Hanping Wu

Formulation and Characterization of Echogenic Lipid–Pluronic Nanobubbles

The advent of microbubble contrast agents has enhanced the capabilities of ultrasound as a medical imaging modality and stimulated innovative strategies for ultrasound-mediated drug and gene delivery. While the utilization of microbubbles as carrier vehicles has shown encouraging results in cancer therapy, their applicability has been limited by a large size which typically confines them to the vasculature. To enhance their multifunctional contrast and delivery capacity, it is critical to reduce bubble size to the nanometer range without reducing echogenicity. In this work, we present a novel strategy for formulation of nanosized, echogenic lipid bubbles by incorporating the surfactant Pluronic, a triblock copolymer of ethylene oxide copropylene oxide coethylene oxide into the formulation. Five Pluronics (L31, L61, L81, L64 and P85) with a range of molecular weights (M(w): 1100 to 4600 Da) were incorporated into the lipid shell either before or after lipid film hydration and before addition of perfluorocarbon gas. Results demonstrate that Pluronic-lipid interactions lead to a significantly reduced bubble size. Among the tested formulations, bubbles made with Pluronic L61 were the smallest with a mean hydrodynamic diameter of 207.9 +/- 74.7 nm compared to the 880.9 +/- 127.6 nm control bubbles. Pluronic L81 also significantly reduced bubble size to 406.8 +/- 21.0 nm. We conclude that Pluronic is effective in lipid bubble size control, and Pluronic M(w), hydrophilic-lipophilic balance (HLB), and Pluronic/lipid ratio are critical determinants of the bubble size. Most importantly, our results have shown that although the bubbles are nanosized, their stability and in vitro and in vivo echogenicity are not compromised. The resulting nanobubbles may be better suited for contrast enhanced tumor imaging and subsequent therapeutic delivery.

Effect of injection site on in situ implant formation and drug release in vivo

In situ forming drug delivery implants offer an attractive alternative to pre-formed implant devices for local drug delivery due to their ability to deliver fragile drugs, simple manufacturing process, and less invasive placement. However, the clinical translation of these systems has been hampered, in part, by poor correlation between in vitro and in vivo drug release profiles. To better understand this effect, the behavior of poly(D,l-lactide-co-glycolide) (PLGA) in situ forming implants was examined in vitro and in vivo after subcutaneous injection as well as injection into necrotic, non-necrotic, and ablated tumor. Implant formation was quantified noninvasively using an ultrasound imaging technique. Drug release of a model drug agent, fluorescein, was correlated with phase inversion in different environments. Results demonstrated that burst drug release in vivo was greater than in vitro for all implant formulations. Drug release from implants in varying in vivo environments was fastest in ablated tumor followed by implants in non-necrotic tumor, in subcutaneous tissue, and finally in necrotic tumor tissue with 50% of the loading drug mass released in 0.7, 0.9, 9.7, and 12.7h respectively. Implants in stiffer ablated and non-necrotic tumor tissue showed much faster drug release than implants in more compliant subcutaneous and necrotic tumor environments. Finally, implant formation examined using ultrasound confirmed that in vivo the process of precipitation (phase inversion) was directly proportional to drug release. These findings suggest that not only is drug release dependent on implant formation but that external environmental effects, such as tissue mechanical properties, may explain the differences seen between in vivo and in vitro drug release from in situ forming implants.

Multimodal In Vivo Imaging Exposes the Voyage of Nanoparticles in Tumor Microcirculation

Tumors present numerous biobarriers to the successful delivery of nanoparticles. Decreased blood flow and high interstitial pressure in tumors dictate the degree of resistance to extravasation of nanoparticles. To understand how a nanoparticle can overcome these biobarriers, we developed a multimodal in vivo imaging methodology, which enabled the noninvasive measurement of microvascular parameters and deposition of nanoparticles at the microscopic scale. To monitor the spatiotemporal progression of tumor vasculature and its vascular permeability to nanoparticles at the microcapillary level, we developed a quantitative in vivo imaging method using an iodinated liposomal contrast agent and a micro-CT. Following perfusion CT for quantitative assessment of blood flow, small animal fluorescence molecular tomography was used to image the in vivo fate of cocktails containing liposomes of different sizes labeled with different NIR fluorophores. The animal studies showed that the deposition of liposomes depended on local blood flow. Considering tumor regions of different blood flow, the deposition of liposomes followed a size-dependent pattern. In general, the larger liposomes effectively extravasated in fast flow regions, while smaller liposomes performed better in slow flow regions. We also evaluated whether the tumor retention of nanoparticles is dictated by targeting them to a receptor overexpressed by the cancer cells. Targeting of 100 nm liposomes showed no benefits at any flow rate. However, active targeting of 30 nm liposomes substantially increased their deposition in slow flow tumor regions (∼12-fold increase), which suggested that targeting prevented the washout of the smaller nanoparticles from the tumor interstitium back to blood circulation.

