Riboflavin-targeted polymers improve tolerance of paclitaxel while maintaining therapeutic efficacy.

Active targeting can enhance precision and efficacy of drug delivery systems (DDS) against cancers. Riboflavin (RF) is a promising ligand for active targeting due to its biocompatibility and high riboflavin-receptor expression in cancers. In this study, RF-targeted 4-arm polyethylene glycol (PEG) stars conjugated with Paclitaxel (PTX), named PEG PTX RF, were evaluated as a targeted DDS. In vitro, PEG PTX RF exhibited higher toxicity against tumor cells compared to the non-targeted counterpart (PEG PTX), while free PTX displayed the highest acute toxicity. In vivo, all treatments were similarly effective, but PEG PTX RF-treated tumors showed fewer proliferating cells, pointing to sustained therapy effects. Moreover, PTX-treated animals' body and liver weights were significantly reduced, whereas both remained stable in PEG PTX and PEG PTX RF-treated animals. Overall, our targeted and non-targeted DDS reduced PTX's adverse effects, with RF targeting promoted drug uptake in cancer cells for sustained therapeutic effect.

Welfare Assessment on Healthy and Tumor-Bearing Mice after Repeated Ultrasound Imaging.

INTRODUCTION: Ultrasound (US) imaging enables tissue visualization in high spatial resolution with short examination times. Thus, it is often applied in preclinical research. Diagnostic US, including contrast-enhanced US (CEUS), is considered to be well-tolerated by laboratory animals although no systematic study has been performed to confirm this claim. Therefore, the aim of this study was to screen for possible effects of US and CEUS examinations on welfare of healthy mice. Additionally, the potential influence of CEUS and molecular CEUS on well-being and therapy response to regorafenib was investigated in breast cancer-bearing mice. MATERIAL AND METHODS: Forty healthy Balb/c mice were randomly assigned for examination with US or CEUS (3×/week) for 4 weeks. Untreated healthy mice and mice receiving only isoflurane anesthesia served as controls (n = 10/group). Ninety-four 4T1 tumor-bearing Balb/c mice were allocated randomly to the following groups: no imaging, isoflurane anesthesia, CEUS, and molecular CEUS. They either received 10 mg/kg regorafenib or vehicle solution daily by oral gavage. Animals were examined three times within 2 weeks. CEUS measurements were performed using phospholipid microbubbles, and phospholipid microbubbles targeting the vascular endothelial growth factor receptor-2 were applied for molecular CEUS. Welfare evaluation was performed by daily observational score sheets, measuring the heart rate, Rotarod performance, and fecal corticosterone metabolites twice a week. On the last day, pathological changes in serum corticosterone concentrations, hemograms, and organ weights were obtained. Moreover, a potential influence of isoflurane anesthesia, CEUS, and molecular CEUS on regorafenib response in tumor-bearing mice was examined. Analysis of variance and Dunnett's post hoc test were performed as statistical analyses. RESULTS: Severity parameters were not altered after repeated US and CEUS examinations of healthy mice, but spleen sizes were significantly lower after isoflurane anesthesia. In tumor-bearing mice, no effect on animal welfare after repeated CEUS and molecular CEUS could be observed. However, leukocyte counts and spleen weights of tumor-bearing mice were significantly lower in animals examined with CEUS and molecular CEUS compared to the control groups. This effect was not visible in regorafenib-treated animals. CONCLUSIONS: Repeated US and (molecular) CEUS have no detectable impact on animal welfare in healthy and tumor-bearing mice. However, CEUS and molecular CEUS in combination with isoflurane anesthesia might attenuate immunological processes in tumor-bearing animals and may consequently affect responses to antitumor therapy.

Influence of Riboflavin Targeting on Tumor Accumulation and Internalization of Peptostar Based Drug Delivery Systems.

Riboflavin carrier protein (RCP) and riboflavin transporters (RFVTs) have been reported to be highly overexpressed in various cancer cells. Hence, targeting RCP and RFVTs using riboflavin may enhance tumor accumulation and internalization of drug delivery systems. To test this hypothesis, butyl-based 3-arm peptostar polymers were synthesized consisting of a lysine core (10 units per arm) and a sarcosine shell (100 units per arm). The end groups of the arms and the core were successfully modified with riboflavin and the Cy5.5 fluorescent dye, respectively. While in phosphate buffered saline the functionalized peptostars showed a bimodal behavior and formed supramolecular structures over time, they were stable in the serum maintaining their hydrodynamic diameter of 12 nm. Moreover, the polymers were biocompatible and the uptake of riboflavin targeted peptostars in A431 and PC3 cells was higher than in nontargeted controls and could be blocked competitively. In vivo, the polymers showed a moderate passive tumor accumulation, which was not significantly different between targeted and nontargeted peptostars. Nonetheless, at the histological level, internalization into tumor cells was strongly enhanced for the riboflavin-targeted peptostars. Based on these results, we conclude that passive accumulation is dominating the accumulation of peptostars, while tumor cell internalization is strongly promoted by riboflavin targeting.

Molecular Ultrasound Imaging.

