Adaptable cascaded registration for personalized maxilla completion and cleft defect volume estimation.

BACKGROUND: Cone-beam computed tomography (CBCT) images provide high-resolution insights into the underlying craniofacial anomaly in patients with cleft lip and palate (CLP), requiring non-negligible annotation costs to measure the cleft defect for the guidance of the clinical secondary alveolar bone graft procedures. Considering the cumbersome volumetric image acquisition, there is a lack of paired CLP CBCTs and normal CBCTs for learning-based anatomical structure restoration models. Nowadays, the registration-based method relieves the annotation burden, though one-shot registration and the regular mask are limited to handling fine-grained shape variations and harmony between restored bony tissues and the defected maxilla. PURPOSE: This study aimed to design and evaluate a novel method for deformable partial registration of the CLP CBCTs and normal CBCTs, enabling personalized maxilla completion and cleft defect volume prediction from CLP CBCTs. METHODS: We proposed an adaptable deep registration framework for personalized maxilla completion and cleft defect volume prediction from CLP CBCTs. The key ingredient was a cascaded partial registration to exploit the maxillary morphology prior and attribute transfer. Cascaded registration with coarse-to-fine registration fields handled morphological variations of cleft defects and fine-grained maxillary restoration. We designed an adaptable cleft defect mask and volumetric Boolean operators for reliable voxel filling of the defected maxilla. A total of 36 clinically obtained CLP CBCTs were used to train and validate the proposed model, among which 22 CLP CBCTs were used to generate a training dataset with 440 synthetic CBCTs by B-spline deformation-based data augmentation and the remaining for testing. The proposed model was evaluated on maxilla completion and cleft defect volume prediction from clinically obtained unilateral and bilateral CLP CBCTs. RESULTS: Extensive experiments demonstrated the effectiveness of the adaptable cleft defect mask and the cascaded partial registration on maxilla completion and cleft defect volume prediction. The proposed method achieved state-of-the-art performances with the Dice similarity coefficient of 0.90 ± $\pm$ 0.02 on the restored maxilla and 0.84 ± $\pm$ 0.04 on the estimated cleft defect, respectively. The average Hausdorff distance between the estimated cleft defect and the manually annotated ground truth was 0.30 ± $\pm$ 0.08 mm. The relative volume error of the cleft defect was 0.09 ± $0.09\pm$ 0.08. The proposed model allowed for the prediction of cleft defect maps that were in line with the ground truth in the challenging unilateral and bilateral CLP CBCTs. CONCLUSIONS: The results suggest that the proposed adaptable deep registration model enables patient-specific maxilla completion and automatic annotation of cleft defects, relieving tedious voxel-wise annotation and image acquisition burdens.

Activation of 5-HT5A receptor in the ventrolateral orbital cortex produces antinociceptive effects in rat models of neuropathic and inflammatory pain.

The ventrolateral orbital cortex (VLO) is identified as an integral component of the endogenous analgesic system comprising a spinal cord - thalamic nucleus submedius - VLO - periaqueductal gray (PAG) - spinal cord loop. The present study investigates the effects of 5-HT5A receptor activation in the VLO on allodynia induced by spared nerve injury and formalin-evoked flinching behavior and spinal c-Fos expression in male SD rats, and further examines whether GABAergic modulation is involved in the effects evoked by VLO 5-HT5A receptor activation. We found an upregulation of 5-HT5A receptor expression in the VLO during neuropathic and inflammatory pain states. Microinjection of the non-selective 5-HT5A receptor agonist 5-CT into the VLO dose dependently alleviated allodynia, and flinching behavior and spinal c-Fos expression, which were blocked by the selective 5-HT5A receptor antagonist SB-699551. Moreover, application of the GABAA receptor antagonist bicuculline in the VLO augmented the analgesic effects induced by 5-CT in neuropathic and inflammatory pain states, whereas the GABAA receptor agonist muscimol attenuated these analgesic effects. Additionally, the 5-HT5A receptors were found to be colocalized with GABAergic neurons in the VLO. These results provide new evidence for the involvement of central 5-HT5A receptors in the VLO in modulation of neuropathic and inflammatory pain and support the hypothesis that activation of 5-HT5A receptors may inhibit the inhibitory effect of GABAergic interneurons on output neurons projecting to the PAG (GABAergic disinhibitory mechanisms), consequently activating the brainstem descending inhibitory system that depresses nociceptive transmission at the spinal cord level.

