*Review* **Dissolution and Absorption of Inhaled Drug Particles in the Lungs**

**Basanth Babu Eedara 1,2 , Rakesh Bastola <sup>1</sup> and Shyamal C. Das 1,\***


**Abstract:** Dry powder inhalation therapy has been effective in treating localized lung diseases such asthma, chronic obstructive pulmonary diseases (COPD), cystic fibrosis and lung infections. In vitro characterization of dry powder formulations includes the determination of physicochemical nature and aerosol performance of powder particles. The relationship between particle properties (size, shape, surface morphology, porosity, solid state nature, and surface hydrophobicity) and aerosol performance of an inhalable dry powder formulation has been well established. However, unlike oral formulations, there is no standard dissolution method for evaluating the dissolution behavior of the inhalable dry powder particles in the lungs. This review focuses on various dissolution systems and absorption models, which have been developed to evaluate dry powder formulations. It covers a summary of airway epithelium, hurdles to developing an in vitro dissolution method for the inhaled dry powder particles, fine particle dose collection methods, various in vitro dissolution testing methods developed for dry powder particles, and models commonly used to study absorption of inhaled drug.

**Keywords:** dissolution; absorption; inhalation; dry powders; fine particle dose

**1. Introduction**

Although pulmonary drug delivery by inhalation has been used for many years, research in dry powder inhalers (DPIs) has undergone rapid advancements during the last decade for both local and systemic delivery of drugs [1–4]. DPIs are monophasic solid particulate mixtures, introduced in the 1970s. DPIs are easy to process, portable, more stable, eco-friendly due to the absence of propellants, patient compliance and cost-effective [5–10]. Most of the DPIs available in the market are suffering from short residence time and low drug bioavailability locally in the lungs, resulting in suboptimal local therapeutic effect [11,12].

The rapid dissolution of micron-sized particles and subsequent absorption of the drug into the systemic circulation is one of the clearance mechanisms of inhaled drug particles from the lungs [13–15]. Therefore, many formulation strategies have been followed to prolong the residence time of inhaled drugs at the site of action with reduced dosing and to avoid unwanted toxicities [16,17]. Some of the approaches to prolong the residence time of the inhaled drug particles in the lung are drug encapsulation in a particulate carrier system (liposomes, polymeric and lipid microparticles), increase the molecular mass of the drug by conjugating with a ligand and decrease the solubility of the drug by conjugation with a low water-soluble, hydrophobic material [18].

In vitro dissolution testing is a traditional and standardized quality control tool in all the pharmacopoeias used to evaluate the batch-to-batch consistency, differentiate immediate and controlled release formulations and also to approximate in vivo release profiles [19]. There are many well-established pharmacopeial dissolution methods for oral solid dosage forms, however, there is no accepted standardized method for inhaled products, although many dissolution methods for testing aerosols have been developed [20–28].

**Citation:** Eedara, B.B.; Bastola, R.; Das, S.C. Dissolution and Absorption of Inhaled Drug Particles in the Lungs. *Pharmaceutics* **2022**, *14*, 2667. https://doi.org/10.3390/ pharmaceutics14122667

Academic Editors: Yuan Huang and Jingyuan Wen

Received: 30 September 2022 Accepted: 25 November 2022 Published: 30 November 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

This review describes the dissolution of inhaled respirable size particles and absorption of dissolved drug through lung epithelium. It covers a summary of airway epithelium, hurdles to develop an in vitro dissolution method for the inhaled dry powder particles, fine particle dose collection methods, various in vitro dissolution testing methods developed for dry powder particles, and various models commonly used to study the absorption of inhaled drugs. dosage forms, however, there is no accepted standardized method for inhaled products, although many dissolution methods for testing aerosols have been developed [20–28]. This review describes the dissolution of inhaled respirable size particles and absorption of dissolved drug through lung epithelium. It covers a summary of airway epithelium, hurdles to develop an in vitro dissolution method for the inhaled dry powder particles, fine particle dose collection methods, various in vitro dissolution testing methods

#### **2. Airway Epithelium** developed for dry powder particles, and various models commonly used to study the absorption of inhaled drugs.

