Preparation of inclusion complex of perfluorocarbon compound with β-cyclodextrin for ultrasound contrast agent

Abstract

FC-77, a kind of perfluorocarbon compound, was inclusion-complexed with β-cyclodextrin (β-CD) in an attempt to improve their stability and bioavailability. Analyses by powder X-ray diffraction (XRD), thermogravimetric analyses (TGA), differential scanning calorimeter (DSC), Fourier transform infrared spectrum (FT-IR), transmission electron microscopy (TEM) and dynamic light scattering (DLS) proved the formation of the inclusion complex between FC-77 and β-CD, and high performance ion chromatography revealed that FC-77 content in the inclusion complex is 9.77% at 5 : 1 molar ratio of β-CD and FC-77. Furthermore, the inclusion constant and capacity were evaluated by UV-vis absorption spectroscopy and double reciprocal method. The results show that the nanoparticle of 1 : 1 FC-77/β-CD inclusion complex with an inclusion constant of 242.7 M⁻¹ was formed. Furthermore, ultrasound imaging experiments were performed to demonstrate biomedical application of FC-77/β-CD inclusion complex, which indicated that FC-77/β-CD inclusion complex could be employed as an ultrasound contrast agent for increasing ultrasound contrast.

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Introduction

Molecular imaging, including ultrasound (US), computed tomography (CT), and magnetic resonance imaging (MRI), is a novel tool which allows noninvasive diagnostic imaging to observe of biological processes at the cellular level, and contrast agents are important to provide diagnostic value for molecular imaging.¹ Recently, several nano- or microparticle systems are in development as contrast agents for diagnostic imaging.^2,3 Among them, perfluorocarbon (PFC) nanoparticles are a unique platform technology that may be applied to multiple clinically relevant imaging modalities.⁴ PFC is synthetic organic compounds in which all or most of the hydrogen atoms have been replaced with fluorine atoms. These molecules have the unique property of being both lipophobic and hydrophobic. Thus, as they are immiscible in aqueous systems, including biological fluids such as plasma and cell culture medium, they have to be modified for intravascular administration and for use in biological reactors.⁵ In the past years, PFC emulsions have become one of the main candidates for PFC applications in the biomedical and bio-technological fields, such as injectable oxygen carriers, contrast agents, drug delivery systems, and cell culture medium supplements. For example, PFC nanoemulsions/nanodroplets have been widely used as US contrast agents.^6–8 However, taking into account the short-term stability of PFC emulsions, the preparation and application of the stable PFC system is still an interest and active task.

Cyclodextrin (CD) are non-toxic cyclic oligosaccharides, consisting of (α-1,4)-linked α-D-glucopyranose units, with a hydrophilic outer surface and hollow hydrophobic interior. As an effective and facile approach, CD is focused as an inclusion host, which has been successfully used to improve the solubility, chemical stability and bioavailability of a number of poorly soluble compounds.^9,10 The most abundant natural cyclodextrins are α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), γ-cyclodextrin (γ-CD) containing six, seven and eight glucopyranose units, respectively. Investigations of molecular recognition have attracted much attention in supramolecular chemistry involving natural and artificial host–guest systems.^11,12 Specially, some reports have mentioned that fluorocarbon-modified polymers or fluorinated surfactants can form inclusion complexes efficiently with β-CD in aqueous solution. For example, Hogen-Esch et al. prepared the complex of β-cyclodextrin and perfluorocarbon-modified water-soluble polymers.¹³ Wilson et al. reported the formation of host–guest complexes of β-cyclodextrin and perfluorooctanoic acid.¹⁴ However, PFC for biomedical application has rarely been included in CDs.

In the present work, FC-77, used as a model medical PFC, is chosen as the object because of its widely application in clinic.^15–18 We report the preparation and characterization of a new type of inclusion complexes formed from FC-77 and β-CD with an aim to improve the stability and bioavailability of FC-77. The obtained FC-77/β-CD inclusion complex can be used as an ultrasound contrast agent to increase ultrasound contrast, which is potential for biomedical engineering in future. The research provides a potential chance in preparing ultrasound contrast agent system in biomedical applications.

Experimental

Materials

β-cyclodextrin (β-CD, ≥99%) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Perfluoro-compound (FC-77, CAS: 52623-00-4, perfluoroalkane (C₈F₁₈) mixture with perfluorocyclic ether (C₈F₁₆O)) was purchased from 3M Corporation. And the chemical structures of perfluoroalkane and perfluorocyclic ether are shown in Fig. 1. All the chemicals were of analytical reagent grade and used without purification.

