Bacteriophages can be stored in two different forms: liquid suspension or solid/dry powder formulation (Chang et al., 2018a). Either option has advantages and disadvantages and can impact the selection of the route of administration. Some considerations for selecting between them are target-specific delivery, phage stability, and clearance by the reticuloendothelial system of the recipient (Loc-Carrillo and Abedon, 2011). Developing optimal formulations for therapeutic phages is thus important for the efficacy and efficiency of phage therapy (Loc-Carrillo and Abedon, 2011). In fact, protein misfolding, denaturalization, and aggregation can affect bacteriophages, leading to a loss of functionality. These detrimental effects are commonly observed when the bacteriophages are exposed to adverse conditions, such as changes in pH, temperature, or moisture content (Parasion et al., 2014).

The conventional preparation of bacteriophages is as a liquid formulation, as it is for many other bioactive compounds. Liquid formulations are easy to prepare and have an average shelf half-life of about a year, depending on the type of phage. However, this method lacks accuracy; moreover, bacterial contaminants, such as exotoxins, endotoxins, or lipopolysaccharide (LPS) from the lysed cells, could damage the purity of bacteriophage formulations (Luong et al., 2020; João et al., 2021).

Despite the fact that the method for extracting bacteriophage in liquid form has been improved, refining this liquid extraction increases the costs and times (Hashemi et al., 2013; Hietala et al., 2013; Bonilla et al., 2016).

Regardless of the procedure followed to create the liquid formulation, a dramatic reduction in bacteriophage titer can occur at any time, depending on the individual stability of the phage (Malik et al., 2017).

Dry powders, through lyophilization or other similar dehydration techniques, show a longer preservation of stability of bioactivity (Baldelli et al., 2022a). Such procedure is introduced in Figure 3. However, some dehydration processes need some additives, such as sugars, amino acids, proteins, and other components, to prevent osmotic damage or phage aggregation. Spray drying could also be used to prepare bacteriophage powders to be sold on the market. Details of spray drying will be shown in the following sections when discussing spray drying in encapsulation.

Several drying techniques would allow the formation of dry powders. For example, thin film freeze drying (TFFD) (AboulFotouh et al., 2022) and lyophilization (Kawasaki et al., 2019) are some of the most straightforward techniques for generating dry powders for respiratory delivery. These, however, show some significant disadvantages for the purpose of proper encapsulation. First, a chemical procedure would be required to encapsulate bioactive compounds. This procedure can produce chemical waste, and be lengthy and costly (Peng et al., 2019). The main drawback of TFFD is its extremely long processing time, which would not allow it to be transferred into large-scale production (Zhang et al., 2020; Ergin, 2022). A bacteriophage has been successfully encapsulated via lyophilization: Golshahi et al. (2011) encapsulated the phages S4-M and ϕKZ for Burkholderia cepacia and Pseudomonas aeruginosa using a mixture of lactose and lactoferrin at 60: 40 w⁄w. However, powders produced by lyophilization tend to have a broad size distribution, resulting in poor target delivery (Shekunov et al., 2007). About 15%–30% of the particles show an average size of 5–0.5 µm; this results in only 45% of the powder reaching areas beyond the throat in the respiratory tract (Golshahi et al., 2011).

Particle engineering theory can assist spray drying and the prevention of damage (Vehring et al., 2007; Vehring, 2008; Baldelli et al., 2022c; Baldelli et al., 2023). The theory of particle engineering explains the formation process of solid micro- and nanoparticles from liquid droplets (Baldelli and Vehring, 2016b). An extensive review article describes the parameters influencing particle formation; for example, temperature, airflow, atomizer diameter, and percentages of the chemical compounds can all influence dry powders’ chemical and morphological properties (Shoyele and Cawthorne, 2006; Vehring et al., 2007; Baldelli et al., 2016; Ferraz-Albani et al., 2017; Baldelli et al., 2022b). Particle engineering is thus the leading theory of some of the most common drying technologies, such as spray drying (SD) (Vehring et al., 2007) and spray freeze drying (SFD) (Baldelli et al., 2022d), as shown in Figure 4. The main difference between the former and latter is the operating temperature: SFD allows colder temperatures (Baldelli et al., 2022d). These drying techniques have been used for several decades to produce dry powders to deliver bioactive compounds to the respiratory system (Baldelli et al., 2022a). SD is the most common technique used for producing dry powders containing encapsulated bioactive compounds for respiratory delivery. Due to the average size of SD microparticles, the lung is the most common deposition location of SD powders. The smaller the diameter of SD microparticles, the deeper in the respiratory system they can be deposited (Baldelli et al., 2015).

