A quality-by-design strategic approach for the development of bedaquiline-pretomanid nanoparticles as inhalable dry powders for TB treatment.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (M.tb) and is the second leading cause of death from an infectious disease globally. The disease mainly affects the lungs and forms granulomatous lesions that encapsulate the bacteria, making treating TB challenging. The current treatment includes oral administration of bedaquiline (BDQ) and pretomanid (PTD); however, patients suffer from severe systemic toxicities, low lung drug concentration, and non-adherence. In this study, we developed BDQ-PTD loaded nanoparticles as inhalable dry powders for pulmonary TB treatment using a Quality-by-Design (QbD) approach. The BDQ-PTD combination showed an additive/synergistic effect for M.tb inhibition in vitro, and the optimized drug ratio (1:4) was successfully loaded into polymeric nanoparticles (PLGA NPs). The QbD approach was implemented by identifying the quality target product profile (QTPPs), critical quality attributes (CQAs), and critical process parameters (CPPs) to develop efficient design space for dry powder preparation using spray drying. The three-factorial and three-level Box-Behnken Design was used to assess the effect of process parameters (CPPs) on product quality (CQAs). The Design of Experiments (DoE) analysis showed different regression models for product quality responses and helped optimize process parameters to meet QTPPs. The optimized dry powder showed excellent yield (72 ± 2 % w/w), high drug (BDQ-PTD) loading, low moisture content (<1% w/w), and spherical morphology. Further, aerosolization performance revealed the suitability of powder for deposition in the respiratory airways of the lungs (MMAD 2.4 µm and FPF > 75 %). In conclusion, the QbD approach helped optimize process parameters and develop dry powder with a suitable quality profile for inhalation delivery in TB patients.

Pulmonary delivery of spray-dried Nisin ZP antimicrobial peptide for non-small cell lung cancer (NSCLC) treatment.

Nisin ZP is an antimicrobial peptide (AMP) produced by the bacterium Lactococcus lactis, and we have previously demonstrated anticancer activity in NSCLC (A549) cells. In this study, we formulated a nisin ZP dry powder (NZSD) using a spray dryer to facilitate inhaled delivery for the treatment of NSCLC. Nisin ZP was spray-dried with mannitol, l-leucine, and trehalose in a ratio of 75:15:10 using Büchi mini spray-dryer B-290 in different drug loadings (10, 20, and 30% w/w). NZSD powder revealed a good powder yield of >55% w/w with ≤3 % w/w moisture content and high nisin ZP drug loading for all the peptide ratios. The NZSD powder particles were irregularly shaped with corrugated morphology. The presence of an endothermic peak in DSC thermograms and attenuated crystalline peaks in PXRD diffractograms confirmed the semi-crystalline powder nature of NZSD. The anticancer activity of nisin ZP was maintained after fabricating it into NZSD powder and showed a similar inhibitory concentration to free nisin ZP. Stability studies indicated that NZSD powders were stable for three months at 4 and 25 ℃ with more than 90% drug content and semi-crystalline nature, as confirmed by DSC and PXRD. Aerosolization studies performed using NGI indicated an aerodynamic diameter (MMAD) within the desired range (1-5 µm) and a high fine particle fraction (FPF > 75%) for all peptide ratios, suggesting powder deposition in the lung's respiratory airways. In conclusion, a dry powder of nisin ZP was formulated using a spray dryer with enhanced storage stability and suitable for inhaled delivery.

Solubility Enhancement and Inhalation Delivery of Cyclodextrin-Based Inclusion Complex of Delamanid for Pulmonary Tuberculosis Treatment.

