Vibropolyfection: coupling polymer-mediated gene delivery to mechanical stimulation to enhance transfection of adherent cells.

BACKGROUND: With the success of recent non-viral gene delivery-based COVID-19 vaccines, nanovectors have gained some public acceptance and come to the forefront of advanced therapies. Unfortunately, the relatively low ability of the vectors to overcome cellular barriers adversely affects their effectiveness. Scientists have thus been striving to develop ever more effective gene delivery vectors, but the results are still far from satisfactory. Therefore, developing novel strategies is probably the only way forward to bring about genuine change. Herein, we devise a brand-new gene delivery strategy to boost dramatically the transfection efficiency of two gold standard nucleic acid (NA)/polymer nanoparticles (polyplexes) in vitro. RESULTS: We conceived a device to generate milli-to-nanoscale vibrational cues as a function of the frequency set, and deliver vertical uniaxial displacements to adherent cells in culture. A short-lived high-frequency vibrational load (t = 5 min, f = 1,000 Hz) caused abrupt and extensive plasmalemma outgrowths but was safe for cells as neither cell proliferation rate nor viability was affected. Cells took about 1 hr to revert to quasi-naïve morphology through plasma membrane remodeling. In turn, this eventually triggered the mechano-activated clathrin-mediated endocytic pathway and made cells more apt to internalize polyplexes, resulting in transfection efficiencies increased from 10-to-100-fold. Noteworthy, these results were obtained transfecting three cell lines and hard-to-transfect primary cells. CONCLUSIONS: In this work, we focus on a new technology to enhance the intracellular delivery of NAs and improve the transfection efficiency of non-viral vectors through priming adherent cells with a short vibrational stimulation. This study paves the way for capitalizing on physical cell stimulation(s) to significantly raise the effectiveness of gene delivery vectors in vitro and ex vivo.

The mode of dexamethasone decoration influences avidin-nucleic-acid-nano-assembly organ biodistribution and in vivo drug persistence.

Avidin-Nucleic-Acid-NanoASsemblies (ANANAS) possess natural tropism for the liver and, when loaded with dexamethasone, reduce clinical progression in an autoimmune hepatitis murine model. Here, we investigated the linker chemistry (hydrazide-hydrazone, Hz-Hz, or carbamate hydrazide-hydrazone, Cb-Hz bond) and length (long, 5 kDa PEG, or short, 5-6 carbons) in biotin-dexamethasone conjugates used for nanoparticle decoration through in vitro and in vivo studies. All four newly synthesized conjugates released the drug at acidic pH only. In vitro, the Hz-Hz and the PEG derivatives were less stable than the Cb-Hz and the short chain ones, respectively. Once injected in healthy mice, dexamethasone location in the PEGylated ANANAS outer layer favors liver penetration and resident macrophages uptake, while drug Hz-Hz, but not Cb-Hz, short spacing prolongs drug availability. In conclusion, the tight modulation of ANANAS decoration can significantly influence the host interaction, paving the way for the development of steroid nanoformulations suitable for different pharmacokinetic profiles.

Micro- and Nanoplastics' Effects on Protein Folding and Amyloidosis.

A significant portion of the world's plastic is not properly disposed of and, through various processes, is degraded into microscopic particles termed micro- and nanoplastics. Marine and terrestrial faunae, including humans, inevitably get in contact and may inhale and ingest these microscopic plastics which can deposit throughout the body, potentially altering cellular and molecular functions in the nervous and other systems. For instance, at the cellular level, studies in animal models have shown that plastic particles can cross the blood-brain barrier and interact with neurons, and thus affect cognition. At the molecular level, plastics may specifically influence the folding of proteins, induce the formation of aberrant amyloid proteins, and therefore potentially trigger the development of systemic and local amyloidosis. In this review, we discuss the general issue of plastic micro- and nanoparticle generation, with a focus on their effects on protein folding, misfolding, and their possible clinical implications.

Vitamin E Phosphate Coating Stimulates Bone Deposition in Implant-related Infections in a Rat Model.

