1411 aptamer (5'-NH2-TTG GTG GTG GTG GTT GTG GTG GTG GTG G-3', 100 µM) was added and stirred for another 6 h, followed by multiple washing with PBS buffer (100 mM, pH 7.4) to form the DNAconjugated liposomes (TSLs-AS1411)26. The concentration of phospholipid and gadolinium in the TSLs were determined by ICP-AES (Perkin-Elmer Optima 8000). The hydrodynamic diameter and zeta potential of TSLs were measured using Zetasizer Nano ZS at 25 oC. Differential scanning calorimetry (DSC) was used to measure the phase transition temperatures of the TSLs27. Each sample was transferred into aluminum crucibles and sealed. Measurements have been performed with differential scanning calorimetry (VP-DSC, Microcal, US). The samples were scanned with an average heating rate of 1 oC /min from 10 to 65 oC.

2.3 MR measurements. The longitudinal relaxation time (T1) of Gd-DTPA and


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TSLs-AS1411 were measured by a 0.5 T NMR-analyzer (GY-PNMR-10). T1 of GdDTPA were measured at Gd concentrations of 0.156, 0.3125, 0.625, 1.25 and 2.5 mM, while T1 of TSLs-AS1411 at 37

o

C and 42

o

C were measured at Gd

concentrations of 0.125, 0.25, 0.5, 1 and 2 mM.

2.4 Temperature sensitivity of TSLs-AS1411 and T1 weighted MR imaging study. For the temperature-dependency study, the T1 measurements of TSLs were performed as follows: the samples in aqueous solution were incubated at 37 oC and 42 oC for 30 min, respectively, then T1 were measured. The T1 weighted MR imagings of TSLs were performed and processed at the 0.5 T NMR-analyzer system. The measurement parameters are as follows: TR=100 mS, TE=8.6 mS, and NS=1.

2.5 Cell culture. MCF-7 cells and 293T cells were cultured in RPMI 1640 and DMEM with 10% fetal bovine serum (FBS), respectively. Both culture media were supplanted with streptomycin at 100 units/mL and penicillin at 100 units/mL, and the cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 in air.

2.6 Cytotoxicity study. The cytotoxicity of TSLs-AS1411 against 293T cells was evaluated by MTT assay. 293T cells were seeded into 96-well plates at a density of 10000 cells/well in 100 µL complete DMEM. When cells achieved 60-70% confluence after 24 h of incubation, Gd-DTPA and TSLs-AS1411 at different concentrations (0.4-3.2 mM) were added to the wells and incubated for further 24 h.


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Then, 20 µL of MTT (5 mg/mL in PBS) was added to each well except for the background wells and incubated for further 4 h. Finally, the medium was removed, and 150 µL DMSO was added. The absorbance was measured at 490 nm using a microplate reader (Perkin-Elmer Victor X4). The relative cell viability was calculated according to the following equation: Relative cell viability (%) = 100 × (ODsample – ODbackground)/(ODcontrol – ODbackground)

2.7 In vitro MR imaging of TSLs. MCF-7 and 293T cells were seeded into culture dishes at a density of 1 × 106 cells per plate in 10 mL of culture medium. After cells achieved 60-70% confluence, the dishes was incubated with TSLs at a Gd concentration of 0.4 mM for 2 h. Then, the medium was removed and cells were washed with 5 mL of PBS three times. After treating with trypsin, the cells were harvested and washed with PBS by centrifuged at 1000 rpm for 5 min to remove medium28. The precipitated cells were transferred into capillary tubes (1.8 mm x 100 mm) which were centrifuged again at 1400 rpm for 5 min to enable cells well packed at the bottom of the capillary for MRI, then, the cells incubated with TSLs were treated at 42 oC for 30 min29. MRI study was performed at the 11.7 T Bruker microMRI system with a 25 mm RF coil. The measurement parameters are as follows: TR = 500.0 ms, TE = 5.2 ms, imaging matrix = 128 × 96, field of view (FOV) = 12 × 12 mm, NEX = 4.

