Nano-sized Hollow Colloidal Organosilica Nanospheres with High

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Nano-sized Hollow Colloidal Organosilica Nanospheres with High Elasticity for Contrast-enhanced Ultrasonography of Tumors Yi Wang, Kun Zhang, Yonghua Xu, and Hangrong Chen ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00779 • Publication Date (Web): 13 Dec 2017 Downloaded from http://pubs.acs.org on December 13, 2017

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ACS Biomaterials Science & Engineering

Nano-sized

Hollow

Colloidal

Organosilica

Nanospheres with High Elasticity for Contrastenhanced Ultrasonography of Tumors Yi Wang,† Kun Zhang,‡,§,*Yong-Hua Xua,†,#,* and Hang-Rong Chen§ †

The Institute of Ultrasound Engineering in Medicine, Chongqing Medical University, 1 Yi-xue-

yuan Road, Chongqing 400016, P.R. China. ‡

Department of Medical Ultrasound, Shanghai Tenth People’s Hospital, Tongji University

School of Medicine, Tongji University, 301 Yan-chang-zhong Road, Shanghai, 200072, P. R. China. §

State Key Laboratory of High Performance Ceramics and Superfine Microstructures, Shanghai

Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, P.R. China. #

Department of Imaging and Interventional Radiology, the Central Hospital of Xuhui District,

966 Huai-hai-zhong Road, Shanghai 200031, P.R. China. *E-mail: [email protected] and [email protected]

ACS Paragon Plus Environment

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ABSTRACT: Despite several reports on using silica hollow spheres as ultrasound contrast agents, they all suffer from their inherent drawbacks, e.g., poor imaging ability caused by highly rigid shell or large particles size that leads to failures of these particles in entering tumor tissues, no mesoporous channels to load other active molecules (e.g., fluorocarbons). In this report, amino groups-functionalized hollow colloidal organosilica nanospheres (HCONs) with approximately 260 nm in diameter are prepared. Depending on the thin and pure organosilica shell, the HCONs feature high elasticity beneficial for acquiring excellent ultrasonic imaging outcomes. Our in vitro experiment shows that the ultrasonic contrast increases 11 times and in vivo experiment also shows that the ultrasound imaging performance of tumor is improved for 1.5 times. More importantly, HCONs can load liquid perfluorohexane (PFH) capable of vaporizing into gas bubbles, which further enhance the ultrasonic imaging outcome. In addition, the organosilica nanospheres have a good biosafety, which is expected to become a new generation of ultrasound contrast agent. KEYWORDS:

ultrasound

contrast

agent,

mesoporous

hollow

silica

nanoparticles,

perfluorohexane, stability, high elasticity


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INTRODUCTION Ultrasound contrast agents (UCAs) have been developed for decades. Most traditional UCAs are organic microbubbles produced by mixing with saline solution.1 It has been widely documented that acoustic-sensitive liquid droplets with organic shell could be transformed into these microbubbles during ultrasound irradiation for enhancing ultrasound imaging.2 However, these organic microbubbles or droplets also suffered from some inevitable disadvantages, e.g., poor structural stability, short-lived ultrasound imaging, and potential blood occlusion risks due to their overlarge size.3 Fortunately, a number of inorganic UCAs have been developed for improving the structural stability.4,5 As a paradigm, Dai et al. engineered a unique polymorphous diagnostic agent, i.e., gold-nano-shelled microcapsules,4 and such microcapsules received an intensified ultrasound contrast imaging (UCI) outcome and significantly improved the methodologies for cancer diagnosis and therapy. However, the micro-level particle size limited its further application. Instead, hollow silica spheres have recently gained increasing attention.6-8 Compared to the traditional organic microbubbles or other inorganic UCAs, the silica hollow spheres features many advantages, e.g., excellent biocompatibility, tunable particle size and composition, facile surface modification, tunable pore volume, varied morphology, homogeneous size distribution, robust shell and superior stability.6 These merits make them become a star platform in the field of nanomedicine, typically such as drug transportation, protein loading, and biomedical imaging.6-10 Very recently, these carriers have been employed as UCAs,11-16 especially after encapsulating gas.12,15 For instance, Fe-SiO2 nano-shells with 500 nm could reflect ultrasound waves under color Doppler ultrasound mode and realize UCI of Py8119 tumor,11 and Martinez et al. reported that an effective color Doppler UCA was synthesized by encapsulating PFP gas in


