MALDI-TOF technique to study process-induced degradation of bioabsorbable polymers

摘要

Introduction: Bioabsorbable polymers like poly(lactic acid) (PLA) are attractive biomatenals for regenerative medicine and tissue engineering applications due to their inherent property of in vivo resorption over time. However, the challenge is to be able to better understand and predict the degradation profile and the formation of by-products for bioabsorbable polymers. The problem is further compounded by the fact that these polymers are sensitive to processing conditions, such as temperature and moisture, which cause pre-mature degradation. In order to study process-induced degradation of PLA, we propose the use of Matrix-Assisted Laser Desorption /lonization Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS). MALDI-TOF technique has been widely used for the characterization of proteins and peptides, but recently, the technique has been used for synthetic polymers as well. In this study, PLA fibers were formed using melt-spinning process and the process parameters were varied. The fibers were then characterized using MALDI-TOF technique to study the extent and the by-products of process-induced degradation. Materials and Methods: NatureWorks PLA polymer (6100 HP) was used for the experimental melt-spinning process to form PLA multifilament yarns. The effects of extrusion temperature (220 °C vs. 250 °C), and the residence time in the extruder (3 minutes vs. 6 minutes) were considered for the study. The choice of the matrix, the solvent for the matrix and the analyte and the ratio of the matrix and the analyte are crucial parameters for good-quality and reliable MALDI measurements. We followed the protocol described here. Individual solutions of the analyte (PLA) and the matrix (2,5-dihydroxybenzoic acid (DHB)) were prepared in tetrahydrofuran (THF) at a concentration of 10 g/L. The analyte and the matrix were mixed in the ratio 1:5 (v/v) and the mass spectra were obtained using AB Sciex 5800 MALDI-TOF mass spectrometer. Results: The MALDI spectrum corresponding to 22 repeat units of PLA is shown in Fig. 1. This is an example that enables the identification of the cyclic and the linear oligomeric species summarized in Table-1. The predominant species found for PLA were, in order of abundance: (ⅰ) cyclic [LA_c]_n. mainly present as Na~+ adducts ([M+Na]~+: m/z 1608.3) and in smaller quantities, H~+ adducts ([M+H]~+: 1585.3 m/z); (ⅱ) linear H-[LA_L]_n-OH, which bears a hydroxyl and a carbonyl end groups ([M+Na]~+: m/z 1626.3 and [M+H]~+: m/z 1604.3); and, (ⅲ) linear H-[LA_L]_n-O-CH_3, which has a methoxyl group and a hydroxyl group bearing ([M+Na]~+: m/z 1640.3 and [M+H]~+: m/z 1618.3), commonly found with PLAs obtained by ring opening polymerization (ROP) due to the use of alkoxydic initiators, which remain in the terminal units, other low predominant species found that might be formed as by-products at higher processing temperatures were: (iv) linear CH3-O-[LAL]n-CH3, bearing two methoxyl groups ([M+Na]~+: m/z 1654.4 and [M+H]~+: m/z 1632.4); and (v) linear CH_3-CO-O-[LA_L]_n-H, which has a hydroxyl and a carboxylic methyl ester groups as end units ([M+Na]~+: m/z 1668.4 and [M+H]~+: m/z 1646.4), that might appear due to esterification reactions from different chain capping routes during polymerization. Different species corresponding to different m/z values would be formed under different processing conditions, which can be used to determine the extent of degradation. Conclusion: Different spectra from PLA samples produced under different processing conditions can be compared to determine the extent of process-induced degradation and analyze the by-products in the form of oligomeric species formed under each processing condition. Thus, the MALDI-TOF technique can facilitate a rapid quantitative study of degradation of bioabsorbable polymers at various stages of processing. A better understanding of process-induced degradation will help to design absorbable biomaterials, such as scaffolds, with better control of their in vivo performance.