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Introduction The use of spinal implants for the treatment of back disorders is largely affected by the insurgence of infections at the implantation site. Antibacterial coatings have been proposed as a viable solution to limit such infections. However, despite being effective at short-term, conventional coatings lack the ability to prevent infections at medium and long-term. Hydrogel-based drug delivery systems may represent a solution controlling the release of the loaded antibacterial agents while improving cell integration. Agarose, in particular, is a biocompatible natural polysaccharide known to improve cell growth and already used in drug delivery system formulations. In this study, an agarose hydrogel-based coating has been developed for the controlled release of gentamicin (GS). Methods Sand blasted Ti6Al4V discs were grafted with dopamine (DOPA) solution. After, GS loaded agarose hydrogels have been produced and additioned with tannic acid (TA) and calcium chloride (CaCl2) as crosslinkers. The different GS-loaded hydrogel formulations were deposited on Ti6Al4V-DOPA surfaces, and allowed to react under UV irradiation. Surface topography, wettability and composition have been analyzed with profilometry, static contact angle measurement, XPS and FTIR spectroscopy analyses. GS release was performed under pseudo-physiological conditions up to 28 days and the released GS was quantified using a specific ELISA test. The cytotoxicity of the produced coatings against human cells have been tested, along with their antibacterial activity against S. aureus bacteria. Results A homogeneous coating was obtained with all the hydrogel formulations. Moreover, the coatings presented a hydrophilic behavior and micro-scale surface roughness. The addition of TA in the hydrogel formulations showed an increase in the release time compared to the normal GS-agarose hydrogels. Moreover, the GS released from these gels was able to significantly inhibit S. aureus growth compared to the GS-agarose hydrogels. The addition of CaCl2 to the gel formulation was able to significantly decrease cytotoxicity of the TA-modified hydrogels. Conclusions Due to their surface properties, low cytotoxicity and high antibacterial effects, the hereby proposed gentamicin-loaded agarose-hydrogels provide new insight, and represent a promising approach for the surface modification of spinal implants, greatly impacting their application in the orthopedic surgical scenario., Natural Science and Engineering Research Council of Canada; Scientific and Technological Research Council of Turkey (TUBITAK) [1059B141700535]; Ege University BAP [16FBE025], This research was partially supported by the Natural Science and Engineering Research Council of Canada (Discovery program, Strategic and INNOV-I2I, CUI projects), by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project Number: 1059B141700535), and by Ege University BAP (Project Number: 16FBE025).

Non-viral gene delivery vectors have increasingly come under the spotlight, but their performaces are still far from being satisfactory. Therefore, there is an urgent need for forecasting tools and screening methods to enable the development of ever more effective transfectants. Here, coarse-grained (CG) models of gold standard transfectant poly(ethylene imine)s (PEIs) have been profitably used to investigate and highlight the effect of experimentally-relevant parameters, namely molecular weight (2 vs. 10 kDa) and topologies (linear vs. branched), protonation state, and ammine-to-phosphate ratios (N/Ps), on the complexation and the gene silencing efficiency of siRNA molecules. The results from the in vitro screening of cationic polymers and conditions were used to validate the in silico platform that we developed, such that the hits which came out of the CG models were of high practical relevance. We show that our in silico platform enables to foresee the most suitable conditions for the complexation of relevant siRNA-polycation assemblies, thereby providing a reliable predictive tool to test bench transfectants in silico, and foster the design and development of gene delivery vectors.

