Systemic Administration of Polymer‐Coated Nano‐Graphene to Deliver Drugs to Glioblastoma

Graphene—2D carbon—has received significant attention thanks to its electronic, thermal, and mechanical properties. Recently, nano‐graphene (nGr) has been investigated as a possible platform for biomedical applications. Here, a polymer‐coated nGr to deliver drugs to glioblastoma after systemic administration is reported. A biodegradable, biocompatible poly(lactide) (PLA) coating enables encapsulation and controlled release of the hydrophobic anticancer drug paclitaxel (PTX), and a hydrophilic poly(ethylene glycol) (PEG) shell increases the solubility of the nGr drug delivery system. Importantly, the polymer coating mediates the interaction of nGr with U‐138 glioblastoma cells and decreases cytotoxicity compared with pristine untreated nGr. PLA‐PEG‐coated nGr is also able to encapsulate PTX at 4.15 wt% and sustains prolonged PTX release for at least 19 d. PTX‐loaded nGr‐PLA‐PEGs are shown to kill up to 20% of U‐138 glioblastoma cells in vitro. Furthermore, nGr‐PLA‐PEG and CNT‐PLA‐PEG, two carbon nanomaterials with different shapes, are able to kill U‐138 in vitro as well as free PTX at significantly lower doses of drug. Finally, in vivo biodistribution of nGr‐PLA‐PEG shows accumulation of nGr in intracranial U‐138 glioblastoma xenografts and organs of the reticuloendothelial system.     

Docetaxel‐Loaded Nanoparticles of Dendritic Amphiphilic Block Copolymer H40‐PLA‐b‐TPGS for Cancer Treatment

A dendritic amphiphilic block copolymer H40‐poly(d,l ‐lactide)‐block‐d‐α‐tocopheryl polyethylene glycol 1000 succinate (H40‐PLA‐b‐TPGS) is synthesized, which is then employed to develop a system of nanoparticles (NPs) loaded with docetaxel (DTX) as a model drug for cancer treatment due to its higher drug‐loading content and drug encapsulation efficiency, smaller particle size, faster drug release, and higher cellular uptake in comparison to the linear PLA polymer NPs and PLA‐b‐TPGS copolymer NPs. The drug‐loaded NPs are prepared by a modified nanoprecipitation method and characterized in terms of size and size distribution, surface morphology, drug release profile, and physical state of DTX. Cellular uptake of coumarin 6‐loaded NPs by MCF‐7 cancer cells is determined by flow cytometry and confocal laser scanning microscopy. The antitumor efficacy of the drug‐loaded NPs is investigated in vitro by MTT assay and in vivo by xenograft tumor model. The 72 h IC50 of the drug formulated in the PLA, PLA‐b‐TPGS, and H40‐PLA‐b‐TPGS NPs is found to be, 1.5 ± 0.3, 0.9 ± 0.1, and 0.15 ± 0.06 μg mL^?1, which are 7.3, 12.2, and 73.3‐fold effective than 11.0 ± 1.2 μg mL^?1 for Taxotere, respectively. Such advantages are further confirmed by the measurement of the tumor size and weight.     

Preparation and in vitro evaluation of an ultrasound-triggered drug delivery system: 10-hydroxycamptothecin loaded PLA microbubbles

10-Hydroxycamptothecin (HCPT) loaded PLA microbubbles, used as an ultrasound-triggered drug delivery system, were fabricated by a double emulsion-solvent evaporation method. The obtained microbubbles were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and confocal laser scanning microscope (CLSM). In addition, the effect of diagnostic ultrasound exposure on BEL-7402 cells combined with HCPT-loaded PLA microbubbles was evaluated using cytotoxicity assay, CLSM and flow cytometry (FCM). It was found that the HCPT-loaded PLA microbubbles showed smooth surface and spherical shape, and the drug was amorphously dispersed within the shell and the drug loading content reached up to 1.69%. Nearly 20% of HCPT was released upon exposure to diagnostic ultrasound at frequency of 3.5 MHz for 10 min. Moreover, HCPT fluorescence in the cells treated only with the HCPT-loaded PLA microbubbles was discernible, but less intense, while those treated with the microbubbles in conjunction with ultrasound exposure was evident and intense, indicating an increased cellular uptake of HCPT by ultrasound exposure. Cytotoxicity test on BEL-7402 cells indicated that the HCPT-loaded PLA microbubbles combined with ultrasound exposure were more cytotoxic than the microbubbles alone. The results suggest that the combination of drug loaded PLA microbubbles and diagnostic ultrasound exposure exhibit an effective intracellular drug uptake by tumor cells, indicating their great potential for antitumor therapy.     

