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.

Microbubble-encapsulated Cobalt Nitrato Complexes for Ultrasound-triggerable Nitric Oxide Delivery.

Cobalt complexes exhibit versatile reactivity with nitric oxide (NO), enabling their utilization in applications ranging from homogeneous catalysis to NO-based modulation of biological processes. However, the coordination geometry around the cobalt center is complex, the therapeutic window of NO is narrow, and controlled NO delivery is difficult. To better understand the complexation of cobalt with NO, we prepared four cobalt nitrato complexes and present a structure-property relationship for ultrasound-triggerable NO release. We hypothesized that modulation of the coordination geometry by ligand-modification would improve responsiveness to mechanical stimuli, like ultrasound. To enable eventual therapeutic testing, we here first demonstrate the in vitro tolerability of [Co(ethylenediamine)2(NO)(NO3)](NO3) in A431 epidermoid carcinoma cells and J774A.1 murine macrophages, and we subsequently show successful encapsulation of the complex in poly(butyl cyanoacrylate) microbubbles. These hybrid Co-NO-containing microbubbles may in the future aid in ultrasound imaging-guided treatment of NO-responsive vascular pathologies.

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.

Hybrid ultrasound and photoacoustic contrast agent designs combining metal phthalocyanines and PBCA microbubbles.

Photoacoustic (PA) imaging is an emerging diagnostic technology that combines the penetration depth of ultrasound (US) imaging and the contrast resolution of optical imaging. Although PA imaging can visualize several endogenous chromophores to obtain clinically-relevant information, multiple applications require the administration of external contrast agents. Metal phthalocyanines have strong PA properties and chemical stability, but their extreme hydrophobicity requires their encapsulation in delivery systems for biomedical applications. Hence, we developed hybrid US/PA contrast agents by encapsulating metal phthalocyanines in poly(butyl cyanoacrylate) microbubbles (PBCA MB), which display acoustic response and ability to efficiently load hydrophobic drugs. Six different metal chromophores were loaded in PBCA MB, showing greater encapsulation efficiency with higher chromophore hydrophobicity. Notably, while the US response of the MB was unaffected by the loading of the chromophores, the PA characteristics varied greatly. Among the different formulations, MB loaded with zinc and cobalt naphthalocyanines showed the strongest PA contrast, as a result of high encapsulation efficiencies and tunable optical properties. The strong US and PA contrast signals of the formulations were preserved in biological environment, as demonstrated by in vitro imaging in serum and whole blood, and ex vivo imaging in deceased mice. Taken together, these findings highlight the advantages of combining highly hydrophobic PA contrast agents and polymeric MB for the development of contrast agents for hybrid US/PA imaging, where different types of information (structural, functional, or potentially molecular) can be acquired by combining both imaging modalities.

CCL2 chemokine inhibition primes the tumor vasculature for improved nanomedicine delivery and efficacy.

Blood vessel functionality is crucial for efficient tumor-targeted drug delivery. Heterogeneous distribution and perfusion of angiogenic blood vessels contribute to suboptimal accumulation of (nano-) therapeutics in tumors and metastases. To attenuate pathological angiogenesis, an L-RNA aptamer inhibiting the CC motif chemokine ligand 2 (CCL2) was administered to mice bearing orthotopic 4T1 triple-negative breast cancer tumors. The effect of CCL2 inhibition on tumor blood vessel functionality and tumor-targeted drug delivery was evaluated via multimodal and multiscale optical imaging, employing fluorophore-labeled polymeric (10 nm) and liposomal (100 nm) nanocarriers. Anti-CCL2 treatment induced a dose-dependent anti-angiogenic effect, reflected by a decreased relative blood volume, increased blood vessel maturity and functionality, and reduced macrophage infiltration, accompanied by a shift in the polarization of tumor-associated macrophages (TAM) towards a less M2-like and more M1-like phenotype. In line with this, CCL2 inhibitor treatment improved the delivery of polymers and liposomes to tumors, and enhanced the antitumor efficacy of free and liposomal doxorubicin. Together, these findings demonstrate that blocking the CCL2-CCR2 axis modulates TAM infiltration and polarization, resulting in vascular normalization and improved tumor-targeted drug delivery.

Transferrin Receptor-Targeted Nonspherical Microbubbles for Blood-Brain Barrier Sonopermeation.

Microbubbles (MB) are widely used for ultrasound (US) imaging and drug delivery. MB are typically spherically shaped, due to surface tension. When heated above their glass transition temperature, polymer-based MB can be mechanically stretched to obtain an anisotropic shape, endowing them with unique features for US-mediated blood-brain barrier (BBB) permeation. It is here shown that nonspherical MB can be surface-modified with BBB-specific targeting ligands, thereby promoting binding to and sonopermeation of blood vessels in the brain. Actively targeted rod-shaped MB are generated via 1D stretching of spherical poly(butyl cyanoacrylate) MB and via subsequently functionalizing their shell with antitransferrin receptor (TfR) antibodies. Using US and optical imaging, it is demonstrated that nonspherical anti-TfR-MB bind more efficiently to BBB endothelium than spherical anti-TfR-MB, both in vitro and in vivo. BBB-associated anisotropic MB produce stronger cavitation signals and markedly enhance BBB permeation and delivery of a model drug as compared to spherical BBB-targeted MB. These findings exemplify the potential of antibody-modified nonspherical MB for targeted and triggered drug delivery to the brain.

