Nanostructures for prevention, diagnosis, and treatment of viral respiratory infections: from influenza virus to SARS-CoV-2 variants.

Viruses are a major cause of mortality and socio-economic downfall despite the plethora of biopharmaceuticals designed for their eradication. Conventional antiviral therapies are often ineffective. Live-attenuated vaccines can pose a safety risk due to the possibility of pathogen reversion, whereas inactivated viral vaccines and subunit vaccines do not generate robust and sustained immune responses. Recent studies have demonstrated the potential of strategies that combine nanotechnology concepts with the diagnosis, prevention, and treatment of viral infectious diseases. The present review provides a comprehensive introduction to the different strains of viruses involved in respiratory diseases and presents an overview of recent advances in the diagnosis and treatment of viral infections based on nanotechnology concepts and applications. Discussions in diagnostic/therapeutic nanotechnology-based approaches will be focused on H1N1 influenza, respiratory syncytial virus, human parainfluenza virus type 3 infections, as well as COVID-19 infections caused by the SARS-CoV-2 virus Delta variant and new emerging Omicron variant.

Co-Delivery of the Human NY-ESO-1 Tumor-Associated Antigen and Alpha-GalactosylCeramide by Filamentous Bacteriophages Strongly Enhances the Expansion of Tumor-Specific CD8+ T Cells.

Tumor-associated antigens (TAAs) represent attractive targets in the development of anti-cancer vaccines. The filamentous bacteriophage is a safe and versatile delivery nanosystem, and recombinant bacteriophages expressing TAA-derived peptides at a high density on the viral coat proteins improve TAA immunogenicity, triggering effective in vivo anti-tumor responses. To enhance the efficacy of the bacteriophage as an anti-tumor vaccine, we designed and generated phage particles expressing a CD8+ peptide derived from the human cancer germline antigen NY-ESO-1 decorated with the immunologically active lipid alpha-GalactosylCeramide (α-GalCer), a potent activator of invariant natural killer T (iNKT) cells. The immune response to phage expressing the human TAA NY-ESO-1 and delivering α-GalCer, namely fdNY-ESO-1/α-GalCer, was analyzed either in vitro or in vivo, using an HLA-A2 transgenic mouse model (HHK). By using NY-ESO-1-specific TCR-engineered T cells and iNKT hybridoma cells, we observed the efficacy of the fdNY-ESO-1/α-GalCer co-delivery strategy at inducing activation of both the cell subsets. Moreover, in vivo administration of fdNY-ESO-1 decorated with α-GalCer lipid in the absence of adjuvants strongly enhances the expansion of NY-ESO-1-specific CD8+ T cells in HHK mice. In conclusion, the filamentous bacteriophage delivering TAA-derived peptides and the α-GalCer lipid may represent a novel and promising anti-tumor vaccination strategy.

Engineering and Development of a Tissue Model for the Evaluation of Microneedle Penetration Ability, Drug Diffusion, Photothermal Activity, and Ultrasound Imaging: A Promising Surrogate to Ex Vivo and In Vivo Tissues.

Driven by regulatory authorities and the ever-growing demands from industry, various artificial tissue models have been developed. Nevertheless, there is no model to date that is capable of mimicking the biomechanical properties of the skin whilst exhibiting the hydrophilicity/hydrophobicity properties of the skin layers. As a proof-of-concept study, tissue surrogates based on gel and silicone are fabricated for the evaluation of microneedle penetration, drug diffusion, photothermal activity, and ultrasound bioimaging. The silicone layer aims to imitate the stratum corneum while the gel layer aims to mimic the water-rich viable epidermis and dermis present in in vivo tissues. The diffusion of drugs across the tissue model is assessed, and the results reveal that the proposed tissue model shows similar behavior to a cancerous kidney. In place of typical in vitro aqueous solutions, this model can also be employed for evaluating the photoactivity of photothermal agents since the tissue model shows a similar heating profile to skin of mice when irradiated with near-infrared laser. In addition, the designed tissue model exhibits promising results for biomedical applications in optical coherence tomography and ultrasound imaging. Such a tissue model paves the way to reduce the use of animals testing in research whilst obviating ethical concerns.

Bioengineered materials with selective antimicrobial toxicity in biomedicine.

