Thermal and Acoustic Stabilization Of Volatile Phase-Change Contrast Agents Via Layer-By-Layer Assembly.

OBJECTIVE: Phase-change contrast agents (PCCAs) are perfluorocarbon nanodroplets (NDs) that have been widely studied for ultrasound imaging in vitro, pre-clinical studies, and most recently incorporated a variant of PCCAs, namely a microbubble-conjugated microdroplet emulsion, into the first clinical studies. Their properties also make them attractive candidates for a variety of diagnostic and therapeutic applications including drug-delivery, diagnosis and treatment of cancerous and inflammatory diseases, as well as tumor-growth tracking. However, control over the thermal and acoustic stability of PCCAs both in vivo and in vitro has remained a challenge for expanding the potential utility of these agents in novel clinical applications. As such, our objective was to determine the stabilizing effects of layer-by-layer assemblies and its effect on both thermal and acoustic stability. METHODS: We utilized layer-by-layer (LBL) assemblies to coat the outer PCCA membrane and characterized layering by measuring zeta potential and particle size. Stability studies were conducted by; 1) incubating the LBL-PCCAs at atmospheric pressure at 37∘C and 45∘C followed by; 2) ultrasound-mediated activation at 7.24 MHz and peak-negative pressures ranging from 0.71 - 5.48 MPa to ascertain nanodroplet activation and resultant microbubble persistence. The thermal and acoustic properties of decafluorobutane gas-condensed nanodroplets (DFB-NDs) layered with 6 and 10 layers of charge-alternating biopolymers, (LBL6NDs and LBL10NDs) respectively, were studied and compared to non-layered DFB-NDs. Half-life determinations were conducted at both 37∘C and 45∘C with acoustic droplet vaporization (ADV) measurements occurring at 23∘C. DISCUSSION: Successful application of up to 10 layers of alternating positive and negatively charged biopolymers onto the surface membrane of DFB-NDs was demonstrated. Two major claims were substantiated in this study; namely, (1) biopolymeric layering of DFB-NDs imparts a thermal stability up to an extent; and, (2) both LBL6NDs and LBL10NDs did not appear to alter particle acoustic vaporization thresholds, suggesting that the thermal stability of the particle may not necessarily be coupled with particle acoustic vaporization thresholds. CONCLUSION: Results demonstrate that the layered PCCAs had higher thermal stability, where the half-lifes of the LBLxNDs are significantly increased after incubation at 37∘C and 45∘C. Furthermore, the acoustic vaporization profiles the DFB-NDs, LBL6NDs, and LBL10NDs show that there is no statistically significant difference between the acoustic vaporization energy required to initiate acoustic droplet vaporization.

Effects of Microencapsulated Sodium Butyrate, Probiotics and Short Chain Fructooligosaccharides in Patients with Irritable Bowel Syndrome: A Study Protocol of a Randomized Double-Blind Placebo-Controlled Trial.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disease in the pathogenesis of which gut dysbiosis may play an important role. Thus, probiotics, prebiotics, or microbiota metabolites, such as butyric acid, are considered to be effective therapy for IBS. However, there are still no trials presenting the efficacy of these three biotic components administered simultaneously. This study aims to evaluate the effects of the product comprising sodium butyrate, probiotics, and short-chain fructooligosaccharides (scFOS) on the severity of clinical IBS symptoms and the quality of life (IBS-QOL). This is a randomized double-blind placebo-controlled trial conducted in 120 adults with IBS diagnosed according to Rome IV criteria. The intervention group (n = 60) will receive a mixture of the following components: 300 mg of colon-targeted microencapsulated sodium butyrate combined with probiotic Lactobacillus strains (L. rhamnosus and L. acidophilus) and Bifidobacterium strains (B. longum, B. bifidum, B. lactis), and 64 mg of prebiotic scFOS. The control group (n = 60) will receive a placebo (maltodextrin). The primary outcomes will be changes in IBS symptoms with the use of the IBS-Severity Scoring System (IBS-SSS), IBS-Global Improvement Scale (IBS-GIS), IBS-Adequate Relief (IBS-AR), and IBS-QOL after 12 weeks of intervention. The secondary outcomes will be the type of stools, patient-recorded symptoms, adverse events, anthropometric and nutritional parameters, and inflammatory cytokine levels. The findings will provide the first evidence of the use of a combination of three biotic compounds in IBS. The study was registered in the clinicaltrials.gov registry under the number NCT05013060.

