Tinghui Yin

Nanobubbles for enhanced ultrasound imaging of tumors

The fabrication and initial applications of nanobubbles (NBs) have shown promising results in recent years. A small particle size is a basic requirement for ultrasound contrast-enhanced agents that penetrate tumor blood vessel pores to allow for targeted imaging and therapy. However, the nanoscale size of the particles used has the disadvantage of weakening the imaging ability of clinical diagnostic ultrasound. In this work, we fabricated a lipid NBs contrast-enhanced ultrasound agent and evaluated its passive targeting ability in vivo. The results showed that the NBs were small (436.8 ± 5.7 nm), and in vitro ultrasound imaging suggested that the ultrasonic imaging ability is comparable to that of microbubbles (MBs). In vivo experiments confirmed the ability of NBs to passively target tumor tissues. The NBs remained in the tumor area for a longer period because they exhibited enhanced permeability and retention. Direct evidence was obtained by direct observation of red fluorescence-dyed NBs in tumor tissue using confocal laser scanning microscopy. We have demonstrated the ability to fabricate NBs that can be used for the in vivo contrast-enhanced imaging of tumor tissue and that have potential for drug/gene delivery.

Tumor-penetrating codelivery of siRNA and paclitaxel with ultrasound-responsive nanobubbles hetero-assembled from polymeric micelles and liposomes

Drug resistance is a big problem in systemic chemotherapy of hepatocellular carcinoma (HCC), and nanomedicines loaded with both chemotherapeutic agents (e.g. paclitaxel, PTX) and siRNAs targeting antiapoptosis genes (e.g. BCL-2) possess the advantages to simultaneously overcome the efflux pump-mediated drug resistance and antiapoptosis-related drug resistance. However, tumor-penetrating drug delivery with this type of nanomedicines is extremely difficult due to their relatively big size compared to the single drug-loaded nanomedicines. Aiming at address this problem, US-responsive nanobubbles encapsulating both anti-cancer drug paclitaxel (PTX) and siRNA (PTX-NBs/siRNA) for HCC treatment were developed by hetero-assembly of polymeric micelles and liposomes in the present study. Utilizing an external low-frequency US force imposed to the tumor site, effective tumor-penetrating codelivery of siRNA and PTX was achieved via tail vein injection of PTX-NBs/siRNA into nude mice bearing human HepG2 xerografts. Consequently, the PTX treatment-inducible antiapoptosis in HepG2 cells was effectively suppressed by the codelivered siRNA targeting an antiapoptosis gene (BCL-2 siRNA) during chemotherapy. Owing to the synergistic anti-cancer effect of two therapeutic agents, tumor growth was completely inhibited using low-dose PTX in animal study. Our results highlight the great potential of this type of US-responsive hetero-assemblies carrying both anti-cancer drug and siRNA as an effective nanomedicinal system for HCC therapy.

Ultrasound-sensitive siRNA-loaded nanobubbles formed by hetero-assembly of polymeric micelles and liposomes and their therapeutic effect in gliomas

Ultrasound (US)-sensitive nanobubble (NB) which may utilize the physical power of US exposure to improve delivery efficiency to target cells is emerging as one of the most promising nanocarriers for drug delivery. On the basis of successfully fabricating NBs with the ability of passively accumulating in tumor tissue, in this study we synthesized a US-sensitive NB bearing siRNA (siRNA-NB) for tumor therapy via a hetero-assembling strategy using the siRNA-complexed polymeric micelles and gas-cored liposomes. The US exposure-aided siRNA transfection effectively enhanced the gene silencing effect of siRNA-NBs both in vitro and in vivo, which resulted in much elevated level of cancer cell apoptosis. Consequently, significantly improved therapeutic effect was achieved in a nude mouse glioma model, using siRNA-NBs bearing siRNA to target the anti-apoptosis gene sirtuin 2 (SIRT2). These results show that, with the aid of US exposure, the US-sensitive siRNA-NB may be an ideal delivery vector to mediate highly effective RNA interference for tumor treatment.

pH-Sensitive Nanomicelles for Controlled and Efficient Drug Delivery to Human Colorectal Carcinoma LoVo Cells

