Perfluorodecalin nanocapsule as an oxygen carrier and contrast agent for ultrasound imaging

Abstract

The preparation of a perfluorodecalin (PFD) encapsulated silica nanocapsules via a one-pot synthesis method using Pluronic F68 (PF68) stabilised PFD in water nanoemulsion as a template is demonstrated herein. The method described here simplifies previous multi-step procedures that required pre-fabrication of silica hollow spheres and post-loading of the perfluorocarbon. The size of the PFD nanoemulsion template can be varied easily by changing the concentration of PF68. Successful incorporation of PFD (78% wt) into the silica nanocapsule core and the mechanical stability of the structure were evident from ¹⁹F nuclear magnetic resonance analysis; these properties were conferred by the silica-PF68 surface structure. This significant improvement in PFD encapsulation results in higher oxygen carrying capacity compared to encapsulation without PFD as observed from oxygen solubility measurements. The nanocapsules did not show cytotoxicity against HepG2 and 3T3 cells after 24 h exposure at a concentration below 0.8 g L⁻¹ and 0.08 g L⁻¹, respectively. Ultrasound imaging of silica nanocapsules in agarose gel showed a greater than two-fold increment in echogenicity as compared to the PFD nanoemulsion at a similar concentration.

---

1. Introduction

Tumour cells that exist in a low oxygen environment (hypoxia) have been shown to be more resistant to radiotherapy and chemotherapy than those under normoxic conditions, resulting in malignant tumour progression.^1,2 While the role of oxygen is still not fully understood, it was postulated that more reactive radicals are produced in radiotherapy when oxygen is present and this is conducive to destroying the tumour cells. Moreover, molecular oxygen can also oxidise the DNA radicals that are produced in radiotherapy; this renders the DNA damage irreparable, resulting in a better therapeutic effect.^3–5 Hyperbaric oxygen treatment has been employed whereby the tumour cells are exposed to oxygen prior to the cancer therapy. However, the cure rate of hyperbaric oxygen treatment is still very low.⁶ This is due to the large interstitial distance (up to 150 μm) between the hypoxic tumor cells and blood vessels, which prevents adequate oxygen diffusion towards the tumour cells.^7,8 Nano-sized oxygen carrying particles present a potential solution to the problem of low oxygen diffusion across the interstitial barrier.⁹ Previously, perfluorocarbon nanoemulsions with size between 100 and 300 nm have been used in a wide range of biomedical applications due to the high oxygen dissolving and delivering capabilities of this group of compounds. These include treating decompression sickness, acute lung injury, and uses in retinal surgery.^10–16 The use of perfluorocarbon emulsions have also been shown to improve tumour killing after radiotherapy.^17,18

The applications of perfluorocarbon emulsions in contrast enhanced ultrasound (CEUS) imaging have also been demonstrated recently.^19–25 Volatile perfluorocarbon gases such as perfluoropropane and perfluorobutane have been shown to be excellent contrast agents for CEUS imaging due to their low diffusion rate across blood capillaries and their ability to generate a significant backscattering ultrasound signal.²⁶ However, gas phase perfluorocarbon nanoemulsions have poor resistance against physical deformation and ultrasound irradiation, resulting in a short in vivo circulating life time.²⁷ For application in tumor imaging and treatment, a long biological life-time is a highly desirable feature for the contrast agent that is used.^{21–23,28,29} Therefore, liquid phase perfluorocarbon (PFC) nanoemulsions have been sought as an alternative in order to extend the circulation life time, thus enabling a longer period of ultrasound diagnosis.^20,22,30 For the purpose of both oxygen delivery and ultrasound imaging, the perfluorocarbon is administered intravenously and therefore has to be miscible with plasma fluid. One way to achieve this property is via emulsification of perfluorocarbon prior to administration. Typically, the size of the emulsion has to be smaller (<5 μm) than the size of blood capillaries and have good dispersion and a stable size during the course of the imaging session. Both micron-size emulsion (>1 μm) and nano-size (<500 nm) emulsions have been shown to be suitable for use as ultrasound contrast agent.^{20,22,29,31–34} Micron size emulsions have been shown to be more effective ultrasound contrast agents, however their use has been limited to intravascular diagnosis such as assessment of myocardial perfusion and detection of vascular thrombosis.³⁵ For application in tumor assessment and treatment using ultrasound imaging, smaller-size emulsion is required due to the higher probability of penetrating most tumor mass which has pore size of between the range of 380 to 780 nm.³⁶ For this reason, there has been wide interest in the facile preparation of perfluorocarbon emulsions in the nano size range with good long term structural stability and ultrasound backscattering properties.

Herein, we report the fabrication of silica coated PFD nanoemulsion via one-pot synthesis to achieve both high PFD loading and improvement in ultrasound backscattering. PFD in water nanoemulsions template were first prepared with PF68, which serves two purposes. Firstly, the PFD droplets are stabilised in water via the central polypropylene oxide (PPO) chain of PF68 which adsorbs onto the droplets surface with the polyethylene oxide (PEO) chains tailing into the continuous aqueous phase. The loop and train conformation of PF68 on the surface of the perfluorocarbon droplets creates steric repulsion that prevents the perfluorocarbon from coalescing, thus forming a stable perfluorocarbon nanoemulsion.^37,38 Secondly, the interfacial hydrophilic layer serves as the template for silanisation reaction to take place. This is unachievable if silica is in direct contact with PFD liquid due to both the hydrophobic and lipophobic nature of the liquid. In our study, the variation of PF68 concentration towards the size of the nanoemulsion was first investigated. Together, the effect of pH towards the morphology, size and stability of silica nanocapsules was also investigated. The success of incorporating a silica shell layer, the presence of PFD in the core of the silica nanocapsules as well as the mechanical stability of the structure was confirmed by ¹⁹F nuclear magnetic resonance measurements and thermogravimetric analysis. Cytotoxicity assays were also performed to confirm the biocompatibility of the silica nanocapsules. Finally, oxygen solubility measurements were undertaken to confirm the oxygen content in the silica nanocapsules and in vitro ultrasound measurements were also carried out to compare the effect of silica incorporation on the ultrasound signal intensity.

