Baptista-Silva, Sara

Borges, Sandra

Freixo, Ricardo

Learmonth, David A.

Oliveira, Ana L.

Silva, Miguel

Soares, Gonçalo C.

English

Repositório Institucional da Universidade Católica Portuguesa

New Insights on Biopolymer Sterilization Using Supercritical CO2 TechnologyGonçalo C. Soaresa, Miguel Silvaa, Sandra Borgesa, Sara Baptista-Silvaa, Ricardo Freixoa, David A. Learmonthb, Mariana C. Vallejob, Rui. A. Sousab, Ana L. Oliveiraa*aUniversidade Católica Portuguesa, CBQF – Centro de Biotecnologia e Química Fina, Laboratório Associado, Escola Superior de Biotecnologia, Porto, PortugalbStemmatters, Biotecnologia e Medicina Regenerativa SA, Parque de Ciência e Tecnologia Avepark, Zona Industrial da Gandra, Guimarães, Portugal.* aloliveira@porto.ucp.ptIntroduction/Resume Materials and MethodsResultsConclusionsAcknowledgementsThe development of biomaterials represents a challenge to existing medical sterilization technologies, since they are oftencomposed of natural polymers which are sensitive to high temperatures. Supercritical CO2 (ScCO2) has recently been identified asan effective technique for the sterilization of thermally and hydrolytically sensitive polymers.1 ScCO2 sterilization can be achievedat low temperature and presents several advantages such as inertness, non-toxicity, high penetration ability and non-flammability.1 According to EN 556–1, a guaranteed sterility assurance level (SAL) is required.2 It has been demonstrated thatScCO2 combined with low molecular volatile additives can markedly improve bacterial endospores’ inactivation to reach therequired SAL3 Although progress has been done in this field demonstrating the efficacy of ScCO2 sterilization of severalbiopolymers, many are still to be studied. In this work, ScCO2 was used for sterilizing two proteins, silk sericin and fibroin and apolysaccharide, gellan gum. The applied ScCO2 sterilization process was modified from Herdegen et al.4, according to each of thestudied polymeric system. The biomaterials were inoculated with Bacillus cereus, a spore-forming bacteria, and after the ScCO2process, the microbiological analysis indicated an effective sterilization.Characterisation of biomaterials before/after ScCO2 sterilization, was performed by Scanning Electron Microscopy (SEM), FourierTransformed Infrared (FTIR), NMR and Fast Protein Liquid Chromatography (FPLC) for proteins. According to these techniques thephysical integrity, molecular structure and biological activity of the tested materials was maintained and no signs of degradationwere detected. The proposed ScCO2 treatments proved efficient for sterilizing the studied biopolymers without compromising itsproperties or performance.ScCO2 has been used to sterilize silk fibroin in the form of porous scaffolds and membranes while silk sericin and gellan gum were tested in theform of powder, after freeze-drying. In order to evaluate the sterilization efficacy, each sample have been inoculated with a bacterial solution ofBacillus cereus NCTC 2599, with a density superior to 106 colony forming unit (CFU)/ mL.ScCO2 sterilization proved to be effective against Bacillus cereus, which was guaranteed by a 10-6reduction of CFU/mL, according to SAL assay (Table 2).Table 2. Microbiological results of the different studied materials after 9 days of incubation at 37 ºC.FTIR analyses revealed that any physicochemical alteration occurred by means of ScCO2sterilization. As can be seen in Figures 2-4, FTIR bands are coincident before and after ScCO2sterilization for every sample tested out.This ScCO2 sterilization process also proved to be gentle with protein samples, since FPLCchromatograms (Figures 5 and 6) show no signs of degradation.1H NMR analyses revealed that no detectable structural alteration to the repeat tetrasaccharidebackbone of gellan gum occurred during ScCO2 sterilization, as clearly seen in Figures 7 (pre-treatment) and 8 (post-treatment).ScCO2 sterilization, under the addition of 2% hydrogen peroxide, successfully inactivated Bacillus cereus. Physicochemical properties and structuralintegrity and stability of fibroin, sericin and gellan gum were not compromised by this sterilization process. Thus, this ScCO2 sterilization process poses asa promising and viable alternative to the most conventional sterilization techniques.Figure 1. Schematic diagram of ScCO2 equipment. Legend: 1. CO2 bottle; 2. Thermostatic bath; 3. Injection pumps; 4. Needle valves; 5. Mass flow meter; 6. Heater resistors; 7. Pressure vessel with a stirrer and a refrigeration circuit, surrounded by a heater resistor; 8. Back pressure regulator valve with heater resistor; 9. Collection cup with heater resistor. #CyclesPressure range (bar)Δ Time (min)Temperature (ºC)Stirring (rpm)Sterilization aid1 0-245 2038 5002% (v/v) H2O2 30%2 80-245 203 80-245 204 80-245 205 80-245 206 80-245 20Table 1. Process conditions used for the ScCO2 sterilization process.Control After SterilizationMaterials: CFU/mL Log(CFU/mL) CFU/mL Log(CFU/mL)Sericin 2.4x107±5.6x106 7.4±0.10 <1.0x101 <1.0Fibroin 8.0x106±7.1x105 6.9±0.04 <1.0x101 <1.0Gellan gum 5.0x107±1.3x107 7.7±0.12 <1.0x101 <1.0Figure 2. FTIR corresponding to the fibroin membrane. Figure 3. FTIR corresponding to sericin.Figure 4. FTIR corresponding to gellan gum.Figure 5. FPLC chromatogram corresponding to fibroin membranes. Figure 6. FPLC chromatogram corresponding to sericin.Figure 7. 1H NMR spectrum of gellan gum before ScCO2sterilisation.Figure 8. 1H NMR spectrum of gellan gum after ScCO2sterilisation.National Funds from FCT - Fundação para a Ciência e a Tecnologia through project UID/Multi/50016/2013; FCT post doc fellowship to Baptista-Silva S. (SFRH/BPD/116024/2016), Program FCT Investigatorto A. L. Oliveira (IF/00411/2013), project SERICAMED (IF/00411/2013/CP1167), and through "Biotherapies- Bioengineered Therapies for Infectious Diseases and Tissue Regeneration", funded byNorte2020.References1. Sikin, A. & Rizvi, S. Recent patents on the sterilization of food and biomaterials by supercritical fluids. Recent Patents Food. Nutr. Agric. 3, 212–225 (2011).2. EN 556-1 Beuth Verlag, Burggrafenstr.3. Bernhardt, A. et al. Improved Sterilization of Sensitive Biomaterials with Supercritical Carbon Dioxide at Low Temperature. PLoS One 10, 1–19 (2015).4. Herdegen, V. et al. Sterilization of medical products from collagen by means of supercritical CO2. Chem. Eng. Technol. 37, 1891–1895 (2014).View publication stats

New insights on biopolymer sterilization using supercritical co2 technology

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New insights on biopolymer sterilization using supercritical co2 technology

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