Experimental study of HIFU incomplete ablation on the damage effect and prognosis of rabbit VX2 breast cancer model.

PURPOSE: High-intensity focused ultrasound (HIFU) represents an emerging noninvasive modality for tumor treatment. While biological responses and immunological change associated with incomplete ablation have not been thoroughly investigated. This study aims to evaluate the damage effect of HIFU incomplete ablation via establishing animal model and further explore its possible mechanism to inhibit tumor growth. METHODS: The rabbit VX2 breast cancer model was established and received HIFU treatment with complete ablation (100% tumor volume) and incomplete ablation (about 80% tumor volume) under real-time B-ultrasound monitoring. Histopathological alterations, dynamics of tumor cell apoptosis and proliferation, expression levels of VEGF, MMP-9, IL-2R, TGF-ß1, HSP-70, IL-6, IL-8, and INF-γ, and the presence of circulating tumor cells (CTCs) were evaluated post-HIFU incomplete ablation. RESULTS: For HIFU 80% ablation group, there was an 85.85% reduction in tumor volume 21 days post-intervention. A marked increase in tumor cell apoptosis and a concomitant decrease in proliferation were observed. Notably, distant tumor metastasis rates, CTC counts, and expression levels of VEGF, MMP-9, IL-2R, TGF-ß1, IL-6, and IL-8 were significantly reduced. In contrast, INF-γ and HSP-70 expressions were notably elevated, aligning with findings from the 100% ablation group. CONCLUSIONS: HIFU incomplete ablation, with an 80% tumor ablation rate, induces substantial tumor damage, augments tumor cell apoptosis, and triggers an anti-tumor immune response, curtailing metastasis. These insights may underpin further investigations into the therapeutic implications of HIFU incomplete ablation.

Low intensity ultrasound-mediated drug-loaded nanoparticles intravaginal drug delivery: an effective synergistic therapy scheme for treatment of vulvovaginal candidiasis.

PURPOSE: Vulvovaginal candidiasis (VVC) is a mucosal infection of the female lower genital tract for which treatment using conventional antifungal drugs shows limited effectiveness. Herein, amphotericin B-loaded poly(lactic-co-glycolic acid)-polyethylene glycol (PLGA-PEG) nanoparticles (AmB-NPs) were fabricated and combined with low intensity ultrasound (US) to mediate AmB-NPs intravaginal drug delivery to achieve productive synergistic antifungal activity in a rabbit model of VVC. METHODS: Polymeric AmB-NPs were fabricated by a double emulsion method and the physical characteristics and biosafety of nanoparticles were analyzed. The distribution and tissue permeability of nanoparticles after intravaginal ultrasound irradiation (1.0 MHz, 1.0 W/cm2, 5 min, 50% duty ratio) were observed in the vagina. The synergistic therapeutic activity of US-mediated AmB-NPs treatment was evaluated using an experimental rabbit model of VVC. Vaginal C. albicans colony counts, the pathological structure of the vagina epithelium, and Th1/Th2/Th17-type cytokine and oxidative stress levels were analyzed to investigate the therapeutic effect in vivo. RESULTS: The prepared AmB-NPs showed an obvious shell and core structure with uniform size and good dispersion and displayed high biosafety and US-sensitive slow drug release. Ultrasound significantly enhanced nanoparticle transport through the mucus and promoted permeability in the vaginal tissue. US-mediated AmB-NPs treatment effectively increased drug sensitivity, even in the presence of the vaginal mucus barrier in vitro. On the seventh day after treatment in vivo, the combination treatment of AmB-NPs and US significantly reduced the fungal load in the vagina, achieving over 95% clearance rates, and also improved the pathological epithelium structural damage and glycogen secretion function. The expression of Th1 (IFN-γ, IL-2) and Th17 (IL-17) cytokines were significantly increased and Th2 (IL-6, IL-10) cytokines significantly decreased in the US + AmB-NP group. Furthermore, US-mediated AmB-NPs treatment effectively increased C. albicans intracellular reactive oxygen species (ROS) levels and promoted vaginal oxidation and antioxidants to normal levels. CONCLUSION: US-mediated drug-loaded nanoparticles with intravaginal drug delivery exhibited a productive synergistic antifungal effect, which may provide a new non-invasive, safe, and effective therapy for acute or recurrent fungal vaginitis.

A synergistic bactericidal effect of low-frequency and low-intensity ultrasound combined with levofloxacin-loaded PLGA nanoparticles on M. smegmatis in macrophages.