Dense core-granulated cells, basal cells, Clara cells, serous cells, ciliated cells, and mucus goblet cells are six distinct cell types present in the epithelium of the respiratory tract (Figure 1). At all levels of the airway, ciliated cells are the most abundant cells. Their primary function is to propel mucus towards the proximal direction, in simple term the process is known as mucociliary clearance. The ciliated cells in the bronchial pseudostratified epithelium are interspersed by secretory cells (mainly mucus-secreting goblet cells), whereas ciliated cells are interspersed mainly by Clara cells in the bronchiolar cuboidal epithelium. Two types of pneumocytes namely, type I and type II pneumocyte alveolar cells are found in the alveolar squamous epithelium (Figure 1). The luminal surface of the alveoli is mainly lined with alveolar type I cells. In addition, alveoli contain alveolar type II pneumocytes that possess microvilli and are cuboidal secretory cells [29]. Epithelial cells in the airway contribute to the secretion of respiratory tract lining fluid (RTLF) that lies on the surfaces of airways from nasal airways to alveolar regions [30]. RTLF is mainly composed of mucins in the conducting airways (trachea, bronchi, bronchioles and terminal bronchioles) whereas it mainly contains phospholipid-rich surfactants in respiratory zone (respiratory bronchioles, alveolar ducts and alveolar sacs) [31]. **2. Airway Epithelium** Dense core-granulated cells, basal cells, Clara cells, serous cells, ciliated cells, and mucus goblet cells are six distinct cell types present in the epithelium of the respiratory tract (Figure 1). At all levels of the airway, ciliated cells are the most abundant cells. Their primary function is to propel mucus towards the proximal direction, in simple term the process is known as mucociliary clearance. The ciliated cells in the bronchial pseudostratified epithelium are interspersed by secretory cells (mainly mucus-secreting goblet cells), whereas ciliated cells are interspersed mainly by Clara cells in the bronchiolar cuboidal epithelium. Two types of pneumocytes namely, type I and type II pneumocyte alveolar cells are found in the alveolar squamous epithelium (Figure 1). The luminal surface of the alveoli is mainly lined with alveolar type I cells. In addition, alveoli contain alveolar type II pneumocytes that possess microvilli and are cuboidal secretory cells [29]. Epithelial cells in the airway contribute to the secretion of respiratory tract lining fluid (RTLF) that lies on the surfaces of airways from nasal airways to alveolar regions [30]. RTLF is mainly composed of mucins in the conducting airways (trachea, bronchi, bronchioles and terminal bronchioles) whereas it mainly contains phospholipid-rich surfactants in respiratory zone (respiratory bronchioles, alveolar ducts and alveolar sacs) [31].

**Figure 1.** Comparison of the tracheobronchial, bronchiolar and alveolar regions of the lungs [32]. Reproduced with permission from Ref. [32]. 2015, McGraw Hill. **Figure 1.** Comparison of the tracheobronchial, bronchiolar and alveolar regions of the lungs [32]. Reproduced with permission from Ref. [32]. 2015, McGraw Hill.

Particles inhaled in the respiratory tract have to overcome the non-epithelial pulmonary barriers (such as RTLF, mucociliary clearance, macrophage uptake) before they come in contact with the epithelial cells. Different types of transport systems occur in the epithelium of the airways such as paracellular transport, receptor-mediated transport and transportermediated transport [33]. Such transport systems translocate inhaled particles into epithelial cells and/or across the epithelia into the interstitium and to the blood and lymph [34].

#### **3. In Vitro Dissolution Testing of Inhalable Dry Powder Particles**

In vitro characterization of dry powder formulations includes the determination of physicochemical nature and aerosol performance of powder particles. The relationship between particle properties (size, shape, surface morphology, porosity, solid state nature, and surface hydrophobicity) and aerosol performance of an inhalable dry powder formulation has been well established [18,35–38]. However, unlike oral formulations, there is no standard dissolution method for evaluating the dissolution behaviour of the inhalable dry powder particles in the lungs.

