phase-change-dimethyldioctadecylammonium-shelled-microdroplets-as

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 ...

University of Rome Tor Vergata

Among different therapeutic applications of Ultrasound (US), transient membrane sonoporation (SP) - a temporary, non-lethal porosity, mechanically induced in cell membranes through US exposure - represents a compelling opportunity towards an efficient and safe drug delivery. Nevertheless, progresses in this field have been limited by an insufficient understanding of the potential cytotoxic effects of US related to the failure of the cellular repair and to the possible activation of inflammatory pathway. In this framework we studied the in vitro effects of very low-intensity US on a human keratinocyte cell line, which represents an ideal model system of skin protective barrier cells which are the first to be involved during medical US treatments. Bioeffects linked to US application at 1 MHz varying the exposure parameters were investigated by fluorescence microscopy and fluorescence activated cell sorting. Our results indicate that keratinocytes undergoing low US doses can uptake drug model molecules with size and efficiency which depend on exposure parameters. According to sub-cavitation SP models, we have identified the range of doses triggering transient membrane SP, actually with negligible biological damage. By increasing US doses we observed a reduced cells viability and an inflammatory gene overexpression enlightening novel healthy relevant strategies.

Differential effects on membrane permeability and viability of human keratinocyte cells undergoing very low intensity megasonic fields.

One of the main assets of crosslinked polymer-shelled microbubbles (MBs) as ultrasound-active theranostic agents is the robustness of the shells, combined with the chemical versatility in modifying the surface with ligands and/or drugs. Despite the long shelf-life, subtle modifications occur in the MB shells involving shifts in acoustic, mechanical and structural properties.

Long-term physical evolution of an elastomeric ultrasound contrast microbubble.

With the aim of developing new drug carriers for inhalation therapy, we report here an in depth investigation of the structure of multilamellar liposomes loaded with two well-established anti-tubercular (anti-TB) drugs, isoniazid (INH) and rifampicin (RIF), by means of small-angle neutron-scattering (SANS) analysis. Unloaded, single drug-loaded and co-loaded liposomes were prepared using different amounts of drugs and characterized regarding size, encapsulation efficiency and drug release. Detailed information on relevant properties of the investigated host-guest structures, namely the steric bilayer thickness, particle dispersion, number of lamellae and drug localization was studied by SANS. Results showed that RIF-liposomes were less ordered than unloaded liposomes. INH induced a change in the inter-bilayer periodical spacing, while RIF-INH co-loading stabilized the multilamellar liposome architecture, as confirmed by the increment of the drug loading capacity. These findings could be useful for the understanding of in vitro and in vivo behavior of these systems and for the design of new drug carriers, intended for inhaled therapy.

Drugs/lamellae interface influences the inner structure of double-loaded liposomes for inhaled anti-TB therapy: An in-depth small-angle neutron scattering investigation.

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.

Phase Change Ultrasound Contrast Agents with a Photopolymerized Diacetylene Shell.

Doxorubicin (DOX) is commonly used to treat several tumor types, but its severe side effects, primarily cardiotoxicity, represent a major limitation for its use in clinical settings. In this study we developed and characterized biodegradable and stable poly(D,L-lactic-co-glycolic) acid (PLGA) submicrocarriers employing an osmosis-based patented methodology, which allowed to optimize the drug loading efficiency up to 99%. Proceeding from this, we evaluated on MCF-7, a human breast cancer cell line, the ability of PLGA to promote the internalization of DOX and to improve its cytotoxicity in vitro. We found that the in vitro uptake efficiency is dramatically increased when DOX is loaded within PLGA colloidal carriers, which adhere to the cell membrane behaving as an efficient drug reservoir. In fact, the particles provide a diffusion-driven, sustained release of DOX across the cell membrane, resulting in high drug concentration. Accordingly, the cytotoxic analysis clearly showed that DOX-loaded PLGA exhibit a lower 50% inhibitory concentration than free DOX. The decay time of cell viability was successfully compared with DOX diffusion time constant from PLGA. The overall in vitro results highlight the potential of DOX-loaded PLGA particles to be employed as vectors with improved antitumor efficacy.

PLGA based particles as "drug reservoir" for antitumor drug delivery: characterization and cytotoxicity studies.

