A new microfluidic platform for the highly reproducible preparation of non-viral gene delivery complexes.

Transfection describes the delivery of exogenous nucleic acids (NAs) to cells utilizing non-viral means. In the last few decades, scientists have been doing their utmost to design ever more effective transfection reagents. These are eventually mixed with NAs to give rise to gene delivery complexes, which must undergo characterization, testing, and further refinement through the sequential reiteration of these steps. Unfortunately, although microfluidics offers distinct advantages over the canonical approaches to preparing particles, the systems available do not address the most frequent and practical quest for the simultaneous generation of multiple polymer-to-NA ratios (N/Ps). Herein, we developed a user-friendly microfluidic cartridge to repeatably prepare non-viral gene delivery particles and screen across a range of seven N/Ps at once or significant volumes of polyplexes at a given N/P. The microchip is equipped with a chaotic serial dilution generator for the automatic linear dilution of the polymer to the downstream area, which encompasses the NA divider to dispense equal amounts of DNA to the mixing area, enabling the formation of particles at seven N/Ps eventually collected in individual built-in tanks. This is the first example of a stand-alone microfluidic cartridge for the fast and repeatable preparation of non-viral gene delivery complexes at different N/Ps and their storage.

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.

Engineering the early bone metastatic niche through human vascularized immuno bone minitissues.

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 2Din vitromodels and animal studies are not effectively reproducing breast cancer cells (CCs) interactions with the bone microenvironment and suffer from species-specific differences, respectively. Moreover, simplifiedin vitromodels 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.

Electrophoretic processing of chitosan based composite scaffolds with Nb-doped bioactive glass for bone tissue regeneration.

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.

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

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.

Prothrombotic activity of cytokine-activated endothelial cells and shear-activated platelets in the setting of ventricular assist device support.

BACKGROUND: We systematically analyzed the synergistic effect of: (i) cytokine-mediated inflammatory activation of endothelial cells (ECs) with and (ii) shear-mediated platelet activation (SMPA) as a potential contributory mechanism to intraventricular thrombus formation in the setting of left ventricular assist device (LVAD) support. METHODS: Intact and shear-activated human platelets were exposed to non-activated and cytokine-activated ECs. To modulate the level of LVAD-related shear activation, platelets were exposed to shear stress patterns of varying magnitude (30, 50, and 70 dynes/cm2, 10 minutes) via a hemodynamic shearing device. ECs were activated via exposure to inflammatory tumor necrosis factor-α (TNF-α 10 and 100 ng/ml, 24 hours), consistent with inflammatory activation recorded in patients on LVAD circulatory support. RESULTS: Adhesivity of shear-activated platelets to ECs was significantly higher than that of intact/unactivated platelets, regardless of the initial activation level (70 dynes/cm2 shear-activated platelets vs intact platelets: +80%, p < 0.001). Importantly, inflammatory activation of ECs amplified platelet prothrombinase activity progressively with increasing shear stress magnitude and TNF-α concentration: thrombin generation of 70 dynes/cm2 shear-activated platelets was 2.6-fold higher after exposure and adhesion to 100 ng/ml TNF-α‒activated ECs (p < 0.0001). CONCLUSIONS: We demonstrated synergistic effect of SMPA and cytokine-mediated EC inflammatory activation to enhance EC‒platelet adhesion and platelet prothrombotic function. These mechanisms may contribute to intraventricular thrombosis in the setting of mechanical circulatory support.

The polyplex, protein corona, cell interplay: Tips and drawbacks.

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.

Bioreducible Hydrophobin-Stabilized Supraparticles for Selective Intracellular Release.

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.

Demineralized dentin and enamel matrices as suitable substrates for bone regeneration.

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 (Ø<1 mm), demineralized and sterilized. Thorough physical-chemical and biochemical characterizations of native and demineralized materials were performed by SEM and EDS analysis and ELISA kits to determine mineral, collagen type I and BMP-2 contents. In addition, MG63 and SAOS-2 cells were seeded on tooth-derived materials and Bio-Oss®, and a comparison of cell responses in terms of adhesion and proliferation was carried out. RESULTS: 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.

