Aerosol pulmonary immune engineering.

Aerosolization of immunotherapies poses incredible potential for manipulating the local mucosal-specific microenvironment, engaging specialized pulmonary cellular defenders, and accessing mucosal associated lymphoid tissue to redirect systemic adaptive and memory responses. In this review, we breakdown key inhalable immunoengineering strategies for chronic, genetic, and infection-based inflammatory pulmonary disorders, encompassing the historic use of immunomodulatory agents, the transition to biological inspired or derived treatments, and novel approaches of complexing these materials into drug delivery vehicles for enhanced release outcomes. Alongside a brief description of key immune targets, fundamentals of aerosol drug delivery, and preclinical pulmonary models for immune response, we survey recent advances of inhaled immunotherapy platforms, ranging from small molecules and biologics to particulates and cell therapies, as well as prophylactic vaccines. In each section, we address the formulation design constraints for aerosol delivery as well as advantages for each platform in driving desirable immune modifications. Finally, prospects of clinical translation and outlook for inhaled immune engineering are discussed.

Inhaled Lipid Nanoparticles Alleviate Established Pulmonary Fibrosis.

Pulmonary fibrosis, a sequela of lung injury resulting from severe infection such as severe acute respiratory syndrome-like coronavirus (SARS-CoV-2) infection, is a kind of life-threatening lung disease with limited therapeutic options. Herein, inhalable liposomes encapsulating metformin, a first-line antidiabetic drug that has been reported to effectively reverse pulmonary fibrosis by modulating multiple metabolic pathways, and nintedanib, a well-known antifibrotic drug that has been widely used in the clinic, are developed for pulmonary fibrosis treatment. The composition of liposomes made of neutral, cationic or anionic lipids, and poly(ethylene glycol) (PEG) is optimized by evaluating their retention in the lung after inhalation. Neutral liposomes with suitable PEG shielding are found to be ideal delivery carriers for metformin and nintedanib with significantly prolonged retention in the lung. Moreover, repeated noninvasive aerosol inhalation delivery of metformin and nintedanib loaded liposomes can effectively diminish the development of fibrosis and improve pulmonary function in bleomycin-induced pulmonary fibrosis by promoting myofibroblast deactivation and apoptosis, inhibiting transforming growth factor 1 (TGFß1) action, suppressing collagen formation, and inducing lipogenic differentiation. Therefore, this work presents a versatile platform with promising clinical translation potential for the noninvasive inhalation delivery of drugs for respiratory disease treatment.

Neutralisation of SARS-CoV-2 by monoclonal antibody through dual targeting powder formulation.

Neutralising monoclonal antibody (mAb) is an important weapon in our arsenal for combating respiratory viral infections. However, the effectiveness of neutralising mAb has been impeded by the rapid emergence of mutant variants. Early administration of broad-spectrum mAb with improved delivery efficiency can potentially enhance efficacy and patient outcomes. WKS13 is a humanised mAb which was previously demonstrated to exhibit broad-spectrum activity against SARS-CoV-2 variants. In this study, a dual targeting formulation strategy was designed to deliver WKS13 to both the nasal cavity and lower airways, the two critical sites of infection caused by SARS-CoV-2. Dry powders of WKS13 were first prepared by spray drying, with cyclodextrin used as stabiliser excipient. Two-fluid nozzle (TFN) was used to produce particles below 5 µm for lung deposition (C-TFN formulation) and ultrasonic nozzle (USN) was used to produce particles above 10 µm for nasal deposition (C-USN formulation). Gel electrophoresis and size exclusion chromatography studies showed that the structural integrity of mAb was successfully preserved with no sign of aggregation after spray drying. To achieve dual targeting property, C-TFN and C-USN were mixed at various ratios. The aerosolisation property of the mixed formulations dispersed from a nasal powder device was examined using a Next Generation Impactor (NGI) coupled with a glass expansion chamber. When the ratio of C-TFN in the mixed formulation increased, the fraction of particles deposited in the lung increased proportionally while the fraction of particles deposited in the nasal cavity decreased correspondingly. A customisable aerosol deposition profile could therefore be achieved by manipulating the mixing ratio between C-TFN and C-USN. Dual administration of C-TFN and C-USN powders to the lung and nasal cavity of hamsters, respectively, was effective in offering prophylactic protection against SARS-CoV-2 Delta variant. Viral loads in both the lung tissues and nasal wash were significantly reduced, and the efficacy was comparable to systemic administration of unformulated WKS13. Overall, dual targeting powder formulation of neutralising mAb is a promising approach for prophylaxis of respiratory viral infections. The ease and non-invasive administration of dual targeting nasal powder may facilitate the widespread distribution of neutralising mAb during the early stage of unpredictable outbreaks.

