Search Results (720)

Background: Pain is a complex phenomenon characterized by unpleasant experiences with profound heterogeneity influenced by biological, psychological, and social factors. According to the National Health Interview Survey, 50.2 million U.S. adults (20.5%) experience pain on most days, with the annual cost of prescription medication for pain reaching approximately USD 17.8 billion. Theranostic pain nanomedicine therefore emerges as an attractive analgesic strategy with the potential for increased efficacy, reduced side-effects, and treatment personalization. Theranostic nanomedicine combines drug delivery and diagnostic features, allowing for real-time monitoring of analgesic efficacy in vivo using molecular imaging. However, clinical translation of these nanomedicines are challenging due to complex manufacturing methodologies, lack of standardized quality control, and potentially high costs. Quality by Design (QbD) can navigate these challenges and lead to the development of an optimal pain nanomedicine. Our lab previously reported a macrophage-targeted perfluorocarbon nanoemulsion (PFC NE) that demonstrated analgesic efficacy across multiple rodent pain models in both sexes. Here, we report PFC-free, biphasic nanoemulsions formulated with a biocompatible and non-immunogenic plant-based coconut oil loaded with a COX-2 inhibitor and a clinical-grade, indocyanine green (ICG) near-infrared fluorescent (NIRF) dye for parenteral theranostic analgesic nanomedicine. Methods: Critical process parameters and material attributes were identified through the FMECA (Failure, Modes, Effects, and Criticality Analysis) method and optimized using a 3 × 2 full-factorial design of experiments. We investigated the impact of the oil-to-surfactant ratio (w/w) with three different surfactant systems on the colloidal properties of NE. Small-scale (100 mL) batches were manufactured using sonication and microfluidization, and the final formulation was scaled up to 500 mL with microfluidization. The colloidal stability of NE was assessed using dynamic light scattering (DLS) and drug quantification was conducted through reverse-phase HPLC. An in vitro drug release study was conducted using the dialysis bag method, accompanied by HPLC quantification. The formulation was further evaluated for cell viability, cellular uptake, and COX-2 inhibition in the RAW 264.7 macrophage cell line. Results: Nanoemulsion droplet size increased with a higher oil-to-surfactant ratio (w/w) but was no significant impact by the type of surfactant system used. Thermal cycling and serum stability studies confirmed NE colloidal stability upon exposure to high and low temperatures and biological fluids. We also demonstrated the necessity of a solubilizer for long-term fluorescence stability of ICG. The nanoemulsion showed no cellular toxicity and effectively inhibited PGE2 in activated macrophages. Conclusions: To our knowledge, this is the first instance of a celecoxib-loaded theranostic platform developed using a plant-derived hydrocarbon oil, applying the QbD approach that demonstrated COX-2 inhibition. Full article

Background/Objectives: Paclitaxel (PTX) is widely used in the treatment of a variety of solid tumours due to its broad-spectrum anti-tumour activity, but its use in brain gliomas is limited by insufficient blood–brain tumour barrier (BBTB) penetration and systemic toxicity. The aim of this study was to develop a Solutol HS-15-based micellar nanoparticle (PSM) to enhance the brain glioma targeting of PTX and reduce toxicity. Methods: PSMs were prepared by solvent injection and characterised for particle size, encapsulation rate, haemolysis rate and in vitro release properties. A C6 in situ glioma mouse model was used to assess the brain targeting and anti-tumour effects of the PSM by in vivo imaging, tissue homogenate fluorescence analysis and bioluminescence monitoring. Meanwhile, its safety was evaluated by weight monitoring, serum biochemical indexes and histopathological analysis. Results: The particle size of PSMs was 13.45 ± 0.70 nm, with an encapsulation rate of 96.39%, and it demonstrated excellent cellular uptake. In tumour-bearing mice, PSMs significantly enhanced brain tumour targeting with a brain drug concentration 5.94 times higher than that of free PTX. Compared with Taxol, PSMs significantly inhibited tumour growth (terminal luminescence intensity <1 × 10⁶ p/s/cm²/Sr) and did not cause significant liver or kidney toxicity or body weight loss. Conclusions: PSMs achieve an efficient accumulation of brain gliomas through passive targeting and EPR effects while significantly reducing the systemic toxicity of PTX. Its simple preparation process and excellent therapeutic efficacy support its use as a potential clinically translational candidate for glioma treatment. Full article