A Stepwise Approach for the Isolation and the Identification of Chemically Reactive Exofacial Protein Thiols.

BACKGROUND: The plasma membrane controls the selective internalization of (macro)molecules through different mechanisms, often relaying on specialized outward-facing carriers such as exofacial proteins thiols (EPTs). Although the interchange of critical thiols and disulphides between EPTs and exogenous cargoes is the first critical event, the identification of specific cell interactors remains to be thoroughly explored. Besides, it is likewise evident that only the relatively little suite of EPTs truly reactive can be considered theranostic targets. OBJECTIVE: We were aimed at developing a stepwise procedure for the isolation and identification of a subset of EPTs, that we named chemically reactive EPS, which are potential theranostic targets. METHOD: In the present study, EPTs that displayed permissive sulfhydryls on the surface of live cells in vitro underwent i) chemo-selective capture by means of thiolated superparamagnetic microbeads (isolation step), followed by ii) their prompt release via disulphide breakage through the addition of DTT reducing agent (elution step) and iii) analysis by means of SDS-PAGE and LCMS/ MS (identification step). RESULTS: In total cell lysates, most of the proteins recovered were intracellular. Conversely, this methodology allowed a 2.6-fold enrichment in chemically reactive EPTs recovered and identified, corresponding up to 37% of the total cellular proteins. The key element of our approach was the reversible chemo-selective capture through disulphide linkages between chemically reactive EPTs and free thiols on microbeads. CONCLUSION: We devised an enabling methodology to selectively pick up, recover and characterize chemically reactive EPTs.

Size matters for in vitro gene delivery: investigating the relationships among complexation protocol, transfection medium, size and sedimentation.

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.

Characterization and Investigation of Redox-Sensitive Liposomes for Gene Delivery.

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.

The study of polyplex formation and stability by time-resolved fluorescence spectroscopy of SYBR Green I-stained DNA.

Polyplexes are nanoparticles formed by the self-assembly of DNA/RNA and cationic polymers specifically designed to deliver exogenous genetic material to cells by a process called transfection. There is a general consensus that a subtle balance between sufficient extracellular protection and intracellular release of nucleic acids is a key factor for successful gene delivery. Therefore, there is a strong need to develop suitable tools and techniques for enabling the monitoring of the stability of polyplexes in the biological environment they face during transfection. In this work we propose time-resolved fluorescence spectroscopy in combination with SYBR Green I-DNA dye as a reliable tool for the in-depth characterization of the DNA/vector complexation state. As a proof of concept, we provide essential information on the assembly and disassembly of complexes formed between DNA and each of three cationic polymers, namely a novel promising chitosan-graft-branched polyethylenimine copolymer (Chi-g-bPEI), one of its building block 2 kDa bPEI and the gold standard transfectant 25 kDa bPEI. Our results highlight the higher information content provided by the time-resolved studies of SYBR Green I/DNA, as compared to conventional steady state measurements of ethidium bromide/DNA that enabled us to draw relationships among fluorescence lifetime, polyplex structural changes and transfection efficiency.

Mapping asbestos-cement roofing with hyperspectral remote sensing over a large mountain region of the Italian Western Alps.

The World Health Organization estimates that 100 thousand people in the world die every year from asbestos-related cancers and more than 300 thousand European citizens are expected to die from asbestos-related mesothelioma by 2030. Both the European and the Italian legislations have banned the manufacture, importation, processing and distribution in commerce of asbestos-containing products and have recommended action plans for the safe removal of asbestos from public and private buildings. This paper describes the quantitative mapping of asbestos-cement covers over a large mountainous region of Italian Western Alps using the Multispectral Infrared and Visible Imaging Spectrometer sensor. A very large data set made up of 61 airborne transect strips covering 3263 km2 were processed to support the identification of buildings with asbestos-cement roofing, promoted by the Valle d'Aosta Autonomous Region with the support of the Regional Environmental Protection Agency. Results showed an overall mapping accuracy of 80%, in terms of asbestos-cement surface detected. The influence of topography on the classification's accuracy suggested that even in high relief landscapes, the spatial resolution of data is the major source of errors and the smaller asbestos-cement covers were not detected or misclassified.