Real-time Monitoring of Radiofrequency Ablation and Postablation Assessment: Accuracy of Contrast-enhanced US in Experimental Rat Liver Model

PURPOSE To examine the accuracy of the unenhanced zone at contrast material-enhanced ultrasonography (US) in predicting coagulative necrosis during and 21 days after radiofrequency (RF) ablation by using radiologic-pathologic comparison. MATERIALS AND METHODS Animal studies were approved by the Institutional Animal Care and Use Committee. The livers of 28 rats underwent US-guided RF ablation. In four animals, contrast-enhanced US was performed during ablation and 2 hours and 2, 7, 14, and 21 days after ablation. The unenhanced zone area on US images was measured. DiI-labeled microbubbles were administered during ablation at 2, 4, and 6 minutes or at 2 hours and 2, 7, 14, and 21 days after ablation in the remaining 24 animals (n = 3 at each time point). One minute later, the animal was euthanized, and the ablated liver was harvested. Tissue samples were imaged to quantify total fluorescence, and NADH staining was performed on the same slice. Hematoxylin-eosin staining was also performed. The findings on fluorescence images, NADH-stained images, and hematoxylin-eosin-stained images were compared. The areas of DiI bubble-negative zones, NADH-negative zones, and lightly NADH-staining zones were measured. Data were analyzed by using one-way analysis of variance. RESULTS The area of the unenhanced zone on contrast-enhanced US images increased during RF ablation and reached a maximum within 2 days after ablation. At histopathologic examination, a transition zone manifested adjacent to the coagulation zone until 2 days after ablation. The DiI-bubble negative zone on fluorescence images and the damaged zone (transition zone plus coagulation zone) on NADH-stained images increased rapidly within 2 hours after ablation, then slowly reached the maximum on day 2. The ratios of the mean areas of these two zones at hour 2 to those at day 2 were 94.6% and 95.6%, respectively. High uniformity between the damaged zone on NADH-stained images and the DiI bubble-negative zone on fluorescence images was noted at all time points. CONCLUSION The temporary transition zone in NADH staining is partially damaged and should transition to nonviability 2 days after ablation. These results demonstrate that contrast-enhanced US can help delineate the maximum area of cell damage (to within 5% of the maximum) as early as 2 hours after ablation. Contrast-enhanced US may be a simple and accurate tool for monitoring the effects of RF ablation and quantifying the size of thermal damage after treatment.

Dynamic Evolutionary Changes in Blood Flow Measured by MDCT in a Hepatic VX2 Tumor Implant over an Extended 28-day Growth Period: Time-Density Curve Analysis

RATIONALE AND OBJECTIVES The enhancement pattern of malignant tumors has been studied in short-term animal models (7-14 days), but the reported results have been variable and inconsistent. The purpose of this study was to investigate the changing blood flow characteristics of VX2 tumors implanted in rabbit livers with contrast-enhanced multidetector computed tomography (MDCT) to establish a predictable pattern of vascular evolution over an extended 28-day growth period. MATERIALS AND METHODS VX2 carcinoma was implanted in livers of 10 male New Zealand White rabbits. Dynamic CT (2/seconds x 60 seconds) was conducted on days 7, 14, 21, and 28 after tumor implantation. Enhancement parameters of time-density curve (TDC), time to start (T0), time to peak (TP), maximum enhancement (DeltaH), slope of enhancement (SLe), and washout (SLw) in tumor center, tumor rim, and normal liver were analyzed. Tumor samples corresponding to CT images of one tumor on days 14 and 21 and seven tumors on day 28 were stained with hematoxylin and eosin and anti-CD31 monoclonal antibody. The relationship between enhancement parameters and histology parameters (thickness of tumor border, extent of blood stasis, and luminar vessel density) was analyzed. RESULTS Consistent growth, appearance, and vascular changes occurred in 7 of 10 animals over the 4-week observation period. Peripheral rim-like enhancement was noted in CT images. TDC analysis showed that tumor rim enhancement was pronounced and more rapid than normal liver initially but this difference diminished with tumor progression. The SLe, SLw, and DeltaH decreased from 10.03 +/- 3.25 Hu/second, 0.42 +/- 0.25 Hu/sec, and 58.00 +/- 25.27 Hu on day 7 to 5.86 +/- 2.73 Hu/second, 0.10 +/- 0.13 Hu/second, and 37.78 +/- 8.89 Hu/second on day 28, respectively. TP increased from 12.71 +/- 4.85 seconds on day 7 to 25.57 +/- 7.75 seconds on day 28. No significant changes were noted on the TDC parameters in normal liver. The maximum density difference between tumor rim and normal liver (D(rim-liver)) appeared 10.5 +/- 2.1 seconds after contrast injection. The maximum D(rim-liver) decreased from 54.33 +/- 37.86 Hu on day 7 to 11.16 +/- 13.03 Hu on day 28. On histological analysis, viable tumor cells were found in tumor rim with few luminar vessels. The tumor border showed desmoplastic reaction, vascular dilation and proliferation, inflammatory cell infiltration, and blood stasis. These findings were more obvious on day 28 than those on day 14. TP showed significant positive correlations with the extent of blood stasis in tumor border and adjacent liver and the maximum thickness of the tumor border (r = 0.945 and 0.893 respectively, P < .05). CONCLUSION The rabbit VX2 liver tumor is a hypovascular tumor with perilesional enhancement over its lifespan as imaged by MDCT. Consistent changes in the measured vascular parameters correlated with the size/age of the tumor implants. These findings suggest that the accuracy of CT enhancement imaging for VX2 liver tumor detection might be decreased with tumor development.

