Advancing nucleic acid delivery through cationic polymer design: non-cationic building blocks from the toolbox

Polymers used for the delivery of nucleic acids (NAs) typically possess ionizable, cationic moieties enabling their electrostatic interactions with negatively charged NAs and form stable polyplexes. However, non-cationic building blocks have been harnessed to design cationic polymers with enhanced delivery of DNA/RNA to tissues, cells, and subcellular compartments while remaining stable in biological fluids. By customizing the chemistry of these functional groups, we can improve cell targeting behavior, uptake, endosomal escape, non-toxicity, and transfection efficiency. Additionally, the physicochemical properties, such as the loading capacity, complexation ability, size and morphology, biodegradability, pH sensitivity, and amphiphilicity, can be adjusted based on the specific application. This review summarizes the role of non-cationic moieties in various biomedical contexts, from therapeutic interventions to gene editing. By unpacking and critically summarizing the existing literature, this review provides valuable insights into the rational integration of these building blocks for designing more effective nanovectors to deliver NAs.The rational integration of non-cationic building blocks into cationic polymers can be devised to enhance the performance of the resulting gene delivery vectors, improving cell targeting behavior, uptake, endosomal escape, toxicity, and transfection efficiency

Mucoadhesive chitosan-methylcellulose oral patches for the treatment of local mouth bacterial infections

: Mucoadhesive buccal patches are dosage forms promising for successful drug delivery. They show the distinctive advantages of long residence time on the oral mucosa and increased in situ drug bioavailability. In this context, electrophoretic deposition (EPD) of chitosan (CS) has been demonstrated as a simple and easily tunable technique to produce mucoadhesive buccal patches. However, CS-based buccal patches may suffer from weak mucoadhesion, which can impair their therapeutic effect. In this work, methylcellulose (MC), a widely investigated biopolymer in the biomedical area, was exploited to increase the mucoadhesive characteristic of pristine CS patches. CS-MC patches were obtained in a one-pot process via EPD, and the possibility of incorporating gentamicin sulfate (GS) as a model of a broad-spectrum antibiotic in the so-obtained patches was investigated. The resulting CS-MC patches showed high stability in a water environment and superior mucoadhesive characteristic (σadh = 0.85 ± 0.26 kPa, Wadh = 1192.28 ± 602.36 Pa mm) when compared with the CS control samples (σadh = 0.42 ± 0.22 kPa, Wadh = 343.13 ± 268.89 Pa mm), due to both the control of the patch porosity and the bioadhesive nature of MC. Furthermore, GS-loaded patches showed no in vitro cytotoxic effects by challenging L929 cells with material extracts and noteworthy antibacterial activity on both Gram-positive and Gram-negative bacterial strains

Scaffold-Hopping Strategies in Aurone Optimization: A Comprehensive Review of Synthetic Procedures and Biological Activities of Nitrogen and Sulfur Analogues

: Aurones, particular polyphenolic compounds belonging to the class of minor flavonoids and overlooked for a long time, have gained significative attention in medicinal chemistry in recent years. Indeed, considering their unique and outstanding biological properties, they stand out as an intriguing reservoir of new potential lead compounds in the drug discovery context. Nevertheless, several physicochemical, pharmacokinetic, and pharmacodynamic (P3) issues hinder their progression in more advanced phases of the drug discovery pipeline, making lead optimization campaigns necessary. In this context, scaffold hopping has proven to be a valuable approach in the optimization of natural products. This review provides a comprehensive and updated picture of the scaffold-hopping approaches directed at the optimization of natural and synthetic aurones. In the literature analysis, a particular focus is given to nitrogen and sulfur analogues. For each class presented, general synthetic procedures are summarized, highlighting the key advantages and potential issues. Furthermore, the biological activities of the most representative scaffold-hopped compounds are presented, emphasizing the improvements achieved and the potential for further optimization compared to the aurone class

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-naIve 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-to100-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

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

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 (Ã\u98<1 mm), demineralized and sterilized. Thorough physicalchemical 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

BMP-2 and type I collagen preservation in human deciduous teeth after demineralization

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

In Silico Design of New Dual Inhibitors of SARS-CoV-2 MPRO through Ligand- and Structure-Based Methods

The viral main protease is one of the most attractive targets among all key enzymes involved in the life cycle of SARS-CoV-2. Considering its mechanism of action, both the catalytic and dimerization regions could represent crucial sites for modulating its activity. Dual-binding the SARS-CoV-2 main protease inhibitors could arrest the replication process of the virus by simultaneously preventing dimerization and proteolytic activity. To this aim, in the present work, we identified two series' of small molecules with a significant affinity for SARS-CoV-2 M-PRO, by a hybrid virtual screening protocol, combining ligand- and structure-based approaches with multivariate statistical analysis. The Biotarget Predictor Tool was used to filter a large in-house structural database and select a set of benzo[b]thiophene and benzo[b]furan derivatives. ADME properties were investigated, and induced fit docking studies were performed to confirm the DRUDIT prediction. Principal component analysis and docking protocol at the SARS-CoV-2 M-PRO dimerization site enable the identification of compounds 1b,c,i,l and 2i,l as promising drug molecules, showing favorable dual binding site affinity on SARS-CoV-2 M-PRO