Fungi and bacteria afflict humans with innumerous pathogen-related infections and ailments. Most of the commonly employed microbicidal agents target commensal and pathogenic microorganisms without discrimination. To distinguish and fight the pathogenic species out of the microflora, novel antimicrobials have been developed that selectively target specific bacteria and fungi. The cell wall features and antimicrobial mechanisms that these microorganisms involved in are highlighted in the present review. This is followed by reviewing the design of antimicrobials that selectively combat a specific community of microbes including Gram-positive and Gram-negative bacterial strains as well as fungi. Finally, recent advances in the antimicrobial immunomodulation strategy that enables treating microorganism infections with high specificity are reviewed. These basic tenets will enable the avid reader to design novel approaches and compounds for antibacterial and antifungal applications.

PLGA microparticle formulations for tunable delivery of a nano-engineered filamentous bacteriophage-based vaccine: in vitro and in silico-supported approach.

Bacteriophages have attracted great attention in the bioengineering field in diverse research areas from tissue engineering to therapeutic and clinical applications. Recombinant filamentous bacteriophage, carrying multiple copies of foreign peptides on protein capsid has been successfully used in the vaccine delivery setting, even if their plasma instability and degradation have limited their use on the pharmaceutical market. Encapsulation techniques in polymeric materials can be applied to preserve bacteriophage activity, extend its half-life, and finely regulate their release in the target environment. The main goal of this study was to provide tunable formulations of the bacteriophage encapsulated in polymeric microparticles (MPs). We used poly (lactic-co-glycolic-acid) as a biocompatible and biodegradable polymer with ammonium bicarbonate as a porogen to encapsulate bacteriophage expressing OVA (257-264) antigenic peptide. We demonstrate that nano-engineered fdOVA bacteriophages encapsulated in MPs preserve their structure and are immunologically active, inducing a strong immune response towards the delivered peptide. Moreover, MP encapsulation prolongs bacteriophage stability over time also at room temperature. Additionally, in this study, we show the ability of in silico-supported approach to predict and tune the release of bacteriophages. These results lay the framework for a versatile bacteriophage-based vaccine delivery system that could successfully generate robust immune responses in a sustained manner, to be used as a platform against cancer and new emerging diseases. Graphical abstract: Synopsis: administration of recombinant bacteriophage-loaded PLGA microparticles for antigen delivery. PLGA microparticles release the bacteriophages, inducing activation of dendritic cells and enhancing antigen presentation and specific T cell response. Bacteriophage-encapsulated microneedles potentially can be administered into human body and generate robust immune responses.

Long-Term Vaccine Delivery and Immunological Responses Using Biodegradable Polymer-Based Carriers.

Biodegradable polymers are largely employed in the biomedical field, ranging from tissue regeneration to drug/vaccine delivery. The biodegradable polymers are highly biocompatible and possess negligible toxicity. In addition, biomaterial-based vaccines possess adjuvant properties, thereby enhancing immune responses. This Review introduces the use of different biodegradable polymers and their degradation mechanism. Different kinds of vaccines, as well as the interaction between the carriers with the immune system, then are highlighted. Natural and synthetic biodegradable micro-/nanoplatforms, hydrogels, and scaffolds for local or targeted and controlled vaccine release are subsequently discussed.

Cell loaded hydrogel containing Ag-doped bioactive glass-ceramic nanoparticles as skin substitute: Antibacterial properties, immune response, and scarless cutaneous wound regeneration.

An ideal tissue-engineered dermal substitute should possess angiogenesis potential to promote wound healing, antibacterial activity to relieve the bacterial burden on skin, as well as sufficient porosity for air and moisture exchange. In light of this, a glass-ceramic (GC) has been incorporated into chitosan and gelatin electrospun nanofibers (240-360 nm), which MEFs were loaded on it for healing acceleration. The GC was doped with silver to improve the antibacterial activity. The bioactive nanofibrous scaffolds demonstrated antibacterial and superior antibiofilm activities against Gram-negative and Gram-positive bacteria. The nanofibrous scaffolds were biocompatible, hemocompatible, and promoted cell attachment and proliferation. Nanofibrous skin substitutes with or without Ag-doped GC nanoparticles did not induce an inflammatory response and attenuated LPS-induced interleukin-6 release by dendritic cells. The rate of biodegradation of the nanocomposite was similar to the rate of skin regeneration under in vivo conditions. Histopathological evaluation of full-thickness excisional wounds in BALB/c mice treated with mouse embryonic fibroblasts-loaded nanofibrous scaffolds showed enhanced angiogenesis, and collagen synthesis as well as regeneration of the sebaceous glands and hair follicles in vivo.

Mesoporous Bioactive Glasses in Cancer Diagnosis and Therapy: Stimuli-Responsive, Toxicity, Immunogenicity, and Clinical Translation.

