Recent advances in ultrasound-based diagnosis and therapy with micro- and nanometer-sized formulations.

Ultrasound (US) is one of the most frequently used imaging methods in the clinic. The broad spectrum of its applications can be increased by the use of gas-filled microbubbles (MB) as ultrasound contrast agents (UCA). In recent years, also nanoscale UCA like nanobubbles (NB), echogenic liposomes (ELIP) and nanodroplets have been developed, which in contrast to MB, are able to extravasate from the vessels into the tissue. New disease-specific UCA have been designed for the assessment of tissue biomarkers and advanced US to a molecular imaging modality. For this purpose, specific binding moieties were coupled to the UCA surface. The vascular endothelial growth factor receptor-2 (VEGFR-2) and P-/E-selectin are prominent examples of molecular US targets to visualize tumor blood vessels and inflammatory diseases, respectively. Besides their application in contrast-enhanced imaging, MB can also be employed for drug delivery to tumors and across the blood-brain barrier (BBB). This review summarizes the development of micro- and nanoscaled UCA and highlights recent advances in diagnostic and therapeutic applications, which are ready for translation into the clinic.

Balancing Passive and Active Targeting to Different Tumor Compartments Using Riboflavin-Functionalized Polymeric Nanocarriers.

Riboflavin transporters (RFTs) and the riboflavin carrier protein (RCP) are highly upregulated in many tumor cells, tumor stem cells, and tumor neovasculature, which makes them attractive targets for nanomedicines. Addressing cells in different tumor compartments requires drug carriers, which are not only able to accumulate via the EPR effect but also to extravasate, target specific cell populations, and get internalized by cells. Reasoning that antibodies are among the most efficient targeting systems developed by nature, we consider their size (∼10-15 nm) to be ideal for balancing passive and active tumor targeting. Therefore, small, short-circulating (10 kDa, ∼7 nm, t1/2 ∼ 1 h) and larger, longer-circulating (40 kDa, ∼13 nm, t1/2 ∼ 13 h) riboflavin-targeted branched PEG polymers were synthesized, and their biodistribution and target site accumulation were evaluated in mice bearing angiogenic squamous cell carcinoma (A431) and desmoplastic prostate cancer (PC3) xenografts. The tumor accumulation of the 10 kDa PEG was characterized by rapid intercompartmental exchange and significantly improved upon active targeting with riboflavin (RF). The 40 kDa PEG accumulated in tumors four times more efficiently than the small polymer, but its accumulation did not profit from active RF-targeting. However, RF-targeting enhanced the cellular internalization in both tumor models and for both polymer sizes. Interestingly, the nanocarriers' cell-uptake in tumors was not directly correlated with the extent of accumulation. For example, in both tumor models the small RF-PEG accumulated much less strongly than the large passively targeted PEG but showed significantly higher intracellular amounts 24 h after iv administration. Additionally, the size of the polymer determined its preferential uptake by different tumor cell compartments: the 10 kDa RF-PEGs most efficiently targeted cancer cells, whereas the highest uptake of the 40 kDa RF-PEGs was observed in tumor-associated macrophages. These findings imply that drug carriers with sizes in the range of therapeutic antibodies show balanced properties with respect to passive accumulation, tissue penetration, and active targeting. Besides highlighting the potential of RF-mediated (cancer) cell targeting, we show that strong tumor accumulation does not automatically mean high cellular uptake and that the nanocarriers' size plays a critical role in cell- and compartment-specific drug targeting.

Histopathological biomarkers for predicting the tumour accumulation of nanomedicines.

The clinical prospects of cancer nanomedicines depend on effective patient stratification. Here we report the identification of predictive biomarkers of the accumulation of nanomedicines in tumour tissue. By using supervised machine learning on data of the accumulation of nanomedicines in tumour models in mice, we identified the densities of blood vessels and of tumour-associated macrophages as key predictive features. On the basis of these two features, we derived a biomarker score correlating with the concentration of liposomal doxorubicin in tumours and validated it in three syngeneic tumour models in immunocompetent mice and in four cell-line-derived and six patient-derived tumour xenografts in mice. The score effectively discriminated tumours according to the accumulation of nanomedicines (high versus low), with an area under the receiver operating characteristic curve of 0.91. Histopathological assessment of 30 tumour specimens from patients and of 28 corresponding primary tumour biopsies confirmed the score's effectiveness in predicting the tumour accumulation of liposomal doxorubicin. Biomarkers of the tumour accumulation of nanomedicines may aid the stratification of patients in clinical trials of cancer nanomedicines.

Multimodal and multiscale optical imaging of nanomedicine delivery across the blood-brain barrier upon sonopermeation.

