Jianwu Xie

Pulsed-High Intensity Focused Ultrasound and Low Temperature– Sensitive Liposomes for Enhanced Targeted Drug Delivery and Antitumor Effect

Purpose: To determine if pulsed-high intensity focused ultrasound (HIFU) could effectively serve as a source of hyperthermia with thermosensitive liposomes to enhance delivery and efficacy of doxorubicin in tumors. Experimental Design: Comparisons in vitro and in vivo were carried out between non–thermosensitive liposomes (NTSL) and low temperature–sensitive liposomes (LTSL). Liposomes were incubated in vitro over a range of temperatures and durations, and the amount of doxorubicin released was measured. For in vivo experiments, liposomes and free doxorubicin were injected i.v. in mice followed by pulsed-HIFU exposures in s.c. murine adenocarcinoma tumors at 0 and 24 h after administration. Combinations of the exposures and drug formulations were evaluated for doxorubicin concentration and growth inhibition in the tumors. Results:In vitro incubations simulating the pulsed-HIFU thermal dose (42°C for 2 min) triggered release of 50% of doxorubicin from the LTSLs; however, no detectable release from the NTSLs was observed. Similarly, in vivo experiments showed that pulsed-HIFU exposures combined with the LTSLs resulted in more rapid delivery of doxorubicin as well as significantly higher i.t. concentration when compared with LTSLs alone or NTSLs, with or without exposures. Combining the exposures with the LTSLs also significantly reduced tumor growth compared with all other groups. Conclusions: Combining low-temperature heat-sensitive liposomes with noninvasive and nondestructive pulsed-HIFU exposures enhanced the delivery of doxorubicin and, consequently, its antitumor effects. This combination therapy could potentially produce viable clinical strategies for improved targeting and delivery of drugs for treatment of cancer and other diseases.

Radiofrequency Ablation Induces Antigen-presenting Cell Infiltration and Amplification of Weak Tumor-induced Immunity

PURPOSE To evaluate the influence of subtotal radiofrequency (RF) ablation on a tumor-specific immune response in a murine tumor model and to explore the role of intratumoral dendritic cells (ITDCs) in mediating this effect. MATERIALS AND METHODS Animal work was performed according to an approved protocol and in compliance with the National Cancer Institute Animal Care and Use Committee guidelines and regulations. A murine urothelial carcinoma (MB49) model expressing the male minor histocompatibility (HY) antigen was inoculated subcutaneously in female mice. Fourteen days later, splenic T cells were analyzed with enzyme-linked immunosorbent spot for HY immune response (n = 57). In subsequent experiments, mice were randomized into control (n = 7), RF ablation, ITDC (n = 9), and RF ablation + ITDC (n = 9) groups and monitored for tumor growth. Eleven days after treatment, tumors were harvested for histologic and immunohistochemical analysis. Animals demonstrating complete tumor regression were rechallenged in the contralateral flank. RESULTS Animals treated with subtotal RF ablation showed significant increases in tumor-specific class I and II responses to HY antigens and tumor regression. RF ablation, ITDC, and combined groups demonstrated similar levels of antigen-presenting cell infiltration; all groups demonstrated greater levels of infiltration compared with untreated controls. ITDC injection also resulted in tumor regression. However, combination therapy did not enhance tumor regression when compared with either treatment alone. Rechallenged mice in RF ablation, ITDC, and combination groups demonstrated significant tumor growth inhibition compared with controls. CONCLUSION Subtotal RF ablation treatment results in enhanced systemic antitumor T-cell immune responses and tumor regression that is associated with increased dendritic cell infiltration. ITDC injection mimics the RF ablation effect but does not increase immune responses when injected immediately after RF ablation.