Protein Aggregates in Inhaled Biologics: Challenges and Considerations.

Pulmonary delivery is the main route of administration for treatment of local lung diseases. Recently, the interest in delivery of proteins through the pulmonary route for treatment of lung diseases has significantly increased, especially after Covid-19 pandemic. The development of an inhalable protein combines the challenges of inhaled as well as biologic products since protein stability may be compromised during manufacture or delivery. For instance, spray drying is the most common technology for manufacture of inhalable biological particles, however, it imposes shear and thermal stresses which may cause protein unfolding and aggregation post drying. Therefore, protein aggregation should be evaluated for inhaled biologics as it could impact the safety and/or efficacy of the product. While there is extensive knowledge and regulatory guidance on acceptable limits of particles, which inherently include insoluble protein aggregates, in injectable proteins, there is no comparable knowledge for inhaled ones. Moreover, the poor correlation between in vitro setup for analytical testing and the in vivo lung environment limits the predictability of protein aggregation post inhalation. Thus, the purpose of this article is to highlight the major challenges facing the development of inhaled proteins compared to parenteral ones, and to share future thoughts to resolve them.

Respiratory mucosal vaccination of peptide-poloxamine-DNA nanoparticles provides complete protection against lethal SARS-CoV-2 challenge.

The ongoing SARS-CoV-2 pandemic represents a brutal reminder of the continual threat of mucosal infectious diseases. Mucosal immunity may provide robust protection at the predominant sites of SARS-CoV-2 infection. However, it remains unclear whether respiratory mucosal administration of DNA vaccines could confer protective immune responses against SARS-CoV-2 challenge due to insurmountable barriers posed by the airway. Here, we applied self-assembled peptide-poloxamine nanoparticles with mucus-penetrating properties for pulmonary inoculation of a COVID-19 DNA vaccine (pSpike/PP-sNp). The pSpike/PP-sNp not only displays superior gene transfection and favorable biocompatibility in the mouse airway, but also promotes a tripartite immunity consisting of systemic, cellular, and mucosal immune responses that are characterized by mucosal IgA secretion, high levels of neutralizing antibodies, and resident memory phenotype T-cell responses in the lungs of mice. Most importantly, immunization with pSpike/PP-sNp completely eliminates SARS-CoV-2 infection in both upper and lower respiratory tracts and enables 100% survival rate of mice following lethal SARS-CoV-2 challenge. Our findings indicate PP-sNp is a promising platform in mediating DNA vaccines to elicit all-around mucosal immunity against SARS-CoV-2.

Inhalable neutralizing antibodies - promising approach to combating respiratory viral infections.

Monoclonal antibodies represent an exciting class of therapeutics against respiratory viral infections. Notwithstanding their specificity and affinity, the conventional parenteral administration is suboptimal in delivering antibodies for neutralizing activity in the airways due to the poor distribution of macromolecules to the respiratory tract. Inhaled therapy is a promising approach to overcome this hurdle in a noninvasive manner, while advances in antibody engineering have led to the development of unique antibody formats which exhibit properties desirable for inhalation. In this Opinion, we examine the major challenges surrounding the development of inhaled antibodies, identify knowledge gaps that need to be addressed and provide strategies from a drug delivery perspective to enhance the efficacy and safety of neutralizing antibodies against respiratory viral infections.

