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.

Profiling target engagement and cellular uptake of cRGD-decorated clinical-stage core-crosslinked polymeric micelles.

Polymeric micelles are increasingly explored for tumor-targeted drug delivery. CriPec® technology enables the generation of core-crosslinked polymeric micelles (CCPMs) based on thermosensitive (mPEG-b-pHPMAmLacn) block copolymers, with high drug loading capacity, tailorable size, and controlled drug release kinetics. In this study, we decorated clinical-stage CCPM with the αvß3 integrin-targeted cyclic arginine-glycine-aspartic acid (cRGD) peptide, which is one of the most well-known active targeting ligands evaluated preclinically and clinically. Using a panel of cell lines with different expression levels of the αvß3 integrin receptor and exploring both static and dynamic incubation conditions, we studied the benefit of decorating CCPM with different densities of cRGD. We show that incubation time and temperature, as well as the expression levels of αvß3 integrin by target cells, positively influence cRGD-CCPM uptake, as demonstated by immunofluorescence staining and fluorescence microscopy. We demonstrate that even very low decoration densities (i.e., 1 mol % cRGD) result in increased engagement and uptake by target cells as compared to peptide-free control CCPM, and that high cRGD decoration densities do not result in a proportional increase in internalization. In this context, it should be kept in mind that a more extensive presence of targeting ligands on the surface of nanomedicines may affect their pharmacokinetic and biodistribution profile. Thus, we suggest a relatively low cRGD decoration density as most suitable for in vivo application.

Bone resorption and body reorganization during maturation induce maternal transfer of toxic metals in anguillid eels.

During their once-in-a-lifetime transoceanic spawning migration, anguillid eels do not feed, instead rely on energy stores to fuel the demands of locomotion and reproduction while they reorganize their bodies by depleting body reserves and building up gonadal tissue. Here we show how the European eel (Anguilla anguilla) breaks down its skeleton to redistribute phosphorus and calcium from hard to soft tissues during its sexual development. Using multiple analytical and imaging techniques, we characterize the spatial and temporal degradation of the skeletal framework from initial to final gonadal maturation and use elemental mass ratios in bone, muscle, liver, and gonadal tissue to determine the fluxes and fates of selected minerals and metals in the eels' bodies. We find that bone loss is more pronounced in females than in males and eventually may reach a point at which the mechanical stability of the skeleton is challenged. P and Ca are released and translocated from skeletal tissues to muscle and gonads, leaving both elements in constant proportion in remaining bone structures. The depletion of internal stores from hard and soft tissues during maturation-induced body reorganization is accompanied by the recirculation, translocation, and maternal transfer of potentially toxic metals from bone and muscle to the ovaries in gravid females, which may have direct deleterious effects on health and hinder the reproductive success of individuals of this critically endangered species.

Application of polymersomes engineered to target p32 protein for detection of small breast tumors in mice.

Triple negative breast cancer (TNBC) is the deadliest form of breast cancer and its successful treatment critically depends on early diagnosis and therapy. The multi-compartment protein p32 is overexpressed and present at cell surfaces in a variety of tumors, including TNBC, specifically in the malignant cells and endothelial cells, and in macrophages localized in hypoxic areas of the tumor. Herein we used polyethylene glycol-polycaprolactone polymersomes that were affinity targeted with the p32-binding tumor penetrating peptide LinTT1 (AKRGARSTA) for imaging of TNBC lesions. A tyrosine residue was added to the peptide to allow for 124I labeling and PET imaging. In a TNBC model in mice, systemic LinTT1-targeted polymersomes accumulated in early tumor lesions more than twice as efficiently as untargeted polymersomes with up to 20% ID/cc at 24 h after administration. The PET-imaging was very sensitive, allowing detection of tumors as small as ∼20 mm3. Confocal imaging of tumor tissue sections revealed a high degree of vascular exit and stromal penetration of LinTT1-targeted polymersomes and co-localization with tumor-associated macrophages. Our studies show that systemic LinTT1-targeted polymersomes can be potentially used for precision-guided tumor imaging and treatment of TNBC.

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.

Micro-computed tomography (µCT) as a novel method in ecotoxicology--determination of morphometric and somatic data in rainbow trout (Oncorhynchus mykiss).