Background: Gastric cancer (GC) is a malignant tumor of the gastrointestinal tract, characterized by high mortality rates and responsible for about one million new cases each year globally. Surgery is the main treatment, but achieving radical resection remains a relevant intraoperative challenge. Fluorescence-guided surgery offers clinicians greater capabilities for real-time detection of tumor nodules and visualization of tumor margins. In this field, the main challenge remains the development of fluorescent dyes that can selectively target tumor tissues. Methods: we examined the expression of the most suitable GC markers, including carcinoembryonic antigen cell adhesion molecule-5 (CEACAM5) and Claudin-4 (CLDN4), in GC cell lines. To further evaluate their expression, we performed immunohistochemistry (IHC) on tumor and healthy tissue samples from 30 GC patients who underwent partial gastrectomy at the Digestive System Surgery Unit, AOU Careggi, Florence. Additionally, we validated anti-CEACAM5 expression on patient-derived organoids. Furthermore, we developed a fluorescent molecule targeting CEACAM5 on the surface of GC cells and assessed its binding properties on patient tissue slices and fragments. Results: in this work, we first identified CEACAM5 as an optimal GC biomarker, and then we developed a fluorescent antibody specific for CEACAM5. We also evaluated its binding specificity for GC cell lines and patient-derived tumor tissue, achieving an optimal ability to discriminate tumor tissue from healthy mucosa. Conclusions: Overall, our results support the development of our fluorescent antibody as a promising tumor-specific imaging agent that, after further in vivo validation, could improve the accuracy of complete tumor resection. Full article

The extensive diversity of volatile organic compounds, along with their minor structural variations, presents significant challenges in the development of chemosensory-based biosensors. Previously, we generated sensor cells expressing insect odorant receptors (ORs) in Sf21 cells, demonstrating their potential as cell-based odorant sensor elements. However, it remains unclear whether the selectivity of cells expressing ORs in vitro for diverse compounds aligns with the receptor’s in vivo performance, aside from the response to target compounds. To address this, we assessed the ligand responses of sensor cells expressing ORs from Drosophila melanogaster using a high-throughput calcium imaging system. Our results demonstrate that in vitro receptor responses exhibit ligand selectivity comparable to in vivo conditions across different chemical categories. Broadly tuned OR-expressing sensor cells (Or13a, Or47a, and Or98a) displayed differential affinities, whereas the narrowly tuned Or56a-expressing sensor cells selectively responded to geosmin. Moreover, cell responses varied with subtle differences in chemical structure, including carbon chain length and functional group positioning. These findings provide valuable insights into insect OR–ligand interactions in vitro, demonstrating that receptor selectivity in sensor cells closely mirrors in vivo conditions. In addition to this consistency, our results highlight the subtle ligand differentiation capabilities of sensor cells enabling fluorescence-based visualization of receptor–ligand interactions. Full article

