ALGINATE MICRO-BIOREACTORS DELIVERING PROTEINS: AN EXAMPLE DRUG DELIVERY SYSTEM

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ALGINATE MICRO-BIOREACTORS DELIVERING PROTEINS: AN EXAMPLE DRUG DELIVERY SYSTEM Chen, Guisang Daphne and Wang, Chi-Hwa Chemical & Biomolecular Engineering National University of Singapore 1 Lower

1 ALGINATE MICRO-BIOREACTORS DELIVERING PROTEINS: AN EXAMPLE DRUG DELIVERY SYSTEM Chen, Guisang Daphne and Wang, Chi-Hwa Chemical & Biomolecular Engineering National University of Singapore 1 Lower Kent Ridge Road, Singapore Introduction In this project, a novel treatment concept against brain tumors is proposed. It comprises of alginate bioreactors encapsulating PEX protein which secretes QM7 cells with anti-angiogenic and anti-tumor properties. These can be implanted intracranially. Focus would be placed on the egress of a similar protein, carbonic anhydrase(cab). QM7 cells are able to survive through a long period of time even in low level of serum, while sustaining protein production to achieve therapeutic effects. These micro-beads consist of an alginate core, coacervated by chitosan, and crosslinked with genipin. They would resemble a core-shell structure under confocal laser scanning microscope. A model of the core-shell structure is illustrated in Figure 1 below. Figure 1: Illustration on a genipin cross-linked alginate-chitosan micro-bead Permeability of the membrane would allow efficient inward diffusion of nutrients and oxygen, egress waste to sustain cell proliferation, permit outward diffusion of secreted proteins into the target site and provide immunoisolation to avoid inflammatory reactions. It can be controlled by the extent of covalent bonding with genipin. Electrohydrodynamic Atomization (EHDA) process would be used to fabricate the alginate beads since it can provide greater control over particle size, distribution and morphology of the beads. In the scope of this project, extensive optimization study has been done to customize the sizes of GCCA (Genipin-Crosslinked-Chitosan- Alginate) micro-beads into 3 different groups (00μm, 500μm and 1000μm) by adjusting operating conditions of EHDA. These three groups of micro-beads are further subjected to 3 different lengths of reaction time with genipin (6hours, 1hours and 18 hours) as shown below. Reaction time with Genipin /hrs Diameters of micro-particles /μm

2 Table 1: Labels of the 9 groups of micro-beads. The objective of the study is to characterise the mass transfer of CAB with respect to the various sizes and membrane thicknesses of the solid core micro-beads. Materials and Methods Materials used The materials used in the experiment are Sodium Alginate of low viscosity (Lot 03K0191) and Carbonic Anhydrase (Lot 058K1031) purchased from Sigma-Aldrich, Calcium Chloride Dehydrate purchased from Merck KGaA, Chitosan 5 (Lot TSQ4638) and Genipin (Lot PEJ5398) purchased from Wako Pure Chemical Industry Ltd, and Sterican (by Braun) needle (G), which was used as the nozzle. Fabrication of micro-beads Calcium-alginate core 1.5% weight by volume sodium alginate solution was prepared by dissolving 0.375g of low viscosity sodium alginate powder in 5ml of deionized (DI) water through means of sonication for 100 hours. Sodium alginate solution was filtered and dripped into % CaCl solution using EHDA setup shown below. Figure : EHDA set-up The various EHDA conditions for the different particle sizes were set as follows: Size of particles/microns V nozzle /kv V ring /kv Flow-rate/ (ml/hr) Table : EHDA operating conditions. 10mg/ml of chitosan was stirred mechanically with 11mg/ml of CaCl in 1% acetic acid, and ph of the solution is adjusted to using 1M NaOH solution. ph of the solution was measured using a ph meter at room temperature. Micro-beads are separated from CaCl gelling bath by filtering out CaCl solution. These microbeads are subsequently thoroughly washed with deionized water. Micro-beads were added into the Petri-dish

3 containing 30ml of chitosan/cacl /acetic acid solution. The petri dishes are placed on the shaker for 15minutes (00μm micro-beads) or 30 minutes (500μm or 1000μm micro-particles). 30ml of chitosan/cacl /acetic acid solution was added to ensure chitosan was supplied in excess for consistency. Thereafter, micro-beads are washed thoroughly using deionized water to remove traces of chitosan on the surface. Cross-linking of chitosan with genipin 5mg/ml genipin solution was prepared by dissolving genipin powder into deionized water through means of sonication for 1 hour. Micro-beads were added into the Petri-dish containing 30ml of genipin solution. The petri-dishes are placed inside the incubator with conditions of 37 o C and 100 rpm for the various desired lengths of reaction time (6hours, 1hours and 18hours). 30ml of genipin solution added in is in excess to ensure the reaction proceeds in a time-dependent manner. Mass transfer study Preparation of micro-tubes Micro-beads of the various sizes with the various incubation hours were collected and transferred to1.5ml micro-tube. Sufficient 1.7mg/ml CAB solution was added to the micro-tube to ensure a 1:1 ratio of volume of micro-particles to volume of CAB solution. Ingress of CAB PBS solution was used as blank in the mass transfer study. CAB solution was sampled and its protein concentration is measured using Protein A80 module in Nanodrop Concentration of the CAB solution was recorded as the initial concentration of the solution outside the micro-beads in the micro-tube. The solution outside the micro-particles was sampled and its relative concentration was noted after equilibrium is reached. Egress of CAB Similar to ingress of CAB, the concentrations of the solution outside the micro-particles in the micro-tube could be noted using Protein A80 module in Nanodrop A syringe was used to remove all the CAB solution in the micro-tube. Sufficient PBS solution was added to the micro-tube to ensure a 1:1 ratio of volume of microparticles to volume of PBS solution. Initial concentration of the solution outside the micro-particles in the micro-tube was taken to be 0mg/ml for all samples. Diffusion of CAB out of the micro-particles was investigated by shaking the micro-tubes at 37 o C, 100 rpm during a one hour period. The solution outside the micro-particles was sampled in 0.5 th, 1 st, nd, 3 rd, 5 th, 8 th, 10 th, 15 th, 0 th, 30 th, 45 th, 60 th minute, with their relative concentrations recorded.

