The application of biomedical nanotechnology holds promise for solving major problems in current tumor chemotherapy related to the severe side effects of chemotherapeutic drugs and development of drug resistance. During the last decade, the advances of nanomedicine have allowed for the combination of various functionalities in one molecular or supramolecular construct, i.e. polymeric bioconjugate or nanoparticle. The nanoparticles include polymeric micelles, liposomes, hollow particles, nano- or microemulsion droplets etc. Various chemotherapeutic drugs, imaging agents, and targeting moieties may be encapsulated in the same nanocontainer. This ability to combine chemotherapeutic agents and imaging markers is especially important in oncological practice potentially allowing early assessment of response to treatment.
Among various imaging modalities, ultrasound is the most cost effective. Ultrasound provides real-time information. The lack of ionizing radiation (in contrast to e.g. CT) is also an advantage. Ultrasound imaging may be combined with ultrasound-mediated drug delivery by ultrasound-responsive nanoparticles. This allows double tumor targeting because nanoparticles accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect1
, followed by local release of the encapsulated drug in tumor tissue in response to tumor-directed ultrasound2–6
. These drug nanocarriers are expected to passively accumulate in tumors because of the enhanced permeability and retention (EPR) effect, which is based on defective tumor microvasculature that allows extravasation of drug-loaded nanoparticles through large inter-endothelial gaps7, 8
. In addition, poor lymphatic drainage of tumors results in longer retention of extravasated particles in tumor tissue. In contrast to tumors, blood vessels in normal tissues have tight inter-endothelial junctions which do not allow extravasation of nanoparticles.
Effective tumor accumulation of nanoparticles via the EPR effect requires sufficient particle residence time in circulation; to provide for this, nanoparticles are commonly coated with poly(ethylene oxide) chains that suppress blood protein adsorption and prevent particle recognition by the cells of the reticulo-endothelial system.
Ultrasound as a drug delivery modality offers a number of important advantages in comparison with other physical modalities. Sonication may be performed non-invasively or minimally invasively through intraluminal, laparoscopic or percutaneous means. For extracorporeal sonication, the transducer is placed in contact with a water-based gel or a water layer on the skin, and no insertion or surgery is required. Ultrasound can be directed toward deeply located body sites and tumor sonication with millimeter precision is feasible.
During the last decade, a number of groups have concentrated their efforts on developing ultrasound-responsive drug delivery systems for oncological applications9–18
. Ultrasound as a component of a drug delivery modality may be applied with a variety of drug carriers. Development of ultrasound-responsive stable liposomes that manifested prolonged circulation time and effective tumor targeting was recently reported. Ultrasound-induced heating triggered phase transition in the phospholipid membrane and released the drug in the target region. Ultrasound-triggered delivery of paclitaxel (PTX) to the monolayers of prostate cancer cells from a phospholipid-coated perfluorohexane nanoemulsion developed by ImaRx was also reported19
Polymeric micelles that combined passive tumor-targeting ability with ultrasound responsiveness were suggested in earlier works of Rapoport’s group2, 6, 20–25
. It was demonstrated that drug-loaded polymeric micelles accumulated passively in tumor tissue26
. Ultrasonic irradiation of the tumor enhanced micelle uptake, triggered drug release from the micelles and transiently altered cell membrane permeability, resulting in effective intracellular drug internalization by tumor cells. Ultrasound irradiation also enhanced drug diffusion throughout tumor volume, thus reducing drug concentration gradients2, 6, 26
. Substantial reduction of the tumor growth rate was achieved using this drug delivery modality6, 27
All therapeutic modalities associated with application of physical stimuli, such as tumor ablation induced by radiofrequency, microwave, or ultrasound as well as local mild hyperthermia require imaging of targeted sites prior to and during treatment. Though various imaging modalities may be used, combining ultrasound-mediated drug delivery with ultrasound imaging appears especially attractive by virtue of offering real time information, versatility, and cost effectiveness. In this context, during the last decade, microbubbles have attracted attention as drug carriers and enhancers of drug and gene delivery. In current clinical practice, microbubbles have been used as ultrasound contrast agents for cardiovascular imaging28
. Several research groups have concentrated their efforts on developing microbubble-based drug delivery systems2, 3, 11–13, 15, 16, 19, 29–42
The most cost-effective way to solve this problem would be to impart drug carrier properties to FDA approved ultrasound contrast agents such as Optison (Amersham Inc.) or Definity (Lanteus Medical Imaging Inc.). However, currently used contrast agents present a number of inherent problems as drug carriers. Their very short circulation time (minutes) and relatively large size (two to ten microns) do not allow effective extravasation into tumor tissue, which is an essential prerequisite for effective drug targeting.
