Sonodisruption of re-assembled casein micelles at different pH values

Casein solutions with different pH values were sonicated at a frequency of 35 kHz and increasing acoustic powers. As the sonication power increased, turbidity of solutions and particle diameter decreased at any given pH value, suggesting particles disruption due to the ultrasonic treatment. The magnitude of decrease in re-assembled micelles diameter was greater at a higher pH, indicating an interaction between pH and sonication power in sonodissociation. This interaction is attributed to a looser structure of micelles at higher pH values which increases the efficiency of ultrasonic disruption and not directly to the increased cavitation efficiency. We argue that increased cavitation efficiency with increasing sonication power, which enhances shear forces is the most likely reason for sonodisruption of re-assembled casein micelles.     

Dual frequency cavitation event sensor with iodide dosimeter

The inertial cavitation activity depends on the sonication parameters. The purpose of this work is development of dual frequency inertial cavitation meter for therapeutic applications of ultrasound waves. In this study, the chemical effects of sonication parameters in dual frequency sonication (40 kHz and 1 MHz) were investigated in the progressive wave mode using iodide dosimetry. For this purpose, efficacy of different exposure parameters such as intensity, sonication duration, sonication mode, duty factor and net ultrasound energy on the inertial cavitation activity have been studied. To quantify cavitational effects, the KI dosimeter solution was sonicated and its absorbance at a wavelength of 350 nm was measured. The absorbance values in continuous sonication mode was significantly higher than the absorbance corresponding to the pulsed mode having duty factors of 20–80% (p < 0.05). Among different combination modes (1 MHz_100% + 40 kHz_100%, 1 MHz_100% + 40 kHz_80%, 1 MHz_80% + 40 kHz_100%, 1 MHz_80% + 40 kHz_80%), the continuous mode for dual frequency sonication is more effective than other combinations (p < 0.05). The absorbance for this combined dual frequency mode was about 1.8 times higher than that obtained from the algebraic summation of single frequency sonications. It is believed that the optimization of dual frequency sonication parameters at low-level intensity (<3 W/cm²) by optically assisted cavitation event sensor can be useful for ultrasonic treatments.     

Drug‐Conjugation Induced Self‐Assembly of Feather Keratin‐Based Prodrug for Tumor Intracellular Reduction Triggered Drug Delivery

As a kind of natural protein, keratin is widely investigated in the biomedical field. Here, for the first time, a keratin‐based prodrug (PK‐SS‐D) is designed for tumor intracellular reduction triggered drug delivery, by conjugating doxorubicin (DOX) onto poly(ethylene glycol) modified keratin (PEGylated keratin, PK) with a bioreducible disulfide linkage. The protein‐drug conjugate prodrug, with a drug content of 20%, can self‐assemble into micelles with a mean hydrodynamic diameter of 175 nm and a narrow distribution. The in vitro controlled release profiles reveal the reduction triggered thiolated DOX (DOX‐SH) release behavior of the PK‐SS‐D micelles, with a cumulative drug release up to 52% within 10 d in the simulated tumor microenvironment in a sustained releasing mode, and a low drug leakage of 17% in the simulated normal physiological medium. The enhanced tumor growth inhibition of the proposed PK‐SS‐D prodrug micelles is revealed by the methyl tetrazolium (MTT) assays, although the released DOX‐SH prodrug possesses a lower tumor growth inhibition than DOX.     

The effect of free radical scavenger and antioxidant on the increase in intracellular adriamycin accumulation induced by ultrasound

Ultrasound could potentiate cytotoxicity of adriamycin on cancer cell line as a result of increased intracellular accumulation ascribed to cavitation. In order to determine which free radical led to increase of drug content, effects of the free radical scavenger and antioxidant on increased intracellular adriamycin accumulation by ultrasound were investigated. The intracellular drug content of adriamycin was lower in the group where histidine was administrated before ultrasound exposure and in the group where mannitol was added after sonication. Drug accumulation was also decreased in groups in which vitamin C administrated either before or after ultrasonic exposure. These results suggested that hydroxyl radical play the leading role in synergism between ultrasound and adriamycin.     

