Hypothalamic NPFFR2 attenuates central insulin signaling and its knockout diminishes metabolic dysfunction in mouse models of diabetes mellitus.

BACKGROUND: The hypothalamus is a crucial brain region that mediates the effects of insulin and leptin signals on peripheral metabolic functions. Previous research has shown that insulin signals in the hypothalamus act via multiple neuronal circuits and anabolic/catabolic pathways that converge on the vagus nerve and sympathetic fibers to coordinate energy metabolism in peripheral organs. Additionally, neuropeptide FF (NPFF) has been identified as a regulator of feeding behaviors and energy homeostasis in the hypothalamus, but the mechanisms underlying its involvement in metabolic control remain unclear. This study aims to explore the underlying mechanisms of NPFF in modulating metabolic disorders. METHODS: In this study, we investigated the physiological role of NPFF in insulin-related energy homeostasis and metabolic health. First, we evaluated the effects of NPFF and its receptors on central insulin signaling using mouse hypothalamic cell lines and Npffr2-overexpressing mice. To further explore the effects of NPFFR2 on insulin-related metabolic disorders, such as diabetes mellitus, we used Npffr2-deleted mice in combination with the streptozotocin (STZ)-induced type 1 diabetes and high-fat diet/STZ-induced type 2 diabetic mouse models. The impacts of central NPFFR2 were demonstrated specifically through Npffr2 overexpression in the hypothalamic arcuate nucleus, which subsequently induced type 2 diabetes. RESULTS: We found that stimulating NPFFR2 in the hypothalamus blocked hypothalamic insulin activity. Npffr2 deletion improved central and peripheral metabolic symptoms in both mouse models of diabetes mellitus, exerting effects on central and systemic insulin resistance, feeding behaviors, glucose and insulin intolerance, lipid metabolism, liver steatosis, and inflammation of white adipose tissues. The overexpression of ARC Npffr2 augmented the metabolic dysregulation in the mouse model of type 2 diabetes. CONCLUSIONS: Our findings demonstrate that hypothalamic NPFFR2 negatively regulates insulin signaling in the central nervous system and plays an important role in maintaining systemic metabolic health, thereby providing valuable insights for potential clinical interventions targeting these health challenges.

Glibenclamide promotes FGF21 secretion in interscapular BAT and attenuates depression-like behaviors in male mice with HFD-induced obesity.

AIMS: Epidemiological evidence suggests that comorbidity of obesity and depression is extremely common and continues to grow in prevalence. However, the mechanisms connecting these two conditions are unknown. In this study, we explored how treatment with KATP channel blocker glibenclamide (GB) or the well-known metabolic regulator FGF21 impact male mice with high-fat diet (HFD)-induced obesity and depressive-like behaviors. MATERIALS AND METHODS: Mice were fed with HFD for 12 weeks and then treated with recombinant FGF21 protein by infusion for 2 weeks, followed by intraperitoneal injection of 3 mg/kg recombinant FGF21 once per day for 4 days. Measurements were made of catecholamine levels, energy expenditure, biochemical endpoints and behavior tests, including sucrose preference and forced swim tests were. Alternatively, animals were infused with GB into brown adipose tissue (BAT). The WT-1 brown adipocyte cell line was used for molecular studies. KEY FINDINGS: Compared to HFD controls, HFD + FGF21 mice exhibited less severe metabolic disorder symptoms, improved depressive-like behaviors, and more extensive mesolimbic dopamine projections. FGF21 treatment also rescued HFD-induced dysregulation of FGF21 receptors (FGFR1 and co-receptor ß-klotho) in the ventral tegmental area (VTA), and it altered dopaminergic neuron activity and morphology in HFD-fed mice. Importantly, we also found that FGF21 mRNA level and FGF21 release were increased in BAT after administration of GB, and GB treatment to BAT reversed HFD-induced dysregulation of FGF21 receptors in the VTA. SIGNIFICANCE: GB administration to BAT stimulates FGF21 production in BAT, corrects HFD-induced dysregulation of FGF21 receptor dimers in VTA dopaminergic neurons, and attenuates depression-like symptoms.

Targeting nanoparticle-conjugated microbubbles combined with ultrasound-mediated microbubble destruction for enhanced tumor therapy.

