Comparative Evaluation of Different Chitosan Species and Derivatives as Candidate Biomaterials for Oxygen-Loaded Nanodroplet Formulations to Treat Chronic Wounds.

Persistent hypoxia is a main clinical feature of chronic wounds. Intriguingly, oxygen-loaded nanodroplets (OLNDs), filled with oxygen-solving 2H,3H-decafluoropentane and shelled with polysaccharides, have been proposed as a promising tool to counteract hypoxia by releasing a clinically relevant oxygen amount in a time-sustained manner. Here, four different types of chitosan (low or medium weight (LW or MW), glycol-(G-), and methylglycol-(MG-) chitosan) were compared as candidate biopolymers for shell manufacturing. The aim of the work was to design OLND formulations with optimized physico-chemical characteristics, efficacy in oxygen release, and biocompatibility. All OLND formulations displayed spherical morphology, cationic surfaces, ≤500 nm diameters (with LW chitosan-shelled OLNDs being the smallest), high stability, good oxygen encapsulation efficiency, and prolonged oxygen release kinetics. Upon cellular internalization, LW, MW, and G-chitosan-shelled nanodroplets did not significantly affect the viability, health, or metabolic activity of human keratinocytes (HaCaT cell line). On the contrary, MG-chitosan-shelled nanodroplets showed very poor biocompatibility. Combining the physico-chemical and the biological results obtained, LW chitosan emerges as the best candidate biopolymer for future OLND application as a skin device to treat chronic wounds.

Dual-responsive magnetic nanodroplets for controlled oxygen release via ultrasound and magnetic stimulation.

Magnetic oxygen-loaded nanodroplets (MOLNDs) are a promising class of nanomaterials dually sensitive to ultrasound and magnetic fields, which can be employed as nanovectors for drug delivery applications, particularly in the field of hypoxic tissue treatment. Previous investigations were primarily focused on the application of these hybrid systems for hyperthermia treatment, exploiting magnetic nanoparticles for heat generation and nanodroplets as carriers and ultrasound contrast agents for treatment progress monitoring. This work places its emphasis on the prospect of obtaining an oxygen delivery system that can be activated by both ultrasound and magnetic fields. To achieve this goal, Fe3O4 nanoparticles were employed to decorate and induce the magnetic vaporization of OLNDs, allowing oxygen release. We present an optimized method for preparing MOLNDs by decorating nanodroplets made of diverse fluorocarbon cores and polymeric coatings. Furthermore, we performed a series of characterizations for better understanding how magnetic decoration can influence the physicochemical properties of OLNDs. Our comprehensive analysis demonstrates the efficacy of magnetic stimulation in promoting oxygen release compared to conventional ultrasound-based methods. We emphasize the critical role of selecting the appropriate fluorocarbon core and polymeric coating to optimize the decoration process and enhance the oxygen release performance of MOLNDs.

Improvement of Hyperthermia Properties of Iron Oxide Nanoparticles by Surface Coating.

Magnetic hyperthermia is an oncological therapy that exploits magnetic nanoparticles activated by radiofrequency magnetic fields to produce a controlled temperature increase in a diseased tissue. The specific loss power (SLP) of magnetic nanoparticles or the capability to release heat can be improved using surface treatments, which can reduce agglomeration effects, thus impacting on local magnetostatic interactions. In this work, Fe3O4 nanoparticles are synthesized via a coprecipitation reaction and fully characterized in terms of structural, morphological, dimensional, magnetic, and hyperthermia properties (under the Hergt-Dutz limit). Different types of surface coatings are tested, comparing their impact on the heating efficacy and colloidal stability, resulting that sodium citrate leads to a doubling of the SLP with a substantial improvement in dispersion and stability in solution over time; an SLP value of around 170 W/g is obtained in this case for a 100 kHz and 48 kA/m magnetic field.

Functional mechanical attributes of natural and synthetic gel-based scaffolds in tissue engineering: strain-stiffening effects on apparent elastic modulus and compressive toughness.