Fig. 1 The chemical structures of perfluoroalkane (a) and perfluorocyclic ether (b).

Synthesis of FC-77/β-CD inclusion complex

An ethanol solution of FC-77 (0.12 mmol, 0.05 g) was added to aqueous solution of β-CD in 50 mL of water. The molar ratios of β-CD and FC-77 were 1 : 1 to 5 : 1. The resulting mixture was stirred at 30 °C for 24 h. After removal of 20 mL of a mixture of ethanol–water under vacuum, the reaction mixture was cooled at 4 °C overnight. The precipitates were collected after being washed with deionized water and absolute ethanol several times, and dried at 30 °C in vacuum.

Characterizations

The crystallinity of the obtained product was determined by powder X-ray diffraction (XRD) (Rigaku D/MAXIIA diffractometer using Cu Kα radiation). Thermogravimetric analyses (TGA) were conducted with a Linseis STA PT1600 thermoanalyzer instrument, with a temperature range of 30–600 °C at a heating rate of 10 °C min⁻¹. Thermal analyses was carried out with a Linseis PT10 differential scanning calorimeter (DSC) instrument, using 10 °C min⁻¹ scanning rate with a temperature range of 30–300 °C. Fourier transform infrared spectroscopy (FT-IR) was recorded on a Nicolet 6700 FT-IR Spectrometer. UV-vis absorption spectra were investigated on PerkinElmer Lambda 950 UV-vis spectrophotometer, and performed in ethanol and aqueous solutions, respectively. The concentration of FC-77 in ethanol is equal to that in synthesis of FC-77/β-CD inclusion complex. Transmission electron microscopy (TEM) images were recorded on a JEOL-2100F instrument using an accelerating voltage of 200 kV. Size distribution was measured by dynamic light scattering (DLS), which was performed on a Malvern Zetasizer Nano ZS. Fluorine content was determined by high performance ion chromatography (Dionex 500, USA) with conductivity detection through oxygen flask combustion (separating column: IonPac AG14-AS14; eluent: NaHCO₃ 0.0010 M + Na₂CO₃ 0.0035 M).

Ultrasound imaging

Ultrasound imaging was performed using Acuson Sequoia 512 linear transducer (Siemens, Mountain View, CA). A dilute solution (1 mg mL⁻¹) of β-CD and FC-77/β-CD inclusion complex was placed in a polyethylene (PE) tube, respectively, examined by a Siemens Acuson Sequoia 512 with a 3.5 MHz linear transducer.

Results and discussion

The powder X-ray diffraction was performed to confirm the complexation between FC-77 and β-CD. The XRD patterns of β-CD and FC-77/β-CD inclusion complex are shown in Fig. 2. The strong and sharp diffraction peaks indicate that commercial β-CD is well-crystallized. In contrast, the intensity of the sharp peak at 12.7° changes from 8400 to 4700 after the addition of FC-77 to β-CD (at 5 : 1 molar ratio of β-CD and FC-77), and the rest peaks also decrease. Meanwhile, some peaks such as the peak at 27.5° and 38° disappeared, which suggest that there is an interaction between FC-77 and β-CD, and it may contributes to a loss of crystallinity or amorphization of the samples.¹⁹ The result implies that there is an interaction between FC-77 and β-CD, and the inclusion complex could be formed.

Fig. 2 XRD patterns of β-CD (a) and FC-77/β-CD inclusion complex at 5 : 1 molar ratio of β-CD and FC-77 (b).