Nitric oxide and its derivatives containing nasal spray and inhalation therapy for the treatment of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a major health concern worldwide and evolved into different variants. SARS-CoV-2 possesses a spike glycoprotein on its envelope that binds to the angiotensin-converting enzyme 2 (ACE-2) receptor of the host cell via the receptor-binding domain (RBD) in the upper respiratory tract. Since the SARS-CoV-2 virus variants changes the seveirity of dieseases and treatment scenarios, repurposing current medicines may provide a quick and appealing method with established safety features. The efficacy and safety of antiviral medicines against the coronavirus disease 2019 (COVID-19) have been investigated, and several of them are now undergoing clinical studies. Recently, it has been found that nitric oxide (NO) shows antiviral properties against SARS-CoV-2 and prevents the virus from binding to a host cell. In addition, NO is a well-known vasodilator and acts as an important coagulation mediator. With the fast-track development of COVID-19 treatments and vaccines, one avenue of research aimed at improving therapeutics is exploring different forms of drug delivery, including intranasal sprays and inhalation therapy. The nasal mucosa is more prone to be the site of infection as it is in more direct contact with the physical environment via air during inhalation and exhalation. Thus, the use of the exogenous nasal NO therapy via the intranasal route displays a distinct advantage. Therefore, the objective of this review is to summarize the relevant actions of NO via intranasal spray and inhalation delivery, its mechanism of action, and its use in the treatment of COVID-19.

Production of Inhalable Ultra-Small Particles for Delivery of Anti-Inflammation Medicine via a Table-Top Microdevice.

A table-top microdevice was introduced in this work to produce ultrasmall particles for drug delivery via inhalation. The design and operation are similar to that of spray-drying equipment used in industry, but the device itself is much smaller and more portable in size, simpler to operate and more economical. More importantly, the device enables more accurate control over particle size. Using Flavopiridol, an anti-inflammation medication, formulations have been developed to produce inhalable particles for pulmonary delivery. A solution containing the desired components forms droplets by passing through an array of micro-apertures that vibrate via a piezo-electrical driver. High-purity nitrogen gas was introduced and flew through the designed path, which included the funnel collection and cyclone chamber, and finally was pumped away. The gas carried and dried the micronized liquid droplets along the pathway, leading to the precipitation of dry solid microparticles. The formation of the cyclone was essential to assure the sufficient travel path length of the liquid droplets to allow drying. Synthesis parameters were optimized to produce microparticles, whose morphology, size, physio-chemical properties, and release profiles met the criteria for inhalation. Bioactivity assays have revealed a high degree of anti-inflammation. The above-mentioned approach enabled the production of inhalable particles in research laboratories in general, using the simple table-top microdevice. The microparticles enable the inhalable delivery of anti-inflammation medicine to the lungs, thus providing treatment for diseases such as pulmonary fibrosis and COVID-19.

Remdesivir powders manufactured by jet milling for potential pulmonary treatment of COVID-19.

Remdesivir is one of the effective drugs proposed for the treatment of coronavirus disease 2019 (COVID-19). However, the study on inhalable regimen is currently limited though COVID-19 is respiratory diseases and infects lung area. This work aims to prepare inhalable remdesivir formulations and verify their effectiveness through in vitro evaluations. Formulations containing different ratios of jet-milled inhalable remdesivir (5, 10, 20,40, and 70%) with excipients were produced and characterized in terms of the particle size distribution, particle morphology, flowability, water content, crystallinity, the water sorption and desorption capabilities, and the aerodynamic performance. Results indicating that drug loading are a vital factor in facilitating the dispersion of remdesivir dry powder, and the ternary excipient plays a negligible role in improving aerosol performance. Besides, the 70% remdesivir with lactose carrier (70% RD-Lac) was physically stable and retain high aerosol performance after conditioned at 40 °C and 75% RH for a month. Therefore, formulation 70% RD-Lac might be recommended as a candidate product for the potential treatment of COVID-19.