Natural reference structures for three-dimensional maxillary regional superimposition in growing patients.

BACKGROUND: Assessment of growth-related or treatment-related changes in the maxilla requires a reliable method of superimposition. Such methods are well established for two-dimensional (2D) cephalometric images but not yet for three-dimensions (3D). The aims of this study were to identify natural reference structures (NRS) for the maxilla in growing patients in 3D, opportunistically using orthodontic mini-screws as reference; and to test the applicability of the proposed NRS for maxillary superimposition by assessing the concordance of this approach with Björk's 'stable reference structures' in lateral projection. METHODS: The stability of the mini-screws was tested on longitudinal pairs of pre- and post-orthodontic cone-beam computed tomography (CBCT) images by measuring the distance changes between screws. After verifying the stability of the mini-screws, rigid registration was performed for aligning the stable mini-screws. Then, non-rigid registration was used to establish the dense voxel-correspondence among CBCT images and calculate the displacement of each voxel belonging to the maxilla relative to the mini-screws. The displacement vectors were transformed to a standardized maxillary template to categorize the stability of the internal structures statistically. Those voxels that displaced less relative to the mini-screws were considered as the natural reference structures (NRS) for the maxilla. Test samples included another dataset of longitudinal CBCT scans. They were used to evaluate the applicability of the proposed NRS for maxillary superimposition. We assessed whether aligning the maxilla with proposed NRS is in concordance with the maxillary internal reference structures superimposition in the traditional 2D lateral view as suggested by Björk. This was quantitively assessed by comparing the mean sagittal and vertical tooth movements for both superimposition methods. RESULTS: The stability of the mini-screws was tested on 10 pairs of pre- and post-orthodontic cone-beam computed tomography (CBCT) images (T1: 12.9 ± 0.8 yrs, T2: 14.8 ± 0.7 yrs). Both the loaded and the unloaded mini-screws were shown to be stable during orthodontic treatment, which indicates that they can be used as reference points. By analyzing the deformation map of the maxilla, we confirmed that the infraorbital rims, maxilla around the piriform foramen, the infrazygomatic crest and the hard palate (palatal vault more than  1 cm distal to incisor foramen except the palatal suture) were stable during growth. Another dataset of longitudinal CBCT scans (T1: 12.2 ± 0.63 yrs, T2: 15.2 ± 0.96 yrs) was used to assess the concordance of this approach with Björk's 'stable reference structures'. The movement of the maxillary first molar and central incisor showed no statistically significant difference when superimposing the test images with the proposed NRS or with the classic Björk maxillary superimposition in the lateral view. CONCLUSIONS: The infraorbital rims, maxilla around the piriform foramen, the infrazygomatic crest and the hard palate (palatal vault more than 1 cm posterior to incisal foramen except the palatal suture) were identified as stable regions in the maxilla. These stable structures can be used for maxillary superimposition in 3D and generate comparable results to Björk superimposition in the lateral view.

Large HBV Surface Protein-Induced Unfolded Protein Response Dynamically Regulates p27 Degradation in Hepatocellular Carcinoma Progression.

Up to 50% of hepatocellular carcinoma (HCC) is caused by hepatitis B virus (HBV) infection, and the surface protein of HBV is essential for the progression of HBV-related HCC. The expression of large HBV surface antigen (LHB) is presented in HBV-associated HCC tissues and is significantly associated with the development of HCC. Gene set enrichment analysis revealed that LHB overexpression regulates the cell cycle process. Excess LHB in HCC cells induced chronic endoplasmic reticulum (ER) stress and was significantly correlated with tumor growth in vivo. Cell cycle analysis showed that cell cycle progression from G1 to S phase was greatly enhanced in vitro. We identified intensive crosstalk between ER stress and cell cycle progression in HCC. As an important regulator of the G1/S checkpoint, p27 was transcriptionally upregulated by transcription factors ATF4 and XBP1s, downstream of the unfolded protein response pathway. Moreover, LHB-induced ER stress promoted internal ribosome-entry-site-mediated selective translation of p27, and E3 ubiquitin ligase HRD1-mediated p27 ubiquitination and degradation. Ultimately, the decrease in p27 protein levels reduced G1/S arrest and promoted the progress of HCC by regulating the cell cycle.

SMURF2 facilitates ubiquitin-mediated degradation of ID2 to attenuate lung cancer cell proliferation.

SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2) functions as either a tumor promoter or tumor suppressor in several tumors. However, the detailed effect of SMURF2 on non-small cell lung cancer has not been fully understood. In this study, SMURF2 expression and its diagnostic value were analyzed. Co-Immunoprecipitation (Co-IP), proximity ligation assay (PLA), chromatin immunoprecipitation (ChIP) and nude mice tumor-bearing model were applied to further clarify the role of SMURF2 in lung cancer. SMURF2 expression was reduced in the tumor tissues of patients with NSCLC and high SMURF2 expression was significantly correlated with favorable outcomes. Furthermore, the overexpression of SMURF2 significantly inhibited lung cancer cell progression. Mechanistically, SMURF2 interacted with inhibitor of DNA binding 2 (ID2), subsequently promoting the poly-ubiquitination and degradation of ID2 through the ubiquitin-proteasome pathway. Downregulated ID2 in lung cells dissociates endogenous transcription factor E2A, a positive regulator of the cyclin-dependent kinase inhibitor p21, and finally induces G1/S arrest in lung cancer cells. This study revealed that the manipulation of ID2 via SMURF2 may control tumor progression and contribute to the development of novel targeted antitumor drugs.

Triglyceride-Rich Lipoprotein-Mediated Polymer Dots for Multimodal Imaging Interscapular Brown Adipose Tissue Capillaries.

Brown adipose tissues (BATs) have been identified as a promising target of metabolism disorders. [18F]FDG-PET (FDG = fluorodeoxyglucose; PET = positron emission tomography) has been predominantly employed for BAT imaging, but its limitations drive the urgent need for novel functional probes combined with multimodal imaging approaches. It has been reported that polymer dots (Pdots) display rapid BAT imaging without additional cold stimulation. However, the mechanism by which Pdots image BAT remains unclear. Here, we made an intensive study of the imaging mechanism and found that Pdots can bind to triglyceride-rich lipoproteins (TRLs). By virtue of their high affinity to TRLs, Pdots selectively accumulate in capillary endothelial cells (ECs) in interscapular brown adipose tissues (iBATs). Compared to poly(styrene-co-maleic anhydride)cumene terminated (PSMAC)-Pdots with a short half-life and polyethylene glycol (PEG)-Pdots with low lipophilicity, naked-Pdots have good lipophilicity, with a half-life of about 30 min and up to 94% uptake in capillary ECs within 5 min, increasing rapidly after acute cold stimulation. These results suggested that the accumulation changes of Pdots in iBAT can reflect iBAT activity sensitively. Based on this mechanism, we further developed a strategy to detect iBAT activity and quantify the TRL uptake in vivo using multimodal Pdots.

Application of a linear interpolation algorithm in radiation therapy dosimetry for 3D dose point acquisition.

Air-vented ion chambers are generally used in radiation therapy dosimetry to determine the absorbed radiation dose with superior precision. However, in ion chamber detector arrays, the number of array elements and their spacing do not provide sufficient spatial sampling, which can be overcome by interpolating measured data. Herein, we investigated the potential principle of the linear interpolation algorithm in volumetric dose reconstruction based on computed tomography images in the volumetric modulated arc therapy (VMAT) technique and evaluated how the ion chamber spacing and anatomical mass density affect the accuracy of interpolating new data points. Plane measurement doses on 83 VMAT treatment plans at different anatomical sites were acquired using Octavius 729, Octavius1500, and MatriXX ion chamber detector arrays, followed by the linear interpolation to reconstruct volumetric doses. Dosimetric differences in planning target volumes (PTVs) and organs at risk (OARs) between treatment planning system and reconstruction were evaluated by dose volume histogram metrics. The average percentage dose deviations in the mean dose (Dmean) of PTVs reconstructed by 729 and 1500 arrays ranged from 4.7 to 7.3% and from 1.5 to 2.3%, while the maximum dose (Dmax) counterparts ranged from 2.3 to 5.5% and from 1.6 to 7.6%, respectively. The average percentage dose/volume deviations of mixed PTVs and OARs in the abdomen/gastric and pelvic sites were 7.6%, 3.5%, and 7.2%, while mediastinum and lung plans showed slightly larger values of 8.7%, 5.1%, and 8.9% for 729, 1500, and MatriXX detector arrays, respectively. Our findings indicated that the smaller the spacing between neighbouring detectors and the more ion chambers present, the smaller the error in interpolating new data points. Anatomical regions with small local mass density inhomogeneity were associated with superior dose reconstruction. Given a large mass density difference in the various human anatomical structures and the characteristics of the linear interpolation algorithm, we suggest that an alternative data interpolation method should be used in radiotherapy dosimetry.