Ultrasound (US) induced transient membrane permeabilisation has emerged as a hugely promising tool for the delivery of exogenous vectors through the cytoplasmic membrane, paving the way to the design of novel anticancer strategies by targeting functional nanomaterials to specific biological sites. An essential step towards this end is the detailed recognition of suitably marked nanoparticles in sonoporated cells and the investigation of the potential related biological effects. By taking advantage of Synchrotron Radiation Fourier Transform Infrared micro-spectroscopy (SR-microFTIR) in providing highly sensitive analysis at the single cell level, we studied the internalisation of a nanoprobe within fibroblasts (NIH-3T3) promoted by low-intensity US. To this aim we employed 20 nm gold nanoparticles conjugated with the IR marker 4-aminothiophenol. The significant Surface Enhanced Infrared Absorption provided by the nanoprobes, with an absorbance increase up to two orders of magnitude, allowed us to efficiently recognise their inclusion within cells. Notably, the selective and stable SR-microFTIR detection from single cells that have internalised the nanoprobe exhibited clear changes in both shape and intensity of the spectral profile, highlighting the occurrence of biological effects. Flow cytometry, immunofluorescence and murine cytokinesis-block micronucleus assays confirmed the presence of slight but significant cytotoxic and genotoxic events associated with the US-nanoprobe combined treatments. Our results can provide novel hints towards US and nanomedicine combined strategies for cell spectral imaging as well as drug delivery-based therapies.

Ultrasound delivery of Surface Enhanced InfraRed Absorption active gold-nanoprobes into fibroblast cells: a biological study via Synchrotron-based InfraRed microanalysis at single cell level.

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.

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

In an in vitro Ca-induced cataract model, the progression of opacification is paralleled by a rapid decrease of the endogenous levels of spermidine (SPD) and an increase of transglutaminase type 2 (TG2, EC 2.3.2.13)-catalyzed lens crystallins cross-linking by protein-bound --(γ-glutamyl) SPD. This pattern was reversed adding exogenous SPD to the incubation resulting in a delayed loss of transparency of the rabbit lens. The present report shows evidence on the main incorporation of SPD by the catalytic activity of TG2, toward H-crystallins and in particular to the B2- and mostly in B3-crystallins. The increase of endogenous SPD in the cultured rabbit lens showed the activation of a flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAO EC 1.5.3.11). As it is known that FAD-PAO degrades the -terminal reactive portion of -(γ-glutamyl) SPD, the protein-bound -(γ-glutamyl) SPD was found the mainly available derivative for the potential formation of B3-crystallins cross-links by protein-bound --(γ-glutamyl)SPD. In conclusion, FAD-PAO degradation of the -terminal reactive residue of the crystallins bound -(γ-glutamyl)SPD together with the increased concentration of exogenous SPD, leading to saturation of glutamine residues on the substrate proteins, drastically reduces --(γ-glutamyl)SPD crosslinks formation, preventing crystallins polymerization and avoiding rabbit lens opacification. The ability of SPD and MDL 72527 to modulate the activities of TG2 and FAD-PAO involved in the mechanism of lens opacification suggests a potential strategy for the prevention of senile cataract.

Polyamine Oxidase Is Involved in Spermidine Reduction of Transglutaminase Type 2-Catalyzed H-Crystallins Polymerization in Calcium-Induced Experimental Cataract.

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.

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

Article	Year
Differential effects on membrane permeability and viability of human keratinocyte cells undergoing very low intensity megasonic fields. Scientific reports\| PubMed ID: 29184110	2017
Long-term physical evolution of an elastomeric ultrasound contrast microbubble. Journal of colloid and interface science\| PubMed ID: 30640066	2019
Drugs/lamellae interface influences the inner structure of double-loaded liposomes for inhaled anti-TB therapy: An in-depth small-angle neutron scattering investigation. Journal of colloid and interface science\| PubMed ID: 30710822	2019
Phase Change Ultrasound Contrast Agents with a Photopolymerized Diacetylene Shell. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 31042396	2019
PLGA based particles as "drug reservoir" for antitumor drug delivery: characterization and cytotoxicity studies. Colloids and surfaces. B, Biointerfaces\| PubMed ID: 31103709	2019
Ultrasound delivery of Surface Enhanced InfraRed Absorption active gold-nanoprobes into fibroblast cells: a biological study via Synchrotron-based InfraRed microanalysis at single cell level... Scientific reports\| PubMed ID: 31413286	2019
Polyvinyl alcohol based hydrogels as new tunable materials for application in the cultural heritage field. Colloids and surfaces. B, Biointerfaces\| PubMed ID: 32004905	2020
Polyamine Oxidase Is Involved in Spermidine Reduction of Transglutaminase Type 2-Catalyzed H-Crystallins Polymerization in Calcium-Induced Experimental Cataract. International journal of molecular sciences\| PubMed ID: 32751462	2020
Microgel Particles with Distinct Morphologies and Common Chemical Compositions: a Unified Description of the Responsivity to Temperature and Osmotic Stress. Gels (Basel, Switzerland)\| PubMed ID: 33081416	2020

Fabio Domenici

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