Versatility of acetalated dextran in nanocarriers targeting respiratory diseases

Acetalated dextran is a chemically modified version of the FDA approved polysaccharide ‘dextran’, which serves as a perspective drug-delivery material for the pulmonary delivery of therapeutics owing to its biodegradability, sensitivity towards acidic pH for stimuli-sensitive drug release, high encapsulation efficacy, chemical conjugation with pharmaceuticals, and potency to cross the mucosal layer. Mainly, the aerosolized dry powder inhalation formulations of drug-loaded acetalated-dextran prove to be the frontrunner candidates for pulmonary delivery for the effective management of chronic respiratory diseases such as lymphangioleiomyomatosis, tularemia, and the contemporary COVID-19 pandemic. The presented communication provides a succinct account of the pulmonary drug delivery applications of acetalated dextran.

Gelatin Stabilizes Nebulized Proteins in Pulmonary Drug Delivery against COVID-19.

Delivering medication to the lungs via nebulization of pharmaceuticals is a noninvasive and efficient therapy route, particularly for respiratory diseases. The recent worldwide severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic urges the development of such therapies as an effective alternative to vaccines. The main difficulties in using inhalation therapy are the development of effective medicine and methods to stabilize the biological molecules and transfer them to the lungs efficiently following nebulization. We have developed a high-affinity angiotensin-converting enzyme 2 (ACE2) receptor-binding domain (RBD-62) that can be used as a medication to inhibit infection with SARS-CoV-2 and its variants. In this study, we established a nebulization protocol for drug delivery by inhalation using two commercial vibrating mesh (VM) nebulizers (Aerogen Solo and PARI eFlow) that generate similar mist size distribution in a size range that allows efficient deposition in the small respiratory airway. In a series of experiments, we show the high activity of RBD-62, interferon-α2 (IFN-α2), and other proteins following nebulization. The addition of gelatin significantly stabilizes the proteins and enhances the fractions of active proteins after nebulization, minimizing the medication dosage. Furthermore, hamster inhalation experiments verified the feasibility of the protocol in pulmonary drug delivery. In short, the gelatin-modified RBD-62 formulation in coordination with VM nebulizer can be used as a therapy to cure SARS-CoV-2.

Bioavailability Enhancement of Cepharanthine via Pulmonary Administration in Rats and Its Therapeutic Potential for Pulmonary Fibrosis Associated with COVID-19 Infection.

Cepharanthine (CEP) has excellent anti-SARS-CoV-2 properties, indicating its favorable potential for COVID-19 treatment. However, its application is challenged by its poor dissolubility and oral bioavailability. The present study aimed to improve the bioavailability of CEP by optimizing its solubility and through a pulmonary delivery method, which improved its bioavailability by five times when compared to that through the oral delivery method (68.07% vs. 13.15%). An ultra-performance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS) method for quantification of CEP in rat plasma was developed and validated to support the bioavailability and pharmacokinetic studies. In addition, pulmonary fibrosis was recognized as a sequela of COVID-19 infection, warranting further evaluation of the therapeutic potential of CEP on a rat lung fibrosis model. The antifibrotic effect was assessed by analysis of lung index and histopathological examination, detection of transforming growth factor (TGF)-ß1, interleukin-6 (IL-6), α-smooth muscle actin (α-SMA), and hydroxyproline level in serum or lung tissues. Our data demonstrated that CEP could significantly alleviate bleomycin (BLM)-induced collagen accumulation and inflammation, thereby exerting protective effects against pulmonary fibrosis. Our results provide evidence supporting the hypothesis that pulmonary delivery CEP may be a promising therapy for pulmonary fibrosis associated with COVID-19 infection.

Inhibition of SARS-CoV-2 replication in the lung with siRNA/VIPER polyplexes.

