Nanoparticle Delivery Platforms for RNAi Therapeutics Targeting COVID-19 Disease in the Respiratory Tract.

Since December 2019, a pandemic of COVID-19 disease, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread across the globe. At present, the Food and Drug Administration (FDA) has issued emergency approval for the use of some antiviral drugs. However, these drugs still have limitations in the specific treatment of COVID-19, and as such, new treatment strategies urgently need to be developed. RNA-interference-based gene therapy provides a tractable target for antiviral treatment. Ensuring cell-specific targeted delivery is important to the success of gene therapy. The use of nanoparticles (NPs) as carriers for the delivery of small interfering RNA (siRNAs) to specific tissues or organs of the human body could play a crucial role in the specific therapy of severe respiratory infections, such as COVID-19. In this review, we describe a variety of novel nanocarriers, such as lipid NPs, star polymer NPs, and glycogen NPs, and summarize the pre-clinical/clinical progress of these nanoparticle platforms in siRNA delivery. We also discuss the application of various NP-capsulated siRNA as therapeutics for SARS-CoV-2 infection, the challenges with targeting these therapeutics to local delivery in the lung, and various inhalation devices used for therapeutic administration. We also discuss currently available animal models that are used for preclinical assessment of RNA-interference-based gene therapy. Advances in this field have the potential for antiviral treatments of COVID-19 disease and could be adapted to treat a range of respiratory diseases.

Airway Exposure to Polyethyleneimine Nanoparticles Induces Type 2 Immunity by a Mechanism Involving Oxidative Stress and ATP Release.

Polyethyleneimine (PEI) induced immune responses were investigated in human bronchial epithelial (hBE) cells and mice. PEI rapidly induced ATP release from hBE cells and pretreatment with glutathione (GSH) blocked the response. PEI activated two conductive pathways, VDAC-1 and pannexin 1, which completely accounted for ATP efflux across the plasma membrane. Moreover, PEI increased intracellular Ca2+ concentration ([Ca2+]i), which was reduced by the pannexin 1 inhibitor, 10Panx (50 µM), the VDAC-1 inhibitor, DIDS (100 µM), and was nearly abolished by pretreatment with GSH (5 mM). The increase in [Ca2+]i involved Ca2+ uptake through two pathways, one blocked by oxidized ATP (oATP, 300 µM) and another that was blocked by the TRPV-1 antagonist A784168 (100 nM). PEI stimulation also increased IL-33 mRNA expression and protein secretion. In vivo experiments showed that acute (4.5 h) PEI exposure stimulated secretion of Th2 cytokines (IL-5 and IL-13) into bronchoalveolar lavage (BAL) fluid. Conjugation of PEI with ovalbumin also induced eosinophil recruitment and secretion of IL-5 and IL-13 into BAL fluid, which was inhibited in IL-33 receptor (ST2) deficient mice. In conclusion, PEI-induced oxidative stress stimulated type 2 immune responses by activating ATP-dependent Ca2+ uptake leading to IL-33 secretion, similar to allergens derived from Alternaria.

Nucleic acid delivery of immune-focused SARS-CoV-2 nanoparticles drives rapid and potent immunogenicity capable of single-dose protection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines may target epitopes that reduce durability or increase the potential for escape from vaccine-induced immunity. Using synthetic vaccinology, we have developed rationally immune-focused SARS-CoV-2 Spike-based vaccines. Glycans can be employed to alter antibody responses to infection and vaccines. Utilizing computational modeling and in vitro screening, we have incorporated glycans into the receptor-binding domain (RBD) and assessed antigenic profiles. We demonstrate that glycan-coated RBD immunogens elicit stronger neutralizing antibodies and have engineered seven multivalent configurations. Advanced DNA delivery of engineered nanoparticle vaccines rapidly elicits potent neutralizing antibodies in guinea pigs, hamsters, and multiple mouse models, including human ACE2 and human antibody repertoire transgenics. RBD nanoparticles induce high levels of cross-neutralizing antibodies against variants of concern with durable titers beyond 6 months. Single, low-dose immunization protects against a lethal SARS-CoV-2 challenge. Single-dose coronavirus vaccines via DNA-launched nanoparticles provide a platform for rapid clinical translation of potent and durable coronavirus vaccines.

