Delivery of gene editing therapeutics.

For the past decades, gene editing demonstrated the potential to attenuate each of the root causes of genetic, infectious, immune, cancerous, and degenerative disorders. More recently, Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9 (CRISPR-Cas9) editing proved effective for editing genomic, cancerous, or microbial DNA to limit disease onset or spread. However, the strategies to deliver CRISPR-Cas9 cargos and elicit protective immune responses requires safe delivery to disease targeted cells and tissues. While viral vector-based systems and viral particles demonstrate high efficiency and stable transgene expression, each are limited in their packaging capacities and secondary untoward immune responses. In contrast, the nonviral vector lipid nanoparticles were successfully used for as vaccine and therapeutic deliverables. Herein, we highlight each available gene delivery systems for treating and preventing a broad range of infectious, inflammatory, genetic, and degenerative diseases. STATEMENT OF SIGNIFICANCE: CRISPR-Cas9 gene editing for disease treatment and prevention is an emerging field that can change the outcome of many chronic debilitating disorders.

Fabrication of Cu2+-loaded phase-transited lysozyme nanofilm on bacterial cellulose: Antibacterial, anti-inflammatory, and pro-angiogenesis for bacteria-infected wound healing.

Bacterial overgrowth in injured wounds causes wound infection and excessive inflammation, leading to delayed wound healing. Successful treatment of delayed infected wound healing demands dressings, which can inhibit bacterial growth and inflammation and simultaneously induce vascularization, collagen deposition, and re-epithelialization of wounds. In this study, bacterial cellulose (BC) deposited with Cu2+-loaded phase-transited lysozyme (PTL) nanofilm (BC/PTL/Cu) was prepared for healing infected wounds. The results confirm that PTL were successfully self-assembled on BC matrix, and Cu2+ were loaded into PTL through electrostatic coordination. The tensile strength and the elongation at break of the membranes were not significantly changed after modification with PTL and Cu2+. Compared with BC, the surface roughness of BC/PTL/Cu significantly increased while the hydrophilicity decreased. Moreover, BC/PTL/Cu displayed slower release rate of Cu2+ compared with BC directly loaded with Cu2+. BC/PTL/Cu exhibited good antibacterial activity against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa. By controlling copper concentration, BC/PTL/Cu were not cytotoxic to mouse fibroblast cell line L929. In vivo, BC/PTL/Cu accelerated wound healing and promoted re-epithelialization, collagen deposition, and angiogenesis while inhibiting inflammation of the infected full-thickness skin wounds of rats. Collectively, these results demonstrate that BC/PTL/Cu composites are promising dressings for healing infected wounds.

Stable Levels of Antibodies Against Unrelated Toxoid Vaccines After COVID-19: COVID-19 Infection Does Not Affect Toxoid Vaccine Antibody Levels.

Background: Lymphopenia is common in COVID-19. This has raised concerns that COVID-19 could affect the immune system akin to measles infection, which causes immune amnesia and a reduction in protective antibodies. Methods: We recruited COVID-19 patients (n = 59) in Helsinki, Finland, and collected plasma samples on 2 to 3 occasions during and after infection. We measured IgG antibodies to diphtheria toxin, tetanus toxoid, and pertussis toxin, along with total IgG, SARS-CoV-2 spike protein IgG, and neutralizing antibodies. We also surveyed the participants for up to 17 months for long-term impaired olfaction as a proxy for prolonged post-acute COVID-19 symptoms. Results: No significant differences were found in the unrelated vaccine responses while the serological response against COVID-19 was appropriate. During the acute phase of the disease, the SARSCoV-2 IgG levels were lower in outpatients when compared to inpatients. SARS-CoV-2 serology kinetics matched expectations. In the acute phase, anti-tetanus and anti-diphtheria IgG levels were lower in patients with prolonged impaired olfaction during follow up than in those without. Conclusions: We could not detect significant decline in overall humoral immunity during or after COVID-19 infection. In severe COVID-19, there appears to be a temporary decline in total IgG levels.

Bacterial cellulose/soybean protein isolate composites with promoted inflammation inhibition, angiogenesis and hair follicle regeneration for wound healing.