SARS-CoV-2 has been the cause of a global pandemic since 2019 and remains a medical urgency. siRNA-based therapies are a promising strategy to fight viral infections. By targeting a specific region of the viral genome, siRNAs can efficiently downregulate viral replication and suppress viral infection. However, to achieve the desired therapeutic activity, siRNA requires a suitable delivery system. The VIPER (virus-inspired polymer for endosomal release) block copolymer has been reported as promising delivery system for both plasmid DNA and siRNA in the past years. It is composed of a hydrophilic block for condensation of nucleic acids as well as a hydrophobic, pH-sensitive block that, at acidic pH, exposes the membrane lytic peptide melittin, which enhances endosomal escape. In this study, we aimed at developing a formulation for pulmonary administration of siRNA to suppress SARS-CoV-2 replication in lung epithelial cells. After characterizing siRNA/VIPER polyplexes, the activity and safety profile were confirmed in a lung epithelial cell line. To further investigate the activity of the polyplexes in a more sophisticated cell culture system, an air-liquid interface (ALI) culture was established. siRNA/VIPER polyplexes reached the cell monolayer and penetrated through the mucus layer secreted by the cells. Additionally, the activity against wild-type SARS-CoV-2 in the ALI model was confirmed by qRT-PCR. To investigate translatability of our findings, the activity against SARS-CoV-2 was tested ex vivo in human lung explants. Here, siRNA/VIPER polyplexes efficiently inhibited SARS-CoV-2 replication. Finally, we verified the delivery of siRNA/VIPER polyplexes to lung epithelial cells in vivo, which represent the main cellular target of viral infection in the lung. In conclusion, siRNA/VIPER polyplexes efficiently delivered siRNA to lung epithelial cells and mediated robust downregulation of viral replication both in vitro and ex vivo without toxic or immunogenic side effects in vivo, demonstrating the potential of local siRNA delivery as a promising antiviral therapy in the lung.

Dual targeting powder formulation of antiviral agent for customizable nasal and lung deposition profile through single intranasal administration.

Unpredictable outbreaks due to respiratory viral infections emphasize the need for new drug delivery strategies to the entire respiratory tract. As viral attack is not limited to a specific anatomic region, antiviral therapy that targets both the upper and lower respiratory tract would be most effective. This study aimed to formulate tamibarotene, a retinoid derivative previously reported to display broad-spectrum antiviral activity against influenza and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), as a novel dual particle size powder formulation that targets both the nasal cavity and the lung by a single route of intranasal administration. Spray freeze drying (SFD) and spray drying (SD) techniques were employed to prepare tamibarotene powder formulations, and cyclodextrin was used as the sole excipient to enhance drug solubility. With the employment of appropriate atomizing nozzles, particles of size above 10 µm and below 5 µm could be produced for nasal and lung deposition, respectively. The aerosol performance of the powder was evaluated using Next Generation Impactor (NGI) coupled with a glass expansion chamber and the powder was dispersed with a nasal powder device. By blending powder of two different particle sizes, a single powder formulation with dual aerosol deposition characteristic in both the nasal and pulmonary regions was produced. The aerosol deposition fractions in the nasal cavity and pulmonary region could be modulated by varying the powder mixing ratio. All dry powder formulations exhibited spherical structures, amorphous characteristics and improved dissolution profile as compared to the unformulated tamibarotene. Overall, a novel dual targeting powder formulation of tamibarotene exhibiting customizable aerosol deposition profile was developed. This exceptional formulation strategy can be adopted to deliver other antimicrobial agents to the upper and lower airways for the prevention and treatment of human respiratory infections.

Inhaled heparin polysaccharide nanodecoy against SARS-CoV-2 and variants.

The heparin polysaccharide nanoparticles block the interaction between heparan sulfate/S protein and inhibit the infection of both wild-type SARS-CoV-2 pseudovirus and the mutated strains through pulmonary delivery.Image 1.

Spray-Dried Powder Formulation of Capreomycin Designed for Inhaled Tuberculosis Therapy.