Cationic lipids (CLs) have gained significant attention among nonviral gene delivery vectors due to their ease of synthesis and functionalization with multivalent moieties. In particular, there is an increasing request for multifunctional CLs having gene delivery capacity and antibacterial activity. Herein, we describe the design and synthesis of a novel class of aminoglycoside (AG)-based multifunctional vectors with high transfection efficiency and noticeable antibacterial properties. Specifically, cationic amphiphiles were built on a triazine scaffold, allowing for an easy derivatization with up to three potentially different substituents, such as neomycin (Neo) that serves as the polar head and one or two lipophilic tails, namely stearyl (ST) and oleyl (OL) alkyl chains and cholesteryl (Chol) tail. With the aim to shed more light on the effect of different types and numbers of lipophilic moieties on the ability of CLs to condense and transfect cells, the performance of Neo–triazine-based derivatives as gene delivery vectors was evaluated and compared. The ability of Neo–triazine-based derivatives to act as antimicrobial agents was evaluated as well. Neo–triazine-based CLs invariably exhibited excellent DNA condensation ability, even at a low charge ratio (CR, +/−). Besides, each derivative showed very good transfection performance at its optimal CR on two different cell lines, along with negligible cytotoxicity. CLs bearing symmetric two-tailed OL proved to be the most effective in transfection. Interestingly, Neo–triazine-based derivatives, used as either free lipids or lipoplexes, exhibited strong antibacterial activity against Gram-negative bacteria, especially in the case of CLs bearing one or two aliphatic chains. Altogether, these results highlight the potential of Neo–triazine-based derivatives as effective multifunctional nonviral gene delivery vectors.

Plasma electrolytic oxidation (PEO) is proven as a scalable method for surface treatment of titanium (Ti) providing a thick oxide layer with porous micro-nano morphology. Despite the lack of antibacterial performance, this modification has potential to improve the osseointegration properties of Ti-based implant. To address this limitation, we demonstrated a new concept, showing that partial incorporation of Graphene Oxide (GO) to porous-PEO Ti-surface can significantly improve its antibacterial performance. Our idea for partial coating compared with a full surface coverage of GO was motivated to create a mixed surface with porous PEO and GO to improve antibacterial ability, while maintaining the osseointegration properties. To achieve these goals, we combined PEO and electrophoretic deposition process (EPD) to deposit GO sheets over the titanium PEO-treated substrate. The SEM, EDS, optical profilometry, XRD and Raman spectroscopy, confirmed the growth of unique multi-structured porous PEO structures decorated with GO patches. The bio-mineralization test provided the evidence of hydroxyapatite formation over the PEO-GO surface, indicating its good bioactivity. Finally, PEO-GO samples demonstrated a superior antibacterial rate of ~80% against E.coli and ~100% against S. aureus. These results indicate that PEO-GO modified titanium substrates are very promising for the development of advanced biomedical implants.

Bone metastases occur in 65%–80% advanced breast cancer patients. Although significant progresses have been made in understanding the biological mechanisms driving the bone metastatic cascade, traditional 2D in vitro models and animal studies are not effectively reproducing breast cancer cells (CCs) interactions with the bone microenvironment and suffer from species-specific differences, respectively. Moreover, simplified in vitro models cannot realistically estimate drug anti-tumoral properties and side effects, hence leading to pre-clinical testing frequent failures. To solve this issue, a 3D metastatic bone minitissue (MBm) is designed with embedded human osteoblasts, osteoclasts, bone-resident macrophages, endothelial cells and breast CCs. This minitissue recapitulates key features of the bone metastatic niche, including the alteration of macrophage polarization and microvascular architecture, along with the induction of CC micrometastases and osteomimicry. The minitissue reflects breast CC organ-specific metastatization to bone compared to a muscle minitissue. Finally, two FDA approved drugs, doxorubicin and rapamycin, have been tested showing that the dose required to impair CC growth is significantly higher in the MBm compared to a simpler CC monoculture minitissue. The MBm allows the investigation of metastasis key biological features and represents a reliable tool to better predict drug effects on the metastatic bone microenvironment.