Intracellular Breakable and Ultrasound‐Responsive Hybrid Microsized Containers for Selective Drug Release into Cancerous Cells

This work reports an efficient and straightforward strategy to fabricate hybrid microsized containers with reduction‐sensitive and ultrasound‐responsive properties. The ultrasound and reductive sensitivity are visualized using scanning electron microscopy, with the results showing structural decomposition upon ultrasound irradiation and in the presence of reducing agent. The ultrasound‐responsive functionalities of hybrid carriers can be used as external trigger for rapid controlled release, while prolonged drug release can be achieved in the presence of reducing agent. To evaluate the potential for targeted drug delivery, hybrid microsized containers are loaded with the anticancer drug doxorubicin (Dox). Such hybrid capsules can undergo structural intracellular degradation after cellular uptake by human cervical cancer cell line (HeLa), resulting in Dox release into cancer cells. In contrast, there is no Dox release when hybrid capsules are incubated with human mesenchymal stem cells (MSCs) as an example of normal human cells. The cell viability results indicate that Dox‐loaded capsules effectively killed HeLa cells, while they have lower cytotoxicity against MSCs as an example of healthy cells. Thus, the newly developed intracellular‐ and ultrasound‐responsive microcarriers obtained via sol‐gel method and layer‐by‐layer technique provide a high therapeutic efficacy for cancer, while minimizing adverse side effect.     

Nonlinear radial oscillations of encapsulated microbubbles subject to ultrasound: the effect of membrane constitutive law

The nonlinear radial oscillations of bubbles that are encapsulated in an elastic shell are investigated numerically subject to three different constitutive laws describing the viscoelastic properties of the shell: the Mooney-Rivlin (MR), the Skalak (SK), and the Kelvin-Voigt (KV) models are used in order to describe strain-softening, strain-hardening and small displacement (Hookean) behavior of the shell material, respectively. Due to the isotropic nature of the acoustic disturbances, the area dilatation modulus is the important parameter. When the membrane is strain softening (MR) the resonance frequency decreases with increasing sound amplitude, whereas the opposite happens when the membrane is strain hardening (SK). As the amplitude of the acoustic disturbance increases the total scattering cross section of a microbubble with a SK membrane tends to decrease, whereas that of a KV or a MR membrane tends to increase. The importance of strain-softening behavior in the abrupt onset of volume pulsations, that is often observed with small insonated microbubbles at moderately large sound amplitudes, is discussed.     

Mathematical modeling of microbubble cavitation at 70 kHz and the importance of the subharmonic in drug delivery from micelles

In order to gain insight into the experimental observation of ultrasound-induced release of drugs from micelles, we modeled the dynamic oscillations of a 10-μm-diameter bubble insonated at 70 kHz. The Parlitz modification of the Keller–Miksis model was employed to generate bubble dynamics over a wide range of mechanical index values. The resulting Poincaré maps and bifurcation diagram show that bubble oscillations bifurcate at a MI value of 0.32, then return apparently to a single mode before displaying a sudden onset of chaotic behavior at 0.35. The experimental release of drug from micelles occurs at a MI value of 0.37 and correlates with the intensity of the subharmonic in (μW/cm²) of the acoustic spectrum. The dynamic model shows the return to single mode at a MI value of 0.43, and bifurcation leading to chaos at values above 0.5. The correlation between the chaotic behavior predicted by the model and drug release hints at insonation conditions that could facilitate drug delivery.     