Nonspherical ultrasound microbubbles.

Surface tension provides microbubbles (MB) with a perfect spherical shape. Here, we demonstrate that MB can be engineered to be nonspherical, endowing them with unique features for biomedical applications. Anisotropic MB were generated via one-dimensionally stretching spherical poly(butyl cyanoacrylate) MB above their glass transition temperature. Compared to their spherical counterparts, nonspherical polymeric MB displayed superior performance in multiple ways, including i) increased margination behavior in blood vessel-like flow chambers, ii) reduced macrophage uptake in vitro, iii) prolonged circulation time in vivo, and iv) enhanced blood-brain barrier (BBB) permeation in vivo upon combination with transcranial focused ultrasound (FUS). Our studies identify shape as a design parameter in the MB landscape, and they provide a rational and robust framework for further exploring the application of anisotropic MB for ultrasound-enhanced drug delivery and imaging applications.

Effect of Cellular and Microenvironmental Multidrug Resistance on Tumor-Targeted Drug Delivery in Triple-Negative Breast cancer.

Multidrug resistance (MDR) reduces the efficacy of chemotherapy. Besides inducing the expression of drug efflux pumps, chemotherapy treatment alters the composition of the tumor microenvironment (TME), thereby potentially limiting tumor-directed drug delivery. To study the impact of MDR signaling in cancer cells on TME remodeling and nanomedicine delivery, we generated multidrug-resistant 4T1 triple-negative breast cancer (TNBC) cells by exposing sensitive 4T1 cells to gradually increasing doxorubicin concentrations. In 2D and 3D cell cultures, resistant 4T1 cells are presented with a more mesenchymal phenotype and produced increased amounts of collagen. While sensitive and resistant 4T1 cells showed similar tumor growth kinetics in vivo, the TME of resistant tumors was enriched in collagen and fibronectin. Vascular perfusion was also significantly increased. Fluorophore-labeled polymeric (∼10 nm) and liposomal (∼100 nm) drug carriers were administered to mice with resistant and sensitive tumors. Their tumor accumulation and penetration were studied using multimodal and multiscale optical imaging. At the whole tumor level, polymers accumulate more efficiently in resistant than in sensitive tumors. For liposomes, the trend was similar, but the differences in tumor accumulation were insignificant. At the individual blood vessel level, both polymers and liposomes were less able to extravasate out of the vasculature and penetrate the interstitium in resistant tumors. In a final in vivo efficacy study, we observed a stronger inhibitory effect of cellular and microenvironmental MDR on liposomal doxorubicin performance than free doxorubicin. These results exemplify that besides classical cellular MDR, microenvironmental drug resistance features should be considered when aiming to target and treat multidrug-resistant tumors more efficiently.

From In Vitro to Perioperative Vascular Tissue Engineering: Shortening Production Time by Traceable Textile-Reinforcement.

BACKGROUND: The production of tissue-engineered vascular graft (TEVG) usually involves a prolonged bioreactor cultivation period of up to several weeks to achieve maturation of extracellular matrix and sufficient mechanical strength. Therefore, we aimed to substantially shorten this conditioning time by combining a TEVG textile scaffold with a recently developed copolymer reinforced fibrin gel as a cell carrier. We further implemented our grafts with magnetic resonance imaging (MRI) contrast agents to allow the in-vitro monitoring of the TEVG's remodeling process. METHODS: Biodegradable polylactic-co-glycolic acid (PLGA) was electrospun onto a non-degradable polyvinylidene fluoride scaffold and molded along with copolymer-reinforced fibrin hydrogel and human arterial cells. Mechanical tests on the TEVGs were performed both instantly after molding and 4 days of bioreactor conditioning. The non-invasive in vitro monitoring of the PLGA degradation and the novel imaging of fluorinated thermoplastic polyurethane (19F-TPU) were performed using 7T MRI. RESULTS: After 4 days of close loop bioreactor conditioning, 617 ± 85 mmHg of burst pressure was achieved, and advanced maturation of extracellular matrix (ECM) was observed by immunohistology, especially in regards to collagen and smooth muscle actin. The suture retention strength (2.24 ± 0.3 N) and axial tensile strength (2.45 ± 0.58 MPa) of the TEVGs achieved higher values than the native arteries used as control. The contrast agents labeling of the TEVGs allowed the monitorability of the PLGA degradation and enabled the visibility of the non-degradable textile component. CONCLUSION: Here, we present a concept for a novel textile-reinforced TEVG, which is successfully produced in 4 days of bioreactor conditioning, characterized by increased ECM maturation and sufficient mechanical strength. Additionally, the combination of our approach with non-invasive imaging provides further insights into TEVG's clinical application.