Expression of tryptophan hydroxylase in gastric mucosa in symptomatic and asymptomatic Helicobacter pylori infection.

INTRODUCTION: Helicobacter pylori infection induces clinical symptoms in 15-20% of subjects, and the reason for this variation is still not clear. The aim of the present study is to evaluate the expression of tryptophan hydroxylase (TpH-1) in gastric mucosa of patients with symptomatic and asymptomatic H. pylori infection in relation to the intensity of bacterial colonization and severity of dyspeptic symptoms. MATERIAL AND METHODS: Ninety subjects (aged 35-49 years) were enrolled in the study and separated into 3 groups of 30 subjects each. Group I - healthy volunteers without H. pylori infection, group II - subjects with asymptomatic H. pylori infection, group III - H. pylori-infected patients with dyspeptic symptoms, mainly fasting and nocturnal epigastric pain. To diagnose H. pylori infection the urea breath test (UBT-13C) and histological analysis were performed. The level of mRNA expression of tryptophan hydroxylase (TpH-1) was estimated in gastric mucosa with RT-PCR. RESULTS: The expression of this enzyme in antral mucosa was 2.69 ±0.97 in group I, 2.28 ±0.69 in group II (p > 0.05) and 4.40 ±1.64 in group III (p < 0.001). The levels of expression of TpH-1 in gastric body mucosa were 2.16 ±0.70, 1.57 ±0.52 (p > 0.05) and 3.40 ±1.51 (p < 0.001), respectively. In group III a positive correlation was found between intensity of H. pylori colonization and TpH-1 expression as well as between TpH-1 expression and severity of dyspeptic symptoms. CONCLUSIONS: Increased expression of TpH-1 in gastric mucosa plays a role in pathogenesis of chronic dyspepsia.

Intraoperative brain tumor resection with indocyanine green using augmented microscopy.

Treatment outcomes for brain cancer have seen dismal improvements over the last two decades as evident in available statistical data. Efforts to address this challenge include development of near-infrared contrast agents for improvements in diagnostic and therapeutic modalities. This creates a need for imaging technologies that can support the intraoperative use of such agents. Here, we report implementation of a recently introduced augmented microscope in combination with indocyanine green (ICG), a near-infrared contrast agent, for surgical image guidance of a glioma resection in a rat model. Luc-C6 cells were implanted in rats in the left-frontal lobe and grown for 22 days. Surgical resection was performed by a neurosurgeon using the augmented microscope with ICG contrast. ICG accumulated in the tumor tissue due to enhanced permeation and retention from the compromised blood-brain barrier. Videos and images were acquired to evaluate image quality and resection margins. The augmented microscope highlighted tumor tissue regions via visualization of ICG fluorescence and was capable of guiding the rat glioma resection.

Near-Infrared Activated Release of Doxorubicin from Plasmon Resonant Liposomes.