Pulsed High-Intensity Focused Ultrasound Enhances Apoptosis and Growth Inhibition of Squamous Cell Carcinoma Xenografts with Proteasome Inhibitor Bortezomib

PURPOSE To investigate whether combining pulsed high-intensity focused ultrasound (HIFU) with the chemotherapeutic drug bortezomib could improve antitumor activity against murine squamous cell carcinoma (SCC) tumors. MATERIALS AND METHODS All experiments were conducted with animal care and use committee approval. Murine SCC cells were implanted subcutaneously in C3H mice. When tumors reached 100 mm(3), mice were randomized to one of three groups for twice weekly intraperitoneal injections of 1.5 mg of bortezomib per kilogram of body weight, a proteasome inhibitor (n = 10); 1.0 mg/kg bortezomib (n = 11); or a control vehicle (n = 12). Within each group, half of the mice received pulsed HIFU exposure to their tumors immediately prior to each injection. The time for tumors to reach 650 mm(3) was compared among groups. Additional tumors were stained with terminal deoxynucledotidyl transferase-mediated dUTP nick end labeling and CD31 to assess apoptotic index and blood vessel density, respectively. RESULTS Tumors in the control group, pulsed HIFU and control group, and 1.0 mg/kg of bortezomib alone group reached the size end point in 5.2 days +/- 0.8 (standard deviation), 5.3 days +/- 0.8, and 5.6 days +/- 1.1, respectively. However, pulsed HIFU and 1.0 mg/kg bortezomib increased the time to end point to 9.8 days +/- 2.9 (P < .02), not significantly different from the 8.8 days +/- 2.1 in tumors treated with 1.5 mg/kg bortezomib alone (P > .05). Combination therapy was also associated with a significantly higher apoptotic index (P < .05). CONCLUSION Treatment of tumors with pulsed HIFU lowered the threshold level for efficacy of bortezomib, resulting in significant tumor cytotoxicity and growth inhibition at lower dose levels.

Rhodamine-RCA in vivo labeling guided laser capture microdissection of cancer functional angiogenic vessels in a murine squamous cell carcinoma mouse model

BackgroundCancer growth, invasion and metastasis are highly related to tumor-associated neovasculature. The presence and progression of endothelial cells in cancer is chaotic, unorganized, and angiogenic vessels are less functional. Therefore, not all markers appearing on the chaotic endothelial cells are accessible if a drug is given through the vascular route. Identifying endothelial cell markers from functional cancer angiogenic vessels will indicate the accessibility and potential efficacy of vascular targeted therapies.ResultsIn order to quickly and effectively identify endothelial cell markers on the functional and accessible tumor vessels, we developed a novel technique by which tumor angiogenic vessels are labeled in vivo followed by Laser Capture Microdissection of microscopically isolated endothelial cells for genomic screening. Female C3H mice (N = 5) with established SCCVII tumors were treated with Rhodamine-RCA lectin by tail vein injection, and after fluorescence microscopy showed a successful vasculature staining, LCM was then performed on frozen section tissue using the PixCell II instrument with CapSure HS caps under the Rhodamine filter. By this approach, the fluorescent angiogenic endothelial cells were successfully picked up. As a result, the total RNA concentration increased from an average of 33.4 ng/ul +/- 24.3 (mean +/- S.D.) to 1913.4 ng/ul +/- 164. Relatively pure RNA was retrieved from both endothelial and epithelial cells as indicated by the 260/280 ratios (range 2.22–2.47). RT-PCR and gene electrophoresis successfully detected CD31 and Beta-Actin molecules with minimal Keratin 19 expression, which served as the negative control.ConclusionOur present study demonstrates that in vivo Rhodamine RCA angiogenic vessel labeling provided a practical approach to effectively guide functional endothelial cell isolation by laser capture microdissection with fluorescent microscopy, resulting in high quality RNA and pure samples of endothelial cells pooled for detecting genomic expression.

In Vitro Investigations Into Enhancement of tPA Bioavailability in Whole Blood Clots Using Pulsed–High Intensity Focused Ultrasound Exposures

Investigations were carried out on the manner by which pulsed-high intensity focused ultrasound (HIFU) enhances the effectiveness of tissue plasminogen activator (tPA) in whole blood clots, in vitro. Scanning electronic microscope (SEM) of the surface of the clots showed that the exposures increased exposed fibrin, as well as the number of openings to more interior regions. These findings were supported by fluorescent antibody labeling of tPA in frozen sections of clots treated post-HIFU. Here, improved accumulation at the surface and penetration of the tPA into the clots were observed in those treated with HIFU. Fluorescence recovery after photobleaching was also performed, indicating that the diffusion coefficient increased 6.3-fold for fluorescently labeled dextrans, comparable in size to tPA, in the HIFU-treated clots. Improved understanding of the manner by which pulsed-HIFU exposures can improve the effectiveness of thrombolytics will help optimize the exposures for this application and potentially facilitate translation to the clinic.