Synthesis of multifunctional PAMAM-aminoglycoside conjugates with enhanced transfection efficiency.

The development of multifunctional vectors for efficient and safe gene delivery is one of the major challenges for scientists working in the gene therapy field. In this context, we have designed a novel type of aminoglycoside-rich dendrimers with a defined structure based on polyamidoamine (PAMAM) in order to develop efficient, nontoxic gene delivery vehicles. Three different conjugates, i.e., PAMAM G4-neamine, -paromomycin, and -neomycin, were synthesized and characterized by nuclear magnetic resonance (NMR) and MALDI analysis. The conjugates were found to self-assemble electrostatically with plasmid DNA, and unlike neamine conjugate, each at its optimum showed increased gene delivery potency compared to PAMAM G4 dendrimer in three different cell lines, along with negligible cytotoxicity. These results all disclosed aminoglycosides as suitable functionalities for tailoring safe and efficient multifunctional gene delivery vectors.

The yin of exofacial protein sulfhydryls and the yang of intracellular glutathione in in vitro transfection with SS14 bioreducible lipoplexes.

Although redox-sensitive transfectants have been considered hitherto as the Holy Grail of gene delivery because of their ability to restrict the release of nucleic acids to intracellular compartments, the reasons for their sometimes lackluster performance do not seem likewise clear. To ascertain the possible influence of extracellular soluble thiols, exofacial protein sulfhydryls (EPTs) and glutathione (GSH) on the overall efficacy of bioreducible lipoplexes, we utilized a cationic gemini surfactant--SS14--in which the two single-chain amphiphiles are held together by a suitable redox-sensitive linkage. We herein draw a big picture whereby the interaction of bioreducible lipoplexes with cells and their internalization are tightly coupled events that ultimately do affect transfection. Specifically, we provide evidence that EPTs entail the reduction-triggered disruption of SS14 lipoplexes in plain Dulbecco's Modified Eagle Medium (DMEM), thereby resulting in a considerable waste (ca. 30%) of nucleic acids and low transgene expression. The DNA release from bioreducible lipoplexes can be blunted to ca. 16% by transfecting cells in complete medium and fully reverted by preincubating them for 1h before delivery in the same culture supernatant (i.e. preconditioning), thus significantly increasing transfection by ca. 3-fold and 10-fold, respectively. These results lead to the proposal of the protein corona as the central mediator in shielding SS14 bioreducible lipoplexes from the action of EPTs in the early phase of delivery and provide a smart solution as to how to increase their efficacy. Besides, we pinpoint associations between intracellular GSH levels and the extent of transfection. All these issues were unique to bioreducible lipoplexes.

A novel antibacterial modification treatment of titanium capable to improve osseointegration.

BACKGROUND: Among the different causes of orthopedic and dental implant failure, infection remains the most serious and devastating complication associated with biomaterial devices. PURPOSE: The aim of this study was to develop an innovative osteointegrative and antibacterial biomimetic coating on titanium and to perform a chemical-physical and in vitro biological characterization of the coating using the SAOS-2 cell line. We also studied the antibacterial properties of the coating against both Gram-positive and Gram-negative bacteria strains. METHODS: An electrochemical solution containing silicon, calcium, phosphorous, sodium, and silver nanoparticles was used to obtain the antibacterial by Anodic Spark Deposition (ASD) treatment. Surface morphology was characterized using SEM and laser profilometry. A qualitative analysis of the chemical composition of the coating was assessed by EDS. The adhesion properties of the coating to the titanium bulk were performed with a 3-point bending test. SAOS-2 osteoblastic cell line spreading and morphology and viability were investigated. The bacterial adhesion and the antibacterial properties were investigated after 3 h and 24 h of incubation with Streptococcus mutans, Streptococcus epidermidis, and Escherichia coli bacterial strains. RESULTS: The proposed anodization treatment created a chemically and morphologically modified, adherent titanium oxide layer, characterized by a microporous morphology enriched by calcium, silicon, phosphorous, and silver. The preliminary biological characterization showed optimal SAOS-2 cell adhesion and proliferation as well as a strong antibacterial effect. CONCLUSIONS: Based on the results of this study, we believe that this novel biomimetic and antibacterial treatment hold promise for enhancing osteointegration while conferring strong antibacterial properties to titanium.