Cancer is one of the top life-threatening dangers to the human survival, accounting for over 10 million deaths per year. Bioactive glasses have developed dramatically since their discovery 50 years ago, with applications that include therapeutics as well as diagnostics. A new system within the bioactive glass family, mesoporous bioactive glasses (MBGs), has evolved into a multifunctional platform, thanks to MBGs easy-to-functionalize nature and tailorable textural properties-surface area, pore size, and pore volume. Although MBGs have yet to meet their potential in tumor treatment and imaging in practice, recently research has shed light on the distinguished MBGs capabilities as promising theranostic systems for cancer imaging and therapy. This review presents research progress in the field of MBG applications in cancer diagnosis and therapy, including synthesis of MBGs, mechanistic overview of MBGs application in tumor diagnosis and drug monitoring, applications of MBGs in cancer therapy ( particularly, targeted delivery and stimuli-responsive nanoplatforms), and immunological profile of MBG-based nanodevices in reference to the development of novel cancer therapeutics.

Exploiting viral sensing mediated by Toll-like receptors to design innovative vaccines.

Toll-like receptors (TLRs) are transmembrane proteins belonging to the family of pattern-recognition receptors. They function as sensors of invading pathogens through recognition of pathogen-associated molecular patterns. After their engagement by microbial ligands, TLRs trigger downstream signaling pathways that culminate into transcriptional upregulation of genes involved in immune defense. Here we provide an updated overview on members of the TLR family and we focus on their role in antiviral response. Understanding of innate sensing and signaling of viruses triggered by these receptors would provide useful knowledge to prompt the development of vaccines able to elicit effective and long-lasting immune responses. We describe the mechanisms developed by viral pathogens to escape from immune surveillance mediated by TLRs and finally discuss how TLR/virus interplay might be exploited to guide the design of innovative vaccine platforms.

Endocytosis of abiotic nanomaterials and nanobiovectors: Inhibition of membrane trafficking.

Humans are exposed to nanoscopical nanobiovectors (e.g. coronavirus SARS-CoV-2) as well as abiotic metal/carbon-based nanomaterials that enter cells serendipitously or intentionally. Understanding the interactions of cell membranes with these abiotic and biotic nanostructures will facilitate scientists to design better functional nanomaterials for biomedical applications. Such knowledge will also provide important clues for the control of viral infections and the treatment of virus-induced infectious diseases. In the present review, the mechanisms of endocytosis are reviewed in the context of how nanomaterials are uptaken into cells. This is followed by a detailed discussion of the attributes of man-made nanomaterials (e.g. size, shape, surface functional groups and elasticity) that affect endocytosis, as well as the different human cell types that participate in the endocytosis of nanomaterials. Readers are then introduced to the concept of viruses as nature-derived nanoparticles. The mechanisms in which different classes of viruses interact with various cell types to gain entry into the human body are reviewed with examples published over the last five years. These basic tenets will enable the avid reader to design advanced drug delivery and gene transfer nanoplatforms that harness the knowledge acquired from endocytosis to improve their biomedical efficacy. The review winds up with a discussion on the hurdles to be addressed in mimicking the natural mechanisms of endocytosis in nanomaterials design.

Self-assembled peptide and protein nanostructures for anti-cancer therapy: Targeted delivery, stimuli-responsive devices and immunotherapy.

Self-assembled peptides and proteins possess tremendous potential as targeted drug delivery systems and key applications of these well-defined nanostructures reside in anti-cancer therapy. Peptides and proteins can self-assemble into nanostructures of diverse sizes and shapes in response to changing environmental conditions such as pH, temperature, ionic strength, as well as host and guest molecular interactions; their countless benefits include good biocompatibility and high loading capacity for hydrophobic and hydrophilic drugs. These self-assembled nanomaterials can be adorned with functional moieties to specifically target tumor cells. Stimuli-responsive features can also be incorporated with respect to the tumor microenvironment. This review sheds light on the growing interest in self-assembled peptides and proteins and their burgeoning applications in cancer treatment and immunotherapy.

Nanobased Platforms for Diagnosis and Treatment of COVID-19: From Benchtop to Bedside.

