Nano Air Seeds Trapped in Mesoporous Janus Nanoparticles

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Nano-Air Seeds Trapped in Mesoporous Janus Nanoparticles Facilitate Cavitation and Enhance Ultrasound Imaging Konstantin Tamarov, Andrey Sviridov, Wujun Xu, Markus Malo, Valery Andreev, Victor Yu. Timoshenko, and Vesa-Pekka Lehto ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11007 • Publication Date (Web): 18 Sep 2017 Downloaded from http://pubs.acs.org on September 19, 2017

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ACS Applied Materials & Interfaces

Nano-Air Seeds Trapped in Mesoporous Janus Nanoparticles Facilitate Cavitation and Enhance Ultrasound Imaging Konstantin Tamarov†§‡, Andrey Sviridov†‡, Wujun Xu*§, Markus Malo§, Valery Andreev†, Victor Timoshenko†, and Vesa-Pekka Lehto§ †M.V. Lomonosov Moscow State University, Faculty of Physics, 119991 Moscow, Russia §University of Eastern Finland, Department of Applied Physics, 70211 Kuopio, Finland

Keywords: porous silicon nanoparticles, Janus nanoparticles, ultrasound contrast agents, selective modification, hydrophobic hydrophilic

Abstract

The current contrast agents utilized in ultrasound (US) imaging are based on microbubbles which, however, suffer from a short lifetime in the systemic circulation. The present study introduces a new type of contrast agent for US imaging based on bioresorbable Janus nanoparticles (NPs) that are able to generate microbubbles in situ under US radiation for

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extended time. The Janus NPs are based on porous silicon (PSi) that was modified via a nanostopper technique. The technique was exploited to prepare PSi NPs which had hydrophobic pore walls (inner face) while the external surfaces of the NPs (outer face) were hydrophilic. As a consequence, when dispersed in an aqueous solution, the Janus NPs contained substantial amount of air trapped in their nanopores. The specific experimental setup was developed to prove that these nano-air seeds were indeed acting as nuclei for microbubble growth during US radiation. Using the setup, the cavitation thresholds of the Janus NPs were compared to their completely hydrophilic counterparts by detecting the subharmonic signals from the microbubbles. These experiments and the numerical simulations of the bubble dynamics demonstrated that the Janus NPs generated microbubbles with a radii of 1.1 µm. Furthermore, the microbubbles generated by the NPs were detected with a conventional medical ultrasound imaging device. Long systemic circulation time was ensured by grafting the NPs with two different PEG polymers, which did not affect adversely the microbubble generation. The present findings represent an important landmark in the development of ultrasound contrast agents which possess the properties for both diagnostics and therapy.

1 Introduction In traditional medical sonography, contrast enhanced ultrasound (CEUS) employs a contrast enhancing medium to visualize poorly resolvable tissues1 and tissue perfusion2,3. Commercially available contrast agents (CAs) are highly echogenic protein4 or lipid5 stabilized microbubbles (MBs) for systemic administration. MBs have been used to assist thermal ablation6–8, drug delivery9,10, gene therapy11,12, sonothrombolysis13,14 and molecular imaging15–17. However,


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although MBs can significantly enhance the signal-to-noise ratio, their systemic circulation lifetime is very limited, being merely a few minutes18,19 due to the large size and poor stability. The large size and low adhesion of MBs limit their penetration through the epithelial cells of the vasculature to target tissues20. After their intravenous injection, MBs ultimately become trapped in the lungs, where gas exchange occurs21. These issues impede the usage of current MBs as theranostic agents. Consequently, there is a clear need for nanoscale CAs which have long circulating times. Several approaches have been proposed to overcome these limitations. In the first approach, liposomes22–24 and nanobubbles25–27 (NBs) containing nanosized gas were fabricated to address the size issue. However, when the size of the particles was decreased, the Laplace pressure increased significantly resulting in much lower echogenicity and stability26. For example, Huynh et al. observed low contrast enhancement after the fracturing of MBs into NBs28. In another approach, CO2 MBs were produced due to the chemical reaction between nanoparticles (NPs) and a slightly acidic tumor microenvironment29–31. In the third approach, NBs were encapsulated as gas precursors; these grew into MBs in response to physical stimulus, such as high temperature32,33 or high intensity focused ultrasound (HIFU)34,35. However, the most promising candidates are the perfluorocarbon nanodroplets36 that are stable and small enough, and can additionally be loaded with drugs37–39. Unfortunately, because of remarkably high vaporization pressure at nanoscale, the transition of droplets into MBs requires HIFU which means that the safety requirements for US imaging are not satisfied. A few attempts have been made to develop CAs that encapsulate nanoportions of air, which act as nuclei for MB growth and contrast enhancement. Yildrim et al.40 fabricated hydrophobic mesoporous silica NPs coated with a polymer to preserve air inside the pores. Although the NPs