Lysozyme microspheres incorporated with anisotropic gold nanorods for ultrasound activated drug delivery

We report on the fabrication of lysozyme microspheres (LyMs) incorporated with gold nanorods (NRs) as a distinctive approach for the encapsulation and release of an anticancer drug, 5-Fluorouracil (5-FU). LyMs with an average size of 4.0 ± 1.0 µm were prepared by a sonochemical method and characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Fourier-transform infrared spectroscopy (FTIR). The LyMs were examined using hydrophobic (nile red) as well as hydrophilic (trypan blue) dyes under confocal laser scanning microscopy (CLSM) to obtain information about the preferential distribution of fluorescent molecules. Notably, the fluorescent molecules were accumulated in the inner lining of LyMs as the core was occupied with air. The encapsulation efficiency of 5-FU for LyMs-NR was found to be ∼64%. The drug release from control LyMs as well as LyMs incorporated with NRs was investigated under the influence of ultrasound (US) at 200 kHz. The total release for control LyMs and LyMs incorporated with gold NRs was found to be ∼70 and 95% after 1 h, respectively. The density difference caused by NR incorporation on the shell played a key role in rupturing the LyMs-NR under US irradiation. Furthermore, 5-FU loaded LyMs-NR exhibited excellent anti-cancer activity against the THP-1 cell line (∼90% cell death) when irradiated with US of 200 kHz. The enhanced anti-cancer activity of LyMs-NR was caused by the transfer of released 5-FU molecules from bulk to the interior of the cell via temporary pores formed on the surface of cancer cells, i.e., sonoporation. Thus, LyMs-NR demonstrated here has a high potential for use as carriers in the field of drug delivery, bio-imaging and therapy.     

Characterization of ultrasound-induced fracture of polymer-shelled ultrasonic contrast agents by correlation analysis

Beyond a characteristic value of the negative peak pressure, ultrasound fracture the shell of ultrasonic contrast agents (UCAs). Existing criteria for ascertaining this threshold value exploit the dependence of the amplitude of the UCA acoustic response on the incident pressure. However, under the common experimental conditions used in this work, these criteria appear to be unreliable when they are applied to UCAs that are stabilized by a thick polymeric shell. An alternative criterion for determining the onset of shell fracture is introduced here, which uses variations of the shape of the acoustic time-domain response of an UCA suspension. Experimental evidence is presented that links the changes of the cross-correlation coefficient between consecutive time-domain signals to the fracture of the shells, and consequent release of air microbubbles. In principle, this criterion may be used to characterize similar properties of other types of particles that cannot undergo inertial cavitation.     

A theoretical investigation of chirp insonification of ultrasound contrast agents

A theoretical investigation of second harmonic imaging of an Ultrasound Contrast Agent (UCA) under chirp insonification is considered. By solving the UCA’s dynamical equation analytically, the effect that the chirp signal parameters and the UCA shell parameters have on the amplitude of the second harmonic frequency are examined. This allows optimal parameter values to be identified which maximise the UCA’s second harmonic response. A relationship is found for the chirp parameters which ensures that a signal can be designed to resonate a UCA for a given set of shell parameters. It is also shown that the shell thickness, shell viscosity and shell elasticity parameter should be as small as realistically possible in order to maximise the second harmonic amplitude. Keller-Herring, Second Harmonic, Chirp, Ultrasound Contrast Agent.     

Stability of alteplase in presence of cavitation

Several experimental studies have demonstrated that ultrasound (US) can accelerate enzymatic fibrinolysis and this effect is further enhanced in the presence of ultrasound contrast agents (UCA). Although UCA have been shown to be safe when administered to ischemic stroke patients, safety information of these agents in the thrombolysis setting is limited. Therefore, in this study we investigated potential adverse effects of acoustic cavitation generated by UCA on alteplase (t-PA), the drug used for treatment of ischemic stroke patients. A volume of 0.9 mL of alteplase was dispensed into a custom-made polyester sample tube. For treatments in the presence or absence of cavitation either 0.1 mL Optison or phosphate buffer saline was combined with alteplase. Three independent samples of each treatment group were exposed to ultrasound of 2 MHz frequency at three different peak negative acoustic pressures of 0.5, 1.7, and 3.5 MPa for a duration of 60 min. All treatments were carried out in a cavitation detection system which was used to insonify the samples and record acoustic emissions generated within the sample. After ultrasound exposure, the treated samples and three untreated drug samples were tested for their enzymatic activity using a chromogenic substrate. The insonified samples containing Optison demonstrated cavitational activity proportional to acoustic pressure. No significant cavitation activity was observed in the absence of Optison. Enzymatic activity of alteplase in both insonified groups was comparable to that in the control group. These tests demonstrated that exposure of alteplase to 60 min of 2 MHz ultrasound at acoustic pressures ranging from 0.5 MPa to 3.5 MPa, in the presence or absence of Optison had no adverse effects on the stability of this therapeutic compound.     