#### *3.1. Hurdles to Develop an In Vitro Dissolution Method for Inhalable Dry Powder Particles*

One region of the lung differs from another in its anatomy and physiology (Figure 1). In addition, the RTLF where the inhaled particles dissolve varies regionally in composition, thickness and volume. A mucus gel (~3–23 µm) covers the airway region (trachea, bronchi, bronchioles) of the lungs over an area of 1–2 m<sup>2</sup> . Composition of the mucus gel includes 95% of water, 2–3% of mucins, 0.3–0.5% lipids, 0.1–0.5% non-mucin proteins and other cellular debris [39,40]. However, an extremely thin (estimated thickness ~0.07 µm) film of the lung surfactant covers the alveolar region (>100 m<sup>2</sup> ) of the lungs. Lung surfactant contains 90.0% lipids (85.0% phospholipids: dipalmitoyl phosphatidylcholine (47.0%), unsaturated phosphatidylcholine (29.3%) and other lipids (23.7%); 5.0% neutral lipids: cholesterol) and 10.0% proteins (surfactant protein-SP) [41–43]. Hydrophilic SP comprises 3–5% SP-A, and <0.5% SP-D whereas hydrophobic SP contains 0.5–1.0% of SP-B and SP-C each. Gradual decrease in the thickness and volume of the RTLF along a respiratory tract is a major challenge for the development of an in vitro dissolution method that can accurately mimic the conditions of the lungs.

#### *3.2. Fine Particle Dose (FPD) Collection*

During inhalation, only a fine particle dose (FPD) with the particle size 1–5 µm deposits in the deeper lung regions [44,45]. Therefore, estimation of the FPD dissolution profile seems to be more applicable than the whole dose of the powder formulation. To this end, the Andersen Cascade Impactor (ACI), Next Generation Impactor (NGI), Twin Stage Impinger (TSI) and PreciseInhale system (Figure 2) have been used to collect the fine particle dose (FPD). Table 1 summarizes the FPD collection methods for dissolution testing of respirable particles. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 4 of 22

**Figure 2.** Various approaches to collect fine particle dose (FPD). (**A**) Andersen Cascade Impactor (ACI), (**B**) Next Generation Impactor (NGI; top- closed view and bottom- open view of NGI), and (**C**) Twin Stage Impinger (TSI). Figures (**A**–**C**) were reproduced with permission from Driving Results in Inhaler Testing [Brochure, 2020 edition] [46], Copley Scientific Limited. **Figure 2.** Various approaches to collect fine particle dose (FPD). (**A**) Andersen Cascade Impactor (ACI), (**B**) Next Generation Impactor (NGI; top- closed view and bottom- open view of NGI), and (**C**) Twin Stage Impinger (TSI). Figures (**A**–**C**) were reproduced with permission from Driving Results in Inhaler Testing [Brochure, 2020 edition] [46], Copley Scientific Limited.

**Table 1.** Summary of the fine particle dose (FPD) collection methods for dissolution testing of dry

Collected onto a GF filter at the connection point of the induction port and inlet of

Collected onto 6 PVDF membranes placed at the stage 4 of ACI operated at an

air flow of 28.3 L/min

Collected onto the RC membrane (pore size 0.45 µm) at standard USP conditions (4 kPa, 4 L)

Collected onto the RC membrane at standard USP conditions (4 kPa, 4 L) using ACI with stage extension between stage 1 and filter stage, and modified/standard

filter stage

[21]

[20]

[25]

[47]

ACI

, FP, 250 µg

, BD, 50–500

(BDP, 350–700

(0.5 to

Flixotide Accuhaler®

Pulmicort Turbuhaler®

Vanceril® and QVAR®

Azmacort®

Aerobid®

1250 µg

µg

µg)

4 mg)

Azmacort®

Pulmicort Turbuhaler, BD, 200 µg

, TA, 200 µg

, FN, 250–2500 µg

Flovent® HFA and Diskus, FP, 150–

Micronized BD, FN, SAD and SAC; spray dried SA (SAA) HandiHaler®

Micronized BD, SAD and SAC; spray

(1 mg (BD, SA) 10 mg (FN))

dried SA (SAA) HandiHaler®

, TA, 200–2000 µg

powder particles.

Budesonide (BD), Fluticasone

Triamcinolone acetonide (TA)

Beclomethasone dipropionate

Substance A dibromide (SAD), Substance A crystalline base

Substance A amorphous base

propionate (FP),

Flunisolide (FN), TA, BD, FP,

Fenoterol HBr (FNH),

BD, SAD, SAC, SAA

(BDP)

BD,

(SAC),

(SAA)

Andersen Cascade Impactor (ACI)


**Table 1.** Summary of the fine particle dose (FPD) collection methods for dissolution testing of dry powder particles.


#### **Table 1.** *Cont*.

CA—cellulose acetate; GF—glass fibre; IPC—isopore polycarbonate; NC—nitrocellulose; PC—polycarbonate; PE—polyester; PTFE—polytetra fluoroethylene; PVDF—polyvinylidene difluoride; RC—regenerated cellulose; USP—united states pharmacopoeia.