Precise control of drug release from nanoparticles can improve efficacy and reduce systemic toxicity associated with administration of certain medications. Here, we combined two phenomena, photothermal conversion in plasmon resonant gold coating and thermal sensitivity of liposome compositions, to achieve a drug delivery system that rapidly releases doxorubicin in response to external stimulus. Methods: Thermosensitive liposomes were loaded with doxorubicin and gold-coated to produce plasmon resonant drug delivery system. Plasmon resonance facilitates release of contents upon near-infrared laser illumination, thus providing spatial and temporal control of the process. This controlled delivery system was compared to thermosensitive liposomes without gold coating and to the FDA-approved Doxil that was gold-coated to create a plasmon resonant coating. Release of doxorubicin from the gold-coated thermosensitive liposomes was further confirmed by tests of cell viability. Results: Upon laser illumination at 760 nm and 88 mW/cm2 power density, permeability of plasmon resonant liposomes increased by three orders of magnitude, from 70×10-12 to 60,000x10-12 cm/s. In control experiments, mild hyperthermia (42°C) increased permeability of these thermosensitive liposomes to just 3,700×10-12 cm/s. Neither hyperthermia nor laser illumination elicit content release from Doxil or plasmon resonant Doxil obtained by gold coating. Laser-induced release of doxorubicin from plasmon resonant thermosensitive liposomes resulted in the loss of cell viability significantly greater than in any of the control groups. Conclusion: Combination of thermosensitive liposomes with plasmon resonant coating enables rapid, controlled release, not currently available in pharmaceutical formulations of anticancer drugs.

Excitation Modulation of Upconversion Nanoparticles for Switch-like Control of Ultraviolet Luminescence.

The ability to control ultraviolet (UV) luminescence intensity in a switch-like manner is demonstrated through the use of 980 nm excitation pulse-width modulation in NaYF4:Yb3+,Tm3+ upconversion nanoparticles (UNPs). Varying the ytterbium doping resulted in a single order of magnitude improvement of UV luminescence intensity. The excitation pulse-width modulation technique applied to these optimized UNPs enables 3 orders of magnitude control over UV luminescence intensity while maintaining NIR luminescence emission at 800 nm. Controlled in the switch-like manner, these UNPs can transfer their UV energy to 9,10-diphenylanthracene (DPA). Independent control of NIR luminescence and UV energy transfer through NIR excitation modulation may find applications in the development of multifunctional theranostic systems.

[Excretion and metabolism of dopamine in patients with functional dyspepsia]. / Wydzielanie i metabolizm dopaminy u osób z dyspepsja czynnosciowa.

UNLABELLED: Dopamine is one of major neurotransmitter in the central and peripheral nervous system. A significant amount of dopamine is also produced in the visceral nervous system and in gastrointestinal tract, where exhibits inhibitory activity on motility. AIM: The aim of the study was to assess the parameters of dopamine secretion and metabolism in patients with functional dyspepsia. MATERIAL AND METHODS: The study was conducted in a group of 30 healthy subjects and 60 patients with functional dyspepsia (FD), that met the Rome Criteria III, for epigastric pain syndrome (EPS) and postprandial distress syndrome (PDS). The severity of dyspeptic symptoms was determined using a 10-point Visual-Analogue Scale (VAS). Fasting plasma concentration of dopamine (DA) and the contents of homovanillic acid (HVA) in the urine collection were determined by ELISA. RESULTS: DA concentration in plasma was similar in both clinical forms FD (EPS - 55.6 pg/ml, in patients with PDS - 63.5 pg/ml, p>0.05). Urine excretion of HVA in patients with PDS - 6.63 mg/24 h (p<0.05) was higher than in heathy subjects - 5.65 mg/24 h (p<0.05) and those with EPS - 5.07 mg/24 h (p<0.001). In the group with PDS severity of dyspeptic symptoms showed a positive correlation with the DA concentration in plasma and HVA excretion in the urine. CONCLUSIONS: Increased secretion of DA may play a significant role in the pathogenesis of PDS.

Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images.

Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures.

Integration of indocyanine green videoangiography with operative microscope: augmented reality for interactive assessment of vascular structures and blood flow.