PURPOSE: Tuberculosis (TB) is a highly infectious disease caused by Mycobacterium tuberculosis (Mtb), which often parasites in macrophages. This study is performed to investigate the bactericidal effect and underlying mechanisms of low-frequency and low-intensity ultrasound (LFLIU) combined with levofloxacin-loaded PLGA nanoparticles (LEV-NPs) on M. smegmatis (a surrogate of Mtb) in macrophages. METHODS AND RESULTS: The LEV-NPs were prepared using a double emulsification method. The average diameter, zeta potential, polydispersity index, morphology, and drug release efficiency in vitro of the LEV-NPs were investigated. M. smegmatis in macrophages was treated using the LEV-NPs combined with 42 kHz ultrasound irradiation at an intensity of 0.13 W/cm2 for 10 min. The results showed that ultrasound significantly promoted the phagocytosis of nanoparticles by macrophages (P < 0.05). In addition, further ultrasound combined with the LEV-NPs promoted the production of reactive oxygen species (ROS) in macrophage, and the apoptosis rate of the macrophages was significantly higher than that of the control (P < 0.05). The transmission electronic microscope showed that the cell wall of M. smegmatis was ruptured, the cell structure was incomplete, and the bacteria received severe damage in the ultrasound combined with the LEV-NPs group. Activity assays showed that ultrasound combined with the LEV-NPs exhibited a tenfold higher antibacterial activity against M. smegmatis residing inside macrophages compared with the free drug. CONCLUSION: These data demonstrated that ultrasound combined with LEV-NPs has great potential as a therapeutic agent for TB.

Synergistic Antifungal Effect of Amphotericin B-Loaded Poly(Lactic-Co-Glycolic Acid) Nanoparticles and Ultrasound against Candida albicans Biofilms.

Candida albicans is a human opportunistic pathogen that causes superficial and life-threatening infections. An important reason for the failure of current antifungal drugs is related to biofilm formation, mostly associated with implanted medical devices. The present study investigated the synergistic antifungal efficacy of low-frequency and low-intensity ultrasound combined with amphotericin B (AmB)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (AmB-NPs) against C. albicans biofilms. AmB-NPs were prepared by a double-emulsion method and demonstrated lower toxicity than free AmB. We then established biofilms and treated them with ultrasound and AmB-NPs separately or jointly in vitro and in vivo The results demonstrated that the activity, biomass, and proteinase and phospholipase activities of biofilms were decreased significantly after the combination treatment of AmB-NPs with 42 kHz of ultrasound irradiation at an intensity of 0.30 W/cm2 for 15 min compared with the controls, with AmB alone, or with ultrasound treatment alone (P < 0.01). The morphology of the biofilms was altered remarkably after joint treatment based on confocal laser scanning microscopy (CLSM), especially in regard to reduced thickness and loosened structure. Furthermore, the same synergistic effects were found in a subcutaneous catheter biofilm rat model. The number of CFU from the catheter exhibited a significant reduction after joint treatment with AmB-NP and ultrasound for seven continuous days, and CLSM and scanning electron microscopy (SEM) images revealed that the biofilm on the catheter surface was substantially eliminated. This method may provide a new noninvasive, safe, and effective therapy for C. albicans biofilm infection.

Bactericidal effects of high intensity focused ultrasound on Bacillus Calmette-Guerin in vivo and in vitro.

Purpose: The objective of this study was to investigate the bactericidal effects of high intensity focused ultrasound (HIFU) on Bacillus Calmette-Guerin (BCG, a substitute for Mycobacterium tuberculosis) in vitro and in vivo, and to explore the underlying mechanisms. Materials and methods: HIFU, at a fixed frequency of 1 MHz, was applied to both BCG culture suspensions and subcutaneous BCG abscesses in rats. Results: HIFU irradiation significantly reduced the bacterial survival rate and caused temperature elevations both in vitro and in vivo. Furthermore, BCG suspensions irradiated for 15 s at 3185 and 6369 W/cm2 had increased cell wall damage, which resulted in morphological changes compared to the untreated control group. Additionally, we observed histological changes in the rat subcutaneous abscesses after HIFU ablation at 6369 W/cm2. H&E staining of infected lesions showed coagulative necrosis with central nucleus dissolution and increased infiltration of inflammatory cells, as well as nuclear pyknosis and nuclear fragmentation in the periphery. The volumes of the subcutaneous abscesses in the HIFU-treated group were significantly lower than those in the sham-treated group. Conclusion: HIFU has the therapeutic potential to treat BCG-infected tissues in rats. We theorize that a combination of mechanical, cavitation, and thermal effects most efficiently inactivate BCG bacteria via HIFU. This study is expected to provide a bio-plausible basis for a noninvasive and effective treatment for tuberculosis.

