High-throughput fabrication of antimicrobial phage microgels and example applications in food decontamination.

Engineered by nature, biological entities are exceptional building blocks for biomaterials. These entities can impart enhanced functionalities on the final material that are otherwise unattainable. However, preserving the bioactive functionalities of these building blocks during the material fabrication process remains a challenge. We describe a high-throughput protocol for the bottom-up self-assembly of highly concentrated phages into microgels while preserving and amplifying their inherent antimicrobial activity and biofunctionality. Each microgel is comprised of half a million cross-linked phages as the sole structural component, self-organized in aligned bundles. We discuss common pitfalls in the preparation procedure and describe optimization processes to ensure the preservation of the biofunctionality of the phage building blocks. This protocol enables the production of an antimicrobial spray containing the manufactured phage microgels, loaded with potent virulent phages that effectively reduced high loads of multidrug-resistant Escherichia coli O157:H7 on red meat and fresh produce. Compared with other microgel preparation methods, our protocol is particularly well suited to biological materials because it is free of organic solvents and heat. Bench-scale preparation of base materials, namely microporous films (the template for casting microgels) and pure concentrated phage suspension, requires 3.5 h and 5 d, respectively. A single production run, that yields over 1,750,000 microgels, ranges from 2 h to 2 d depending on the rate of cross-linking chemistry. We expect that this platform will address bottlenecks associated with shelf-stability, preservation and delivery of phage for antimicrobial applications, expanding the use of phage for prevention and control of bacterial infections and contaminants.

Functionalization of 3D Printed Scaffolds Using Polydopamine and Silver Nanoparticles for Bone-Interfacing Applications.

The prevention of bacterial colonization and the stimulation of osseointegration are two major requirements for bone-interfacing materials to reduce the incidence of complications and promote the restoration of the patient's health. The present investigation developed an effective, two-step functionalization of 3D printed scaffolds intended for bone-interfacing applications using a simple polydopamine (PDA) dip-coating method followed by the formation of silver nanoparticles (AgNPs) after a second coating step in silver nitrate. 3D printed polymeric substrates coated with a ∼20 nm PDA layer and 70 nm diameter AgNPs proved effective in hindering Staphylococcus aureus biofilm formation, with a 3000-8000-fold reduction in the number of bacterial colonies formed. The implementation of porous geometries significantly accelerated osteoblast-like cell growth. Microscopy characterization further elucidated homogeneity, features, and penetration of the coating inside the scaffold. A proof-of-concept coating on titanium substrates attests to the transferability of the method to other materials, broadening the range of applications both in and outside the medical sector. The antibacterial efficiency of the coating is likely to lead to a decrease in the number of bacterial infections developed after surgery in the presence of these coatings on prosthetics, thus translating to a reduction in revision surgeries and improved health outcomes.

Spray Drying and Particle Engineering in Dosage Form Design for Global Vaccines.

Vaccines are a very important tool in the effort to reduce the global burden of infectious diseases. Modern vaccines can be formulated in several ways to induce specific immunity, including through the use of live bacteria, subunit antigens, and even genetic material. However, vaccines typically need to be transported and stored under controlled refrigerated or frozen conditions to maintain potency. This strict temperature control is incompatible with the available infrastructure in many developing countries. One method of improving the thermostability of a vaccine is through drying of a liquid presentation into a dry dosage form. In addition to enhancing the capability for distribution in resource-poor settings, these dry vaccine forms are more suitable for long-term stockpiling. Spray drying is a drying method that has been successfully used to stabilize many experimental vaccines into a dry form for storage above refrigerated temperatures. Additionally, the use of spray drying allows for the production of engineered particles suitable for respiratory administration. These particles can be further designed for increased out-of-package robustness against high humidity. Furthermore, there are already commercial dry powder delivery devices available that can be used to safely deliver vaccines to the respiratory system. The research in this field demonstrates that the resources to develop highly stable vaccines in flexible dosage forms are available and that these presentations offer many advantages for global vaccination campaigns.

Stress Exposure of Evolved Bacteriophages under Laboratory versus Food Processing Conditions Highlights Challenges in Translatability.

Bacterial viruses, or bacteriophages, are highly potent, target-specific antimicrobials. Bacteriophages can be safely applied along the food production chain to aid control of foodborne pathogens. However, bacteriophages are often sensitive to the environments encountered in food matrices and under processing conditions, thus limiting their applicability. We sought to address this challenge by exposing commercially available Listeria monocytogenes bacteriophage, P100, to three stress conditions: desiccation, elevated temperature, and low pH, to select for stress-resistant bacteriophages. The stressed bacteriophage populations lost up to 5.1 log10 in infectivity; however, the surviving subpopulation retained their stress-resistant phenotype through five passages with a maximum of 2.0 log10 loss in infectivity when exposed to the same stressor. Sequencing identified key mutation regions but did not reveal a clear mechanism of resistance. The stress-selected bacteriophage populations effectively suppressed L. monocytogenes growth at a modest multiplicity of infection of 0.35-0.43, indicating no trade-off in lytic ability in return for improved survivability. The stressed subpopulations were tested for survival on food grade stainless steel, during milk pasteurization, and within acidic beverages. Interestingly, air drying on stainless steel and pasteurization in milk led to significantly less stress and titer loss in bacteriophage compared to similar stress under model lab conditions. This led to a diminished benefit for stress-selection, thus highlighting a major challenge in real-life translatability of bacteriophage adaptational evolution.