Contrast-enhanced ultrasound (CEUS) imaging is a valuable tool for preclinical and clinical diagnostics. The most frequently used ultrasound contrast agents are microbubbles. Besides them, novel nano-sized materials are under investigation, which are briefly discussed in this chapter. For molecular CEUS, the ultrasound contrast agents are modified to actively target disease-associated molecular markers with a site-specific ligand. The most common markers for tumor imaging are related to neoangiogenesis, like the vascular endothelial growth factor receptor-2 (VEGFR2) and αvß3 integrin. In this chapter, applications of molecular ultrasound to longitudinally monitor receptor expression during tumor growth, to detect neovascularization, and to evaluate therapy responses are described. Furthermore, we report on first clinical trials of molecular CEUS with VEGFR2-targeted phospholipid microbubbles showing promising results regarding patient safety and its ability to detect tumors of prostate, breast, and ovary. The chapter closes with an outlook on ultrasound theranostics, where (targeted) ultrasound contrast agents are used to increase the permeability of tumor tissues and to support drug delivery.

In vitro and in vivo evaluation of biohybrid tissue-engineered vascular grafts with transformative 1H/19F MRI traceable scaffolds.

Biohybrid tissue-engineered vascular grafts (TEVGs) promise long-term durability due to their ability to adapt to hosts' needs. However, the latter calls for sensitive non-invasive imaging approaches to longitudinally monitor their functionality, integrity, and positioning. Here, we present an imaging approach comprising the labeling of non-degradable and degradable TEVGs' components for their in vitro and in vivo monitoring by hybrid 1H/19F MRI. TEVGs (inner diameter 1.5 mm) consisted of biodegradable poly(lactic-co-glycolic acid) (PLGA) fibers passively incorporating superparamagnetic iron oxide nanoparticles (SPIONs), non-degradable polyvinylidene fluoride scaffolds labeled with highly fluorinated thermoplastic polyurethane (19F-TPU) fibers, a smooth muscle cells containing fibrin blend, and endothelial cells. 1H/19F MRI of TEVGs in bioreactors, and after subcutaneous and infrarenal implantation in rats, revealed that PLGA degradation could be faithfully monitored by the decreasing SPIONs signal. The 19F signal of 19F-TPU remained constant over weeks. PLGA degradation was compensated by cells' collagen and α-smooth-muscle-actin deposition. Interestingly, only TEVGs implanted on the abdominal aorta contained elastin. XTT and histology proved that our imaging markers did not influence extracellular matrix deposition and host immune reaction. This concept of non-invasive longitudinal assessment of cardiovascular implants using 1H/19F MRI might be applicable to various biohybrid tissue-engineered implants, facilitating their clinical translation.

Repeated Contrast-Enhanced Micro-CT Examinations Decrease Animal Welfare and Influence Tumor Physiology.

OBJECTIVES: Computed tomography (CT) imaging is considered relatively safe and is often used in preclinical research to study physiological processes. However, the sum of low-dose radiation, anesthesia, and animal handling might impact animal welfare and physiological parameters. This is particularly relevant for longitudinal studies with repeated CT examinations. Therefore, we investigated the influence of repeated native and contrast-enhanced (CE) CT on animal welfare and tumor physiology in regorafenib-treated and nontreated tumor-bearing mice. MATERIAL AND METHODS: Mice bearing 4T1 breast cancer were divided into 5 groups: (1) no imaging, (2) isoflurane anesthesia only, (3) 4 mGy CT, (4) 50 mGy CT, and (5) CE-CT (iomeprol). In addition, half of each group was treated with the multikinase inhibitor regorafenib. Mice were imaged 3 times within 1 week under isoflurane anesthesia. Behavioral alterations were investigated by score sheet evaluation, rotarod test, heart rate measurements, and fecal corticosterone metabolite analysis. Tumor growth was measured daily with a caliper. Tumors were excised at the end of the experiment and histologically examined for blood vessel density, perfusion, and cell proliferation. RESULTS: According to the score sheet, animals showed a higher burden after anesthesia administration and in addition with CT imaging ( P < 0.001). Motor coordination was not affected by native CT, but significantly decreased after CE-CT in combination with the tumor therapy ( P < 0.001). Whereas tumor growth and blood vessel density were not influenced by anesthesia or imaging, CT-scanned animals had a higher tumor perfusion ( P < 0.001) and a lower tumor cell proliferation ( P < 0.001) for both radiation doses. The most significant difference was observed between the control and CE-CT groups. CONCLUSION: Repeated (CE-) CT imaging of anesthetized animals can lead to an impairment of animal motor coordination and, thus, welfare. Furthermore, these standard CT protocols seem to be capable of inducing alterations in tumor physiology when applied repetitively. These potential effects of native and CE-CT should be carefully considered in preclinical oncological research.

A century later: Adaptive plasticity and rapid evolution contribute to geographic variation in invasive mosquitofish.