Damage Effects on Bacille Calmette-Guérin by Low-Frequency, Low-Intensity Ultrasound: A Pilot Study.

OBJECTIVES: To perform an in vitro experimental study of the possible damage effects on Bacille Calmette-Guérin (BCG) by low-frequency (42-kHz) ultrasound (US) irradiation at low spatially and temporally averaged intensities and different exposure times. METHODS: A 2-mL BCG suspension was added to the wells of a 24-well cell culture plate. Then the samples were randomly divided into 4 groups, each group including 3 wells, with group 1 as a control group and groups 2, 3, and 4, as US treatment groups. The samples for groups 2, 3, and 4 were irradiated with US at 0.13 W/cm(2) for 5 minutes, 0.13 W/cm(2) for 15 minutes, and 1.53 W/cm(2) for 15 minutes, respectively. After irradiation, the temperature, ratio of damage, and structure of the bacteria were examined. The cavitation effect of the device was detected by the passive cavitation detection method. RESULTS: After US irradiation at the different doses (intensity and exposure time), no significant temperature change was found in all sample suspensions. The ratio of bacterial damage tested by flow cytometry and the optical density of the suspensions as assayed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric method showed that the US-irradiated groups were significantly different from the control group. The BCG damage ratio reached 28% at the intensity of 1.53 W/cm(2). Transmission electron microscopic results showed that the bacterial structure of BCG could be destroyed by low-frequency, low-intensity US. CONCLUSIONS: Low-frequency, low-intensity US can cause acute injury to BCG, and the degree of injury is closely correlated with the US dose applied.

Oxygen-producing and pH-responsive targeted DNA nanoflowers for enhanced chemo-sonodynamic therapy of lung cancer.

Lung cancer is the deadliest kind of cancer in the world, and the hypoxic tumor microenvironment can significantly lower the sensitivity of chemotherapeutic drugs and limit the efficacy of different therapeutic approaches. In order to overcome these problems, we have designed a drug-loaded targeted DNA nanoflowers encoding AS1411 aptamer and encapsulating chemotherapeutic drug doxorubicin and oxygen-producing drug horseradish peroxidase (DOX/HRP-DFs). These nanoflowers can release drugs in response to acidic tumor microenvironment and alleviate tumor tissue hypoxia, enhancing the therapeutic effects of chemotherapy synergistic with sonodynamic therapy. Owing to the encoded drug-loading sequence, the doxorubicin loading rate of DNA nanoflowers reached 73.24 ± 3.45%, and the drug could be released quickly by disintegrating in an acidic environment. Furthermore, the AS1411 aptamer endowed DNA nanoflowers with exceptional tumor targeting properties, which increased the concentration of chemotherapeutic drug doxorubicin in tumor cells. It is noteworthy that both in vitro and in vivo experiments demonstrated DNA nanoflowers could considerably improve the hypoxia of tumor cells, which enabled the generation of sufficient reactive oxygen species in combination with ultrasound, significantly enhancing the therapeutic effect of sonodynamic therapy and evidently inhibiting tumor growth and metastasis. Overall, this DNA nanoflowers delivery system offers a promising approach for treating lung cancer.

An Oxygen Supply Strategy for Sonodynamic Therapy in Tuberculous Granuloma Lesions Using a Catalase-Loaded Nanoplatform.

Purpose: Tuberculosis (TB) is a chronic disease caused by Mycobacterium tuberculosis (MTB) that remains a major global health challenge. One of the main obstacles to effective treatment is the heterogeneous microenvironment of TB granulomas. This study aimed to investigate the potential of a hypoxic remission-based strategy to enhance the outcome of tuberculosis treatment when implemented in combination with ultrasound. Methods: A PLGA nanoparticle (LEV@CAT-NPs) loaded with levofloxacin (LEV) and catalase (CAT) was fabricated by a double emulsification method, and its physical characteristics, oxygen production capacity, drug release capacity, and biosafety were thoroughly investigated. The synergistic therapeutic effects of ultrasound (US)-mediated LEV@CAT-NPs were evaluated using an experimental mouse model of subcutaneous tuberculosis granuloma induced by Bacille Calmette-Guérin (BCG) as a substitute for MTB. Results: LEV@CAT-NPs exhibited excellent oxygen production capacity, biosafety, and biocompatibility. Histological analysis revealed that ultrasound-mediated LEV@CAT-NPs could effectively remove bacteria from tuberculous granulomas, significantly alleviate the hypoxia state, reduce the necrotic area and inflammatory cells within the granuloma, and increase the penetration of dyes in granuloma tissues. The combined treatment also reduced the serum levels of inflammatory cytokines (eg, TNF-α, IL-6, and IL-8), and significantly downregulated the expression of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF). These results suggested that the synergistic treatment of ultrasound-mediated LEV@CAT-NPs effectively eradicated the bacterial infection and reversed the hypoxic microenvironment of tuberculous granulomas, further promoting tissue repair. Conclusion: This study provides a non-invasive and new avenue for treating refractory tuberculosis infections. The potential role of regulating hypoxia within infected lesions as a therapeutic target for infection deserves further exploration in future studies.