A search for sonoluminescence in vivo in the human cheek

In a previous experiment, sonoluminescence was observed in aerated water, especially at the pressure antinodes in the standing-wave field of a physiotherapeutic ultrasound device (Therasonic 1030). Mammalian cells in vitro showed growth inhibition when placed at the pressure antinodes but not at adjacent pressure nodes. In the light of these results, we looked for sonoluminescence in vivo when a similar standing-wave field was set up. To detect luminescence, a light guide was held against the inner surface of the human cheek. This would channel any luminescence photons to a cooled, red-sensitive photomultiplier which would quantify the light. Direct insonation of the cheek produced no detectable luminescence. Similarly when a water bag was placed against the outer surface of the cheek, and the latter was insonated through the bag, no luminescence was detected. Sonoluminescence from the water bag was, however, detected when the bag was placed against the inner surface of the cheek, showing that absorption of sound by the cheek tissue was not preventing cavitation. Further analysis showed that if cavitation had been occurring in the cheek without detection using the system employed, then the resulting sonoluminescence would have to be at most 0.025 times as intense as that produced by an equivalent volume of aerated water.     

超声造影剂微泡非线性动力学响应的机理及相关应用

随着生命科学及现代医学的发展, 一体化无创精准诊疗已经日益成为人们关注的焦点问题, 而关于超声造影剂微泡的非线性效应的相关机理、动力学建模及其在超声医学领域中的应用研究也得到了极大的推动. 本文对下列课题进行了总结和讨论, 包括: 1)基于Mie散射技术和流式细胞仪对造影剂微泡参数进行定征的一体化解决方案; 2)通过对微泡包膜的黏弹特性进行非线性修正, 构建新的包膜微泡动力学模型; 3)探索造影剂惯性空化阈值与其包膜参数之间的相关性; 以及4)研究超声联合造影剂微泡促进基因/药物转染效率并有效降低其生物毒性的相关机理.     

The use of models in "target" theory to evaluate the survival curves of human ovarian carcinoma cell line exposure to adriamycin combined with ultrasound

The models of "target" theory in radiation biology were used to evaluate the survival curves of human ovarian carcinoma cell line after exposure to adriamycin combined with ultrasound. 3AO cells were exposed to adriamycin in group ADR, to adriamycin after ultrasound exposure in group US+ADR, and to adriamycin prior to insonation in group ADR+US. The cell survival was determined by a clonogenic assay. The dose-response curves were fitted to two models, (1) single-hit, single-target model, (2) single-hit, multi-target model. The single-hit, multi-target model could fit the survival curve in group ADR, but it could only describe the survivals in groups US+ADR and ADR+US while the concentration of adriamycin was less than 0.05 mug/ml. These suggested that the single-hit, multi-target model could be conditionally used to describe the survival while cells were exposed to the combination of adriamycin and acoustic exposure. The models also were tools to understand the potentiation attributable to insonation.     

Simultaneous grayscale and subharmonic ultrasound imaging on a modified commercial scanner

Objective

To demonstrate the feasibility of simultaneous dual fundamental grayscale and subharmonic imaging on a modified commercial scanner.

Motivation

The ability to generate signals at half the insonation frequency is exclusive to ultrasound contrast agents (UCA). Thus, subharmonic imaging (SHI; transmitting at f₀ and receiving at f₀/2) provides improved visualization of UCA within the vasculature via suppression of the surrounding tissue echoes. While this capability has proven useful in a variety of clinical applications, the SHI suppression of surrounding tissue landmarks (which are needed for sonographic navigation) also limits it use as a primary imaging modality. In this paper we present results using a commercial ultrasound scanner modified to allow imaging in both grayscale (f₀ = 4.0 MHz) and SHI (f₀ = 2.5 MHz, f₀/2 = 1.25 MHz) modes in real time.