Tuberculosis (TB) is a contiguous airborne disease caused by Mycobacterium tuberculosis (M.tb), primarily affecting the human lungs. The progression of drug-susceptible TB to drug-resistant strains, MDR-TB and XDR-TB, has become a global challenge toward eradicating TB. Conventional TB treatment involves frequent dosing and prolonged treatment regimens predominantly by an oral or invasive route, leading to treatment-related systemic adverse effects and patient's noncompliance. Pulmonary delivery is an attractive option as we could reduce dose, limit systemic side-effects, and achieve rapid onset of action. Delamanid (DLD), an antituberculosis drug, has poor aqueous solubility, and in this study, we aim to improve its solubility using cyclodextrin complexation. We screened different cyclodextrins and found that HP-ß-CD resulted in a 54-fold increase in solubility compared to a 27-fold and 13-fold increase by SBE-ß-CD and HP-É£-CD, respectively. The stability constant (265 ± 15 M-1) and complexation efficiency (8.5 × 10-4) suggest the formation of a stable inclusion complex of DLD and HP-ß-CD in a 2:1 ratio. Solid-state characterization studies (DSC, PXRD, and NMR) further confirmed successful complexation of DLD in HP-ß-CD. The nebulized DLD-CD complex solution showed a mass median aerodynamic diameter of 4.42 ± 0.62 µm and fine particle fraction of 82.28 ± 2.79%, suggesting deposition in the respiratory airways. In bacterial studies, minimum inhibitory concentration of DLD-CD complex was significantly reduced (four-fold) compared to free DLD in M.tb (H37Ra strain). Furthermore, accelerated stability studies confirmed that the inclusion complex was stable for 4 weeks with 90%w/w drug content. In conclusion, we increased the aqueous solubility of DLD through cyclodextrin complexation and improved its efficacy in vitro.

Anticancer activity of D-LAK-120A, an antimicrobial peptide, in non-small cell lung cancer (NSCLC).

Non-small cell lung cancer (NSCLC) is a major cause of global cancer mortalities and accounts for approximately 80-85% of reported lung cancer cases. Conventional chemotherapeutics show limited application because of poor tumor selectivity and acquired drug resistance. Antimicrobial peptides (AMPs) have gained much attention as potential anticancer therapeutics owing to their high potency and high target selectivity and specificity with limited scope for drug resistance. In this study, D-LAK (D-LAK-120A), a cationic AMP, was evaluated for its anticancer efficacy in various NSCLC cell lines. D-LAK peptide demonstrated enhanced cytotoxicity in A549, H358, H1975, and HCC827 cell lines with inhibitory concentrations between 4.0 and 5.5 µM. An increase in the lactate dehydrogenase (LDH) levels and propidium iodide (PI) uptake across compromised membrane suggested membranolytic activity as an inhibition pathway. In addition, we found D-LAK induced lung cancer cell apoptosis and arrested cells in the S phase (DNA synthesis) of cell cycle. Moreover, a decreased mitochondrial membrane potential and elevated ROS levels were observed after D-LAK treatment, suggesting induction of mitochondria-mediated apoptosis. Additionally, D-LAK inhibited single cell proliferation and cancer cell migration in vitro. The tumor reduction observed in the 3D spheroid assay further suggests the potential use of D-LAK as an anticancer agent for NSCLC treatment. Our results postulate innovative insights on the anticancer mechanism of D-LAK, which may contribute to its further development into preclinical studies and a potential therapeutic.

Nisin ZP, an Antimicrobial Peptide, Induces Cell Death and Inhibits Non-Small Cell Lung Cancer (NSCLC) Progression in vitro in 2D and 3D Cell Culture.

Lung cancer is the leading cause of cancer deaths globally with most of the reported cases (> 85%) associated with non-small cell lung cancer (NSCLC). Current therapies have enhanced the overall survival rate of patients but treatment-related adverse effects and increase in drug-resistance limit the success of these treatment options. Antimicrobial peptides (AMPs) have gained interest as anticancer agents as they selectively target cancer cells and decrease the possibility of resistance. Nisin ZP is a polycyclic antimicrobial peptide produced by the Gram-positive bacterium, Lactococcus lactis and is commonly used as a food preservative. Nisin ZP has recently demonstrated anticancer activity in melanoma, head and neck squamous cell carcinoma, hepatic, colon, and blood cancer. In this study, we evaluated the anticancer potential of nisin ZP and assessed the underlying mechanisms in NSCLC cells. The results revealed that nisin ZP induced selective toxicity in cancer (A549 and H1299) cells compared to healthy (HEK293) cells after 48 h of treatment. Nisin ZP exposure induced apoptosis and cell cycle arrest (G0/G1 phase) in NSCLC cells irrespective of tumor protein p53 expression. The cancer cell proliferation was inhibited via non-membranolytic pathways by mitochondrial membrane depolarization and elevation in reactive oxygen species (ROS) generation. Furthermore, nisin ZP decreased cancer cells' clonal expansion and migration, demonstrating potential use against highly metastatic NSCLC. The 3D spheroid growth and cell viability of the A549 cells were significantly inhibited by nisin ZP compared to control. Overall, the results suggest an excellent antitumor potential in vitro and, thus, can further be developed as a novel therapeutic for NSCLC.