Synergistic antibacterial effects of ultrasound combined nanoparticles encapsulated with cellulase and levofloxacin on Bacillus Calmette-Guérin biofilms.

Tuberculosis is a chronic infectious disease, the treatment of which is challenging due to the formation of cellulose-containing biofilms by Mycobacterium tuberculosis (MTB). Herein, a composite nanoparticle loaded with cellulase (CL) and levofloxacin (LEV) (CL@LEV-NPs) was fabricated and then combined with ultrasound (US) irradiation to promote chemotherapy and sonodynamic antimicrobial effects on Bacillus Calmette-Guérin bacteria (BCG, a mode of MTB) biofilms. The CL@LEV-NPs containing polylactic acid-glycolic acid (PLGA) as the shell and CL and LEV as the core were encapsulated via double ultrasonic emulsification. The synthesized CL@LEV-NPs were uniformly round with an average diameter of 196.2 ± 2.89 nm, and the zeta potential of -14.96 ± 5.35 mV, displaying high biosafety and sonodynamic properties. Then, BCG biofilms were treated with ultrasound and CL@LEV-NPs separately or synergistically in vivo and in vitro. We found that ultrasound significantly promoted biofilms permeability and activated CL@LEV-NPs to generate large amounts of reactive oxygen species (ROS) in biofilms. The combined treatment of CL@LEV-NPs and US exhibited excellent anti-biofilm effects, as shown by significant reduction of biofilm biomass value and viability, destruction of biofilm architecture in vitro, elimination of biofilms from subcutaneous implant, and remission of local inflammation in vivo. Our study suggested that US combined with composite drug-loaded nanoparticles would be a novel non-invasive, safe, and effective treatment modality for the elimination of biofilm-associated infections caused by MTB.

Mucus-Permeable Sonodynamic Therapy Mediated Amphotericin B-Loaded PEGylated PLGA Nanoparticles Enable Eradication of Candida albicans Biofilm.

Background: Candida albicans (C. albicans) forms pathogenic biofilms, and the dense mucus layer secreted by the epithelium is a major barrier to the traditional antibiotic treatment of mucosa-associated C. albicans infections. Herein, we report a novel anti-biofilm strategy of mucus-permeable sonodynamic therapy (mp-SDT) based on ultrasound (US)-mediated amphotericin B-loaded PEGylated PLGA nanoparticles (AmB-NPs) to overcome mucus barrier and enable the eradication of C. albicans biofilm. Methods: AmB-NPs were fabricated using ultrasonic double emulsion method, and their physicochemical and sonodynamic properties were determined. The mucus and biofilm permeability of US-mediated AmB-NPs were further investigated. Moreover, the anti-biofilm effect of US-mediated AmB-NPs treatment was thoroughly evaluated on mucus barrier abiotic biofilm, epithelium-associated biotic biofilm, and C. albicans-induced rabbit vaginal biofilms model. In addition, the ultrastructure and secreted cytokines of epithelial cells and the polarization of macrophages were analyzed to investigate the regulation of local cellular immune function by US-mediated AmB-NPs treatment. Results: Polymeric AmB-NPs display excellent sonodynamic performance with massive singlet oxygen (1O2) generation. US-mediated AmB-NPs could rapidly transport through mucus and promote permeability in biofilms, which exhibited excellent eradicating ability to C. albicans biofilms. Furthermore, in the vaginal epithelial cells (VECs)-associated C. albicans biofilm model, the mp-SDT scheme showed the strongest biofilm eradication effect, with up to 98% biofilm re-formation inhibition rate, improved the ultrastructural damage, promoted local immune defense enhancement of VECs, and regulated the polarization of macrophages to the M1 phenotype to enhance macrophage-associated antifungal immune responses. In addition, mp-SDT treatment exhibited excellent therapeutic efficacy against C. albicans-induced rabbit vaginitis, promoted the recovery of mucosal epithelial ultrastructure, and contributed to the reshaping of a healthier vaginal microbiome. Conclusion: The synergistic anti-biofilm strategies of mp-SDT effectively eradicated C. albicans biofilm and simultaneously regulated local antifungal immunity enhancement, which may provide a new approach to treat refractory drug-resistant biofilm-associated mucosal candidiasis.