Spray drying procedures tend to apply thermal energy to the evaporating solvent for a few milliseconds. This thermal stress is commonly intense, though limited, but can last throughout the spraying procedure; even at lower temperatures than the inlet ones, a process taking a few seconds can jeopardize the stability of bioactive compounds. Another possible source of damage can be shear stress, which occurs when spraying the liquid feed over the nozzle and atomizing it into small droplets (Ma et al., 2015). To bypass these types of stress, and thus the possibility of damaging the bacteriophage during the delivery process, encapsulation can be used using SD or SFD. This can be achieved by adding additional materials to spray the formulation. These materials have been called “wall” and “bulk” (Baldelli et al., 2022d; Ahad et al., 2023; Baldelli et al., 2023; Nimbkar et al., 2023). Wall material is the outer layer of micro- or nanoparticles containing an encapsulated compound. The purpose of this material is obvious: it creates a shell that covers and protects a bioactive compound. This shell is useful for reasons other than the increase of bioavailability of the encapsulated bioactive compound. The wall material is usually a polymer with a higher molecular weight than the bioactive compound to be encapsulated. This characteristic allows the formation of the shell at an early stage in the particle formation process (Baldelli et al., 2015). Forming a shell for each sprayed droplet ensures a high bioavailability of the bioactive compound and, thus, a long half-shell life (Risch, 1995; Baldelli et al., 2022a). The properties of the dry powder are highly dependent on the properties of the shell (Shamaei et al., 2017). For instance, if the polymer is known to show mucoadhesive properties, then the dry powders are expected to have a high residence time on the mucosa in the respiratory tract (Baldelli et al., 2022b). Adhesion forces can also be influenced by the type of material used to form the spray-dried microparticle wall (Fahs et al., 2010). However, the roughness of the microparticles can impact the adhesion forces. This property can be tuned by both the wall material, the spraying conditions, and the presence of a bulk material (Baldelli and Vehring, 2016b). The bulk material is considered a cushion for the bioactive compound for protection greater than that provided by a shell alone. Depending on the quantity, the presence of a bulk material has a great influence on the morphology of spray-dried microparticles (Baldelli et al., 2022a). In some cases, wall and bulk materials are not the only components of a spray-dried formation to encapsulate bioactive compounds—amino acids, such as leucine, are widely used to encapsulate a bioactive compound via spraying techniques (Lechuga-Ballesteros et al., 2008; Ordoubadi et al., 2021; Wang et al., 2021; Baldelli et al., 2022a). The use of amino acids is justified by the promotion of shell formation. Previous research has found that the presence of leucine, at weight percentages between 6 and 22, facilitates shell formation in a variety of spray-dried formulations (Chow et al., 2017). Leucine recrystallizes on the surface of the particles, generating a shell to decrease inter-particle interaction and boost the dispersibility of the powder (Leung et al., 2016). Other amino acids have been used, but there is poor available knowledge on the comparison of these other amino acids with leucine (Carrigy et al., 2020). This review article on designing encapsulated bacteriophage thus focuses on the selection of bulk and wall materials.

While the use of bacteriophages in treating bacterial infections is well-established, the use of spraying techniques as a method for encapsulating bacteriophages for nose or lung delivery is fairly recent. Therefore, the encapsulation of bacteriophages using spraying techniques has been demonstrated in only a limited quantity of research (Table 1). Most of the references cited in Table 1 use a common laboratory-scale spray dryer, Buchi 290, with an atomizer nozzle of 0.6–0.7 mm in diameter. In addition to Buchi 290, other similar products, such as Buchi 191 and 90, were used in previous studies. The reason for this common use is the popularity of this spray dryer and its ability to scale up results (Vehring et al., 2007). In addition, spray drying produces microparticles of about 0.5–20 μm, which is suitable for delivery to the respiratory tract (Baldelli et al., 2015). Furthermore, twin-fluid atomizers have been shown to create lower stress than other types, such as vibrational ones, on the encapsulation of bacteriophages (Carrigy et al., 2020). Another connection between most previous research on the encapsulation of bacteriophages for respiratory delivery is the target: about 90% of previous research focuses on P. aeruginosa. The phages of this bacterium seem to be more stable than others; for instance, they appear stable for temperatures between 25°C and 50°C and pH between 3 and 11 (Akremi et al., 2022). In addition, the antibiotic resistance of P. aeruginosa is one of the highest, attracting the attention of the research community (Golshahi et al., 2011).