Pulsed focused ultrasound exposures enhance locally administered gene therapy in a murine solid tumor model

Gene therapy by intratumoral injection is a promising approach for treating solid tumors. However, this approach has limited success due to insufficient distribution of gene vectors used for gene delivery. Previous studies have shown that pulsed-focused ultrasound (pFUS) can enhance both systemic and local delivery of therapeutic agents in solid tumors and other disease models. Here, murine squamous cell carcinoma flank tumors were treated with single intratumoral injection of naked tumor necrosis factor-alpha (TNF-α) plasmid, either with or without a preceding pFUS exposure. The exposures were given at 1 MHz, at a spatial average, temporal peak intensity of 2660 W cm(-2), using 50 ms pulses, given at a pulse repetition frequency of 1 Hz. One hundred pulses were given at individual raster points, spaced evenly over the projected surface of the tumor at a distance of 2 mm. Exposures alone had no effect on tumor growth. Significant growth inhibition was observed with injection of TNF-α plasmid, and tumor growth was further inhibited with pFUS. Improved results with pFUS correlated with larger necrotic regions in histological sections and improved distribution and penetration of fluorescent surrogate nanoparticles. Electron microscopy demonstrated enlarged gaps between cells in exposed tissue, and remote acoustic palpation showed decreases in tissue stiffness after pFUS. Combined, these results suggest pFUS effects may be reducing barriers for tissue transport and additionally lowering interstitial fluid pressure to further improve delivery and distribution of injected plasmid for greater therapeutic effects. This suggests that pFUS could potentially be beneficial for improving local gene therapy treatment of human malignancies.

Pulsed-high intensity focused ultrasound (HIFU) exposures for enhanced delivery of therapeutics: Mechanisms and applications

The majority of focused ultrasound applications today involve long, continuous exposures that produce significant temperature elevations for tissue ablation and irreversible coagulative necrosis. Comparatively little has been done with non‐continuous (or, pulsed) exposures that can produce primarily mechanical effects with only minimal heat. Our investigations have shown that pulsed‐HIFU exposures can non‐invasively and non‐destructively enhance the delivery of both systemically and locally injected materials (e.g. imaging agents, optical probes, and plasmid DNA) in both normal and cancerous tissues. It is hypothesized that the enhancing effects are directly linked to tissue displacement from locally‐generated radiation forces. In normal tissue, it is thought that shear forces are produced between adjacent tissue regions experiencing non‐uniform displacement. The resulting strain opens cellular junctions in both the vasculature and the parenchyma, increasing extravasation and interstitial diffusion, respe...

Live cell imaging of the endocytosis and the intracellular trafficking of multifunctional lipid nanoparticles

Artificial lipid nanoparticles have drawn great attention due to their potential in medicine. Linked with targeting ligands, they can be used as probes and/or gene delivery vectors for specific types of target cells. Therefore, they are very promising agents in early detection, diagnosis and treatment of cancers and other genetic diseases. However, there are several barriers blocking the applications. Controlling the cellular uptake of the lipid nanoparticles is an important technical challenge to overcome. Understanding the mechanism of the endocytosis and the following intracellular trafficking is very important for improving the design and therefore the efficiency as a drug delivery system. By using fluorescence microscopy methods, we studied the endocytosis of lipid nanoparticles by live M21 cells. The movements of the nanoparticles inside the cell were quantitatively characterized and classified based on the diffusion behavior. The trajectories of nanoparticles movement over the cell membrane revealed hop-diffusion behavior prior to the endocytosis. Fast movement in large steps is observed in intracellular trafficking and is attributed to active movement along microtubule. These observations help to understand the mechanism of the endocytosis and the pathway of the particles in cells.