Multifunctional Theranostic Nanoparticles for Enhanced Tumor Targeted Imaging and Synergistic FUS/Chemotherapy on Murine 4T1 Breast Cancer Cell.

Purpose: Triple negative breast cancer (TNBC) is challenging for effective remission due to its very aggressive, extremely metastatic and resistant to conventional chemotherapy. Herein, a multifunctional theranostic nanoparticle was fabricated to enhance tumor targeted imaging and promote focused ultrasound (FUS) ablation and chemotherapy and sonodynamic therapy (SDT). A multi-modal synergistic therapy can improve the therapeutic efficacy and prognosis of TNBC. Methods: AS1411 aptamer modified PEG@PLGA nanoparticles encapsulated with perfluorohexane (PFH) and anti-cancer drug doxorubicin (DOX) were constructed (AS1411-DOX/PFH-PEG@PLGA) to enhance tumor targeted imaging to guide ablation and synergistic effect of FUS/chemotherapy. FUS was utilized to trigger the co-release of doxorubicin and simultaneously PFH phase transition and activate DOX for SDT effect. The physicochemical, phase-changeable imaging capability, biosafety of nanoparticles and multi-mode synergistic effects on growth of TNBC were thoroughly evaluated in vivo and in vitro. Results: The synthesized AS1411-DOX/PFH-PEG@PLGA (A-DPPs) nanoparticles are uniformly round with an average diameter of 306.03 ± 5.35 nm and the zeta potential of -4.05 ± 0.13 mV, displaying high biosafety and FUS-responsive drug release in vitro and in vivo. AS1411 modified NPs specifically bind to 4T1 cells and elevate the ultrasound contrast agent (UCA) image contrast intensity via PFH phase-transition after FUS exposure. Moreover, the combined treatment of A-DPPs nanoparticles with FUS exhibited significantly higher apoptosis rate, stronger inhibitory effect on 4T1 cell invasion in vitro, induced more reactive oxygen species (ROS), and enhanced anti-tumor effect compared to a single therapy (p < 0.05). Additionally, the joint strategy resulted in more intense cavitation effect and larger ablated areas and reduced energy efficiency factor (EEF) both in vitro and in vivo. Conclusion: The multifunctional AS1411-DOX/PFH-PEG@PLGA nanoparticles can perform as a marvelous synergistic agent for enhanced FUS/chemotherapy, promote real-time contrast enhanced US imaging and improve the therapeutic efficacy and prognosis of TNBC.

Diagnosis and Etiological Analysis of Gastroesophageal Reflux Disease by Gastric Filling Ultrasound and GerdQ Scale.

Objective: To investigate the diagnosis and etiological analysis of GERD by gastric filling ultrasound and GerdQ scale. Methods: The clinical data of 100 suspected GERD patients were selected for retrospective analysis. The selection time was from June 2016 to June 2019. According to the gold standard (endoscopy) results, they were divided into the gastroesophageal reflux group (positive, n = 62) and the nongastroesophageal reflux group (negative, n = 38); both gastric filling ultrasound and GerdQ scale examination were performed to compare the positive predictive value and negative predictive value, evaluate the abdominal esophageal length, His angle, and GerdQ scale score, and analyze the AUC value, sensitivity, specificity, and Youden index of His angle, length of abdominal esophagus, combined ultrasound parameters, and GerdQ scale in the diagnosis of GERD. Results: 100 patients with suspected GERD were diagnosed as GERD by endoscopy; in a total of 62 cases, the percentage was 62.00%. Among them, 28 cases were caused by the abnormal structure and function of the antireflux barrier, accounting for 45.16%, 18 cases were caused by the reduction of acid clearance of the esophagus, accounting for 29.03%, and 16 cases were caused by the weakening of the esophageal mucosal barrier, accounting for 25.81%. After ultrasound detection, the positive predictive value was 88.71% and the negative predictive value was 81.58%; after the GerdQ scale was tested, the positive predictive value was 71.43% and the negative predictive value was 54.05%. The length of the abdominal esophagus in the gastroesophageal reflux group was lower than that of the nongastroesophageal reflux group, while the scores of His angle and GerdQ scale were higher than those in the gastroesophageal reflux group (P < 0.05). ROC curve analysis showed that the AUC values of His angle, length of abdominal esophagus, combined ultrasound parameters, and GerdQ scale to diagnose GERD were 0.957, 0.861, 0.996, and 0.931 (P < 0.05), their sensitivity was 93.5%, 98.40%, 98.40%, and 90.30%, and the specificity was 92.10%, 63.20%, 100.00%, and 92.10%, respectively. Conclusion: Both gastric filling ultrasound and GerdQ scale have a certain application value in the diagnosis of GERD, but the former has a higher accuracy rate, and it is more common for gastroesophageal reflux caused by abnormal structure and function of antireflux barrier in etiological analysis.