Methods

A Logiq 9 ultrasound scanner (GE Healthcare, Milwaukee, WI) with a 4C curvilinear probe was modified to provide this capability. Four commercially available UCA (Definity, Lantheus Medical Imaging, North Billerica, MA; Optison, GE Healthcare, Princeton, NJ; SonoVue, Bracco Imaging, Milan, Italy; and Sonazoid, GE Healthcare, Oslo, Norway) were all investigated in vitro over an acoustic output range of 3.34 MPa. In vivo the subharmonic response of Sonazoid was investigated in the portal veins of four canines (open abdominal cavity) and four patients with suspected portal hypertension.

Results

In vitro, the four UCA showed an average maximum subharmonic amplitude of 44.1 ± 5.4 dB above the noise floor with a maximum subharmonic amplitude of 48.6 ± 1.6 dB provided by Sonazoid. The average in vivo maximum signal above the noise floor from Sonazoid was 20.8 ± 2.3 dB in canines and 33.9 ± 5.2 dB in humans. Subharmonic amplitude as a function of acoustic output in both groups matched the S-curve behavior of the agent observed in vitro. The dual grayscale imaging provided easier sonographic navigation, while the degree of tissue suppression in SHI mode varied greatly on a case by case basis.

Conclusions

These results demonstrate the feasibility of dual grayscale and SHI on a modified commercial scanner. The ability to simultaneously visualize both imaging modes in real time should improve the applicability of SHI as a future primary clinical imaging modality.     

多层膜磁性微泡的非线性声振动特性

超顺磁性氧化铁纳米粒子与造影剂微泡结合形成磁性微泡,用于产生多模态造影剂,以增强医学超声和磁共振成像.将装载有纳米磁性颗粒的微泡包膜层看作由磁流体膜与磷脂膜组合而成的双层膜结构,同时考虑磁性纳米颗粒体积分数a对膜密度及黏度的影响,从气泡动力学基本理论出发,构建多层膜结构磁性微泡非线性动力学方程.数值分析了驱动声压和频率等声场参数、颗粒体积分数、膜层厚度以及表面张力等膜壳参数对微泡声动力学行为的影响.结果表明,当磁性颗粒体积分数较小且a≤0.1时,磁性微泡声响应特性与普通包膜微泡相似,微泡的声频响应与其初始尺寸和驱动压有关;当驱动声场频率f为磁性微泡共振频率f0的2倍(f=2f0)时,微泡振动失稳临界声压最低;磁性颗粒的存在抑制了泡的膨胀和收缩但抑制效果非常有限;磁性微泡外膜层材料的表面张力参数K及膜层厚度d也会影响微泡的振动,当表面张力参数及膜厚取值分别为0.2—0.4 N/m及50—150 nm时,可观察到气泡存在不稳定振动响应区.     

Nonlinear response of ultrasound contrast agent microbubbles:From fundamentals to applications

Modelling and biomedical applications of ultrasound contrast agent(UCA) microbubbles have attracted a great deal of attention. In this review, we summarize a series of researches done in our group, including(i) the development of an all-in-one solution of characterizing coated bubble parameters based on the light scattering technique and flow cytometry;(ii) a novel bubble dynamic model that takes into consideration both nonlinear shell elasticity and viscosity to eliminate the dependences of bubble shell parameters on bubble size;(iii) the evaluation of UCA inertial cavitation threshold and its relationship with shell parameters; and(iv) the investigations of transfection efficiency and the reduction of cytotoxicity in gene delivery facilitated by UCAs excited by ultrasound exposures.     

超声造影剂微气泡的包膜黏弹特性的定量表征研究

包膜黏弹特性显著影响微气泡超声造影剂的诊断及治疗应用效果. 本文结合原子力显微镜技术及声衰减特性测量提出了一种对微气泡造影剂包膜黏弹特性定量表征的新方法. 首先采用原子力显微镜技术进行机械特性分析得到包膜微气泡的有效硬度及体弹性模量; 然后测量声衰减特性, 基于微气泡动力学理论, 计算包膜微气泡的体黏度系数. 为验证方法的有效性, 实验制备了直径为1-5 μm的白蛋白包膜微气泡造影剂, 原子力显微镜测量的有效硬度和体弹性模量分别为0.149±0.012 N/m和8.31±0.667 MPa, 并与粒径无关. 声衰减特性测量和动力学理论拟合的包膜微气泡的体黏度系数为0.374±0.003 Pa·s. 该方法可推广至其他种类包膜微气泡的黏弹特性表征, 对超声造影剂的制备及其诊断和治疗应用有积极意义.     