2. Experimental

2.1. Materials

Perfluorodecalin (PFD, 95%, cis- and trans-mixture) and 3,5-dichloro-2-hydroxybenzenesulfonic acid (DCHBS, 99%) were purchased from Alfa Aesar. Pluronic® F68 (PF68, M_w = 8350 g mol⁻¹), tetramethylorthosilicate (TMOS, 98%), 4-aminoantipyrine (AAP), glucose oxidase (GO, cat. #G7141) and horseradish peroxidase (HRP, cat. #77332) were purchased from Sigma Aldrich. 50 kDa MWCO cellulose ester dialysis membrane was purchased from Spectra/Por® Biotech. Dulbecco's Modified Eagle Medium–Nutrient F-12 (DMEM–F-12, 1 : 1 mixture with GlutaMax™), penicillin (pen, 5000 units per mL), streptomycin (strep, 5000 μg mL⁻¹), phosphate buffer saline (PBS, pH 7.2), fetal bovine serum (FBS), Trypsin–EDTA (10X, 0.5%) and Hank's Balanced Salt Solution 1X (HBSS, cat. #14175) were purchased from Life Technologies. Agarose (cat. #9010E) was sourced from Scientifix. Type 1 water (ISO 3696) was produced by Millipore's Milli-Q Gradient integral water purification system and used in the preparation of all reagents.

2.2. Synthesis of silica nanocapsules

PFD nanoemulsions were prepared by sonicating (Misonix Sonicator S-4000) a mixture of 12 mL of PF68 solution (5% w/v) and PFD (32% w/v) for 5 min to produce a bluish and semi-transparent solution, which indicates the formation of nano-size emulsion. The mixture was then added drop-wise into 12 mL of pre-hydrolysed TMOS (0.28 M). The mixture was stirred for 24 h before it was transferred into a 50 kDa MWCO dialysis tube and dialysed against frequently replaced deionised water for 3 days to remove excess PF68 and TMOS.

2.3. Characterisation

2.3.1. Transmission electron microscopy. Transmission Electron Microscopy (TEM) was used to characterise the morphology and size of the silica nanocapsules. Briefly, a drop of solution containing the nanocapsules was deposited onto a carbon coated copper grid and air dried. The sample was imaged with a TEM (JEOL JEM-1400) operating at an accelerating voltage of 100 kV and a beam current of 55 μA.

2.3.2. Dynamic light scattering. Dynamic light scattering (90Plus, Brookhaven) was used to determine the hydrodynamic size of the silica nanocapsule aggregates when suspended in water at pH 7. The size of the silica nanocapsules was measured over 240 min after ultrasonic dispersion for 1 min.

2.3.3. Zeta potential. Phase analysis light scattering (ZetaPALS, Brookhaven) was used to determine the zeta potential of the silica nanocapsules in a 3 mM potassium chloride solution at pH 7.

2.3.4. Nuclear magnetic resonance. Nuclear Magnetic Resonance (NMR) spectroscopy was used to quantify the retaining PFD in silica nanocapsules in water. In brief, a mixture of 71 μL silica nanocapsule suspensions in water (9.8 g L⁻¹), 559 μL of deionised water and 70 μL of D₂O was pipetted into a NMR tube to yield particles with silica concentration of 0.8 g L⁻¹ in 10% D₂O. To quantify the amount of retained PFD in the silica nanocapsules, a standard calibration curve was constructed using known amount of PFD nanoemulsion in 10% v/v D₂O. ¹⁹F-NMR spectra of the nanocapsule suspensions before the suspensions were centrifuged at 14 000 × g, and after the pellet was re-dispersed, spectra were determined using 600 MHz FT-NMR (Bruker Avance III 600 MHz).

2.3.5. Thermogravimetric analysis. Thermogravimetric analysis (Q500, TA Instruments) was used to determine the amount of PF68 in the silica nanocapsules. Briefly, dried silica nanocapsules powder was subjected to heating from room temperature up to 800 °C in air flowing at 25 mL min⁻¹. The weight percent of PF68 was calculated from the following equation
where W_{150 °C} and W_{800 °C} corresponded to the weight of the sample at 150 °C and 800 °C, respectively.

2.4. Oxygen solubility measurement

Oxygen solubility measurements were performed using a fluorescence lifetime micro oxygen monitoring system (Instech Laboratories, Inc), according to the method reported by Fraker et al.³⁹ Briefly, an ‘enzyme solution’ was prepared by dissolving 10.5 U mL⁻¹ GO and 5 U mL⁻¹ HRP in Ca²⁺ and Mg²⁺ free Dulbecco's PBS. Together, 3,5-dichloro-2-hydroxybenzenesulfonic acid and 4-aminoantipyrine were dissolved in HBSS solution to yield a final concentration of 80 and 20 g L⁻¹, respectively and this is termed as substrate solution. 10 μL of the substrate solution was added 180 μL of appropriate amount of silica nanocapsules or PFD nanoemulsion, dispersed in HBSS solution. A 190 μL aliquot of the mixture was then added into the microchamber and the oxygen partial pressure was monitored until it is stable. A 10 μL aliquot of the enzyme solution was subsequently added and the chamber was quickly sealed with an acrylic plug. The drop in oxygen partial pressure with time was recorded every 2 s inside the sealed microchamber, regulated at 25 °C. The drop in oxygen partial pressure of either the PFD nanoemulsion or the silica encapsulated PFD solution inside the sealed microchamber is due to the consumption of dissolved oxygen and glucose according to the following reactions:

The rate of oxygen decrease from 140 mmHg to 40 mmHg was normalised to that of the particles free solution, thus yielding the oxygen solubility, measured in fold base increase.