Bioactive glasses (BGs), due to their ability to influence osteogenic cell functions, have become attractive materials to improve loaded and unloaded bone regeneration. BG systems can be easily doped with several metallic ions (e.g., Ag, Sr, Cu, Nb) in order to confer antibacterial properties. In particular, Nb, when compared with other metal ions, has been reported to be less cytotoxic and possess the ability to enhance mineralization process in human osteoblast populations. In this study, we co-deposited, through one-pot electrophoretic deposition (EPD), chitosan (CS), gelatin (GE) and a modified BG containing Nb to obtain substrates with antibacterial activity for unloaded bone regeneration. Self-standing composite scaffolds, with a defined porosity (15–90 μm) and homogeneous dispersion of BGs were obtained. TGA analysis revealed a BG loading of about 10% in the obtained scaffolds. The apatite formation ability of the scaffolds was evaluated in vitro in simulated body fluid (SBF). SEM observations, XRD and FT-IR spectra showed a slow (21–28 days) yet effective nucleation of CaP species on BGs. In particular, FT-IR peak around 603 cm−1 and XRD peak at 2θ = 32°, denoted the formation of a mineral phase after SBF immersion. In vitro biological investigation revealed that the release of Nb from composite scaffolds had no cytotoxic effects. Interestingly, BG-doped Nb scaffolds displayed antibacterial properties, reducing S. lutea and E. coli growth of ≈60% and ≈50%, respectively. Altogether, the obtained results disclose the produced composite scaffolds as promising materials with inherent antibacterial activity for bone tissue engineering applications.

Transfection by means of non-viral gene delivery vectors is the cornerstone of modern gene delivery. Despite the resources poured into the development of ever more effective transfectants, improvement is still slow and limited. Of note, the performance of any gene delivery vector in vitro is strictly dependent on several experimental conditions specific to each laboratory. The lack of standard tests has thus largely contributed to the flood of inconsistent data underpinning the reproducibility crisis. A way researchers seek to address this issue is by gauging the effectiveness of newly synthesized gene delivery vectors with respect to benchmarks of seemingly well-known behavior. However, the performance of such reference molecules is also affected by the testing conditions. This survey points to non-standardized transfection settings and limited information on variables deemed relevant in this context as the major cause of such misalignments. This review provides a catalog of conditions optimized for the gold standard and internal reference, 25 kDa polyethyleneimine, that can be profitably replicated across studies for the sake of comparison. Overall, we wish to pave the way for the implementation of standardized protocols in order to make the evaluation of the effectiveness of transfectants as unbiased as possible.

The grail of gene delivery is the development of delivery vectors as effective and non-cytotoxic as possible. In this regard, there is an urgent need of new tools for the straightforward and quantitative assessment of transfection efficiency and cytotoxicity simultaneously. We herein reported the development and validation of an easy-to-use lab-on-chip platform to perform cell transfection assays for unbiased, high-throughput selection of more and more effective gene delivery vectors by using two commercially sourced lipids, Lipofectamine 2000® and FuGene® 6. A single PDMS-layer platform was endowed with: i) a chaotic serial dilution generator, designed for the automatic generation of a linear lipoplex dilution (from 100% to 0% with 25% steps) independently delivered to; and ii) the downstream culture and transfection module consisting in five units, each composed of 33 serially connected and fluidically connected culture chambers for trapping small populations of ≈10 cells/chamber. In the absence of any transfectant, cells spread and duplicated up to 2 days. Besides, cells were transfected with EGFP-encoding reporter gene. The very facile visual inspection of the microdevice by means of a microscope and a semi-automated analytical method allowed pinpointing the best transfection conditions in terms of efficiency, cytotoxicity, cell doubling rates, and morphological changes at once.

Ayurvedic medicine is one of the oldest medical systems. It is an example of a coherent traditional system which has a time-tested and precise algorithm for medicinal plant selection, based on several ethnopharmacophore descriptors which knowledge endows the user to adequately choose the optimal plant for the treatment of certain pathology. This work aims for linking traditional knowledge with biomedical science by using traditional ayurvedic plants extracts with antimicrobial effect in form of thin films for implant protection. We report on the transfer of novel composites from bioactive glass mixed with antimicrobial plants extracts and polymer by matrix-assisted pulsed laser evaporation into uniform thin layers onto stainless steel implant-like surfaces. The comprehensive characterization of the deposited films was performed by complementary analyses: Fourier transformed infrared spectroscopy, glow discharge optical emission spectroscopy, scanning electron microscopy, atomic force microscopy, electrochemical impedance spectroscopy, UV–VIS absorption spectroscopy and antimicrobial tests. The results emphasize upon the multifunctionality of these coatings which allow to halt the leakage of metal and metal oxides into the biological fluids and eventually to inner organs (by polymer use), to speed up the osseointegration (due to the bioactive glass use), to exert antimicrobial effects (by ayurvedic plants extracts use) and to decrease the implant price (by cheaper stainless steel use).