Regardless of the target, the number of bacteriophages used needs to be above 10¹⁰ pfu/mL since, assuming possible losses during the encapsulation procedure, the required quantity for an effective treatment against most respirator bacteria is 10⁶ pfu/mL (Su et al., 1998; Danelishvili et al., 2006; Chhibber et al., 2008; Chegini et al., 2020). The optimization of the encapsulation procedure could potentially reduce the losses of the bacteriophages. Such optimization can be achieved by tuning the atomization conditions and the materials selected. Having the greatest effect on the stability of sprayed bioactive compounds are the atomization liquid and airflow speed rates and the spraying temperatures (Baldelli et al., 2022e). The gas flow levels of the feed are between 1.8 and 3 mL/min and of the gas are between 11 and 13 L/min. Within these values, bacteriophages appear not to undergo the lowest stress. Lower levels of feed flow rate do not allow the liquid to form droplets and evaporate into dry microparticles (Sheu and Rosenberg, 1995). Higher levels of gas flow rate can reduce the spraying yields and increase the stress applied to bacteriophages (Carrasquillo et al., 2001). An elevated atomizing airflow (12 compared 5 L/min) showed a higher reduction of phage titer (Vandenheuvel et al., 2013).

The lower the temperature, the less damage occurs to a bioactive compound through a spraying procedure (Baldelli et al., 2022d). Bacteriophages have been encapsulated using spray drying at temperatures of the following ranges: inlet at 60°C and outlet at 40°C. For example, one of the lowest titer reductions (0.11) of P. aeruginosa phages has been achieved using these inlet and outlet temperature conditions (Li et al., 2021).

Atomization conditions can have an impact on the stability of bioactive compounds, but the efficiency of the encapsulation procedure is mostly dependent on material selection. When no materials are added to the formulation of bacteriophages, the lowest titer reduction is achieved. Therefore, adding another component to the matrix ensures the survival of most of the bacteriophages. Sugars have been the most common additive used (Shoyele and Cawthorne, 2006). Without these, spray-dried phages were unable to retain their bioactivity during the spray drying process, resulting in log10 6.7 loss or complete titer reduction [118]. In addition, sugars and carbohydrates are able to replace the bioactive compound water hydrogen bonds due to their relatively high molecular weight and structure (Chang et al., 2017).

The most popular sugars are trehalose, mannitol, and lactose, which are also the most commonly used in respiratory delivery (Vehring, 2008). For instance, lactose and trehalose are commonly used to shield biomaterials against desiccation in spray drying. Both saccharides show a high glass transition temperature when in anhydrous amorphous form (108°C and 115°C for lactose and trehalose, respectively). For instance, the majority of PEV1, PEV20, and PEV61 phages spray dried with 12 mg/mL trehalose or lactose resulted in less than log 10 1.5 titer loss (Chang et al., 2017). However, amorphous lactose is hygroscopic; T _g decreases when exposed to succeeding uptake of moisture. The hygroscopic nature of amorphous lactose could generate a lack of phage protection. Amorphous powders are thermodynamically unstable; they can, thus, recrystallize when exposed to moisture (Chang et al., 2017). Thus, trehalose seems more efficient as a stabilizer since it is not influenced by high residual moisture contents (Vandenheuvel et al., 2013). When trehalose is used as an excipient when drying a bioactive compound, the compound's immobilization in a glassy matrix hinders the denaturalization of the bioactive compound. Furthermore, trehalose is highly attracted to water and can act as a lyoprotectant (Ly et al., 2019). For instance, after spray drying, P. aeruginosa’s phage shows a total 1.3 log titer reduction in formulations containing 40%, 60%, and 80% trehalose, and 2.4 and 5.1 log reductions in formulations containing 20% and no trehalose, respectively (Leung et al., 2017).