基于NMR的4T1荷瘤小鼠脾脏受不同给药方式影响的代谢组学研究

联合阿霉素和紫杉醇治疗肿瘤的策略在临床上已有广泛应用,为了进一步借助纳米载体对肿瘤组织的靶向优势,以良好生物相容性纳米载体包埋阿霉素和紫杉醇的新型给药方式得以发展.虽然前期研究结果显示纳米给药方式相对传统给药治疗有更好的抑制肿瘤生长的效果,但是它们在分子代谢水平对机体的影响并没有被确切研究.本文采用4T1荷瘤乳腺癌模型小鼠,通过比较荷瘤小鼠分别在不处理、传统给药方式和通过纳米载体给药处理的条件下与健康小鼠脾脏代谢组的差异,发现传统给药处理对小鼠肠道微生物相关的代谢紊乱有较好的恢复效果,而通过纳米载体的给药处理可以更好的调节荷瘤小鼠的葡萄糖和延胡索酸等与能量供应相关的代谢途径.此外,这两种给药方式都不能有效改善荷瘤小鼠显著的脾脏肿大症状,也不能改善脾脏免疫调节功能和供应肿瘤生长而导致的氨基酸、有机酸、核酸和胆碱等一系列代谢物变化.这可能与脾脏对外来肿瘤的免疫反应较为激烈而有效药物治疗时间较短有关.该研究为更全面认识荷瘤小鼠脾脏代谢表型变化和不同给药方式对脾脏代谢组的影响提供了基础数据.     

Effect of ultrasound on binding interaction between emodin and micellar casein and its microencapsulation at various temperatures

Emodin is a bioactive compound with strong anti-inflammatory and antioxidant properties. Micellar casein is casein concentrates close to the native state of casein micelles. The interaction of emodin and micellar casein under heat treatment in the absence and presence of ultrasound was investigated, and the properties of microencapsulated emodin in micellar casein were compared. Fluorescence experiments proved that the major interaction between emodin and micellar casein was through hydrophobic forces under heat treatment in the absence and presence of ultrasound. However, ΔH, ΔS and ΔG of emodin-casein complexation without sonication were higher than those with sonication, in contradiction to binding constants. The particle sizes of emodin-casein complexes in the presence of ultrasound were smaller than those without sonication, while the specific surface area showed an opposite trend. As to encapsulation, emodin-casein capsules under heat-sonication treatment showed higher antioxidant properties than those of heat treatment alone under similar experimental conditions. Interestingly, micellar casein-emodin encapsulation in the presence of ultrasound showed a lower release rate of emodin in gastrointestinal conditions than that without ultrasound at the emdoin concentration of 10 μmol per gram casein. Ultrasound has been shown to be a potential processing technology for customizing the release kinetics of bioactive compounds.     

Suppressing chaotic oscillations of a spherical cavitation bubble through applying a periodic perturbation

Nonlinear dynamics of a spherical cavitation bubble was studied. A method based on applying a periodic perturbation to suppress chaotic oscillations is introduced. The relation between this method and dual frequency ultrasonic irradiation is correlated to prove its applicability in applications involving cavitation phenomena. Results indicated its strong impact on reducing the chaotic oscillations to regular ones. The governing parameters are the secondary frequency value and the phase difference between the secondary frequency and the fundamental one. In the end, the possible application of this method in high intensity focused ultrasound tumor ablation as an instance, is discussed accounting for both free bubbles and microbubbles.     