Correlation between patient-specific quality assurance in volumetric modulated arc therapy and 2D dose image features.

In radiotherapy, air-filled ion chamber detectors are ubiquitously used in routine dose measurements for treatment planning. However, its use has been restricted by intrinsic low spatial resolution barriers. We developed one procedure for patient-specific quality assurance (QA) in arc radiotherapy by coalescing two adjacent measurement images into a single image to improve spatial resolution and sampling frequency, and investigated how different spatial resolutions affect the QA results. PTW 729 and 1500 ion chamber detectors were used for dosimetric verification via coalescing two measurements with 5 mm-couch shift and the isocenter, and only isocenter measurement, which we call coalescence and standard acquisition (SA). Statistical process control (SPC), process capability analysis (PCA), and receiver operating characteristic (ROC) curve were used to compare the performance of the two procedures in determining tolerance levels and identifying clinically relevant errors. By analyzing 1256 γ values calculated on interpolated data points, our results indicated that detector 1500 showed higher averages in coalescence cohorts at different tolerance criteria and the dispersion degrees were spread out smaller. Detector 729 yielded a slightly lower process capability of 0.79, 0.76, 1.10, and 1.34, but detector 1500 exhibited somewhat different results of 0.94, 1.42, 1.19, and 1.60 in magnitude. The results of SPC individual control chart showed that cases in coalescence cohorts with γ values lowering its lower control limit (LCL) were greater than those in SA cohorts for detector 1500. A combination of the width of multi-leaf collimator (MLC) leaf, the cross-sectional area of the single detector, and the spacing between adjacent detectors might lead to discrepancies in percent γ values across diverse spatial resolution scenarios. The accuracy of reconstructed volume dose is mainly determined by the interpolation algorithm used in dosimetric systems. The magnitude of filling factor in the ion chamber detectors determined its ability to detect dose deviations. SPC and PCA results indicated that coalescence procedure could detect more potential failure QA results than SA while enhancing action thresholds.

Integrative and Comprehensive Pan-Cancer Analysis of Lymphocyte-Specific Protein Tyrosine Kinase in Human Tumors.

Lymphocyte-specific protein tyrosine kinase (LCK) is common in a variety of hematologic malignancies but comparatively less common in solid tumors. This study aimed to explore the potential diagnostic and prognostic value of LCK across tumors through integrative and comprehensive pan-cancer analysis, as well as experimental validation. Multiple databases were used to explore the expression, alteration, prognostic value, association with immune infiltration, and potential functional pathways of LCK in pan-cancers. The results were further validated by western blotting and qPCR of patient samples as well as tumor cell lines. High LCK expression typically represents a better prognosis. Notably, drug sensitivity prediction of LCK identified P-529 as a candidate for drug development. Gene Annotations (GO) and KEGG analyses showed significant enrichment of PD-L1 and the T-cell receptor pathway. The results from patient samples and tumor cell lines confirmed these conclusions in LIHC. In conclusion, LCK is differentially expressed in multiple tumors and normal tissues. Further analysis highlighted its association with prognostic implications, pan-cancer genetic alterations, and immune signatures. Our data provide evidence for a diagnostic marker of LCK and the possible use of LCK as a target for the treatment of tumors.

CD147-specific chimeric antigen receptor T cells effectively inhibit T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is invasive and heterogeneous, and existing therapies are sometimes unsuccessful. Chimeric antigen receptor (CAR) T cell therapy is a breakthrough tumor treatment method, particularly for B cell acute lymphoblastic leukemia. We found that CD147 was highly expressed in tumor T cells of T-ALL patients and T cell lymphoma. Therefore, CD147-CAR T cells that contain a humanized single-chain variable fragment targeting human CD147 and a second-generation CAR frame were constructed for treating T-ALL. CD147-CAR T cells were able to maintain a healthy proliferation rate, preserving a subset of CD62L+/CCR7+ memory T cells. CD147-CAR T cells showed a potent anti-tumor activity against human T-ALL cell line and T-ALL blasts, releasing high level of cytokines in the process. However, CD147-CAR T cells exhibited potential safety toward human normal cells and CD147-deficent cells. NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt mice were used to establish a T-ALL xenograft model and CD147-CAR T cells conferred robust protection against T-ALL progression and significantly improved survival in mice. Overall, we found that CD147 is a potential antigen target of CAR T cell therapy for T-ALL.