BACKGROUND: Implant-related infections are associated with impaired bone healing and osseointegration. In vitro antiadhesive and antibacterial properties and in vivo antiinflammatory effects protecting against bone loss of various formulations of vitamin E have been demonstrated in animal models. However, to the best of our knowledge, no in vivo studies have demonstrated the synergistic activity of vitamin E in preventing bacterial adhesion to orthopaedic implants, thus supporting the bone-implant integration. QUESTIONS/PURPOSES: The purpose of this study was to test whether a vitamin E phosphate coating on titanium implants may be able to reduce (1) the bacterial colonization of prosthetic implants and (2) bone resorption and osteomyelitis in a rat model of Staphylococcus aureus-induced implant-related infection. METHODS: Twelve rats were bilaterally injected in the femurs with S aureus UAMS-1-Xen40 and implanted with uncoated or vitamin E phosphate-coated titanium Kirschner wires without local or systemic antibiotic prophylaxis. Eight rats represented the uninfected control group. A few hours after surgery, two control and three infected animals died as a result of unexpected complications. With the remaining rats, we assessed the presence of bacterial contamination with qualitative bioluminescence imaging and Gram-positive staining and with quantitative bacterial count. Bone changes in terms of resorption and osteomyelitis were quantitatively analyzed through micro-CT (bone mineral density) and semiquantitatively through histologic scoring systems. RESULTS: Six weeks after implantation, we found only a mild decrease in bacterial count in coated versus uncoated implants (Ti versus controls: mean difference [MD], -3.705; 95% confidence interval [CI], -4.416 to -2.994; p < 0.001; TiVE versus controls: MD, -3.063; 95% CI, -3.672 to -2.454; p < 0.001), whereas micro-CT analysis showed a higher bone mineral density at the knee and femoral metaphysis in the vitamin E-treated group compared with uncoated implants (knee joint: MD, -11.88; 95% CI, -16.100 to -7.664; p < 0.001 and femoral metaphysis: MD, -19.87; 95% CI, -28.82 to -10.93; p < 0.001). We found decreased osteonecrosis (difference between medians, 1.5; 95% CI, 1-2; p < 0.002) in the infected group receiving the vitamin E-coated nails compared with the uncoated nails. CONCLUSIONS: These preliminary findings indicate that vitamin E phosphate implant coatings can exert a protective effect on bone deposition in a highly contaminated animal model of implant-related infection. CLINICAL RELEVANCE: The use of vitamin E coatings may open new perspectives for developing coatings that can limit septic loosening of infected implants with bacterial contamination. However, a deeper insight into the mechanism of action and the local release of vitamin E as a coating for orthopaedic implants is required to be used in clinics in the near future. Although this study cannot support the antimicrobial properties of vitamin E, promising results were obtained for bone-implant osseointegration. These preliminary results will require further in vivo investigations to optimize the host response in the presence of antibiotic prophylaxis.

Biocompatible Polymer Nanoformulation To Improve the Release and Safety of a Drug Mimic Molecule Detectable via ICP-MS.

Fluorescent poly(ε-caprolactone)-based nanoparticles (NPs) have been synthesized and successfully loaded with a titanium organometallic compound as a mimic of a water-insoluble drug. The nature of this nanovector enabled us to combine the quantification of the metal in tissues after systemic administration in healthy immunocompetent mice by inductively coupled plasma mass spectroscopy (ICP-MS) followed by the visualization of NPs in organ sections by confocal microscopy. This innovative method of nanodrug screening has enabled us to elucidate the crucial parameters of their kinetics. The organometallic compound is a good mimic of most anticancer drugs, and this approach is an interesting starting point to design the relevance of a broad range of nanoformulations in terms of safety and targeted delivery of the cargoes.

Single particle extinction and scattering optical method unveils in real time the influence of the blood components on polymeric nanoparticles.

Here we report the quantitative in situ characterization of size distribution evolution of polymeric nanoparticles incubated in murine serum, filtered and unfiltered murine blood. We used an analytical optical approach, named Single Particle Extinction and Scattering (SPES), which relies on the measurements of two independent parameters of single particles. SPES is based on a robust self-reference interference optical scheme which allows a rejection of the spurious signals coming from the background caused by the medium. We employed polystyrene nanoparticles as reference system and polydisperse poly(lactic-co-glycolic acid) nanoparticles. Our results demonstrate that SPES can be used for carrying out ex vivo analysis of nanoparticles to evaluate the modifications that NPs undergo in vivo following different routes of entry. Conversely, Dynamic Light Scattering is not able to provide reliable results for these systems due to the presence of the biological components in solution.

Internalization of nanopolymeric tracers does not alter characteristics of placental cells.