On the feasibility of spray-dried eudragit-trehalose microparticles for enteric drug delivery.

Enteric infections have long constituted a silent epidemic responsible for hundreds of thousands of deaths around the world every year. Because of the global rise in antibiotic-resistant bacteria and the slow development of new small-molecule antibiotics, alternatives such as bacteriophage therapy have become a much sought-after option in the treatment of enteric infections. However, the administration of therapeutics through the oral route to target gastrointestinal infections poses challenges to dosage formulation because these active ingredients, particularly relatively fragile biological entities, require protection from the stomach's harsh acids. Encapsulation of the therapeutics within a pH-responsive coating capable of surviving low pH conditions has the potential to provide such protection. In this study, we developed a spray-dried powder vehicle capable of withstanding low pH comparable to stomach conditions, using Eudragit® S100 as a protective particle coating and trehalose as a stabilizing excipient for a possible active component. A particle formation model and a monodisperse droplet chain technique were initially used to study the formation process of Eudragit-trehalose composite microparticles at different ratios and in different ratios of water-ethanol solvent, which showed formation of particles with Eudragit shells varying in thickness from 0.13 µm to 0.75 µm. Promising Eudragit-trehalose formulations were subsequently spray-dried and their survival in acidic and alkaline environments studied using a new shadowgraphic imaging method. The results demonstrated that Eudragit was capable of creating a protective shell in the particles irrespective of the type of solvent used to prepare the formulations. The trehalose cores of particles with higher than 5% w/w of Eudragit remained protected after one hour of exposure at pH 2, indicating the potential of Eudragit-trehalose formulations for enteric delivery of drugs.

Evaluation of the stability of a spray-dried tuberculosis vaccine candidate designed for dry powder respiratory delivery.

Particle engineering via spray drying was used to develop a dry powder presentation of an adjuvanted tuberculosis vaccine candidate. This presentation utilizing a trileucine-trehalose excipient system was designed to be both thermostable and suitable for respiratory delivery. The stability of the spray-dried vaccine powder was assessed over one year at various storage temperatures (-20, 5, 25, 40, 50 °C) in terms of powder stability, adjuvant stability, and antigen stability. A formulation without trileucine was included as a control. The results showed that the interior particle structure and exterior particle morphology of the powder was maintained for one year at 40 °C, while the control case exhibited a small extent of particle fusing under the same storage conditions. Moisture content was maintained, and powder solid state remained amorphous for all storage temperatures. Aerosol performance was assessed with a commercial dry powder inhaler in combination with a human mouth-throat model. The emitted dose and lung dose were maintained for all samples after one year at temperatures up to 40 °C. Nanoemulsion size and oil content of the adjuvant system were maintained after one year at temperatures up to 40 °C, and the agonist content was maintained after one year at temperatures up to 25 °C. The antigen was completely degraded in the control formulation at seven months of storage at 40 °C; by contrast, 45% of the antigen was still present in the trehalose-trileucine formulation after one year of storage at 50 °C. Comparatively, the antigen was completely degraded in a liquid sample of the vaccine candidate after only one month of storage at 37 °C. The spray-dried trehalose-trileucine vaccine powder clearly maintained its inhalable properties after one year's storage at high temperatures and improved overall thermostability of the vaccine.

Microparticle encapsulation of a tuberculosis subunit vaccine candidate containing a nanoemulsion adjuvant via spray drying.

Spray drying is a technique that can be used to stabilize biopharmaceuticals, such as vaccines, within dry particles. Compared to liquid pharmaceutical products, dry powder has the potential to reduce costs associated with refrigerated storage and transportation. In this study, spray drying was investigated for processing an adjuvanted tuberculosis subunit vaccine, formulated as an oil-in-water nanoemulsion, into a dry powder composed of microparticles. Applying in-silico approaches to the development of formulation and processing conditions, successful encapsulation of the adjuvanted vaccine within amorphous microparticles was achieved in only one iteration, with high retention (>90%) of both the antigen and adjuvant system. Moisture-controlled stability studies on the powder were conducted over 26 months at temperatures up to 40 °C. Results showed that the powder was physically stable after 26 months of storage for all tested temperatures. Adjuvant system integrity was maintained at temperatures up to 25 °C after 26 months and after one month of storage at 40 °C. The spray-dried product demonstrated improved antigen thermostability when stored above refrigerated temperatures as compared to the liquid product. These results demonstrate the feasibility of spray drying as a method of encapsulating and stabilizing an adjuvanted vaccine.