Gene therapy targeted to vascular cells represents a promising approach for prevention and treatment of pathological conditions such as intimal hyperplasia, in-stent and post-angioplasty restenosis. In this context, polymeric non-viral gene delivery systems are a safe alternative to viral vectors but a further improvement in efficiency and cytocompatibility is needed to improve their clinical success. Herein, a library of 24 branched polyethylenimine (bPEI) derivatives modified with hydrophobic moieties was synthesised, characterised and tested in vitro on primary vascular cells, aiming to identify delivery agents with superior transfection efficiency and low cytotoxicity. Low molecular weight PEIs (0.6, 1.2 and 2 kDa) were grafted with long (C18) and short (C3) aliphatic chains, featuring different unsaturation degrees and degrees of substitution. 0.6 kDa bPEI-based derivatives were generally ineffective in transfection on vascular smooth muscle cells (VSMCs), while among the other derivatives some promising vectors were identified. Forcing polyplexes on the cell surface by means of centrifugation invariably boosted transfection levels but increased cytotoxicity as well. Of note, a propionyl-substituted derivative (PEI2-PrA1, C3:0) was the most effective on both VSMCs and endothelial cells (ECs), with higher and more sustained gene expression in combination with markedly lower cytotoxicity with respect to the gold standard 25 kDa bPEI. In addition, a linoleoyl-substituted derivative (PEI1.2-LA6, C18:2) owing to its high efficiency in VSMCs and relative inefficacy in ECs, combined with tolerable cytotoxicity was proposed as a vector for specific VSMCs targeting.

Current COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has put a spotlight on the spread of infectious diseases brought on by pathogenic airborne bacteria and viruses. In parallel with a relentless search for therapeutics and vaccines, considerable effort is being expended to develop ever more powerful technologies to restricting the spread of airborne microorganisms in indoor spaces through the minimization of health- and environment-related risks. In this context, UV-based and photocatalytic oxidation (PCO)-based technologies (i.e., the combined action of ultraviolet (UV) light and photocatalytic materials such as titanium dioxide (TiO2)) represent the most widely utilized approaches at present because they are cost-effective and ecofriendly. The virucidal and bactericidal effect relies on the synergy between the inherent ability of UV light to directly inactivate viral particles and bacteria through nucleic acid and protein damages, and the production of oxidative radicals generated through the irradiation of the TiO2 surface. In this literature survey, we draw attention to the most effective UV radiations and TiO2-based PCO technologies available and their underlying mechanisms of action on both bacteria and viral particles. Since the fine tuning of different parameters, namely the UV wavelength, the photocatalyst composition, and the UV dose (viz, the product of UV light intensity and the irradiation time), is required for the inactivation of microorganisms, we wrap up this review coming up with the most effective combination of them. Now more than ever, UV- and TiO2-based disinfection technologies may represent a valuable tool to mitigate the spread of airborne pathogens.

One of the main hurdles in nanomedicine is the low stability of drug-nanocarrier complexes as well as the drug delivery efficiency in the region-of-interest. Here, we describe the use of the film-forming protein hydrophobin HFBII to organize dodecanethiol-protected gold nanoparticles (NPs) into well-defined supraparticles (SPs). The obtained SPs are exceptionally stable in vivo and efficiently encapsulate hydrophobic drug molecules. The HFBII film prevents massive release of the encapsulated drug, which, instead, is activated by selective SP disassembly triggered intracellularly by glutathione reduction of the protein film. As a consequence, the therapeutic efficiency of an encapsulated anticancer drug is highly enhanced (2 orders of magnitude decrease in IC50). Biodistribution and pharmacokinetics studies demonstrate the high stability of the loaded SPs in the bloodstream and the selective release of the payloads once taken up in the tissues. Overall, our results provide a rationale for the development of bioreducible and multifunctional nanomedicines.