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were stable for a long time in water, HIFU was needed to achieve contrast enhancement. The utilization of HIFU corresponded to the mechanical index (MI) of 9.4, which is far beyond the imaging safety requirements (MI < 1.9). Jin et al. prepared superhydrophobic silica NPs which achieved a safe level of MI in diagnostic US41. However, due to the hydrophobic outer surface, the NPs aggregated rapidly in biologically relevant liquids and the contrast enhancement disappeared after 10 min. These studies mainly focused on the development of the NPs assuming that the contrast would be enhanced due to the generation of MBs, but it would be difficult to verify their presence using the B-scans obtained by clinical equipment. Furthermore, the existence of MBs, their dynamics and formation requirements still remain poorly investigated. Recently, several methods for selective modification were introduced for controllable engineering of the inner pore walls and outer surfaces of mesoporous silica42,43 and silicon materials44,45. Such approaches make it possible to prepare NPs with two distinct surfaces, for example, with hydrophobic pores that can trap nano-seeds of air inside the pores and hydrophilic outer surface. The present study aimed to develop a new hydrophilic-hydrophobic CA and investigate microbubble dynamics both experimentally through the detection of cavitation and theoretically by conducting numerical simulation. The CA is based on Janus porous silicon (J-PSi) NPs, which were selectively modified45 to have hydrophobic inner pore surfaces, while the external surfaces were oxidized to become hydrophilic. Additionally, the NPs were conjugated with two different polyethylene glycol (PEG) polymers to secure a prolonged blood circulation time46. The specific experimental setup was developed to prove that the air inside the pores of PSi NPs acted as nuclei for microbubble growth and lowered the cavitation threshold, i.e. the cavitating bubbles are responsible for the contrast enhancement. Furthermore, the numerical simulation of


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an air bubble dynamics was applied to reveal the bubble sizes involved in the cavitation process. Thus, the results of this work can become as a foundation for the design and development of next generation CAs for US diagnostics and therapy.

2 Experimental section 2.1 Materials Si wafers (diameter 20 cm, p+ (100), 0.01-0.02 Ω·cm, Okmetic Inc.), ethanol (EtOH, absolute, Altia Oyj), ammonium hydroxide (NH4OH, 28%, VWR), hydrogen peroxide (H2O2, > 30% w/v, Fisher Scientific), n-hexane (≥ 99%, Merck), toluene (anhydrous, 99.8%, Alfa Aesar), hydrofluoric acid (38%, Merck), 0.5 kDa methoxy-PEG-silane (90%, 0.5 kDa mPEG, Fluorochem Ltd.), 2 kDa methoxy-PEG-silane (2 kDa mPEG, Laysan Bio Inc.) were used as received. 2.2 Preparation of J-PSi NPs First, the free-standing PSi (pore size ~10 nm) films were prepared by anodizing p+-type silicon wafers (100) with the resistivity of 0.01-0.02 Ω·cm in a HF (38%)-ethanol mixture47. After drying at 65 °C for 1 h, the obtained films were ball milled in ethanol to produce PSi NPs. In order to prepare J-PSi NPs, hexane was used as a nanostopper45 to protect hydrogen terminated (hydrophobic) pore walls that were the result of etching. Second, the unprotected outer surface was selectively oxidized in NH4OH:H2O2(30%):H2O 1:1:6 at RT for 15 minutes and in HCl:H2O2(30%):H2O 1:1:6 for 15 minutes, subsequently. This is possible since hexane is a nonpolar solvent and does not mix with aqueous solutions. Next, PSi NPs were washed in water and stored in absolute ethanol. Finally, dual PEGylation48 of J-PSi NPs was applied to improve their biocompatibility and blood circulation time46. Briefly, 50 µl 0.5 kDa methoxy-