3. RESULTS AND DISCUSSION


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3.1 Preparation and Characterization of TSLs-AS1411. The major challenge in designing liposomes as high efficient and safety contrast agents is to improve the stability of liposomes and the loading amount of contrast agents. Otherwise, enduing the liposomes with target capability will enhance the cellular uptake of CA and reduce the dose of CA. The aim of this study was to develop a magnetic resonance imaging detectable liposome with high relaxivity and stability. Therefore, we designed a PEG-shielded thermosensitive liposome encapsulating with Gd-DTPA, followed by conjugating with AS1411. A conceptual scheme for Gd-DTPA release from AS1411 modified thermosensitive liposome at Tm was shown in Scheme 1. The resulting liposomes were extensively characterized such as contrast agent efficiency and structural properties. The molar ratio of Gd and lipid was 1:10 which was measured by ICP-AES. The size and zeta potential were measured using a Zetasizer Nano ZS. The size distribution of TSLs-AS1411 loaded with Gd-DTPA was shown in Figure 1A. The as-prepared liposomes of TSL-COOH and TSL-AS1411 had an average diameter around 123.2 nm and 136.1 nm, respectively. Meanwhile, Zeta potential of TSLs-COOH and TSLs-AS1411 were measured to further verify the successful coupling of AS1411 on the surface of TSLs (Figure 1B). The TSLs encapsulated with Gd-DTPA were conjugated with aptamer AS1411. The prepared TSL-AS1411 sample was purified with Vivaspin ultrafiltration spin column (MW 10kDa) to remove the unreacted AS1411. To confirm that the unreacted AS1411 was completely removed, the filtrate was detected at 260 nm by UV-vis spectroscopy every time. The results showed there was almost no absorption after the


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eighth filtration. The results were shown in Fig. S3. The UV-vis absorption spectra of TSLs-COOH and TSLs-AS1411 were shown in Figure 1C. The appearance of the peak at 260 nm for TSLs-AS1411 was attributed to DNA absorbance, indicating the successful modification of DNA onto the TSLs-COOH surface. Fluorescence analysis of the FAM modified sequence (FAM-CS) which was complementary to AS1411 further confirmed the successful conjugation of AS1411 onto TSLs (Figure 1D). To quantify the aptamer bound on the surface of TSL, an organic dye Thioflavin T (Tht) was chosen to determine the concentration of the aptamer. Tht could induce AS1411 to form G-quadruplex to enhance the fluorescence of Tht. The fluorescence of Tht was measured and the concentration was determined according to a standard linear calibration curve of Tht. Based on the concentration of lipid measured by ICP-AES, there is approximately ~4.17 nM DNA aptamer with 1.325 µM lipid. The coupling efficiency of aptamer on DSPE-PEG2000-carboxylic acid was calculated to be 8% (Fig. S1). DSC was performed to investigate the temperature sensitivity and the stability of CA-loaded TSLs. As shown in Figure 1E, the DSC thermogram of TSLs showed that the main phase transition peak was at 42 oC, and TSLs could be stable at 37 oC. The transition temperature observed with TSLs was due to the presence of MSPC lysolipid in the lipid bilayer. The MSPC lysolipid tends to stabilize the bilayer in gel phase at low temperature and destabilize the bilayer in disordered structure at high temperature. The average liposome size in the TEM image of TSL-AS1411 was around 100 nm and the result was shown in Fig. S2.