#### 3.2.1. Andersen Cascade Impactor (ACI)

The Andersen cascade impactor (ACI, Figure 2A) is one of the high flow rate cascade impactors used to assess the aerodynamic size distribution of particles for both pharmaceutical and toxicological applications [60]. It consists of a standard tubular induction port (IP) with a 90◦ curvature, stages from 0–7 and a filter stage (stage-F). Each impactor stage comprises several nozzles with a decreasing size as the stage number increases, which directs air and particles onto the collection plates.

Davies and Feddah, 2003 [21] collected dry powder particles onto a glass fibre filter at the connection point of the induction port and inlet part of ACI using a custom designed stainless steel ring with a stainless steel screen support filter. The ACI assembly consists of an induction port and base of the impactor with only stage number zero. The main drawback of this collection method is that the whole emitted dose is collected over the filter, which does not mimic the size of the particles deposited in the deeper lung regions.

Later, Arora et al., 2010 [20] collected aerodynamically classified particles with diameters of 4.7–5.8 µm and 2.1–3.3 µm on the filter membranes from stage 2 and stage 4 of ACI. In this study, they used a 8-stage ACI with stage 2 and stage 4 collecting plates turned upside down to arrange six polyvinylidene difluoride (PVDF) filter membranes (25 mm in diameter; 0.22 µm pore size) for dose collection.

In another study, May et al., 2012 [25] collected particles onto the regenerated cellulose membrane filters at stage-F of an abbreviated ACI. In this study, the ACI assembly consisted of an induction port, pre-separator, stages 0, 1 and F. Later they modified this assembly by adding a cylindrical stage extension of 5.8 cm in between stage 1 and stage-F to attain a homogenous particle distribution on the membrane [47]. Further, a modified filter stage comprising of only three small bars was used to change the flow and deposition pattern of aerosolized particles. The modifications in ACI resulted in homogenous deposition of particles on the membrane compared to unmodified ACI.

Rohrschneider et al., 2015 [26], collected aerosolized particles onto the filter papers positioned at stage 4 of an 8-stage ACI connected to an external humidifier to maintain the humidity.

#### 3.2.2. Next Generation Impactor (NGI)

Next generation impactor (NGI, Figure 2B) is a high flow rate cascade impactor specially designed for pharmaceutical inhaler testing. NGI was constructed with seven distinct stages plus a micro-orifice collector (MOC, a final filter) with a minimum stage overlap. The airflow passes with increasing velocity in a saw tooth pattern through a series of nozzles containing progressively reducing jet diameters. Out of seven, five stages give a particle size cut-off diameter of 0.54–6.13 µm at flow rates from 30 to 100 L min−<sup>1</sup> .

Son et al., 2009 [28], collected aerodynamically separated particles on a polycarbonate membrane (PC) using a modified NGI. At each collection plate of NGI, a polycarbonate (PC) membrane was placed and covered with a plate-shaped wax paper which consists of a rectangular opening (2.0 × 2.5 cm) at the centre. The powder samples were dispersed into the NGI using an Aerolizer® device at an air flow rate of 60 L min−<sup>1</sup> for 15 s per capsule. The limited size of the prototype holder frame only collects a fraction of powder particles over a rectangular area of the membrane. To overcome this, they designed a special membrane holder which fit in with the NGI cup and collected the whole dispersed particles [51,61]. However, the particles collected using the NGI either with a prototype holder frame or a special membrane holder were not homogenously distributed over the membrane.

#### 3.2.3. Twin Stage Impinger (TSI)

The Twin stage impinger (TSI, Figure 2C) is a simplified device to multistage liquid impinger with only two stages. It was developed to assess drug delivery from meter dose inhalers. TSI is made up of a series of glassware components such as an inlet, a glass bulb which simulates oropharynx, upper (stage 1) and lower (stage 2) impinger stages. TSI separates the actuated aerosol into a coarse oropharyngeal fraction (non-respirable fraction) and a fine pulmonary fraction with an aerodynamic diameter of ≤6.4 µm [60].

