Peptide-Functionalized Hydrogel Cubes for Active Tumor Cell Targeting.

Conjugation of bioactive targeting molecules to nano- or micrometer-sized drug carriers is a pivotal strategy to improve their therapeutic efficiency. Herein, we developed pH- and redox-sensitive hydrogel particles with a surface-conjugated cancer cell targeting ligand for specific tumor-targeting and controlled release of the anticancer drug doxorubicin. The poly(methacrylic acid) (PMAA) hydrogel cubes of 700 nm and 2 µm with a hepsin-targeting (IPLVVPL) surface peptide are produced through multilayer polymer assembly on sacrificial cubical mesoporous cores. Direct peptide conjugation to the disulfide-stabilized hydrogels through a thiol-amine reaction does not compromise the structural integrity, hydrophilicity, stability in serum, or pH/redox sensitivity but does affect internalization by cancer cells. The cell uptake kinetics and the ultimate extent of internalization are controlled by the cell type and hydrogel size. The peptide modification significantly promotes the uptake of the 700 nm hydrogels by hepsin-positive MCF-7 cells due to ligand-receptor recognition but has a negligible effect on the uptake of 2 µm PMAA hydrogels. The selectivity of 700 nm IPLVVPL-PMAA hydrogel cubes to hepsin-overexpressing tumor cells is further confirmed by a 3-10-fold higher particle internalization by hepsin-positive MCF-7 and SK-OV-3 compared to that of hepsin-negative PC-3 cells. This work provides a facile method to fabricate enhanced tumor-targeting carriers of submicrometer size and improves the general understanding of particle design parameters for targeted drug delivery.

Theranostic Multilayer Capsules for Ultrasound Imaging and Guided Drug Delivery.

Despite the accessibility of ultrasound, the clinical potential of ultrasound-active theranostic agents has not been fully realized because it requires combining sufficient imaging contrast, high encapsulation efficiency, and ultrasound-triggered release in one entity. We report on theranostic polymer microcapsules composed of hydrogen-bonded multilayers of tannic acid and poly(N-vinylpyrrolidone) that produce high imaging contrast and deliver the anticancer drug doxorubicin upon low-power diagnostic or high-power therapeutic ultrasound irradiation. These capsules exhibit excellent imaging contrast in both brightness and harmonic modes and show prolonged contrast over six months, unlike commercially available microbubbles. We also demonstrate low-dose gradual and high-dose fast release of doxorubicin from the capsules by diagnostic (∼100 mW/cm2) and therapeutic (>10 W/cm2) ultrasound irradiation, respectively. We show that the imaging contrast of the capsules can be controlled by varying the number of layers, polymer type (relatively rigid tannic acid versus more flexible poly(methacrylic acid)), and polymer molecular weight. In vitro studies demonstrate that 50% doxorubicin release from ultrasound-treated capsules induces 97% cytotoxicity to MCF-7 human cancer cells, while no cytotoxicity is found without the treatment. Considering the strong ultrasound imaging contrast, high encapsulation efficiency, biocompatibility, and tunable drug release, these microcapsules can be used as theranostic agents for ultrasound-guided chemotherapy.

Molecular Ultrasound Imaging of Tissue Inflammation Using an Animal Model of Acute Kidney Injury.

PURPOSE: The objective of this study was to evaluate the use of molecular ultrasound (US) imaging for monitoring the early inflammatory effects following acute kidney injury. PROCEDURES: A population of rats underwent 30 min of renal ischemia (acute kidney injury, N = 6) or sham injury (N = 4) using established surgical methods. Animals were divided and molecular US imaging was performed during the bolus injection of a targeted microbubble (MB) contrast agent to either P-selectin or vascular cell adhesion molecule 1 (VCAM-1). Imaging was performed before surgery and 4 and 24 h thereafter. After manual segmentation of renal tissue space, the molecular US signal was calculated as the difference between time-intensity curve data before MB injection and after reaching steady-state US image enhancement. All animals were terminated after the 24 h imaging time point and kidneys excised for immunohistochemical (IHC) analysis. RESULTS: Renal inflammation was analyzed using molecular US imaging. While results using the P-selectin and VCAM-1 targeted MBs were comparable, it appears that the former was more sensitive to biomarker expression. All molecular US imaging measures had a positive correlation with IHC findings. CONCLUSIONS: Acute kidney injury is a serious disease in need of improved noninvasive methods to help diagnose the extent of injury and monitor the tissue throughout disease progression. Molecular US imaging appears well suited to address this challenge and more research is warranted.

Synergistic ablation of liver tissue and liver cancer cells with high-intensity focused ultrasound and ethanol.

We investigated the combined effect of ethanol and high-intensity focused ultrasound (HIFU), first, on heating and cavitation bubble activity in tissue-mimicking phantoms and porcine liver tissues and, second, on the viability of HepG2 liver cancer cells. Phantoms or porcine tissues were injected with ethanol and then subjected to HIFU at acoustic power ranging from 1.2 to 20.5 W (HIFU levels 1-7). Cavitation events and the temperature around the focal zone were measured with a passive cavitation detector and embedded type K thermocouples, respectively. HepG2 cells were subjected to 4% ethanol solution in growth medium (v/v) just before the cells were exposed to HIFU at 2.7, 8.7 or 12.0 W for 30 s. Cell viability was measured 2, 24 and 72 h post-treatment. The results indicate that ethanol and HIFU have a synergistic effect on liver cancer ablation as manifested by greater temperature rise and lesion volume in liver tissues and reduced viability of liver cancer cells. This effect is likely caused by reduction of the cavitation threshold in the presence of ethanol and the increased rate of ethanol diffusion through the cell membrane caused by HIFU-induced streaming, sonoporation and heating.

PDMS well platform for culturing millimeter-size tumor spheroids.

Multicellular tumor spheroids are widely used as in vitro models for testing of anticancer drugs. The advantage of this approach is that it can predict the outcome of a drug treatment on human cancer cells in their natural three-dimensional environment without putting actual patients at risk. Several methods were utilized in the past to grow submillimeter-size tumor spheroids. However, these small models are not very useful for preclinical studies of tumor ablation where the goal is the complete destruction of tumors that can reach several centimeters in diameter in the human body. Here, we propose a PDMS well method for large tumor spheroid culture. Our experiments with HepG2 hepatic cancer cells show that three-dimensional aggregates of tumor cells with a volume as large as 44 mm(3) can be grown in cylindrical PDMS wells after the initial culture of tumor cells by the hanging drop method. This is a 350 times more than the maximum volume of tumor spheroids formed inside hanging drops (0.125 mm(3)).