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3.2 Temperature dependent relaxivity profile of TSLs-AS1411 encapsulating Gd-DTPA. To design a thermosensitive liposome, the lipid composition should be considered first. Banno’s group has studied the function of lysolipid content in TSL system30. The result indicated that leakage of molecules was highly dependent on the content of lysolipid in the formulation. Needham’s group also showed that liposome containing lysolipid displayed a dramatic enhancement in the permeability rate about two degrees below the calorimetric peak of the thermal transition13,14. Based on these results, the content of lysolipid MSPC was chosen to be 10% in the composition of TSLs. After incubating for 10 min at 42 oC, the leakage of Gd-DTPA began to appear, and with increasing the incubating time, the leakage of Gd-DTPA increased, and the incubating time was determined according to the previous work31,32. The longitudinal relaxation rate of TSLs-AS1411 encapsulating Gd-DTPA and a commercial contrast agent (Magnevist) for comparison were measured on a 0.5T MRI scanner. As shown in Figure 2. The r1 value of Magnevist calculated from the slope was 4.014 mM−1 s−1, which is consistent to the value reported in the literatures. However, the r1 value of liposome encapsulated with Gd-DTPA was 0.98 mM−1 s−1, which is much lower than Magnevist. With the temperature increasing, the r1 value of liposome encapsulated with Gd-DTPA went up to 3.92 mM-1 s-1. The increase of r1 value was attributed to the change of permeability rate of liposome, which resulted in the rapid release of Gd-DTPA from liposome at Tm. Figure 3 showed T1-weighted MRI images of TSLs-AS1411 at different Gd3+


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concentration. Obviously, TSLs-AS1411 produced much brighter images with the improvement of the Gd concentration from 0.25 mM to 1 mM at 37 oC, in accordance with the decreasing of the T1 relaxation time from 2126.57 ms to 841.35 ms. After increasing the temperature to 42 oC, the brightness of MRI images was obviously enhanced at the same Gd concentration (T1 for 0.25 mM: 730.24 ms; for 1 mM: 234.62 ms). These results further confirmed the improvement of permeability rate of liposome and release of Gd-DTPA. Below Tm, the MRI contrast agent remains within the liposome and the T1-relaxation enhancement is limited by the transmembrane water exchange rate, which resulted in a low r1 value. Upon heating, the T1-based contrast enhancement went through a maximum at Tm because of the release of the Gd(III) complex from the lumen of the liposome.

3.3 Cytotoxicity Assay. Cytotoxicity is an important parameter in MRI diagnosis. MTT assay was performed to evaluate the cytotoxicity of TSLs-AS1411 and GdDTPA against 293T cells. Different concentration of samples was incubated with 293T cells, the result was shown in Figure 4. The viability of 293T cells did not decrease obviously with increasing Gd concentration. The cell viability of TSLsAS1411 and Gd-DTPA against 293T cells was found to be nearly 100% even at the Gd concentration of 2 mM. This result illustrates that sample possesses well biocompatibility.

3.4 Cellular MRI of TSLs-AS1411 encapsulating Gd-DTPA. To further determine


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the impact of the mild hyperthermia on tumor, in vitro MR imaging studies were performed. MCF-7 cells were selected as cell lines which over expressed nucleolin receptors. 293T and H2O were used as controls. As shown in Figure 5, the images produced by TSLs-COOH were found to be no obvious differences between the 293T cells (Figure 5B) and MCF-7 cells (Figure 5D) in 37 oC. However, the images produced by TSLs-AS1411 in MCF-7 (Figure 5H) cells was slightly brighter than that in 293T cells (Figure 5F) in 37 oC, which illustrated that the AS1411 aptamer enhanced the interaction of tumor cells and aptamer modified liposomes, and resulted in a brighter image. Furthermore, a much brighter image (Figure 5I) was observed at a little higher temperature. The preparation methods of producing Figure 5I and Figure 5H were nearly the same except the temperature. The image of Figure 5H produced by TSLs-AS1411 was treated with 37 oC, while the image of Figure 5I produced by TSLs-AS1411 was treated with 42 oC for 30 min. The random DNA sequence (random DNA, 5'-NH2-CCT CCT TCC TTC AAA ACA ACC AAC CACC-3') modified liposomes (TSLs-randomDNA) were incubated with MCF-7 cell line and treated with 37 oC and 42 oC as part of control groups (Fig. S4). The images showed that there was no targeting capability for the TSLs-randomDNA sample. The images of all control groups with 42 oC were brighter than groups with 37 oC. The results illustrated that the Gd-DTPA was released from the liposomes at Tm, and then resulted in the enhancement of relaxivity.

