Freeze-Drying of Encapsulated Bacteriophage T4 to Obtain Shelf-Stable Dry Preparations for Oral Application

Therapeutic application of bacterial viruses (phage therapy) has in recent years been rediscovered by many scientists, as a method which may potentially replace conventional antibacterial strategies. However, one of the main problems related to phage application is the stability of bacterial viruses. Though many techniques have been used to sustain phage activity, novel tools are needed to allow long-term phage storage and application in versatile forms. In this study, we combined two well-known methods for bacteriophage immobilization.

First, encapsulated phages were obtained by means of extrusion–ionic gelation, and then alginate microspheres were dried using the lyophilization process (freeze-drying). To overcome the risk of phage instability upon dehydration, the microspheres were prepared with the addition of 0.3 M mannitol. Bacteriophage-loaded microspheres were stored at room temperature for 30 days and subsequently exposed to simulated gastric fluid (SGF). The survival of encapsulated phages after drying was significantly higher in the presence of mannitol.

The highest number of viable bacteriophages exceeding 4.8 log₁₀ pfu/mL in SGF were recovered from encapsulated and freeze-dried microspheres, while phages in lyophilized lysate were completely inactivated. Although the method requires optimization, it may be a promising approach for the immobilization of bacteriophages in terms of practical application.

1. Introduction

Antimicrobial resistance of bacteria has in recent decades become a worsening global problem. In light of the limited efficacy of frontline antibiotics and the decreasing number of new antibacterial drugs introduced onto the market each year, innovative antibacterial strategies are urgently needed. In this context, the application of bacteriophages may be a promising approach.

The potential of bacteriophages in the treatment of bacterial infections was perceived over a century ago; however, in recent years, a real boost in the development of phage-based antibacterial strategies has been observed. Phage activity against multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) bacterial strains has been confirmed in many studies [1,2], and the potential of phage therapy in the “post-antibiotic era” has been noted [3,4,5,6].

Despite evidence of the efficiency of bacteriophages and the undisputed advantages of phage treatment over pharmacology-based antibacterial strategies, there are no specific regulatory agreements around phages at the moment [4]. Currently, no phage products are approved for human use by the European Medicines Agency (EMA) or the Food and Drug Administration (FDA) [7], and phage therapy is mostly practiced on a compassionate basis [8]. During the last two decades, significant progress in phage studies has been evident, but we still need to identify and fill gaps in the knowledge, complement experience, and optimize methods and practical tools to make this form of treatment accessible for patients.

One of the limitations in the application of phage preparations is the stability of the viruses during passage through the gastrointestinal tract. Although phage preparations may be easily used in the forms of aerosols, compresses, and ear or eye drops, the oral administration of bacteriophages is associated with the increased risk of treatment inefficiency due to phage inactivation in the stomach environment [9]. This increased sensitivity of bacteriophages to adverse stomach conditions entails the selection of highly resistant phages or the administration of drugs neutralizing acidic pH during therapy [10,11]. Another aspect is phage stability during storage. Phages, as with other protein-based macromolecules, are prone to protein misfolding, aggregation, and denaturation [12]. Storage in liquid forms at 4 °C seems to be the most effective; however, it may not be convenient for commercial approaches. Moreover, considering the literature data and laboratory experience, survivability in these conditions may also be highly variable across bacteriophages [12].
Research in recent years has shown that using different techniques of immobilization may significantly improve phage stability in the stomach environment as well as during storage [13,14]. However, the golden mean is needed—a method which would provide broadly defined phage stability and at the same time a formulation which is convenient and patient-friendly.

The objective of the study was to develop a method for obtaining a phage preparation in the form of a dry powder, making it possible to retain phage viability during storage and subsequently in an acidic stomach environment. We have combined two well-known techniques for phage immobilization. In the first stage, we encapsulated phages with an extrusion method. Then, to obtain the dry formulation, we used lyophilization (freeze-drying), a technique which is frequently used for the immobilization of molecules and microorganisms [14]. Since the dehydration of bacteriophage preparations may relate to significant titer loss, at the encapsulation stage, we used mannitol as an excipient, the protective properties of which we have demonstrated in previous studies [15]. Although the proposed method requires further optimization, we believe it may be a promising approach for the immobilization of bacteriophages in terms of practical application.

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2.2. Encapsulation of Bacteriophage T4

Ca-alginate microspheres were prepared using an extrusion method adapted from [15] and modified. Bacteriophages were encapsulated in alginate (Sigma-Aldrich, Darmstadt, Germany) or alginate with mannitol (Sigma Aldrich, Germany) capsules using encapsulator B-390 (Büchi Labortechnik AG, Flawil, Switzerland). Some 30 mL of T4 lysate was mixed with 90 mL of 2% water solution of sodium alginate. Then, the mixture was extruded through the nozzle with a diameter of 150 µm, 300 µm or 450 µm into the 0.1 M CaCl₂ or 0.1 M CaCl₂ supplemented with 0.3 M mannitol at 23 °C. The following parameters of the encapsulation process were used: air pressure 150 mbar, frequency 800 Hz, and stream height 30 cm. The beads were kept in the solution for 20 min and were allowed to harden. Then, the formed beads were washed with distilled water. The shape and diameter of the capsules were investigated under the light microscope (Zeiss Axio Vert.A1 for wet microspheres and Leica DMI1 for dry microspheres).

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Śliwka, P.; Skaradziński, G.; Dusza, I.; Grzywacz, A.; Skaradzińska, A. Freeze-Drying of Encapsulated Bacteriophage T4 to Obtain Shelf-Stable Dry Preparations for Oral Application. Pharmaceutics 2023, 15, 2792.
https://doi.org/10.3390/pharmaceutics15122792

excipients formulation