Dry powder inhaler (DPI) therapy has been shown to be effective in treating chronic obstructive pulmonary disease (COPD), asthma, and other respiratory conditions like bronchitis and emphysema (Barnes, 2002, Patton and Byron, 2007, Ye et al., 2022, Muralidharan et al., 2015, Spahn et al., 2022, Pasteka et al., 2022). Due to the lack of commercially available generics, DPI products are often expensive and hence significantly increase the cost burden to patients and health systems. DPI products had an estimated market value of 18.9bn US dollars in 2021 and are expected to increase to 29.6bn US dollars by 2031 (FutureMarketInsights, 2022). The acceptance and integration of generic DPI have been hindered by their inherent complexity. Recognizing a pressing medical demand and emerging treatment possibilities, there is a necessity for extensive research and development in the field of DPI pharmaceuticals. Establishing equivalence between generic and brand-name DPI demands the creation of tools and methodologies capable of comprehensively characterizing product performance, considering the multitude of influencing factors (Newman and Witzmann, 2020).

Characterizing individual pharmaceutical aerosol particles, including internal nanodomains, promises to substantially improve understanding of the relationship between physicochemical properties of the drug/excipient formulation and stability, safety, and therapeutic efficacy (Patton and Byron, 2007, Goh and Lane, 2022, Zhang et al., 2023). For a DPI formulation, particle size, distribution and interactions between drug and excipient, polymorphism, degree of crystallinity, and stability are all known to influence the product performance (Khanal et al., 2022, Chan and Chew, 2003, Ke et al., 2020). To improve the powder flowability, fluidization, and dose metering, drugs are often formulated with coarser carrier particles like α-lactose monohydrate (Khanal et al., 2022). Distribution of drugs on the carrier particle is a crucial factor influencing the aerosolization performance and therapeutic activity of a DPI formulation. Drug distribution and composition of individual DPI particles are even more important for formulations containing two or three drug combinations, such as the commercial product Advair Diskus®/Seretide® which contains two drugs, fluticasone propionate (FP) and salmeterol xinafoate (SX). Studies have confirmed that the co-deposition of FP and SX at the same location in the lung may lead to enhanced clinical benefits (Nelson et al., 2003, Theophilus et al., 2006). This is often related to the therapeutic synergies of drugs with molecular mechanisms at the cellular level (Jetzer et al., 2017, Baraniuk et al., 1997). For the FP and SX drug combination studied herein, salmeterol has been shown to slow the transport of fluticasone across mucous-coated Calu-3 cell monolayers (Haghi et al., 2013). This process may increase the residence time of fluticasone, which would prolong the anti-inflammatory effects. Transcriptomic analysis has demonstrated that in human bronchial airway epithelial BEAS-2B cells salmeterol and fluticasone interact together at the level of gene expression (Rider et al., 2018). Thus, the extent of co-delivery of the two drugs and their relative dissolution rates is important for the observed biological effects.

Techniques like aerosol-specific mass spectrometry, morphology-directed Raman spectroscopy (MDRS), X-ray computed tomography, and time-of-flight secondary ion mass spectrometry (TOF-SIMS) have been utilized to assess the qualitative and quantitative surface composition of DPI particles. Despite their effectiveness in certain analyses, these techniques fall short in providing spatially localized thermal and chemical mapping for individual particles (Gajjar et al., 2023, Alhajj et al., 2021, Mohan et al., 2022, Shur and Price, 2012, Newman et al., November). The melting point (Tm) of dry powder inhaler particles is a fundamental property that is correlated with drug dissolution rate, directly impacting therapeutic efficacy (Batisai et al., 2014, Churakov et al., 2017, Alshaikh et al., 2019, Teleki et al., 2020, Luo and Sun, 2013, Chan and Grant, 1989). The weakening of intermolecular bonds leads to melting point depression and subsequent enhanced dissolution rate (Alshaikh et al., 2019). The accurate and rapid determination of melting points of individual drug aerosols will be a powerful tool for dissolution rate estimation (Batisai et al., 2014). These measurements can now be made at a submicron particle level using nanothermal (nanoTA) analysis. This method uses a specialized microfabricated silicon probe (AFM tip) with a miniscule heater to evaluate local thermal properties of a sample with a spatial resolution up to 20 nm (Zhang et al., 2009). NanoTA analysis can be applied to identify the drug and/or excipient (Royall et al., 1999), detect polymorphs (Sanders et al., 2000), and distinguish between crystalline and amorphous content (Royall et al., 2001). In addition, temperature-induced phase transition in a material, melting point determination, glass transition (Tg), and crystallization of a material can be ascertained using this technique (Zhang et al., 2009, Qi et al., 2013, Dai et al., 2012). Particulate level and spatially resolved information offer a powerful new approach to ensure product quality by both formulation developers and government regulatory bodies. This information may be used to understand the similarity between two or more formulations containing the same drug combinations, e.g., for comparing generics with reference products for life-saving medicines.

In this paper, we present an innovative combination of nanoTA, AFM-IR, and nanomechanical methods to probe the nanothermal domains of a spray dried aerosol formulation containing a mixture of the widely used long acting β-2 agonist (SX), inhaled corticosteroid (FP), and excipient lactose. Spray drying was employed to help control co-localization of drugs within individual particles as the FP:SX ratio was varied from 7:1, as used in commercial inhalers, to 2:1 to enable a better understanding of the SX response in this initial study. By combining this data with nanochemical data obtained from AFM-IR, we can begin to develop an understanding of how the drug-drug and drug-excipient ratios within individual particles impact the values of Tm and/or Tg. This nanoTA-based melting point/glass transition data provides insight into the expected range of dissolution rates among individual particles as well as the heterogeneity of dissolution rates within a given micron scale particle.