Rationale: The blood-brain barrier (BBB) is a major obstacle for drug delivery to the brain. Sonopermeation, which relies on the combination of ultrasound and microbubbles, has emerged as a powerful tool to permeate the BBB, enabling the extravasation of drugs and drug delivery systems (DDS) to and into the central nervous system (CNS). When aiming to improve the treatment of high medical need brain disorders, it is important to systematically study nanomedicine translocation across the sonopermeated BBB. To this end, we here employed multimodal and multiscale optical imaging to investigate the impact of DDS size on brain accumulation, extravasation and penetration upon sonopermeation. Methods: Two prototypic DDS, i.e. 10 nm-sized pHPMA polymers and 100 nm-sized PEGylated liposomes, were labeled with fluorophores and intravenously injected in healthy CD-1 nude mice. Upon sonopermeation, computed tomography-fluorescence molecular tomography, fluorescence reflectance imaging, fluorescence microscopy, confocal microscopy and stimulated emission depletion nanoscopy were used to study the effect of DDS size on their translocation across the BBB. Results: Sonopermeation treatment enabled safe and efficient opening of the BBB, which was confirmed by staining extravasated endogenous IgG. No micro-hemorrhages, edema and necrosis were detected in H&E stainings. Multimodal and multiscale optical imaging showed that sonopermeation promoted the accumulation of nanocarriers in mouse brains, and that 10 nm-sized polymeric DDS accumulated more strongly and penetrated deeper into the brain than 100 nm-sized liposomes. Conclusions: BBB opening via sonopermeation enables safe and efficient delivery of nanomedicine formulations to and into the brain. When looking at accumulation and penetration (and when neglecting issues such as drug loading capacity and therapeutic efficacy) smaller-sized DDS are found to be more suitable for drug delivery across the BBB than larger-sized DDS. These findings are valuable for better understanding and further developing nanomedicine-based strategies for the treatment of CNS disorders.

Tumor targeting via EPR: Strategies to enhance patient responses.

The tumor accumulation of nanomedicines relies on the enhanced permeability and retention (EPR) effect. In the last 5-10 years, it has been increasingly recognized that there is a large inter- and intra-individual heterogeneity in EPR-mediated tumor targeting, explaining the heterogeneous outcomes of clinical trials in which nanomedicine formulations have been evaluated. To address this heterogeneity, as in other areas of oncology drug development, we have to move away from a one-size-fits-all tumor targeting approach, towards methods that can be employed to individualize and improve nanomedicine treatments. To this end, efforts have to be invested in better understanding the nature, the complexity and the heterogeneity of the EPR effect, and in establishing systems and strategies to enhance, combine, bypass and image EPR-based tumor targeting. In the present manuscript, we summarize key studies in which these strategies are explored, and we discuss how these approaches can be employed to enhance patient responses.

Decationized polyplexes as stable and safe carrier systems for improved biodistribution in systemic gene therapy.

Many polycation-based gene delivery vectors show high transfection in vitro, but their cationic nature generally leads to significant toxicity and poor in vivo performance which significantly hampers their clinical applicability. Unlike conventional polycation-based systems, decationized polyplexes are based on hydrophilic and neutral polymers. They are obtained by a 3-step process: charge-driven condensation followed by disulfide crosslinking stabilization and finally polyplex decationization. They consist of a disulfide-crosslinked poly(hydroxypropyl methacrylamide) (pHPMA) core stably entrapping plasmid DNA (pDNA), surrounded by a shell of poly(ethylene glycol) (PEG). In the present paper the applicability of decationized polyplexes for systemic administration was evaluated. Cy5-labeled decationized polyplexes were evaluated for stability in plasma by fluorescence single particle tracking (fSPT), which technique showed stable size distribution for 48 h unlike its cationic counterpart. Upon the incubation of the polymers used for the formation of polyplexes with HUVEC cells, MTT assay showed excellent cytocompatibility of the neutral polymers. The safety was further demonstrated by a remarkable low teratogenicity and mortality activity of the polymers in a zebrafish assay, in great contrast with their cationic counterpart. Near infrared (NIR) dye-labeled polyplexes were evaluated for biodistribution and tumor accumulation by noninvasive optical imaging when administered systemically in tumor bearing mice. Decationized polyplexes exhibited an increased circulation time and higher tumor accumulation, when compared to their cationic precursors. Histology of tumors sections showed that decationized polyplexes induced reporter transgene expression in vivo. In conclusion, decationized polyplexes are a platform for safer polymeric vectors with improved biodistribution properties when systemically administered.

In Vivo Nanotoxicity Testing using the Zebrafish Embryo Assay.

Nanoparticles are increasingly used for biomedical purposes. Many different diagnostic and therapeutic applications are envisioned for nanoparticles, but there are often also serious concerns regarding their safety. Given the fact that numerous new nanomaterials are being developed every day, and that not much is known about the long-term toxicological impact of exposure to nanoparticles, there is an urgent need to establish efficient methods for nanotoxicity testing. The zebrafish (Danio rerio) embryo assay has recently emerged as an interesting 'intermediate' method for in vivo nanotoxicity screening, enabling (semi-) high-throughput analyses in a system significantly more complex than cultured cells, but at the same time also less 'invasive' and less expensive than large-scale biocompatibility studies in mice or rats. The zebrafish embryo assay is relatively well-established in the environmental sciences, but it has not yet gained wide notice in the nanomedicine field. Using prototypic polymeric drug carriers, gold-based nanodiagnostics and nanotherapeutics, and iron oxide-based nanodiagnostics, we here show that toxicity testing using zebrafish embryos is easy, efficient and informative, and faithfully reflects, yet significantly extends, cell-based toxicity testing. We therefore expect that the zebrafish embryo assay will become a popular future tool for in vivo nanotoxicity screening.