Spinal 5-HT2A receptor is involved in electroacupuncture inhibition of chronic pain.

Knee osteoarthritis (KOA) is a highly prevalent, chronic joint disorder, and it is a typical disease which can develop chronic pain. Our previous study has proved that endocannabinoid (2-AG)-CB1R-GABA-5-HT pathway is involved in electroacupuncture (EA) mediated inhibition of chronic pain. However, it is still unclear which among the 5-HT receptor subtype is involved in EA evoked 5-HT mediated inhibition of chronic pain in the dorsal spinal cord. 5-HT2A is a G protein-coupled receptor and it is involved in 5-HT descending pain modulation system. We found that EA treatment at frequency of 2 Hz +1 mA significantly increased the expression of 5-HT2A receptor in the dorsal spinal cord and intrathecal injection of 5-HT2A receptor antagonist or agonist reversed or mimicked the analgesic effect of EA in each case respectively. Intrathecal injection of a selective GABAA receptor antagonist Bicuculline also reversed the EA effect on pain hypersensitivity. Additionally, EA treatment reversed the reduced expression of GABAA receptor and KCC2 in the dorsal spinal cord of KOA mice. Furthermore, we demonstrated that intrathecal 5-HT2A receptor antagonist/agonist reversed or mimicked the effect of EA up-regulate of KCC2 expression, respectively. Similarly, intrathecal injection of PLC and PKC inhibitors prevented both anti-allodynic effect and up-regulation of KCC2 expression by EA treatment. Our data suggest that EA treatment up-regulated KCC2 expression through activating 5-HT2A-Gq-PLC-PKC pathway and enhanced the inhibitory function of GABAA receptor, thereby inhibiting chronic pain in a mouse model of KOA.

Active demethylation upregulates CD147 expression promoting non-small cell lung cancer invasion and metastasis.

Non-small cell lung cancer (NSCLC) is a fatal disease, and its metastatic process is poorly understood. Although aberrant methylation is involved in tumor progression, the mechanisms underlying dynamic DNA methylation remain to be elucidated. It is significant to study the molecular mechanism of NSCLC metastasis and identify new biomarkers for NSCLC early diagnosis. Here, we performed MeDIP-seq and hMeDIP-seq analyses to detect the genes regulated by dynamic DNA methylation. Comparison of the 5mC and 5hmC sites revealed that the CD147 gene underwent active demethylation in NSCLC tissues compared with normal tissues, and this demethylation upregulated CD147 expression. Significantly high levels of CD147 expression and low levels of promoter methylation were observed in NSCLC tissues. Then, we identified the CD147 promoter as a target of KLF6, MeCP2, and DNMT3A. Treatment of cells with TGF-ß triggered active demethylation involving loss of KLF6/MeCP2/DNMT3A and recruitment of Sp1, Tet1, TDG, and SMAD2/3 transcription complexes. A dCas9-SunTag-DNMAT3A-sgCD147-targeted methylation system was constructed to reverse CD147 expression. The targeted methylation system downregulated CD147 expression and inhibited NSCLC proliferation and metastasis in vitro and in vivo. Accordingly, we used cfDNA to detect the levels of CD147 methylation in NSCLC tissues and found that the CD147 methylation levels exhibited an inverse relationship with tumor size, lymphatic metastasis, and TNM stage. In conclusion, this study clarified the mechanism of active demethylation of CD147 and suggested that the targeted methylation of CD147 could inhibit NSCLC invasion and metastasis, providing a highly promising therapeutic target for NSCLC.

5-HT7 Receptor Is Involved in Electroacupuncture Inhibition of Chronic Pain in the Spinal Cord.