Grainger et al., 2009 [55] modified the TSI (mTSI) to deposit the respirable particles of dextrans onto the Calu-3 bronchial epithelial cells in a Transwell® insert. A Transwell® insert containing the cells was attached in the place of an adapter piece to the TSI conducting tube in the lower stage without any medium. The powder (~5 mg) was loaded into the dry powder insufflator and aerosolised at 60 L min−<sup>1</sup> air flow rate for 5 s. The particles collected using the TSI are homogenously distributed with a geometric diameter of <6.4 µm. Later they used the same mTSI to collect the beclomethasone dipropionate (BDP) respirable particles for in vitro dissolution testing [56]. The BDP particles emitted from each of the commercial pressurized metered dose inhalers [pMDI): QVAR and Sanasthmax, were collected (1.2 ± 0.12 mg) on a hydrated nitrocellulose membrane (0.45 µm pore size).

Haghi et al., 2012 [57] collected the micronized salbutamol base (SB) and salbutamol sulfate (SS) particles using the mTSI as described by Grainger et al., 2009 [55] for in vitro dissolution studies using the Franz cell. Five milligrams of powder sample was actuated using a Cyclohaler DPI device at 60 L min−<sup>1</sup> for 4 s. The emitted particles were collected in a Transwell® polyester insert (0.4 µm pore size and 0.33 cm<sup>2</sup> area) at stage-2 of mTSI.

Eedara et al., 2019 [62] modified the stage 2 (lower impingement chamber) of TSI (mTSI, Figure 3) with a screw cap at its bottom to collect aerosolized powder particles onto the glass coverslips. Magnetic passe-partouts were used to hold glass coverslip (24 mm diameter) in position as it makes a boundary to collect the particles over an area of ~200 mm<sup>2</sup> (16 mm diameter) during aerosolization. Hard gelatin PEG capsule (size 3; Qualicaps, Osaka, Japan) was used to fill the drug powders (20 mg). Capsule was dispersed using an Aerolizer® device (Novartis, Surrey, UK) at a flow rate of 60 L min−<sup>1</sup> for 4 s into stage 1 (upper impingement chamber) filled with 7 mL of water. The non-respirable fraction of dose gets separated in the stage 1 of TSI. Three capsules were actuated one after another and the mTSI was disassembled to collect coverslips with deposited powders.

**Figure 3.** A modified Twin Stage Impinger (mTSI) to collect fine particle dose (FPD). Reproduced with permission from Eedara et al., 2019 [62], Springer Nature. **Figure 3.** A modified Twin Stage Impinger (mTSI) to collect fine particle dose (FPD). Reproduced with permission from Eedara et al., 2019 [62], Springer Nature.

In a recent study, a modified version of Twin Stage Impinger and in vitro dissolution experiment were used to examine in vitro in vivo correlation of budesonide and salbutamol. Comparison using both the actual and predicted in vivo pharmacokinetic values of the mentioned drugs and the pattern of their Concentration-Time profiles illustrated a good similarity [63]. In a recent study, a modified version of Twin Stage Impinger and in vitro dissolution experiment were used to examine in vitro in vivo correlation of budesonide and salbutamol. Comparison using both the actual and predicted in vivo pharmacokinetic values of the mentioned drugs and the pattern of their Concentration-Time profiles illustrated a good similarity [63].

#### 3.2.4. PreciseInhale System The PreciseInhale system (Inhalation Sciences, Sweden) is a new aerosol delivery 3.2.4. PreciseInhale System

technique that is able to generate a dry powder aerosol in a free flowing state [64]. This system is a combination of a highly efficient aerosol generator and a precision dosing aerosol exposure unit. In brief, the powder to be aerosolized is placed in a powder chamber The PreciseInhale system (Inhalation Sciences, Sweden) is a new aerosol delivery technique that is able to generate a dry powder aerosol in a free flowing state [64]. This system is a combination of a highly efficient aerosol generator and a precision dosing aerosol exposure unit. In brief, the powder to be aerosolized is placed in a powder chamber and suspended in a compressed gas passing from a pressure chamber to the powder chamber. The suspended powder agglomerates in the powder chamber ejects through the narrow conduit into a holding chamber with an ambient pressure and produces an aerosol cloud of deagglomerated particles. The aerosol of deagglomerated particles is transferred by airflow to the animal or collected for analysis.

Gerde et al., 2017 [23] collected the aerosolized dry powder particles of budesonide (BD) and fluticasone propionate (FP) on circular microscope glass coverslips using the PreciseInhale aerosol generator for in vitro dissolution testing by the Dissolv*It* system. Nine circular glass coverslips (13 mm diameter) were placed in a ring-shaped holder and covered with a thin steel passe-partout to limit the area of powder coating to the surface that will be in contact with the model barrier during the dissolution study. The coverslips were exposed to a single generation cycle of the powder (2.5 mg) aerosol produced using the PreciseInhale system at an air flow rate of 1.2 L min−<sup>1</sup> . The amount of drug deposited on the coverslips was in the range from 0.99 to 1.20 mg with a mass median aerodynamic diameter (MMAD) of 1.7 mm for BD and of 3.4 mm for FP.