This paper contains original data supporting the antibacterial activities of Gallium (Ga3+)-doped pro-osteointegrative titanium alloys, obtained via Anodic Spark Deposition (ASD), as described in “The effect of silver or gallium doped titanium against the multidrug resistant Acinetobacter baumannii” (Cochis et al. 2016) [1].In this article we included an indirect cytocompatibility evaluation towards Saos2 human osteoblasts and extended the microbial evaluation of the Ga3+ enriched titanium surfaces against the biofilm former Escherichia coli and Staphylococcus epidermidis strains. Cell viability was assayed by the Alamar Blue test, while bacterial viability was evaluated by the metabolic colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Finally biofilm morphology was analyzed by Scanning Electron Microscopy (SEM). Data regarding Ga3+ activity were compared to Silver. Keywords: E. coli, S. epidermidis, Biofilm, Gallium, Silver, Titanium, Anodic Spark Deposition

Background: Great interest has recently been focused on tooth and tooth derivatives as suitable substrates for the treatment of alveolar bone defects. Here, we propose the use of demineralized baby teeth (BT) as potential grafting materials for bone augmentation procedures. Methods: Particles of human BT (Ø < 1 mm) were demineralized by means of a chemical/thermal treatment. Demineralized BT particles were thoroughly characterized by scanning electron microscopy/energy dispersive X-ray analyses to evaluate the effects of the demineralization on BT topography and mineral phase composition, and by enzyme-linked immunosorbent assays (ELISA) to quantify collagen and bone morphogenetic protein-2 (BMP-2) protein contents. The response of SAOS-2 cells to exogenous BMP-2 stimulation was evaluated to identify the minimum BMP-2 concentration able to induce osteodifferentiation in vitro (alkaline phosphatase (ALP) activity). Results: The demineralization treatment led to a dramatic decrease in relative Ca and P content (%) of ≈75% with respect to the native BT particles, while preserving native protein conformation and activity. Interestingly, the demineralization process led to a rise in the bioavailability of BMP-2 in BT particles, as compared to the untreated counterparts. The BMP-2 content found in demineralized BT was also proved to be very effective in enhancing ALP activity, thus in the osteodifferentiation of SAOS-2 cells in vitro, as confirmed by cell experiments performed upon exogenously added BMP-2. Conclusions: In this study we demonstrate that the BMP-2 content found in demineralized BT is very effective in inducing cell osteodifferentiation, and strengthens the idea that BTs are very attractive bioactive materials for bone-grafting procedures.

One of the tightest bottlenecks in vascular tissue engineering (vTE) is the lack of strength and elasticity of engineered vascular wall models caused by limited elastic fiber deposition. In this study, flat and tubular collagen gel-based scaffolds were cellularised with vascular smooth muscle cells (SMCs) and supplemented with human plasma fibronectin (FN), a known master organizer of several extracellular matrix (ECM) fiber systems. The consequences of FN on construct maturation was investigated in terms of geometrical contraction, viscoelastic mechanical properties and deposition of core elastic fiber proteins. FN was retained in the constructs and promoted deposition of elastin by SMCs as well as of several proteins required for elastogenesis such as fibrillin-1, lysyl oxidase, fibulin-4 and latent TGF-β binding protein-4. Notably, gel contraction, tensile equilibrium elastic modulus and elasticity were strongly improved in tubular engineered tissues, approaching the behaviour of native arteries. In conclusion, this study demonstrates that FN exerts pivotal roles in directing SMC-mediated remodeling of scaffolds toward the production of a physiological-like, elastin-containing ECM with excellent mechanical properties. The developed FN-supplemented systems are promising for tissue engineering applications where the generation of mature elastic tissue is desired and represent valuable advanced in vitro models to investigate elastogenesis.