Development and Testing of a Spray-Dried Tuberculosis Vaccine Candidate in a Mouse Model.

Converting a vaccine into a thermostable dry powder is advantageous as it reduces the resource burden linked with the cold chain and provides flexibility in dosage and administration through different routes. Such a dry powder presentation may be especially useful in the development of a vaccine towards the respiratory infectious disease tuberculosis (TB). This study assesses the immunogenicity and protective efficacy of spray-dried ID93+GLA-SE, a promising TB vaccine candidate, against Mycobacterium tuberculosis (Mtb) in a murine model when administered via different routes. Four administration routes for the spray-dried ID93+GLA-SE were evaluated along with relevant controls-1) reconstitution and intramuscular injection, 2) reconstitution and intranasal delivery, 3) nasal dry powder delivery via inhalation, and 4) pulmonary dry powder delivery via inhalation. Dry powder intranasal and pulmonary delivery was achieved using a custom nose-only inhalation device, and optimization using representative vaccine-free powder demonstrated that approximately 10 and 44% of the maximum possible delivered dose would be delivered for intranasal delivery and pulmonary delivery, respectively. Spray-dried powder was engineered according to the different administration routes including maintaining approximately equivalent delivered doses of ID93 and GLA. Vaccine properties of the different spray-dried lots were assessed for quality control in terms of nanoemulsion droplet diameter, polydispersity index, adjuvant content, and antigen content. Our results using the Mtb mouse challenge model show that both intranasal reconstituted vaccine delivery as well as pulmonary dry powder vaccine delivery resulted in Mtb control in infected mice comparable to traditional intramuscular delivery. Improved protection in these two vaccinated groups over their respective control groups coincided with the presence of cytokine-producing T cell responses. In summary, our results provide novel vaccine formulations and delivery routes that can be harnessed to provide protection against Mtb infection.

Development of a formulation platform for a spray-dried, inhalable tuberculosis vaccine candidate.

Protection against primarily respiratory infectious diseases, such as tuberculosis (TB), can likely be enhanced through mucosal immunization induced by direct delivery of vaccines to the nose or lungs. A thermostable inhalable dry powder vaccine offers further advantages, such as independence from the cold chain. In this study, we investigate the formulation for a stable, inhalable dry powder version of ID93 + GLA-SE, an adjuvanted subunit TB vaccine candidate, containing recombinant fusion protein ID93 and glucopyranosyl lipid A (GLA) in a squalene emulsion (SE) as an adjuvant system, via spray drying. The addition of leucine (20% w/w), pullulan (10%, 20% w/w), and trileucine (3%, 6% w/w) as dispersibility enhancers was investigated with trehalose as a stabilizing agent. Particle morphology and solid state, nanoemulsion droplet size, squalene and GLA content, ID93 presence, and aerosol performance were assessed for each formulation. The results showed that the addition of leucine improved aerosol performance, but increased aggregation of the emulsion droplets was demonstrated on reconstitution. Addition of pullulan preserved emulsion droplet size; however, the antigen could not be detected after reconstitution. The trehalose-trileucine excipient formulations successfully stabilized the adjuvant system, with evidence indicating retention of the antigen, in an inhalable dry powder format suitable for lung delivery.

Skin Stretch Enhances Illusory Movement in Persons with Lower-Limb Amputation.

Performance of lower limb prostheses is related not only to the mechanical design and the control scheme, but also to the feedback provided to the user. Proprioceptive feedback, which is the sense of position and movement of one's body parts, can improve the utility as well as facilitate the embodiment of the prosthetic device. Recent studies have shown that proprioceptive kinesthetic (movement) sense can be elicited when non-invasively vibrating a muscle tendon proximal to the targeted joint. However, consistency and quality of the elicited sensation depend on several parameters and muscle tendons after lower limb amputation may not always be accessible. In this study, we developed an experimental protocol to quantitatively and qualitatively assess the elicited proprioceptive kinesthetic illusion when non-invasively vibrating a muscle belly. Furthermore, we explored ways to improve consistency and strength of the illusion by integrating another non-invasive feedback method, namely cutaneous information manipulation via skin stretch. Our preliminary results from tests conducted with a person with transtibial (below knee) amputation show that stretching skin while vibrating a muscle belly on the residual limb provided a stronger and more consistent kinesthetic illusion (90%) than only vibrating the muscle (50%). In addition, we found that stretching skin enhances the range (1.5 times) and speed (3.5 times) of the illusory movement triggered by muscle vibration. These findings may enable the development of mechanisms for controlling feedback parameters in closing the control loop for various walking routines, which may improve performance of lower limb prostheses.