The accurate identification and determination of elastic modulus and toughness, as well as other functional mechanical attributes of artificial tissues, are of paramount importance in several fields of tissue science, tissue engineering and technology, since biomechanical and biophysical behavior is strongly linked to biological features of the medical implants and tissue-engineering scaffolds. When soft or ultra-soft materials are investigated, a relevant dispersion of elastic modulus values can be achieved, due to the strain-stiffening effects, inducing a typical non-linear behavior of these materials, as a function of strain-range. In this short communication, the Apparent elastic modulus strain-range dependence is estimated from a segmentation of the strain stiffening curve, and the related compressive toughness is investigated and discussed, based on experimental evidence, for 6 different kinds of gels, used for artificial tissue fabrication; experimental results are compared to mechanical properties of native human tissues.

Dextran-shelled oxygen-loaded nanodroplets modulate macrophages killing and inflammatory response to Enterococcus faecalis.

Chronic wounds are associated with inflammation, infections, and hypoxic environment. Macrophages play a crucial role in wound healing removing bacteria and secreting signal molecules to coordinate tissue repair. Recently, dextran-shelled Oxygen-Loaded NanoDroplets (OLNDs) have been proposed as new tools to counteract hypoxia in chronic wounds. Here we investigated the effects of OLNDs on Enterococcus faecalis (E. faecalis) killing and the secretion of inflammatory and angiogenic factors by murine (BMDM) and human (dTHP-1, differentiated THP-1) macrophages, in normoxia and hypoxia. Both OLNDs and Oxygen-Free NanoDroplets (OFNDs) significantly increased reactive oxygen species production by BMDM in normoxia (4.1 and 4 fold increase by 10% OLNDs and OFNDs, respectively, after 120 min) and hypoxia (3.8 and 4 fold increase by 10% OLNDs and OFNDs respectively) but not by dTHP-1. Moreover, only OLNDs induced nitric oxide secretion by BMDM in normoxia. Consequently, both nanodroplets improved E. faecalis killing by BMDM in normoxia (% of killing OLNDs = 44.2%; p < 0.01; OFNDs = 41.4%; p < 0.05) and hypoxia (% of killing OLNDs = 43.1%; p < 0.01; OFNDs = 37.7%; p < 0.05), while dTHP-1-mediated killing was not affected. The secretion of the inflammatory cytokines (TNFα, IL-6, IL-1ß) induced by E. faecalis infection in dTHP-1 was reduced by both types of nanodroplets, suggesting a novel anti-inflammatory activity of the dextran shell. Instead, the increase of VEGF induced by hypoxia was reduced only by OLNDs. These data provide new knowledge on the effects of OLNDs as innovative adjuvant in chronic wounds healing promoting bacterial killing and reducing inflammation.

3D Extrusion Printing of Biphasic Anthropomorphic Brain Phantoms Mimicking MR Relaxation Times Based on Alginate-Agarose-Carrageenan Blends.

The availability of adapted phantoms mimicking different body parts is fundamental to establishing the stability and reliability of magnetic resonance imaging (MRI) methods. The primary purpose of such phantoms is the mimicking of physiologically relevant, contrast-creating relaxation times T1 and T2. For the head, frequently examined by MRI, an anthropomorphic design of brain phantoms would imply the discrimination of gray matter and white matter (WM) within defined, spatially distributed compartments. Multichannel extrusion printing allows the layer-by-layer fabrication of multiple pastelike materials in a spatially defined manner with a predefined shape. In this study, the advantages of this method are used to fabricate biphasic brain phantoms mimicking MR relaxation times and anthropomorphic geometry. The printable ink was based on purely naturally derived polymers: alginate as a calcium-cross-linkable gelling agent, agarose, ι-carrageenan, and GdCl3 in different concentrations (0-280 µmol kg-1) as the paramagnetic component. The suggested inks (e.g., 3Alg-1Agar-6Car) fulfilled the requirements of viscoelastic behavior and printability of large constructs (>150 mL). The microstructure and distribution of GdCl3 were assessed by scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDX). In closely monitored steps of technological development and characterization, from monophasic and biphasic samples as printable inks and cross-linked gels, we describe the construction of large-scale phantom models whose relaxation times were characterized and checked for stability over time.