Pulmonary delivery of osimertinib liposomes for non-small cell lung cancer treatment: formulation development and in vitro evaluation.

Osimertinib (OB) is a third-generation irreversible tyrosine kinase inhibitor targeting the epidermal growth factor receptor (EGFR), overexpressed in non-small cell lung cancer. Systemic administration of drug often results in poor drug levels at the primary tumor in the lungs and is associated with systemic side effects. In this study, we developed inhalable OB liposomes that can locally accumulate at the tumor site thereby limiting systemic toxicity. OB was loaded into liposomes via active and passive loading methods. The OB active liposomes achieved a higher encapsulation (78%) compared to passive liposomes (25%). The liposomes (passive and active) exhibited excellent aerosolization performance with an aerodynamic diameter of 4 µm and fine particle fraction of 82%. In H1975 cells, OB active and passive liposomes reduced IC50 by 2.2 and 1.2-fold, respectively, compared to free drug. As the OB active liposomes demonstrated higher cytotoxicity compared to OB passive liposomes, they were further investigated for in vitro anti-cancer activity. The OB active liposomes inhibited tumor cell migration and colonization as determined by the scratch assay and clonogenic assay, respectively. Furthermore, the 3D spheroid studies showed that the liposomes were successful in inhibiting tumor growth. These results highlight the potential of OB liposomes to suppress lung cancer. Owing to these attributes, the inhalable OB liposomes can potentially promote better therapeutic outcomes with limited systemic toxicity.

Inhalable bedaquiline-loaded cubosomes for the treatment of non-small cell lung cancer (NSCLC).

Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths globally. Treatment-related adverse effects and development of drug resistance limit the available treatment options for most patients. Therefore, newer drug candidates and drug delivery systems that have limited adverse effects with significant anti-cancer efficacy are needed. For NSCLC treatment, delivering drugs via inhalation is highly beneficial as it requires lower doses and limits systemic toxicity. Bedaquiline (BQ), an FDA-approved anti-tuberculosis drug has previously shown excellent anti-cancer efficacy. However, poor aqueous solubility limits its delivery via the lungs. In this project, we developed inhalable BQ-loaded cubosome (BQLC) nanocarriers against NSCLC. The BQLC were prepared using a solvent evaporation technique with the cubosomal nanocarriers exhibiting a particle size of 150.2 ± 5.1 nm, zeta potential of (+) 35.4 ± 2.3 mV, and encapsulation efficiency of 51.85 ± 4.83%. The solid-state characterization (DSC and XRD) confirmed drug encapsulation and in an amorphous form within the cubosomes. The BQLC nanocarriers showed excellent aerodynamic properties after nebulization (MMAD of 4.21 ± 0.53 µm and FPF > 75%). The BQLC displayed enhanced cellular internalization and cytotoxicity with a ~ 3-fold reduction in IC50 compared to free BQ in NSCLC (A549) cells, after 48 h treatment. The BQLC suppressed cell proliferation via apoptotic pathway, further inhibited colony formation, and cancer metastasis in vitro. Additionally, 3D-tumor simulation studies established the anti-cancer efficacy of cubosomal nanocarriers as compared to free BQ. This is the first study exploring the potential of cubosomes as inhalation therapy of repurposed drug, BQ and the results suggest that BQLC may be a promising NSCLC therapy due to excellent aerosolization performance and enhanced anti-cancer activity.

Aerosolized nanoliposomal carrier of remdesivir: an effective alternative for COVID-19 treatment in vitro.

