A Preliminary Investigation of Radiation-Sensitive Ultrasound Contrast Agents for Photon Dosimetry.

Radiotherapy treatment plans have become highly conformal, posing additional constraints on the accuracy of treatment delivery. Here, we explore the use of radiation-sensitive ultrasound contrast agents (superheated phase-change nanodroplets) as dosimetric radiation sensors. In a series of experiments, we irradiated perfluorobutane nanodroplets dispersed in gel phantoms at various temperatures and assessed the radiation-induced nanodroplet vaporization events using offline or online ultrasound imaging. At 25 °C and 37 °C, the nanodroplet response was only present at higher photon energies (≥10 MV) and limited to <2 vaporization events per cm2 per Gy. A strong response (~2000 vaporizations per cm2 per Gy) was observed at 65 °C, suggesting radiation-induced nucleation of the droplet core at a sufficiently high degree of superheat. These results emphasize the need for alternative nanodroplet formulations, with a more volatile perfluorocarbon core, to enable in vivo photon dosimetry. The current nanodroplet formulation carries potential as an innovative gel dosimeter if an appropriate gel matrix can be found to ensure reproducibility. Eventually, the proposed technology might unlock unprecedented temporal and spatial resolution in image-based dosimetry, thanks to the combination of high-frame-rate ultrasound imaging and the detection of individual vaporization events, thereby addressing some of the burning challenges of new radiotherapy innovations.

PVA-Microbubbles as a Radioembolization Platform: Formulation and the In Vitro Proof of Concept.

This proof-of-concept study lays the foundations for the development of a delivery strategy for radioactive lanthanides, such as Yttrium-90, against recurrent glioblastoma. Our appealing hypothesis is that by taking advantage of the combination of biocompatible polyvinyl alcohol (PVA) microbubbles (MBs) and endovascular radiopharmaceutical infusion, a minimally invasive selective radioembolization can be achieved, which can lead to personalized treatments limiting off-target toxicities for the normal brain. The results show the successful formulation strategy that turns the ultrasound contrast PVA-shelled microbubbles into a microdevice, exhibiting good loading efficiency of Yttrium cargo by complexation with a bifunctional chelator. The selective targeting of Yttrium-loaded MBs on the glioblastoma-associated tumor endothelial cells can be unlocked by the biorecognition between the overexpressed αVß3 integrin and the ligand Cyclo(Arg-Gly-Asp-D-Phe-Lys) at the PVA microbubble surface. Hence, we show the suitability of PVA MBs as selective Y-microdevices for in situ injection via the smallest (i.e., 1.2F) neurointerventional microcatheter available on the market and the accumulation of PVA MBs on the HUVEC cell line model of integrin overexpression, thereby providing ~6 × 10-15 moles of Y90 per HUVEC cell. We further discuss the potential impact of using such versatile PVA MBs as a new therapeutic chance for treating glioblastoma multiforme recurrence.

Ultrasound-assisted carbon ion dosimetry and range measurement using injectable polymer-shelled phase-change nanodroplets: in vitro study.

Methods allowing for in situ dosimetry and range verification are essential in radiotherapy to reduce the safety margins required to account for uncertainties introduced in the entire treatment workflow. This study suggests a non-invasive dosimetry concept for carbon ion radiotherapy based on phase-change ultrasound contrast agents. Injectable nanodroplets made of a metastable perfluorobutane (PFB) liquid core, stabilized with a crosslinked poly(vinylalcohol) shell, are vaporized at physiological temperature when exposed to carbon ion radiation (C-ions), converting them into echogenic microbubbles. Nanodroplets, embedded in tissue-mimicking phantoms, are exposed at 37 °C to a 312 MeV/u clinical C-ions beam at different doses between 0.1 and 4 Gy. The evaluation of the contrast enhancement from ultrasound imaging of the phantoms, pre- and post-irradiation, reveals a significant radiation-triggered nanodroplets vaporization occurring at the C-ions Bragg peak with sub-millimeter shift reproducibility and dose dependency. The specific response of the nanodroplets to C-ions is further confirmed by varying the phantom position, the beam range, and by performing spread-out Bragg peak irradiation. The nanodroplets' response to C-ions is influenced by their concentration and is dose rate independent. These early findings show the ground-breaking potential of polymer-shelled PFB nanodroplets to enable in vivo carbon ion dosimetry and range verification.