Although the use of lactose alone seems to be not as functional as trehalose in encapsulating bioactive compounds, the addition of leucine makes the lactose–leucine combination efficient. Leucine is a hydrophobic amino acid that is often used in spray-dried and respirable formulations to enhance powder flowability and dispersibility (Wang et al., 2022). Moreover, leucine can also influence stability against moisture for spray-dried inhalable powders stored at elevated humidities. In the evaporation process, the high hydrophobicity of leucine allows its distribution on the surface, forming a crystalline shell and enhancing the protection of the bacteriophage. For example, spray-dried PEV20 powder containing lactose and L-leucine remains stable for 1 year at 20°C when stored inside an aluminum pouch (Lin et al., 2018). Furthermore, the highest lung dose is obtained with solutions involving 17 mg/mL of lactose and 8 mg/mL of leucine for PEV1 and PEV61, and 20 mg/mL of lactose and 5 mg/mL of leucine for PEV20 (mostly because of the morphology) (Chang et al., 2017). The combination of lactose, trehalose, and leucine can further enhance the protection of the bioactive compound. Formulations containing 40% trehalose, 40% mannitol, and 20% leucine had better storage stability, under storage conditions of −4°C and 30% relative humidity, of P. aeruginosa phage with no further phage loss after 1 month and <1 log storage loss (Yan et al., 2021). The presence of leucine also supports the storage and of phages. Spray-dried powder containing 80% lactose, 20% leucine, and podovirus phage can be stored and handled below 60% RH to prevent crystallization; they stay stable for 1 year at 15% humidity and humidity 20° (Yan et al., 2021).

Other materials used in previous research focusing on encapsulation via spray drying of bacteriophages are mannitol, dextran, and pullulan. Of these, dextran has the highest molecular weight, allowing its distribution across the surface of an evaporating droplet, thus forming a shell. Moreover, dextran is used for spray drying biological materials, preventing P. aeruginosa from connecting to lung epithelial cells and for improving CF sputum clearance (Chang et al., 2021).

Spray drying is the leading technique in encapsulating a bacteriophage for respiratory delivery. The relevant conditions are the inlet and outlet temperatures. For any bacteriophage, these should be below 80°C and 40°C, respectively. When using water as a solvent, it is a challenge to ensure the formation of a shell during the particle formation process. Therefore, as explained in the previous section, leucine, and similar amino acids are valid alternatives since they have been shown to promote shell formation. Together with an amino acid, a common selection is to use a sugar, such as lactose, sucrose, trehalose, or mannitol. Lactose and sucrose have low glass transition temperatures and tend to be more susceptible to crystallization upon room temperature storage (Thiyagarajan et al., 2021). Avoiding crystallization could be beneficial to reducing the risk of damaging the phages. Therefore, as demonstrated by previous research (Ma et al., 2015), trehalose could be a valid option. However, mannitol could be another option when both the procedure and storage temperatures are kept above water’s freezing point. At warm temperatures, mannitol tends not to crystallize (Littringer et al., 2012). The combination of mannitol and trehalose appears to be favorable for phage encapsulation. As a crystalline bulking agent, mannitol can avert the failure of a powder cake, which facilitates drying at higher temperatures and acts as an excipient in inhibiting trehalose from crystallizing (Su et al., 2017). Mannitol is projected to crystallize during the drying stage and form a supporting skeleton for the particles to avoid further mobility of the trehalose particle structure when drying (Chang et al., 2021).

The amount of sugar needed may vary according to the shape and length of the phages. The larger the phage, the more material might be needed to preserve the phage’s bioavailability. For instance, lytic bacteriophages Dp-1 and Cp-1 are roughly 6 µm long. Spray-dried microparticles larger than this would be expected in order to enclose the bacteriophage without major damage. However, a wall material could be used to enhance the protection of the bacteriophages. This could complicate the formulation matrix, but it could ensure shell formation, control the adhesion forces between multiple sprayed microparticles, and improve adhesion to the mucus layer in the respiratory tract (Baldelli and Vehring, 2016b). Moreover, the presence of a hydrophobic polymer on the surface of sprayed microparticles could reduce the impact of humidity on the half-shelf life of powders. In particular, a thicker layer on the surface could increase half-shelf life (Vandenheuvel et al., 2013; Baldelli et al., 2023). Humidity causes the crystallization of any amorphous matrix, ruining the embedded phages. Therefore, reducing its effect on the phages could greatly enhance the half-shelf life of powders. Another process absent in the literature is spray freeze drying. Even though this can be more challenging to design and scale up than spray drying, it can maintain the procedure’s temperatures below 10°C. These low temperatures commonly show a lower chance of jeopardizing the bioavailability of bioactive compounds (Baldelli et al., 2022d).