In the cell therapy scenario, efficient tracing of transplanted cells is essential for investigating cell migration and interactions with host tissues. This is fundamental to provide mechanistic insights which altogether allow for the understanding of the translational potential of placental cell therapy in the clinical setting. Mesenchymal stem/stromal cells (MSC) from human placenta are increasingly being investigated for their potential in treating patients with a variety of diseases. In this study, we investigated the feasibility of using poly (methyl methacrylate) nanoparticles (PMMA-NPs) to trace placental MSC, namely those from the amniotic membrane (hAMSC) and early chorionic villi (hCV-MSC). We report that PMMP-NPs are efficiently internalized and retained in both populations, and do not alter cell morphofunctional parameters. We observed that PMMP-NP incorporation does not alter in vitro immune modulatory capability of placental MSC, a characteristic central to their reparative/therapeutic effects in vitro. We also show that in vitro, PMMP-NP uptake is not affected by hypoxia. Interestingly, after in vivo brain ischaemia and reperfusion injury achieved by transient middle cerebral artery occlusion (tMCAo) in mice, iv hAMSC treatment resulted in significant improvement in cognitive function compared to PBS-treated tMCAo mice. Our study provides evidence that tracing placental MSC with PMMP-NPs does not alter their in vitro and in vivo functions. These observations are grounds for the use of PMMP-NPs as tools to investigate the therapeutic mechanisms of hAMSC and hCV-MSC in preclinical models of inflammatory-driven diseases.

Fate of PLA and PCL-Based Polymeric Nanocarriers in Cellular and Animal Models of Triple-Negative Breast Cancer.

An integrated platform to assess the interaction between nanocarriers and biological matrices has been developed by our group using poly methyl-methacrylate nanoparticles. In this study, we exploited this platform to evaluate the behavior of two biodegradable formulations, poly-ε-caprolactone (PCL3) and poly lactic-acid (PLA8), respectively, in cellular and animal models of triple-negative breast cancer (TNBC). Both NPs shared the main physicochemical parameters (size, shape, ζ-potential) and exclusively differentiated on the material on which they are composed. Our results showed that (1) PLA8 NPs, systemically injected in mice, underwent rapid degradation without penetration into tumors; (2) PLA8 NPs were not internalized in the human TNBC cell line (MDA-MB-231); (3) PCL3 NPs had a longer bioavailability, reached the tumor parenchyma, and efficiently penetrated in MDA-MB-231 cells. Our data highlight the relevance of the material selection to both improve bioavailability and target tropism, and make PCL3 NPs an interesting tool for the development of nanodrugs against TNBC.

Organ Distribution and Bone Tropism of Cellulose Nanocrystals in Living Mice.

Their physicochemical properties and relatively low cost make cellulose nanocrystals (CNCs) a potential candidate for future large-scale production in many fields including nanomedicine. Prior to a sustained and responsible development as theranostic agents, robust and reliable data concerning their safety, biocompatibility, and tissue distribution should be provided. In the present study, CNCs were extracted from Whatman filters functionalized with a fluorescent dye, and their interaction with living organisms has been thoroughly assessed. Our experimental evidence demonstrated that CNCs (1) are well tolerated by healthy mice after systemic injection; (2) are rapidly excreted, thus avoiding bioaccumulation in filter organs such as the kidneys and liver; (3) transiently migrate in bones; and (4) are able to penetrate in the cytoplasm of cancer cells without inducing material-related detrimental effects in terms of cell survival. Our results strongly suggest that the peculiar tropism to the bones is due to the chemical interaction between the Ca(2+) of the bone matrix and the active surface of negatively-charged CNCs. This feature, together with the ability to penetrate cancer cells, makes CNCs a potential nanodevice for theranostics in bone tumors.

Integrated multiplatform method for in vitro quantitative assessment of cellular uptake for fluorescent polymer nanoparticles.

Studies of cellular internalization of nanoparticles (NPs) play a paramount role for the design of efficient drug delivery systems, but so far they lack a robust experimental technique able to quantify the NP uptake in terms of number of NPs internalized in each cell. In this work we propose a novel method which provides a quantitative evaluation of fluorescent NP uptake by combining flow cytometry and plate fluorimetry with measurements of number of cells. Single cell fluorescence signals measured by flow cytometry were associated with the number of internalized NPs, exploiting the observed linearity between average flow cytometric fluorescence and overall plate fluorimeter measures, and previous calibration of the microplate reader with serial dilutions of NPs. This precise calibration has been made possible by using biocompatible fluorescent NPs in the range of 20-300 nm with a narrow particle size distribution, functionalized with a covalently bonded dye, Rhodamine B, and synthesized via emulsion free-radical polymerization. We report the absolute number of NPs internalized in mouse mammary tumor cells (4T1) as a function of time for different NP dimensions and surface charges and at several exposure concentrations. The obtained results indicate that 4T1 cells incorporated 10(3)-10(4) polymer NPs in a short time, reaching an intracellular concentration 15 times higher than the external one.

Mucosa-penetrating liposomes for esophageal local drug delivery.