Due to the growth in the application of antibacterial nanomaterials (NMs), there is an increased potential for ingestion by humans. Evidence shows that NMs can induce dysbiosis in the gut microbiota in vivo. However, in vitro investigation of the antibacterial activity of NMs on gut-relevant, commensal bacteria has been neglected, with studies predominantly assessing NM toxicity against pathogenic bacteria. The current study investigates the antibacterial activity of copper oxide (CuO) NMs to Escherichia coli K12, Enterococcus faecalis, and Lactobacillus casei using a combination of approaches and evaluates the importance of reactive oxygen species (ROS) production as a mechanism of toxicity. The impact of CuO NMs (100, 200, and 300 μg/mL) on the growth and viability of bacterial strains was assessed via plate counts, optical density (OD) measurements, well and disc diffusion assays, and live/dead fluorescent imaging. CuO NMs reduced the viability of all bacteria in a concentration-dependent manner in all assays except the diffusion assays. The most sensitive methods were OD measurements and plate counts. The sensitivity of bacterial strains varied depending on the method, but overall, the results suggest that E. coli K12 is the most sensitive to CuO NM toxicity. The production of ROS by all bacterial strains was observed via DCFH-DA fluorescent imaging following exposure to CuO NMs (300 μg/mL). Overall, the data suggests that CuO NMs have antibacterial activity against gut-relevant bacteria, with evidence that NM-mediated ROS production may contribute to reductions in bacterial viability. Our findings suggest that the use of a combination of assays provides a robust assessment of the antibacterial properties of ingested NMs, and in particular, it is recommended that plate counts and OD measurements be prioritised in the future when screening the antibacterial properties of NMs. Full article

The development of fluorophores with high-fluorescence quantum yields is highly desirable. To regulate photophysical properties, previous fumaronitrile-core fluorophore designs primarily employed electron-donating structure modifications and π-conjugation extension strategies. Here, we report a novel strategy to enhance the fluorescence performance of fluorophores by introducing methyl groups into fumaronitrile phenyl rings. The introduction of methyl groups reduces the ability to generate reactive oxygen species while enhancing the fluorescence quantum yield. Notably, after encapsulating DSPE-PEG2000 to form nanoparticles, TFN-Me nanoparticles exhibited superior fluorescence performance than previously reported fluorophores and successfully applied in in vivo tumor fluorescence imaging. This study indicates that the methyl introduction strategy holds the potential to become a powerful tool for developing high-brightness fluorophores with fumaronitrile structure. Full article

Developments of nanostructured materials have a significant impact in various areas, such as energy technology and biomedical use. Examples include solar cells, energy management, environmental control, bioprobes, tissue engineering, biological marking, cancer diagnosis, therapy, and drug delivery. Currently, researchers are designing multifunctional nanodrugs that combine in vivo imaging (using fluorescent nanomaterials) with targeted drug delivery, aiming to maximize therapeutic efficacy while minimizing toxicity. These fascinating nanoscale “magic bullets” should be available in the near future. Inorganic nanovehicles are flexible carriers to deliver drugs to their biological targets. Most commonly, mesoporous nanostructured silica, carbon nanotubes, gold, and iron oxide nanoparticles have been thoroughly studied in recent years. Opposite to polymeric and lipid nanostructured materials, inorganic nanomaterial drug carriers are unique because they have shown astonishing theranostic (therapy and diagnostics) effects, expressing an undeniable part of future use in medicine. This review summarizes research from development to the most recent discoveries in the field of nanostructured materials and their applications in drug delivery, including promising metal-based complexes, platinum, palladium, ruthenium, titanium, and tin, to tumor cells and possible use in theranostics. Full article