Polyplexes (PX) are soft materials, obtained by blending polycations and nucleic acids, designed for gene delivery applications. While much is known about the transfection properties of PX, their protein corona, the biomolecules interacting with colloids once in a biological environment, represents an underlooked parameter in gene transfection. In this study, linear and branched polyethylenimines (lPEI and bPEI), the golden standard among non-viral vectors, were selected and used throughout the work: their physicochemical properties and protein corona when complexed to DNA were studied and linked to the toxicity and transfection efficiency arisen upon their delivery to cells. Interestingly, lPEIDNA and bPEIDNA complexes were characterized by similar physicochemical features, but different biological behavior. In fact, the biological milieu where cells and PX interact greatly influences their size, stability and transfection abilities. Using PX as a soft material model system, we spotlighted structure-activity relationships and methodologies that can help interpret their biological behavior and guide future studies in the field.

Zinc-doped hydroxyapatite (HA + Zn) has aroused increasing interest in bone contact applications, for its capacity to modulate osteoblast activity and for the antibacterial properties of zinc ions. In this study, HA + Zn coatings deposited by the Solution Precursor Plasma Spraying (SPPS) process were characterized. This method employs solution feedstocks providing simple and effective ion doping and resulting in the deposition of lamellae of smaller size than the conventional Atmospheric Plasma Spraying method. After a comprehensive microstructural characterization of the coatings, their bioactivity, cytotoxicity, and antibacterial efficacy were investigated. The coatings' bioactivity was confirmed by soaking them in Simulated Body Fluid (SBF), a commonly-used protocol to evaluate samples' bioactivity in vitro. Further, the coatings were non-cytotoxic against human osteoblast Saos-2-cells and, additionally, they showed antibacterial effects against Escherichia coli and Staphylococcus aureus. The HA + Zn coatings obtained showed higher efficacy against the gram-positive S. aureus than against the gram-negative Escherichia coli. These findings demonstrate the potential of the SPPS method for fabricating Zn-doped hydroxyapatite coatings, and show that Zn ions impart antibacterial properties against E. coli and S. aureus bacteria.

The risk of bacterial colonization of abiotic surfaces of biomedical devices poses important challenges for the pharmaceutical and biomaterials science fields. In this scenario, antibacterial coatings have been developed, using a number of different molecules and materials. Among them, chitosan is a non-cytotoxic, biocompatible biopolymer with an inherent antimicrobial activity that has been already used in a wide variety of healthcare and industrial applications. Herein, chitosan-based antibacterial coatings are critically surveyed, with a special emphasis on their production methods, pharmaceutical and biomedical applications, along with their pros and cons, and finally highlighting the key challenges to be faced and future perspectives in this field.

Background In recent decades, tooth derivatives such as dentin (D) and enamel (E) have been considered as potential graft biomaterials to treat bone defects. This study aimed to investigate the effects of demineralization on the physical-chemical and biological behavior of D and E. Methods Human D and E were minced into particles (ØResults The sterilization process, as a combination of chemical and thermal treatments, was found to be effective for all materials. On the other hand, D demineralization allowed preserving the collagen content, while increasing BMP-2 bioavailability. D and demineralized D (dD) displayed excellent biocompatibility, even greater than Bio-Oss®. Conversely, the high mineral content displayed by E, as confirmed by EDS analysis, inhibited cell proliferation. Of note, even though the demineralization process was somehow less effective in E than in D, demineralized E (dE) displayed increased BMP-2 bioavailability and improved performance in vitro compared with native E. Conclusions Our results substantiate the idea that the demineralization process lead to an increase of BMP-2 bioavailability, thus paving the way toward development of more effective, osteoinductive tooth-derived materials for bone regeneration and replacement.

Although branched and linear polyethylenimines (bPEIs and lPEIs) are gold standard transfectants, a systematic analysis of the effects of the preparation protocol of polyplexes and the composition of the transfection medium on their physicochemical behaviour and effectiveness in vitro have been much neglected, undermining in some way the identification of precise structure-function relationships. This work aimed to address these issues. bPEI/DNA and lPEI/DNA, prepared using two different modes of addition of reagents, gave rise to polyplexes with exactly the same chemical composition but differing in dimensions. Upon dilution in serum-free medium, the size of any kind of polyplex promptly rose over time while remained invariably stable in complete DMEM. Of note, the bigger the dimension of polyplexes (in the nano- to micrometer range), the greater their efficiency in vitro. Besides, centrifugal sedimentation of polyplexes displaying different dimensions to speed up and enhance their settling onto cells boosted transfection efficiencies. Conversely, transgene expression was significantly blunted in cells held upside-down and transfected, definitively pointing out the impact of gravitational sedimentation of polyplexes on their transfection efficiency. Overall, much more attention must be paid to the actual polyplex size that relies on the complexation conditions and the transfection medium.