2.5. Ultrasound imaging

Ultrasound imaging was performed using Vevo® 770 (Visualsonics) equipped with a RMV 708 scan head, and operating at 55 MHz and frame rate of 46 Hz. Imaging phantoms were prepared by dispersing the silica nanocapsules in agarose gel. Briefly, silica nanocapsules with PFD concentrations ranging from 0 to 34.6% w/v were prepared by sonicating the particle with appropriate concentration for 1 min using an ultrasonic probe (Misonix Sonicator S-4000). This was followed by a 1 : 1 dilution with 1% w/v agarose solution at 80 °C. The mixture was vortexed for 5 s and poured into a plastic tray (2 cm × 3 cm). The tray was then placed in an ice bath for rapid setting, and left overnight before ultrasound imaging was carried out.

The mean gray scale ultrasound contrast intensity can be quantified as reported by Delogu et al.⁴⁰ Briefly, the average intensity (0–255 with 255 being the brightest) corresponds to the 8-bit grayscale image of the ultrasound image at the region of interest (ROI) was determined and compared against particle-free solution in agarose gel.

2.6. Cytotoxicity study of PFD encapsulated silica nanocapsules

3T3 mouse fibroblasts were cultured in DMEM/F-12 supplemented with 10% FBS, penicillin/streptomycin (1%) in 75 cm² size culture flasks. The cells were incubated at 37 °C in 95% humidity and 5% CO₂ environment and removed by treatment with Trypsin-EDTA (1X, 10%) upon 90% confluency. The cytotoxicity of silica nanocapsules towards the 3T3 mouse fibroblasts was evaluated using fluorescence spectroscopy. Briefly, 5000 cells were seeded in 96 well plates for 24 h followed by 24 h exposure to the silica nanocapsules at silica concentrations ranging from 0.008 g L⁻¹ up to 0.8 g L⁻¹. After 24 h, the particles were removed and washed twice with PBS. CellTiter-Blue® (Promega) and DMEM/F-12 at a ratio of 1 : 5 was then introduced into the 96 well plates. The 96 well plates were covered with aluminium foil and incubated at 37 °C in 95% humidity and 5% CO₂ environment for another 4 h prior to recording the fluorescence at 560_Ex/590_Em using FLUOstar Omega fluorescence plate reader.

3. Results and discussion

3.1. Effects of PF68 concentration and time on the size of PFD nanoemulsion template

The ultrasonication process used in this work was shown to produce a PFD in water emulsion with a PFD concentration of 16% w/v. Fig. 1a shows that the hydrodynamic size of nanoemulsion can be varied by altering the amount of PF68 that was used. It was found that the size of the PFD emulsion decreases with respect to the concentration of PF68 until the concentration of the latter reaches 5% w/v, whereby a minimum size of 269 nm was obtained. The reduction in size is due to the decrease in the surface tension of PFD droplets upon increasing PF68 concentration.⁴¹ Increasing the concentration of PF68 beyond this 5% w/v value results in an increase in the hydrodynamic size of PFD nanoemulsion. This can be attributed to concentration induced Ostwald ripening. Excess PF68 creates smaller PFD droplets and smaller droplets have higher Ostwald ripening rate, thus promoting the growth of nanoemulsion.⁴²

Fig. 1 (a) Hydrodynamic size of PFD nanoemulsion with increasing PF68 concentration (% w/v); (b) hydrodynamic size of nanoemulsion prepared at concentration of 2.5% w/v (◆) and 5% w/v (■) of PF68 over period of 24 h at 25 °C. In both cases, the concentration of PFD was maintained at 16% w/v. Error bars represent standard deviation of at least three repeated measurements.

Over time, the size of the nanoemulsions also increases due to Ostwald ripening.²⁵ Fig. 1b shows the kinetic stability of the PFD nanoemulsion over a period of 24 h as measured by dynamic light scattering. It was observed that PFD nanoemulsion coalesces linearly within this time frame. Furthermore, a high concentration of PF68 (5% w/v) was found to result in faster coalescence with the nanoemulsion reaching a similar size as nanoemulsion prepared from a lower concentration (2.5% w/v) of PF68 after a period of more than 10 h. The faster growth rate of the nanoemulsion at the higher PF68 concentration is evidence of the higher surface energy of smaller PFD nanoemulsion droplets which coalesces at a higher Ostwald ripening rate.

3.2. Synthesis of PFD encapsulated silica nanocapsules

3.2.1. Effects of pre-hydrolysis pH on silica nanocapsule size and morphology. The smallest PFD nanoemulsion obtained from 5% w/v PF68 was chosen in the subsequent study as the template for preparing PFD encapsulated silica nanocapsules. Fig. 2 shows the TEM images of silica nanocapsules synthesised at pre-hydrolysis pH (pH_h) values ranging from 1 to 11. The figure shows that the nanocapsules have a wrinkled surface structure which indicates the formation of a soft and thin silica shell around the PFD emulsion droplet. The addition of the PFD nanoemulsion into a solution of pre-hydrolysed TMOS resulted in the adsorption of silanol species onto the surface of the nanoemulsion through covalent bonding with PEO via an esterification (Si–O–C) reaction,⁴³ followed by condensation reaction between the adjacent silanol to form the silica shell.⁴⁴ Zeta potential measurements show an increase in the zeta potential of PFD nanoemulsion from −3 ± 12 mV to between −11 ± 9 to −21 ± 8 mV after silica encapsulation. Note that the silica nanocapsules were collected and then re-suspended in water at pH 7 prior to the size and zeta potential measurements. The negative zeta potential after silica encapsulation is due to changes in the surface environment from non-ionic PF68 loop and train conformation to a partially ionic silica-PF68 interface.