Spatiotemporal Distribution of Nanodroplet Vaporization in a Proton Beam Using Real-Time Ultrasound Imaging for Range Verification.

The potential of proton therapy to improve the conformity of the delivered dose to the tumor volume is currently limited by range uncertainties. Injectable superheated nanodroplets have recently been proposed for ultrasound-based in vivo range verification, as these vaporize into echogenic microbubbles on proton irradiation. In previous studies, offline ultrasound images of phantoms with dispersed nanodroplets were acquired after irradiation, relating the induced vaporization profiles to the proton range. However, the aforementioned method did not enable the counting of individual vaporization events, and offline imaging cannot provide real-time feedback. In this study, we overcame these limitations using high-frame-rate ultrasound imaging with a linear array during proton irradiation of phantoms with dispersed perfluorobutane nanodroplets at 37°C and 50°C. Differential image analysis of subsequent frames allowed us to count individual vaporization events and to localize them with a resolution beyond the ultrasound diffraction limit, enabling spatial and temporal quantification of the interaction between ionizing radiation and nanodroplets. Vaporization maps were found to accurately correlate with the stopping distribution of protons (at 50°C) or secondary particles (at both temperatures). Furthermore, a linear relationship between the vaporization count and the number of incoming protons was observed. These results indicate the potential of real-time high-frame-rate contrast-enhanced ultrasound imaging for proton range verification and dosimetry.

Ultrasound-assisted investigation of photon triggered vaporization of poly(vinylalcohol) phase-change nanodroplets: A preliminary concept study with dosimetry perspective.

PURPOSE: We investigate the vaporization of phase-change ultrasound contrast agents using photon radiation for dosimetry perspectives in radiotherapy. METHODS: We studied superheated perfluorobutane nanodroplets with a crosslinked poly(vinylalcohol) shell. The nanodroplets' physico-chemical properties, and their acoustic transition have been assessed firstly. Then, poly(vinylalcohol)-perfluorobutane nanodroplets were dispersed in poly(acrylamide) hydrogel phantoms and exposed to a photon beam. We addressed the effect of several parameters influencing the nanodroplets radiation sensitivity (energy/delivered dose/dose rate/temperature). The nanodroplets-vaporization post-photon exposure was evaluated using ultrasound imaging at a low mechanical index. RESULTS: Poly(vinylalcohol)-perfluorobutane nanodroplets show a good colloidal stability over four weeks and remain highly stable at temperatures up to 78 °C. Nanodroplets acoustically-triggered phase transition leads to microbubbles with diameters <10 µm and an activation threshold of mechanical index = 0.4, at 7.5 MHz. A small number of vaporization events occur post-photon exposure (6MV/15MV), at doses between 2 and 10 Gy, leading to ultrasound contrast increase up to 60% at RT. The nanodroplets become efficiently sensitive to photons when heated to a temperature of 65 °C (while remaining below the superheat limit temperature) during irradiation. CONCLUSIONS: Nanodroplets' core is linked to the degree of superheat in the metastable state and plays a critical role in determining nanodroplet' stability and sensitivity to ionizing radiation, requiring higher or lower linear energy transfer vaporization thresholds. While poly(vinylalcohol)-perfluorobutane nanodroplets could be slightly activated by photons at ambient conditions, a good balance between the degree of superheat and stability will aim at optimizing the design of nanodroplets to reach high sensitivity to photons at physiological conditions.

Phase Change Dimethyldioctadecylammonium-Shelled Microdroplets as a Promising Drug Delivery System: Results on 3D Spheroids of Mammalian Tumor Cells.