A possible way to solve the above problem may consist in developing drug-loaded, nano-scaled microbubble precursors that would effectively accumulate in tumor tissue and then convert into microbubbles in situ after tumor accumulation. With this in mind, we have recently developed block copolymer stabilized echogenic (i.e. ultrasound contrast generating) perfluoropentane (PFP) nanoemulsions that convert into microbubbles under ultrasound irradiation2, 3, 43, 44
. The nanoemulsions are produced from drug-loaded poly(ethylene oxide)-co-poly(L-lactide) (PEG-PLLA) or poly(ethylene oxide-co-polycaprolactone (PEG-PCL) micelles by introducing a phase-shift perfluorocarbon compound, perfluoropentane (PFP). The PFP has a boiling temperature of 29 °C. However, PFP nanoemulsions manifest remarkable thermal stability due to the excessive pressure called Laplace pressure inside nanodroplets4, 43
. The thermal stability of PFP nanoemulsions prevents their conversion into microbubbles in circulation. They maintain their nanoscale size, which allows tumor accumulation by extravasation through leaky tumor microvasculature (i.e. passive targeting)2, 4
. Without ultrasound, the drug is tightly retained inside the droplet walls, which is important for effective tumor targeting4
. After tumor accumulation, droplet-to-bubble conversion is triggered by tumor-directed ultrasound. This effect is called acoustic droplet vaporization (ADV)45
. Tumor irradiation by therapeutic ultrasound induces localized drug release from nanodroplets and microbubbles and effective intracellular drug uptake by tumor cells, which in turn, results in effective tumor regression. In addition, primary small microbubbles coalesce into larger microbubbles and provide a long-lasting ultrasonic contrast2–4
. Using this treatment modality, effective tumor regression was observed in mouse models of breast and ovarian cancer2–4
; the first promising results for pancreatic cancer were presented in ref.4
Pancreatic ductal adenocarcinoma (PDA) is the fourth most common cause of cancer death in the United States. The annual incidence rate of pancreatic cancer is almost identical to the mortality rate; approximately 37,000 new cases are diagnosed each year in the United States, and approximately 33,000 patients die from this disease. Only 4% of patients are alive 5 years after the time of diagnosis. Most PDA presentations are inoperable at the time of diagnosis due to the extensive tumor burden, local invasion, poor general health, and multiple aggressive micrometastases that are resistant to chemotherapy and radiation treatment. About 40% of patients have a dismal prognosis; median survival time is only 3–6 months46
The only FDA-approved chemotherapeutic agent for PDA is a nucleoside analogue gemcitabine (GEM), but the partial response rate to chemotherapy is well below 10%47, 48
most probably due to the development of GEM resistance in the course of chemotherapy. Despite low effectiveness, GEM remains the cornerstone of neoadjuvant and adjuvant chemotherapy in pancreatic cancer and imparts a progression-free survival interval ranging from 0.9 to 4.2 months49
. Neither biotherapies based on the targeted gene therapy, use of antibodies against Vascular Endothelial Growth factor (VEGF) or Epithelial Growth Factor (EGF-R) receptors50
nor high-intensity ultrasonic tumor ablation51–54
proved successful. An attempt was made to treat pancreatic cancer by extracorporeal high intensity focused ultrasound (HIFU), with only marginal success55
New approaches to PDA treatment are urgently needed. In this context, ultrasound-mediated chemotherapy by polymeric micelles and/or nanoemulsion/microbubble encapsulated drugs may offer an innovative approach to PDA treatment. Important physical properties of PFP nanoemulsions allow drug encapsulation, tumor-targeting, enhanced intracellular drug delivery, and enhanced tumor visibility. The success of our pilot studies4
suggests that drug delivery in PFP nanoemulsions combined with non-invasive or minimally invasive ultrasound-mediated chemotherapy may allow development of curative rather than palliative therapy for pancreatic cancer. Here we present and discuss advantages and limitations of this approach for therapy and imaging of human MiaPaCa-2 pancreatic cancer xenografts inoculated in nu/nu mice. Cancer cells were transfected with red fluorescent protein (RFP) to allow comparison of RFP and ultrasound imaging data.