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hollow silica/boron micro-shells.12 Taken all together, although these inorganic particles with good stability could be achieved at higher MI for longer duration, these silica particles suffered from some inevitable drawbacks. Firstly, no mesoporous channels mean no post-loading of liquid PFCs or drugs. Secondly, overlarge particle size (＞300 nm) determines the failure of crossing the blood vessels. Last but the important one, the silica composition was completely derived from the polymerization of TEOS and no organosilane added during polymerization, which, therefore, inevitably caused the robust rigidity of shell and endangered the UCI performance in spite of improving the stability. Therefore, mesoporous hollow silica nanoparticles with a diameter of less than 300 nm that simultaneously share excellent stability and high elasticity is desirable. In early work, we developed an ‘in-situ hydrophobic layer-protected selective etching’ method to yield hollow mesoporous silica for UCI.17 Despite the presence of mesopores, the large size (400 nm) and rigid shell obtained via the co-copolymerization of dominant TEOS and supplementary organosilane precursors (5:2) that contains a short carbon chains (3 carbon length) still needs to be addressed, since over 80 MHz of resonance frequency make the ultrasonic scattering weaker.3 In this study, a thin-walled and mesoporous hollow colloidal organosilica nanospheres (HCONs) with 260 nm in diameter and 10 nm thickness of a shell were successfully fabricated by a modified in-situ hydrophobic shell protected selective etching strategy.17 The organosilica composition in shell completely originated from the hydrolysis and polymerization of pure N-[3-(trimethoxysilyl) propyl] ethylenedi-amine (TSD), a long carbon chain-contained organosilane precursor, determining HCONs a low elastic modulus (153 MPa) via atomic force microscopy. Acting as UCAs, the elastic shell of HCONs could effectively carry out ultrasonic reflection/scattering, thereby exhibiting good ultrasound contrast-enhanced


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outcome in vitro even at high mechanical index. More importantly, the interior cavity of mesoporous could load liquid fluorocarbon due to the mesoporous channels in shell, thus the UCI performance can be expected to be further enhanced. MATERIALS AND METHODS Materials Tetraethoxysilane (TEOS, A.R), N-[3-(trimethoxysilyl) propyl] ethylenedi-amine (TSD), Perfluorohexane (PFH) ammonia solution (NH3•H2O) (25, 28%, A.R), and sodium carbonate anhydrous (Na2CO3, A.R) were attained from Shanghai Lingfeng Chemical Reagent Co.LTD. Ethyl alcohol absolute (EtOH) was acquired from Shanghai Zhenxing No.1 Chemical Plant. In all experiments of this research, we applied deionized water. Cetyltrimethyl Ammonium Bromide (CTAB) was taken from Sigma-Aldrich. Fluorescein isothiocyanate isomeric (FITC, 90%) was acquired from ACROS ORGANICS. Characterization Analysis of all samples was implemented with a JEM 2100 F electron microscope operated at 200 kV. Nitrogen adsorption–desorption isotherms of HCON and HCON-PFH were analyzed at 77 K by use of a Micromeritics Tristar 3000 analyzer. The size distributions of pores for all samples were estimated by use of adsorption isotherm branches by the BJH method. Pore volume and specific surface region were measured by utilizing BJH and BET ways, respectively. By utilizing a field-emission JEOL JSM-6700F microscope, SEM images were acquired. The ultrasonic images were acquired from Philips IU22, and their average gray values were got by means of image processing software, (SONOMATH-DICOM). Particle size distributions of all subjects with diluted concentrations were calculated by dynamic light scattering (DLS) on