Soybean protein, as a safe and low-cost alternative to animal protein, attracts increasing attention in wound healing. In the present study, beta-conglycinin (7S) and glycinin (11S) with high solubility were obtained through separation of soybean protein. Afterward, 7S or 11S modified bacterial cellulose (BC) composites were produced by self-assembly method. Results confirmed the successful self-assembly of soybean protein isolates on the nanofibers of BC. The surface roughness and hydrophilicity of BC/7S and BC/11S decreased compared with native BC. Soybean protein could be steadily released from BC/7S and BC/11S and BC/11S released more soybean proteins than BC/7S. In vitro, BC/7S and BC/11S supported fibroblasts attachment and promoted fibroblasts proliferation and type I collagen expression. In vivo, BC/7S and BC/11S facilitated wound healing and collagen deposition, enhanced angiogenesis and hair follicle regeneration, as well as reduced scar formation and inflammation in full-thickness skin wounds of rats. Moreover, wounds treated with BC/11S showed a faster wound healing rate and more collagen depositions than those of BC/7S, which may be attributed to the larger considerable amount of soybean protein released by BC/11S. These results indicate that BC/7S and BC/11S are potential candidates for wound dressings.

Inhibition of SARS-CoV-2 Alpha Variant and Murine Noroviruses on Copper-Silver Nanocomposite Surfaces.

With the continued scenario of the COVID-19 pandemic, the world is still seeking out-of-the-box solutions to break its transmission cycle and contain the pandemic. There are different transmission routes for viruses, including indirect transmission via surfaces. To this end, we used two relevant viruses in our study. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the pandemic and human norovirus (HuNV), both known to be transmitted via surfaces. Several nanoformulations have shown attempts to inhibit SARS-CoV-2 and other viruses. However, a rigorous, similar inactivation scheme to inactivate the cords of two tedious viruses (SARS-CoV-2 Alpha variant and HuNV) is lacking. The present study demonstrates the inactivation of the SARS-CoV-2 Alpha variant and the decrease in the murine norovirus (MNV, a surrogate to HuNV) load after only one minute of contact to surfaces including copper-silver (Cu-Ag) nanocomposites. We thoroughly examined the physicochemical characteristics of such plated surfaces using diverse microscopy tools and found that Cu was the dominanting element in the tested three different surfaces (~56, ~59, and ~48 wt%, respectively), hence likely playing the major role of Alpha and MNV inactivation followed by the Ag content (~28, ~13, and ~11 wt%, respectively). These findings suggest that the administration of such surfaces within highly congested places (e.g., schools, public transportations, public toilets, and hospital and live-stock reservoirs) could break the SARS-CoV-2 and HuNV transmission. We suggest such an administration after an in-depth examination of the in vitro (especially on skin cells) and in vivo toxicity of the nanocomposite formulations and surfaces while also standardizing the physicochemical parameters, testing protocols, and animal models.

Evaluation of the anti-biofilm activities of bacterial cellulose-tannic acid-magnesium chloride composites using an in vitro multispecies biofilm model.

Chronic wounds are a serious worldwide problem, which are often accompanied by wound infections. In this study, bacterial cellulose (BC)-based composites introduced with tannic acid (TA) and magnesium chloride (BC-TA-Mg) were fabricated for anti-biofilm activities. The prepared composites' surface properties, mechanical capacity, thermal stability, water absorption and retention property, releasing behavior, anti-biofilm activities and potential cytotoxicity were tested. Results showed that TA and MgCl2 particles closely adhered to the nanofibers of BC membranes, thus increasing surface roughness and hydrophobicity of the membranes. While the introduction of TA and MgCl2 did not influence the transparency of the membranes, making it beneficial for wound inspection. BC-TA and BC-TA-Mg composites displayed increased tensile strength and elongation at break compared to pure BC. Moreover, BC-TA-Mg exhibited higher water absorption and retention capacity than BC and BC-TA, suitable for the absorption of wound exudates. BC-TA-Mg demonstrated controlled release of TA and good inhibitory effect on both singly cultured Staphylococcus aureus and Pseudomonas aeruginosa biofilm and co-cultured biofilm of S. aureus and P. aeruginosa. Furthermore, the cytotoxicity grade of BC-TA-6Mg membrane was eligible based on standard toxicity classifications. These indicated that BC-TA-Mg is potential to be used as wound dressings combating biofilms in chronic wounds.