Fig. 2 TEM images showing the morphology and size of silica nanocapsules synthesised at pH (a) 1, (b) 3, (c) 5, (d) 7 and (e) 11. Broken structure of silica nanocapsules synthesised at pH 11 was also observed as indicated by the dark arrow.

It is important that the TMOS was pre-hydrolysed to create a homogenous silanol solution prior to PFD nanoemulsion addition, and the concentration of TMOS is maintained at approximately 0.141 M. Doubling the concentration of TMOS to 0.282 M was shown to result in the formation of a gel network due to the trapping of water by an excess amount of silanol species. Conversely, if the concentration of TMOS is too low, leached PFD droplets were observed after dialysis indicating that insufficient adsorption of silanol to fully encapsulate the PFD nanoemulsion. It is also important that the PFD-in-water nanoemulsion is added drop-wise into this silanol rich solution, and the solution is stirred vigorously to ensure the even distribution of the silanol species around the nanoemulsion droplets. Otherwise, inhomogeneous silica condensation will occur, resulting in a mixture of silica nanospheres and silica nanocapsules. The sizes of single silica nanocapsules were much smaller than the size of the PFD nanoemulsion when the pre-hydrolysis pH is in the range of 1 to 7 (Fig. 2).

Fig. 2 further shows the effect of pre-hydrolysis pH on the diameter of the silica nanocapsules. The size distribution of 300 or more silica nanocapsules was determined from the TEM images. At pre-hydrolysis pH of 7 and below, the average size of individual silica nanocapsules were approximately the same, varying between 41 ± 11 nm to 59 ± 16 nm. The similar size of the nanocapsules can be explained by the similar silanol condensation rate at similar final pH values of just above and below the point of zero charge (PZC) of silica, i.e. pH 2 upon mixing with PFD nanoemulsion.⁴⁵ When the pre-hydrolysis pH was increased to 11, the size of the nanocapsules doubled to approximately 117 ± 36 nm. The doubling in size was accompanied by a broader size distribution. The increase in the size of individual silica nanocapsules can be explained by a reduction in the rate of silica deposition at higher pH and the simultaneous growth of the PFD nanoemulsions due to Ostwald ripening over time as shown in Fig. 1b.⁴⁵ Due to the low deposition rate at higher pH, broken shells were frequently observed under TEM, indicating a loss of structural integrity at high pH (Fig. 2e, dark arrow). Under a highly alkaline condition (pH > 13), no silica nanocapsules formation was observed and the TEM grid contains only debris-like material. It is likely that at this high pH, silanol solution is deprotonated into SiO⁻ and H⁺, thus reduces the interaction with PF68 and the deprotonated SiO⁻ preferentially self-aggregates and that results in no silica nanocapsules formation.⁴⁶

The change in the hydrodynamic size of silica nanocapsules aggregates was also monitored over time and the result is shown in Fig. 3. The nanocapsules exhibit excellent stability for at least 240 min in water, with the exception of silica nanocapsules prepared at a pre-hydrolysis pH of 11. The excellent stability of the nanocapsules prepared at pH 1 to 7 can be attributed to both the electrostatic repulsion of silica shell and steric stabilisation from the excess PF68 adsorbed. The observed decrease in the aggregate size from 294 ± 2 to 260 ± 2 nm of nanocapsules synthesised at pH 11 suggests that aggregation and sedimentation occurs. This is due to the smaller steric stabilisation by having less adsorbed PF68 with silica nanocapsules at high pH as observed by Sarkar et al.⁴⁶ However, the silica nanocapsules were shown to be unstable over a 24 h period in cell culture media (DMEM) supplemented with 10% v/v FBS. Others have reported a similar observation with silica nanoparticles that have not been modified with polymers such as PEG and PVP.⁴⁷

Fig. 3 Aggregate size of silica nanocapsules in water over period of 240 min. The sizes of silica nanocapsules, prepared at hydrolysis pH of 1 to 7 are approximate between 230 and 250 nm during this period. Decreasing aggregate size from 294 ± 2 to 260 ± 2 nm after 240 min was observed when the pH is raised to 11 suggesting the aggregation and sedimentation of the particles due to smaller steric stabilisation by having less adsorbed PF68 (Fig. 2e). Error bars represent standard deviation of at least three repeated measurements.

3.3. Composition of silica nanocapsules

¹⁹F-NMR was used to verify the confinement of PFD within the silica nanocapsules and quantify the amount of PFD. A silica nanocapsule suspensions in 10% D₂O was first measured to identify the presence of PFD. After that, silica nanocapsules were centrifuged; the supernatant and the re-suspended pellets (in equal amount of supernatant) were analysed separately for PFD signal. Fig. 4a shows the NMR spectrum of a silica nanocapsule suspension. PFD has a three equivalents fluorine environment: (1,4,5,8), (2,3,6,7) and (9,10) designated with number 1,2 and 9 in Fig. 4. The figure also reveals that there are four doublets between −120 and −150 ppm and one doublet upfield of −180 ppm which correspond to the axial (a) and equatorial (e) fluorine environment 1 and 2, and fluorine environment 9 respectively confirming the presence of PFD. Fig. 4b shows that no PFD signal could be obtained from the supernatant, while Fig. 4c shows that the re-suspended pellet has an intensity signal that is almost equivalent to the silica nanocapsule suspensions. The NMR analysis confirms that PFD is strongly associated with the silica nanocapsules and there was no detectable PFD present in the continuous aqueous phase. Together with the TEM image observed (see Fig. 2), the result confirms the successful encapsulation of the PFD inside silica nanocapsules, and the structural integrity of the silica nanocapsules.