4. CONCLUSION


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In this study, Gd-DTPA was successfully encapsulated into a thermosensitive liposome, followed by conjugating with AS1411. The TSLs-AS1411 displayed temperature-dependent membrane-disruptive capabilities, resulting in release of encapsulated DTPA-Gd at Tm, and then the longitudinal relaxivity of TSLs-AS1411 increased from 0.98 to 3.92. Importantly, the MTT result showed that no obviously cytotoxicity of TSLs-AS1411 was observed even at the Gd concentration of 2 mM. The AS1411 targeted TSLs showed enhanced imaging effect on MCF-7 cells in response to a mild hyperthermic treatment. This novel aptamer modified thermosensitive liposome with targeting capability and admirable biocompatibility shows excellent potential as an efficient MR imaging probe.

Supporting Information Available TEM images and standard curve for quantification of DNA aptamer concentration were shown in supporting information. This material is available at free of charge via the Internet at http://pubs.acs.org

ACKNOWLEDGMENTS: This work was financially supported by National Natural Science Foundation of China (21304106) and the CAS Hundred Talents program.

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(38) Meyre, M. E.; Raffard, G.; Franconi, J. M.; Duguet, E.; Lambert, O.; Faure, C. Nanomedicine: Nanotechnology, Biology, and Medicine. 2011, 7, 18-21. (39) He, Y. N.; Zhang, L. H.; Zhu, D. W.; Song, C. X. International Journal of Nanomedicine. 2014, 9, 4055-4066. (40) Cheng, Z. L.; Zaki, A. A.; Jones, I. W.; Hall, H. K.; Aspinwall, C. A.; Tsourkas, A. Chem. Commun. 2014, 50, 2502-2504.


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Biomacromolecules

Scheme 1 Illustration of Gd-DTPA release from AS1411 modified thermosensitive liposome.

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Figure 1 (A) The size distribution of TSLs-COOH and TSLs-AS1411. (B) Zeta potential of TSLs-COOH and TSLs-AS1411. (C) UV-vis spectra of TSLs-COOH and TSLs-AS1411. (D) Fluorescence spectra of TSLs-COOH and TSLs-AS1411 after reacting with FAM-CS, λex=492 nm. (E) Differential scanning calorimetry of TSLs-AS1411.

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Figure 2 Longitudinal relaxivity (r1) of Magnevist (A) and TSLs with encapsulated Gd-DTPA at 37 oC and 42 oC (B).


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Figure 3 T1-weighted MR images of TSLs-AS1411 at different Gd3+ concentrations: 0.25 mM (A), 0.5 mM (B) and 1mM (C) at 37 oC, and 0.25 mM (D), 0.5 mM (E) and 1 mM (F) at 42 oC, all the measurements were taken under 0.5 T NMR-analyzer. T1 relaxation was carried out by inversion recovery method, while the T1-weighted images were obtained by SE sequence. For T1-weighted images, TR=100 mS, TE=8.6 mS, and NS=1.

Figure 4 Cytotoxicity profile of Gd-DTPA and TSLs-AS1411 with encapsulated Gd-DTPA against 293T cells.


Biomacromolecules

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Figure 5 T1-weithted images of 293T cell and MCF-7 cells. (A) Water, (B) 293T cells incubated with TSLs-COOH, (C) 293T cells incubated with TSLs-COOH, followed by incubating at 42 oC, (D) MCF-7 cells incubated with TSLs-COOH, (E) MCF-7 cells incubated with TSLs-COOH, followed by incubating at 42 oC, (F) 293T cells incubated with TSLs-AS1411, (G) 293T cells incubated with TSLs-AS1411, followed by incubating at 42 oC, (H) MCF-7 cells incubated with TSLs-AS1411, (I) MCF-7 cells incubated with TSLs-AS1411, followed by incubating at 42 oC. All the measurements were taken under 11.7T Bruker micro-MRI system.

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