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PEG-silane and 50 mg of 2 kDa methoxy-PEG-silane were dissolved in 2 ml of anhydrous toluene, and 5 mg of J-PSi NPs was added to the solution. The dispersion was sonicated for 3 min and bubbled with nitrogen (N2) gas for 20 min to deoxygenate the dispersion. The dispersion was heated overnight (∼18 h) at 120oC under reflux. At the end of the reaction, toluene was evaporated and the PEGylated J-PSi NPs (denoted as PEG-J-PSi NPs) were rinsed in ethanol for 3 times with 5–10 min sonication to remove any physically adsorbed PEG. As a reference sample, fully hydrophilic nanoparticles were prepared through non-selective oxidization of PSi nanoparticles. The oxidized particles without and with PEG coating were prepared as described previously48 and are denoted as O-PSi and PEG-O-PSi, respectively. 2.3 Characterization of PSi NPs The surface composition at each step of the surface modification was verified with FTIR (Thermo Nicolet Nexus 8700) measurements of KBr tablets containing the PSi NPs in the transmittance geometry. The pore sizes and pore volumes were determined with N2 adsorption/desorption (Micrometrics Tristar II). The morphology of PSi NPs was imaged with transmission electron microscopy (TEM) in a JEOL JEM-2100F microscope. Dynamic light scattering (DLS, Malvern Instruments Zetasizer Nano ZS) was used to measure the zeta potentials and the hydrodynamic diameters of the NPs samples in different aqueous solutions. The dilute suspensions (< 0.2 mg/ml) were equilibrated at 25°C for 5 min before the measurements. The PEG content of PSi NPs was evaluated with thermogravimetry (TG) (Q50 TGA, TA Instruments). TG measurements were performed under N2 flow of 200 ml/min. The samples were first heated from ambient temperature up to 80°C and equilibrated for 20 min to remove water. The samples were then heated to 700°C at a heating rate of 20°C/min. 2.4 Colloidal stability of PSi NPs


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Before the colloidal stability studies, the PSi NPs were rinsed twice with distilled water. To evaluate the long term stability of the particles in vitro, NPs were suspended in a mixture of blood serum and phosphate buffered saline (PBS, pH 7.0) in the proportion of 1:1 and incubated at 37oC. The colloidal stability was monitored with DLS by measuring the average particle size at different time points. 2.5 Detection of cavitation Cavitating air bubbles can be detected by the presence of the subharmonic signal in the spectrum obtained from a hydrophone49. A specific setup was developed for the initiation and detection of cavitation in the suspensions of PSi NPs. The core part of the setup consisted of a cylindrical sample chamber with two US transparent windows located between a flat transducer and a hydrophone immersed into the water tank (Figure 1). The external diameter of the chamber was 25 mm and the length was 30 mm. The diameter of the windows and the inner diameter of the chamber was smaller resulting in a volume of 14.7 ml. The distance between the transducer and the center of chamber was 30 mm; the distance from the center of the chamber to the hydrophone was 65 mm. In order to specifically amplify the subharmonic signal, the central frequency of the hydrophone was half (1.04 MHz) of the central frequency of the transducer (2.08 MHz). The transducer was connected to an US amplifier, which amplified the sinusoidal signal from a generator (Tektronix AFG 3021B). An oscilloscope (Tektronix TDS 3032B) averaged every 32 samples of the signal received from the hydrophone and performed fast Fourier transform (FFT) to obtain the signal spectrum. The generator and oscilloscope were connected to a PC with a custom NI LabVIEW 2013 (National Instruments Corp., Austin, TX) TM

program to control the experiment. The cavitation thresholds were measured by detecting the growth of subharmonic amplitude, which was extracted from the spectrum along with the


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fundamental and second harmonics. The voltage on the transducer was increased stepwise to provide a change of acoustic pressure in the range of 0.1 – 0.45 MPa (Figure S1, Suppl. Inf.). At each pressure step, the harmonics were measured for 30 seconds and the average values for this period were calculated to obtain the mean amplitude. The pause after each pressure step was 15 seconds. The presence of air bubbles was considered to be detected when the subharmonic amplitude was at least twice the level of the background noise level.