Background: Gallbladder hypomotility is a key pathogenic factor in cholelithiasis. Non-invasive interventions to enhance gallbladder contractility remain limited. Ultrasound therapy has shown promise in various muscular disorders, but its effects on gallbladder function are unexplored. Methods: This study employed low-intensity pulsed ultrasound (LIPUS) at a 3 MHz frequency and 0.8 W/cm² intensity with a 20% duty cycle to irradiate the gallbladder region of fasting guinea pigs. Gallbladder contractile function was evaluated through multiple complementary approaches: in vivo assessment via two-dimensional/three-dimensional ultrasound imaging to monitor volumetric changes; quantitative functional evaluation using nuclear medicine scintigraphy (^99mTc-HIDA); and ex vivo experiments including isolated gallbladder muscle strip tension measurements, histopathological analysis, α-smooth muscle actin (α-SMA) immunohistochemistry, and intracellular calcium fluorescence imaging. Results: Ultrasound significantly enhanced gallbladder emptying, evidenced by the volume reduction and increased ejection fraction. Scintigraphy confirmed accelerated bile transport in treated animals. Ex vivo analyses demonstrated augmented contractile force, amplitude, and frequency in ultrasound-treated smooth muscle. Histological examination revealed smooth muscle hypertrophy, α-SMA upregulation, and elevated intracellular calcium levels. Extended ultrasound exposure produced sustained functional improvements without tissue damage. Conclusions: Ultrasound effectively enhances gallbladder contractile function through mechanisms involving smooth muscle structural modification and calcium signaling modulation. These findings establish the experimental foundation for ultrasound as a promising non-invasive therapeutic approach to improve gallbladder motility and potentially prevent gallstone formation. Full article

Background: Rectal cancer (RC) patients with a clinical complete response (cCR) after neoadjuvant chemoradiotherapy (nCRT) may qualify for a watch-and-wait (W&W) approach. However, a 20–30% local tumor regrowth rate highlights challenges in identifying true responders. This study explores markers for future near-infrared fluorescence tumor imaging by endoscopy to differentiate responders and the effect of nCRT on their expression. Methods: RC samples (n = 51) were collected from both pre-treatment biopsies and corresponding post-treatment surgical specimens. Samples were categorized by treatment response and determined using tumor regression grade (TRG) scoring. Immunohistochemistry assessed the expression of CEA, EpCAM, EGFR, and c-MET in tumors and adjacent normal tissues. Expression levels were quantified using H-scores (0–3), combining the percentage and intensity of stained cells. Pre- and post-treatment H-scores were compared to evaluate the impact of nCRT. Results: CEA, EpCAM, and c-MET were overexpressed in tumor tissue as compared to adjacent healthy mucosa in 100% (51/51), 98.4% (50/51), and 92% (47/51) of tumor biopsies, respectively, while EGFR showed no overexpression. A tumor-to-normal (T/N) ratio ≥ 2 was considered sufficient for differentiation in molecular fluorescence imaging. In pre-treatment biopsy samples, c-MET showed the highest T/N expression ratio (53% of the samples ≥ 2), followed by CEA (26.3%) and EpCAM (16%). Following nCRT treatment, CEA and c-MET maintained a ≥ 2 differential expression in 45% of all samples, whereas EpCAM exhibited this difference in only 9.2% of cases. Neoadjuvant therapy even significantly improved the T/N expression ratio for CEA and c-MET (p < 0.01) and EpCAM (p < 0.05), while EGFR expression remained lower than adjacent normal tissue. Significant increases in all marker expressions were observed in minimal responders (TRG4/5, p < 0.01–0.001), while near-complete responders (TRG2) exhibited non-significant changes in CEA, c-MET, and EGFR expression. Conclusions: c-MET and CEA emerged as optimal tumor imaging targets, showing sustained differential expression after nCRT. In vivo fluorescence-guided endoscopy using probes against these markers could play a role in future clinical decision-making. Full article

Chemophototherapy (CPT) is an emerging cancer treatment that leverages the synergistic effects of photodynamic therapy (PDT) and chemotherapy. This approach utilizes photosensitizers like Porphyrin-Phospholipid (PoP) and combined with chemotherapeutic like Doxorubicin (Dox) to enable light-triggered drug release and targeted tumor destruction. Here, we present the validation of a wide-field laparoscopic spatial frequency domain imaging (SFDI) system in an ovarian cancer model. The system allows quantitative fluorescence imaging to obtain absolute drug concentrations in vivo to obtain the absolute concentrations of PoP and Dox fluorescence by correcting for tissue absorption and scattering effects. Fluorescence imaging revealed a significant reduction (~25%, p < 0.001) in PoP concentration in tumor regions post-illumination, demonstrating PDT-mediated photobleaching. Next, the Dox release experiment showed an increase of ~13 µg/mL Dox concentration at the local site. The ability to quantify both PoP and Dox fluorescence concentrations with a laparoscopic system underscores its potential for intraoperative monitoring of CPT efficacy. These findings indicate wide-field laparoscopic SFDI as a promising tool for guiding minimally invasive PDT and targeted drug delivery in preclinical and future clinical settings. Full article