Effect of Intratumoral Injection of Carboplatin Combined with Pluronic P85 or L61 on Experimental Colorectal Carcinoma in Rats

Pluronic, a poly(ethylene oxide)-poly(propylene oxide)-poly (ethylene oxide) block copolymer, has been shown to enhance the cytotoxic activity of anticancer drugs in various cell lines. In the current study the effect of Pluronic P85 (P85) and Pluronic L61 (L61) on the intratumoral chemotherapy of an experimental adenocarcinoma in rats was examined. A total of 120 subcutaneous tumors (4 per rat) were inoculated in 30 BDIX rats and were treated weekly for 4 weeks with intratumoral injection of carboplatin (CPt) alone or with either P85 or L61. Tumors were monitored weekly and were excised at the endpoint for histologic evaluation. The effect of Pluronic on levels of intracellular ATP was explored as a possible mechanism of sensitization. Results showed that tumors treated with low-dose CPt (2.8 mg/kg) and P85 or L61 exhibited significant reductions in tumor volume after 28 days relative to Day 0 (112.7% ± 34.4%, n = 15; 131.3% ± 55.6%, n = 8) compared with tumors treated with free drug (339.4% ± 75.0%, n = 16). Control tumors treated with either P85 or L61 alone or with saline showed volume increases of 1079.4% ± 143.6% (n = 16), 729.4% ± 202.2% (n = 7), and 1119.2% ± 6.1% (n = 16), respectively. Treatment with high-dose CPt (20.7 mg/kg) led to a 79.3% ± 4.2% reduction in tumor volume, and no differences were noted with addition of P85 or L61. In vitro ATP measurements showed that 28.0 mg/kg of P85 significantly reduced levels of intracellular ATP to 44.7% ± 1.5% of controls, whereas L61 at this concentration depleted ATP levels completely. Results confirm that Pluronic P85 and L61 act as potent sensitizers to carboplatin chemotherapy of the experimental colorectal carcinoma, leading to a significant reduction of tumor growth compared to carboplatin alone. ATP depletion is a possible mechanism for these observed differences.

Improving performance of nanoscale ultrasound contrast agents using N,N-diethylacrylamide stabilization

The design of nanoscale yet highly echogenic agents for imaging outside of the vasculature and for ultrasound-mediated drug delivery remains a formidable challenge. We have previously reported on formulation of echogenic perfluoropropane gas nanobubbles stabilized by a lipid-pluronic surfactant shell. In the current work we describe the development of a new generation of these nanoparticles which consist of perfluoropropane gas stabilized by a surfactant and lipid membrane and a crosslinked network of N,N-diethylacrylamide. The resulting crosslinked nanobubbles (CL-PEG-NB) were 95.2±25.2nm in diameter and showed significant improvement in stability and retention of echogenic signal over 24h. In vivo analysis via ultrasound and fluorescence mediated tomography showed greater tumor extravasation and accumulation with CL-PEG-NB compared to microbubbles. Together these results demonstrate the capabilities and advantages of a new, more stable, nanometer-scale ultrasound contrast agent that can be utilized in future work for diagnostic scans and molecular imaging.

Ultrasound molecular imaging of ovarian cancer with CA-125 targeted nanobubble contrast agents

Ultrasound is frequently utilized in diagnosis of gynecologic malignancies such as ovarian cancer. Because epithelial ovarian cancer (EOC) is often characterized by overexpression of cancer antigen 125 (CA-125), ultrasound contrast agents able to target this molecular signature could be a promising complementary strategy. In this work, we demonstrate application of CA-125-targeted echogenic lipid and surfactant-stabilized nanobubbles imaged with standard clinical contrast harmonic ultrasound for imaging of CA-125 positive OVCAR-3 tumors in mice. Surface functionalization of the nanobubbles with a CA-125 antibody achieved rapid significantly (P < 0.05) enhanced tumor accumulation, higher peak ultrasound signal intensity and slower wash out rates in OVCAR-3 tumors compared to CA-125 negative SKOV-3 tumors. Targeted nanobubbles also exhibited increased tumor retention and prolonged echogenicity compared to untargeted nanobubbles. Data suggest that ultrasound molecular imaging using CA-125 antibody-conjugated nanobubbles may contribute to improved diagnosis of EOC.