Figure 1. The experimental setup used for cavitation detection. The sample chamber, the US transducer and the hydrophone are immersed into the water tank. The voltage generator is used to generate sinusoidal electromagnetic wave, which is then amplified by the US amplifier. The piezoelectric material of the ultrasound transducer converts the electrical signal in to mechanical vibration leading to ultrasonic irradiation of the sample. The oscilloscope collects data from the hydrophone and performs FFT. The PC reads data from the oscilloscope, feedbacks the amplitude for the voltage generator and controls the measurement time.

2.6 Ultrasound imaging


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The US imaging was performed with a Philips Envisor HD ultrasound system (Philips Medical Systems, Bothell, Washington). Two ml Eppendorf tubes containing suspensions of PSi NPs were placed into a custom-made gelatin phantom and B-mode imaged with a linear array transducer (model L12-3, 3-12 MHz, 160 element linear array) having a central frequency of 7.5 MHz (gain 80 dB, MI 1.4, Figure S2 of Suppl. Inf.). Using the 23 Hz frame rate, 230 frames were recorded for each concentration of PSi NPs. US B-mode images were analyzed with a custom MATLAB R2015a (Mathworks, Natick, MA) program, where the relative average intensities were calculated within a circular region of interest inside the tubes having a diameter of 5 mm.

3 Results and Discussion 3.1 Nanoparticles and their properties An improvement of the ultrasonographic contrast can be achieved by enhanced scattering of US by air bubbles. In our case, PSi NPs were selectively modified using the nanostopper approach45 to keep the pore surfaces hydrophobic, while the outer surfaces of the particles were made hydrophilic. This specific modification of J-PSi NPs allowed them to preserve air inside the pores when J-PSi NPs were suspended in water (Figure 2). The hydrophilic (oxidized) external surfaces with hydroxyl groups enabled their further functionalization. Accordingly, the outer surfaces were dual PEGylated48 (PEG-J-PSi NPs) to improve the colloidal stability of J-PSi NPs in biologically relevant fluids and to prolong the blood circulation time46. The trapped air acts as nuclei for microbubble growth and cavitation both of which improve the contrast in US images. J-PSi and PEG-J-PSi NPs throughout the study were compared to their fully oxidized hydrophilic counterparts denoted as O-PSi and PEG-O-PSi NPs.


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Figure 2. Fabrication route of Janus PSi NPs. Illustration of the selective modification procedure, PEGylation and the schematics of a microbubble growth and contrast enhancement under US irradiation.

Both the O-PSi and the J-PSi NPs typically had irregular shapes (Figure 3a,b, Figure S3 of Suppl. Inf.) due to the top-down fabrication method50; this has been considered to be beneficial for the adhesion of nanoparticles on blood vessel walls51. The sizes of all the investigated NPs


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were in the range of 150 ± 50 nm (Figure S4 of Suppl. Inf.), while the pore size of J-PSi NPs was 12 ± 3 nm. This value was slightly lower in the case of O-PSi (Figure 3c) due to the additional oxide layer on the pore walls. FTIR measurements revealed the preservation of Si-Hx bonds after the selective modification confirming the success of the modification method (Figure 3d). The pristine material of PSi displayed intense bands between 2000 and 2170 cm–1 due to the surface Si–Hx (x = 1–3) groups52. After the complete surface oxidation (O-PSi), an intense peak at 1000– 1200 cm–1 of Si–Ox bonds appeared, while the peak from Si–Hx disappeared completely. However, in the case of the J-PSi NPs, the intensity of Si–Ox was lower, and a small amount of Si–Hx bands were still present, indicating the preservation of hydrogen termination of the inner pore surfaces. PEGylation gave rise to the additional absorption at 2872 cm–1 revealing the successful conjugation of PEG onto the J-PSi NPs. PEG coating was also confirmed by thermogravimetry (Figure 3e), which was used to evaluate the amount of PEG. The mass loss after PEGylation amounted to 32 wt.% and 22 wt.% for the PEG-O-PSi and the PEG-J-PSi nanoparticles, respectively. The PEG-J-PSi NPs had a lower amount of PEG, because PEG was conjugated only on the external surface, while the PEG-O-PSi NPs had also the polymer inside the pores. The PEG coating significantly increased the colloidal stability of the PEG-O-PSi and PEG-J-PSi NPs (up to several days) compared to non-PEGylated counterparts (less than 15 min) in blood plasma:PBS 1:1 mixture (Figure 3f). Thus, according to previous studies46, the PEGylated NPs can circulate long enough in the bloodstream to allow them to reach their target. It is known, that the contact of Si hydride species on the pristine PSi surface with moisture leads to the production of hydrogen and silane53. However, in the present study, hydrogen production was not noticed when the J-PSi NPs were dispersed in water, suggesting that the