Human respiratory viral infections are the leading cause of morbidity and mortality around the world. Among the various respiratory viruses, coronaviruses (e.g., SARS-CoV-2) have created the greatest challenge and most frightening health threat worldwide. Human coronaviruses typically infect the upper respiratory tract, causing illnesses that range from common cold-like symptoms to severe acute respiratory infections. Several promising vaccine formulations have become available since the beginning of 2021. Nevertheless, achievement of herd immunity is still far from being realized. Social distancing remains the only effective measure against SARS-CoV-2 infection. Nanobiotechnology enables the design of nanobiosensors. These nanomedical diagnostic devices have opened new vistas for early detection of viral infections. The present review outlines recent research on the effectiveness of nanoplatforms as diagnostic and antiviral tools against coronaviruses. The biological properties of coronavirus and infected host organs are discussed. The challenges and limitations encountered in combating SARS-CoV-2 are highlighted. Potential nanodevices such as nanosensors, nanobased vaccines, and smart nanomedicines are subsequently presented for combating current and future mutated versions of coronaviruses.

Nonspherical Metal-Based Nanoarchitectures: Synthesis and Impact of Size, Shape, and Composition on Their Biological Activity.

Metal-based nanoentities, apart from being indispensable research tools, have found extensive use in the industrial and biomedical arena. Because their biological impacts are governed by factors such as size, shape, and composition, such issues must be taken into account when these materials are incorporated into multi-component ensembles for clinical applications. The size and shape (rods, wires, sheets, tubes, and cages) of metallic nanostructures influence cell viability by virtue of their varied geometry and physicochemical interactions with mammalian cell membranes. The anisotropic properties of nonspherical metal-based nanoarchitectures render them exciting candidates for biomedical applications. Here, the size-, shape-, and composition-dependent properties of nonspherical metal-based nanoarchitectures are reviewed in the context of their potential applications in cancer diagnostics and therapeutics, as well as, in regenerative medicine. Strategies for the synthesis of nonspherical metal-based nanoarchitectures and their cytotoxicity and immunological profiles are also comprehensively appraised.

Viral Emerging Diseases: Challenges in Developing Vaccination Strategies.

In the last decades, a number of infectious viruses have emerged from wildlife or re-emerged, generating serious threats to the global health and to the economy worldwide. Ebola and Marburg hemorrhagic fevers, Lassa fever, Dengue fever, Yellow fever, West Nile fever, Zika, and Chikungunya vector-borne diseases, Swine flu, Severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and the recent Coronavirus disease 2019 (COVID-19) are examples of zoonoses that have spread throughout the globe with such a significant impact on public health that the scientific community has been called for a rapid intervention in preventing and treating emerging infections. Vaccination is probably the most effective tool in helping the immune system to activate protective responses against pathogens, reducing morbidity and mortality, as proven by historical records. Under health emergency conditions, new and alternative approaches in vaccine design and development are imperative for a rapid and massive vaccination coverage, to manage a disease outbreak and curtail the epidemic spread. This review gives an update on the current vaccination strategies for some of the emerging/re-emerging viruses, and discusses challenges and hurdles to overcome for developing efficacious vaccines against future pathogens.

Recombinant Filamentous Bacteriophages Encapsulated in Biodegradable Polymeric Microparticles for Stimulation of Innate and Adaptive Immune Responses.

Escherichia coli filamentous bacteriophages (M13, f1, or fd) have attracted tremendous attention from vaccinologists as a promising immunogenic carrier and vaccine delivery vehicle with vast possible applications in the development of vaccines. The use of fd bacteriophage as an antigen delivery system is based on a modification of bacteriophage display technology. In particular, it is designed to express multiple copies of exogenous peptides (or polypeptides) covalently linked to viral capsid proteins. This study for the first time proposes the use of microparticles (MPs) made of poly (lactic-co-glycolic acid)(PLGA) to encapsulate fd bacteriophage. Bacteriophage-PLGA MPs were synthesized by a water in oil in water (w1/o/w2) emulsion technique, and their morphological properties were analyzed by confocal and scanning electron microscopy (SEM). Moreover, phage integrity, encapsulation efficiency, and release were investigated. Using recombinant bacteriophages expressing the ovalbumin (OVA) antigenic determinant, we demonstrated the immunogenicity of the encapsulated bacteriophage after being released by MPs. Our results reveal that encapsulated bacteriophages are stable and retain their immunogenic properties. Bacteriophage-encapsulated PLGA microparticles may thus represent an important tool for the development of different bacteriophage-based vaccine platforms.

Arming Filamentous Bacteriophage, a Nature-Made Nanoparticle, for New Vaccine and Immunotherapeutic Strategies.