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Malvern. The irritation and emanation spectra were marked on a Shimadzu RF-5301PC spectrofluorophotometer. Cell viability of HCONs was implemented on BD FACSCalibur. Synthesis of HCONs This experimental process showed that 60 ml of ethanol dissolved in 20 ml aqueous ammonia solution and then this mixture was stirred lasting 30 min. After the above process, another solution of TEOS (2 ml) in ethanol (18 ml) was put immediately into the above mentioned mixture, and then the chemical reaction between these substances proceeded for 18 min, obtaining s-SiO2. Subsequently, a solution containing TSD (2 ml) and ethanol (2 ml) was added dropwise, and another 4 h reaction was carried out, harvesting s-SiO2/h-SiO2. Afterwards, the s-SiO2/h-SiO2 nanoparticles were divided into triple and collected via high-speed centrifugation method and further re-dispersed in Na2CO3 aqueous solution (0.4 M, 50 ml). The etching process was carried out at 80 °C for 40 min. Finally, the prepared HCONs were centrifuged and rinsing for three times, and afterwards frozen in a vacuum for overnight. PFP Loading After lyophilizing, the HCONs (20 mg) were put into a plugged bottle with rubber (5 ml). The bottle was pumped into the vacuum. Then PFP (100 µl) was put into the vacuum bottle. After ultrasonic oscillating in the ice water for 2 min, PFP-loading HCONs (HCON-PFH) were oscillated in the freeze water for 2 min, and then dispersed in PBS for the following experiment. In vitro ultrasonic imaging The HCONs or HCON-PFH with a certain concentration (5 mg/mL) were added in a sealed rubber container, and then the set of equipment was immersed into a container outside a basin with distilled water, the transducer with tunable MI but fixed 8 MHz frequency was operated on the elastic rubber container packed with solution.


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In vitro cytotoxicity of HCONs We chose a 96-well plate at a density of 104 cells per well in a 100 µL volume to sow Mesenchymal stem cell line. After treatment with HCONs, Cells were continuously preserved at 37 °C for 24 h and 48 h. By using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay (MTT cell growth assay kit, Chemicon, USA) ， Cell viability was then examined. The HCON- treated cells were hatched in the MTT for 4 h, and 100 ml of DMSO was separately put into each well and continuously incubated for 30 min, and the absorbance of 570 nm was observed and recorded. To ensure the accuracy, every concentration process was repeated three times, and the results are typically stated as percentages. In vivo ultrasound imaging of samples in VX2 tumor-bearing New Zealand rabbits New Zealand white rabbits (2.5-3.0 kg) bearing VX2 liver tumor implanted in muscle of thigh were provided by Laboratory Animals Center of Chongqing Medical University. The experiment conformed by the ethics committee of University and abided by technical process for Laboratory Animals in China. Animals were kept in a state of abrosia for 24 h before experiments and their abdomen and back were given a skin preparation. After the rabbit was anesthetized and fixed, the HCONs at the dose of 15 mg was intravenously injected into six VX2 tumor-bearing New Zealand rabbit separately. Ultrasonic images before and after administrating HCONs, of which various scatterings were recorded under B fundamental imaging mode, THI mode and contrast harmonic imaging mode. Under THI mode, MI is 0.7 and the frame frequency (FR) is 15 Hz; under BFI mode, MI is 0.6 and FR is 28 Hz. In vivo distribution of HCON-PFH in main organs and VX2 tumor of New Zealand rabbit New Zealand white rabbits (2.5-3.0 kg) bearing VX2 liver tumor implanted in muscle of thigh were provided by Laboratory Animals Center of Chongqing Medical University. The


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experiment conformed by the ethics committee of University and abided by technical process for Laboratory Animals in China. After the rabbit was anesthetized and fixed, the HCONs at the dose of 15 mg was intravenously injected into six VX2 tumor-bearing New Zealand rabbit separately. At different time intervals (12 h and 24 h), rabbits were euthanasia via injecting excessive anesthetic (5% pentobarbital). Main organs and tumors were taken out after dissection and weighted to measure the concentration distribution of HCONs. Each organ was dissolved by the mixture of HNO3 and HClO4 (3:1 in volume ratio) at 120 ℃. Then, inductively coupled plasma-atom emission spectrometry (ICP-OES, Agilent) was applied to measure the Si concentration. RESULTS and DISCUSSION Synthesis Process and Characterization of HCONs The synthetic procedure of HCONs is indicated in Figure 1 and they can be obtained by using a modified selective etching protocol.17 The principle of this protocol lies in the fact that the organic carbon chains in shell will spontaneously form hydrophobic layers that protect shell from etching by alkaline solution. In principle, Si-O-Si bonds of the silica shell should be weaker than those of solid silica cores. This intriguing phenomenon could lead to lower extending wavenumber in shells than that in inner cores, which suggests the Si-O-Si bonds of the outer shell, in theory, are more susceptible to alkaline etching agents comparative to inner cores and will break when they are subjected to initial etching. Actually, we found that solid silica inner cores were more susceptible and more easily corrosive than the outer shells. Thus, we speculated that there could be some protective mechanisms, which prevented the outer shell from prior erosion by the alkaline solution. Therefore, the in-situ hydrophobic shell protected selective etching strategy was rationally used to yield HCONs.