Insight into Sonoluminescence Augmented by ZnO-Functionalized Nanoparticles.

Recent advances in optical imaging techniques rely on the use of nanosized contrast agents for in vitro and in vivo applications. We report on an imaging method based on the inertial cavitation of ultrasound-irradiated water solutions that lead to sonoluminescence (SL), here, newly proposed in combination with semiconductor nanoparticles, in particular, aminopropyl-functionalized zinc oxide nanocrystals. The obtained measurements confirm the ability of such nanocrystals to increase the sonoluminescence emission, together with the ability to modify the SL spectrum when compared to the pure water behavior. In particular, it is shown that the UV component of SL is absorbed by the semiconductor behavior that is also confirmed in different biologically relevant media. Finally, optical images of nanocrystal-assisted SL are acquired for the first time, in particular, in biological buffers, revealing that at low ultrasound intensities, SL is measurable only when the nanocrystals are present in solution. All of these results witness the role of amine-functionalized zinc oxide nanocrystals for sonoluminescence emission, which makes them very good candidates as efficient nanocontrast agents for SL imaging for biological and biomedical applications.

Antibacterial and Antifungal Efficacy of Medium and Low Weight Chitosan-Shelled Nanodroplets for the Treatment of Infected Chronic Wounds.

Purpose: Medium versus low weight (MW vs LW) chitosan-shelled oxygen-loaded nanodroplets (cOLNDs) and oxygen-free nanodroplets (cOFNDs) were comparatively challenged for biocompatibility on human keratinocytes, for antimicrobial activity against four common infectious agents of chronic wounds (CWs) - methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Candida albicans and C. glabrata - and for their physical interaction with cell walls/membranes. Methods: cNDs were characterized for morphology and physico-chemical properties by microscopy and dynamic light scattering. In vitro oxygen release from cOLNDs was measured through an oximeter. ND biocompatibility and ability to promote wound healing in human normoxic/hypoxic skin cells were challenged by LDH and MTT assays using keratinocytes. ND antimicrobial activity was investigated by monitoring upon incubation with/without MW or LW cOLNDs/cOFNDs either bacteria or yeast growth over time. The mechanical interaction between NDs and microorganisms was also assessed by confocal microscopy. Results: LW cNDs appeared less toxic to keratinocytes than MW cNDs. Based on cell counts, either MW or LW cOLNDs and cOFNDs displayed long-term antimicrobial efficacy against S. pyogenes, C. albicans, and C. glabrata (up to 24 h), whereas a short-term cytostatic effects against MRSA (up to 6 h) was revealed. The internalization of all ND formulations by all four microorganisms, already after 3 h of incubation, was showed, with the only exception to MW cOLNDs/cOFNDs that adhered to MRSA walls without being internalized even after 24 h. Conclusion: cNDs exerted bacteriostatic and fungistatic effects, due to the presence of chitosan in the outer shell and independently of oxygen addition in the inner core. The duration of such effects strictly depends on the characteristics of each microbial species, and not on the molecular weight of chitosan in ND shells. However, LW chitosan was better tolerated by human keratinocytes than MW. For these reasons, the use of LW NDs should be recommended in future research to assess cOLND efficacy for the treatment of infected CWs.

Antimicrobial oxygen-loaded nanobubbles as promising tools to promote wound healing in hypoxic human keratinocytes.