Fish are important sentinel organisms for the assessment of water quality and play a central role in ecotoxicological research. Of particular importance to the assessment of health and fitness of fish stocks in response to environmental conditions or pollution are morphometric (e.g. Fulton's condition index) and somatic indices (e.g. hepatosomatic, and gonadosomatic index). Standard measurements of somatic indices are invasive and require, by definition, the sacrifice of examined animals, thus prohibiting longitudinal studies and relocation of animals captured in the field. As a potential solution, in the present study, we propose the use of micro-computed tomography (µCT) as imaging modality to non-invasively tomographically image rainbow trout (Oncorhynchus mykiss) exposed to different sediment suspensions. We here demonstrate that µCT can be used as a tool to reliably measure the volumes of different organs, which could then be applied as a substitute of their weights in calculation of somatic indices. To the best of our knowledge, this study is the first to report the results of µCT analyses in the context of ecotoxicological research in rainbow trout. It has the potential to greatly increase the information value of experiments conducted with fish and also to potentially reduce the number of animals required for studying temporal effects through facilitating longitudinal studies within the same individuals.

Image-guided drug delivery: preclinical applications and clinical translation.

Image-guided drug delivery refers to the combination of drug targeting and imaging. Preclinically, image-guided drug delivery can be used for several different purposes, including for monitoring biodistribution, target site accumulation, off-target localization, drug release and drug efficacy. Clinically, it holds significant potential for preselecting patients. In this editorial, we briefly summarize the main principles of image-guided drug delivery, and we describe its potential for facilitating, furthering and personalizing nanomedicine treatments.

Liposomal delivery of dexamethasone attenuates prostate cancer bone metastatic tumor growth in vivo.

BACKGROUND: The inflammatory tumor microenvironment, and more specifically the tumor-associated macrophages, plays an essential role in the development and progression of prostate cancer towards metastatic bone disease. Tumors are often characterized by a leaky vasculature, which - combined with the prolonged circulation kinetics of liposomes - leads to efficient tumor localization of these drug carriers, via the so-called enhanced permeability and retention (EPR) -effect. In this study, we evaluated the utility of targeted, liposomal drug delivery of the glucocorticoid dexamethasone in a model of prostate cancer bone metastases. METHODS: Tumor-bearing Balb-c nu/nu mice were treated intravenously with 0.2-1.0-5.0 mg/kg/week free- and liposomal DEX for 3-4 weeks and tumor growth was monitored by bioluminescent imaging. RESULTS: Intravenously administered liposomes localize efficiently to bone metastases in vivo and treatment of established bone metastases with (liposomal) dexamethasone resulted in a significant inhibition of tumor growth up to 26 days after initiation of treatment. Furthermore, 1.0 mg/kg liposomal dexamethasone significantly outperformed 1.0 mg/kg free dexamethasone, and was found to be well-tolerated at clinically-relevant dosages that display potent anti-tumor efficacy. CONCLUSIONS: Liposomal delivery of the glucocorticoid dexamethasone inhibits the growth of malignant bone lesions. We believe that liposomal encapsulation of dexamethasone offers a promising new treatment option for advanced, metastatic prostate cancer which supports further clinical evaluation.

Fluorescent cell-traceable dexamethasone-loaded liposomes for the treatment of inflammatory liver diseases.

Liposomes are routinely used carrier materials for delivering drug molecules to pathological sites. Besides in tumors and inflammatory sites, liposomes also strongly accumulate in liver and spleen. The potential of using liposomes to treat acute and chronic liver disorders, however, has not yet been evaluated. We here explored the therapeutic potential of dexamethasone (Dex)-loaded liposomes for inflammatory liver diseases, using experimental models of acute and chronic liver injury in mice. Fluorescently labeled liposomes predominantly accumulated in hepatic phagocytes, but also in T cells. Importantly, Dex-loaded liposomes reduced T cells in blood and liver, more effectively than free Dex, and endorsed the anti-inflammatory polarization of hepatic macrophages. In experimental chronic liver damage, Dex-loaded liposomes significantly reduced liver injury and liver fibrosis. In immune-mediated acute hepatitis Dex-loaded liposomes, but not free Dex, significantly reduced disease severity. T cells, not macrophages, were significantly depleted by Dex liposomes in liver disease models in vivo, as further supported by mechanistic cell death in vitro studies. Our data indicate that Dex liposomes may be an interesting treatment option for liver diseases, in particular for immune-mediated hepatitis. The depletion of T cells might represent the major mechanism of action of Dex liposomes, rather than their macrophage-polarizing activities.