Significant improvement of phase-change perfluorocarbon microdroplets (MDs) in the vast theranostic scenario passes through the optimization of the MDs composition with respect to synthesis efficiency, stability, and drug delivery capability. To this aim, decafluoropentane (DFP) MDs stabilized by a shell of dimethyldioctadecylammonium bromide (DDAB) cationic surfactant were designed. A high concentration of DDAB-MDs was readily obtained within a few seconds by pulsed high-power insonation, resulting in low polydisperse 1 µm size droplets. Highly positive ζ-potential, together with a long, saturated hydrocarbon chains of the DDAB shell, are key factors to stabilize the droplet and the drug cargo therein. The high affinity of the DDAB shell with cell plasma membrane allows for localized chemotherapeutics delivery by increasing the drug concentration at the tumor cell interface and boosting the uptake. This would turn DDAB-MDs into a relevant drug delivery tool exhibiting high antitumor activity at very low drug doses. In this work, the efficacy of such an approach is shown to dramatically improve the effect of doxorubicin against 3D spheroids of mammalian tumor cells, MDA-MB-231. The use of three-dimensional (3D) cell cultures developed in the form of multicellular tumor spheroids (i.e., densely packed cells in a spherical shape) has numerous advantages compared to 2D cell cultures: in addition to have the potential to bridge the gap between conventional in vitro studies and animal testing, it will improve the ability to perform more predictive in vitro screening assays for preclinical drug development or evaluate the potential of off-label drugs and new co-targeting strategies.

Modulating ultrasound contrast generation from injectable nanodroplets for proton range verification by varying the degree of superheat.

PURPOSE: Despite the physical benefits of protons over conventional photon radiation in cancer treatment, range uncertainties impede the ability to harness the full potential of proton therapy. While monitoring the proton range in vivo could reduce the currently adopted safety margins, a routinely applicable range verification technique is still lacking. Recently, phase-change nanodroplets were proposed for proton range verification, demonstrating a reproducible relationship between the proton range and generated ultrasound contrast after radiation-induced vaporization at 25°C. In this study, previous findings are extended with proton irradiations at different temperatures, including the physiological temperature of 37°C, for a novel nanodroplet formulation. Moreover, the potential to modulate the linear energy transfer (LET) threshold for vaporization by varying the degree of superheat is investigated, where the aim is to demonstrate vaporization of nanodroplets directly by primary protons. METHODS: Perfluorobutane nanodroplets with a shell made of polyvinyl alcohol (PVA-PFB) or 10,12-pentacosadyinoic acid (PCDA-PFB) were dispersed in polyacrylamide hydrogels and irradiated with 62 MeV passively scattered protons at temperatures of 37°C and 50°C. Nanodroplet transition into echogenic microbubbles was assessed using ultrasound imaging (gray value and attenuation analysis) and optical images. The proton range was measured independently and compared to the generated contrast. RESULTS: Nanodroplet design proved crucial to ensure thermal stability, as PVA-shelled nanodroplets dramatically outperformed their PCDA-shelled counterpart. At body temperature, a uniform radiation response proximal to the Bragg peak is attributed to nuclear reaction products interacting with PVA-PFB nanodroplets, with the 50% drop in ultrasound contrast being 0.17 mm ± 0.20 mm (mean ± standard deviation) in front of the proton range. Also at 50°C, highly reproducible ultrasound contrast profiles were obtained with shifts of -0.74 mm ± 0.09 mm (gray value analysis), -0.86 mm ± 0.04 mm (attenuation analysis) and -0.64 mm ± 0.29 mm (optical analysis). Moreover, a strong contrast enhancement was observed near the Bragg peak, suggesting that nanodroplets were sensitive to primary protons. CONCLUSIONS: By varying the degree of superheat of the nanodroplets' core, one can modulate the intensity of the generated ultrasound contrast. Moreover, a submillimeter reproducible relationship between the ultrasound contrast and the proton range was obtained, either indirectly via the visualization of secondary reaction products or directly through the detection of primary protons, depending on the degree of superheat. The potential of PVA-PFB nanodroplets for in vivo proton range verification was confirmed by observing a reproducible radiation response at physiological temperature, and further studies aim to assess the nanodroplets' performance in a physiological environment. Ultimately, cost-effective online or offline ultrasound imaging of radiation-induced nanodroplet vaporization could facilitate the reduction of safety margins in treatment planning and enable adaptive proton therapy.

Microgel Particles with Distinct Morphologies and Common Chemical Compositions: a Unified Description of the Responsivity to Temperature and Osmotic Stress.