Chronic wounds (CWs) are typically characterized by persistent hypoxia, exacerbated inflammation, and impaired skin tissue remodeling. Additionally, CWs are often worsened by microbial infections. Oxygen-loaded nanobubbles (OLNBs), displaying a peculiar structure based on oxygen-solving perfluorocarbons such as perfluoropentane in the inner core and polysaccharydes including chitosan in the outer shell, have proven effective in delivering oxygen to hypoxic tissues. Antimicrobial properties have been largely reported for chitosan. In the present work chitosan/perfluoropentane OLNBs were challenged for biocompatibility with human skin cells and ability to promote wound healing processes, as well as for their antimicrobial properties against methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans. After cellular internalization, OLNBs were not toxic to human keratinocytes (HaCaT), whereas oxygen-free NBs (OFNBs) slightly affected their viability. Hypoxia-dependent inhibition of keratinocyte migratory ability after scratch was fully reversed by OLNBs, but not OFNBs. Both OLNBs and OFNBs exerted chitosan-induced short-term bacteriostatic activity against MRSA (up to 6 h) and long-term fungistatic activity against C. albicans (up to 24 h). Short-term antibacterial activity associated with NB prolonged adhesion to MRSA cell wall (up to 24 h) while long-term antifungal activity followed NB early internalization by C. albicans (already after 3 h of incubation). Taken altogether, these data support chitosan-shelled and perfluoropentane-cored OLNB potential as innovative, promising, non-toxic, and cost-effective antimicrobial devices promoting repair processes to be used for treatment of MRSA- and C. albicans-infected CWs.

Leveraging re-chargeable nanobubbles on amine-functionalized ZnO nanocrystals for sustained ultrasound cavitation towards echographic imaging.

Nanoparticles able to promote inertial cavitation when exposed to focused ultrasound have recently gained much attention due to their vast range of possible applications in the biomedical field, such as enhancing drug penetration in tumor or supporting ultrasound contrast imaging. Due to their nanometric size, these contrast agents could penetrate through the endothelial cells of the vasculature to target tissues, thus enabling higher imaging resolutions than commercial gas-filled microbubbles. Herein, Zinc Oxide NanoCrystals (ZnO NCs), opportunely functionalized with amino-propyl groups, are developed as novel nanoscale contrast agents that are able, for the first time, to induce a repeatedly and over-time sustained inertial cavitation as well as ultrasound contrast imaging. The mechanism behind this phenomenon is investigated, revealing that re-adsorption of air gas nanobubbles on the nanocrystal surface is the key factor for this re-chargeable cavitation. Moreover, inertial cavitation and significant echographic signals are obtained at physiologically relevant ultrasound conditions (MI < 1.9), showing great potential for low side-effects in in-vivo applications of the novel nanoscale agent from diagnostic imaging to gas-generating theranostic nanoplatforms and to drug delivery.

The Synergistic Effect of Nanocrystals Combined With Ultrasound in the Generation of Reactive Oxygen Species for Biomedical Applications.

Reactive oxygen species (ROS) effects on living cells and tissues is multifaceted and their level or dose can considerably affect cell proliferation and viability. It is therefore necessary understand their role also designing ways able to regulate their amount inside cells, i.e., using engineered nanomaterials with either antioxidant properties or, for cancer therapy applications, capable to induce oxidative stress and cell death, through tunable ROS production. In this paper, we report on the use of single-crystalline zinc oxide (ZnO) round-shaped nanoparticles, yet ZnO nanocrystals (NCs) functionalized with amino-propyl groups (ZnO-NH2 NCs), combined with pulsed ultrasound (US). We show the synergistic effects produced by NC-assisted US which are able to produce different amount of ROS, as a result of inertial cavitation under the pulsed US exposure. Using Passive Cavitation Detection (PCD) and Electron Paramagnetic Resonance (EPR) spectroscopy, we systematically study which are the key parameters, monitoring, and influencing the amount of generated ROS measuring their concentration in water media and comparing all the results with pure water batches. We thus propose a ROS generation mechanism based on the selective application of US to the ZnO nanocrystals in water solutions. Ultrasound B-mode imaging is also applied, proving in respect to pure water, the enhanced ecographic signal generation of the aqueous solution containing ZnO-NH2 NCs when exposed to pulsed ultrasound. Furthermore, to evaluate the applicability of ZnO-NH2 NCs in the biomedical field, the ROS generation is studied by interposing different tissue mimicking materials, like phantoms and ex vivo tissues, between the US transducer and the sample well. As a whole, we clearly proof the enhanced capability to produce ROS and to control their amount when using ZnO-NH2 NCs in combination with pulsed ultrasound anticipating their applicability in the fields of biology and health care.