Local drug delivery to the esophagus is hampered by rapid transit time and poor permeability of the mucosa. If some strategies aimed to improve the residence time have been proposed, non-invasive approaches to increase the drug penetration in the mucosa have not been described so far. Herein, we designed mucosa-penetrating liposomes to favor the penetration and retention of curcumin (CURC) in the esophagus. A novel mucosa penetrating peptide (MPP), SLENKGP, was selected by Phage Display and conjugated to pegylated liposomes at different PEG and MPP's surface densities. Pegylation assured a long residence time of liposomes (at least 30 min) in the esophagus in vivo, but it did not favor the penetration of CURC in the mucosa. MPP-decorated liposomes instead delivered a significant higher amount of CURC in the mucosa compared to naked pegylated liposomes. Confocal microscopy studies showed that naked pegylated liposomes remain confined in the superficial layers of the mucosa whereas MPP-decorated liposomes penetrate the whole epithelium. In vitro, MPP reduced the interaction of PEG with mucin, meanwhile favoring the paracellular penetration of liposomes across epithelial cell multilayers. In conclusion, pegylated liposomes represent a valid approach to target the esophagus and the surface functionalization with MPP enhances their penetration in the mucosa.

Biocompatible fluorescent nanoparticles for in vivo stem cell tracking.

Efficient application of stem cells to the treatment of neurodegenerative diseases requires safe cell tracking to follow stem cell fate over time in the host environment after transplantation. In this work, for the first time, fluorescent and biocompatible methyl methacrylate (MMA)-based nanoparticles (fluoNPs) were synthesized through a free-radical co-polymerization process with a fluorescent macromonomer obtained by linking Rhodamine B and hydroxyethyl methacrylate. We demonstrate that the fluoNPs produced by polymerization of MMA-Rhodamine complexes (1) were efficient for the labeling and tracking of multipotent human amniotic fluid cells (hAFCs); (2) did not alter the main biological features of hAFCs (such as viability, cell growth and metabolic activity); (3) enabled us to determine the longitudinal bio-distribution of hAFCs in different brain areas after graft in the brain ventricles of healthy mice by a direct fluorescence-based technique. The reliability of our approach was furthermore confirmed by magnetic resonance imaging analyses, carried out by incubating hAFCs with both superparamagnetic iron oxide nanoparticles and fluoNPs. Our data suggest that these finely tunable and biocompatible fluoNPs can be exploited for the longitudinal tracking of stem cells.

Cellulose nanocrystals: a multimodal tool to enhance the targeted drug delivery against bone disorders.

Aim: We investigated the use of cellulose nanocrystals (CNCs) as drug nanocarriers combining an anti-osteoporotic agent, alendronate (ALN), and an anti-cancer drug, doxorubicin (DOX). Materials & methods: CNC physicochemical characterization, in vivo imaging coupled with histology and in vitro uptake and toxicity assays were carried out. Results:In vivo CNC-ALN did not modify bone tropism and lung penetration, whereas its liver and kidney accumulation was slightly higher compared with CNCs alone. In vitro studies showed that CNC-ALN did not impair ALN's effect on osteoclasts, whereas CNC-DOX confirmed the therapeutic potential against bone metastatic cancer cells. Conclusions: This study provides robust proof of the potential of CNCs as easy, flexible and specific carriers to deliver compounds to the bone.

Repeated administration of the food additive E171 to mice results in accumulation in intestine and liver and promotes an inflammatory status.

Titanium dioxide (TiO2) is widely used in pharmaceuticals preparations, cosmetics, and as a food additive (E171). It contains microparticles and a fraction of nanoparticles (NPs) which can be absorbed systemically by humans after ingestion. Increasing concern has been aroused about the impact of oral exposure to TiO2 NPs from dietary and non-dietary sources on human health. In spite of several toxicological studies conducted in recent years, a solid risk assessment of oral exposure to E171 has not been satisfactorily achieved. We investigated whether repeated oral administration of E171 to mice at a dose level (5 mg/kg body weight for 3 days/week for 3 weeks) comparable to estimated human dietary exposure, results in TiO2 deposition in the digestive system and internal organs, and in molecular and cellular alterations associated with an inflammatory response. To reproduce the first phase of digestion, a new administration approach involving the dripping of the E171 suspension into the mouth of mice was applied. Significant accumulation of titanium was observed in the liver and intestine of E171-fed mice; in the latter a threefold increase in the number of TiO2 particles was also measured. Titanium accumulation in liver was associated with necroinflammatory foci containing tissue monocytes/macrophages. Three days after the last dose, increased superoxide production and inflammation were observed in the stomach and intestine. Overall, the present study indicates that the risk for human health associated with dietary exposure to E171 needs to be carefully considered.