Drug Loading of Biomedical Polymers Using Supercritical Carbon Dioxide

Rui Ping Hoo, Laura Poole-Warren, Fariba Dehghani and Neil Foster
Research Theme: Health, Sport & Entertainment

1. Introduction

Tissue engineering requires a highly interconnected porous scaffold to carry bioactive agents to the targeted sites without altering its activity.¹ Therefore, the fabrication method for the scaffold must take into consideration potential exposure to heat or organic solvents that might denature the biological agents. In this project, chitosan is used as the scaffold due to its excellent muco-adhesiveness, biocompatibility in animal tissues, and biodegradable properties.²

Supercritical fluids, which exhibit density and solubility properties similar to liquid and diffusivity and viscosity properties similar to gases, provides a novel processing method for porous polymers. SCF technology is a solvent free approach. The polymer is saturated with the SCF at high pressure, and then forced into a supersaturated state by depressurization and this leads to nucleation and growth of gas bubbles that are dispersed throughout the matrix.³ Hence, pore formation occurs.

There are several methods for loading protein into a polymer scaffold in tissue engineering applications, such as particulate leaching, emulsion freeze drying and others. However, these techniques involve organic solvents, which increase the possibility of leaving toxic residues and initiates biocompatibility problems.⁴ Moreover, bioactive agents are generally sensitive to pH and temperature.⁵ Therefore, SCF processing is highly recommended in fabricating porous polymer and drug loading applications.

2. Aim

• To examine the optimum processing parameters (temperature and depressurization time) for maximum porosity in fabricating chitosan scaffold
• To study two drug loading paths and to compare their efficiency

3. Method

Chitosan flakes were dissolved in acetic acid and filtered. The solution was then cast and dried under laminar flow. A thin film was formed and allowed to swell in water. The next step involved soaking in gradually increasing ethanol concentration to assist solvent exchange. The chitosan was then soaked in 100% ethanol overnight. After soaking, the chitosan was placed in a high pressure vessel (see Figure 1) and pressurized till 150bar. After that, the chitosan was isolated for 1 hour and prior to depressurization, the flow rate of SCF was increased to 10ml/min to wash out the ethanol. Lastly, the system was depressurized (for parameters refer Table 1).

Figure 1: High pressure stirred view vessel

The porosity of the chitosan was characterized with BET analysis and confirmed with SEM imaging. SEM images were taken at magnification 30,000× by Hitachi S4500.

When the optimum parameters were obtained, they were used for drug loading.

Method 1: 25mg lysozyme was loaded in 20% water, 80% ethanol, followed by supercritical fluid treatment.
Method 2: A cotton swab saturated with 20ml lysozyme (25mg) solution was placed on top of chitosan matrix for pressurizing and depressurizing.⁵

The amount of lysozyme loaded was quantified by UV spectrophotometry at 280nm.

4. Results and Discussion

BET Analysis and SEM Imaging

BET analysis measures the pore volume (for pore size less than 300nm) and the average pore size for a given processing parameter (refer Table 1).

Table 1: Pore volume and average pore diameter within Chitosan scaffold at various conditions. Operating pressure for all experiments was 150bar prior to depressurisation

Figure 2 shows the pores within the chitosan scaffold at different processing parameters.

  (a)

  (b)

  (c)

Figure 2: SEM image (30K magnification) of Chitosan processed at 40°C (a) 1min and (b)30 mins depressuristion, (c) at 50°C, 1 min depressurisation.

Drug Loading

Lysozyme was successfully loaded into chitosan scaffolds. The amount of lysozyme impregnated is shown as Table 2.

Table 2: Comparison of efficiency of two drug loading methods

The exchange solvent method is more effective as 6 times more lysozyme was impregnated compared with the cotton swab method.

5. Conclusion

• SCF processing method is solvent free and fast
• The optimum porosity is achieved when chitosan was processed at 40°C and depressurised in 30 minutes
• For drug loading, the exchange solvent method is more effective than the cotton swab method

6. Reference

1. Quirk R.A et al, 2003, ‘Supercritical Fluid Technologies and Tissue Engineering Scaffolds’, Current Opinion in Solid State and Materials Science, 313-321
2. Agnihotri S et al, 2004, ‘Recent Advances on Chitosan-based Micro and Nanoparticles in Drug Delivery, Journal of Controlled Release, 5-28
3. Barry J. J.A et al, 2003, ‘Porous methacrylate Scaffolds: Supercritical Fluid Fabrication and In Vitro Chondrocyte Responses’, Biomaterials, 3559-3568
4. Mathieu L.M. et al, 2005, ‘Bioresorbable Composites Prepared by Supercritical Fluid Foaming’, Wiley Interscience, 89-97
5. Sproule T.L. et al, 2003, ‘Bioactive Polymer Surfaces Via Supercritical Fluids’, Journal of Supercritical Fluids, 241-248

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