The stress of the abnormal stromal matrix of solid tumors is a major limiting factor that prevents drug penetration. Controlled, accurate, and efficient delivery of theranostic agents into tumor cells is crucial. Combining ultrasound with nanocarrierbased drug delivery systems have become a promising approach for targeted drug delivery in preclinical cancer therapy. In this study, to ensure effective tumor barrier penetration, access to the tumor microenvironment, and local drug release, we designed targeted nanoparticle (NP)-conjugated microbubbles (MBs); ultrasound could then help deliver acoustic energy to release the NPs from the MBs. The ultrasound-targeted MB destruction (UTMD) system of negatively charged NPs was conjugated with positively charged MBs using an ionic gelation method. We demonstrated the transfer of targeted NPs and their entry into gastric cancer cells through ligand-specific recognition, followed by enhanced cell growth inhibition owing to drug delivery-induced apoptosis. Moreover, the UTMD system combining therapeutic and ultrasound image properties can effectively target gastric cancer, thus significantly enhancing antitumor activity, as evident by tumor localization in an orthotopic mouse model of gastric cancer. The combination of ultrasound and NP-based drug delivery systems has become a promising approach for targeted drug delivery in preclinical cancer therapy.

Targeting Tumor Cells with Nanoparticles for Enhanced Co-Drug Delivery in Cancer Treatment.

Gastric cancer (GC) is a fatal malignant tumor, and effective therapies to attenuate its progression are lacking. Nanoparticle (NP)-based solutions may enable the design of novel treatments to eliminate GC. Refined, receptor-targetable NPs can selectively target cancer cells and improve the cellular uptake of drugs. To overcome the current limitations and enhance the therapeutic effects, epigallocatechin-3-gallate (EGCG) and low-concentration doxorubicin (DX) were encapsulated in fucoidan and d-alpha-tocopherylpoly (ethylene glycol) succinate-conjugated hyaluronic acid-based NPs for targeting P-selectin-and cluster of differentiation (CD)44-expressing gastric tumors. The EGCG/DX-loaded NPs bound to GC cells and released bioactive combination drugs, demonstrating better anti-cancer effects than the EGCG/DX combination solution. In vivo assays in an orthotopic gastric tumor mouse model showed that the EGCG/DX-loaded NPs significantly increased the activity of gastric tumors without inducing organ injury. Overall, our EGCG/DX-NP system exerted a beneficial effect on GC treatment and may facilitate the development of nanomedicine-based combination chemotherapy against GC in the future.

Correction to: The knockdown of Neuropeptide FF receptor 2 inhibits capsaicin-induced CGRP Upregulation in mouse trigeminal ganglion.

An amendment to this paper has been published and can be accessed via the original article.

Neuropeptide FF receptor 2 inhibits capsaicin-induced CGRP Upregulation in mouse trigeminal ganglion.

BACKGROUND: Stimulation of trigeminovascular pathway is widely used to establish the headache animal model. Headache is a common neurological disorder, in which symptomatic attacks are mediated by calcitonin-gene-related peptide (CGRP). CGRP is synthesized and released from the trigeminal ganglion to transmit pain signals under stimulation. On the other hand, Neuropeptide FF (NPFF) is a candidate transmitter/modulator for migraine, and stimulation of its receptor, NPFFR2, increases the expression and release of CGRP in mice sensory neurons. Here, we investigate the impact of NPFFR2 on trigeminal CGRP level in a capsaicin-induced headache mouse model. METHODS: Mice were intracisternally injected with capsaicin into the cisterna magna to activate the trigeminovascular pathway and induce headache symptoms. Mice pretreated with Npffr2-shRNA or NPFFR2 knockouts were adopted to test the impact of NPFFR2 on capsaicin-induced CGRP upregulation in trigeminal ganglion. The gene silencing effect of Npffr2-shRNA in trigeminal ganglion was confirmed by real-time PCR. Trigeminal CGRP level was determined by immunofluorescence staining, and the percentage of CGRP-positive cell was calculated after setting the signal intensity threshold by Image J software. Amount of trigeminal CGRP in NPFFR2 overexpressed mice was also measured by CGRP ELISA. FINDINGS: Infusion of capsaicin into the cisterna magna upregulated the CGRP in trigeminal ganglion and induced spontaneous pain behaviors including the reduction of locomotor activity and the increase of freezing behavior. Intracisternal injection of Npffr2-shRNA reduced the mRNA of Npffr2 in trigeminal ganglion. Mice pretreatment with Npffr2-shRNA prevented capsaicin-induced CGRP upregulation in trigeminal ganglion. Similarly, CGRP upregulation was also reduced in NPFFR2 knockout mice. On the contrary, trigeminal CGRP was increased in NPFFR2 overexpressed mice. CONCLUSIONS: Reducing the level of NPFFR2 leads to the downregulation of capsaicin-induced CGRP in trigeminal ganglion, which would consequently attenuate the activation of trigeminovascular pathway. Thus, NPFFR2 could serve as a potential target for neuromodulation of cephalic pain.