Spectral Properties and Solubilization Location of 2'-Ethylhexyl 4-(N,N-Dimethylamino)benzoate in Micelles

在阳离子、非离子和阴离子表面活性剂胶束溶液中,研究了4-(N,N-二甲氨基)苯甲酸2'-乙基己基酯(EHDMAB)的双重荧光和紫外吸收．当EHDMAB增溶在不同的胶束溶液中,紫外吸收增强,在离子型胶束溶液中,可观察到具有较长波长的EHDMAB分子内扭转电荷转移(TICT)荧光,相反,在非离子型胶束溶液中,可观察到具有较短波长TICT荧光,特别是位于阳离子胶束Stern层中的吡啶阳离子可强烈猝灭EHDMAB分子的双重荧光,所吸收的紫外辐射主要通过TICT荧光和非辐射去活化衰减．按照EHDMAB分子TICT荧光在有机溶剂中的极性依赖性,EHDMAB分子的4-(N,N-二甲氨基)在离子型胶束和非离子型胶束中处于不同的极性环境;根据EHDMAB和表面活性剂分子的结构和大小分析,EHDMAB分子的4-(N,N-二甲氨基)应朝向胶束的极性头基团,而2'-乙基己基链则朝向疏水性的胶束内核．动态荧光猝灭测量为EHDMAB分子在不同胶束中的位置进一步提供了佐证．     

Small angle neutron scattering study of doxorubicin-surfactant complexes encapsulated in block copolymer micelles

Self-assembling behaviour of block copolymers and their ability to evade the immune system through polyethylene oxide stealth makes it an attractive candidate for drug encapsulation. Micelles formed by polyethylene oxide-polypropylene oxide- polyethylene oxide triblock copolymers (PEO-PPO-PEO), pluronic P123, have been employed for encapsulating the anti-cancer drug doxorubicin hydrochloride. The binding affinity of doxorubicin within the micelle carrier is enhanced through complex formation of drug and anionic surfactant, aerosol OT (AOT). Electrostatic binding of doxorubicin with negatively charged surfactants leads to the formation of hydrophobic drug-surfactant complexes. Surfactant-induced partitioning of the anti-cancer drug into nonpolar solvents such as chloroform is investigated. SANS measurements were performed on pluronic P123 micelles in the presence of drug-surfactant complex. No significant changes in the structure of the micelles are observed upon drug encapsulation. This demonstrates that surfactant- drug complexes can be encapsulated in block copolymer micelles without disrupting the structure of aggregates.      

Identification of micellar stability zones and structural inversion process of thermoresponsive polymeric micelles by dissipative particle dynamics simulations

The stimuli-sensitive polymeric micelles have huge applications to industrial and technological level, know their behaviour under the influence of a pure stimulus, such as the temperature is an important aspect to control and amplify its application field. In this paper, we investigate the micellar stability zones together with the structural inversion process of thermoresponsive polymeric micelles formed by a diblock copolymer (poly(N-isopropylacrylamide-b-3-[N-(3-methacrylamidopropyl)-N,N-dimethyl]ammoniopropane sulphonate (PNIPA-b-PSPP)) in an aqueous environment employing dissipative particle dynamics simulations. Our outcomes show that the PNIPA-b-PSPP copolymer has the ability to form thermodynamically stable micelles with different core-shell structure (PSPP-core/PNIPA-shell and PNIPA-core/PSPP-shell) depending on the direction of the applied stimulus (low or high temperature), the duality of this micellar behaviour is controlled by temperature effect and by the double hydrophilic character that exhibit the PNIPA and PSPP polymeric segments. Four micellar stability zones and one zone where only exist free unimer chains were detected during the thermal scan. The micellar inversion process is triggered by purely temperature effect, is totally reversible and involves three main stages: (1) micellar dissociation, (2) stabilisation of free unimer chains and (3) formation of inverse micelles, all transitory and metastable stages of micellar inversion process are described and analysed in this paper.     