Background The triblock copolymers PEG-P(Asp-DIP)-P(Lys-Ca) (PEALCa) of polyethylene glycol (PEG), poly(N-(N’,N’-diisopropylaminoethyl) aspartamide) (P(Asp-DIP)), and poly (lysine-cholic acid) (P(Lys-Ca)) were synthesized as a pH-sensitive drug delivery system. In neutral aqueous environment such as physiological environment, PEALCa can self-assemble into stable vesicles with a size around 50-60 nm, avoid uptake by the reticuloendothelial system (RES), and encase the drug in the core. However, the PEALCa micelles disassemble and release drug rapidly in acidic environment that resembles lysosomal compartments. Methodology/Principal Findings The anticancer drug Paclitaxel (PTX) and hydrophilic superparamagnetic iron oxide (SPIO) were encapsulated inside the core of the PEALCa micelles and used for potential cancer therapy. Drug release study revealed that PTX in the micelles was released faster at pH 5.0 than at pH 7.4. Cell culture studies showed that the PTX-SPIO-PEALCa micelle was effectively internalized by human colon carcinoma cell line (LoVo cells), and PTX could be embedded inside lysosomal compartments. Moreover, the human colorectal carcinoma (CRC) LoVo cells delivery effect was verified in vivo by magnetic resonance imaging (MRI) and histology analysis. Consequently effective suppression of CRC LoVo cell growth was evaluated. Conclusions/Significance These results indicated that the PTX-SPION-loaded pH-sensitive micelles were a promising MRI-visible drug release system for colorectal cancer therapy.

Ultrasound-responsive microbubbles for sonography-guided siRNA delivery

RNA interfering is a gene therapeutic approach of great potential for cancer. However, tumor-targeted delivery of small interfering RNA (siRNA) solely based on the enhanced permeability and retention effect of nanocarriers is often insufficient. To address this challenge, siRNA encapsulated ultrasound-responsive microbubble (MB) was developed from polymeric siRNA micelles and liposomal MBs using hetero-assembling strategy. 1MHz low-frequency ultrasound exposure of the tumor site after intratumoral injection of XIAP siRNA/MBs led to enhanced permeability for much more siRNA delivery into deep tumor regions. Significant improvement of XIAP gene silencing and cleaved caspase-3 activation was achieved, resulting in good therapeutic effect on human cervical cancer xenograft model in nude mice. Moreover, real-time US monitoring of the tumor was also possible using the siRNA/MBs as a contrast agent during the therapeutic process. These results show that the multi-functional siRNA/MBs are a promising theranostic system for cancer gene therapy.

Ultrasound Imaging based on Molecular Targeting for Quantitative Evaluation of Hepatic Ischemia Reperfusion Injury

The aim of the present study was to quantitatively diagnose and monitor the therapy response of hepatic ischemia–reperfusion injury (IRI) with the use of targeted ultrasound (US) imaging. Targeted microbubbles (MBs) were fabricated, and the binding of intracellular adhesion molecule 1 (ICAM‐1) antibodies to MBs was observed. To establish a quantitative method based on targeted US imaging, contrast‐enhanced US was applied for IRI rats. After andrographolide treatment, the IRI rats were subjected to the quantitative targeted US imaging for a therapeutic effect. Effective binding of ICAM‐1 antibodies to MBs was observed. According to the quantitative targeted US imaging, the ICAM‐1 normalized intensity difference (NID) in the IRI rats (38.74 ± 15.08%) was significantly higher than that in the control rats (10.08 ± 2.52%, p = 0.048). Further, different degrees of IRI (mild IRI, moderate to severe IRI) were distinguished by the use of the NID (37.14 ± 2.14%, 22.34 ± 1.08%, p = 0.002). Analysis of mRNA expression demonstrated the accuracy of analyzing the NID by using quantitative targeted US imaging (R2 = 0.7434, p < 0.001). Andrographolide treatment resulted in an obviously weakened NID of ICAM‐1 (17.7 ± 4.8% vs 34.2 ± 6.6%, p < 0.001). The study showed the potential of the quantitative targeted US imaging method for the diagnosis and therapeutic monitoring of IRI.

Porphyrin-grafted Lipid Microbubbles for the Enhanced Efficacy of Photodynamic Therapy in Prostate Cancer through Ultrasound-controlled In Situ Accumulation