Monitoring of acoustic cavitation in microbubble-presented focused ultrasound exposure using gradient-echo MRI.

BACKGROUND: Gadolinium-based contrast agents can be used to identify the blood-brain barrier (BBB) opening after inducing a focused ultrasound (FUS) cavitation effect in the presence of microbubbles. However, the use of gadolinium may be limited for frequent routine monitoring of the BBB opening in clinical applications. PURPOSE: To use a gradient-echo sequence without contrast agent administration for monitoring of acoustic cavitation. STUDY TYPE: Animal and phantom prospective. PHANTOM/ANIMAL MODEL: Static and flowing gel phantoms; six normal adult male Sprague-Dawley rats. FIELD STRENGTH/SEQUENCE: 3T, 7T; fast low-angle shot sequence. ASSESSMENT: Burst FUS with acoustic pressures = 1.5, 2.2, 2.8 MPa; pulse repetition frequencies = 1, 10,100 Hz; and duty cycles = 2%, 5%, 10% were transmitted to the chamber of a static phantom with microbubble concentrations = 10%, 1%, 0.1%. MR slice thicknesses = 3, 6, 8 mm were acquired. In flowing phantom experiments, 0.1%, 0.25%, 0.5%, 0.75%, and 1% microbubbles were infused and transmitted by burst FUS with an acoustic pressure = 0.4 and 1 MPa. In in vivo experiments, 0.25% microbubbles was infused and 0.8 MPa burst FUS was transmitted to targeted brain tissue beneath the superior sagittal sinus. The mean signal intensity (SI) was normalized using the mean SI from pre-FUS. STATISTICAL TESTS: Two-tailed Student's t-test. P < 0.05 was considered statistically significant. RESULTS: In the static phantom, the time courses of normalized SI decreases to minimum SI levels of 70-80%. In the flowing phantom, substantial normalized SI of 160-230% was present with variant acoustic pressures and microbubble concentrations. Compared with in vivo control rats, the brain tissue of experimental rats with transmission of FUS pulses exhibited considerable decreases of normalized SI (P < 0.001) because of the cavitation-induced perturbation of flow. DATA CONCLUSION: Observing gradient-echo SI changes can help monitor the targeted location of microbubble-enhanced FUS, which in turn assists the monitoring of the BBB opening. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2020;51:311-318.

Focused Ultrasound-Induced Blood-Brain Barrier Opening Enhances GSK-3 Inhibitor Delivery for Amyloid-Beta Plaque Reduction.

Alzheimer's disease (AD) is a neurodegenerative disease that is the leading cause of age-related dementia. Currently, therapeutic agent delivery to the CNS is a valued approach for AD therapy. Unfortunately, the CNS penetration is greatly hampered by the blood-brain barrier (BBB). Focused-ultrasound (FUS) has been demonstrated to temporally open the BBB, thus promoting therapeutic agent delivery to the CNS. Recently, the BBB opening procedure was further reported to clear the deposited Aß plaque due to microglia activation. In this study, we aimed to evaluate whether the use of FUS-induced BBB opening to enhance GSK-3 inhibitor delivery, which would bring additive effect of Aß plaque clearance by FUS with the reduction of Aß plaque synthesis by GSK-3 inhibitor in an AD mice model. FUS-induced BBB opening on APPswe/PSEN1-dE9 transgenic mice was performed unilaterally, with the contralateral hemisphere serving as a reference. GSK-3 level was confirmed by immunohistochemistry (IHC) and autoradiography (ARG) was also conducted to quantitatively confirm the Aß plaque reduction. Results from IHC showed GSK-3 inhibitor effectively reduced GSK-3 activity up to 61.3% with the addition of FUS-BBB opening and confirming the proposed therapeutic route. ARG also showed significant Aß-plaque reduction up to 31.5%. This study reveals the therapeutic potentials of ultrasound to AD treatment, and may provide a useful strategy for neurodegenerative disease treatment.

Drug-carrying microbubbles as a theranostic tool in convection-enhanced delivery for brain tumor therapy.