The rapid pace of modern life has contributed to a significant decline in sleep quality, which has become an urgent global public health issue. Melatonin, an endogenous hormone that regulates circadian rhythms, is vital in maintaining normal sleep cycles. While oral melatonin supplementation is widely used, transdermal delivery systems present advantages that include the avoidance of first-pass metabolism effects and enhanced bioavailability. In this study, a novel melatonin transdermal delivery system was successfully developed using a thermosensitive poly(N-vinylcaprolactam) [p(NVCL)]-based carrier. The p(NVCL) polymer was synthesized through free radical polymerization and characterized for its structural properties and phase transition temperature, in alignment with skin surface conditions. Orthogonal optimization experiments identified 3% azone, 3% menthol, and 4% borneol as the optimal enhancer combination for enhanced transdermal absorption. The formulation demonstrated exceptional melatonin loading characteristics with high encapsulation efficiency and stable physicochemical properties, including an appropriate pH and optimal moisture content. Comprehensive in vivo evaluation using normal mouse models revealed significant sleep quality improvements, specifically a shortened sleep latency and extended non-rapid eye movement sleep duration, with elevated serum melatonin and serotonin levels. Safety assessments including histopathological examination, biochemical analysis, and 28-day continuous administration studies confirmed excellent biocompatibility with no adverse reactions or systemic toxicity. Near-infrared fluorescence imaging provided direct evidence of enhanced transdermal absorption and superior biodistribution compared to oral administration. These findings indicate that the p(NVCL)-based melatonin transdermal gel system offers a safe, effective and convenient non-prescription option for sleep regulation, with promising potential for clinical translation as a consumer sleep aid. Full article

In prostate cancer (PCa) surgery, precise tumor margin identification remains challenging despite advances in surgical techniques. This study evaluates the combination of tumor-specific near-infrared imaging with the PSMA-targeting molecule PSMA-914 and optical endomicroscopy (NIR-pCLE) for single-cell-level tumor identification in a preclinical proof of concept. Methods: NIR-pCLE imaging of varying PSMA-914 concentrations was performed on PSMA-positive LNCaP and PSMA-negative PC-3 cells using Cellvizio^® 100 with pCLE Confocal Miniprobes™. To identify optimal PSMA-914 dosing for in vivo imaging, different doses (0–10 nmol) were evaluated using NIR-pCLE, Odyssey CLx imaging, and confocal microscopy in an LNCaP tumor-bearing xenograft model. A proof of concept mimicking a clinical workflow was performed using 5 nmol [⁶⁸Ga]Ga-PSMA-914 in LNCaP and PC-3 tumor xenografts, including PET/MRI, in/ex vivo NIR-pCLE imaging, and microscopic/macroscopic imaging. Results: NIR-pCLE detected PSMA-specific fluorescence at concentrations above 30 nM in vitro. The optimal dose was identified as 5 nmol PSMA-914 for NIR-pCLE imaging with cellular resolution in LNCaP xenografts. PET/MRI confirmed high tumor uptake and a favorable distribution profile of PSMA-914. NIR-pCLE imaging enabled real-time, single-cell-level detection of PSMA-positive tissue, visualizing tumor heterogeneity, confirmed by ex vivo microscopy and imaging. Conclusions: This preclinical proof of concept demonstrates the potential of intraoperative PSMA-specific NIR-pCLE imaging to visualize tissue structures in real time at cellular resolution. Clinical implementation could provide surgeons with valuable additional information, potentially advancing PCa patient care through improved surgical precision. Full article