Aim: To formulate an aerosolized nanoliposomal carrier for remdesivir (AL-Rem) against coronavirus disease 2019. Methods: AL-Rem was prepared using modified hydration technique. Cytotoxicity in lung adenocarcinoma cells, stability and aerodynamic characteristics of developed liposomes were evaluated. Results: AL-Rem showed high encapsulation efficiency of 99.79%, with hydrodynamic diameter of 71.46 ± 1.35 nm and surface charge of -32 mV. AL-Rem demonstrated minimal cytotoxicity in A549 cells and retained monolayer integrity of Calu-3 cells. AL-Rem showed sustained release, with complete drug release obtained within 50 h in simulated lung fluid. Long-term stability indicated >90% drug recovery at 4°C. Desirable aerosol performance, with mass median aerodynamic diameter of 4.56 ± 0.55 and fine particle fraction of 74.40 ± 2.96%, confirmed successful nebulization of AL-Rem. Conclusion: AL-Rem represents an effective alternative for coronavirus disease 2019 treatment.

Lay abstract Remdesivir is one of the first drugs approved for the treatment of coronavirus disease 2019. Currently, it is administered via an injection into the bloodstream. This means that the drug circulates around the entire body and only a limited amount reaches the diseased site ­ the lungs. Frequent dosing is therefore required, which needs expert personnel and multiple hospital visits and can result in serious side effects. In this study, the authors developed specialized, nanosized particles containing the drug remdesivir that can be administered directly into the lungs. This could drastically minimize side effects, enhance efficacy and allow easy self-administration at home. The results of the study are promising but require additional investigation.

An Overview of Nanocarrier-Based Adjuvants for Vaccine Delivery.

The development of vaccines is one of the most significant medical accomplishments which has helped to eradicate a large number of diseases. It has undergone an evolutionary process from live attenuated pathogen vaccine to killed whole organisms or inactivated toxins (toxoids), each of them having its own advantages and disadvantages. The crucial parameters in vaccination are the generation of memory response and protection against infection, while an important aspect is the effective delivery of antigen in an intelligent manner to evoke a robust immune response. In this regard, nanotechnology is greatly contributing to developing efficient vaccine adjuvants and delivery systems. These can protect the encapsulated antigen from the host's in-vivo environment and releasing it in a sustained manner to induce a long-lasting immunostimulatory effect. In view of this, the present review article summarizes nanoscale-based adjuvants and delivery vehicles such as viral vectors, virus-like particles and virosomes; non-viral vectors namely nanoemulsions, lipid nanocarriers, biodegradable and non-degradable nanoparticles, calcium phosphate nanoparticles, colloidally stable nanoparticles, proteosomes; and pattern recognition receptors covering c-type lectin receptors and toll-like receptors.

Microbes as Medicines: Harnessing the Power of Bacteria in Advancing Cancer Treatment.

Conventional anti-cancer therapy involves the use of chemical chemotherapeutics and radiation and are often non-specific in action. The development of drug resistance and the inability of the drug to penetrate the tumor cells has been a major pitfall in current treatment. This has led to the investigation of alternative anti-tumor therapeutics possessing greater specificity and efficacy. There is a significant interest in exploring the use of microbes as potential anti-cancer medicines. The inherent tropism of the bacteria for hypoxic tumor environment and its ability to be genetically engineered as a vector for gene and drug therapy has led to the development of bacteria as a potential weapon against cancer. In this review, we will introduce bacterial anti-cancer therapy with an emphasis on the various mechanisms involved in tumor targeting and tumor suppression. The bacteriotherapy approaches in conjunction with the conventional cancer therapy can be effective in designing novel cancer therapies. We focus on the current progress achieved in bacterial cancer therapies that show potential in advancing existing cancer treatment options and help attain positive clinical outcomes with minimal systemic side-effects.

Exosomes as drug delivery systems: A brief overview and progress update.

Exosomes are intracellular membrane-based vesicles with diverse compositions that are involved in biological and pathological processes. Since the discovery of exosomes, they have been used as diagnostic biomarkers and as potential drug delivery vehicles based on their size and competence to transfer biological materials to recipient cells. The properties of exosomes such as biocompatibility, preferred tumor homing, adjustable targeting efficiency, and stability make them striking and excellent drug delivery vehicles for use in various diseases and cancer therapy. In this article, we provide a brief overview of the biogenesis, functions, and contents of exosomes along with the separation and characterization techniques. Our major focus is on the recent progress made in application of exosomes as drug delivery systems involving delivery of small molecules, macromolecules, and nucleotides. Further, we discuss the challenges faced when using exosomes as a drug delivery vehicle.