Nebulization of Model Hydrogel Nanoparticles to Macrophages at the Air-Liquid Interface.

Nanoparticle evaluation within the pulmonary airspace has increasingly important implications for human health, with growing interest from drug delivery, environmental, and toxicology fields. While there have been widespread investigations of nanoparticle physiochemical properties following many routes of administration, nanoparticle behavior at the air-liquid interface (ALI) is less well-characterized. In this work, we fabricate two formulations of poly(ethylene)-glycol diacrylate (PEGDA)-based model nanoparticles to establish an in vitro workflow allowing evaluation of nanoparticle charge effects at the ALI. Both cationic and anionic PEGDA formulations were synthesized with similar hydrodynamic diameters around ~225 nm and low polydispersity, with expected surface charges corresponding with the respective functional co-monomer. We find that both formulations are readily nebulized from an aqueous suspension in a commercial Aeroneb® Lab Nebulizer, but the aqueous delivery solution served to slightly increase the overall hydrodynamic and geometric size of the cationic particle formulation. However, nanoparticle loading at 50 µg/ml of either formulation did not influence the resultant aerosol diameter from the nebulizer. To assess aerosol delivery in vitro, we designed a 3D printed adapter capable of ensuring aerosol delivery to transwell 24-well culture plates. Nanoparticle uptake by macrophages was compared between traditional cell culture techniques and that of ALI-cultured macrophages following aerosol delivery. Cell viability was unaffected by nanoparticle delivery using either method. However, only traditional cell culture methods demonstrated significant uptake that was dependent on the nanoparticle surface charge. Concurrently, ALI culture resulted in lower metabolic activity of macrophages than those in traditional cell culture, leading to lower overall nanoparticle uptake at ALI. Overall, this work demonstrates that base-material similarities between both particle formulations provide an expected consistency in aerosol delivery regardless of the nanoparticle surface charge and provides an important workflow that enables a holistic evaluation of aerosolizable nanoparticles.

A Pediatric Upper Airway Library to Evaluate Interpatient Variability of In Silico Aerosol Deposition.

The airway of pediatric patients' changes through development, presenting a challenge in developing pediatric-specific aerosol therapeutics. Our work aims to quantify geometric variations and aerosol deposition patterns during upper airway development in subjects between 3.5 months-6.9 years old using a library of 24 pediatric models and 4 adult models. Computational fluid-particle dynamics was performed with varying particle size (0.1-10 µm) and flow rate (10-120 Lpm), which was rigorously analyzed to compare anatomical metrics (epiglottis angle (θE), glottis to cricoid ring ratio (GC-ratio), and pediatric to adult trachea ratio (H-ratio)), inhaler metrics (particle diameter, [Formula: see text], and flow rate, Q), and clinical metrics (age, sex, height, and weight) against aerosol deposition. Multivariate non-linear regression indicated that all metrics were all significantly influential on resultant deposition, with varying influence of individual parameters. Additionally, principal component analysis was employed, indicating that [Formula: see text], Q, GC-ratio, θE, and sex accounted for 90% of variability between subject-specific deposition. Notably, age was not statistically significant among pediatric subjects but was influential in comparing adult subjects. Inhaler design metrics were hugely influential, thus supporting the critical need for pediatric-specific inhalable approaches. This work not only improves accuracy in prescribing inhalable therapeutics and informing pediatric aerosol optimization, but also provides a framework for future aerosol studies to continue to strive toward optimized and personalized pediatric medicine.

Spatial aerosol deposition correlated to anatomic feature development in 6-year-old upper airway computational models.

The upper airways of children undergo developmental changes around age 6, yielding differences between adult and pediatric anatomies. These differences include the cricoid ring area shape, the location of narrowest constriction, and the angle of the epiglottis, all of which are expected to alter local fluid dynamic profiles and subsequent upper airway deposition and downstream aerosol delivery of inhaled therapeutics. In this work, we quantify "pediatric"-like and "adult"-like geometric and fluid dynamic features of two computed tomography (CT)-scan derived models of 6-year-old upper airways in healthy subjects and compare to an idealized model. The two CT-scan models had a mixture of "adult"- and "pediatric"-like anatomic features, with Subject B exhibiting more "pediatric"-like features than Subject A, while the idealized model exhibited entirely "adult"-like features. By computational fluid-particle dynamics, these differences in anatomical features yielded distinct local fluid profiles with altered aerosol deposition between models. Notably, the idealized model better predicted deposition characteristics of Subject A, the more "adult"-like model, including the relationship between the impaction parameter, dp2Q and the fraction of deposition across a range of flow rates and particle diameters, as well as deposition of an approximate pharmaceutical particle size distribution model. Our results with even this limited dataset suggest that there are key personalized metrics that are influenced by anatomical development, which should be considered when developing pediatric inhalable therapeutics. Quantifying anatomical development and correlating to aerosol deposition has the potential for high-throughput developmental characterization and informing desired aerosol characteristics for pediatric applications.

Intracellular delivery of protein drugs with an autonomously lysing bacterial system reduces tumor growth and metastases.