Identifying parathyroid glands during surgery is challenging and time-consuming due to their small size (3–5 mm) and camouflaged appearance in the background of the thyroid, lymph nodes, fat, and other neck structures. For the gland itself, it is also important to differentiate abnormal ones from normal ones. Accidental damage or removal of the normal glands can result in complications like hypocalcemia, which may necessitate lifelong medication dependence, and, in extreme cases, lead to death. The study of autofluorescence optical properties of normal and abnormal parathyroid glands and the surrounding tissue will be helpful for developing non-invasive detection devices. The near-infrared (NIR) autofluorescence characteristics of parathyroid and thyroid tissues have been studied extensively and are now used for parathyroid gland detection during surgery. Additionally, there have been a few reports on the UV-visible light-excited autofluorescence characteristics of these tissues with a focus on spectroscopy. However, there is a lack of high-resolution, side-by-side autofluorescence imaging comparisons of both tissue types under various excitation wavelengths, ranging from visible to NIR. We developed a standalone tabletop autofluorescence imaging system to acquire images of ex vivo specimens in the operating room under different excitation wavelengths: visible 405 nm, 454 nm, 520 nm, 628 nm, and NIR 780 nm. Autofluorescence imaging features of parathyroid adenomas for each excitation wavelength were described and compared. It was found that visible light excites much stronger autofluorescence from parathyroid adenoma tissue compared to NIR light. However, NIR excitation provides the best intensity difference/contrast between parathyroid adenoma and thyroid tissue, making it optimal for differentiating these two tissue types, and detecting parathyroid adenoma during surgery. The high fluorescent site under the NIR 780 nm excitation also generates high fluorescence under visible excitation wavelengths. Heterogeneous fluorescence patterns were observed in most of the parathyroid adenoma cases across all the excitation wavelengths. Full article

In recent years, an increasing number of studies have shown that novel metal complexes with bio-imaging capabilities could enhance precision oncology, particularly through optimized photosensitizer (PS) design for subcellular organelle targeting photodynamic therapy (PDT). Based on this, we successfully developed a two-photon (TP) fluorescent Zn(II) complex, LIFM–ZY–3, characterized by the specifical targeting capability of lysosome. This complex was capable of monitoring dual changes in pH and viscosity. Additionally, our findings indicated that the complex could generate multiple reactive oxygen species (ROS), including singlet oxygen (¹O₂), hydroxyl radicals (•OH), and superoxide anion radicals (O₂⁻) under white light irradiation in vivo and in vitro. These findings underscored the remarkable versatility of LIFM–ZY–3 as an advanced multifunctional PS for both microenvironment monitoring and tumor therapy. Full article

Conditional transgenic systems and multi-copy target transgenes were used to produce transient fluorescent protein expression in adult Drosophila melanogaster, with the goal of developing an in vivo assay of protein turnover. Free-moving flies were assayed at multiple time points using video, and decay in fluorescence was used to calculate protein half-life. Additional experiments involved image capture of anesthetized flies. The half-life of eGFP was increased by the proteasome inhibitor bortezomib, both in vivo and in vitro, indicating proteasomal degradation of eGFP. The accumulation of eGFP in vivo was decreased by the protein synthesis inhibitor cycloheximide, without affecting half-life. The half-lives of several fluorescent proteins were determined, using both tissue-general and tissue-specific expression, in flies of both sexes and varying ages. Typical half-life values varied by fluorescent protein. DsRED showed a greater half-life than eGFP, and little if any degradation was detected for mCherry. Half-life also varied by tissue, with greater eGFP half-life observed in muscle relative to other tissues. Increased half-life with age was detected for DsRED but not for eGFP. Limited effects were observed for sex and female mating status. Taken together, the data indicate the in vivo assays are promising tools for the study of protein degradation regulated by protein sequence, subcellular compartment, tissue and small molecules. Full article