The pharmaceutical use of bacteriophages as safe and inexpensive therapeutic tools is collecting renewed interest. The use of lytic phages to fight antibiotic-resistant bacterial strains is pursued in academic and industrial projects and is the object of several clinical trials. On the other hand, filamentous bacteriophages used for the phage display technology can also have diagnostic and therapeutic applications. Filamentous bacteriophages are nature-made nanoparticles useful for their size, the capability to enter blood vessels, and the capacity of high-density antigen expression. In the last decades, our laboratory focused its efforts in the study of antigen delivery strategies based on the filamentous bacteriophage 'fd', able to trigger all arms of the immune response, with particular emphasis on the ability of the MHC class I restricted antigenic determinants displayed on phages to induce strong and protective cytotoxic responses. We showed that fd bacteriophages, engineered to target mouse dendritic cells (DCs), activate innate and adaptive responses without the need of exogenous adjuvants, and more recently, we described the display of immunologically active lipids. In this review, we will provide an overview of the reported applications of the bacteriophage carriers and describe the advantages of exploiting this technology for delivery strategies.

An Ig Transmembrane Domain Motif Improves the Function of TCRs Transduced in Human T Cells: Implications for Immunotherapy.

Adoptive transfer of T lymphocytes (ACT) engineered with T-cell receptors (TCRs) of known antitumor specificity is an effective therapeutic strategy. However, a major constraint of ACT is the unpredictable interference of the endogenous TCR α and ß chains in pairing of the transduced TCR. This effect reduces the efficacy of the genetically modified primary T cells and carries the risk of generating novel TCR reactivities with unintended functional consequences. Here, we show a powerful approach to overcome these limitations. We engineered TCR α and ß chains with mutations encompassing a conserved motif (FXXXFXXS) required to stabilize the pairing of immunoglobulin heavy chain transmembrane domains. Molecular modeling supported the preferential pairing of mutated TCR and impaired pairing between mutated and wild-type TCRs. Expression of the mutated TCR was similar to wild type and conferred the expected specificity. Fluorescence resonance energy transfer analysis in mouse splenocytes transduced with mutated or wild-type TCRs showed a higher proximity of the former over the latter. Importantly, we show that mutated TCRs effectively outcompete endogenous TCRs and improve in vitro antitumor cytotoxicity when expressed in ex vivo isolated human T cells. This approach should contribute to improving current protocols of anticancer immunetherapy protocols.

Immunostimulatory Phosphatidylmonogalactosyldiacylglycerols (PGDG) from the Marine Diatom Thalassiosira weissflogii: Inspiration for a Novel Synthetic Toll-Like Receptor 4 Agonist.

An unprecedented phosphatidylmonogalactosyldiacylglycerol pool (PGDG, 1) rich in polyunsaturated fatty acids was isolated from the marine diatoms Thalassiosira weissflogii. Here we report for the first time the NMR characterization of this rare lipid from marine organisms along with a synthetic strategy for the preparation of a PGDG analog (2). PGDG 1 exhibited immunostimulatory activity in human dendritic cells (DCs) and the synthetic PGDG 2 was prepared to explore its mechanism of action. A Toll-like receptor-4 (TLR-4) agonistic activity was evidenced in human and murine DCs underlying the antigen-specific T-cell activation of this class of molecules.

Vectorized Delivery of Alpha-GalactosylCeramide and Tumor Antigen on Filamentous Bacteriophage fd Induces Protective Immunity by Enhancing Tumor-Specific T Cell Response.

We have exploited the properties of filamentous bacteriophage fd to deliver immunologically active lipids together with antigenic peptides. Filamentous bacteriophages resemble for size, capability to be permeable to blood vessels, and high density antigen expression, a nature-made nanoparticle. In addition, their major coat protein pVIII, which is arranged to form a tubular shield surrounding the phage genome, has a high content of hydrophobic residues promoting lipid association. We conjugated bacteriophages to alpha-GalactosylCeramide (α-GalCer), a lipid antigen-stimulating invariant natural killer T (iNKT) cells and capable of inducing their anti-tumoral activities. We found that bacteriophage fd/α-GalCer conjugates could repeatedly stimulate iNKT cells in vitro and in vivo, without inducing iNKT anergy. Moreover, co-delivery of α-GalCer and a MHC class I restricted tumor-associated antigenic determinant to antigen-presenting cells via bacteriophages strongly boosted adaptive CD8+ T cell response and efficiently delayed tumor progression. Co-delivery of a tumor antigen and iNKT-stimulatory lipid on the surface of filamentous bacteriophages is a novel approach to potentiate adaptive anti-cancer immune responses, overcoming the current limitations in the use of free α-GalCer and may represent an attractive alternative to existing delivery methods, opening the path to a potential translational usage of this safe, inexpensive, and versatile tool.