Purpose: In spite of increased effort in control, tuberculosis remains one of the world most devastating diseases. As such, it has been communicated in public health circles, that efficient control of the disease will depend on a combination of effective vaccination and treatment. However, vaccine formulation development studies on Mycobacterium tuberculosis have been found to be very challenging. This is because the bacterium has a generation time of about 24 hours and requires four to six weeks to yield appreciable colonies. In addition, the biosafety level 3 containment imposes stringent laboratory restrictions, as such, research on the bacterium is restricted to a few well equipped facilities. As a solution, Mycobacterium smegmatis, a saprophytic member of the species has been used as surrogate in vitro model for Mycobacterium tuberculosis in a number of studies. It offers technical benefits such as shorter generation time and has none of the pathogenic properties of Mycobacterium tuberculosis and therefore, poses negligible risk to laboratory workers. Microparticle technology is an established technique that has been used to deliver several different types of drugs, including antigens by systemic and mucosal administration. This strategy allows the targeting of the immunizing antigens, incorporated in the microparticles, to appropriate antigen presenting cells (APCs). The microparticles can function as an adjuvant by presenting the antigens in a particulate form and, depending on its size (1-10 μm), the capability of being more readily taken up by professional antigen presenting cells, such as dendritic cells and macrophages. However, for effective use of microparticles to deliver cells and proteins, the formulation strategy must be such that the active therapeutic agent must be stable during the formulation process and over the shelf-life of the product until it reaches the target organ. The objective of this project, therefore, was to encapsulate Mycobacterium smegmatis in Chitosan/Sodium alginate microparticles and to evaluate, the physico-chemical characteristics of the microparticles, the ability of the bacterium to survive the formulation process, and the bacterium’s cell viability in the non-lyophilized microparticles at various storage temperatures. The aim is to evaluate the possibility of using the formulation process as a platform for systemic and mucosal vaccine delivery system for Mycobacterium tuberculosis antigens. Chitosan and Sodium alginate are biodegradable and biocompatible polymers with mucoadhesive properties that are ideal for both systemic and mucosal delivery of vaccines.
Methods: Mycobacterium smegmatis (ATCC® 14468TM strain and cell count of 3 x 108 CFU) was encapsulated in sodium alginate and chitosan co-polymer matrix at a ratio 4.23:1 respectively through polyionic complexation method at high speed stirring with TPP as the crosslinking agent. Tween 80 at a concentration of 1% was added to the polymer matrix to produce defined microparticles and to de-aggregate the cells before use. This formulation method avoids the employment of high temperatures or organic solvents that have the potential to damage the activity of biopharmaceutical drug candidates such as living cells. After the formulation, the particle sizes and size distribution, zeta potential, and product yield of the microparticles as well as the encapsulation efficiency of the bacterial cells in the microparticles were determined. The microparticles were stored at 40C and -200C without lyophilization and cryoprotectants after zero-time subculturing. Preliminary cell viability studies were done by sub-culturing the encapsulated cells at zero time, 72 hours and 8 days.
Results: The average particle size of 4.74 ± 0.79 microns, size range of 3.23 to 9.55 microns, and zeta potential of -28.3 ± 6.4 mV is within the range recommended for uptake by antigen-presenting cells in vaccine delivery. The use of normal magnetic stirrer instead of ultra-sonication resulted in large size range. The product yield was 78.6%. The high yield percentage shows that most of the chitosan was used up. The encapsulation efficiency was determined to be 84.0 ± 3.7 %. This means that most of the cells were captured in the polymer matrix. The microparticle cultures showed cell growth at all the storage temperatures for the 8 days. Plate count showed no significant difference in cell count for all the time points and storage temperatures.
Conclusion: The cell viability studies showed that the formulation process did not have any negative effect on the viability of the encapsulated cells. The study also showed that microparticles without cryoprotectants are capable of maintaining the cell viability at fridge temperature for 8 days. The physico-chemical properties of the microparticles are ideal for delivery of mycobacterium cells for vaccine administration.
1. Caetano et al (2016): Effect of experimental parameters on Alginate/Chitosan microparticles for BCG encapsulation. Marine Drugs, 14:90
2. Yeboah KG and D’Souza MJ (2009) Evaluation off albumin microspheres as oral delivery system for Mycobacterium tuberculosis vaccines
Jordan Stubblefied– PharmD candidate, Harding University, Searcy, Arkansas
Jacob Hatvany– Teaching Assistant, Harding University College of Pharmacy, Searcy, Arkansas
Amber Hug– Assistant Professor, Harding University, Searcy, Arkansas
Aladin Siddig– Associate Professor, University of Charleston, Charleston, West Virginia