Silymarin/curcumin loaded albumin nanoparticles coated by chitosan as muco-inhalable delivery system observing anti-inflammatory and anti COVID-19 characterizations in oleic acid triggered lung injury and in vitro COVID-19 experiment.

Respiratory infected by COVID-19 represents a major global health problem at moment even after recovery from virus corona. Since, the lung lesions for infected patients are still sufferings from acute respiratory distress syndrome including alveolar septal edema, pneumonia, hyperplasia, and hyaline membranes Therefore, there is an urgent need to identify additional candidates having ability to overcome inflammatory process and can enhance efficacy in the treatment of COVID-19. The polypenolic extracts were integrated into moeties of bovine serum albumin (BSA) and then were coated by chitosan as a mucoadhesion polymer. The results of interleukin-6, and c-reactive protein showed significant reduction in group treated by Encap. SIL + CUR (64 ± 0.8 Pg/µL & 6 ± 0.5 µg/µL) compared to group treated by Cham. + CUR (102 ± 0.8 Pg/µL & 7 ± 0.5 µg/µL) respectively and free capsules (with no any drug inside) (148 ± 0.6 Pg/µL & 10 ± 0.6 µg/µL) respectively. Histopathology profile was improved completely. Additionally, encapsulating silymarin showed anti-viral activity in vitro COVID-19 experiment. It can be summarized that muco-inhalable delivery system (MIDS) loaded by silymarin can be used to overcome inflammation induced by oleic acid and to overcome COVID-19.

Polyaminoacid Based Core@shell Nanocarriers of 5-Fluorouracil: Synthesis, Properties and Theranostics Application.

Cancer is one of the most important health problems of our population, and one of the common anticancer treatments is chemotherapy. The disadvantages of chemotherapy are related to the drug's toxic effects, which act on cancer cells and the healthy part of the body. The solution of the problem is drug encapsulation and drug targeting. The present study aimed to develop a novel method of preparing multifunctional 5-Fluorouracil (5-FU) nanocarriers and their in vitro characterization. 5-FU polyaminoacid-based core@shell nanocarriers were formed by encapsulation drug-loaded nanocores with polyaminoacids multilayer shell via layer-by-layer method. The size of prepared nanocarriers ranged between 80-200 nm. Biocompatibility of our nanocarriers as well as activity of the encapsulated drug were confirmed by MTT tests. Moreover, the ability to the real-time observation of developed nanocarriers and drug accumulation inside the target was confirmed by fluorine magnetic resonance imaging (19F-MRI).

Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses.

Adjuvants are critical for improving the quality and magnitude of adaptive immune responses to vaccination. Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have shown great efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanism of action of this vaccine platform is not well-characterized. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, we demonstrated that our LNP formulation has intrinsic adjuvant activity that promotes induction of strong T follicular helper cell, germinal center B cell, long-lived plasma cell, and memory B cell responses that are associated with durable and protective antibodies in mice. Comparative experiments demonstrated that this LNP formulation outperformed a widely used MF59-like adjuvant, AddaVax. The adjuvant activity of the LNP relies on the ionizable lipid component and on IL-6 cytokine induction but not on MyD88- or MAVS-dependent sensing of LNPs. Our study identified LNPs as a versatile adjuvant that enhances the efficacy of traditional and next-generation vaccine platforms.

Protein and peptide delivery to lungs by using advanced targeted drug delivery.