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partial oxidation covered sufficiently the surface and moisture did not reach the hydrophobic walls.


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Figure 3. Material characterization of the PSi NPs. (a,b) The TEM images of J-PSi NPs and PEG-J-PSi NPs, respectively; the insets show the NPs at a smaller scale. (c) Pore size distributions of O- and J-PSi NPs before the PEGylation. (d) FTIR spectra of (1) hydrogen terminated pristine PSi NPs, (2) O-PSi NPs, (3) PEG-O-PSi NPs, (4) J-PSi NPs and (5) PEG-JPSi NPs; the inset shows spectra at a smaller scale. (e) Thermogravimetric analysis of PEG content of PSi NPs. (f) Colloidal stability of the NPs; the average particle size was measured with dynamic light scattering.

3.2 Detection of cavitation and cavitation thresholds The cavitation thresholds in suspensions of the PSi NPs were measured by detecting the growth of the subharmonics amplitude, which indicates the presence of cavitating air bubbles49. The suspensions of PSi NPs were placed in the chamber and irradiated with continuous wave US of different acoustic pressures for 30 s (Figure S5 of Suppl. Inf.). The cavitation was considered to be detected when the amplitude of the subharmonic signal in the spectrum was at least twice as high as the noise level at the beginning of the 30 s period, and the corresponding value of the acoustic pressure was recognized as the cavitation threshold. The results of the threshold measurements for the suspensions of 0.05 mg/ml concentration are summarized in Figure 4. Figure 4a shows the dependence of the subharmonic amplitude on the acoustic pressure produced by the transducer. In deionized water, air bubbles appeared in a steady manner when the acoustic pressure was increased up to 265 ± 10 kPa. The presence of the O-PSi and PEG-OPSi NPs slightly lowered the threshold to 240 ± 10 kPa and 250 ± 10 kPa, respectively, because solid NPs can enhance the nucleation probability of air bubbles54. Nonetheless, when the J-PSi and PEG-J-PSi NPs were introduced, the threshold declined to 170 ± 10 kPa.


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Figure 4. Detection of cavitation threshold. (a) Dependence of the subharmonic amplitudes on the acoustic pressure for deionized H2O and the suspensions of the O-PSi, PEG-O-PSi, J-PSi and PEG-J-PSi NPs (0.05 mg/ml); the connecting lines are a visual aid. (b) Dependence of the threshold time for cavitation appearance as a function of acoustic pressure. The dashed lines indicate the cavitation threshold.


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It is also important cavitation appears immediately after the US radiation is switched on. Figure 4b depicts the time spans passing between switching the transducer on and the moment when the subharmonic amplitude began to exceed the noise level. Here, similar to Figure 4a, threshold values were obtained. Cavitation was detected at similar pressure levels for all the studied concentrations in the range of 0.05 – 1 mg/ml (Figure 5). Moreover, similar threshold pressures were detected with 10 times lower concentration of 5 µg/ml demonstrating, that even small amount of NPs is sufficient to produce air bubbles (Figures S6 and S7 of Suppl. Inf.). The threshold pressure did not depend on the concentration, because the concentration affects exclusively the amplitude of the subharmonic signal: the increase of concentration leads to the increase of the number of bubbles that collapsed and emitted subharmonic signal. This effect is important when using nanoparticles as contrast agents, since the ultrasound image contrast increases with the number of cavitating bubbles in the irradiated region. These results demonstrate that even a minor amount of J-PSi NPs was sufficient to significantly lower the cavitation threshold. This is due to the preservation of air inside the pores of the J-PSi NPs suspended in water – the nanoparticles act as nuclei for bubble growth from the pores and cavitation. It was previously demonstrated, that PSi NPs with randomly distributed hydrophobic sites can lower the cavitation threshold54. Those findings are consistent with the present study, since nanosized air nuclei can be preserved on hydrophobic surfaces in water55. However, the relatively high concentration of 0.5 mg/ml and presence of hydrophobic sites on the outer surface might limit their application as CAs in that case. Herein, the selective modification procedure made it possible for J-PSi NPs with low concentration of 5 µg/ml to reduce the cavitation