Knee osteoarthritis (KOA) is a common and disabling condition characterized by attacks of pain around the joints, and it is a typical disease that develops chronic pain. Previous studies have proved that 5-HT1, 5-HT2, and 5-HT3 receptors in the spinal cord are involved in electroacupuncture (EA) analgesia. The 5-HT7 receptor plays antinociceptive role in the spinal cord. However, it is unclear whether the 5-HT7 receptor is involved in EA analgesia. The 5-HT7 receptor is a stimulatory G-protein (Gs)-coupled receptor that activates adenylyl cyclase (AC) to stimulate cyclic adenosine monophosphate (cAMP) formation, which in turn activates protein kinase A (PKA). In the present study, we found that EA significantly increased the tactile threshold and the expression of the 5-HT7 receptor in the dorsal spinal cord. Intrathecal injection of 5-HT7 receptor agonist AS-19 mimicked the analgesic effect of EA, while a selective 5-HT7 receptor antagonist reversed this effect. Moreover, intrathecal injection of AC and PKA antagonists prior to EA intervention prevented its anti-allodynic effect. In addition, GABAA receptor antagonist bicuculline administered (intrathecal, i.t.) prior to EA intervention blocked the EA effect on pain hypersensitivity. Our data suggest that the spinal 5-HT7 receptor activates GABAergic neurons through the Gs-cAMP-PKA pathway and participates in EA-mediated inhibition of chronic pain in a mouse model of KOA.

NIR/photoacoustic imaging of multitype gallbladder cancer using carboxyl/amino functionalized polymer dots.

Gallbladder cancer has high incidence and mortality and a low early diagnosis rate and requires rapid and efficient diagnosis. Herein, carboxyl/amino functionalized polymer dots (Pdots) were designed to enhance cellular internalization and tumor accumulation. The prepared Pdots were 40-50 nm in diameter, contained no toxic metal, exhibited long circulation time and high stability, and produced strong NIR emission and photoacoustic signals. Different cellular uptake and distribution of functionalized Pdots in eight gallbladder cell lines were quantitatively investigated using flow cytometry and super-resolution microscopy. In vivo NIR fluorescence imaging showed that the functional Pdots had high accumulation in the tumor after 30 minutes of injection and remained there for up to 6 days. In addition, photoacoustic imaging found that the abundant blood vessels around the tumor microenvironment and Pdots entered the tumor through the blood vessels. Furthermore, a high heterogeneity of vascular networks was visualized in real-time and high resolution by probe-based confocal laser endomicroscopy imaging. These results offer a new avenue for the development of functional Pdots as a probe for multi-modal and multi-scale imaging of gallbladder cancer in small animals.

The Effect of Polymer Dots During Mammalian Early Embryo Development and Their Biocompatibility on Maternal Health.

Conjugated polymer dots have excellent fluorescence properties in terms of their structural diversity and functional design, showing broad application prospects in the fields of biological imaging and biosensing. Polymer dots contain no heavy metals and are thought to be of low toxicity and good biocompatibility. Therefore, systematic studies on their potential toxicity are needed. Herein, the biocompatibility of poly[(9,9-dioctylfluorenyl-2,7diyl)-co-(1,4-benzo-{2,1',3}-thiadiazole)],10% benzothiadiazole(y) (PFBT) and poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) polymer dots on early embryo development as well as maternal health is studied in detail. The results show that prepared polymer dots are dose-dependently toxic to preimplantation embryos, and low-dose polymer dots can be used for cell labeling of early embryos without affecting the normal development of embryos into blastocysts. In addition, the in vivo distribution data show that the polymer dots accumulate mainly in the maternal liver, spleen, kidney, placenta, ovary, and lymph nodes of the pregnant mice. Histopathological examination and blood biochemical tests demonstrate that exposure of the maternal body to polymer dots at a dosage of 14 µg g-1 does not affect the normal function of the maternal organs and early fetal development. The research provides a safe basis for the wide application of polymer dots.

Fluorescent Polymer Dots for Tracking SKOV3 Cells in Living Mice with Probe-Based Confocal Laser Endomicroscopy.

PURPOSE: Probe-based confocal laser endomicroscopy (pCLE) is a novel technique allowing real-time and high-resolution imaging in vivo. It provides microscopic images and increases the penetration depth of tissues compared with conventional white light endoscopy. The aim of the present study was to track ovarian cancer cells in organs by fluorescent polymer dots based on pCLE. PROCEDURES: SKOV3-mCherry cells were incubated with polymer dots for 24 h in a serum-free culture medium. Labeled cells were administrated to nude mice via intravenous, intraperitoneal, and lymph node injection. The fluorescent signals of labeled cells in organs were observed by pCLE. Furthermore, the results were confirmed by frozen section analysis. RESULTS: pCLE displayed fluorescence signals of labeled cells in the vessels of organs. Besides, the accumulations of labeled cells visualized in detoxification organs like the spleen and kidney were increased with time. CONCLUSIONS: In this article, we present a real-time and convenient method for tracking SKOV3-mCherry in living mice by combined fluorescent polymer dots with pCLE.