Monitoring the Remodeling of Biohybrid Tissue-Engineered Vascular Grafts by Multimodal Molecular Imaging.

Tissue-engineered vascular grafts (TEVGs) with the ability to grow and remodel open new perspectives for cardiovascular surgery. Equipping TEVGs with synthetic polymers and biological components provides a good compromise between high structural stability and biological adaptability. However, imaging approaches to control grafts' structural integrity, physiological function, and remodeling during the entire transition between late in vitro maturation and early in vivo engraftment are mandatory for clinical implementation. Thus, a comprehensive molecular imaging concept using magnetic resonance imaging (MRI) and ultrasound (US) to monitor textile scaffold resorption, extracellular matrix (ECM) remodeling, and endothelial integrity in TEVGs is presented here. Superparamagnetic iron-oxide nanoparticles (SPION) incorporated in biodegradable poly(lactic-co-glycolic acid) (PLGA) fibers of the TEVGs allow to quantitatively monitor scaffold resorption via MRI both in vitro and in vivo. Additionally, ECM formation can be depicted by molecular MRI using elastin- and collagen-targeted probes. Finally, molecular US of αv ß3 integrins confirms the absence of endothelial dysfunction; the latter is provocable by TNF-α. In conclusion, the successful employment of noninvasive molecular imaging to longitudinally evaluate TEVGs remodeling is demonstrated. This approach may foster its translation from in vitro quality control assessment to in vivo applications to ensure proper prostheses engraftment.

Strategies to Maximize Anthracycline Drug Loading in Albumin Microbubbles.

Human serum albumin (HSA) microbubbles (MBs) are attracting increasing attention as image-guided and stimuli-responsive drug delivery systems. To better understand and maximize drug encapsulation in HSA MBs, we investigated the impact of the loading strategy and the drugs' physicochemical properties on their entrapment in the MB shell. Regarding loading strategy, we explored preloading, i.e., incubating drugs with HSA prior to MB formation, as well as postloading, i.e., incubating drugs with preformed MB. Both strategies were utilized to encapsulate six anthracyclines with different physicochemical properties. We demonstrate that drug loading in the HSA MB shell profits from preloading as well as from employing drugs with high intrinsic HSA binding affinity. These findings exemplify the potential of exploiting the natural bioconjugation interactions between drugs and HSA to formulate optimally loaded MBs, and they promote the development of HSA MBs for ultrasound-triggered drug delivery.

Molecular magnetic resonance imaging of Alpha-v-Beta-3 integrin expression in tumors with ultrasound microbubbles.

Microbubbles (MB) are used as ultrasound (US) contrast agents and can be efficiently targeted against markers of angiogenesis and inflammation. Due to their gas core, MB locally alter susceptibilities in magnetic resonance imaging (MRI), but unfortunately, the resulting contrast is low and not sufficient to generate powerful molecular MRI probes. Therefore, we investigated whether a potent molecular MR agent can be generated by encapsulating superparamagnetic iron oxide nanoparticles (SPION) in the polymeric shell of poly (n-butylcyanoacrylate) (PBCA) MB and targeted them against αvß3 integrins on the angiogenic vasculature of 4T1 murine breast carcinomas. SPION-MB consist of an air core and a multi-layered polymeric shell enabling efficient entrapment of SPION. The mean size of SPION-MB was 1.61 ± 0.32 µm. Biotin-streptavidin coupling was employed to functionalize the SPION-MB with cyclic RGDfK (Arg-Gly-Asp) and RADfK (Arg-Ala-Asp) peptides. Cells incubated with RGD-SPION-MB showed enhanced transverse relaxation rates compared with SPION-MB and blocking αvß3 integrin receptors with excess free cRGDfK significantly reduced RGD-SPION-MB binding. Due to the fast binding of RGD-SPION-MB in vivo, dynamic susceptibility contrast MRI was employed to track their retention in tumors in real-time. Higher retention of RGD-SPION-MB was observed compared with SPION-MB and RAD-SPION-MB. To corroborate our MRI results, molecular US was performed the following day using the destruction-replenishment method. Both imaging modalities consistently indicated higher retention of RGD-SPION-MB in angiogenic vessels compared with SPION-MB and RAD-SPION-MB. Competitive blocking experiments in mice further confirmed that the binding of RGD-SPION-MB to αvß3 integrin receptors is specific. Overall, this study demonstrates that RGD-SPION-MB can be employed as molecular MR/US contrast agents and are capable of assessing the αvß3 integrin expression in the neovasculature of malignant tumors.