Insight into ultrasound-mediated reactive oxygen species generation by various metal-porphyrin complexes.

Ultrasound is used to trigger the cytotoxicity of chemical compounds, known as sonosensitisers, in an approach called sonodynamic therapy (SDT), which is under investigation herein. The generation of reactive oxygen species (ROS) has been proposed as the main biological occurrence that leads to the cytotoxic effects, which are achieved via the synergistic action of two components: the energy-absorbing sonosensitiser and ultrasound (US), which are both harmless per se. Despite some promising results, a lack of investigation into the mechanisms behind US sonosensitiser-mediated ROS generation has prevented SDT from reaching its full potential. The aim of this work is to investigate the US-responsiveness of a variety of metal-porphyrin complexes, free-base porphyrin and Fe(III), Zn(II) and Pd(II) porphyrin, by analyzing their ROS generation under US exposure and related bio-effects. All experiments were also carried out under light exposure and the results were used as references. Our results show that porphyrin ultrasound-responsiveness depends on the metal ion present, with Zn(II) and Pd(II) porphyrin being the most efficient in generating singlet oxygen and hydroxyl radicals. ROS production efficiency is lower after ultrasound exposure than after light exposure, because of the various physico-chemical mechanisms involved in sensitiser activation. US and porphyrin-mediated ROS generation is oxygen-dependent and the activation of porphyrin by US appears to be more compatible with sonoluminescence-based photo-activation rather than a radical path process that occurs via the homolytic bond rupture of water. Notably, the cytotoxicity results reported herein, which are mirrored by ex-cellulo data, confirm that the type of ROS generation achieved by the US activation of intracellular porphyrins is pivotal to the effectiveness of cancer cell killing.

Strain-rate and temperature dependent material properties of Agar and Gellan Gum used in biomedical applications.

Agar and Gellan Gum are biocompatible polymers extensively used in several fields of tissue engineering research (e.g. tissue replacement, tissue support, tissue mimicking), due to their mechanical behaviour effectively representative of actual biological tissues. Since mechanical properties of artificial tissues are related to biocompatibility and functionality of medical implants and significantly influence adhesion, growth and differentiation of cells in tissue-engineering scaffolds, an accurate characterization of Young׳s modulus and relaxation time processes is needed. In this study, the strain-rate and temperature dependent material properties of Agarose and one among the numerous kind of Gellan Gum commercially available, known as Phytagel(®), have been investigated. Nine hydrogel samples have been realized with different mechanical properties: the first one Agar-based as a reference material, the further eight samples Gellan Gum based in which the effect of dispersed solid particles like kieselguhr and SiC, as enhancing mechanical properties factors, have been investigated as a function of concentration. Stress-strain has been investigated in compression and relaxation time has been evaluated by means of the Kohlrausch-Williams-Watts time decay function. Mechanical properties have been measured as a function of temperature between 20 °C and 35 °C and at different strain rates, from ~10(-3)s(-1) and ~10(-2)s(-1) (or deformation rate from ~0.01 mms(-1) to ~0.1 mms(-1)). From experimental data, the combined temperature and strain-rate dependence of hydrogels Young׳s modulus is determined on the basis of a constitutive model. In addition to a dependence of Young׳s modulus on temperature, a remarkable influence of strain-rate has been observed, especially in the sample containing solid particles; in same ranges of temperature and strain-rate, also relaxation time variations have been monitored in order to identify a possible dependence of damping properties on temperature and strain-rate. The result is the impossibility to determine univocally mechanical properties of studied biomaterials without a proper definition of boundary conditions at which they have been obtained.