In this work, laser micropolishing (LμP) was employed to reduce the surface roughness and waviness of cold-rolled AISI 304 stainless steel sheets. A pulsed fibre laser operating in the ns regime was used and the influence of laser parameters in a N2-controlled atmospheres was evaluated. In the optimal conditions, the surface remelting induced by the process allowed to reduce the surface roughness by closing cracks and defects formed during the rolling process. Other conditions that did not improve the surface quality were analysed for defect typology. Moreover, laser treatments allowed the production of more hydrophobic surfaces, and no surface chemistry modification was identified. Surface cleanability was investigated with Escherichia coli (E. coli), evaluating the number of residual bacteria adhering to the substrate after a washing procedure. These results showed that LμP is a suitable way to lower the average surface roughness by about 58% and average surface waviness by approximately 38%. The LμP process proved to be effective on the bacteria cleanability as approximately five times fewer bacteria remained on the surfaces treated with the optimized LμP parameters compared to the untreated surfaces.

PEGylation is widely used to improve the stability of gene delivery vectors through the decrease of non-specific binding to serum proteins. In order to provide “stealth” vectors with targeting properties, the grafting of specific ligands is mandatory. For instance, the tripeptide arginine-glycine-aspartic acid (RGD) has been shown to confer selectivity towards some integrin-overexpressing tumor cells. Unfortunately, owing to the different RGD grafting degrees, some difference in starting materials and experimental conditions, the literature displays contradictory findings about the efficacy of this strategy. Starting from branched poly(ethylene imine) ( b PEI) transfectant and the heterobifunctional linker N -hydroxy-succinimide-poly(ethylene glycol)-maleimide, a series of variably substituted PEI-PEGs were synthesized and functionalized with linear and cyclic RGDs. b PEI-PEG-RGDs were as effective as b PEI in complexing DNA while derivatization did affect the physicochemical properties of polyplexes. A degree of substitution of 1.31% led to enhanced targeting of cognate receptor-expressing cells without impairing the transfection efficiency of b PEI, even in the presence of serum. Of note, both RGDs were equally effective to confer selectivity to polyplexes. These results highlight the key role of the degree of substitution on the effectiveness and selectivity of b PEI-PEG-RGDs, suggesting that a systematic approach is needed for the development of more effective targeted transfectants.

International audience; Small interfering RNAs (siRNAs) are double strand RNA fragments of short sequence ([similar]20 bp). RNA interference came into focus only 13 years ago as a major biological breakthrough and, since then, many studies have described the involvement of siRNA in gene silencing. Application to gene therapy is extremely promising, provided that appropriate vectors are used. Optimising transfection efficacy strongly relies on the knowledge and tuning of physicochemical properties of transfection complexes, such as size, surface charge and internal interactions, which govern in vitro and in vivo stability. Here we report a study on siRNA complexation with micelles of two types of divalent cationic surfactants, i.e. three Gemini bis(quaternary ammonium) bromide with variable spacer length (12-3-12, 12-6-12, 12-12-12) and one weak electrolyte surfactant with a triazine polar head. The process of complex formation was followed by SANS, DLS and zeta potential. Charge density on micelles and counterion exchange were key factors in determining the extent of complexation, as it happens to polymer electrolytes interacting with micelles. A description of complex formation was given in terms of liquid-liquid micro-phase separation, due to internally structured coacervates progressively nucleating from the micelle solution upon siRNA addition. An affinity order between surfactants and siRNA could be established on the basis of the obtained results and their comparison.