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threshold. Furthermore, the subsequent dual PEGylation significantly prolonged the colloidal stability of PEG-J-PSi NPs in simulated biological conditions.

Figure 5. The concentration dependence of the cavitation threshold for H2O (black), O-PSi NPs (green), PEG-O-PSi NPs (magenta), J-PSi NPs (red) and PEG-J-PSi NPs (blue). 3.3 Calculation of bubble size The cavitation dynamics of an air bubble developed from the pores in nanoparticles to a submicron size can be described by the Gilmore model, which takes into account the surface tension forces, the compressibility and the dependence of the speed of sound on the acoustic pressure56,57. The main equation of this model can be obtained from the Navier-Stokes equation and was used in the following form:

1 1 1 1 ,

(1)

where R is the bubble radius, c is the speed of sound in liquid, H is the liquid enthalpy on the bubble wall, and one and two “overdots” denote the first and second time derivative,


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respectively. The enthalpy was calculated with a modified Tait equation58. Once Equation 1 is solved for the time-dependent bubble size and bubble wall velocity, the pressure radiated by the bubble can be calculated, from which the subharmonic signal can be extracted. Detailed information concerning the model and numerical calculation of bubble dynamics can be found in Suppl. Inf. (Figures S8, S9). To estimate the air bubble sizes responsible for the cavitation in nanoparticle suspensions generated in response to the US stimulus, the experimental data were compared with the simulation results. The normalized subharmonic amplitudes for the suspensions of the O-PSi and J-PSi NPs shown in Figure 4a were plotted first as a function of acoustic pressure. Then, for the same values of acoustic pressures, the dependencies of the subharmonic signal for the individual air bubbles of the equilibrium radii in the range of 0.5 – 1.2 µm were calculated. Subsequently, those equilibrium bubble radii were distinguished, for which the corresponding dependencies of subharmonic amplitudes were closest to the experimental values in terms of the cavitation threshold (Figure 6a). The closest bubble radii that promoted the cavitation similar to the experimentally observed value were found to be around 0.7 µm and 1.1 µm in the cases of the OPSi and J-PSi NPs, respectively.


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Figure 6. Estimation of the air bubble sizes for the experimentally observed cavitation threshold values. (a) The normalized amplitude of subharmonic signals in suspensions of the O-PSi NPs (top) and J-PSi NPs (bottom) as a function of the acoustic pressure. The symbols correspond to the experimental data and the lines correspond to the simulated data for the bubbles of 0.7 and 1.1 µm in radius. (b) Theoretical dependence of the cavitation threshold on the radius of the air bubble. The cyan area corresponds to the range of bubble sizes contributing to the subharmonic signal at the acoustic pressure levels used in the experiment.

Even though there are several reasons for the discrepancies between the experimental and simulated data (Figure 6a, see also Suppl. Inf.), it was possible to reliably estimate the range of bubble sizes that would contribute to the observed cavitation process. Clarification was obtained as shown in Figure 6b which revealed the dependence of the cavitation threshold on the air bubble radius. It is evident that the threshold value declines with an increase in the bubble radius, reaching a minimum value in the vicinity of resonant size59 (~2 µm for 2.08 MHz, see Suppl.


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Inf.). Thus, at a certain pressure level, there exists a critical minimal bubble size, to which the air nuclei inside NP must grow in order to affect the subharmonic amplitude. The process of bubble growth from a nucleus is a well-known phenomenon of rectified diffusion60, which has its own threshold pressure values and is defined by several parameters (dissolved gas concentration, diffusion coefficient, initial nucleus size, etc.).