Copper-Silver Nanohybrids: SARS-CoV-2 Inhibitory Surfaces.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a severe health threat. The COVID-19 infections occurring in humans and animals render human-animal interfaces hot spots for spreading the pandemic. Lessons from the past point towards the antiviral properties of copper formulations; however, data showing the "contact-time limit" surface inhibitory efficacy of copper formulations to contain SARS-CoV-2 are limited. Here, we show the rapid inhibition of SARS-CoV-2 after only 1 and 5 min on two different surfaces containing copper-silver (Cu-Ag) nanohybrids. We characterized the nanohybrids' powder and surfaces using a series of sophisticated microscopy tools, including transmission and scanning electron microscopes (TEM and SEM) and energy-dispersive X-ray spectroscopy (EDX). We used culturing methods to demonstrate that Cu-Ag nanohybrids with high amounts of Cu (~65 and 78 wt%) and lower amounts of Ag (~7 and 9 wt%) inhibited SARS-CoV-2 efficiently. Collectively, the present work reveals the rapid SARS-CoV-2 surface inhibition and the promising application of such surfaces to break the SARS-CoV-2 transmission chain. For example, such applications could be invaluable within a hospital or live-stock settings, or any public place with surfaces that people frequently touch (i.e., public transportation, shopping malls, elevators, and door handles) after the precise control of different parameters and toxicity evaluations.

Could Nanotheranostics be the Answer to the Coronavirus Crisis?

The COVID-19 pandemic is expanding worldwide. This pandemic associated with COVID-19 placed the spotlight on how bacterial (e.g., methicillin-resistant Staphylococcus aureus) co-infections may impact responses to coronavirus. In this review the ways in which nanoparticles can contain and rapidly diagnose COVID-19 under the umbrella of nanotheranostics (i.e., smart, single agents combining nanodiagnostics and nanotherapeutics) are elaborated. The present work provides new insights into the promising incorporation of antiviral nanotheranostics into nanostructured materials, including electrospun fibers with tailored pore sizes and hydrophobicity, namely "superhydrophobic self-disinfecting electrospun facemasks/fabrics (SSEF)." SSEFs are proposed as smart alternatives to address the drawbacks of N95 respirators. The challenges of coronavirus containment are underscored, literature is reviewed, and "top-five suggestions" for containing COVID-19 are offered, including: i) preventive appraisals-avoiding needless hospital admission and practicing frequent hand washing (from 20 to 60 s). ii) Diagnostics-highly recommending nanodiagnostics, detecting COVID-19 within 10 min. iii) Therapeutics-expanding nanotherapeutics to treat COVID-19 and bacterial co-infections after safety assessments and clinical trials. iv) Multipronged and multinational, including China, collaborative appraisals. v) Humanitarian compassion to traverse this pandemic in a united way.

COVID-19 Pandemic: What about the Safety of Anti-Coronavirus Nanoparticles?

Every day, new information is presented with respect to how to best combat the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This manuscript sheds light on such recent findings, including new co-factors (i.e., neuropilin-1) and routes (i.e., olfactory transmucosal) allowing cell entry of SARS-CoV-2 and induction of neurological symptoms, as well as the new SARS-CoV-2 variants. We highlight the SARS-CoV-2 human-animal interfaces and elaborate containment strategies using the same vaccination (i.e., nanoparticle "NP" formulations of the BNT162b2 and mRNA-1273 vaccines) for humans, minks, raccoon dogs, cats, and zoo animals. We investigate the toxicity issues of anti-CoV NPs (i.e., plasmonic NPs and quantum dots) on different levels. Namely, nano-bio interfaces (i.e., protein corona), in vitro (i.e., lung cells) and in vivo (i.e., zebrafish embryos) assessments, and impacts on humans are discussed in a narrative supported by original figures. Ultimately, we express our skeptical opinion on the comprehensive administration of such antiviral nanotheranostics, even when integrated into facemasks, because of their reported toxicities and the different NP parameters (e.g., size, shape, surface charge, and purity and chemical composition of NPs) that govern their end toxicity. We believe that more toxicity studies should be performed and be presented, clarifying the odds of the safe administration of nanotoxocological solutions and the relief of a worried public.