Fig. 4 ¹⁹F-NMR spectra at 600 MHz of (a) PFD encapsulated silica nanocapsules dispersed in D₂O, (b) the supernatant of silica nanocapsule suspensions and (c) the suspension after re-dispersion of the pellet.

For PFD quantification, a standard linear curve based on a series dilution of PFD nanoemulsion was constructed by integrating the signal intensity from NMR spectra ranging from −110 to −150 ppm. The concentration was estimated from the linear standard curve to be 17.6 ± 0.4 g L⁻¹ when dry weight of 1 g L⁻¹ silica nanocapsules after drying at 150 °C (measured after removal of PFD) was used.

To quantify the amount of PF68 within the silica shell, thermogravimetric analysis (TGA) was performed. Fig. 5a shows a derivative thermogravimetric curve of silica nanocapsules and PFD nanoemulsion that have been placed in an oven at 50 °C overnight to remove the PFD via evaporation. TGA analysis shows that dried PFD nanoemulsion has a decomposition temperature peaking at 271 °C that can be attributed to PF68. However, PFD-free silica nanocapsules have a decomposition temperature peaking at ∼177 °C which can be attributed to the oxidation of PF68 and condensation of hydrolysed silica network. The relatively lower decomposition temperature of the PF68 can be explained by the catalytic decomposition of the PF68 dispersed within silica network which suggests the formation of strong silica-PF68 interaction.^48,49

Fig. 5 (a) Derivative thermogravimetric curve of PFD nanoemulsion and silica nanocapsules. (b) TGA curve of silica nanocapsules. Samples were dried at 50 °C overnight.

Fig. 5b shows the TGA profile of the PFD-free silica nanocapsules. The amount of organic removal was estimated by measuring the weight loss of dried silica nanocapsules, when the temperature was increased from 150 to 800 °C. The result shows that dried silica nanocapsules consist of ∼63% wt of PF68, equivalent to 0.0371 mol of PF68 per mol of silica. Together with ¹⁹F-NMR study, this suggested that 78% wt of the particles consist of PFD, and the remaining mass consist of the thin silica-polymeric shell.

The high PF68 content in the silica nanocapsules and strong PF68-silica interaction suggest that PF68 plays an important role in maintaining the nanocapsule structure by intercalating with silica layer, keeping the PFD intact even under strong centrifugation of 14 000 × g.⁵⁰ When the silica nanocapsules were exposed to absolute ethanol, leakage of PFD liquid droplets was observed which can be attributed to a breakdown of PF68 loop and train structure. This further signifies the importance of PF68 in maintaining the intact of silica shell in the encapsulated PFD nanocapsules.

3.4. Oxygen solubility measurement

For the purpose of tumour cells oxygenation, the ability to carry an adequate amount of dissolved oxygen is important. We followed the method for determining oxygen content in the PFD loaded silica nanocapsules that was described by Fraker et al.³⁹ Fig. 6 shows the measured oxygen solubility of PFD containing silica nanocapsules and PFD nanoemulsion, normalised to a solution containing no PFD. The oxygen solubility, in terms of fold base increase, of silica nanocapsules and PFD nanoemulsion are similar within the error of measurement. The fold base increase is also comparable to values reported by Fraker et al.³⁹ The fact that there is no significant difference in the fold base increase of the oxygen curve between PFD nanoemulsion and silica nanocapsules suggests that the silica-PF68 shell did not affect the mass transfer of oxygen from PFD into the bulk solution.

Fig. 6 PFD containing silica nanocapsules (◆) shows a similar increase in oxygen solubility as PFD nanoemulsion (■) upon increasing the % weight of PFD encapsulated, suggesting high oxygen carrying property of silica nanocapsules. Error bars represent standard deviation of at least three repeated measurements.

3.5. In vitro assessment of silica nanocapsules’ cytotoxicity

The biocompatibility of PFD loaded silica nanocapsules was also assessed via an in vitro cytotoxicity study. Fig. 7 shows the cytotoxicity evaluation of HepG2 and 3T3 cells after 24 h exposure in vitro to different concentrations of PFD loaded silica nanocapsules. The concentration reported is with respect to the concentration of silica content in g L⁻¹, that is without considering the weight of PFD. Concentration which is above 0.8 g L⁻¹ silica nanocapsules was not considered in the study as the silica nanocapsule suspensions become too viscous and require strong sonication to produce a good dispersion of the nanocapsules in water.

Fig. 7 Cytotoxicity assessment of silica nanocapsules on 3T3 mouse fibroblast and HepG2 human hepatocarcinoma cell lines. The silica nanocapsules concentration was varied from 0.008 to 0.8 g L⁻¹. Positive effect was observed with silica nanocapsules at all concentration with HepG2 while 3T3 cells experience reduction in cell viability at the highest silica concentration of 0.8 g L⁻¹ (one-way Anova, *P < 0.05, **P < 0.01 vs. 0 g L⁻¹ silica concentration, taken from three independent experiments).

