In vitro and in vivo behavior of nanoparticles (NPs) is often studied by tracing the NPs with fluorescent dyes. This requires stable incorporation of dyes within the NPs, as dye leakage may give a wrong interpretation of NP biodistribution, cellular uptake and intracellular distribution. Furthermore, NP labeling with trace amounts of dye should not alter NP properties such as interactions with cells or tissues.To allow for versatile NP studies with a variety of fluorescence-based assays, labeling of NPs with different dyes is desirable. Hence, when new dyes are introduced, simple and fast screening methods to assess labeling stability and NP-cell interactions are needed. For this purpose we have used a previously described generic flow cytometry assay; incubation of cells with NPs at 4°C and 37°C. Cell-NP interaction is confirmed by cellular fluorescence after 37°C incubation, and NP dye retention is confirmed when no cellular fluorescence is detected at 4°C.Three different NP-platforms labeled with six different dyes were screened, and a great variability in dye retention was observed. Surprisingly, incorporation of trace amounts of certain dyes was found to reduce, or even inhibit NP uptake. This work highlights the importance of thoroughly evaluating every dye-NP combination before pursuing fluorescent NP applications.3

Compared with conventional chemotherapy, encapsulation of drugs in nanoparticles can improve efficacy and reduce toxicity. However, delivery of nanoparticles is often insufficient and heterogeneous because of various biological barriers and uneven tumor perfusion. We investigated a unique multifunctional drug delivery system consisting of microbubbles stabilized by polymeric nanoparticles (NPMBs), enabling ultrasound-mediated drug delivery. The aim was to examine mechanisms of ultrasound-mediated delivery and to determine if increased tumor uptake had a therapeutic benefit. Cellular uptake and toxicity, circulation and biodistribution were characterized. After intravenous injection of NPMBs into mice, tumors were treated with ultrasound of various pressures and pulse lengths, and distribution of nanoparticles was imaged on tumor sections. No effects of low pressures were observed, whereas complete bubble destruction at higher pressures improved tumor uptake 2.3 times, without tissue damage. An enhanced therapeutic effect was illustrated in a promising proof-of-concept study, in which all tumors exhibited regression into complete remission.

IntroductionUltrasound in combination with microbubbles can make cells and tissues more accessible for drugs and thereby achieve improved therapeutic activity. In this review we establish the term "sonopermeation", covering mechanisms such as pore formation (sonoporation), opening of tight junctions, stimulated endocytosis/transcytosis, altered perfusion and changes in stromal compartment. Sonopermeation has gained a lot of interest in the last decade, especially for delivering drugs through the otherwise impermeable blood-brain barrier, but also to tumors. Areas coveredIn this review we summarize various in vitro assays and in vivo setups that have been employed to unravel the fundamental mechanisms involved in ultrasound-enhanced drug delivery, as well as clinical trials that are ongoing in patients with brain, pancreatic, liver and breast cancer. We summarize the basic principles of sonopermeation, describe recent findings obtained in (pre-) clinical trials, and discuss future directions. Expert OpinionWe suggest that an improved mechanistic understanding, and microbubbles and ultrasound equipment specialized for drug delivery (and not imaging) are key aspects to create more effective treatment regimens by sonopermeation. Real time feedback and tools to stratify which tumors will benefit from sonopermeation will be important for clinical success.

Microbubbles (MBs) are routinely used as contrast agents for ultrasound imaging. The use of ultrasound in combination with MBs has also attracted attention as a method to enhance drug delivery.We have developed a technology platform incorporating multiple functionalities, including imaging and therapy in a single system consisting of MBs stabilized by polyethylene glycol (PEG) coated polymeric nanoparticles (NPs). The NPs, containing lipophilic drugs and/or contrast agents, are composed of the widely used poly(butyl cyanoacrylate) (PBCA) polymer and prepared in a single step. MBs stabilized by these NPs are subsequently prepared by self-assembly of NPs at the MB air/liquid interface. Here we show that these MBs can act as contrast agents for conventional ultrasound imaging. Successful encapsulation of iron oxide NPs inside the PBCA NPs is demonstrated, potentially enabling the NPs/MBs to be used as magnetic resonance imaging (MRI) and/or molecular ultrasound imaging contrast agents. By precise tuning of the applied ultrasound pulse, the MBs burst and the NPs constituting the shell are released. This could result in increased local deposit of NPs into target tissue providing improved therapy and imaging contrast compared to freely distributed NPs.

Preclinical research has demonstrated that nanoparticles and macromolecules can accumulate in solid tumors due to the enhanced permeability and retention effect. However, drug loaded nanoparticles often fail to show increased efficacy in clinical trials. A better understanding of how tumor heterogeneity affects nanoparticle accumulation could help elucidate this discrepancy and help in patient selection for nanomedicine therapy. Here we studied five human tumor models with varying morphology and evaluated the accumulation of 100 nm polystyrene nanoparticles. Each tumor model was characterized in vivo using micro-computed tomography, contrast-enhanced ultrasound and diffusion-weighted and dynamic contrast-enhanced magnetic resonance imaging. Ex vivo, the tumors were sectioned for both fluorescence microscopy and histology. Nanoparticle uptake and distribution in the tumors were generally heterogeneous. Density of functional blood vessels measured by fluorescence microscopy correlated significantly (p = 0.0056) with nanoparticle accumulation and interestingly, inflow of microbubbles measured with ultrasound also showed a moderate but significant (p = 0.041) correlation with nanoparticle accumulation indicating that both amount of vessels and vessel morphology and perfusion predict nanoparticle accumulation. This indicates that blood vessel characterization using contrast-enhanced ultrasound imaging or other methods could be valuable for patient stratification for treatment with nanomedicines.

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Encapsulation of drugs in nanoparticles can enhance the accumulation of drugs in tumours, reduce toxicity toward healthy tissue, and improve pharmacokinetics compared to administration of free drug. To achieve efficient delivery and release of drugs at the target site, mechanisms of interaction between the nanoparticles and cells and the mechanism of delivery of the encapsulated drug are crucial to understand. Our aim was to determine the mechanisms for cellular uptake of a fluorescent hydrophobic model drug from poly(butylcyanoacrylate) nanoparticles. Prostate adenocarcinoma cells were incubated with Nile Red-loaded nanoparticles or free Nile Red. Uptake and intracellular distribution were evaluated by flow cytometry and confocal laser scanning microscopy. The nanoparticles mediated a higher intracellular level and more rapid uptake of encapsulated Nile Red compared to model drug administered alone. The main mechanism for delivery was not by endocytosis of nanoparticles but by nanoparticle-cell contact-mediated transfer directly to the cytosol and, to a smaller extent, release of payload from nanoparticles into the medium followed by diffusion into cells. The payload thus avoids entering the endocytic pathway, evading lysosomal degradation and instead gains direct access to intracellular targets. The nanoparticles are promising tools for efficient intracellular delivery of hydrophobic anticancer drugs; therefore, they are clinically relevant for improved cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12645-014-0008-4) contains supplementary material, which is available to authorized users.

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