The Clinicians' Satisfaction with Clinical Pathway Implementation: Preliminary Development of an Assessment Scale in China.

PURPOSE: To develop a satisfaction scale of Chinese clinicians with Clinical Pathway (CP) implementation and evaluate its validity, reliability and item discrimination. MATERIALS AND METHODS: Literature review, in-person interviews, and Delphi were used to design the scale. Data were collected in two phases using random sampling on the spot and an online survey. In the first phase, data from 239 clinicians were investigated in exploratory factor analysis. In the second phase, 513 valid questionnaires were collected and used for confirmatory factor analysis. RESULTS: The scale developed in this study has three dimensions (organization support, process identity, and effect perception) and a total of 21 items. Cronbach's alpha of each dimension was higher than 0.9. The 3-factor model had enough fitness (χ2/df = 5.602, NFI = 0.926, IFI = 0.938, CFI = 0.938, RFI = 0.914, TLI = 0.929, RMSEA = 0.095, RMR = 0.045). The standardized factor loadings of 21 variables were between 0.742 and 0.949. The average variance extracted (AVE) of each dimension was higher than 0.7, and the construct reliability (CR) of the dimensions was higher than 0.9. The Chi-square difference test results showed that the difference value between the unlimited and limited model of each two potential constructs was higher than 3.84 (P < 0.001). CONCLUSION: The clinicians' satisfaction scale developed in this study has good construct validity, convergent validity, discriminant validity, internal consistency, and item discrimination. This suggests its usefulness as a tool to assess the satisfaction of clinicians in the implementation of CP in China.

The enhancing antifungal effect of AD1 aptamer-functionalized amphotericin B-loaded PLGA-PEG nanoparticles with a low-frequency and low-intensity ultrasound exposure on C.albicans biofilm through targeted effect.

The prevalence and fatality rates with fungal biofilm-associated infections urgently need to develop targeted therapeutic approaches to augment the action of antifungal drugs. This study developed amphotericin B-loaded PLGA-PEG nanoparticles (AmB-NPs) with AD1 aptamer conjugation on its surface via an EDC/NHS technique. Their high nuclease resistance of the conjugation was confirmed by PAGE gel electrophoresis. The targeting and toxicity of AD1-AmB-NPs in the subcutaneous C. albicans infection model were evaluated. AD1-AmB-NPs can bind to different morphological forms(including yeast cells, germ tubes, hyphae) of C. albicans biofilms and extracellular matrix material. Low-frequency and low-intensity ultrasound (LFU, with a fixed frequency of 42 kHz, at the intensity of 0.30 W/cm2 for 15 min) significantly promoted permeability of the biofilm and allowed AD1-AmB-NPs into the deepest layers of the biofilm. After 7 days of treatment, the combination treatment of AD1-AmB-NPs and LFU, kills at least 99% of the biofilm fungal population in vivo comparison with ultrasound alone or AD1-AmB-NPs alone, and returned to normal subcutaneously. Our data suggest that the combined strategy of AD1-AmB-NPs and ultrasound treatment selective delivered of therapeutic drugs to the infection site and exhibited significant synergistic antifungal effects.

Levofloxacin-Loaded Nanosonosensitizer as a Highly Efficient Therapy for Bacillus Calmette-Guérin Infections Based on Bacteria-Specific Labeling and Sonotheranostic Strategy.