Dissolution and reconstitution of casein micelle containing dairy powders by high shear using ultrasonic and physical methods

The effect of shear on the solubilization of a range of dairy powders was investigated. The rate of solubilization of low solubility milk protein concentrate and micellar casein powders was examined during ultrasonication, high pressure homogenization and high-shear rotor–stator mixing and compared to low-shear overhead stirring. The high shear techniques were able to greatly accelerate the solubilization of these powders by physically breaking apart the powder agglomerates and accelerating the release of individual casein micelles into solution. This was achieved without affecting the structure of the solubilized proteins. The effect of high shear on the re-establishment of the mineral balance between the casein micelles and the serum was examined by monitoring the pH of the reconstituted skim milk powder after prior exposure to ultrasonication. Only minor differences in the re-equilibration of the pH were observed after sonication for up to 3 min, suggesting that the localized high shear forces exerted by sonication did not significantly affect the mass transfer of minerals from within the casein micelles.     

Impact of process parameters in the generation of novel aspirin nanoemulsions – Comparative studies between ultrasound cavitation and microfluidizer

In the present investigation, the operating efficiency of a bench-top air-driven microfluidizer has been compared to that of a bench-top high power ultrasound horn in the production of pharmaceutical grade nanoemulsions using aspirin as a model drug. The influence of important process variables as well as the pre-homogenization and drug loading on the resultant mean droplet diameter and size distribution of emulsion droplets was studied in an oil-in-water nanoemulsion incorporated with a model drug aspirin. Results obtained show that both the emulsification methods were capable of producing very fine nanoemulsions containing aspirin with the minimum droplet size ranging from 150 to 170 nm. In case of using the microfluidizer, it has been observed that the size of the emulsion droplets obtained was almost independent of the applied microfluidization pressure (200–600 bar) and the number of passes (up to 10 passes) while the pre-homogenization and drug loading had a marginal effect in increasing the droplet size. Whereas, in the case of ultrasound emulsification, the droplet size was generally decreased with an increase in sonication amplitude (50–70%) and period of sonication but the resultant emulsion was found to be dependent on the pre-homogenization and drug loading. The STEM microscopic observations illustrated that the optimized formulations obtained using ultrasound cavitation technique are comparable to microfluidized emulsions. These comparative results demonstrated that ultrasound cavitation is a relatively energy-efficient yet promising method of pharmaceutical nanoemulsions as compared to microfluidizer although the means used to generate the nanoemulsions are different.     

The effect of acoustically-induced cavitation on the permeance of a bullfrog urinary bladder

It is well known that ultrasound enhances drug delivery to tissues, although there is not a general consensus about the responsible mechanisms. However, it is known that the most important factor associated with ultrasonically-enhanced drug permeance through tissues is cavitation. Here we report results from research conducted using a experimental approach adapted from single bubble sonoluminescence experiments which generates very well defined acoustic fields and allows controlled activation and location of cavitation. The experimental design requires that a biological tissue be immersed inside a highly degassed liquid media to avoid random bubble nucleation. Therefore, live frog bladders were used as the living tissue due to their high resistance to hypoxia. Tissue membrane permeance was measured using radiolabeled urea. The results show that an increase in tissue permeance only occurs when cavitation is present near the tissue membrane. Moreover, confocal microscopy shows a direct correlation between permeance increases and physical damage to the tissue.     

New approach in synthesis,characterization and release study of pH-sensitive polymeric micelles,based on PLA-Lys-<Emphasis Type="Italic">b</Emphasis>-PEGm,conjugated with doxorubicin