3.4 Ultrasound contrast enhancement The bubble sizes around 1-2 µm, which can grow from the pores of the J-PSi NPs, are echogenic and can enhance the contrast for US imaging. Here, B-mode images of the suspensions of oxidized and Janus PSi NPs of different concentrations were captured using a conventional diagnostic scanner. Figure 7a shows the representative images of the suspensions for MI = 1.4. Clearly, J-PSi NPs exhibited a significant contrast intensity, while both the O-PSi NPs and PEG-O-PSi NPs displayed poorer contrast under the same conditions. The contrast intensities were further quantified by averaging the contrast of 230 images which were normalized to the original signal intensity according to the scale bar of 80 dB. As shown in Figure 7b, Janus NPs produced significantly higher contrast than their fully oxidized counterparts. The increase of NP concentration resulted in the growth of a higher number of cavitating bubbles and a subsequent increase of the contrast in B-mode images in the volume irradiated by US.


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Figure 7. Ultrasound imaging of suspensions of PSi NPs. (a) Representative B-mode images of (1) O-PSi NPs, (2) PEG-O-PSi NPs (3) J-PSi NPs (4) PEG-J-PSi NPs of various concentrations (mg/ml) in water. (b) The concentration dependence of average intensities calculated from the Bmode images. Recently,

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exosome-like61

and

superhydrophobic silica41 nanoparticles, silica62 and gold63 nanoshell microparticles, and carbon nanotubes64 have been suggested to enhance the contrast for ultrasound imaging. Unfortunately, only Jin et al.41 reported the B-mode contrast in dB units, which makes it possible to be compared with the present study. Their superhydrophobic silica NPs gave the signal intensity of 29.0 ± 9.2 dB in water, which however decreased quickly in serum. Instead PEG-J-PSi NPs are supposed to stay for a long time in the systemic circulation due to the PEG coating46. The contrasting effectiveness of J-PSi NPs was also compared with the commercially available CA SonoVue® prepared according to the manufacturer’s instructions and measured using MI = 0.6 to prevent the rupture of MBs (Figure S10 of Suppl. Inf.). Since MBs and NPs have


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different size and density, the comparison was made using the number of MBs and NPs in 1 ml. Here, the number concentration of MBs was 2·109 ml-1, while it was 0.1·1012 ml-1 for the NPs with the average size of 200 nm, porosity of 60% and 0.5 mg/ml mass concentration. The calculated average intensity of SonoVue® MBs was 13.7 ± 1.5 dB, making them much more echogenic than J-PSi NPs. However, one should note, that in case of J-PSi NPs, air bubbles have to first grow from the pores, and these grown bubbles produce the contrast during B-mode imaging. Since the imaging is usually performed in the pulsed mode, it is rather difficult for MBs to grow from nano-air seeds trapped inside the pores of J-PSi NPs. On the contrary, the cavitation threshold measurements were performed under continuous US irradiation and the presence of air bubbles was detected at as low as 5 µg/ml PEG-J-PSi NPs concentration. Thus, to obtain B-scans with better contrast, presumably, continuous wave US should be first applied to grow MBs from J-PSi NPs.

4 Conclusion The selective modification method was applied to ensure that the inner pore walls of the mesoporous silicon nanoparticles remained hydrophobic while the external surface was made hydrophilic through oxidation. Such a modification made it possible to preserve nano-air seeds inside the pores when the nanoparticles were immersed into water. The pores filled with air were demonstrated to act as nuclei for the growth of the microbubbles and cavitation. The dynamics of an individual cavitating air bubble was simulated to estimate the minimal bubble size required to have the same cavitation threshold values as observed in the experiment. The generated air bubbles were sufficiently echogenic and were considered as the primary source of the contrast during ultrasound imaging. Furthermore, PEGylation of the outer surface did not reduce the


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acoustic properties of the nanoparticles significantly, but improved their stability in biologically relevant fluids. Thus, the present study described a totally new strategy to make nanoparticles visible to ultrasound and highlighted their feasibility for ultrasound imaging. The numerical simulation sufficiently well predicted the cavitation thresholds observed in the experiment and gave the bubble sizes that grow from Janus particles. The developed contrast agent is the first of its kind that utilizes a bioresorbable mesoporous inorganic core and polymer shell. Due to the nature of the material, the size of the agent can be varied over a wide range, the agent can be loaded with several cargos and the external surface can be easily chemically modified, e.g., for targeting purposes.