PURPOSE: The rapid emergence of multidrug-resistant Mycobacterium tuberculosis (MTB) poses a significant challenge to the treatment of tuberculosis (TB). Sonodynamic antibacterial chemotherapy (SACT) combined with sonosensitizer-loaded nanoparticles with targeted therapeutic function is highly expected to eliminate bacteria without fear of drug resistance. This study aimed to investigate the antibacterial effect and underlying mechanism of levofloxacin-loaded nanosonosensitizer with targeted therapeutic function against Bacillus Calmette-Guérin bacteria (BCG, an MTB model). METHODS: This study developed levofloxacin-loaded PLGA-PEG (poly lactide-co-glycolide-polyethylene glycol) nanoparticles with BM2 aptamer conjugation on its surface using the crosslinking agents EDC and NHS (BM2-LVFX-NPs). The average diameter, zeta potential, morphology, drug-loading properties, and drug release efficiency of the BM2-LVFX-NPs were investigated. In addition, the targeting and toxicity of BM2-LVFX-NPs in the subcutaneous BCG infection model were evaluated. The biosafety, reactive oxygen species (ROS) production, cellular phagocytic effect, and antibacterial effect of BM2-LVFX-NPs in the presence of ultrasound stimulations (42 kHz, 0.67 W/cm2, 5 min) were also systematically evaluated. RESULTS: BM2-LVFX-NPs not only specifically recognized BCG bacteria in vitro but also gathered accurately in the lesion tissues. Drugs loaded in BM2-LVFX-NPs with the ultrasound-responsive feature were effectively released compared to the natural state. In addition, BM2-LVFX-NPs exhibited significant SACT efficiency with higher ROS production levels than others, resulting in the effective elimination of bacteria in vitro. Meanwhile, in vivo experiments, compared with other options, BM2-LVFX-NPs also exhibited an excellent therapeutic effect in a rat model with BCG infection after exposure to ultrasound. CONCLUSION: Our work demonstrated that a nanosonosensitizer formulation with LVFX could efficiently translocate therapeutic drugs into the cell and improve the bactericidal effects under ultrasound, which could be a promising strategy for targeted therapy for MTB infections with high biosafety.

Multiparameter prediction control of in vitro drug delivery into mycobacterium smegmatis induced by microbubble-enhanced sonoporation.

The antibacterial method induced by microbubble-enhanced sonoporation has shown its great potential in facilitating drug delivery into thallus. The enhanced drug delivery induced by microbubble-enhanced sonoporation is a complex event which can be affected by various physical parameters. How to determine the correlation between experimental parameters and the drug delivery efficiency to give the instruction on reasonably choosing the parameters and achieve the control of drug delivery efficiency is impeding further investigations for this complex biophysical process. In the present work, we have explored a number of key parameters affecting the drug delivery efficiency induced by microbubble-enhanced sonoporation using multivariate biological experiments. To achieve the control of the drug delivery efficiency, a multiparameter prediction control method based on modified artificial neural network is presented in this paper. This method is a new modeling method based on combined back-propagation neural network and the multiple model idea to establish quantitative relationship between experimental parameters and drug delivery efficiency. By analyzing the experimental samples, a mapping relationship expression can be deduced to determine the input and output variables of artificial neural network models. Experimental samples were divided into training and test samples. We trained models based on back-propagation neural network to establish their quantitative relationship. In this model, the multiple model idea was introduced into the selection of training samples to modify the traditional back-propagation neural network model to avoid model mismatch caused by poor training sample selection. Numerical experiments results have shown that compared with the traditional model, the identification results obtained by modified model are more closed to experimental results. It is elucidated that an appropriately trained network can act as a good alternative for costly and time-consuming experiments. The findings of this study indicate that this approach can realize the prediction of drug delivery efficiency induced by microbubble-enhanced sonoporation under different experimental parameters, and then achieve the control of drug delivery efficiency through reasonable parameter selection, finally achieve the purpose of efficiently killing bacteria.

Effects of low-intensity and low-frequency ultrasound combined with tobramycin on biofilms of extended-spectrum beta-lactamases (ESBLs) Escherichia coli.

Low-intensity and low-frequency ultrasound (LILFU) can enhance the bactericidal action of antibiotics against various sensitive bacterial species. The current study investigated the effects of LILFU combined with tobramycin on extended-spectrum beta-lactamases (ESBLs) Escherichia coli biofilms (a multi-drug resistant bacteria). The biofilms of ESBLs E. coli were established and treated with ultrasound (42 kHz and ISATA of 0.66 W/cm2) continuously for 0.5 h with and without tobramycin. The bacterial viability, the morphology and the antibiotic penetration of ESBLs E. Coli biofilms were investigated. The results demonstrated that the bacterial viability of biofilms significantly declined and the diameter of the inhibition zone was significantly increased after treatment with ultrasound combined with tobramycin compared with the controls (P < 0.05). Confocal laser scanning microscopy showed that the bacterial viability was affected most in the outer layer of ESBLs E. coli biofilms after joint treatment. The morphological structure of the biofilms was altered remarkably after joint treatment based on scanning electron microscopy, especially in regard to reduced thickness and loosened structure. These results suggest that the combination of ultrasound and tobramycin can exert synergistic bactericidal effects against biofilms formed by ESBLs E. coli.

