Po-Hung Hsu

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 (1×109 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.

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.

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.

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.

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.

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 (R2 = 0.93) and inductively coupled plasma-atomic emission spectroscopy (R2 = 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 (Aβ42) 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 Aβ42. In this work, a reusable biosensor based on magnetic nitrogen-doped graphene (MNG) modified Au electrode for the detection of Aβ42 has been developed. The antibodies of Aβ 1–28 (Aβab) are used as the specific biorecognition element for Aβ42 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 Aβ42 and a detection limit of 5 pg mL−1 had been achieved. Furthermore, the fabricated biosensor for Aβ42 detection not only improves the detection performance but also reduces the cost and shortens the response time, demonstrating its potential in diagnosing applications.

Evaluation of Prognostic Integrin α2β1 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 α2β1 might be a prognostic biomarker for advanced phenotype of cancers. In this study, a novel (68)Ga-labeled integrin α2β1-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 α2β1, 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 α2β1 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 α2β1 expression and can be embedded in MB lipid shell for enhanced delivery and controlled release by sonoporation.

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

Abstract 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. HighlightsActivation of NPFFR2 increases the expression and release of CGRP in the DRG.NPFFR2‐evoked CGRP enhances the pain transmission that leads to hyperalgesia.Algesic effect of NPFFR2 and its linkage with CGRP are independent of MOR.NPFFR2‐mediated hyperalgesia could be ameliorated by a CGRP antagonist.