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s-SiO2

s-SiO2/h-SiO2

HCON

Figure 1. Synthesis and structure evolution of HCONs. Schematic diagram of preparation on how to prepare HCONs, wherein step 1 aims at producing s-SiO2/h-SiO2 nanoparticle, and step 2 is the etching process available for producing HCONs. In detail, solid silica spheres were firstly fabricated by the hydrolysis of tetraethoxysilane (denoted TEOS) dissolved in alkaline mixed solution consisting of ethanol and water using the typical Stöber method,18 wherein ammonia served as the catalyst. Subsequently, the solid silica spheres were coated by an organosilica shell, prepared by the hydrolysis and condensation of TSD that is long chains-contained organic silane coupling agent (SCA). Ultimately, the alkaline etching agent, i.e., Na2CO3, was used to completely remove the interior solid silica cores, leaving the mesoporous HOCNs. We control the particle size, coating thickness, porosity and the amount of HCONs via varying synthetic parameters.17 Depending on the presence of mesoporous channels, post-loading of liquid PFCs or drugs is feasible. The organosilica shell hydrolyzed from TSD containing long chains enables the high backscattering, which will benefit the contrast-enhanced ultrasound imaging.


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Transmission electron microscopy (TEM) images (Figure 2a,) of HCONs show that the average size of HCONs is around 260 nm in diameter, and their shell is 10 nm in thickness. In particular, the particle size of HCONs can be adjusted to 500 nm when varying the pH and precursor amount, as shown in Figure S1. In addition, TEM images further indicate that these particles are hollow. In the SEM images (Figure 2b), the particles share a well-defined spherical morphology and display a uniform and mono-dispersed distribution. According to dynamic light scattering (DLS) data (Figure 2c), HCONs have exhibited a narrow size distribution with an average hydrated diameter of 260 nm. In Figure 2a, disordered pores are observed in HCONs, which is further demonstrated by N2 adsorption-desorption techniques and SEM imaging. According to the N2 adsorption-desorption isotherms of HCONs, the BET surface area of HCONs is 281 m2/g. In addition, N2 adsorption isotherm is a common and effective technique that can determine the pore diameter of mesoporous material. From N2 adsorption-desorption isotherms (Figure 2d, e), when relative pressure of P/Po is greater than 0.8, an exact hysteresis loop can be noticed which corresponds to the most likely peak pore size of 20 nm. As shown in Figure S2, there is an evident characteristic stretching vibration peak of -NH2 at 2250 cm-1 in the UV-vis absorbance window, suggesting the probable positively-charged zeta potential of HCONs. DLS method was monitored to test it. As expected, the potential of HCONs is measured to be 37.5 mV, as exhibited in Figure S3, which is indeed consistent with the result that -NH2 carries positive charges in the surface of HCONs.17 The protective layer for the Si-O-Si framework in organosilica shell is the hydrophobic, which can effectively withstand the attack by the alkaline solution (i.e., Na2CO3 solution). As a comparison, the solid silica inner cores failed to generate hydrophobic coatings or polymers, since they were easily eroded by alkaline solution. The presence of hydrogen bonding can make


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the hydrophobic layer more compact and withstand the erosion from Na2CO3 solution, as shown in Figure 2f.