Poly(N-isopropylacrylamide) (PNIPAM) hydrogel microparticles with different core-shell morphologies have been designed, while maintaining an unvaried chemical composition: a morphology with (i) an un-crosslinked core with a crosslinked shell of PNIPAM chains and (ii) PNIPAM chains crosslinked to form the core with a shell consisting of tethered un-crosslinked PNIPAM chains to the core. Both morphologies with two different degrees of crosslinking have been assessed by confocal microscopy and tested with respect to their temperature responsivity and deformation by applying an osmotic stress. The thermal and mechanical behavior of these architectures have been framed within a Flory-Rehner modified model in order to describe the microgel volume shrinking occurring as response to a temperature increase or an osmotic perturbation. This study provides a background for assessing to what extent the mechanical features of the microgel particle surface affect the interactions occurring at the interface of a microgel particle with a cell, in addition to the already know ligand/receptor interaction. These results have direct implications in triggering a limited phagocytosis of microdevices designed as injectable drug delivery systems.

In vitro analysis of the trajectories of adhesive microbubbles approaching endothelial cells.

Adhesion is a key process when ultrasound contrast agents, i.e. microbubbles, approach pathological tissues. A way to accomplish tumour targeting is to tether surface engineered microbubbles to endothelial cells of the up-regulated vascularization of cancer tissues. This can be achieved by coupling the microbubbles surface with the Arginine-Glycine-Aspartate, RGD, sequence. Such molecule interacts with the integrin receptors placed on the endothelial cells. Stability and trajectories of RGD modified lipid shelled MBs have been analysed in vitro using microchannels coated with human umbilical vein endothelial cells, HUVEC. In the microchannels realistic conditions, close to the physiological ones, were reproduced replicating shear rate, roughness comparable to the endothelium and channel size mimicking the postcapillary venules. In these conditions, the analysis of the trajectories close to the walls highlights a substantial difference between the modified MBs and the plain ones. Moreover, MBs adhesion has dynamic features recalling the motion of neutrophils engaged near the substrate such as rolling, translations and transient detachments. These findings are useful for the optimization of in vivo imaging and targeting functions.

Polyvinyl alcohol based hydrogels as new tunable materials for application in the cultural heritage field.

Hydrogel-based cleaning of paper artworks is an increasingly widespread process in the cultural heritage field. However, the search for tuned (compatible, highly retentive and not perishable) hydrogels is a challenging open question. In this paper, a complete characterization of chemical hydrogels based on polyvinyl alcohol (PVA) crosslinked with telechelic PVA and their remarkable performances as gels for cleaning paper artworks are reported. The rheological properties, porosity, water content of these gels were determined and analyzed as a function of the components concentration during synthesis. Due mechanical and retentive properties, the reported gels are optimum candidates for paper cleaning applications. The efficacy of these PVA-based gels has been demonstrated applying them on the surface of the sheets of several paper artworks, and characterizing the samples before and after the cleaning process by means of a multidisciplinary approach involving spectroscopic and chromatographic tests.

Proton range verification with ultrasound imaging using injectable radiation sensitive nanodroplets: a feasibility study.

Technologies enabling in vivo range verification during proton therapy are actively sought as a means to reduce the clinical safety margins currently adopted to avoid tumor underdosage. In this contribution, we applied the semi-empirical theory of radiation-induced vaporization of superheated liquids to coated nanodroplets. Nanodroplets are injectable phase-change contrast agents that can vaporize into highly echogenic microbubbles to provide contrast in ultrasound images. We exposed nanodroplet dispersions in aqueous phantoms to monoenergetic proton beams of varying energies and doses. Ultrasound imaging of the phantoms revealed that radiation-induced droplet vaporization occurred in regions proximal to the proton Bragg peak. A statistically significant increase in contrast was observed in irradiated regions for doses as low as 2 Gy and found to be proportional to the proton fluence. The absence of enhanced response in the vicinity of the Bragg peak, combined with theoretical considerations, suggest that droplet vaporization is induced by high linear energy transfer (LET) recoil ions produced by nuclear reactions with incoming protons. Vaporization profiles were compared to non-elastic cross sections and LET characteristics of oxygen recoils. Shifts between the ultrasound image contrast drop and the expected proton range showed a sub-millimeter reproducibility. These early findings confirm the potential of superheated nanodroplets as a novel tool for proton range verification.

Antifolate SERS-active nanovectors: quantitative drug nanostructuring and selective cell targeting for effective theranostics.