Critical cancer pathways often cannot be targeted because of limited efficiency crossing cell membranes. Here we report the development of a Salmonella-based intracellular delivery system to address this challenge. We engineer genetic circuits that (1) activate the regulator flhDC to drive invasion and (2) induce lysis to release proteins into tumor cells. Released protein drugs diffuse from Salmonella containing vacuoles into the cellular cytoplasm where they interact with their therapeutic targets. Control of invasion with flhDC increases delivery over 500 times. The autonomous triggering of lysis after invasion makes the platform self-limiting and prevents drug release in healthy organs. Bacterial delivery of constitutively active caspase-3 blocks the growth of hepatocellular carcinoma and lung metastases, and increases survival in mice. This success in targeted killing of cancer cells provides critical evidence that this approach will be applicable to a wide range of protein drugs for the treatment of solid tumors.

Realizing Lobe-Specific Aerosol Targeting in a 3D-Printed In Vitro Lung Model.

Background: Delivery of aerosols to isolated lobes of the lungs would be beneficial for diseases that have lobe-specific effects, such as cancer, pneumonia, and chronic obstructive pulmonary disorder. Recent computational fluid-particle dynamic (CFPD) modeling has demonstrated that in low flow rates, the inlet location of a particle at the mouth dictates the lobe into which it will deposit. However, realization of this lobe-specific deposition has yet to be attempted experimentally or in the clinic. To address this, we sought to develop a proof-of-concept in vitro model and targeting device for achieving lobe-specific delivery. Methods: Using 3D printing, a lung replica was created from a computed tomography scan of a healthy 47-year-old male volunteer and connected to a flow setup to control inlet flow rate and outlet airflow distribution to each lobe. A device was designed and fabricated that directs particles to an inlet location that is 5% of the total inlet area and described by radial coordinates (r,θ). Filter paper at sampling ports for each lobe was used to capture fluorescent polystyrene particles to quantify particle collection. We evaluated lobe-specific targeting at varied inlet coordinates, particle diameters, inlet flow rates, and disease lobe flow rate distribution profiles. Results: Guided by CFPD modeling, inlet locations were identified that increased particle collection to a target lobe between 63% and 90%. For example, release of fluorescent particles at the inlet location r = 4.67 mm, θ = 252° with respect to the center of the inlet using 1 µm particles, 1 L/min inlet flow rate, and healthy subject lobe flow distribution profile yielded 90% of the aerosol dose to the right upper lobe, corresponding to an increase of 4.6 × above the non-targeted percent particle collection. Particle size, inlet flow rate, and disease airflow distributions were all shown to generally decrease the efficiency of lobe-specific targeting. Conclusions: Our results indicate that aerosol targeting of a specific lobe is possible in vitro under optimized conditions and that controlling inlet locations could be a potentially useful method for treatment of lobe-specific diseases. This is the first demonstration of lobe-specific particle collection in a physical lung model and illuminates numerous challenges that will be faced as this method is translated to clinical applications.

Check the gap: Facemask performance and exhaled aerosol distributions around the wearer.

Current facemask research focuses on material characterization and efficiency; however, facemasks are often not tested such that aerosol distributions are evaluated from the gaps in the sides, bottom, and nose areas. Poor evaluation methods could lead to misinformation on optimal facemasks use; a high-throughput, reproducible method which illuminates the issue of fit influencing aerosol transmission is needed. To this end, we have created an in vitro model to quantify particle transmission by mimicking exhalation aerosols in a 3D printed face-nose-mouth replica via a nebulizer and quantifying particle counts using a hand-held particle counter. A sewn, sewn with pipe cleaner nose piece, and sewn with a coffee filter facemask were used to evaluate current common homemade sewn facemask designs, benchmarked against industry standard surgical, N95 respirator tightly fit, and N95 respirator loosely fit facemasks. All facemasks have significantly reduced particle counts in front of the facemask, but the side and top of the facemask showed increases in particle counts over the no facemask condition at that same position, suggesting that some proportion of aerosols are being redirected to these gaps. An altered size distribution of aerosols that escape at the vulnerable positions was observed; escaped particles have larger count median diameters, with a decreased ratio of smaller to larger particles, possibly due to hygroscopic growth or aggregation. Of the homemade sewn facemasks, the facemask with a coffee filter insert performed the best at reducing escaped aerosols, with increased efficiency also observed for sewn masks with a pipe cleaner nose piece. Importantly, there were minimal differences between facemasks at increasing distances, which supports that social distance is a critical element in reducing aerosol transmission. This work brings to light the importance of quantifying particle count in positions other than directly in front of the facemask and identifies areas of research to be explored.

Evaluating Regional Pulmonary Deposition using Patient-Specific 3D Printed Lung Models.

Development of targeted therapies for pulmonary diseases is limited by the availability of preclinical testing methods with the ability to predict regional aerosol delivery. Leveraging 3D printing to generate patient-specific lung models, we outline the design of a high-throughput, in vitro experimental setup for quantifying lobular pulmonary deposition. This system is made with a combination of commercially available and 3D printed components and allows the flow rate through each lobe of the lung to be independently controlled. Delivery of fluorescent aerosols to each lobe is measured using fluorescence microscopy. This protocol has the potential to promote the growth of personalized medicine for respiratory diseases through its ability to model a wide range of patient demographics and disease states. Both the geometry of the 3D printed lung model and the air flow profile setting can be easily modulated to reflect clinical data for patients with varying age, race, and gender. Clinically relevant drug delivery devices, such as the endotracheal tube shown here, can be incorporated into the testing setup to more accurately predict a device's capacity to target therapeutic delivery to a diseased region of the lung. The versatility of this experimental setup allows it to be customized to reflect a multitude of inhalation conditions, enhancing the rigor of preclinical therapeutic testing.