We still have a long way to go to effectively deliver genes!

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.

Chitosan-graft-branched polyethylenimine copolymers: influence of degree of grafting on transfection behavior.

BACKGROUND: Successful non-viral gene delivery currently requires compromises to achieve useful transfection levels while minimizing toxicity. Despite high molecular weight (MW) branched polyethylenimine (bPEI) is considered the gold standard polymeric transfectant, it suffers from high cytotoxicity. Inversely, its low MW counterpart is less toxic and effective in transfection. Moreover, chitosan is a highly biocompatible and biodegradable polymer but characterized by very low transfection efficiency. In this scenario, a straightforward approach widely exploited to develop effective transfectants relies on the synthesis of chitosan-graft-low MW bPEIs (Chi-g-bPEI(x)) but, despite the vast amount of work that has been done in developing promising polymeric assemblies, the possible influence of the degree of grafting on the overall behavior of copolymers for gene delivery has been largely overlooked. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of providing a comprehensive evaluation of the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of copolymeric vectors, we have synthesized seven Chi-g-bPEI(x) derivatives with a variable amount of bPEI grafts (minimum: 0.6%; maximum: 8.8%). Along the Chi-g-bPEI(x) series, the higher the degree of grafting, the greater the ζ-potential and the cytotoxicity of the resulting polyplexes. Most important, in all cell lines tested the intermediate degree of grafting of 2.7% conferred low cytotoxicity and higher transfection efficiency compared to other Chi-g-bPEI(x) copolymers. We emphasize that, in transfection experiments carried out in primary articular chondrocytes, Chi-g-bPEI(2.7%) was as effective as and less cytotoxic than the gold standard 25 kDa bPEI. CONCLUSIONS/SIGNIFICANCE: This work underlines for the first time the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of Chi-g-bPEI(x) copolymers. Crucially, we have demonstrated that, along the copolymer series, the fine tuning of the degree of grafting directly affected the overall charge of polyplexes and, altogether, had a direct effect on cytotoxicity.

Trends in biomedical engineering: focus on Regenerative Medicine.

Regenerative medicine is a critical frontier in biomedical and clinical research. The major progresses in the last few years were driven by a strong clinical need which could benefit from regenerative medicine outcomes for the treatment of a large number of conditions including birth defects, degenerative and neoplastic diseases, and traumatic injuries. Regenerative medicine applies the principles of engineering and life sciences to enhance the comprehension of the fundamental biological mechanisms underlying the structure-function relationships in physiologic and pathologic tissues and to accomplish alternative strategies for developing in vitro biological substitutes which are able to restore, maintain, or improve tissue, and organ function. This paper reviews selected approaches currently being investigated at Politecnico di Milano in the field of regenerative medicine. Specific tissue-oriented topics are divided in three sections according to each developmental stage: in vitro study, pre-clinical study, and clinical application. In vitro studies investigate the basic phenomena related to gene delivery, stem cell behavior, tissue regeneration, and to explore dynamic culture potentiality in different applications: cardiac and skeletal muscle, cartilage, hematopoietic system, peripheral nerve, and gene delivery. Specific fields of regenerative medicine, i.e., bone, blood vessels, and ligaments engineering have already reached the preclinical stage providing promising insights for further research towards clinical applications. The translation of the results obtained during in vitro and preclinical steps into clinical organ replacement is a very challenging issue, which can offer a valid alternative to fight morbidity, organ shortage, and ethical-social problems associated with allotransplantation as shown in the clinical case reported in this review.