One of the frontiers of nanomedicine is the rational design of theranostic nanovectors. These are nanosized materials combining diagnostic and therapeutic capabilities, i.e. capable of tracking cancer cells and tissues in complex environments, and of selectively acting against them. We herein report on the preparation and application of antifolate plasmonic nanovectors, made of functionalized gold nanoparticles conjugated with the folic acid competitors aminopterin and methotrexate. Due to the overexpression of folate binding proteins on many types of cancer cells, these nanosystems can be exploited for selective cancer cell targeting. The strong surface enhanced Raman scattering (SERS) signature of these nanovectors acts as a diagnostic tool, not only for tracing their presence in biological samples, but also, through a careful spectral analysis, to precisely quantify the amount of drug loaded on a single nanoparticle, and therefore delivered to the cells. Meanwhile, the therapeutic action is implemented based on the strong toxicity of antifolate drugs. Remarkably, supplying the drug in the nanostructured form, rather than as a free molecule, enhances its specific toxicity. The selectivity of the antifolate nanovectors can be optimized by the design of a hybrid folate/antifolate coloaded nanovector for the specific targeting of folate receptor α, which is overexpressed on numerous cancer cell types.

Phase Change Ultrasound Contrast Agents with a Photopolymerized Diacetylene Shell.

Phase change contrast agents for ultrasound (US) imaging consist of nanodroplets (NDs) with a perfluorocarbon (PFC) liquid core stabilized with a lipid or a polymer shell. Liquid ↔ gas transition, occurring in the core, can be triggered by US to produce acoustically active microbubbles (MBs) in a process named acoustic droplet vaporization (ADV). MB shells containing polymerized diacetylene moiety were considered as a good trade off between the lipid MBs, showing optimal attenuation, and the polymeric ones, displaying enhanced stability. This work reports on novel perfluoropentane and perfluorobutane NDs stabilized with a monolayer of an amphiphilic fatty acid, i.e. 10,12-pentacosadiynoic acid (PCDA), cured with ultraviolet (UV) irradiation. The photopolymerization of the diacetylene groups, evidenced by the appearance of a blue color due to the conjugation of ene-yne sequences, exhibits a chromatic transition from the nonfluorescent blue color to a fluorescent red color when the NDs are heated or the pH of the suspension is basic. An estimate of the molecular weights reached by the polymerized PCDA in the shell, poly(PCDA), has been obtained using gel permeation chromatography and MALDI-TOF mass spectrometry. The poly(PCDA)/PFC NDs show good biocompatibility with fibroblast cells. ADV efficiency and acoustic properties before and after the transition were tested using a 1 MHz probe, revealing a resonance frequency between 1 and 2 MHz similar to other lipidic MBs. The surface of PCDA shelled NDs can be easily modified without influencing the stability and the acoustic performances of droplets. As a proof of concept we report on the conjugation of cyclic RGD and PEG chains of the particles to support targeting ability toward endothelial cells.

Biofabrication of genipin-crosslinked peptide hydrogels and their use in the controlled delivery of naproxen.

The synthesis and optimization of peptide-based hydrogel materials have gained growing interest in the last years, thanks to their properties, that make them appealing for diverse biotechnological applications, with a particular focus in the field of biomedicine. The self-assembling abilities of low molecular weight peptides make them ideal for designing advanced materials using mild reaction conditions. In this work, a biocatalytic approach has been used for the synthesis of an Fmoc-tripeptide that is able to self-assemble in water affording a self-supporting hydrogel. The mechanical properties of this material have been enhanced through chemical crosslinking by using a natural compound, genipin, that allows to minimize cytotoxic effects. Moreover, we have tested the potential of the prepared materials to be employed as drug delivery systems using naproxen as an anti-inflammatory model drug, and studying its release kinetics in aqueous medium. The cytotoxicity of the hydrogels has been evaluated, and their mechanical and morphological properties have been studied by rheology and SEM microscopy.

Graphene Meets Microbubbles: A Superior Contrast Agent for Photoacoustic Imaging.