BACKGROUND: Preservation of adequate blood flow and exclusion of flow from lesions are key concepts of vascular neurosurgery. Indocyanine green (ICG) fluorescence videoangiography is now widely used for the intraoperative assessment of vessel patency. OBJECTIVE: Here, we present a proof-of-concept investigation of fluorescence angiography with augmented microscopy enhancement: real-time overlay of fluorescence videoangiography within the white light field of view of conventional operative microscopy. METHODS: The femoral artery was exposed in 7 anesthetized rats. The dissection microscope was augmented to integrate real-time electronically processed near-infrared filtered images with conventional white light images seen through the standard oculars. This was accomplished by using an integrated organic light-emitting diode display to yield superimposition of white light and processed near-infrared images. ICG solution was injected into the jugular vein, and fluorescent femoral artery flow was observed. RESULTS: Fluorescence angiography with augmented microscopy enhancement was able to detect ICG fluorescence in a small artery of interest. Fluorescence appeared as a bright-green signal in the ocular overlaid with the anatomic image and limited to the anatomic borders of the femoral artery and its branches. Surrounding anatomic structures were clearly visualized. Observation of ICG within the vessel lumens permitted visualization of the blood flow. Recorded video loops could be reviewed in an offline mode for more detailed assessment of the vasculature. CONCLUSION: The overlay of fluorescence videoangiography within the field of view of the white light operative microscope allows real-time assessment of the blood flow within vessels during simultaneous surgical manipulation. This technique could improve intraoperative decision making during complex neurovascular procedures.

The Diagnosis of Sigmoid Liposarcoma in a Young Male with Metabolic Syndrome.

The clinical picture and risk factors are decisive in differential diagnosis. It was proved that patients with metabolic syndrome have increased incidence of malignant tumours. The visceral adipose tissue releases active proteins that promote oncogenesis. We are presenting a case of 34-year-old male with metabolic syndrome suffering from pain in left iliac fossa with accompanying variable stool pattern. At first, the sigmoid diverticulosis was suspected. Patient's condition after the treatment has improved. The ambulatory, partial colonoscopy revealed a cauliflower-like, balloting, wide-base growth in the sigmoid that narrowed its lumen. The biopsy did not reveal atypical growth characteristics. Because of tumour type and enlarged regional lymph nodes seen in abdomen CT scan, the segmental colon resection and end-to-end anastomosis was performed in the area of sigmoid-rectal junction. Macroscopically, there were no visible metastases in the operation field. The surgery and postoperative period ran without any complications. On histopathological examination, the removed tumour was a well differentiated liposarcoma (WDL) stemming unusually from adipose tissue of colonic submucosa in an obese male. WDL is a hard to diagnose tumour, especially in early stages of its growth. In the presented case, the tumour was completely resected.

Focal activation of cells by plasmon resonance assisted optical injection of signaling molecules.

Experimental methods for single cell intracellular delivery are essential for probing cell signaling dynamics within complex cellular networks, such as those making up the tumor microenvironment. Here, we show a quantitative and general method of interrogation of signaling pathways. We applied highly focused near-infrared laser light to optically inject gold-coated liposomes encapsulating bioactive molecules into single cells for focal activation of cell signaling. For this demonstration, we encapsulated either inositol trisphosphate (IP3), an endogenous cell signaling second messenger, or adenophostin A (AdA), a potent analogue of IP, within 100 nm gold-coated liposomes, and injected these gold-coated liposomes and their contents into the cytosol of single ovarian carcinoma cells to initiate calcium (Ca(2+)) release from intracellular stores. Upon optical injection of IP3 or AdA at doses above the activation threshold, we observed increases in cytosolic Ca(2+) concentration within the injected cell initiating the propagation of a Ca(2+) wave throughout nearby cells. As confirmed by octanol-induced inhibition, the intercellular Ca(2+) wave traveled via gap junctions. Optical injection of gold-coated liposomes represents a quantitative method of focal activation of signaling cascades of broad interest in biomedical research.

Simultaneous multiplane imaging of human ovarian cancer by volume holographic imaging.