The thermal method is widely used to characterize cyclodextrins and their inclusion complexes. It can provide evidence for evaluating the inclusion process by any differences in the number and/or position of peaks between the physical mixture and the putative inclusion compound. Fig. 3 illustrates the DSC and TG curve of the FC-77/β-CD inclusion complex. As seen from Fig. 3a, the typical endothermic peaks of β-CD at about 120 °C and 265 °C are observed which is considered to be a dehydration process. When FC-77 is added, the bands of endothermic peak are decreased to 98 °C and 256 °C, respectively (at 5 : 1 molar ratio of β-CD and FC-77). Meanwhile, both of endothermic peaks become broader, which is probably due to the destruction of the cyclodextrin framework. Additionally, TG curves provide further information about the thermal property of the FC-77/β-CD complex (Fig. 3b). There is a weight loss before 130 °C, which is due to the release of water molecules from the outside or/and inside of β-CD cavity. After kept in a very wide temperature range, the samples undergo a rapid decomposition, which owns to the thermal decomposition process of the β-CD, and the survived β-CD is left. Compared to the starting temperature for mass loss of β-CD at 282.5 °C, the FC-77/β-CD inclusion complexes decomposed at 237.2 °C, which suggests that it starts to lose material at a temperature much lower than that of β-CD. Furthermore, when the temperature reached 600 °C, the weights loss of β-CD and FC-77/β-CD inclusion complexes are 86% and 99%, respectively. The addition of the guest (FC-77) will affect the structure of arrangement or the accumulation form of β-CD, as the results show that FC-77 molecule makes β-CD less stable during heating, which confirms the formation of the inclusion complex. Moreover, fluorine content in the inclusion complex is 7.39% determined by high performance ion chromatography. After conversion, FC-77 content in the inclusion complex is 9.77%.

Fig. 3 (a) DSC thermograms and (b) TG thermograms of β-CD and FC-77/β-CD inclusion complex at 5 : 1 molar ratio of β-CD and FC-77.

The complexation between FC-77 and β-CD is further evidenced by FT-IR spectra in Fig. 4. In the spectra of FC-77, the –CF₂ group gives the characteristic stretching vibration bands at 1206, 1142 and 1120 cm⁻¹, whereas the stretching vibration bands of –CF₃ group is at 1052 cm⁻¹. In addition, the bands at 1078 and 995 cm⁻¹ are absorption of C–C of liner china and C–O–C of the rings.²⁰ Furthermore, in the spectra of β-CD, the band at 1240 cm⁻¹ is due to OH bending of physically adsorbed water. Bands at 1152, 1078 and 1020 cm⁻¹ are assigned to stretching vibrations of C–O, C–O/C–C and C–O–C of glucose units, respectively, while bands at 995 and 943 cm⁻¹ are attributed to absorption of C–O–C of the rings.²¹ Compared with the spectra of FC-77 and β-CD, not all changes of the stretching frequency of FC-77 can be observed in the spectra of FC-77/β-CD inclusion complex when FC-77 was encapsulated with β-CD (at 5 : 1 molar ratio of β-CD and FC-77), because parts of the characteristic bands of FC-77 are overlapped with those of β-CD. Closer observation reveals the band at 1206 cm⁻¹, the typical –CF₂ group absorption can be found in the spectra of FC-77/β-CD inclusion complex, which suggests the interaction between FC-77 molecules and β-CD. However, the intensity of the bands at 1206, 1142 and 1120 cm⁻¹ is decreased, which is probably due to the interaction is taking place in the cavity of the cyclodextrin.

Fig. 4 FT-IR spectra of FC-77, β-CD and FC-77/β-CD inclusion complex at 5 : 1 molar ratio of β-CD and FC-77.

UV-vis absorption spectra are used to evaluate the complexation of FC-77 titrated with β-CD. As shown in Fig. 5a, with the increasing concentration of β-CD, the absorption maximum of FC-77 red-shifts from 208 nm to 218 nm, and the intensity decreases gradually, that can be explained by the high electron density of the β-CD cavity, which induce electron migration of the FC-77 and finally lead to the red-shifts of the absorption maximum of FC-77 and the intensity decreases gradually through noncovalent interactions.²² The results further indicate the formation of inclusion complex between FC-77 and β-CD.

Fig. 5 (a) UV-vis absorption spectra of FC-77 in solution with different molar ratio β-CD and FC-77; (b) double reciprocal plot for the determination of the inclusion constant of FC-77/β-CD inclusion complex. Molar ratio β-CD and FC-77: (1) 0, (2) 1 : 1, (3) 2 : 1, (4) 3 : 1, (5) 4 : 1 and (6) 5 : 1.