Figure 2. Structure characterization of HCONs. (a) TEM image of HCONs; (b) SEM image of HCONs; (c) Size distribution of HCONs obtained by measuring the sizes of 100 nanoparticles in HCON TEM images; (d) corresponding pore size distribution; (e) the N2 adsorption– desorption isotherms of HCONs; (f) an integral HCON. In vitro Ultrasound Imaging In the past several decades, considerable advance has been made in investigating how to enhance imaging outcome of inorganic UCAs, mainly referring to the highly-biocompatible hollow and/or solid silica nanoparticles.3,11 However, the absence of mesopores in the outer shells limited their utilization in loading and carrying drug, protein/enzyme, and biomedical imaging,8-10 and overlarge (＞300 nm) determined the dominant blood imaging of these types of hollows silica spheres.19 More significantly, the robust rigidity remains an unresolved problem. In contrast, the elastic modulus of HCONs in this report is 153 MPa obtained according to the Hertzian contact model via the atomic force microscopy,20,21 and this value is much lower than that (above 2 GPa) of hollow silica nano-spheres in previous report wherein hollow silica nano-


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spheres were obtained via the hydrolysis and condensation of pure TEOS.21 Therefore, excellent UCI using HCONs as UCAs is expected.

Figure 3. Structural superiority of HCON in intensifying US imaging. (a) Ultrasound image using degassed water; (b) ultrasound image using HCONs as UCAs; (c) the average gray value obtained from degassed water and HCONs; Notes ‘***’ represents p ＜ 0.001. (d) The preliminary scattering signal spectra of degassed water control, HHSC. It is well accepted that B fundamental imaging (BFI) mode can directly reflect the ultrasound imaging ability of UCAs,3 since it directly receives the backscattering signal from UCAs. The in vitro UCI using HCONs as UCAs was carried out under BFI mode. Before operation, the adsorbed gas and encapsulated gas in hollow cavity of HOCNs were removed by ultra-sonication for 30 min so as to exclude the influence of gas on UCI. As a contrast, degassed


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water was used as the control group. As can be found in Figure 3a, b, the contrast HCONs as UCAs are significantly improved in comparison to that using degassed water. Accordingly, the average calculated gray values (Figure 3c) increase from 5 corresponding to degassed water to 54 corresponding to HCONs, exhibiting more than 10-fold increase in gray value. These results sufficiently suggest the occurrence of backscattering by HCONs. To visually validate it, scattering measurements of HCONs and degassed water were carried out, wherein the emitting transducer simultaneously served as the receiving transducer of scattered signals. It is clearly found that HCONs indeed reflected more US waves than degassed water, as shown in Figure 3d. Ultrasonic Imaging under Different Mechanical Index (MI) Values Despite accommodating high elasticity, excellent structure stability is also necessary, especially on the occasion of applying high MI that is also within the safe window of clinical application, since detection for different tissues needs different MI.22 In this report, the influence of different MI values on UCI was evaluated. It is found that as the MI value increase, the contrast and average gray value of ultrasound contrast image accordingly increase, exhibiting an approximate linear relationship between MI value and average grey value, as demonstrated in Figure 4a-f. To evaluate it stability, the clinical UCAs (SonoVue) were employed as the contrast group (Figure 4f). SonoVue was a sulfur hexafluoride microbubble that was encapsulated by a phospholipid. When MI is larger than 1.2, SonoVue was susceptible to mechanical forces and subjected to collapse.23 In contrast, HCONs can withstand the mechanical forces when MI is above 1.2 (Figure 4f), suggesting the structure stability of HCONs satisfies the clinical demand. Even though, the MI value increases to 1.9, the backscattering signal still exists, meaning the application field of HCONs as UCAs can be further broadened. This excellent ultrasound


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contrast imaging of HCONs is probably attributed to the high elasticity and high stability caused by the special organic/inorganic hybrid composition deriving from condensation of TSD, which holds great promise in future ultrasonic diagnosis of tumor.