The challenges and difficulties associated with conventional drug delivery systems have led to the emergence of novel, advanced targeted drug delivery systems. Therapeutic drug delivery of proteins and peptides to the lungs is complicated owing to the large size and polar characteristics of the latter. Nevertheless, the pulmonary route has attracted great interest today among formulation scientists, as it has evolved into one of the important targeted drug delivery platforms for the delivery of peptides, and related compounds effectively to the lungs, primarily for the management and treatment of chronic lung diseases. In this review, we have discussed and summarized the current scenario and recent developments in targeted delivery of proteins and peptide-based drugs to the lungs. Moreover, we have also highlighted the advantages of pulmonary drug delivery over conventional drug delivery approaches for peptide-based drugs, in terms of efficacy, retention time and other important pharmacokinetic parameters. The review also highlights the future perspectives and the impact of targeted drug delivery on peptide-based drugs in the coming decade.

Comparison of the safety and immunogenicity of a novel Matrix-M-adjuvanted nanoparticle influenza vaccine with a quadrivalent seasonal influenza vaccine in older adults: a phase 3 randomised controlled trial.

BACKGROUND: Improved seasonal influenza vaccines for older adults that can induce broadly cross-reactive antibodies and enhanced T-cell responses, particularly against A H3N2 viruses, while avoiding egg-adaptive antigenic changes, are needed. We aimed to show that the Matrix-M-adjuvanted quadrivalent nanoparticle influenza vaccine (qNIV) was immunologically non-inferior to a licensed, standard-dose quadrivalent inactivated influenza vaccine (IIV4) in older adults. METHODS: This was a phase 3 randomised, observer-blinded, active-comparator controlled trial done across 19 US community-based clinical research sites during the 2019-20 influenza season. Participants were clinically stable and community-dwelling, aged at least 65 years, and were randomised in a 1:1 ratio using an interactive web response system to receive a single intramuscular dose of qNIV or IIV4. The primary objective was to describe safety and show that qNIV was immunologically non-inferior to IIV4. The primary outcomes were adverse events by treatment group and comparative haemagglutination-inhibiting antibody responses (assayed with egg-propagated virus) on day 28, summarised in terms of the ratio of geometric mean titres (GMTRqNIV/IIV4) and seroconversion rate (SCR) difference between participants receiving qNIV or IIV4 for all four vaccine homologous influenza strains. The immunogenicity outcome was measured in the per-protocol population. Non-inferiority was shown if the lower bound of the two-sided 95% CI on the GMTRqNIV/IIV4 was at least 0·67 and the lower bound of the two-sided 95% CI on the SCR difference -was at least -10%. The study is registered with clinicaltrials.gov, NCT04120194, and is active and not recruiting. FINDINGS: 2742 adults were assessed for eligibility and 2654 were enrolled and randomised between Oct 14, 2019, and Oct 25, 2019; 1333 participants were randomised to the qNIV group and 1319 to the IIV4 group (two participants withdrew consent before being assigned to a group). qNIV showed immunological non-inferiority to IIV4: GMTRqNIV/IIV4 for the four vaccine homologous influenza strains was A/Brisbane 1·09 (95% CI 1·03 to 1·15), A/Kansas 1·19 (1·11 to 1·27), B/Maryland 1·03 (0·99 to 1·07), and B/Phuket 1·23 (1·16 to 1·29); and SCR difference was A/Brisbane 5·0 (95% CI 1·9 to 8·1), A/Kansas 7·3 (3·6 to 11·1), B/Maryland 0·5 (-1·9 to 2·9), and B/Phuket 8·5 (5·0 to 11·9). 659 (49·4%) of 1333 of participants in the qNIV group and 551 (41·8%) of 1319 participants in the IIV4 group had at least one treatment-emergent adverse event. More solicited adverse events were reported by participants in the qNIV group (551 [41·3%] of 1333) than in the IIV4 group (420 [31·8%] of 1319), and were comprised primarily of mild to moderate transient injection site pain (341 [25·6%] in the qNIV group vs 212 [16·1%] in the IIV4 group). INTERPRETATION: qNIV was well tolerated and produced qualitatively and quantitatively enhanced humoral and cellular immune response in older adults compared with IIV4. qNIV might enhance the effectiveness of seasonal influenza vaccination, and future studies to show clinical efficacy are planned. FUNDING: Novavax.

What Do COVID-19 Vaccines Tell Us About Nucleic Acid Delivery In Vivo?