Fig. 7 shows that the silica nanocapsules did not demonstrate any cytotoxic effect towards the HepG2 cell line for all concentrations tested. Conversely, the presence of the particles increased the proliferation of the cells by 23 to 28%. Similarly, 0.08 g L⁻¹ and 0.008 g L⁻¹ of particles result in increase of cell proliferation of 23 and 19% respectively. The increase in proliferation is attributed to oxygen transfer from PFD in the silica nanocapsules to the cells.^51,52 Significant toxicity was, however, observed at the highest concentration of 0.8 g L⁻¹ which leads to a 45% reduction in viable cells. This suggests that HepG2 cells have better tolerance towards silica nanocapsules compared to 3T3 cells. A similar observation was made by Selvan et al. who found a larger reduction in cell viability in 3T3 cells than HepG2 cells upon exposure to silica coated particles.⁵³

A potential source of the toxicity at the highest silica nanocapsules loading could come from the elevated levels of exposure to PF68 as it has been shown in literature that the high PF68 concentrations (of more than 2% w/v) is toxic to some cells.⁵⁴ Based on the calculation of the composition of the silica nanocapsules samples at 0.8 g L⁻¹ exposure, the total PF68 concentration is approximately 0.4% wt. It has been observed that exposure to this level of PF68 could result in a 50% reduction in cell viability.⁵⁵ Furthermore, PF68, when used as a cell culture additive, typically does not exceed a concentration of 0.1% w/v.

3.6. In vitro assessment of ultrasound imaging in agarose gel

The ultrasound contrast enhancement of PFD loaded silica nanocapsules and PFD nanoemulsion in agarose gel was compared. Fig. 8a–d shows the ultrasound images of agarose gel containing PFD nanoemulsion or silica encapsulated PFD nanocapsules at concentrations between 0 and 10.8% w/v. Enhanced ultrasound contrast was observed with silica encapsulated PFD nanocapsules as compared to PFD nanoemulsion at the same PFD concentration of 10.8% w/v as indicated by the bright white spots (Fig. 8a and b). There is no observable difference in the ultrasound contrast produced by PFD nanoemulsion (10.8% w/v) and PFD free aqueous solution (Fig. 8b and d). This is contrary to the observation of Mattrey et al., who reported enhanced ultrasound signal for tumour imaging with Fluosol Da (20%), a similar perfluorocarbon emulsion.⁵⁶ A plausible explanation of the ultrasound enhancement observed by Mattrey et al. is due to accumulation of the nanoemulsion around the tumour cells as suggested by the transmission line model whereas in our study, the nanoemulsions are well dispersed in agarose gel.⁵⁷ The increase in ultrasound contrast was also observed as the concentration increases from 2.2 to 10.8% w/v, suggesting that the ultrasound contrast enhancement is also concentration dependent (Fig. 8a and c).

Fig. 8 In vitro contrast mode ultrasound images comparing (a) 0% w/v of PFD, (b) 10.8% w/v of PFD in nanoemulsion, (c) 2.2% w/v PFD in silica nanocapsule suspensions and (d) 10.8% w/v PFD in silica nanocapsule suspensions dispersed in agarose gel. The area of approximately 1 mm² as shown with blue circles in the images was used as the region of interest to determine the mean gray scale intensity of silica nanocapsules and PFD nanoemulsion. (e) Comparing the normalised mean gray scale intensity of PFD nanoemulsion and PFD encapsulated silica nanocapsules at various PFD concentration, measured in % w/v. Significant enhancement was observed when the PFD concentration is 5.4% w/v and above for PFD loaded silica nanocapsules (one-way ANOVA, *P < 0.05, **P < 0.01 vs. 0% w/v PFD, taken from three independent experiments). Silica nanocapsules at 5.4% w/v PFD and 10.8% w/v show better ultrasound contrast enhancement as compared to the PFD nanoemulsion of the same concentration (one-way ANOVA, ^###P < 0.05, taken from three independent experiments).

Fig. 8e compares the change in mean gray scale intensity with PFD concentration of silica nanocapsules and PFD nanoemulsion. Regardless of the PFD concentration in PFD nanoemulsions, no increase in echogenicity was observed. Moreover, no ultrasound measurement could be carried at PFD concentrations beyond this threshold concentration due to destabilisation of the PFD nanoemulsion upon attempt to concentrate the emulsion. Conversely, silica nanocapsules show significant increase in mean gray scale intensity to 43 and 45 at 5.4 and 10.8% w/v PFD respectively (P < 0.05 vs. PFD nanoemulsion). Unlike PFD nanoemulsion, further increases in PFD loading in silica nanocapsules by concentrating the particles solution did not result in destabilisation of the particles. However, this did not result in substantial ultrasound echogenicity enhancement. This is due to signal saturation, caused by increasing multiple scattering at high concentration.²¹

The two fold increase in the normalised mean gray scale intensity when silica encapsulated PFD nanocapsules were used as a contrast agent suggests that the presence of a thin silica-PF68 shell improves the ultrasound backscattering. The acoustic impedance of silica, PFD and agarose gel are reported to be 1320 × 10⁻³ kg s⁻¹ m⁻², 1.34 × 10⁶ kg s⁻¹ m⁻² and 1.52 to 1.76 × 10⁶ kg s⁻¹ m⁻² respectively.^57–60 Despite having only 14% w/v of silica content (see Fig. 5b), the large acoustic impedance mismatch between agarose gel and silica shell can result in strong ultrasound enhancement. It is also worth noting that there is no significant difference in the acoustic impedance of PFD and agarose gel, thus justifying the insignificant ultrasound backscattering as observed in Fig. 8b.