Theoretical gas body pulsation in relation to empirical gas-body destabilization and to cell membrane damage thresholds

Contrast agent gas bodies attached to phagocytic monolayer cells pulsate in response to ultrasound exposure and damage the cells above thresholds, which increase in proportion to frequency. This study considered the physical basis for the thresholds and their frequency dependence. Theory for the pulsation was evaluated using empirical pulse waveforms acquired at thresholds for 1.0, 2.25, 3.5, 5.0, 7.5, and 10 MHz. For optimum-sized gas bodies, the amplitudes calculated at the thresholds were about 11% of the initial radii. At the cell membrane damage thresholds, theoretical negative shell stresses were approximately constant with frequency at about 50 MPa. This stress appears to be sufficient to induce failure of the shell, and gas body destabilization was confirmed by increases in transmission of ultrasound pulses through the monolayer and by microscopically-observed shrinkage of the gas bodies. A model of acoustic microstreaming was used to calculate the shear stress during the pulses. The maximum shear stress increased from about 1500 to 4500 Pa from 1 to 10 MHz, sufficient for the cell membrane damage. This theoretical analysis shows that both the gas body destabilization and the cell membrane damage could be expected at similar peak rarefactional pressure amplitudes, with thresholds having the observed proportionality to frequency.     

Nanoengineering of methylene blue loaded silica encapsulated magnetite nanospheres and nanocapsules for photodynamic therapy

Core–shell nanostructures have emerged as an important class of functional materials with potential applications in diverse fields, especially in health sciences. In this article, nanoengineering of novel magnetic colloidal dispersion containing surface modifiable silica with a core of single domain magnetite nanoparticles loaded with photosensitizer (PS) drug “Methylene blue” (MB) has been described. Magnetite core is produced by the well-established chemical coprecipitation technique and silica shell is formed over it by the modified hydrolysis and condensation of TEOS (tetraethyl orthosilicate). Conditions for reaction kinetics have been established to tailor the core–shell structures in the form of nanospheres and nanocapsules. MB is loaded into the nanostructures by demethylation reaction. The major conclusion drawn from this study is that the synthesis route yields stable, non-aggregated MB loaded superparamagnetic magnetite-silica nanostructures with tailored morphology, tunable loading, and excellent magnetic properties.     

A comparison of the fragmentation thresholds and inertial cavitation doses of different ultrasound contrast agents

Contrast bubble destruction is important in several new diagnostic and therapeutic applications. The pressure threshold of destruction is determined by the shell material, while the propensity for of the bubbles to undergo inertial cavitation (IC) depends both on the gas and shell properties of the ultrasound contrast agent (UCA). The ultrasonic fragmentation thresholds of three specific UCAs (Optison, Sonazoid, and biSpheres), each with different shell and gas properties, were determined under various acoustic conditions. The acoustic emissions generated by the agents, or their derivatives, characteristic of IC after fragmentation, was also compared, using cumulated broadband-noise emissions (IC "dose"). Albumin-shelled Optison and surfactant-shelled Sonazoid had low fragmentation thresholds (mean = 0.13 and 0.15 MPa at 1.1 MHz, 0.48 and 0.58 MPa at 3.5 MHz, respectively), while polymer-shelled biSpheres had a significant higher threshold (mean = 0.19 and 0.23 MPa at 1.1 MHz, 0.73 and 0.96 MPa for thin- and thick-shell biSpheres at 3.5 MHz, respectively, p<0.01). At comparable initial concentrations, surfactant-shelled Sonazoid produced a much larger IC dose after shell destruction than did either biSpheres or Optison (p<0.01). Thick-shelled biSpheres had the highest fragmentation threshold and produced the lowest IC dose. More than two and five acoustic cycles, respectively, were necessary for the thin- and thick-shell biSpheres to reach a steady-state fragmentation threshold.