Role of Pluronic block copolymers in modulation of heat shock protein 70 expression

Purpose: The goal of this study was to evaluate the relationship between previously demonstrated thermosensitising effects of the block copolymer, Pluronic, and heat shock protein 70 (Hsp70) expression in an experimental colorectal cancer model in vitro and in vivo. Materials and methods: Rat colorectal carcinoma cells were treated with low-grade hyperthermia (43°C) alone or in combination with Pluronics L10 (3 mg/mL), L61 (0.3 mg/mL), or L64 (0.5 mg/mL) for 20 min. Adinosine triphosphate (ATP) levels and cell viability were determined using standard assays. Hsp70 expression was quantified by western blot for cells treated with L10, L61, and L64 at doses specified above and Pluronic P85 (10 mg/mL) alone and in combination with heat. BDIX rats with flank tumours were used to study the effect of L61 and hyperthermia on Hsp70 expression in vivo. Results: In vitro, treatment with L10, L61, and L64 plus low-grade hyperthermia lead to depletion of ATP levels to between 8 and 66% of untreated control after 24 h. Maximum expression of Hsp70 was observed at 9 h following hyperthermia alone. The combination of low-grade hyperthermia and Pluronic treatment reduced Hsp70 expression for up to 6 hours, and L10 appeared to completely inhibit the Hsp70 expression. In vivo, Hsp70 expression was increased 5 h after hyperthermia in BDIX rat tumour models and no Hsp70 expression was observed in L61 pre-treated and control groups. Conclusion: Pluronic effectively improves hyperthermic and low-grade hyperthermic treatment in part due to reduction of Hsp70 expression.

Radiofrequency ablation: post-ablation assessment using CT perfusion with pharmacological modulation in a rat subcutaneous tumor model.

RATIONALE AND OBJECTIVES Inflammatory reaction surrounding the ablated area is a major confounding factor in the early detection of viable tumor after radiofrequency (RF) ablation. A difference in the responsiveness of normal and tumor blood vessels to vasoactive agents may be used to distinguish these regions in post-ablation follow-up. The goal of this study was to examine longitudinal perfusion changes in untreated viable tumor and the peripheral hyperemic rim of RF-ablated tumor in response to a vasoconstrictor (phenylephrine) or vasodilator (hydralazine) in a subcutaneous rat tumor model. MATERIALS AND METHODS Bilateral subcutaneous shoulder tumors were inoculated in 24 BDIX rats and evenly divided into two groups (phenylephrine and hydralazine groups). One tumor in each animal was completely treated with RF ablation (at 90 +/- 2 degrees C for 3 minutes), and the other remained untreated. Computed tomographic perfusion scans before and after phenylephrine (10 microg/kg) or hydralazine (5 mg/kg) administration were performed 2, 7, and 14 days after ablation. Four rats per group were euthanized on each scan day, and pathologic evaluation was performed. The changes of blood flow in the peripheral rim of ablated tumor and untreated viable tumor in response to phenylephrine or hydralazine at each time point were compared. The diagnostic accuracy of viable tumor using the percentage change of blood flow in response to phenylephrine and hydralazine was compared using receiver-operating characteristic analysis. RESULTS The peripheral rim of ablated tumor presented with a hyperemic reaction with dilated vessels and congestion on day 2 after ablation, numerous inflammatory vessels on day 7, and granulation tissue formation on day 14. Phenylephrine significantly decreased the blood flow in the peripheral hyperemic rim of ablated tumor on days 2, 7, and 14 by 16.3 +/- 9.7% (P = .001), 24.0 +/- 22.6% (P = .007), and 31.1 +/- 25.4% (P = .045), respectively. In untreated viable tumor, the change in blood flow after phenylephrine was irregular and insignificant. Hydralazine decreased the blood flow in the peripheral rim of both ablated tumor and untreated viable tumor. Receiver-operating characteristic analysis showed that reliable tumor diagnosis using the percentage change of blood flow in response to phenylephrine was noted on days 2 and 7, for which the areas under the curve were 0.82 (95% confidence interval, 0.64-1.00) and 0.81 (95% confidence interval, 0.56-1.00), respectively. However, tumor diagnosis using the blood flow change in response to hydralazine was unreliable. CONCLUSION Phenylephrine markedly decreased blood flow in the peripheral hyperemic rim of ablated tumor but had little effect on the untreated viable tumor. Computed tomographic perfusion with phenylephrine may be useful in the long-term treatment assessment of RF ablation.