Figure 4. Ultrasonic imaging under different MI values. (a-d) Ultrasonic images applying HCONs (10 mg/ml) as UCAs got under different MIs: 0.6, 0.8, 1.0, 1.6, respectively; (e) the average gray scale values of a-d at varied MIs; (f) Image uses a MI scale to illustrate the insonation-pressure amplitude range for contrast enhancement. The enhancement of MI range is clarified by the bracketed region for the MI at which microbubble blasting prevails over highlighted area (shaded area): lipid capsules MI>1.0; HCON MI>1.5. In vivo Ultrasound Contrast Imaging The mesoporous channels in their organosilica shell could endow HCONs with the ability of encapsulating and continuously releasing guest molecules, since mesoporous silica nanoparticles (MSN) have been extensively investigated in biomedicines.7-10 Herein, we employed HCONs as the carriers to load a biocompatible compound, liquid perfluorohexane (PFH) featuring temperature-sensitive evaporation, denoted as HCON-PFH. Such a UCA combines the merits of HCONs (e.g., high stability, tunable particle sizes and facile surface modification, sustained release, robust shell, varied morphologies) and PFH (optimal liquid-gas


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boiling point and good biocompatibility).24 PFH is expected to enhance UCI via the acoustic droplets vaporization (ADV) process, as stated in many reports.2,25 It is clearly observed that no bubbles were observed in bright-field confocal microscopic image of HCONs (Figure 5a) upon exposure to ultrasound irradiation. In contrast a large number of micro-sized bubbles (Figure 5b) are observed in the group of HCON-PFH upon exposure to ultrasound irradiation, suggesting occurrence of the liquid-gas phase transition of PFH in HCON-PFH nano-capsules via the ADV process. Therefore, depending on the special ADV process and subsequent PFH bubbles, HCON-PFH achieves a larger enhancement under BFI mode in contrast and average gray value in comparison to HCONs alone due to the presence of larger scattering interface caused by the generated PFH bubbles, as demonstrated in Figure 5c-e. This result also enables the highlyeffective in vivo ultrasound imaging using HCON-PFH as UCAs.

Figure 5. Characterization of HCON-PFH nanoparticles. (a) Confocal microscopic image of HCONs; (b) Confocal microscopic image of HCON-PFH under heating condition; (c) Ultrasound image using HCON; (d) Ultrasound image using HCON-PFH; (e) the average gray value obtained from HCON and HCON-PFH, Notes ‘***’ represents p＜0.001.


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Before in vivo ultrasound imaging, the cytotoxicity of the HCON-PFH was evaluated on mesenchymal stem cells using methyl thiazolyl tetrazolium (MTT) assay. The cells without incubation with HCON-PFH were used as the control group. It is fund that the cell viability remained above 98% even at the concentration of 1000 µg/mL when the incubation time is 24 h. Especially after incubation for over 48 h, the viability is still above 96% (Figure S4). Even at a high concentration of 1000 µg/mL (Figure S4), no significant cytotoxicity of HCON-PFH was observed for mesenchymal stem cells, proving a good biocompatibility of HCON-PFH. Subsequently, the bio-distribution of HCON-PFH in main organs (heat, liver, spleen, lung, kidney) and VX2 tumors of New Zealand white rabbits were evaluated, because sufficient accumulation of HCON-PFH is a guarantee for acquiring excellent in vivo UCI. To study the bio-distribution of HCON-PFH in the body, the quantification of the Si content was investigated as the detection barker of HCON-PFH via the ICP-AES method. After injecting HCON-PFH into rabbits through their ear veins, the main organs and tumor were harvested after 12 h and 24 h post-incubation. The liver and spleen show the highest Si accumulation at both 12 h and 24 h among all organs, as shown in Figure S5. The more accumulation in the liver and spleen could be considered related to the clearance of HCONs from the blood by cells of the mononuclear phagocytic system. The heart and lung (black and magenta boxes) exhibited the lower uptake of Si at 12 h, and their uptake slightly decreased at 24 h, demonstrating that HCONs were removed from heart and lung. In particular, depending on the positively-charged amino groups in HCONs platform, the accumulation of Si in tumor are also sufficient, since positive charge-modified nanoparticles could be efficiently uptake by tumor cells,26 which will benefit the in vivo UCI of in-situ VX2 tumor implanted on.


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Figure 6. In vivo ultrasonic imaging of HCON and HCON-PFH nanoparticles. (a, b) In vivo ultrasonic images of VX2 liver tumor in the rabbit model under BFI mode before (a) and after (b) administrating HCON particles; (d, e) In vivo ultrasonic images of VX2 liver tumor in rabbit model under THI mode before (d) and after (e) administrating HCON particles; (c, f) Enhanced average gray values of before and after administrating HCON particles under BFI, THI modes in regions of interest (circled by dotted ellipse). (g-h) In vivo ultrasonic images of VX2 liver tumor in the rabbit model under BFI mode before (g) and after (h) administrating HCON-PFH particles; (j, k) In vivo ultrasonic images of VX2 liver tumor in rabbit model under THI mode before (j) and after (k) administrating HCON-PFH particles; (i, l) Enhanced average gray values before and after administrating HCON-PFH particles. Notes ‘**’ and ‘***’ represent p＜0.005 and 0.001, respectively.