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.

The Theranostic Path to Personalized Nanomedicine.

Advances in nanotechnology and chemical engineering have led to the development of many different drug delivery systems. These 1-100(0) nm-sized carrier materials aim to increase drug concentrations at the pathological site, while avoiding their accumulation in healthy non-target tissues, thereby improving the balance between the efficacy and the toxicity of systemic (chemo-) therapeutic interventions. An important advantage of such nanocarrier materials is the ease of incorporating both diagnostic and therapeutic entities within a single formulation, enabling them to be used for theranostic purposes. We here describe the basic principles of using nanomaterials for targeting therapeutic and diagnostic agents to pathological sites, and we discuss how nanotheranostics and image-guided drug delivery can be used to personalize nanomedicine treatments.

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.

Recent progress in nanomedicine: therapeutic, diagnostic and theranostic applications.

In recent years, the use of nanomedicine formulations for therapeutic and diagnostic applications has increased exponentially. Many different systems and strategies have been developed for drug targeting to pathological sites, as well as for visualizing and quantifying important (patho-) physiological processes. In addition, ever more efforts have been undertaken to combine diagnostic and therapeutic properties within a single nanomedicine formulation. These so-called nanotheranostics are able to provide valuable information on drug delivery, drug release and drug efficacy, and they are considered to be highly useful for personalizing nanomedicine-based (chemo-) therapeutic interventions.

Personalized nanomedicine.

Personalized medicine aims to individualize chemotherapeutic interventions on the basis of ex vivo and in vivo information on patient- and disease-specific characteristics. By noninvasively visualizing how well image-guided nanomedicines-that is, submicrometer-sized drug delivery systems containing both drugs and imaging agents within a single formulation, and designed to more specifically deliver drug molecules to pathologic sites-accumulate at the target site, patients likely to respond to nanomedicine-based therapeutic interventions may be preselected. In addition, by longitudinally monitoring how well patients respond to nanomedicine-based therapeutic interventions, drug doses and treatment protocols can be individualized and optimized during follow-up. Furthermore, noninvasive imaging information on the accumulation of nanomedicine formulations in potentially endangered healthy tissues may be used to exclude patients from further treatment. Consequently, combining noninvasive imaging with tumor-targeted drug delivery seems to hold significant potential for personalizing nanomedicine-based chemotherapeutic interventions, to achieve delivery of the right drug to the right location in the right patient at the right time.

In vitro and in vivo anticancer activity of extracts, fractions, and eupomatenoid-5 obtained from Piper regnellii leaves.

Despite numerous studies with the Piper genus, there are no previous results reporting in vitro or in vivo Piper regnellii (Miq.) C. DC. var. regnellii anticancer activity. The aim of this study was to investigate P. regnellii in vitro and in vivo anticancer activity and further identify its active compounds. In vitro antiproliferative activity was evaluated in 8 human cancer cell lines: melanoma (UACC-62), breast (MCF7), kidney (786-0), lung (NCI-H460), prostate (PC-3), ovary (OVCAR-3), colon (HT29), and leukemia (K-562). Total growth inhibition (TGI) values were chosen to measure antiproliferative activity. Among the cell lines evaluated, eupomatenoid-5 demonstrated better in vitro antiproliferative activity towards prostate, ovary, kidney, and breast cancer cell lines. In vivo studies were carried out with Ehrlich solid tumor on Balb/C mice treated with 100, 300, and 1000 mg/kg of P. regnellii leaves dichloromethane crude extract (DCE), with 30 and 100 mg/kg of the active fraction (FRB), and with 30 mg/kg of eupomatenoid-5. The i. p. administration of DCE, FRB, and eupomatenoid-5 significantly inhibited tumor progression in comparison to control mice (saline). Therefore, this study showed that neolignans of Piper regnellii have promising anticancer activity. Further studies will be undertaken to determine the mechanism of action and toxicity of these compounds.