Ovarian cancer is the most deadly gynecologic cancer, a fact which is attributable to poor early detection and survival once the disease has reached advanced stages. Intraoperative laparoscopic volume holographic imaging has the potential to provide simultaneous visualization of surface and subsurface structures in ovarian tissues for improved assessment of developing ovarian cancer. In this ex vivo ovarian tissue study, we assembled a benchtop volume holographic imaging system (VHIS) to characterize the microarchitecture of 78 normal and 40 abnormal tissue specimens derived from ovarian, fallopian tube, uterine, and peritoneal tissues, collected from 26 patients aged 22 to 73 undergoing bilateral salpingo-oophorectomy, hysterectomy with bilateral salpingo-oophorectomy, or abdominal cytoreductive surgery. All tissues were successfully imaged with the VHIS in both reflectance- and fluorescence-modes revealing morphological features which can be used to distinguish between normal, benign abnormalities, and cancerous tissues. We present the development and successful application of VHIS for imaging human ovarian tissue. Comparison of VHIS images with corresponding histopathology allowed for qualitatively distinguishing microstructural features unique to the studied tissue type and disease state. These results motivate the development of a laparoscopic VHIS for evaluating the surface and subsurface morphological alterations in ovarian cancer pathogenesis.

Protective role of melatonin in liver damage.

The liver plays a key role in the detoxification of numerous molecules which results in the formation of an excessive number of toxic reactive oxygen species. This results in oxidative damage to the hepatocytes, which when severe, compromises the function of this critical organ. A variety of antioxidants protect the liver from free radical-mediated damage, one of the best of which is melatonin. Clinical studies have confirmed the melatonin, as well as it precursor tryptophan, protect the liver from non-alcoholic liver disease and also during the surgical procedure of partial liver resection.

Multiphoton Imaging of Upconverting Lanthanide Nanoparticles in Three Dimensional Models of Cancer.

While upconverting lanthanide nanoparticles have numerous advantages over other exogenous contrast agents used in scanned multiphoton imaging, their long luminescence lifetimes cause images collected with non-descanned detection to be greatly blurred. We demonstrate herein the use of Richardson-Lucy deconvolution to deblur luminescence images obtained via multiphoton scanning microscopy. Images were taken of three dimensional models of colon and ovarian cancer following incubation with NaYF4:Yb,Er nanoparticles functionalized with an antibody for EGFR and folic acid respectively. Following deconvolution, images had a lateral resolution on par with the optimal performance of the imaging system used, ~1.2 µm, and an axial resolution of ~5 µm. Due to the relatively high multiphoton excitation efficiency of these nanoparticles, it is possible to follow binding of individual particles in tissue. In addition, their extreme photostability allows for prolonged imaging without significant loss in luminescence signal. With these advantageous properties in mind, we also discuss the potential application of upconverting lanthanide nanoparticles for tracking of specific, cancer relevant receptors in tissue.

Light-activated content release from liposomes.

Successful integration of diagnostic and therapeutic actions at the level of individual cells requires new materials that combine biological compatibility with functional versatility. This review focuses on the development of liposome-based functional materials, where payload release is activated by light. Methods of sensitizing liposomes to light have progressed from the use of organic molecular moieties to the use of metallic plasmon resonant structures. This development has facilitated application of near infrared light for activation, which is preferred for its deep penetration and low phototoxicity in biological tissues. Presented mechanisms of light-activated liposomal content release enable precise in vitro manipulation of minute amounts of reagents, but their use in clinical diagnostic and therapeutic applications will require demonstration of safety and efficacy.

NIR-activated content release from plasmon resonant liposomes for probing single-cell responses.

Technological limitations have prevented the interrogation and manipulation of cellular activity in response to bioactive molecules within model and living systems that is required for the development of diagnostic and treatment modalities for diseases, such as cancer. In this work, we demonstrate that gold-coated liposomes are capable of encapsulation and on-demand release of signaling molecules with a spatial and temporal resolution leading to activation of individual cells. As a model system, we used cells modified to overexpress a certain G-protein coupled receptor, the CCK2 receptor, and achieved its activation in a single cell via the localized release of its agonist. This content release was triggered by illumination of the liposomes at wavelengths corresponding to the plasmon resonance of the gold coating. The use of plasmon resonant liposomes may enable on-demand release of a broad range of molecules using biologically safe near-infrared light and without molecule chemical modification. In combination with the spectral tunability of plasmon resonant coating, this technology may allow for multiplexed interrogation of complex and diverse signaling pathways in model or living tissues with unprecedented spatial and temporal control.