ASSOCIATED CONTENT Supporting Information The following files are available free of charge. Characterization of 2.08 MHz ultrasound transducer, scheme of B-mode imaging of nanoparticles, additional characterization of nanoparticles, more information of cavitation threshold measurement technique, detailed description of numerical simulation of air bubble dynamics (PDF) B-mode imaging video of the suspensions of PEG-O-PSi NPs (AVI) B-mode imaging video of the suspensions of PEG-J-PSi NPs (AVI) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]


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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. The authors declare no competing financial interest. Funding Sources Russian Science Foundation (Grant No16-13-10145) ACKNOWLEDGMENT K.T., A.S., V.A. and V.T. acknowledge the financial support from the Russian Science Foundation (Grant No16-13-10145). W.X and V.-P. Lehto acknowledge the financial support from Academy of Finland (Grant No 288531). REFERENCES (1)

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Table of Contents Image 83x35mm (300 x 300 DPI)



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The experimental setup used for cavitation detection. The sample chamber, the US transducer and the hydrophone are immersed into the water tank. The voltage generator is used to generate sinusoidal electromagnetic wave, which is then amplified by the US amplifier. The piezoelectric material of the ultrasound transducer converts the electrical signal in to mechanical vibration leading to ultrasonic irradiation of the sample. The oscilloscope collects data from the hydrophone and performs FFT. The PC reads data from the oscilloscope, feedbacks the amplitude for the voltage generator and controls the measurement time. 170x60mm (300 x 300 DPI)


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Fabrication route of Janus PSi NPs. Illustration of the selective modification procedure, PEGylation and the schematics of a microbubble growth and contrast enhancement under US irradiation. 170x140mm (300 x 300 DPI)



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Material characterization of the PSi NPs. (a,b) The TEM images of J-PSi NPs and PEG-J-PSi NPs, respectively; the insets show the NPs at a smaller scale. (c) Pore size distributions of O- and J-PSi NPs before the PEGylation. (d) FTIR spectra of (1) hydrogen terminated pristine PSi NPs, (2) O-PSi NPs, (3) PEG-O-PSi NPs, (4) J-PSi NPs and (5) PEG-J-PSi NPs; the inset shows spectra at a smaller scale. (e) Thermogravimetric analysis of PEG content of PSi NPs. (f) Colloidal stability of the NPs; the average particle size was measured with dynamic light scattering. 170x180mm (300 x 300 DPI)


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Detection of cavitation threshold. (a) Dependence of the subharmonic amplitudes on the acoustic pressure for deionized H2O and the suspensions of the O-PSi, PEG-O-PSi, J-PSi and PEG-J-PSi NPs (0.05 mg/ml); the connecting lines are a visual aid. (b) Dependence of the threshold time for cavitation appearance as a function of acoustic pressure. The dashed lines indicate the cavitation threshold. 170x160mm (300 x 300 DPI)



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The concentration dependence of the cavitation threshold for H2O (black), O-PSi NPs (green), PEG-O-PSi NPs (magenta), J-PSi NPs (red) and PEG-J-PSi NPs (blue). 1065x476mm (96 x 96 DPI)


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Estimation of the air bubble sizes for the experimentally observed cavitation threshold values. (a) The normalized amplitude of subharmonic signals in suspensions of the O-PSi NPs (top) and J-PSi NPs (bottom) as a function of the acoustic pressure. The symbols correspond to the experimental data and the lines correspond to the simulated data for the bubbles of 0.7 and 1.1 µm in radius. (b) Theoretical dependence of the cavitation threshold on the radius of the air bubble. The cyan area corresponds to the range of bubble sizes contributing to the subharmonic signal at the acoustic pressure levels used in the experiment. 85x43mm (300 x 300 DPI)



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Ultrasound imaging of suspensions of PSi NPs. (a) Representative B-mode images of (1) O-PSi NPs, (2) PEGO-PSi NPs (3) J-PSi NPs (4) PEG-J-PSi NPs of various concentrations (mg/ml) in water. (b) The concentration dependence of average intensities calculated from the B-mode images. 171x78mm (300 x 300 DPI)


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