4. Conclusion

In summary, we demonstrated a facile and robust method for synthesising PFD loaded silica nanocapsules. The size of PFD nanoemulsion that serves as template can be varied by changing the PF68 concentration, thus allowing control over the size of the silica nanocapsules. The PFD loaded silica nanocapsules can also be prepared over a broad range of pH values without changing the physical properties. High PFD loading capacity was evident from TGA analysis and ¹⁹F-NMR analysis, and the silica shell is strong enough to withstand strong centrifugation. Oxygen solubility profile of the silica nanocapsules and the PFD nanoemulsion suggests that there is little difference in their respective oxygen carrying capacity and oxygen mass transfer properties. The particles are shown to be not only biocompatible with 3T3 and HepG2 cells but also promote cell proliferation under some conditions. The incorporation of silica also shows improvement in the ultrasound contrast enhancement to an almost non-echnogenic PFD nanoemulsion. Findings from this study demonstrate the significance of tuning surface shell chemistry for designing a potential theranostic contrast agent based on the silica nanocapsules containing PFD developed in this work.

Acknowledgements

The work was financially supported by Australian Research Council (ARC). The authors also acknowledge the UNSW Mark Wainwright Analytical Centre for access to the facility in obtaining TEM and ¹⁹F-NMR results.

References

1. A. X. Meng, F. Jalali, A. Cuddihy, N. Chan, R. S. Bindra, P. M. Glazer and R. G. Bristow, Radiother. Oncol., 2005, 76, 168–176.
2. A. Padhani, K. Krohn, J. Lewis and M. Alber, Eur. J. Radiol., 2007, 17, 861–872.
3. M. Yoshimura, S. Itasaka, H. Harada and M. Hiraoka, BioMed Res. Int., 2013, 2013, 13.
4. L. H. Gray, A. D. Conger, M. Ebert, S. Hornsey and O. C. A. Scott, Br. J. Radiol., 1953, 26, 638–648.
5. J. M. Brown and W. R. Wilson, Nat. Rev. Cancer, 2004, 4, 437–447.
6. D. G. Hirst, Int. J. Radiat. Oncol., Biol., Phys., 1986, 12, 1271–1277.
7. D. J. Chaplin, P. L. Olive and R. E. Durand, Cancer Res., 1987, 47, 597–601.
8. J. Han, M. Yu, M. Dai, P. Cui, H. Li, J. Zhang, Q. Liu and R. Xiu, Artif. Cells, Blood Substitutes, Biotechnol., 2008, 36, 431–438.
9. C. J. Cheng and W. M. Saltzman, Nat. Nanotechnol., 2012, 7, 346–347.
10. S. Crafoord, J. Larsson, L.-J. Hansson, J.-O. Carlsson and S. Stenkula, Acta Ophthalmol. Scand., 1995, 73, 442–445.
11. L. Konstantinidis and T. Wolfensberger, Eye, 2009, 24, 1109–1111.
12. R. T. Mahon, C. R. Auker, S. G. Bradley, A. Mendelson and A. A. Hall, Spinal Cord, 2012, 51, 188–192.
13. L. A. Forgiarini Jr, L. F. Forgiarini, D. P. da Rosa, R. Mariano, J. M. Ulbrich and C. F. Andrade, J. Surg. Res., 2013, 183, 835–840.
14. B. P. Fuhrman, in Ventilator-induced lung injury, ed. D. Dreyfuss, G. Saumon and R. Hubmayr, Taylor & Francis, New York, 1st edn, 2006, pp. 656–676.
15. A. Doctor, M. Mazzoni, U. DelBalzo and J. H. Arnold, Pediatr. Res., 1997, 41, 32.
16. L. Lindgren, C. Marshall and B. E. Marshall, Acta Physiol. Scand., 1985, 123, 335–338.
17. B. A. Teicher, T. S. Herman and S. M. Jones, Cancer Res., 1989, 49, 2693–2697.
18. M. Guichard, Radiother. Oncol., 1991, 20, 59–64.
19. Y. Zhou, Z. Wang, Y. Chen, H. Shen, Z. Luo, A. Li, Q. Wang, H. Ran, P. Li, W. Song, Z. Yang, H. Chen, Z. Wang, G. Lu and Y. Zheng, Adv. Mater., 2013, 25, 4123–4130.
20. R. Díaz-López, N. Tsapis and E. Fattal, Pharm. Res., 2010, 27, 1–16.
21. E. Pisani, N. Tsapis, B. Galaz, M. Santin, R. Berti, N. Taulier, E. Kurtisovski, O. Lucidarme, M. Ourevitch, B. T. Doan, J. C. Beloeil, B. Gillet, W. Urbach, S. L. Bridal and E. Fattal, Adv. Funct. Mater., 2008, 18, 2963–2971.
22. R. Díaz-López, N. Tsapis, D. Libong, P. Chaminade, C. Connan, M. M. Chehimi, R. Berti, N. Taulier, W. Urbach, V. Nicolas and E. Fattal, Biomaterials, 2009, 30, 1462–1472.
23. H. P. Martinez, Y. Kono, S. L. Blair, S. Sandoval, J. Wang-Rodriguez, R. F. Mattrey, A. C. Kummel and W. C. Trogler, MedChemComm, 2010, 1, 266–270.
24. J. M. Janjic, M. Srinivas, D. K. K. Kadayakkara and E. T. Ahrens, J. Am. Chem. Soc., 2008, 130, 2832–2841.
25. J. M. Janjic and E. T. Ahrens, Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol., 2009, 1, 492–501.