Molecular catch and release: controlled delivery using optical trapping with light-responsive liposomes.

Gold-coated liposomes are maneuvered using an optical trap to achieve precise delivery of encapsulated molecular cargo. Movement and payload release from these plasmon resonant nanocapsules are independently controlled using a pulsed trapping beam. This technology enables in vitro delivery of a payload to a selected cell and may be applied to the interrogation of individual cells within their biological microenvironment.

Scanning two-photon microscopy with upconverting lanthanide nanoparticles via Richardson-Lucy deconvolution.

The use of upconverting lanthanide nanoparticles in fast-scanning microscopy is hindered by a long luminescence decay time, which greatly blurs images acquired in a nondescanned mode. We demonstrate herein an image processing method based on Richardson-Lucy deconvolution that mitigates the detrimental effects of their luminescence lifetime. This technique generates images with lateral resolution on par with the system's performance, ∼1.2 µm, while maintaining an axial resolution of 5 µm or better at a scan rate comparable with traditional two-photon microscopy. Remarkably, this can be accomplished with near infrared excitation power densities of 850 W/cm(2), several orders of magnitude below those used in two-photon imaging with molecular fluorophores. By way of illustration, we introduce the use of lipids to coat and functionalize these nanoparticles, rendering them water dispersible and readily conjugated to biologically relevant ligands, in this case epidermal growth factor receptor antibody. This deconvolution technique combined with the functionalized nanoparticles will enable three-dimensional functional tissue imaging at exceptionally low excitation power densities.

Melatonin levels in serum and ascitic fluid of patients with hepatic encephalopathy.

Cirrhotic patients exhibit disturbed melatonin homeostasis, which may lead to sleep disturbances, but an influence on the hepatic encephalopathy has not been elucidated. Aim. In the present study, the association of melatonin levels in serum and ascitic fluid and ammonia concentration related to the intensity of hepatic encephalopathy (HE) was investigated. Material and Methods. The study included 90 alcoholic patients with hepatic encephalopathy and 30 healthy volunteers (C). Patients were divided in three groups according to 0-4 West-Haven Score: HE(1) (n = 28), HE(2) (n = 30), and HE(3) (n = 32). Melatonin was measured by radioimmune assay. Results. In fasting patients with hepatic encephalopathy we noted higher melatonin serum levels [pg/mL] than in healthy subjects groups: C-11.3 ± 3.9, HE(1) - 34.3 ± 12.2 (P < 0.01), HE(2)-54.8 ± 23.9, and HE(3)-119.8 ± 96.4 (P < 0.001). No correlation between melatonin and ammonia levels was found. Melatonin was detected in ascetic fluid in 24 patients of group HE(2) and 27 patients of group HE(2) of hepatic encephalopathy. Conclusions. Our results suggest that high blood levels of melatonin in cirrhotic liver patients may account for some of the clinical manifestations of hepatic encephalopathy, for example, daytime sleepiness, fatigue.

Toward the Use of Two-Color Emission Control in Upconverting NaYF4:Er3+, Yb3+ Nanoparticles for Biomedical Imaging.

In the interest of generating new biomedical sensing techniques as well as improving those that currently exist, a great deal of attention has been given to upconverting lanthanide nanoparticles in recent years. In order to develop these nanoparticles for use in multiplexed and ratiometric sensing techniques, many recent studies have focused on experimental control of their emission wavelengths. Here we describe a new method for controlling the relative intensity of green and red emission bands in NaYF4:Yb3+, Er3+ nanoparticles via control of the excitation pulse repetition rate. Using this method, particles of the same composition may be tuned to produce red and green light in user-defined ratios. We discuss the mechanism behind this control as well as potential applications that could make use of this property, specifically in super resolution imaging techniques.