Convection-enhanced delivery (CED) is a promising technique for infusing a therapeutic agent through a catheter with a pressure gradient to create bulk flow for improving drug spread into the brain. So far, gadopentetate dimeglumine (Gd-DTPA) is the most commonly applied surrogate agent for predicting drug distribution through magnetic resonance imaging (MRI). However, Gd-DTPA provides only a short observation duration, and concurrent infusion provides an indirect measure of the exact drug distribution. In this study, we propose using microbubbles as a contrast agent for MRI monitoring, and evaluate their use as a drug-carrying vehicle to directly monitor the infused drug. Results show that microbubbles can provide excellent detectability through MRI relaxometry and accurately represent drug distribution during CED infusion. Compared with the short half-life of Gd-DTPA (1-2 hours), microbubbles allow an extended observation period of up to 12 hours. Moreover, microbubbles provide a sufficiently high drug payload, and glioma mice that underwent a CED infusion of microbubbles carrying doxorubicin presented considerable tumor growth suppression and a significantly improved survival rate. This study recommends microbubbles as a new theranostic tool for CED procedures.

Activation of NPFFR2 leads to hyperalgesia through the spinal inflammatory mediator CGRP in mice.

Neuropeptide FF (NPFF) is recognized as an opioid modulating peptide that regulates morphine-induced analgesia. The aim of this study was to delineate the role of NPFFR2 in pain transmission. We found the expression levels of NPFF and NPFFR2 were increased in the lumbar dorsal horn of animals with CFA- and carrageenan-induced inflammation and both NPFFR2 over-expressing transgenic (NPFFR2-Tg) and NPFFR2 agonist-treated mice displayed hyperalgesia. BOLD signals from functional MRI showed that NPFFR2-Tg mice exhibited increased activation of pain-related brain regions after painful stimulation when compared to WT mice. Inflammatory mediators within the spinal cord, calcitonin gene-related peptide (CGRP) and substance P (SP), were up-regulated in NPFFR2-Tg and chronic NPFFR2 agonist-treated mice. In DRG cultures, treatment with an NPFFR2 agonist induced the expression and release of CGRP, an action which was blocked by NPFFR2 siRNA. Furthermore, treatment with a CGRP antagonist ameliorated the pain hyperalgesia in NPFFR2-Tg mice, returning the pain threshold to a control level. However, treatment with a SP antagonist reduced the pain responses in both WT and NPFFR2-Tg mice and did not suppress pain hypersensitivity in NPFFR2-Tg mice. Together, these results demonstrate that NPFFR2 activation modulates pain transmission by up-regulating the pain mediator CGRP, leading to hyperalgesia.

Ultrasound/Magnetic Targeting with SPIO-DOX-Microbubble Complex for Image-Guided Drug Delivery in Brain Tumors.

One of the greatest challenges in the deployment of chemotherapeutic drugs against brain tumors is ensuring that sufficient drug concentrations reach the tumor, while minimizing drug accumulation at undesired sites. Recently, injection of therapeutic agents following blood-brain barrier (BBB) opening by focused ultrasound (FUS) with microbubbles (MBs) has been shown to enhance drug delivery in targeted brain regions. Nevertheless, the distribution and quantitative deposition of agents delivered to the brain are still hard to estimate. Based on our previous work on superparamagnetic iron oxide (SPIO)-loaded MBs, we present a novel theranostic complex of SPIO-Doxorubicin (DOX)-conjugated MB (SD-MB) for drug delivery to the brain. Magnetic labeling of the drug enables direct visualization via magnetic resonance imaging, and also facilitates magnetic targeting (MT) to actively enhance targeted deposition of the drug. In a rat glioma model, we demonstrated that FUS sonication can be used with SD-MBs to simultaneously facilitate BBB opening and allow dual ultrasound/magnetic targeting of chemotherapeutic agent (DOX) delivery. The accumulation of SD complex within brain tumors can be significantly enhanced by MT (25.7 fold of DOX, 7.6 fold of SPIO). The change in relaxation rate R2 (1/T2) within tumors was highly correlated with SD deposition as quantified by high performance liquid chromatography (R(2) = 0.93) and inductively coupled plasma-atomic emission spectroscopy (R(2) = 0.94), demonstrating real-time monitoring of DOX distribution. Our results suggest that SD-MBs can serve as multifunction agents to achieve advanced molecular theranostics.