Sonochemistry and bubble dynamics.

The details of bubble behaviour in chemically active cavitation are still not sufficiently well understood. Here we report on experimental high-speed observations of acoustically driven single-bubble and few-bubble systems with the aim of clarification of the connection of their dynamics with chemical activity. Our experiment realises the sonochemical isomerization reaction of maleic acid to fumaric acid, mediated by bromine radicals, in a bubble trap set-up. The main result is that the reaction product can only be observed in a parameter regime where a small bubble cluster occurs, while a single trapped bubble stays passive. Evaluations of individual bubble dynamics for both cases are given in form of radius-time data and numerical fits to a bubble model. A conclusion is that a sufficiently strong collapse has to be accompanied by non-spherical bubble dynamics for the reaction to occur, and that the reason appears to be an efficient mixing of liquid and gas phase. This finding corroborates previous observations and literature reports on high liquid phase sonochemical activity under distinct parameter conditions than strong sonoluminescence emissions.

Acoustical characterization of polysaccharide polymers tissue-mimicking materials.

Tissue-mimicking phantoms play a crucial role in medical ultrasound research because they can simulate biological soft tissues. In last years, many types of polymeric tissues have been proposed and characterized from an acoustical and a thermal point of view, but, rarely, a deep discussion about the quality of the measurements, in terms of the uncertainty evaluation, has been reported. In this work, considering the necessity to develop laboratory standards for the measurement of ultrasonic exposure and dose quantities, a detailed description of the experimental apparatuses for the sound speed and the attenuation coefficient measurements is given, focusing the attention on the uncertainty evaluation both of the results and analysis algorithms. In particular, this algorithm reveals a novel empirical relation, fixing a limit to the energy content (therefore limits the number of cycles) of the three parts in which the authors have proposed to divide the acoustical signal. Furthermore, the realisation of multi-components phantoms, Agar and Phytagel based tissue-mimicking gels along with others long chain molecules (dextrane or polyvinyl alcohol) and scattering materials (silicon carbide and kieselguhr) are investigated. This paper reports accurate speed of sound and attenuation coefficient measurements. Speed of sound is measured by a pulse-echo technique in far-field condition, using an optical glass buffer rod; while attenuation coefficient is determined by an insertion technique, using demineralized water as reference material. The experimental sound speed results are subjected to an overall estimated relative uncertainty of about 1.5% and the attenuation coefficient uncertainty is less than 2.5%. For the development of laboratory standards, a detailed analysis of the measurement uncertainty is fundamental to make sample properties comparable. The authors believe this study could represent the right direction to make phantoms characterizations referable and traceable.

2H,3H-decafluoropentane-based nanodroplets: new perspectives for oxygen delivery to hypoxic cutaneous tissues.

Perfluoropentane (PFP)-based oxygen-loaded nanobubbles (OLNBs) were previously proposed as adjuvant therapeutic tools for pathologies of different etiology sharing hypoxia as a common feature, including cancer, infection, and autoimmunity. Here we introduce a new platform of oxygen nanocarriers, based on 2H,3H-decafluoropentane (DFP) as core fluorocarbon. These new nanocarriers have been named oxygen-loaded nanodroplets (OLNDs) since DFP is liquid at body temperature, unlike gaseous PFP. Dextran-shelled OLNDs, available either in liquid or gel formulations, display spherical morphology, ~600 nm diameters, anionic charge, good oxygen carrying capacity, and no toxic effects on human keratinocytes after cell internalization. In vitro OLNDs result more effective in releasing oxygen to hypoxic environments than former OLNBs, as demonstrated by analysis through oxymetry. In vivo, OLNDs effectively enhance oxy-hemoglobin levels, as emerged from investigation by photoacoustic imaging. Interestingly, ultrasound (US) treatment further improves transdermal oxygen release from OLNDs. Taken together, these data suggest that US-activated, DFP-based OLNDs might be innovative, suitable and cost-effective devices to topically treat hypoxia-associated pathologies of the cutaneous tissues.