The utilization of the mRNA-based Pfizer-BioNTech and Moderna coronavirus disease 2019 (COVID-19) vaccines represents the culmination of many years of nonviral nucleic acid delivery, but more importantly, they signify a massive clinical scientific success. Scientists working in the area of nucleic acid delivery using lipid nanoparticles will undoubtedly be energized by the success of these vaccines and begin to collect much needed data in the realm of nonviral-based RNA and DNA delivery, specifically, the use of lipid nanoparticles, the immune response, safety, and efficacy. It is easily conceivable that in the future we can utilize these data to help streamline our approach for the delivery of DNA for gene therapy and regulatory RNAs for therapeutic and regenerative medicine (ie, wound repair) applications.

The oral vaccine based on self-replicating RNA lipid nanoparticles can simultaneously neutralize both SARS-CoV-2 variants alpha and delta.

The aim of this study was to evaluate self-replicating RNA lipid nanoparticles (saRNA LNPs) to neutralize SARS-CoV-2 variants delta (B.1.617 lineage) and alpha (B.1.1.7 lineage). Before immunization of mice with saRNA LNPs, we saw high expression of S-protein at both mRNA and protein levels after transfection of HEK293T/17 cells with saRNA LNPs. After oral immunization of BALB/c mice with 0.1 - 10 µg saRNA LNPs , a high quantity of SARS-CoV-2 specific IgG and IgA antibodies were seen with a dose-dependent pattern. Importantly, the ratio of IgG2a/IgG1 in serum of vaccinated mice showed Th1/Th2 skewing response. We also found that the secreted antibodies could neutralize SARS-CoV-2 variants delta (B.1.617 lineage) and alpha (B.1.1.7 lineage). Re-stimulated splenocytes of vaccinated mice showed high secretion of IFN-γ, IL-6, and TNF- α . The authors think that although the preclinical study confirmed the efficacy of saRNA LNPs against SARS-CoV-2, the actual efficacy and safety of the oral vaccine must be evaluated in clinical trials.

Characteristics of SARS-CoV2 that may be useful for nanoparticle pulmonary drug delivery.

As a non-invasive method of local and systemic drug delivery, the administration of active pharmaceutical ingredients (APIs) via the pulmonary route represents an ideal approach for the therapeutic treatment of pulmonary diseases. The pulmonary route provides a number of advantages, including the rapid absorption which results from a high level of vascularisation over a large surface area and the successful avoidance of first-pass metabolism. Aerosolization of nanoparticles (NPs) is presently under extensive investigation and exhibits a high potential for targeted delivery of therapeutic agents for the treatment of a wide range of diseases. NPs need to possess specific characteristics to facilitate their transport along the pulmonary tract and appropriately overcome the barriers presented by the pulmonary system. The most challenging aspect of delivering NP-based drugs via the pulmonary route is developing colloidal systems with the optimal physicochemical parameters for inhalation. The physiochemical properties of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have been investigated as a template for the synthesis of NPs to assist in the formulation of virus-like particles (VLPs) for pharmaceutical delivery, vaccine production and diagnosis assays.

How Far Are Non-Viral Vectors to Come of Age and Reach Clinical Translation in Gene Therapy?

Efficient delivery of genetic material into cells is a critical process to translate gene therapy into clinical practice. In this sense, the increased knowledge acquired during past years in the molecular biology and nanotechnology fields has contributed to the development of different kinds of non-viral vector systems as a promising alternative to virus-based gene delivery counterparts. Consequently, the development of non-viral vectors has gained attention, and nowadays, gene delivery mediated by these systems is considered as the cornerstone of modern gene therapy due to relevant advantages such as low toxicity, poor immunogenicity and high packing capacity. However, despite these relevant advantages, non-viral vectors have been poorly translated into clinical success. This review addresses some critical issues that need to be considered for clinical practice application of non-viral vectors in mainstream medicine, such as efficiency, biocompatibility, long-lasting effect, route of administration, design of experimental condition or commercialization process. In addition, potential strategies for overcoming main hurdles are also addressed. Overall, this review aims to raise awareness among the scientific community and help researchers gain knowledge in the design of safe and efficient non-viral gene delivery systems for clinical applications to progress in the gene therapy field.