Focused Ultrasound Enhances Central Nervous System Delivery of Bevacizumab for Malignant Glioma Treatment.

Purpose To demonstrate that magnetic resonance (MR) imaging-monitored transcranial focused ultrasound can enhance the delivery of the antiangiogenic monoclonal antibody bevacizumab into the central nervous system (CNS) for glioblastoma multiforme (GBM) treatment. Materials and Methods All animal experiments were approved by the animal committee and adhered to experimental animal care guidelines. Transcranial focused ultrasound exposure in the presence of microbubbles was used to open the blood-brain barrier (BBB) to enhance bevacizumab penetration into the CNS in healthy and glioma-bearing mice. Bevacizumab concentration was quantitated with high-performance liquid chromatography, and Western blot testing was performed to confirm the specific biologic form in the CNS. Penetration of bevacizumab into brain tissue was estimated in vivo by means of contrast material-enhanced MR imaging and quantitative gallium 68 ((68)Ga)-bevacizumab micro-positron emission tomography, and glioma progression was longitudinally followed with T2-weighted MR imaging. Hematoxylin-eosin staining and cluster of differentiation 31 immunostaining were used to assess morphologic changes and vascular inhibition at histologic examination. The two-tailed Student t test and the Mantel-Cox log-rank test were used for statistical analyses, with a significance level of .05. Results Focused ultrasound significantly enhanced bevacizumab penetration into the CNS by 5.7- to 56.7-fold compared with that in nonexposed brain (both P < .0001). Contrast-enhanced MR imaging indexes correlated with bevacizumab concentration (r = 0.748-0.857) in vivo. Focused ultrasound-enhanced bevacizumab delivery significantly retarded glioma progression, with a significantly increased median survival (median increase in survival time = 135% in the group treated with bevacizumab and focused ultrasound, P < .0001; as compared with 48% in the group treated with bevacizumab alone, P = .0002). Conclusion Focused ultrasound-enhanced bevacizumab delivery can provide an antivascularization normalization effect to suppress glioma. (©) RSNA, 2016 Online supplemental material is available for this article.

Non-invasive screening for early Alzheimer's disease diagnosis by a sensitively immunomagnetic biosensor.

Amyloid-beta peptide 1-42 (Aß42) is considered as a reliable biomarker for the early diagnosis of Alzheimer's disease (AD). Thus, it is urgent to develop a simple and efficient method for the detection of Aß42. In this work, a reusable biosensor based on magnetic nitrogen-doped graphene (MNG) modified Au electrode for the detection of Aß42 has been developed. The antibodies of Aß 1-28 (Aßab) are used as the specific biorecognition element for Aß42 that were conjugated on the surface of MNG. In the presence of magnetic nanoparticles on MNG, the electrode coating material, the biosensor can be quickly constructed, without requiring an electrode drying process, which reduce the analysis time and is convenient for proceeding to detection. The reusable biosensor with good reproducibility and stability was linear within the range from 5 pg mL(-1) to 800 pg mL(-1), covering the cut-off level of Aß42 and a detection limit of 5 pg mL(-1) had been achieved. Furthermore, the fabricated biosensor for Aß42 detection not only improves the detection performance but also reduces the cost and shortens the response time, demonstrating its potential in diagnosing applications.

A theranostic nrGO@MSN-ION nanocarrier developed to enhance the combination effect of sonodynamic therapy and ultrasound hyperthermia for treating tumor.

Sonodynamic therapy (SDT), which induces activation of sonosensitizers in cancer cells through ultrasound irradiation, has emerged as an alternative and promising noninvasive therapeutic approach to kill both superficial and deep parts of tumors. In this study, mesoporous silica (MSN) grown on reduced graphene oxide nanosheet (nrGO) capped with Rose Bengal (RB)-PEG-conjugated iron-oxide nanoparticles (IONs), nrGO@MSN-ION-PEG-RB, was strategically designed to have targeted functionality and therapeutic efficacy under magnetic guiding and focused ultrasound (FUS) irradiation, respectively. The singlet oxygen produced by ultrasound-activated RB and the ultrasound-induced heating effect was enhanced by rGO and IONs, which improved the cytotoxic effect in cancer cells. In an animal experiment, we demonstrated that the combination of sonodynamic/hyperthermia therapy with magnetic guidance using this nanocomposite therapeutic agent can produce remarkable efficacious therapy in tumor growth inhibition. Furthermore, the combination effect induced by FUS irradiation produces significant damage to both superficial and deep parts of the targeted tumor.