Ultrasonic exposure parameters screening in permeability of mycobacterium smegmatis cytoderm induced by cavitation based on artificial neural network identification.

The low intensity ultrasound has been adopted by researchers to enhance the bactericidal effect against bacteria in vitro and in vivo. Although the mechanism is not completely understood, one dominant opinion is that the permeability increases because of acoustic cavitation. However, the relationship between ultrasonic exposure parameters and cavitation effects is not definitely addressed. In this paper, by establishing a modified artificial neural network (ANN) model between ultrasonic parameters and cavitation effects, the cavitation effects can be predicted and inversely the direction for choosing parameters can be given despite of different ultrasonic systems. Compared with the generic model, the computational results obtained by modified model are more close to experimental results with low calculation cost. It means that as an efficient solution, the validity of the new model has been proved. Although the research is of preliminary stage, the new method may have great value and significance because of reducing the experimental expense. The next step of this research is to explore an optimization method to obtain the most suitable parameters based on this identification model. We hope it can give a guideline for future applications in ultrasonic therapy.

Measurement of free concentrations of highly protein-bound warfarin in plasma by ultra performance liquid chromatography-tandem mass spectrometry and its correlation with the international normalized ratio.

BACKGROUND: A novel method has been developed for the determination of free warfarin in human plasma by ultra performance liquid chromatography combined with tandem mass spectrometry (UPLC-MS/MS). METHODS: The plasma filtrate was prepared by a high-speed ultrafiltration (UF), and was then extracted by a liquid/liquid extraction (LLE) with ethyl acetate. The chromatographic separation was performed on an Acquity UPLC BEH C(18) (2.1 mm x 50 mm, 1.7 microm, Waters) with a mobile phase consisting of 0.1% formic acid/acetonitrile (50:50, v/v). The analyses were carried out by multiple reaction monitoring (MRM) using the precursor-to-product combinations of m/z 307.3-->161.2 for warfarin and m/z 347.2-->161.3 for Cl-War. RESULTS: The lower limit of quantification (LLOQ) was 0.25 ng/ml and the assay exhibited a linear range of 0.25-16 ng/ml and gave a correlation coefficient (r) of > or = 0.9999. Quality control samples (0.5, 2, 8 ng/ml) in 5 replicates from 3 different runs of analysis demonstrated intra-assay CVs of 3.0-10.2%, inter-assay CVs of 4.2-12.0%, and an overall accuracy of 85-115%. CONCLUSIONS: The method can be applied to analyze the correlation of free concentration of warfarin and the international normalized ratio (INR).

[Changes of blood pressure and pulse rate and the microstructure of celiac ganglion in rabbits with damaged celiac ganglion induced by high intensity focused ultrasound and alcohol].

OBJECTIVE: To observe the changes of blood pressure (BP), pulse rate (PR) and the microstructure of celiac ganglion (CG) in rabbits with damaged CGs induced by alcohol and high intensity focused ultrasound. METHODS: Fourteen rabbits were randomly divided into two groups. The CGs of the rabbits in group A and group B were damaged by alcohol and high intensity focused ultrasound respectively. The changes of BP and PR 0, 1, 3, 5, and 10 minutes after the damage were recorded and compared. The microstructure changes of the damaged CGs were examined under optics microscope and electron microscope. RESULTS: Ganglionic morphology changes were obvious in both groups, with moved and concentrated karyons. In the CGs damaged by alcohol, the nucleolus still existed; some organelles could be identified; the myelination nerve fibre lost its myelin sheath or delaminated while the unmyelination nerve fibre exhibited vacuole formation. In the CGs damaged by high intensity focused ultrasound, all nucleolus disappeared, vacuole formed, intracellular membrane disappeared, axone locally necrotized. The BPs of the rabbits started to decrease three minutes after the alcohol treatment (P < 0.01), one minute after the high intensity focused ultrasound (P < 0.01). Significant differences of BP decline were observed between the two groups one minute after the CG damages. (P < 0.01). The PRs of the rabbits increased 5 minutes and 10 minutes after the high intensity focused ultrasound (P < 0.05, P < 0.01). CONCLUSION: Using high intensity focused ultrasound to damage CGs has more significant impacts on BPs and PRs than alcohol.