One century after their introduction to Europe, eastern mosquitofish (Gambusia holbrooki) represent a natural experiment to determine the relative contributions of adaptive plasticity and rapid evolutionary change in creating large-scale geographic variation in phenotypes. We evaluated the population-genetic structure and invasion history based on allele length polymorphisms of 15 nuclear microsatellites, which we quantified for N = 660 individuals from 23 populations sampled in 2013 across the invasive range of G. holbrooki in Europe. We analysed body-shape and life-history variation in N = 1331 individuals from 36 populations, sampled in 2013 and 2017, and tested heritability of phenotypic differences in a subset of four populations using a common-garden experiment. The genetic structure of wild-caught individuals suggested a single introduction for all European mosquitofish, which were genetically impoverished compared to their native counterparts. We found some convergent patterns of phenotypic divergence across native and invasive climatic gradients (e.g., increased body size in colder/more northern populations); however, several phenotypic responses were not consistent between sampling years, pointing towards plastic phenotypes. Our analysis of common-garden reared individuals uncovered moderate heritability estimates only for two measures of male body size (intraclass correlation coefficient, ICC = 0.628 and 0.556) and offspring fat content (ICC = 0.734), while suggesting high levels of plasticity in most other phenotypic traits (ICC ≤ 0.407). Our results highlight the importance of phenotypic plasticity in invasive species during range expansions and demonstrate that strong selective pressures-in this case towards increased body size in colder environments-simultaneously promote rapid evolutionary divergence.

Influence of MRI Examinations on Animal Welfare and Study Results.

OBJECTIVES: Magnetic resonance imaging (MRI) is considered to be well tolerated by laboratory animals. However, no systematic study has been performed yet, proving this assumption. Therefore, the aim of this study was to investigate the possible effects of longitudinal native and contrast-enhanced (CE) 1-T and 7-T MRI examinations on mouse welfare as well as 4T1 breast cancers progression and therapy response. MATERIAL AND METHODS: Forty-seven healthy and 72 breast cancer-bearing mice (4T1) were investigated. One-Tesla (ICON) and 7-T (Biospec) MRI measurements were performed thrice per week under isoflurane anesthesia in healthy BALB/c mice for 4 weeks and 3 times within 2 weeks in tumor-bearing animals. Animal welfare was examined by an observational score sheet, rotarod performance, heart rate measurements, and assessment of fecal corticosterone metabolites. Furthermore, we investigated whether CE-MRI influences the study outcome. Therefore, hemograms and organ weights were obtained, and 4T1 tumor growth, perfusion, immune cell infiltration, as well as response to the multikinase inhibitor regorafenib were investigated. Statistical comparisons between groups were performed using analysis of variance and Tukey or Bonferroni post hoc tests. RESULTS: Mice showed no alterations in the observational score sheet rating, rotarod performance, heart rate, and fecal corticosterone metabolites (P > 0.05) after repeated MRI at both field strengths. However, spleen weights were reduced in all healthy mouse groups that received isoflurane anesthesia (P < 0.001) including the groups investigated by 1-T and 7-T MRI (P = 0.02). Neither tumor progression nor response to the regorafenib treatment was affected by isoflurane anesthesia or CE-MRI monitoring. Furthermore, immunohistological tumor analysis did not indicate an effect of isoflurane and MRI on macrophage infiltration of tumors, perfusion of tumor vessels, and apoptotic cell rate (P > 0.05). CONCLUSIONS: Repeated MRI did not influence the welfare of mice and did not affect tumor growth and therapy response of 4T1 tumors. However, systemic immunological effects of isoflurane anesthesia need to be considered to prevent potential bias.

Predation risk and abiotic habitat parameters affect personality traits in extremophile populations of a neotropical fish (Poecilia vivipara).

Understanding whether and how ambient ecological conditions affect the distribution of personality types within and among populations lies at the heart of research on animal personality. Several studies have focussed on only one agent of divergent selection (or driver of plastic changes in behavior), considering either predation risk or a single abiotic ecological factor. Here, we investigated how an array of abiotic and biotic environmental factors simultaneously shape population differences in boldness, activity in an open-field test, and sociability/shoaling in the livebearing fish Poecilia vivipara from six ecologically different lagoons in southeastern Brazil. We evaluated the relative contributions of variation in predation risk, water transparency/visibility, salinity (ranging from oligo- to hypersaline), and dissolved oxygen. We also investigated the role played by environmental factors for the emergence, strength, and direction of behavioral correlations. Water transparency explained most of the behavioral variation, whereby fish from lagoons with low water transparency were significantly shyer, less active, and shoaled less than fish living under clear water conditions. When we tested additional wild-caught fish from the same lagoons after acclimating them to homogeneous laboratory conditions, population differences were largely absent, pointing toward behavioral plasticity as a mechanism underlying the observed behavioral differences. Furthermore, we found correlations between personality traits (behavioral syndromes) to vary substantially in strength and direction among populations, with no obvious associations with ecological factors (including predation risk). Altogether, our results suggest that various habitat parameters simultaneously shape the distribution of personality types, with abiotic factors playing a vital (as yet underestimated) role. Furthermore, while predation is often thought to lead to the emergence of behavioral syndromes, our data do not support this assumption.