A multitheragnostic nanobubble system to induce blood-brain barrier disruption with magnetically guided focused ultrasound.

A novel magnetically guidable nanobubble is designed for disrupting the blood-brain barrier (BBB) by combining magnetic guidance with focused ultrasound in vivo. The magnetic-nanobubble platform also demonstrates the potential to serve as a unique theranostic tool via performing focused ultrasound (FUS)-induced BBB disruption and magnetic resonance imaging (MRI)/ultrasound dual-modality contrast-agent imaging to improve the drug delivery of therapeutic substances or gene therapy into the central nervous system.

Evaluation of prognostic integrin α2ß1 PET tracer and concurrent targeting delivery using focused ultrasound for brain glioma detection.

The ability to early detect and assess the treatment response of recurrent and/or disseminated metastatic glioblastoma is critical for the effective management of this group of patients. Accumulating experimental evidence indicates that integrin α2ß1 might be a prognostic biomarker for advanced phenotype of cancers. In this study, a novel (68)Ga-labeled integrin α2ß1-targeted PET tracer (68)Ga-NOTA-PEG4-cyclo (GDGEAyK) ((68)Ga-A2B1) was designed and evaluated for the potential prognostic imaging of glioblastoma tumor in preclinical model. To prospectively verify the prognostic value of integrin α2ß1, the in vitro Western blot and flow cytometry studies were performed to validate the integrin expression level of human glioblastoma (U87MG) cells. Extremely high expression level of integrin α2ß1 justifies its role as a potential targeting marker. Thus, (68)Ga-A2B1 positron emission tomography was performed in subcutaneous U87MG tumor bearing athymic mice at 15 min postinjection after injection of 7-8MBq tracers. The receptor targeting specificity was confirmed in a competition blocking experiment. The tumor uptake of (68)Ga-A2B1 in the control and blockage groups was 1.57 ± 0.13 %ID/g (n = 3) and 0.96 ± 0.23 %ID/g** (n = 3), respectively. However, because of the quick renal washout rate and labile nature of peptide tracers in circulation conditions, the focus ultrasound (FUS) mediated delivery method was adopted to enhance tumor uptake and retention of tracers. To test the FUS delivery efficacy in vivo, three experimental arms were designed as follows: tumor bearing mice were administrated with (68)Ga-A2B1 only or microbubbles (MBs) with FUS treatment ((68)Ga-A2B1 + FUS + MBs) or embedded (68)Ga-A2B1-microbubbles ((68)Ga-A2B1-MBs + FUS) followed with FUS sonication. The average radioactivity accumulation within a tumor was quantified from the multiple region of interest volumes using the %ID/g value and was analyzed in accordance with the ex vivo autoradiographic and pathologic data. The significant tumor uptake in (68)Ga-A2B1 + FUS +MBs group (n = 6) and (68)Ga-A2B1-MBs + FUS group (n = 4) following FUS treatment were calculated as 2.25 ± 0.50 %ID/g* and 2.6 ± 0.49 %ID/g**, comparing with (68)Ga-A2B1 only group 1.48 ± 0.42 %ID/g (n = 10). These results suggest that there is significant difference in (68)Ga-A2B1 tumor uptake by FUS treatment either with or without tracer integration with microbubbles, which demonstrate a promising delivery strategy and critical multimodal setting for phenotyping imaging of aggressive glioma tumor. In conclusion, (68)Ga labeled (68)Ga-A2B1 allows noninvasive imaging of tumor-associated α2ß1 expression and can be embedded in MB lipid shell for enhanced delivery and controlled release by sonoporation.

Non-invasive synergistic treatment of brain tumors by targeted chemotherapeutic delivery and amplified focused ultrasound-hyperthermia using magnetic nanographene oxide.

The combination of chemo-thermal therapy is the best strategy to ablate tumors, but how to heat deep tumor tissues effectively without side-damage is a challenge. Here, a systemically delivered nanocarrier is designed with multiple advantages, including superior heat absorption, highly efficient hyperthermia, high drug capacity, specific targeting ability, and molecular imaging, to achieve both high antitumor efficacy and effective amplification of hyperthermia with minimal side effects.

Noninvasive and targeted gene delivery into the brain using microbubble-facilitated focused ultrasound.