Amphiphilic block copolymers are well established as building blocks for the preparation of micellar drug carriers. The functional polymer micelles possess several advantages, such as high drug efficiency, targeted delivery, and minimized cytotoxicity. The synthesis of block copolymers using nano-structured templates has emerged as a useful and versatile approach for preparing drug carriers. Here, we report the synthesis of a smart polymeric compound of a diblock PLA-Lys-b-PEG copolymer containing doxorubicin. We have synthesized functionalized diblock copolymers, with lysinol, poly(lactide) and monomethoxy poly(ethylene glycol) via thermal ring-opening polymerization and a subsequent six-step substitution reaction. A variety of spectroscopic methods were employed here to verify the product of our synthesis. ¹H-Nuclear magnetic resonance and Fourier transform infrared studies validated the expected synthesis of copolymers. Doxorubicin is chemically loaded into micelles, and the ex vitro release can be evaluated either in weak acidic or in SBF solution by UV–vis spectroscopy. Dynamic light scattering, thermo gravimetric analysis, and size exclusion chromatography have also been used.     

Thioflavin T Displays Enhanced Fluorescence Selectively Inside Anionic Micelles and Mammalian Cells

Thioflavin T (ThT) has been widely employed to detect amyloid fibrils in tissues and recently in presence of SDS micelles. However, the contribution of membranes or micelles to ThT fluorescence has never been investigated. In this paper, we show for the first time that the anionic micellar microenvironment of SDS has a profound impact on the absorption and fluorescence spectra of ThT in sharp contrast to cationic (CTAB) and neutral micelles (Triton X-100 & Tween 20). Unlike CTAB or Triton X-100 or Tween 20 micelles, formation of SDS micelles shifts the λ_max for ThT absorption from 412 nm in buffer to 428 nm inside the micelle, with a 28% increase in the peak molar absorptivity and a ∼13 fold increase in ThT fluorescence (λ_max = 489 nm). Extending these observations to cell plasma membranes, we show that ThT can quickly enter and appear selectively fluorescent inside mammalian cells like BHK21 and HT29, against a dark background owing to negligible fluorescence from free ThT in aqueous medium. The above results suggest that ThT can be a useful probe for live cell imaging and for selectively labeling micelles on the basis of the charge in the polar headgroup. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pulsed-wave low-dose ultrasound hyperthermia selectively enhances nanodrug delivery and improves antitumor efficacy for brain metastasis of breast cancer

The clinical application of chemotherapeutics for brain tumors remains a challenge due to limitation of blood-brain barrier/blood-tumor barrier (BBB/BTB). In this study, we investigated the effects of low-dose focused ultrasound hyperthermia (UH) on the delivery and therapeutic efficacy of pegylated liposomal doxorubicin (PLD) for brain metastasis of breast cancer. Murine breast cancer cells (4T1-luc2) expressing firefly luciferase were implanted into mouse striatum as a brain tumor model. The mice were intravenously injected with PLD with/without transcranial pulsed-wave/continuous-wave UH (pUH/cUH) treatment on day-6 after tumor implantation. pUH (frequency: 500 kHz, PRF: 1000 Hz, duty cycle: 50%) was conducted under equal acoustic power (2.2-Watt) and sonication duration (10-min) as cUH. The amounts of doxorubicin accumulated in the normal brain and tumor tissues were measured with fluorometry. The tumor growth responses for the control, pUH, PLD, PLD + cUH, and PLD + pUH groups were evaluated with IVIS. The PLD distribution and cell apoptosis were assessed with immunofluorescence staining. The results showed that pUH significantly enhanced the PLD delivery into brain tumors and the tumor growth was further inhibited by PLD + pUH without damaging the sonicated normal brain tissues. This indicates that low-dose transcranial pUH is a promising method to selectively enhance nanodrug delivery and improve the brain tumor treatment.     