Gene therapy is emerging as a revolutionary alternative to conventional therapeutic approaches. However, its clinical application is still hampered by the lack of safe and effective gene delivery techniques. Among the plethora of diverse approaches used to ferry nucleic acids into target cells, non-viral vectors represent promising and safer alternatives to viruses and physical techniques. Both cationic lipids and polymers spontaneously wrap and shrink the genetic material in complexes named lipoplexes and polyplexes, respectively, thereby protecting it and shielding its negative charges. The development of non-viral vectors commenced more than two decades ago. Since then, some major classes of interesting molecules have been identified and modified to optimize their properties. However, the way towards the final goal of gene delivery, i.e. protein expression or gene silencing, is filled with obstacles and current non-viral carriers still have concerns about their overall efficiency. We strongly believe that the future of non-viral gene delivery relies on the development of multifunctional vectors specifically tailored with diverse functionalities that act more like viruses. Although these vectors are still a long way from clinical practice they are the ideal platform to effectively shuttle the genetic material to target cells in a safe and controlled way. In this review, after briefly introducing the basis of gene delivery and therapeutic applications we discuss the main polymeric and lipidic vectors utilized for gene delivery, focusing on the strategies adopted to overcome the major weaknesses inherent to their still limited activity, on the way towards ideal multifunctional vectors.

Vascular tissue engineering combines cells with scaffold materials in vitro aiming the development of physiologically relevant vascular models. For natural scaffolds such as collagen gels, where cells can be mixed with the material solution before gelation, cell seeding density is a key parameter that can affect extracellular matrix deposition and remodeling. Nonetheless, this parameter is often overlooked and densities sensitively lower than those of native tissues, are usually employed. Herein, the effect of seeding density on the maturation of tubular collagen gel-based scaffolds cellularized with smooth muscle cells is investigated. The compaction, the expression, and deposition of key vascular proteins and the resulting mechanical properties of the constructs are evaluated up to 1 week of maturation. Results show that increasing cell seeding density accelerates cell-mediated gel compaction, enhances elastin expression (more than sevenfold increase at the highest density, Day 7) and finally improves the overall mechanical properties of constructs. Of note, the tensile equilibrium elastic modulus, evaluated by stress-relaxation tests, reach values comparable to native arteries for the highest cell density, after a 7-day maturation. Altogether, these results show that higher cell seeding densities promote the rapid maturation of collagen gel-based vascular constructs toward structural and mechanical properties better mimicking native arteries.

A number of smart nonviral gene delivery vectors relying on bioresponsiveness have been introduced in the past few years to overcome the limits of the first generation of gene carriers. Among them, redox-sensitive lipidic and polymeric vectors exploit the presence of disulfide bonds in their structure to take advantage of the highly reductive intracellular milieu and to promote complex unpacking and nucleic acids release after cellular uptake (disulfide linker strategy). Glutathione (GSH) has been often identified as the leading actor in the intracellular reduction of bioreducible vectors but their actual mechanisms of action have been rarely investigated in depth and doubts about the real effectiveness of the disulfide linker strategy have been raised. Herein, we outline a simple protocol for the preparation and investigation of nano-sized reducible cationic liposomes, focusing on their thorough characterization and optimization as gene delivery vectors. In addition, we carefully describe the techniques and procedures necessary for the assessment of the bioreducibility of the vectors and to demonstrate that the GSH-mediated intracellular cleavage of disulfide bonds is a pivotal step in their transfection process. Liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), and of the reducible cationic lipid SS14 are reported as a practical example but the proposed protocol can be easily shifted to other formulations of reducible lipids/liposomes and to reducible polymers.

In this work laser micro polishing (LMP) of cold rolled 0.3 mm thick 304 stainless steel with a pulsed fibre laser is studied, for applications where antibacterial properties are required. Due to its production method, the initial surface roughness of the tested material was considerably low (Sa=85.3±2.8 μm), rendering a demanding case for the laser polishing process. Accordingly, process feasibility under three different atmospheric conditions, namely ambient, Ar and N 2 atmosphere, was investigated. A large set of process parameter combinations was tested and initial analysis was carried out to identify the polishing feasibility by inspection under an optical microscope. Once the feasibility window was determined, a primary characterization was made on selected surfaces for roughness and waviness. Results show that in some process conditions belonging to the explored feasibility range, surface roughness could be decreased by 50%.