Photodynamic therapy (PDT) holds promise for focal therapy of prostate cancer (PCa). However, the therapeutic efficacy needs improvement, and further development of PDT for PCa has challenges, including uncertainty of photosensitizers (PSs) accumulation at the tumor site and difficulty in visualizing lesions using conventional ultrasound (US) imaging. We have developed novel porphyrin-grafted lipid (PGL) microbubbles (MBs; PGL-MBs) and propose a strategy to integrate PGL-MBs with US imaging to address these limitations and enhance PDT efficacy. Methods: PGL-MBs have two functions: imaging guidance by contrast-enhanced ultrasound (CEUS) and targeted delivery of PSs by ultrasound targeted microbubble destruction (UTMD). PGL-MBs were prepared and characterized before and after low-frequency US (LFUS) exposure. Then, in vitro studies validated the efficacy of PDT with PGL-MBs in human prostate cancer PC3 cells. PC3-xenografted nude mice were used to validate CEUS imaging, accumulation at the tumor site, and in vivo PDT efficacy. Results: PGL-MBs showed good contrast enhancement for US imaging and were converted into nanoparticles upon LFUS exposure. The resulting uniquely structured nanoparticles avoided porphyrin fluorescence quenching and efficiently accumulated at the tumor site through the sonoporation effect created with the assistance of US to achieve excellent PDT efficacy. Conclusions: This is the first preclinical investigation of MBs applied in PDT for PCa. PGL-MBs possess favorable CEUS imaging effects to enhance the localization of tumors. PGL-MBs with LFUS control PS accumulation at the tumor site to achieve highly effective PDT of PCa. This strategy carries enormous clinical potential for PCa management.

Highly uniform ultrasound-sensitive nanospheres produced by a pH-induced micelle-to-vesicle transition for tumor-targeted drug delivery

Although gas-filled microbubbles with high echogenicity are widely applied inclinical ultrasonography, the micron scale particle size impedes their use in the treatment of solid tumors,which are accessible to objects less than several hundred nanometers. We herein propose an unusual approach involving apH-induced core–shell micelle-to-vesicle transition to prepare ultrasound-sensitive polymeric nanospheres (polymersomes in structure) possessing multiple features, including nanosize, monodispersity, and incorporation of a phase-transitional imaging agent into the aqueous lumen. These features are not achievable via the conventional double-emulsion method for polymersome preparation. The nanospheres were constructed based on a novel triblock copolymer with dual pH sensitivity. The liquid-to-gas phase transition of the imaging agent induced by external low-frequency ultrasound may destroy the nanospheres for a rapid drug release, with simultaneous tissue-penetrating drug delivery inside a tumor. These effects may provide new opportunities for the development of an effective cancer therapy with few adverse effects.

Noninvasive quantification of intrarenal allograft C4d deposition with targeted ultrasound imaging

Antibody‐mediated rejection (AMR) has emerged as a major cause of renal allograft dysfunction. C4d, a specific marker for AMR diagnosis, was strongly recommended for routine surveillance; however, currently, C4d detection is dependent upon tissue biopsy, which is invasive and provides only local semi‐quantitative data. Targeted ultrasound imaging has been used extensively for noninvasive and real‐time molecular detection with advantages of high specificity and sensitivity. In this study, we designed C4d‐targeted microbubbles (MBC4d) using a streptavidin‐biotin conjugated method and detected C4d deposition in vivo in a rat model of AMR by enhanced ultrasound imaging. This noninvasive procedure allowed successful acquisition of the first qualitative image of C4d deposition in a wide renal allograft section, which reflected real‐time C4d distribution in grafts. Moreover, we introduced normal intensity difference for quantitative analysis, which exhibited a nearly linear correlation with the grade of C4d deposition according to pathologic analysis. In addition, this approach showed no influence on survival rates and pathologic features in the microbubble injection groups, thereby demonstrating its safety. These findings demonstrated a simple, noninvasive, quantitative, and safe evaluation method for C4d, with the utility of this approach potentially preventing patients from having to undergo an invasive biopsy.

Size-Modulable Nanoprobe for High-Performance Ultrasound Imaging and Drug Delivery against Cancer

Among medical imaging modalities available in the clinic, ultrasonography is the most convenient, inexpensive, ionizing-radiation-free, and most common. Micrometer-size perfluorocarbon bubbles have been used as efficient contrast for intravascular ultrasonography, but they are too big for tumor penetration. Nanodroplets (250-1000 nm) encapsulating both perfluorocarbon and drug have been used as an ultrasound-triggered release drug delivery platform against cancer, but they are generally not useful as a tumor imaging agent. The present study aims to develop a type of pH-sensitive, polymersome-based, perfluorocarbon encapsulated ultrasonographic nanoprobe, capable of maintaining at 178 nm during circulation and increasing to 437 nm at the acidic tumor microenvironment. Its small size allowed efficient tumor uptake. At the tumor site, the nanoparticle swells, resulting in lowering of the vaporization threshold for the perfluorocarbon, efficient conversion of nanoprobes to echogenic nano/microbubbles for ultrasonic imaging, and eventual release of doxorubicin from the theranostic nanoprobe for deep tissue chemotherapy, triggered by irradiation with low-frequency ultrasound.