Nanotheranostics: A Possible Solution for Drug-Resistant Staphylococcus aureus and their Biofilms?

Staphylococcus aureus is a notorious pathogen that colonizes implants (orthopedic and breast implants) and wounds with a vicious resistance to antibiotic therapy. Methicillin-resistant S. aureus (MRSA) is a catastrophe mainly restricted to hospitals and emerged to community reservoirs, acquiring resistance and forming biofilms. Treating biofilms is problematic except via implant removal or wound debridement. Nanoparticles (NPs) and nanofibers could combat superbugs and biofilms and rapidly diagnose MRSA. Nanotheranostics combine diagnostics and therapeutics into a single agent. This comprehensive review is interpretative, utilizing mainly recent literature (since 2016) besides the older remarkable studies sourced via Google Scholar and PubMed. We unravel the molecular S. aureus resistance and complex biofilm. The diagnostic properties and detailed antibacterial and antibiofilm NP mechanisms are elucidated in exciting stories. We highlight the challenges of bacterial infections nanotheranostics. Finally, we discuss the literature and provide "three action appraisals". (i) The first appraisal consists of preventive actions (two wings), avoiding unnecessary hospital visits, hand hygiene, and legislations against over-the-counter antibiotics as the general preventive wing. Our second recommended preventive wing includes preventing the adverse side effects of the NPs from resistance and toxicity by establishing standard testing procedures. These standard procedures should provide breakpoints of bacteria's susceptibility to NPs and a thorough toxicological examination of every single batch of synthesized NPs. (ii) The second appraisal includes theranostic actions, using nanotheranostics to diagnose and treat MRSA, such as what we call "multifunctional theranostic nanofibers. (iii) The third action appraisal consists of collaborative actions.

Bacterial Cellulose: Functional Modification and Wound Healing Applications.

Significance: Wound dressings are frequently used for wound covering and healing. Ideal wound dressings should provide a moist environment for wounds and actively promote wound healing and skin recovery. The materials used as ideal wound dressings should possess specific properties, thus accelerating skin tissue regeneration process. Recent Advances: Bacterial cellulose (BC) is a natural polymer synthesized by some bacteria. As a kind of natural biopolymer, BC shows good biological activity, biodegradability, and biological adaptability. It has many unique physical, chemical, and biological properties, such as ultrafine nanofiber network, high crystallinity, high water absorption and retention capacity, and high tensile strength and elastic modulus. These excellent properties of BC have laid the foundation for its application as dressing in wound healing. Critical Issues: To optimize the biocompatibility and antimicrobial activity of BC, different methods including microbial fermentation, physical modification, chemical modification, and compound modification have been adopted to modify BC to ensure a better application in wound healing. BC-based wound dressings have been applied in infected wounds, acute traumatic injuries, burns, and diabetic wounds, showing remarkable therapeutic effects on promoting wound healing. Furthermore, there have been some commercial BC-based dressings and they have been utilized in clinical practice. Future Directions: Because of its excellent physicochemical characteristics and biological properties, BC shows high clinical value to be used as a wound dressing for skin tissue regeneration.

Silica-gentamicin nanohybrids: combating antibiotic resistance, bacterial biofilms, and in vivo toxicity.

INTRODUCTION: Antibiotic resistance is a growing concern in health care. Methicillin-resistant Staphylococcus aureus (MRSA), forming biofilms, is a common cause of resistant orthopedic implant infections. Gentamicin is a crucial antibiotic preventing orthopedic infections. Silica-gentamicin (SiO2-G) delivery systems have attracted significant interest in preventing the formation of biofilms. However, compelling scientific evidence addressing their efficacy against planktonic MRSA and MRSA biofilms is still lacking, and their safety has not extensively been studied. MATERIALS AND METHODS: In this work, we have investigated the effects of SiO2-G nanohybrids against planktonic MRSA as well as MRSA and Escherichia coli biofilms and then evaluated their toxicity in zebrafish embryos, which are an excellent model for assessing the toxicity of nanotherapeutics. RESULTS: SiO2-G nanohybrids inhibited the growth and killed planktonic MRSA at a minimum concentration of 500 µg/mL. SiO2-G nanohybrids entirely eradicated E. coli cells in biofilms at a minimum concentration of 250 µg/mL and utterly deformed their ultrastructure through the deterioration of bacterial shapes and wrinkling of their cell walls. Zebrafish embryos exposed to SiO2-G nanohybrids (500 and 1,000 µg/mL) showed a nonsignificant increase in mortality rates, 13.4±9.4 and 15%±7.1%, respectively, mainly detected 24 hours post fertilization (hpf). Frequencies of malformations were significantly different from the control group only 24 hpf at the higher exposure concentration. CONCLUSION: Collectively, this work provides the first comprehensive in vivo assessment of SiO2-G nanohybrids as a biocompatible drug delivery system and describes the efficacy of SiO2-G nanohybrids in combating planktonic MRSA cells and eradicating E. coli biofilms.