Prophylactic vaccine delivery systems against epidemic infectious diseases.

Prophylactic vaccines have evolved from traditional whole-cell vaccines to safer subunit vaccines. However, subunit vaccines still face problems, such as poor immunogenicity and low efficiency, while traditional adjuvants are usually unable to meet specific response needs. Advanced delivery vectors are important to overcome these barriers; they have favorable safety and effectiveness, tunable properties, precise location, and immunomodulatory capabilities. Nevertheless, there has been no systematic summary of the delivery systems to cover a wide range of infectious pathogens. We herein summarized and compared the delivery systems for major or epidemic infectious diseases caused by bacteria, viruses, fungi, and parasites. We also included the newly licensed vaccines (e.g., COVID-19 vaccines) and those close to licensure. Furthermore, we highlighted advanced delivery systems with high efficiency, cross-protection, or long-term protection against epidemic pathogens, and we put forward prospects and thoughts on the development of future prophylactic vaccines.

Antimicrobial Activity of Curcumin in Nanoformulations: A Comprehensive Review.

Curcumin (CUR) is a natural substance extracted from turmeric that has antimicrobial properties. Due to its ability to absorb light in the blue spectrum, CUR is also used as a photosensitizer (PS) in antimicrobial Photodynamic Therapy (aPDT). However, CUR is hydrophobic, unstable in solutions, and has low bioavailability, which hinders its clinical use. To circumvent these drawbacks, drug delivery systems (DDSs) have been used. In this review, we summarize the DDSs used to carry CUR and their antimicrobial effect against viruses, bacteria, and fungi, including drug-resistant strains and emergent pathogens such as SARS-CoV-2. The reviewed DDSs include colloidal (micelles, liposomes, nanoemulsions, cyclodextrins, chitosan, and other polymeric nanoparticles), metallic, and mesoporous particles, as well as graphene, quantum dots, and hybrid nanosystems such as films and hydrogels. Free (non-encapsulated) CUR and CUR loaded in DDSs have a broad-spectrum antimicrobial action when used alone or as a PS in aPDT. They also show low cytotoxicity, in vivo biocompatibility, and improved wound healing. Although there are several in vitro and some in vivo investigations describing the nanotechnological aspects and the potential antimicrobial application of CUR-loaded DDSs, clinical trials are not reported and further studies should translate this evidence to the clinical scenarios of infections.

Nanoparticle-mediated surfactant therapy in patients with severe COVID-19: a perspective.

Coronavirus disease 2019 (COVID-19) is an RNA virus-based disease that can be deadly. For critically ill patients, mechanical ventilation is an important life-saving treatment. However, mechanical ventilation shows a trade-off between supporting respiratory function and ventilator-induced lung injury (VILI). Surfactant therapy is a medical administration of exogenous surfactant to supplement or replace deficient or dysfunctional endogenous surfactant. Surfactant therapy can be used to postpone or shorten the use of mechanical ventilation to minimize or avoid VILI, because surfactants can reduce surface tension, improve lung compliance, and enhance oxygenation. In addition, nanotechnology can be applied to improve the therapeutic effect and reduce the adverse effects of surfactants. In this perspective, we discussed how nanoparticles deliver surfactants through intravenous injection and inhalation to the expected lung disease regions where surfactants are mostly needed, and discussed the prospects of nanoparticle-mediated surfactant therapy in the treatment of patients with severe COVID-19.

Carbohydrate-containing nanoparticles as vaccine adjuvants.