4 RESULTS AND DISCUSSION Governing equation for mass transfer The following equation is used for curve fitting as the governing equation accounting for the egress of CAB (1) Where, is the protein concentration in the dispersing medium at time t (mg/ml), Assumptions is the protein concentration in the dispersing medium at the equilibrium state (mg/ml), is the characteristic time (min), and t is the time (min). 1. The interior of the microbeads can be assumed to be well mixed with identical solute concentration, C everywhere within the microbeads at any time.. No marker adsorption on the surface of the microbeads. Determination of τ τ is the characteristic time which can be expressed as: () It can be observed from equation () that the diffusion constant, D m, is inversely proportional to the characteristic time, τ, and directly proportional to the radius, r, and thickness of the membrane, l. Matlab provides a curve fitting toolbox that can be used to determine τ. Therefore, the experimental data is entered into Matlab. A graph of / against t is plotted and is fitted with equation (1). From this curve fitting toolbox, τ and R can be obtained in which R will show the goodness of the fit of equation (1) for the experimental data obtained τ (min) R Discussion on τ values obtained Table 3: Values of τ and R for Egress of CAB Studies has shown that the greater the duration of cross-linking of chitosan with genipin, the denser the cross-linking of the chitosan-genipin membranes on the micro-beads will be. With denser cross-linked membrane, diffusion coefficient will be lower and thus, τ will be higher. This is correctly reflected in most of the data obtained, except for micro-beads 51. This could be due to experimental errors due to the NanoDrop equipment or the sample used in this particular study is not of the correct size or the sample has a very wide size

5 distribution. Several readings should be taken while using the NanoDrop equipment so as to reduce random errors. Discussion on R values obtained R values obtained are useful for evaluating the goodness of fit of equation (1) for the experimental data obtained. The closer the R value obtained is to 1, the better the obtained data is described by equation (1). From Table 3, it can be observed that most of the τ values for the egress for CAB have R values that are close to 1, reflecting reasonable accuracy in the τ values obtained from Matlab. However, this is not true for micro-beads with size of 1000 microns. Discussion on the micro-beads with size of 1000 microns It was hypothesized that the core resistance of the largest micro-particles among the samples has a significant effect on the egress of CAB due to their large core. Therefore, samples of 1000 microns sized micro-beads without membranes were formed and tested. The results obtained are as follows: Cs(t)/ mg/ml t/min Figure : C s (t)/ mg/ml versus t/min for 1000 microns micro-particles without membrane The non-linear graph above proves that the hypothesis is right that the core resistance of 1000 microns size micro-particles caused the data of the egress of CAB for micro-particles with 1000microns size to be poorly described by equation (1), resulting in R values obtained to deviate from 1 significantly. On the other hand, while there are still core resistance effects on the micro-particles with 00 and 500 microns sizes, they do not cause the R values to differ from 1 by a large extent as the size of the micro-particles are relatively much smaller than the 1000microns sized ones. This is because smaller micro-particles have smaller cores, and thus smaller and less significant core resistances. Conclusion In the scope of this project, main interests have been developed to characterise the mass transfer of CAB with respect to the various sizes and membrane cross-linking densities of the solid core micro-beads. This aim has been achieved by finding the characteristic time, τ, and R values from the experimental results obtained for the egress of CAB. In addition, efforts also have been made to further study on the exceptional results obtained for the micro-particles of 1000microns in size. It can be inferred that from the results of mass transfer study, 506 is deemed most suitable for cell encapsulation based on its best fit to theoretical equation (1). Future study could be made to explore on the use of liquid alginate core. This may eliminate the core resistance factor affecting the egress of CAB.

6 Acknowledgements This work was supported by BMRC grant (R ). I thank the colleagues and collaborators who have contributed to the development of this work and Mr. Sudhir Ranganath who has reviewed the manuscript. References 1. Insight into permeability of protein through microcapsule membranes Journal of Membrane Science, Volume 69, Issues 1-, 1 February 006, Pages Wentao Qi, Juan Ma, Yingwei Liu, Xiudong Liu, Ying Xiong, Yubing Xie, Xiaojun Ma. Permeability of azo-dye through poly(urea-urethane) microcapsule membrane Journal of Membrane Science, Volume 13, Issues 1-, 1 March 003, Pages 5-31 Takashi Sato, Takao Yamamoto, Seiji Shibako, Kimio Ichikawa, Toshiaki Dobashi

Drug Delivery with Alginate Dr. J. Vernengo and Dr. S. Farrell

Objectives Drug Delivery with Alginate Dr. J. Vernengo and Dr. S. Farrell Define a hydrogel. Define the chemical structure and ionic crosslinking of alginate to form hydrogels. Discuss the role of hydrogels