#### *3.3. In Vitro Dissolution Methods*

In vitro dissolution studies by conventional dissolution methods using USP apparatus 1 (basket) [65,66] and 2 (paddle) [27,67–69] have several limitations. Primarily these methods provide well-stirred environments contrasting with the in vivo condition in the alveolar region of the lungs. Homogenous dispersion of the particles into the vessel/basket is challenging, and dispersed particles adhere to the dissolution apparatus components and inadvertently enter the aliquots during the sampling procedure. To make up for some of the deficits of commercial USP 1 and 2 dissolution systems, various in vitro dissolution methods using compendial (USP 2) paddle apparatus, flow-through cell apparatus, dialysis bag, Franz diffusion cell, Transwell® and DissolvIt systems (Table 2) have been developed and applied to evaluate the drug release characteristics of the inhaled dry powder even though they are limited in mimicking the in vivo situation.


**Table 2.** Summary of various in vitro dissolution testing methods for dry powder particles.


#### **Table 2.** *Cont*.

DPPC—di-palmitoylphosphatidylcholine; HBSS—Hanks balanced salt solution; PB—phosphate buffer; PBS phosphate-buffered saline; PEO—polyethylene oxide; SDS—sodium dodecyl sulfate; SLF—simulated lung fluid; SLS—sodium lauryl sulfate.

#### 3.3.1. Modified USP 2 (Paddle over Disc) Apparatus

The paddle-over-disc dissolution setup consists of a round bottom glass vessel of 150 mL capacity with rotating mini-paddles and a membrane cassette. The membrane cassette is a powder holding device contains two membranes with sandwiched powder particles inside a modified histology cassette frame. Son and McConville 2009 [28], evaluated the dissolution properties of hydrocortisone inhalable powders using a mini-paddle dissolution apparatus containing a membrane cassette (Figure 4). Aerodynamically classified particles collected over a polycarbonate membrane (PC) using NGI were sandwiched using another pre-soaked PC membrane and inserted into the cassette frame. Then, this membrane cassette was placed into a dissolution vessel containing 100 mL of dissolution medium (SLF and mSLF; 37 ◦C), and drug release was evaluated at a paddle rotation speed of 50 rpm. In this method, the sandwiched dispersed particles in the membrane cassette undergo dissolution in the small volumes of the medium that enters through the pores in the membrane followed by diffusion of the dissolved drug into the bulk reservoir medium. This new method of dissolution studies showed a significant difference in the dissolution profiles between bulk hydrocortisone (HC) and an aerodynamically classified HC. However, the dissolution tests were performed for only a portion of dose collected on the rectangular portion of the membrane due to the holder frame limitations. is a powder holding device contains two membranes with sandwiched powder particles inside a modified histology cassette frame. Son and McConville 2009 [28], evaluated the dissolution properties of hydrocortisone inhalable powders using a mini-paddle dissolution apparatus containing a membrane cassette (Figure 4). Aerodynamically classified particles collected over a polycarbonate membrane (PC) using NGI were sandwiched using another pre-soaked PC membrane and inserted into the cassette frame. Then, this membrane cassette was placed into a dissolution vessel containing 100 mL of dissolution medium (SLF and mSLF; 37 °C), and drug release was evaluated at a paddle rotation speed of 50 rpm. In this method, the sandwiched dispersed particles in the membrane cassette undergo dissolution in the small volumes of the medium that enters through the pores in the membrane followed by diffusion of the dissolved drug into the bulk reservoir medium. This new method of dissolution studies showed a significant difference in the dissolution profiles between bulk hydrocortisone (HC) and an aerodynamically classified HC. However, the dissolution tests were performed for only a portion of dose collected on the rectangular portion of the membrane due to the holder frame limitations.