Effects of silica-gentamicin nanohybrids on osteogenic differentiation of human osteoblast-like SaOS-2 cells.

INTRODUCTION: In recent years, there has been an increasing interest in silica (SiO2) nanoparticles (NPs) as drug delivery systems. This interest is mainly attributed to the ease of their surface functionalization for drug loading. In orthopedic applications, gentamicin-loaded SiO2 NPs (nanohybrids) are frequently utilized for their prolonged antibacterial effects. Therefore, the possible adverse effects of SiO2-gentamicin nanohybrids on osteogenesis of bone-related cells should be thoroughly investigated to ensure safe applications. MATERIALS AND METHODS: The effects of SiO2-gentamicin nanohybrids on the cell viability and osteogenic differentiation of human osteoblast-like SaOS-2 cells were investigated, together with native SiO2 NPs and free gentamicin. RESULTS: The results of Cell Count Kit-8 (CCK-8) assay show that both SiO2-gentamicin nanohybrids and native SiO2 NPs reduce cell viability of SaOS-2 cells in a dose-dependent manner. Regarding osteogenesis, SiO2-gentamicin nanohybrids and native SiO2 NPs at the concentration range of 31.25-125 µg/mL do not influence the osteogenic differentiation capacity of SaOS-2 cells. At a high concentration (250 µg/mL), both materials induce a lower expression of alkaline phosphatase (ALP) but an enhanced mineralization. Free gentamicin at concentrations of 6.26 and 9.65 µg/mL does not significantly influence the cell viability and osteogenic differentiation capacity of SaOS-2 cells. CONCLUSIONS: The results of this study suggest that both SiO2-gentamicin nanohybrids and SiO2 NPs show cytotoxic effects to SaOS-2 cells. Further investigation on the effects of SiO2-gentamicin nanohybrids on the behaviors of stem cells or other regular osteoblasts should be conducted to make a full evaluation of the safety of SiO2-gentamicin nanohybrids in orthopedic applications.

Nanosilver-Silica Composite: Prolonged Antibacterial Effects and Bacterial Interaction Mechanisms for Wound Dressings.

Infected superficial wounds were traditionally controlled by topical antibiotics until the emergence of antibiotic-resistant bacteria. Silver (Ag) is a kernel for alternative antibacterial agents to fight this resistance quandary. The present study demonstrates a method for immobilizing small-sized (~5 nm) silver nanoparticles on silica matrix to form a nanosilver-silica (Ag-SiO₂) composite and shows the prolonged antibacterial effects of the composite in vitro. The composite exhibited a rapid initial Ag release after 24 h and a slower leaching after 48 and 72 h and was effective against both methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli ( E . coli ). Ultraviolet (UV)-irradiation was superior to filter-sterilization in retaining the antibacterial effects of the composite, through the higher remaining Ag concentration. A gauze, impregnated with the Ag-SiO₂ composite, showed higher antibacterial effects against MRSA and E . coli than a commercial Ag-containing dressing, indicating a potential for the management and infection control of superficial wounds. Transmission and scanning transmission electron microscope analyses of the composite-treated MRSA revealed an interaction of the released silver ions with the bacterial cytoplasmic constituents, causing ultimately the loss of bacterial membranes. The present results indicate that the Ag-SiO₂ composite, with prolonged antibacterial effects, is a promising candidate for wound dressing applications.