The inclusion constant, which represents the inclusion capacity, is also determined in the study. The double reciprocal method was used to estimate the inclusion constant of the complex, which can be obtained from the Benesi–Hildebrand equation:²³

1/[A − A₀] = 1/a + 1/aK[CD]₀

Here, A, A₀, a, K, and [CD]₀ are the absorbance of FC-77 in the presence of β-CD, that in the absence of β-CD, a constant, the inclusion constant for the formation of 1 : 1 FC-77/β-CD inclusion complex, and the initial concentration of β-CD, respectively. The inclusion constant K can be calculated by the ratio of intercept over slope. Fig. 5b exhibits a double reciprocal plot for the UV-vis intensity of FC-77 in aqueous solution containing β-CD. The result shows the good linearity, which suggests that 1 : 1 FC-77/β-CD inclusion complex has formed. Furthermore, the inclusion constant K can be calculated by the ratio of intercept over slope. The determined K value for β-CD is 242.7 M⁻¹, implying weak inclusion happened between the FC-77 and β-CD in the water.²⁴ Various noncovalent interactions between host and guest, such as dipole–dipole, hydrophobic, electrostatic, van der Waals, and hydrogen-bonding interaction, cooperatively contribute to the inclusion process.²⁵ Thus, the dipolar or hydrogen bonding interactions may mainly influence the stability of the complex owing to the great polarity of FC-77.

The size and morphology of the obtained products were characterized. As shown in Fig. 6a, the average hydrodynamic size of the assembly of FC-77/β-CD inclusion complex is 350 nm, 530 nm and 695 nm at 1 : 1, 3 : 1 and 5 : 1 molar ratio of β-CD and FC-77, respectively. The results reveal that the hydrodynamic size of the assembly of FC-77/β-CD inclusion complex increases with the addition of β-CD due to the hydrophilicity of β-CD. Furthermore, TEM image of the assembly of FC-77/β-CD inclusion complex at 5 : 1 molar ratio of β-CD and FC-77 is presented in Fig. 6b. The nanoparticles about 50 nm are obtained although some of them have been agglomerated. The result is similar to the reported literature, which CD or CD inclusion complex nanoparticles could be produced in organic and aqueous mixed solvent through the reprecipitation mechanism.^26,27

Fig. 6 (a) DLS data of obtained at the 1 : 1, 3 : 1 and 5 : 1 molar ratio β-CD and FC-77 and (b) TEM image of obtained FC-77/β-CD inclusion complex at 5 : 1 molar ratio of β-CD and FC-77.

To demonstrate that FC-77 inclusion-complexed β-CD can be used as an ultrasound contrast agent, ultrasound imaging experiments were performed. The results are shown in Fig. 7, which shows the ultrasound images of PE tube with and without FC-77/β-CD inclusion complex in cross-section. As shown in Fig. 7a, the weak signal could attribute to the bubbles coming from shakiness of tube. However, FC-77/β-CD inclusion complex with 5 : 1 reaction ratio of β-CD and FC-77 shows relatively strong ultrasound contrast due to large differences in acoustic impedances between perfluorocarbons and water (Fig. 7b). Furthermore, the visible smearing of the signal from FC-77/β-CD inclusion complex suggests that some disruption of inclusion complex in the ultrasound condition. The results reveal that FC-77/β-CD inclusion complex is potential for biomedical engineering in molecular imaging.

Fig. 7 Ultrasound images of (a) pure β-CD and (b) FC-77/β-CD inclusion complex at 5 : 1 molar ratio of β-CD and FC-77 in PE tube.

Conclusions

The inclusion complex of FC-77 with β-CD is prepared successfully. XRD patterns, DSC and TG thermograms and FT-IR spectra prove the formation of the inclusion complex and high performance ion chromatography reveals that FC-77 content in the inclusion complex is 9.77% at 5 : 1 molar ratio of β-CD and FC-77. Furthermore, TEM and DLS show that the nanoparticle of FC-77/β-CD inclusion complex could be obtained and their hydrodynamic size is in nanometer and increases with the addition of β-CD. Besides, UV-vis absorption spectra reveal the 1 : 1 stoichiometry ratio and an inclusion constant of 242.7 M⁻¹, which deduces that the dipolar or hydrogen bonding interactions is the driving force for the formation of FC-77/β-CD inclusion complex. Furthermore, ultrasound imaging experiments proves FC-77/β-CD inclusion complex can be used as an ultrasound contrast agent to increase ultrasound contrast. The promising results imply that β-CD can be used as the active ingredients of perfluorocarbons and give the possibility of enhancing and widening the usage of perfluorocarbons.

Acknowledgements

The work is supported by National Key Technology Research and Development Program (no. 2014BAK05B02) and National Natural Science Foundation of China (no. 51303135).

Notes and references

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Footnote

† These authors contributed equally to this work.

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