流体控制方程的超声空化泡动力学模拟*

本文基于流体动力学控制方程和VOF模型,在FLUENT 14.5软件环境下对超声空化泡进行数值模拟。首先研究了超声空化泡一个周期内的形态变化,并且利用空化泡形态变化的最大面积、最小面积、膨胀时间、收缩时间等数值结果分析超声参数对空化效果的影响。同时探究了双频超声作用下空化泡运动的变化,计算结果表明:在其他条件相同的情况下,在1~5MPa范围内,超声声压幅值为3MPa时空化效果最好;当超声频率大于20kHz时,空化效果随着超声频率的增大而降低。对于频率相同的双频超声,较声压幅值为其两倍的单频超声有更好的空化效果;对于频率不同的双频超声,空化效果受到频率差的影响。     

Photon correlation spectroscopy in micellar solutions of sodium and potassium oleate

Photon correlation spectroscopy was applied to concentrated micellar solutions of sodium and potassium oleate. In rod-like micellar phases the relaxation times of the concentration fluctuation were measured as a function of temperature. A remarkable temperature dependence of the relaxation time was found in the isotropic phase of rod-like micelles. This suggests that the rotational diffusion is strongly reduced by the entanglement effect. The thermal expansion coefficient of rod-like micelles was determined.     

Spatial distribution of sonoluminescence and sonochemiluminescence generated by cavitation bubbles in 1.2 MHz focused ultrasound field

An intensified charge coupled device (ICCD) camera was used to observe the spatial distribution of sonoluminescence (SL) and sonochemiluminescence (SCL) generated by cavitation bubbles in a 1.2 MHz focused ultrasound (FU) field in order to investigate the mechanisms of acoustic cavitation under different sonication conditions for FU therapeutic applications.It was found that SL emissions were located in the post-focal region. When the intensity of SL and SCL increased as the power rose, the growth of SCL was much higher than that of SL. In the post-focal region, the SCL emissions moved along specific paths and formed branch-like streamers. At the beginning of the ultrasound irradiation, cavitation bubbles generated SCL in both the pre-focal and the post-focal region. When the electrical power or the sonication time increased, the SCL in the post-focal region increased and became higher than that in the pre-focal region. The intensity of SCL in the focal region is usually the weakest because of “oversaturation”.The spatial distribution of SCL near a tissue boundary differed from that obtained in free fields. It organized into special structures under different acoustic amplitudes. When the electrical power was relatively low, the SCL emission was conical shape which suggested a standing wave formation at the tissue-fluid boundary. When the electrical power exceeded a certain threshold, only a bright spot could be captured in the focus. The cavitation bubbles which centralized in the focus concentrated energy and hindered the formation of standing waves. With rising electrical power at high levels, besides a bright spot in the focus, there were some irregular light spots in pre-focal region, which indicated some cavitation bubbles or small bubble clusters achieved the threshold of SCL and induced the reaction with the luminol solution.     

Multifunctional antitumor magnetite/chitosan-<Emphasis Type="SmallCaps">l</Emphasis>-glutamic acid (core/shell) nanocomposites

The development of anticancer drug delivery systems based on biodegradable nanoparticles has been intended to maximize the localization of chemotherapy agents within tumor interstitium, along with negligible drug distribution into healthy tissues. Interestingly, passive and active drug targeting strategies to cancer have led to improved nanomedicines with great tumor specificity and efficient chemotherapy effect. One of the most promising areas in the formulation of such nanoplatforms is the engineering of magnetically responsive nanoparticles. In this way, we have followed a chemical modification method for the synthesis of magnetite/chitosan-l-glutamic acid (core/shell) nanostructures. These magnetic nanocomposites (average size ≈340 nm) exhibited multifunctional properties based on its capability to load the antitumor drug doxorubicin (along with an adequate sustained release) and its potential for hyperthermia applications. Compared to drug surface adsorption, doxorubicin entrapment into the nanocomposites matrix yielded a higher drug loading and a slower drug release profile. Heating characteristics of the magnetic nanocomposites were investigated in a high-frequency alternating magnetic gradient: a stable maximum temperature of 46 °C was successfully achieved within 40 min. To our knowledge, this is the first time that such kind of stimuli-sensitive nanoformulation with very important properties (i.e., magnetic targeting capabilities, hyperthermia, high drug loading, and little burst drug release) has been formulated for combined antitumor therapy against cancer.