Coupling graphene with a soft polymer surface offers the possibility to build hybrid constructs with new electrical, optical, and mechanical properties. However, the low reactivity of graphene is a hurdle in the synthesis of such systems which is often bypassed by oxidizing its carbon planar structure. However, the defects introduced with this process jeopardize the properties of graphene. In this paper we present a different approach, applicable to many different polymer surfaces, which uses surfactant assisted ultrasonication to exfoliate, and simultaneously suspend, graphene in water in its intact form. Tethering pristine graphene sheets to the surfaces is accomplished by using suitable reactive functional groups of the surfactant scaffold. We focused on applying this approach to the fabrication of a hybrid system, made of pristine graphene tethered to poly(vinyl alcohol) based microbubbles (PVA MBs), designed for enhancing photoacoustic signals. Photoacoustic imaging (PAI) is a powerful preclinical diagnostic tool which provides real time images at a resolution of 40 µm. The leap toward clinical imaging has so far been hindered by the limited tissues penetration of near-infrared (NIR) pulsed laser radiation. Many academic and industrial research laboratories have met this challenge by designing devices, each with pros and cons, to enhance the photoacoustic (PA) signal. The major advantages of the hybrid graphene/PVA MBs construct, however, are (i) the preservation of graphene properties, (ii) biocompatibility, a consequence of the robust anchoring of pristine graphene to the bioinert surface of the PVA bubble, and (iii) a very good enhancement in a NIR spectral region of the PA signal, which does not overlap with the signals of PA active endogenous molecules such as hemoglobin.

"Soft" confinement of graphene in hydrogel matrixes.

Graphene plays as protagonist among the newly discovered carbon nanomaterials on the laboratory bench. Confinement of graphene, combined with enhanced exchange properties within aqueous environment, is key for the development of biosensors, biomedicine devices, and water remediation applications. Such confinement is possible using hydrogels as soft matrixes. Many entrapment methods focused on the modification of the graphene structure. In this paper, however, we address a confinement method that leaves unchanged the graphene structure, although intimately participating in the buildup of a network of polyvinyl alcohol (PVA) chains. PVA is a polymer known as biomaterial for its hydrophilicity, biocompatibility, and chemical versatility. A robust hybrid PVA-graphene construct was obtained starting from a surfactant-assisted sonication of an aqueous dispersion of graphite. Stable graphene sheets suspension was photopolymerized in a methacryloyl-grafted PVA, using the vinyl moiety present on the surfactant scaffold. This method can allow the incorporation in the polymer network of oligomers of N-(isopropylacrylammide), p(NiPAAm). These chains display in aqueous solution a low critical solution temperature, LCST, around 33 °C and trigger a volume phase transition when incorporated in a hydrophilic network around the physiological temperature. Raman analysis was used to characterize the state of hydrogel embedded graphene single sheets. Evidence for an intimate interaction of graphene sheets and polymer matrix was collected. Release of the anticancer drug doxorubicin showed the active role of the graphene/PVA/p(NiPAAm) construct in the drug delivery.

Biosynthesis and Characterization of Cross-Linked Fmoc Peptide-Based Hydrogels for Drug Delivery Applications.

Recently, scientific and technological interest in the synthesis of novel peptide-based hydrogel materials have grown dramatically. Applications of such materials mostly concern the biomedical field with examples covering sectors such as drug delivery, tissue engineering, and production of scaffolds for cell growth, thanks to their biocompatibility and biodegradability. In this work we synthesized Fmoc-Phe3 based hydrogels of different chirality by using a biocatalytic approach. Moreover, we investigated the possibility of employing a crosslinker during the biosynthetic process and we studied and compared some chemico-physical features of both crosslinked and non-crosslinked hydrogels. In particular, we investigated the rheological properties of such materials, as well as their swelling ability, stability in aqueous medium, and their structure by SEM and AFM analysis. Crosslinked and non-crosslinked hydrogels could be formed by this procedure with comparable yields but distinct chemico-physical features. We entrapped dexamethasone within nanopolymeric particles based on PLGA coated or not with chitosan and we embedded these nanoparticles into the hydrogels. Dexamethasone release from such a nanopolymer/hydrogel system was controlled and sustained and dependent on genipin crosslinking degree. The possibility of efficiently coupling a drug delivery system to hydrogel materials seem particularly promising for tissue engineering applications, where the hydrogel could provide cells the necessary support for their growth, while nanoparticles could favor cell growth or differentiation by providing them the necessary bioactive molecules.