Recombinant adeno-associated viral (rAAV) vectors are potentially powerful tools for gene therapy of CNS diseases, but their penetration into brain parenchyma is severely limited by the blood-brain barrier (BBB) and current delivery relies on invasive stereotactic injection. Here we evaluate the local, targeted delivery of rAAV vectors into the brains of mice by noninvasive, reversible, microbubble-facilitated focused ultrasound (FUS), resulting in BBB opening that can be monitored and controlled by magnetic resonance imaging (MRI). Using this method, we found that IV-administered AAV2-GFP (green fluorescence protein) with a low viral vector titer (1×10(9) vg/g) can successfully penetrate the BBB-opened brain regions to express GFP. We show that MRI monitoring of BBB-opening could serve as an indicator of the scale and distribution of AAV transduction. Transduction peaked at 3 weeks and neurons and astrocytes were affected. This novel, noninvasive delivery approach could significantly broaden the application of AAV-viral-vector-based genes for treatment of CNS diseases.

Gold-nanorod contrast-enhanced photoacoustic micro-imaging of focused-ultrasound induced blood-brain-barrier opening in a rat model.

In this study, we develop a novel photoacoustic imaging technique based on gold nanorods (AuNRs) for quantitatively monitoring focused-ultrasound (FUS) induced blood-brain barrier (BBB) opening in a rat model in vivo. This study takes advantage of the strong near-infrared absorption (peak at ≈ 800 nm) of AuNRs and the extravasation tendency from BBB opening foci due to their nano-scale size to passively label the BBB disruption area. Experimental results show that AuNR contrast-enhanced photoacoustic microscopy (PAM) successfully reveals the spatial distribution and temporal response of BBB disruption area in the rat brains. The quantitative measurement of contrast enhancement has potential to estimate the local concentration of AuNRs and even the dosage of therapeutic molecules when AuNRs are further used as nano-carrier for drug delivery or photothermal therapy. The photoacoustic results also provide complementary information to MRI, being helpful to discover more details about FUS induced BBB opening in small animal models.

Paramagnetic perfluorocarbon-filled albumin-(Gd-DTPA) microbubbles for the induction of focused-ultrasound-induced blood-brain barrier opening and concurrent MR and ultrasound imaging.

This paper presents new albumin-shelled Gd-DTPA microbubbles (MBs) that can concurrently serve as a dual-modality contrast agent for ultrasound (US) imaging and magnetic resonance (MR) imaging to assist blood-brain barrier (BBB) opening and detect intracerebral hemorrhage (ICH) during focused ultrasound brain drug delivery. Perfluorocarbon-filled albumin-(Gd-DTPA) MBs were prepared with a mean diameter of 2320 nm and concentration of 2.903×10(9) MBs ml(-1) using albumin-(Gd-DTPA) and by sonication with perfluorocarbon (C(3)F(8)) gas. The albumin-(Gd-DTPA) MBs were then centrifuged and the procedure was repeated until the free Gd(3+) ions were eliminated (which were detected by the xylenol orange sodium salt solution). The albumin-(Gd-DTPA) MBs were also characterized and evaluated both in vitro and in vivo by US and MR imaging. Focused US was used with the albumin-(Gd-DTPA) MBs to induce disruption of the BBB in 18 rats. BBB disruption was confirmed with contrast-enhanced T(1)-weighted turbo-spin-echo sequence MR imaging. Heavy T(2)*-weighted 3D fast low-angle shot sequence MR imaging was used to detect ICH. In vitro US imaging experiments showed that albumin-(Gd-DTPA) MBs can significantly enhance the US contrast in T(1)-, T(2)- and T(2)*-weighted MR images. The r(1) and r(2) relaxivities for Gd-DTPA were 7.69 and 21.35 s(-1)mM(-1), respectively, indicating that the MBs represent a positive contrast agent in T(1)-weighted images. In vivo MR imaging experiments on 18 rats showed that focused US combined with albumin-(Gd-DTPA) MBs can be used to both induce disruption of the BBB and detect ICH. To compare the signal intensity change between pure BBB opening and BBB opening accompanying ICH, albumin-(Gd-DTPA) MB imaging can provide a ratio of 5.14 with significant difference (p = 0.026), whereas Gd-DTPA imaging only provides a ratio of 2.13 and without significant difference (p = 0.108). The results indicate that albumin-(Gd-DTPA) MBs have potential as a US/MR dual-modality contrast agent for BBB opening and differentiating focused-US-induced BBB opening from ICH, and can monitor the focused ultrasound brain drug delivery process.