Later this research group designed a new, easy to use membrane holder (Figure 4B) to evaluate the dissolution behaviour of the whole dose collected in each NGI plate [50,51]. This new membrane holder consists of a NGI dissolution cup with a removable impaction insert, a securing ring, two sealing o-rings, and a PC membrane. A pre-soaked PC membrane was placed over the impaction insert with dispersed particles and secured in the designed membrane holder. The secured membrane holder with sandwiched particles was transferred into the dissolution vessel containing 300 mL of dissolution medium with membrane side up facing the rotating paddle. Later this research group designed a new, easy to use membrane holder (Figure 4B) to evaluate the dissolution behaviour of the whole dose collected in each NGI plate [50,51]. This new membrane holder consists of a NGI dissolution cup with a removable impaction insert, a securing ring, two sealing o-rings, and a PC membrane. A pre-soaked PC membrane was placed over the impaction insert with dispersed particles and secured in the designed membrane holder. The secured membrane holder with sandwiched particles was transferred into the dissolution vessel containing 300 mL of dissolution medium with membrane side up facing the rotating paddle.

#### 3.3.2. Dialysis Bag 3.3.2. Dialysis Bag

medium.

Dialysis is a separation technique which works by diffusion, a process that results from the thermal motion of molecules in solution from a region of higher to lower concentration until an equilibrium is reached. A dialysis bag made of a semipermeable membrane and had small pores. The bag filled with solid dry powder particles is suspended in a dialysate medium (Figure 5A). The large dry powder particles cannot pass through the pores of the membrane. Upon dissolution of the dry powder particles, the drug molecules are small enough to diffuse through the pores of the membrane into the dialysate Dialysis is a separation technique which works by diffusion, a process that results from the thermal motion of molecules in solution from a region of higher to lower concentration until an equilibrium is reached. A dialysis bag made of a semipermeable membrane and had small pores. The bag filled with solid dry powder particles is suspended in a dialysate medium (Figure 5A). The large dry powder particles cannot pass through the pores of the membrane. Upon dissolution of the dry powder particles, the drug molecules are small enough to diffuse through the pores of the membrane into the dialysate medium.

**Figure 5.** Schematic diagrams of (**A**) dialysis bag method, (**B**) flow-through cell [21], (**C**) Franz diffusion cell [27] and (**D**) Transwell® system [20].. (**B**) reproduced with permission from Davies and Feddah 2003 [21], Elsevier. (**C**) reproduced with permission from Salama et al., 2008 [27], Elsevier. (**D**) reproduced with permission from Arora et al., 2010 [20], Springer Nature. **Figure 5.** Schematic diagrams of (**A**) dialysis bag method, (**B**) flow-through cell [21], (**C**) Franz diffusion cell [27] and (**D**) Transwell® system [20].. (**B**) reproduced with permission from Davies and Feddah 2003 [21], Elsevier. (**C**) reproduced with permission from Salama et al., 2008 [27], Elsevier. (**D**) reproduced with permission from Arora et al., 2010 [20], Springer Nature.

Arora et al., 2015 [72] investigated the voriconazole release from the polylactide microparticles by the dialysis bag (MWCO: 12 kDa) method using 20 mL of phosphate-buffered saline (pH 7.4, 10 mM) containing 0.1% Tween 80 at 37 ± 0.5 °C and 900 rpm. Several other researchers also used dialysis bag method to evaluate the drug release from dry powder particles [73–75]. Arora et al., 2015 [72] investigated the voriconazole release from the polylactide microparticles by the dialysis bag (MWCO: 12 kDa) method using 20 mL of phosphatebuffered saline (pH 7.4, 10 mM) containing 0.1% Tween 80 at 37 ± 0.5 ◦C and 900 rpm. Several other researchers also used dialysis bag method to evaluate the drug release from dry powder particles [73–75].

#### 3.3.3. Flow-Through Cell Apparatus 3.3.3. Flow-Through Cell Apparatus

The flow-through dissolution system (Figure 5B) was introduced in 1957 as a flowing medium dissolution apparatus [83] and in 1990, it was officially accepted by the United States, and European Pharmacopoeia for the evaluation of drug release behaviour from various dosage forms. The flow-through cell apparatus is a modified USP apparatus 4 which comprises a filter holder containing a membrane with the loaded powder particles and a pump that forces the dissolution medium from a reservoir into a vertically positioned flow cell. The flow-through system has several advantages: maintenance of sink conditions by the continuous flow of fresh medium; reduced influence of diffusion during dissolution testing [84]. The flow-through dissolution system (Figure 5B) was introduced in 1957 as a flowing medium dissolution apparatus [83] and in 1990, it was officially accepted by the United States, and European Pharmacopoeia for the evaluation of drug release behaviour from various dosage forms. The flow-through cell apparatus is a modified USP apparatus 4 which comprises a filter holder containing a membrane with the loaded powder particles and a pump that forces the dissolution medium from a reservoir into a vertically positioned flow cell. The flow-through system has several advantages: maintenance of sink conditions by the continuous flow of fresh medium; reduced influence of diffusion during dissolution testing [84].