High resolution tracking of macrophage cells in deep organs and lymphatics using fluorescent polymer dots.

In vivo cell tracking can provide information on cell migration and accumulation in the organs. Here, both folate and amino modified polymer dots were synthesized and screened for in vitro and in vivo tracking of macrophage Ana-1 cells. Flow cytometry analysis demonstrated that prepared polymer dots showed cellular uptake of approximately 98% within a short incubation time of 2 h, and these polymer dots maintained a cell labeling rate over 97% after 2 d. Moreover, a CCK-8 assay suggested that these polymer dots increased Ana-1 cell viabilities up to 110% at concentrations from 5 to 50 µg mL-1. Furthermore, the in vivo real time imaging of labelled Ana-1 cells in the alveolus of lung and lymph nodes were clearly detected by probe-based confocal laser endomicroscopy (pCLE). This study demonstrates a unique approach using polymer dots for real-time high resolution tracking of macrophage cells in deep organs and the lymphatic system.

Multiscale Imaging of Brown Adipose Tissue in Living Mice/Rats with Fluorescent Polymer Dots.

Brown adipose tissue (BAT) has been identified as a promising target for the treatment of obesity, diabetes, and relevant metabolism disorders because of the adaptive thermogenesis ability of this tissue. Visualizing BAT may provide an essential tool for pathology study, drug screening, and efficacy evaluation. Owing to limitations of current nuclear and magnetic resonance imaging approaches for BAT detection, fluorescence imaging has advantages in large-scale preclinical research on small animals. Here, fast BAT imaging in mice is conducted based on polymer dots as fluorescent probes. As early as 5 min after the intravenous injection of polymer dots, extensive fluorescence is detected in the interscapular BAT and axillar BAT. In addition, axillar and inguinal white adipose tissues (WAT) are recognized. The real-time in vivo behavior of polymer dots in rodents is monitored using the probe-based confocal laser endomicroscopy imaging, and the preferred accumulation in BAT over WAT is confirmed by histological assays. Moreover, the whole study is conducted without a low temperature or pharmaceutical stimulation. The imaging efficacy is verified at the cellular, histological, and whole-body levels, and the present results indicate that fluorescent polymer dots may be a promising tool for the visualization of BAT in living subjects.

Facilely synthesized pH-responsive fluorescent polymer dots entrapping doped and coupled doxorubicin for nucleus-targeted chemotherapy.

Doxorubicin (Dox) functionalization methods can affect the Dox loading efficiency and release capability in nanosized drug delivery systems. Herein, different Dox-functionalized (doping, coupling, and doping and coupling) fluorescent poly(9,9-dioctylfluorene-alt-benzothiadiazole) (PFBT) polymer dots were designed and their application performance was evaluated. Polyethylene glycol-modified doxorubicin (PEG-Dox) was synthesized using a responsive hydrazone linker. PEG-DOX-doped polymer dots were self-assembled using a co-precipitation method and free PEG-Dox was further coupled on the surface of the polymer dots via an EDC-NHS coupling to prepare the PEG-Dox doped and coupled PFBT polymer dots. The hydrazone linker of PEG-Dox is responsive to the acidic environment, resulting in the doped Dox being released into the cell nucleus and the coupled Dox changes the structure of the polymer dots and accelerates the release of the doped Dox. Moreover, the coupled Dox linked via an amide bond was still on the surface of the polymer dots and retained their cytoplasmic toxicity for a synergistic effect. A high Dox carrying efficiency (weight of the loaded Dox/weight of PFBT) was achieved using the PEG-Dox doped and coupled PFBT polymer dots: 107% and 82% of Dox was released in vitro within 24 h at pH 5.5. The cytotoxicity and cell imaging were investigated in three cancer cell lines: cervical cancer cells (HeLa), lung cancer cells (NCI-H292), and glioma cells (U87-MG); the results indicate that the PEG-Dox doped and coupled PFBT polymer dots show a distinct killing efficacy and nucleus targeted capability. Moreover, in vivo tumor suppression was observed in lung tumor-bearing mice over 20 days and no weight loss and damage or inflammation of the major organs were detected using the PEG-Dox doped and coupled PFBT polymer dots.