26. S. A. Wickline, M. Hughes, F. C. Ngo, C. S. Hall, J. N. Marsh, P. A. Brown, J. S. Allen, M. D. McLean, M. J. Scott, R. W. Fuhrhop and G. M. Lanza, Acad. Radiol., 2002, 9, S290–S293.
27. L. Mullin, R. Gessner, J. Kwan, M. Kaya, M. A. Borden and P. A. Dayton, Contrast Media Mol. Imaging, 2011, 6, 126–131.
28. X. Wang, H. Chen, Y. Chen, M. Ma, K. Zhang, F. Li, Y. Zheng, D. Zeng, Q. Wang and J. Shi, Adv. Mater., 2012, 24, 785–791.
29. N. Rapoport, K.-H. Nam, R. Gupta, Z. Gao, P. Mohan, A. Payne, N. Todd, X. Liu, T. Kim and J. Shea, J. Controlled Release, 2011, 153, 4–15.
30. F. C. Ngo, C. S. Hall, J. N. Marsh, R. W. Fuhrhop, J. S. Allen, P. Brown, M. D. McLean, M. J. Scott, S. A. Wickline and G. M. Lanza, Proc. – IEEE Ultrason. Symp., 2000, 2, 1931–1934.
31. N. Y. Rapoport, A. M. Kennedy, J. E. Shea, C. L. Scaife and K.-H. Nam, J. Controlled Release, 2009, 138, 268–276.
32. N. Reznik, R. Williams and P. N. Burns, Ultrasound Med. Biol., 2011, 37, 1271–1279.
33. G. M. Lanza, D. R. Abendschein, C. S. Hall, M. J. Scott, D. E. Scherrer, A. Houseman, J. G. Miller and S. A. Wickline, J. Am. Soc. Echocardiogr., 2000, 13, 608–614.
34. E. C. Unger, T. Porter, W. Culp, R. Labell, T. Matsunaga and R. Zutshi, Adv. Drug Delivery Rev., 2004, 56, 1291–1314.
35. H. Wu, N. G. Rognin, T. M. Krupka, L. Solorio, H. Yoshiara, G. Guenette, C. Sanders, N. Kamiyama and A. A. Exner, Ultrasound Med. Biol., 2013, 39, 2137–2146.
36. X. Zhanwen, W. Jinrui, K. Hengte, Z. Bo, Y. Xiuli, D. Zhifei and L. Jibin, Nanotechnology, 2010, 21, 145607.
37. C. Washington, S. M. King and R. K. Heenan, J. Phys. Chem., 1996, 100, 7603–7609.
38. C. Washington and S. M. King, Langmuir, 1997, 13, 4545–4550.
39. C. A. Fraker, A. J. Mendez and C. L. Stabler, J. Phys. Chem. B, 2011, 115, 10547–10552.
40. L. G. Delogu, G. Vidili, E. Venturelli, C. Ménard-Moyon, M. A. Zoroddu, G. Pilo, P. Nicolussi, C. Ligios, D. Bedognetti, F. Sgarrella, R. Manetti and A. Bianco, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 16612–16617.
41. T. M. Krupka, L. Solorio, R. E. Wilson, H. Wu, N. Azar and A. A. Exner, Mol. Pharm., 2009, 7, 49–59.
42. Y. De Smet, L. Deriemaeker and R. Finsy, Langmuir, 1999, 15, 6745–6754.
43. N. A. Alcantar, E. S. Aydil and J. N. Israelachvili, J. Biomed. Mater. Res., 2000, 51, 343–351.
44. H. Zhang, X. Wang and D. Wu, J. Colloid Interface Sci., 2010, 343, 246–255.
45. C. J. Brinker, J. Non-Cryst. Solids, 1988, 100, 31–50.
46. B. Sarkar, V. Venugopal, M. Tsianou and P. Alexandridis, Colloids Surf., A, 2013, 422, 155–164.
47. E. Izak-Nau, M. Voetz, S. Eiden, A. Duschl and V. Puntes, Part. Fibre Toxicol., 2013, 10, 56.
48. D. Zhao, Q. Huo, J. Feng, B. F. Chmelka and G. D. Stucky, J. Am. Chem. Soc., 1998, 120, 6024–6036.
49. F. Bérubé and S. Kaliaguine, Microporous Mesoporous Mater., 2008, 115, 469–479.
50. B. Tian, X. Liu, L. A. Solovyov, Z. Liu, H. Yang, Z. Zhang, S. Xie, F. Zhang, B. Tu, C. Yu, O. Terasaki and D. Zhao, J. Am. Chem. Soc., 2003, 126, 865–875.
51. S. F. Khattak, K.-S. Chin, S. R. Bhatia and S. C. Roberts, Biotechnol. Bioeng., 2007, 96, 156–166.
52. K. Chin, S. F. Khattak, S. R. Bhatia and S. C. Roberts, Biotechnol. Prog., 2008, 24, 358–366.
53. S. T. Selvan, T. T. Tan and J. Y. Ying, Adv. Mater., 2005, 17, 1620–1625.
54. C. A. Fraker, A. J. Mendez, L. Inverardi, C. Ricordi and C. L. Stabler, Colloids Surf., B, 2012, 98, 26–35.
55. R. Singh, H. H. Tonnesen, S. Kristensen and K. Berg, Photochem. Photobiol. Sci., 2013, 12, 559–575.
56. R. F. Mattrey, G. Strich, R. E. Shelton, B. B. Gosink, G. R. Leopold, T. Lee and J. Forsythe, Radiology, 1987, 163, 339–343.
57. J. N. Marsh, C. S. Hall, M. J. Scott, R. W. Fuhrhop, P. J. Gaffneyi, S. A. Wickline and G. M. Lanza, IEEE Trans. Sonics Ultrason., 2002, 49, 29–38.
58. J. Capilla, J. Olivares, M. Clement, J. Sangrador, E. Iborra and A. Devos, Frequency Control and the European Frequency and Time Forum (FCS), 2011 Joint Conference of the IEEE International, 2011.
59. H. Azhari, in Basics of Biomedical Ultrasound for Engineers, John Wiley & Sons, Inc., 2010, pp. 313–314.
60. M. O. Culjat, D. Goldenberg, P. Tewari and R. S. Singh, Ultrasound Med. Biol., 2010, 36, 861–873.

---