Kanapilly et al., 1973 [85] evaluated the in vitro dissolution patterns of radioactive aerosol particles using two flowing systems (a flow-through system and parallel flow system) with adjustable solvent flow rates and a static system. In the flow-through systems, the aerosol particles were collected on a 0.8 μm membrane filter (sample filter) using a 7 stage round jet cascade impactor and sandwiched between a 0.1 μm membrane (backup filter) and 0.8 μm membrane filter (top cover filter) in a high-pressure steel filter holder. The solvent was pumped vertically to flow through the top cover filter, sample filter, and Kanapilly et al., 1973 [85] evaluated the in vitro dissolution patterns of radioactive aerosol particles using two flowing systems (a flow-through system and parallel flow system) with adjustable solvent flow rates and a static system. In the flow-through systems, the aerosol particles were collected on a 0.8 µm membrane filter (sample filter) using a 7-stage round jet cascade impactor and sandwiched between a 0.1 µm membrane (backup filter) and 0.8 µm membrane filter (top cover filter) in a high-pressure steel filter holder. The solvent was pumped vertically to flow through the top cover filter, sample filter, and the backup filter.

the backup filter. Davies and Feddah 2003 [21] adapted the flow through system for in vitro dissolution testing of dry powder particles. In this study, aerodynamically classified aerosol particles were collected over membrane filters using ACI as described earlier and sandwiched Davies and Feddah 2003 [21] adapted the flow through system for in vitro dissolution testing of dry powder particles. In this study, aerodynamically classified aerosol particles were collected over membrane filters using ACI as described earlier and sandwiched using

another membrane with a Teflon ring (1 mm thickness) in between the membranes. These sandwiched membranes were placed into the flow through cell held by two stainless steel support filters at both ends and dissolution medium was pumped at a flow rate of 0.7 mL/min in the upward direction to flow through the particles sandwiched in between the membranes.

Taylor et al., 2006 [76] prepared sustained release respirable spray coated particles of ipratropium bromide and evaluated for in vitro drug release behaviour using a flowthrough cell method. In this study, the powder samples were placed directly onto a wire mesh screen and inserted into a flow cell of 22.6 mm in diameter. Deionized water (pH 5.5; 37 ◦C) was passed through the flow cell using a Sotax CY7 piston pump.

#### 3.3.4. Franz Diffusion Cell

Another method which has been commonly used to investigate the in vitro dissolution of inhaled dry powder particles is the Franz diffusion cell. The Franz diffusion cell consists of two compartments, donor and receptor compartments, separated by a membrane as shown in Figure 5C. The donor compartment is exposed to the air while the receptor compartment filled with dissolution medium. The dissolution medium in the receptor compartment is continuously mixed with a magnetic stir bar. An advantage of the Franz diffusion cell is that it provides an air-liquid interface, as present in the lung [25]. However, in a Franz diffusion cell, it may be difficult to distinguish between dissolution rate and diffusion effects through the membrane [86].

A modified Franz diffusion cell was used by Salama et al. [27] to conduct the dissolution test for controlled released microparticles containing disodium cromoglycate (DSCG) and polyvinyl alcohol (PVA) for inhalation. This study compared several different methods of dissolution and found that a modified Franz diffusion cell was able to discriminate dissolution rates better than the flow through cell and the USP apparatus 2.

In another study, May et al. [25] conducted dissolution studies for unnamed drug substance A dibromide and amorphous base, fenoterol and budesonide in PBS (pH 7.4, 1 L) at 37 ◦C using a modified Franz diffusion cell. A regenerated cellulose membrane filter with a pore size of 0.45 µm was placed into the membrane holder, with the particles collected using the NGI facing upwards. In contrast to the results of Salama et al. [27], this study found that although the Franz diffusion cell was able to discriminate between substances of different solubilities in the dissolution media, it was not as sensitive and as reproducible as the USP dissolution apparatus 2. They speculated that the possible reasons for the variation in the results might be due to the difference in the method set up, membrane type and thickness, and loading dose.