Introduction: Adjuvants are essential to vaccines for immunopotentiation in the elicitation of protective immunity. However, classical and widely used aluminum-based adjuvants have limited capacity to induce cellular response. There are increasing needs for appropriate adjuvants with improved profiles for vaccine development toward emerging pathogens. Carbohydrate-containing nanoparticles (NPs) with immunomodulatory activity and particulate nanocarriers for effective antigen presentation are capable of eliciting a more balanced humoral and cellular immune response.Areas covered: We reviewed several carbohydrates with immunomodulatory properties. They include chitosan, ß-glucan, mannan, and saponins, which have been used in vaccine formulations. The mode of action, the preparation methods, characterization of these carbohydrate-containing NPs and the corresponding vaccines are presented.Expert opinion: Several carbohydrate-containing NPs have entered the clinical stage or have been used in licensed vaccines for human use. Saponin-containing NPs are being evaluated in a vaccine against SARS-CoV-2, the pathogen causing the on-going worldwide pandemic. Vaccines with carbohydrate-containing NPs are in different stages of development, from preclinical studies to late-stage clinical trials. A better understanding of the mode of action for carbohydrate-containing NPs as vaccine carriers and as immunostimulators will likely contribute to the design and development of new generation vaccines against cancer and infectious diseases.

Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs.

Nucleoside-modified messenger RNA (mRNA)-lipid nanoparticles (LNPs) are the basis for the first two EUA (Emergency Use Authorization) COVID-19 vaccines. The use of nucleoside-modified mRNA as a pharmacological agent opens immense opportunities for therapeutic, prophylactic and diagnostic molecular interventions. In particular, mRNA-based drugs may specifically modulate immune cells, such as T lymphocytes, for immunotherapy of oncologic, infectious and other conditions. The key challenge, however, is that T cells are notoriously resistant to transfection by exogenous mRNA. Here, we report that conjugating CD4 antibody to LNPs enables specific targeting and mRNA interventions to CD4+ cells, including T cells. After systemic injection in mice, CD4-targeted radiolabeled mRNA-LNPs accumulated in spleen, providing ∼30-fold higher signal of reporter mRNA in T cells isolated from spleen as compared with non-targeted mRNA-LNPs. Intravenous injection of CD4-targeted LNPs loaded with Cre recombinase-encoding mRNA provided specific dose-dependent loxP-mediated genetic recombination, resulting in reporter gene expression in about 60% and 40% of CD4+ T cells in spleen and lymph nodes, respectively. T cell phenotyping showed uniform transfection of T cell subpopulations, with no variability in uptake of CD4-targeted mRNA-LNPs in naive, central memory, and effector cells. The specific and efficient targeting and transfection of mRNA to T cells established in this study provides a platform technology for immunotherapy of devastating conditions and HIV cure.

Multivalent nanoparticle-based vaccines protect hamsters against SARS-CoV-2 after a single immunization.

The COVID-19 pandemic continues to wreak havoc as worldwide SARS-CoV-2 infection, hospitalization, and death rates climb unabated. Effective vaccines remain the most promising approach to counter SARS-CoV-2. Yet, while promising results are emerging from COVID-19 vaccine trials, the need for multiple doses and the challenges associated with the widespread distribution and administration of vaccines remain concerns. Here, we engineered the coat protein of the MS2 bacteriophage and generated nanoparticles displaying multiple copies of the SARS-CoV-2 spike (S) protein. The use of these nanoparticles as vaccines generated high neutralizing antibody titers and protected Syrian hamsters from a challenge with SARS-CoV-2 after a single immunization with no infectious virus detected in the lungs. This nanoparticle-based vaccine platform thus provides protection after a single immunization and may be broadly applicable for protecting against SARS-CoV-2 and future pathogens with pandemic potential.

Future considerations for the mRNA-lipid nanoparticle vaccine platform.

Vaccines based on mRNA-containing lipid nanoparticles (LNPs) pioneered by Katalin Karikó and Drew Weissman at the University of Pennsylvania are a promising new vaccine platform used by two of the leading vaccines against coronavirus disease in 2019 (COVID-19). However, there are many questions regarding their mechanism of action in humans that remain unanswered. Here we consider the immunological features of LNP components and off-target effects of the mRNA, both of which could increase the risk of side effects. We suggest ways to mitigate these potential risks by harnessing dendritic cell (DC) biology.