Rotello, Vincent M.

siRNA delivery to target cellular processes is an appealing area in lung diseases such as asthma. To realize this potential requires efficient, targeted cytosolic delivery without cellular toxicity or siRNA degradation. We demonstrate the efficacy of PONI-Guan nanoparticles in accessing airway epithelial and smooth muscle cells with specific siRNA targeting of mRNAs of interest while maintaining barrier integrity and avoiding cellular toxicity.

Abstract Rationale : Airway remodeling in asthma involves increased airway smooth muscle (ASM) mass, changes to bronchial epithelial cell (BEC) layer, and increased fibrosis. ASM and BECs actively contribute by secreting growth factors and cytokines, regulating extracellular matrix (ECM) composition and enhancing cell proliferation/migration. Altering mechanisms which modulate remodeling presents a key to novel therapeutic strategies. BDNF increases ASM [Ca2+]i and contractility, and potentiates the effects of inflammation. Importantly, studies show that ASM is not only a target, but also a source of BDNF, raising the possibility of local autocrine/paracrine effects. Arginase 1 and 2 are expressed by BECs, but their roles in asthma are still under investigation with studies showing arginase 1 inhibition blunting epithelial remodeling and ASM hyperplasia. Knockdown of appealing targets such as ASM BDNF and epithelial arginases using small interfering RNA (siRNA) is a promising approach to combating inflammatory lung remodeling. Polymer based nanoparticles have been constructed which demonstrate cell-type specificity and high efficiency in delivering siRNA. Thus, we hypothesize that BDNF and arginases are central to airway remodeling and can be targeted via nanopartiacles.Methods: Primary human ASM and BEC cells from non-asthmatic vs. asthmatic individuals were obtained post-surgery from lung tissues after approval Mayo Clinic IRB. PONI-Guan NP were generated using block copolymer engineering. PCR, immunoblotting and ELISA were performed using standard techniques. Contractility studies were conducted in adult mouse precision cut lung slices. In vivo lung function studies were conducted in 6-8 week old mice, with or without mixed allergen (MA) treatment and Scireq Flexivent system. Results: PONI-Guan NP uptake demonstrates cell-type specificity in BEC/ASM co-culture and mouse lung. Arginase I/II and ECM expression is upregulated in BECs when exposed to BDNF and cytokines. In ASM, BDNF is increased in asthmatics compared to non-asthmatic controls; ECM expression is increased in response to BDNF and TNF-α. In BEC, PONI-Guan NP/siRNA inhibits Arg I/II expression, even in the presence of TNF-α. In ASM, TNF-α increases in BDNF secretion and ECM deposition are blunted by NP-delivered siRNA. PCLS and mouse lung function studies show contractility to MCh: effects suppressed by NP-delivered BDNF and Arg I siRNA. Conclusions: Our studies show the importance of BDNF/Arginase pathways in airway remodeling and efficacy of PONI-based siRNA delivery vectors in blunting remodeling processes.

Fungal infections represent a significant global health challenge. Candida albicans is a particularly widespread pathogen, with both molecular and biofilm-based mechanisms making it resistant to or tolerant of available antifungal drugs. This study reports a combination therapy, active against C. albicans, utilizing terbinafine and essential oils incorporated into a gelatin-based nanoemulsion system (T-GNE). Eugenol and methyl eugenol/terbinafine T-GNEs had an additive efficacy, while carvacrol (CT-GNE) worked synergistically with terbinafine, providing effective antifungal treatment with minimal mammalian cell toxicity. Confocal microscopy demonstrated that CT-GNE penetrated the dense C. albicans biofilm and disrupted the fungal cell membrane. Overall, the combination of essential oils with terbinafine in GNE provided a promising treatment for fungal biofilms.

Asthma is a heterogeneous chronic lung disease that affects nearly 340 million people globally. Airway hyperresponsiveness, remodeling (thickening and fibrosis), and mucus hypersecretion are some hallmarks of asthma. With several current treatments having serious side effects from long-term use and a proportion of patients with uncontrolled asthma, there is an urgent need for new therapies. With an increasing understanding of asthma pathophysiology, there is a recognized need to target therapies to specific cell types of the airway, which necessitates the identification of delivery systems that can overcome increased mucus and thickened airways. Nanoparticles (NPs) that are highly customizable (material, size, charge, and surface modification) are a potential solution for delivery systems of a wide variety of cargoes (nucleic acids, proteins, and/or small molecules), as well as sole therapeutics for asthma. However, there is a need to consider the safety of the NPs in terms of potential for inflammation, toxicity, nonspecific targets, and accumulation in organs. Ongoing clinical trials using NPs, some FDA-approved for therapeutics in other diseases, provide confidence regarding the potential safety and efficacy of NPs in asthma treatment. This review highlights the current state of the use of NPs in asthma, identifying opportunities for further improvements in NP design and utilization for targeting this chronic lung disease.

Biodegradable and all-natural gelatin-based nanoemulsions encapsulate and stabilize antifungal essential oils to provide efficient penetration and eradication of drug-resistant fungal biofilms without resistance generation.

Biofilm infections are a global public health threat, necessitating new treatment strategies. Biofilm formation also contributes to the development and spread of multidrug-resistant (MDR) bacterial strains. Biofilm-associated chronic infections typically involve colonization by more than one bacterial species. The co-existence of multiple species of bacteria in biofilms exacerbates therapeutic challenges and can render traditional antibiotics ineffective. Polymeric nanoparticles offer alternative antimicrobial approaches to antibiotics, owing to their tunable physico-chemical properties. Here, we report the efficacy of poly(oxanorborneneimide) (PONI)-based antimicrobial polymeric nanoparticles (PNPs) against multi-species bacterial biofilms. PNPs showed good dual-species biofilm penetration profiles as confirmed by confocal laser scanning microscopy. Broad-spectrum antimicrobial activity was observed, with reduction in both bacterial viability and overall biofilm mass. Further, PNPs displayed minimal fibroblast toxicity and high antimicrobial activity in an in vitro co-culture model comprising fibroblast cells and dual-species biofilms of Escherichia coli and Pseudomonas aeruginosa. This study highlights a potential clinical application of the presented polymeric platform.

All-natural cross-linked nanoemulsions provide effective treatment of wound biofilm infections.

Prodrug strategies use chemical modifications to improve the pharmacokinetic properties and therefore therapeutic effects of parent drugs. Traditional prodrug approaches use endogenous enzymes for activation. Bioorthogonal catalysis uses processes that endogenous enzymes cannot access, providing a complementary strategy for prodrug uncaging. Site-selective activation of prodrugs to drugs (uncaging) using synthetic catalysts is a promising strategy for localized drug activation. We discuss here recent studies that incorporate metal catalysts into polymers and nanoparticle scaffolds to provide biocompatible “enzyme-like” catalysts that can penetrate bacterial biofilms and activate prodrug antibiotics in situ, affording a new strategy to treat bacterial biofilm infections with the potential for reduced off-target effects.