Multi-drug-resistant tuberculosis (MDR-TB) is a huge public health problem. The treatment regimen of MDR-TB requires prolonged chemotherapy with multiple drugs including second-line anti-TB agents associated with severe adverse effects. Capreomycin, a polypeptide antibiotic, is the first choice of second-line anti-TB drugs in MDR-TB therapy. It requires repeated intramuscular or intravenous administration five times per week. Pulmonary drug delivery is non-invasive with the advantages of local targeting and reduced risk of systemic toxicity. In this study, inhaled dry powder formulation of capreomycin targeting the lung was developed using spray drying technique. Among the 16 formulations designed, the one containing 25% capreomycin (w/w) and spray-dried at an inlet temperature of 90 °C showed the best overall performance with the mass median aerodynamic diameter (MMAD) of 3.38 µm and a fine particle fraction (FPF) of around 65%. In the pharmacokinetic study in mice, drug concentration in the lungs was approximately 8-fold higher than the minimum inhibitory concentration (MIC) (1.25 to 2.5 µg/mL) for at least 24 h following intratracheal administration (20 mg/kg). Compared to intravenous injection, inhaled capreomycin showed significantly higher area under the curve, slower clearance and longer mean residence time in both the lungs and plasma.

Liposomal remdesivir inhalation solution for targeted lung delivery as a novel therapeutic approach for COVID-19.

Strong infectivity enables coronavirus disease 2019 (COVID-19) to rage throughout the world. Moreover, the lack of drugs with definite therapeutic effects further aggravates the spread of the pandemic. Remdesivir is one of the most promising anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) drugs. However, the limited clinical effects make its therapeutic effect controversial, which may result from the poor accumulation and activation of remdesivir in the lung. Therefore, we developed lyophilized remdesivir liposomes (Rdv-lips) which can be reconstituted as liposomal aerosol for pulmonary delivery to improve the in vivo behavior of existing remdesivir cyclodextrin conclusion compound (Rdv-cyc) injections. Liposome encapsulation endowed remdesivir with much higher solubility and better biocompatibility. The in vitro liposomal aerosol characterization demonstrated that Rdv-lips possessed a mass median aerodynamic diameter of 4.118 µm and fine particle fraction (<5 µm) higher than 50%, indicating good pulmonary delivery properties. Compared to the Rdv-cyc intravenous injection group, the Rdv-lips inhalation group displayed a nearly 100-fold increase in the remdesivir-active metabolite nucleotide triphosphate (NTP) concentration and better NTP accumulation in the lung than the Rdv-cyc inhalation group. A faster transition from remdesivir to NTP of Rdv-lips (inhalation) could also be observed due to better cell uptake. Compared to other preparations, the superiority of Rdv-lips was further evidenced by the results of an in vivo safety study, with little possibility of inducing inflammation. In conclusion, Rdv-lips for pulmonary delivery will be a potent formulation to improve the in vivo behavior of remdesivir and exert better therapeutic effects in COVID-19 treatment.

In Vitro Investigations on Optimizing and Nebulization of IVT-mRNA Formulations for Potential Pulmonary-Based Alpha-1-Antitrypsin Deficiency Treatment.

In vitro-transcribed (IVT) mRNA has come into focus in recent years as a potential therapeutic approach for the treatment of genetic diseases. The nebulized formulations of IVT-mRNA-encoding alpha-1-antitrypsin (A1AT-mRNA) would be a highly acceptable and tolerable remedy for the protein replacement therapy for alpha-1-antitrypsin deficiency in the future. Here we show that lipoplexes containing A1AT-mRNA prepared in optimum conditions could successfully transfect human bronchial epithelial cells without significant toxicity. A reduction in transfection efficiency was observed for aerosolized lipoplexes that can be partially overcome by increasing the initial number of components. A1AT produced from cells transfected by nebulized A1AT-mRNA lipoplexes is functional and could successfully inhibit the enzyme activity of trypsin as well as elastase. Our data indicate that aerosolization of A1AT-mRNA therapy constitutes a potentially powerful means to transfect airway epithelial cells with the purpose of producing functional A1AT, while bringing along the unique advantages of IVT-mRNA.