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In vivo ultrasonic images of the transplanted VX2 tumor implanted in muscle of rabbits were obtained under different imaging modes, as shown in Figure 6. Relying on the synergistic effect of HCONs and PFH in enhancing UCI and sufficient accumulation in tumor, we observed a remarkable improvement in average gray scale value at the site of VX2 tumor after injecting HCON-PFH nanoparticles under the BFI mode (Figure 6g, h). For a comparison, we observed an unconspicuous improvement in average gray scale value after injecting HCON nanoparticles under the BFI mode (Figure 6a, b). The quantitative evaluation method also indicated that the average gray value increases from 30 to 50 (Figure 6i) after injecting HCON-PFH, while that from 28 to 36 (Figure 6c) after injecting HCON. Additionally, under tissue harmonic imaging (THI) mode,27,28 the increase of contrast and average gray value was more obvious after injecting HCON-PFH (Figure 6j-l) compared to injecting HCON (Figure 6d-f) in in vivo evaluations. This phenomenon probably resulted from the movement of HCON nanoparticles actuated under sound pressure and the variation of sound velocity.3.29 This result directly suggests HCON-PFH can perform as a potential UCA for tumor detection. CONCLUSIONS In summary, HCONs with a particle of less than 300 nm in diameter have been successfully obtained via a modified in-situ hydrophobic shell protected selective etching strategy. The asprepared HCONs shared high elasticity and robust stability that enable high-efficient and longlived UCI even at high MI due to the organic/inorganic hybrid framework caused by condensation of pure organosilane, namely TSD. More importantly, the HCON can be used to load liquid PFH, leading to a significant increase in contrast and gray value of ultrasonic images due to PFH evaporation via the ADV process. In addition, HCON-PFH exhibited a sufficient accumulation in VX2 liver tumor due to its positively-charged surface, which makes HCON-


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PFH perform well in in vivo UCI for VX2 tumor. Therefore, such special UCAs hold great potential in future tumor diagnosis. ASSOCIATED CONTENT Supporting Information. Supplementary figures from Figure S1 to Figure S5, including TEM image of HCONs, UV-vis absorption spectra of HCONs, The size distribution and Zeta potential of positively charged HCONs, Cell viability analysis of mesenchymal stem cells hatched with HCON-PFH at different concentration for 24 and 48 h, and In vivo Si content distributions in different organs of rabbits bearing VX2 tumor after administrating HCONs for 12 h and 24 h, respectively

AUTHOR INFORMATION ORCID Kun Zhang: 0000-0002-8709-1800 Yong-hua Xu: 0000-0002-3632-8900 Author Contributions Y. W, K. Z, Y. X designed the experiments and wrote the main manuscript text and Y. W, K. Z prepared all figures. Y.W, K. Z and Y. Z performed the experiments. All authors reviewed the manuscript. Funding Sources


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This work was supported by grants from National Natural Science Foundation of China (Grant No. 81771836 and 81501473), and grants from Guangxi Collaborative Innovation Center of Biomedicine (Grant No. GCICB-SR-201703). Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors especially thank State Key Laboratory of High Performance Ceramics and Superfine Microstructures for providing the experimental support. This work was supported by grants from National Natural Science Foundation of China (Grant No. 81771836 and 81501473), and grants from Guangxi Collaborative Innovation Center of Biomedicine (Grant No. GCICB-SR-201703). ABBREVIATIONS UCAs, Ultrasound contrast agents; UCI, Ultrasound contrast imaging; HCONs, mesoporous hollow colloidal organosilica nano-spheres; TSD, N-[3-(trimethoxysilyl) propyl] ethylenediamine; TEOS, Tetraethoxysilane; PFH, Perfluorohexane; NH3•H2O, ammonia solution; Na2CO3, sodium carbonate anhydrous; EtOH, Ethyl alcohol absolute; CTAB, Cetyltrimethyl Ammonium Bromide; FITC, Fluorescein isothiocyanate isomeric; DLS, dynamic light scattering; FR, frame frequency; SCA, silane coupling agent; TEM, Transmission electron microscopy; BFI, B fundamental imaging; MI, Mechanical Index; MSN, mesoporous silica nanoparticles; ADV, acoustic droplets vaporization; MTT, methyl thiazolyl tetrazolium. REFERENCES (1) Klibanov, A. L.; Rasche, P. T.; Hughes, M. S.; Wojdyla, J. K.; Galen, K. P.; Wible, J. H.; Brandenburger, G. H. Detection of individual microbubbles of ultrasound contrast agents:


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For Table of Contents Use Only Nano-sized Hollow Colloidal organosilica Nanospheres with High Elasticity for Contrastenhanced Ultrasonography of Tumors Yi Wang,† Kun Zhang,‡, §*Yong-Hua Xua,†, # * and Hang-Rong Chen§


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Figure 1. Synthesis and structure evolution of HCONs. Schematic diagram of preparation on how to prepare HCONs, wherein step 1 aims at producing s-SiO2/h-SiO2 nanoparticle, and step 2 is the etching process available for producing HCONs. 390x189mm (150 x 150 DPI)



Figure 2 Structure characterization. (a) TEM image of HCONs; (b) SEM image of HCONs; (c) Size distribution of HCONs obtained by measuring the sizes of 100 nanoparticles in HCON TEM images; (d) corresponding pore size distribution; (e) the N2 adsorption–desorption isotherms of HCONs; (f) an integral HCON. 481x226mm (150 x 150 DPI)


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Figure 3 Structural superiority of HCON in intensifying US imaging. (a) Ultrasound image using degassed water; (b) ultrasound image using HCONs as UCAs; (c) the average gray value obtained from degassed water and HCONs; Notes ‘***’ represents p＜0.001. (d) The preliminary scattering signal spectra of degassed water control, HHSC. 312x241mm (150 x 150 DPI)



Figure 4 Ultrasonic imaging under different MI values. (a-d) Ultrasonic images applying HCONs (10 mg/ml ) as UCAs got under different MIs: 0.6, 0.8, 1.0, 1.6, respectively; (e) the average gray scale values of a-d at varied MIs; (f) Image uses a MI scale to illustrate the insonation-pressure amplitude range for contrast enhancement. The enhancement of MI range is clarified by the bracketed region for the MI at which microbubble blasting prevails over highlighted area (shaded area): lipid capsules MI>1.0; HCON MI>1.5. 360x181mm (150 x 150 DPI)


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Figure 5 Characterization of HCON-PFH nanoparticles. (a) Confocal microscopic image of HCONs; (b) Confocal microscopic image of HCON-PFH under heating condition; (c) Ultrasound image using HCON; (d) Ultrasound image using HCON-PFH; (e) the average gray value obtained from HCON and HCON-PFH, Notes ‘***’ represents p＜0.001. 464x234mm (150 x 150 DPI)



Figure 6. In vivo ultrasonic imaging of HCON and HCON-PFH nanoparticles. (a, b) In vivo ultrasonic images of VX2 liver tumor in the rabbit model under BFI mode before (a) and after (b) administrating HCON particles; (d, e) In vivo ultrasonic images of VX2 liver tumor in rabbit model under THI mode before (d) and after (e) administrating HCON particles; (c, f) Enhanced average gray values of before and after administrating HCON particles under BFI, THI modes in regions of interest (circled by dotted ellipse). (g-h) In vivo ultrasonic images of VX2 liver tumor in the rabbit model under BFI mode before (g) and after (h) administrating HCON-PFH particles; (j, k) In vivo ultrasonic images of VX2 liver tumor in rabbit model under THI mode before (j) and after (k) administrating HCON-PFH particles; (i, l) Enhanced average gray values before and after administrating HCON-PFH particles. Notes ‘**’ and ‘***’ represent p＜0.005 and 0.001, respectively.

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Nano-sized Hollow Colloidal Organosilica Nanospheres with High

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