Effect of polymer molecular weight
The impact of the polymer molecular weight was investigated by comparing the following 50/50, “low” and “intermediate” molecular weight, acid-terminated PLGAs which were formulated into thin films: Expansorb® DLG 50-2A, Expansorb® DLG 50-3A, Expansorb® DLG 50-5A, Resomer® RG 502H, Resomer® RG 503H, and Resomer® RG 504H (Table 1). For each of these polymers, the water uptake was generally similar by 3 weeks of incubation, but the lower molecular weight films revealed an increased water uptake during the first two weeks (Fig. 1a,d). Expansorb® DLG 50-5A, the highest molecular weight of the three, appeared to have two phases of molecular weight decline, a slower decline in the first 7 days followed by a relatively faster decline for the next two weeks. Comparing the apparent first order rate of degradation of Expansorb® DLG 50-5A with Expansorb® DLG 50-3A, and Expansorb® DLG 50-2A films in the initial phase determined over the first 7 days of incubation using a least squared linear regression analysis (data not shown), as the starting molecular weight decreased, the initial apparent first order rates of degradation increased, 0.072, 0.091 and 0.092 days− 1, respectively. Over the entire incubation period, the overall rate of degradation for Expansorb® DLG 50-5A films increased, 0.086 days− 1, resulting in similar degradation half-lives for all three polymer films, ~8 days (Fig. 1a-c, Table 2). The lower molecular weight of Expansorb® DLG 50-2A had a significant effect on the onset of erosion (ton), with a ton=1.3 days compared to Expansorb® DLG 50-5A, ton=11.8 days (Table 3). Similar trends were observed with the three Resomer® polymers (Fig. 1d-f). These results are expected, as the effect of molecular weight on polymer degradation and erosion is well-known . We expect the degradation rate of PLGA to remain roughly constant (i.e., log(Mw) linear with time) with a decline in molecular weight until the molecular weight falls below some critical value (e.g., in the vicinity of ~20 kDa as previously reported ), and the polymer chains become far more mobile and the time to reach a soluble polymer oligomer shortens. Hussman et al. previously observed that regardless of the initial molecular weight, polymer microspheres or implants reach a critical molecular weight, around 10-20 kDa, at which point the formulations collapse and undergo significant constant erosion [28–30]. The starting molecular weight does not appear to influence this critical molecular weight, but the time that it takes to reach the critical molecular weight increases with increasing molecular weight. We observed similar behaviors with our polymer formulations, the critical molecular weight ranged from 5-20 kDa (data not shown) and polymers with similar properties behaved similarly. The time to reach this critical molecular weight for our formulations ranged from 7-28 days with low molecular weight, acid-terminated, 50/50 films and 75/25 implants having a shorter time, and higher molecular weight, 75/25 ester-terminated films as well as PLA, acid-terminated implants taking longer to reach this critical molecular weight. Certainly, almost any desired polymer molecular weight can be achieved with “fine-tuning” the synthesis process. Here, when investigating the effects of other variables, we are only comparing formulations that have similar starting molecular weights.
Effect of polymer manufacturer
To compare the effects of different manufacturers, we focused on formulations that had the same L/G ratio, end-capping, and similar starting molecular weights to keep all variables as constant as possible. Therefore, 1) Expansorb® DLG 50-5A, Resomer® RG 504H, and Purasorb® PDLG 5004A films and 2) Expansorb® DLG 50-2A and Resomer® RG 502H films were compared. The water uptake between these films was generally very similar (Fig. 4a,e), but they diverged in their initial first order degradation constants, determined over the first three days of incubation using a least squared linear regression analysis (data not shown). Expansorb® DLG 50-5A and Purasorb® PDLG 5004A films both had faster initial degradation rate constants, kdeg=0.108 days− 1 and 0.105 days− 1, respectively, than Resomer® RG 504H, kdeg=0.078 days− 1. Expansorb® DLG 50-2A films initial degradation rate constant, kdeg=0.13 days− 1, was only slightly faster than Resomer® RG 502H films, kdeg=0.108 days− 1. Our lab previously analyzed the monomer sequence distribution of these same polymers using high resolution 13CNMR spectroscopy and determined their blockiness values, Rc, which describes the presence of glycolic linkages  and their block lengths, LG, which describes the length of the glycolide sequences  by determining relative intensities of the glycolyl and lactyl carbonyls . The PLGAs discussed here are polymers with random monomer distribution along the polymer chain with block lengths > 2, thus, cannot be considered as having a “truly alternating” sequence with block lengths of 1, such as L-G-L-G, or even L-L-G-G-L-L-G-G, which would result from a perfect ring opening of lactide and glycolide and have block lengths of 2. In the case of Expansorb® DLG 50-5A, Resomer® RG 504H, and Purasorb® PDLG 5004A films, both an increased glycolic blockiness and glycolic block length value appeared to be influential . Expansorb® DLG 50-5A polymer had a higher glycolic block length, LG=4.0, than both Purasorb® PDLG 5004A, LG=3.2, and Resomer® RG 504H, LG=2.9, polymers . While Purasorb® PDLG 5004A had a slightly lower block length, it had a higher glycolic blockiness value, Rc=1.6, than Expansorb® DLG 50-5A, Rc=0.7, and Resomer® RG 504H, Rc=1.3 . Expansorb® DLG 50-2A polymer had higher glycolic block length and blockiness values, LG=4.2 and Rc=1.9, than Resomer® RG 502H, LG=3.0 and Rc=1.4 . Both the presence of glycolic linkages and the longer length of these glycolic sequences can influence the faster initial rate of degradation. Expansorb® DLG 50-5A films showed an accelerated onset of erosion compared to the other two polymer formulations, Resomer® RG 504H and Purasorb® PDLG 5004A, but a significantly slower overall erosion rate compared to Resomer® RG 504H (Table 3), so the faster degradation may have led to a faster initial onset of erosion within the polymer film. Similarly, Expansorb® DLG 50-2A had a faster onset of erosion but a slower overall rate of erosion compared to Resomer® RG 502H (Table 3). Although the erosion profiles (Fig. 4c,g) do not clearly show these differences due to inherent variability in weighing the degraded films, we have more definitive evidence from 1HNMR determining the lactic content remaining in the samples after incubation. Fig. 4d,h shows the lactic content remaining in the films after incubation for 7, and 21 or 28 days. Expansorb® DLG 50-5A films revealed a higher increase in lactic content (or loss in glycolic content) relative to their starting contents, ~10%, than Resomer® RG 504H, ~7%, and Purasorb® PDLG 5004A, ~8%, after 28 days. Expansorb® DLG 50-2A films also had a higher increase in lactic content, ~12%, than Resomer® RG 502H films, ~10%, after 21 days. In both cases, the Expansorb® films lost glycolic units faster than the Resomer® films, as expected with their higher glycolic block lengths and faster initial degradation rate. This can also explain why both erosion and degradation rates eventually slow down because these polymers had slightly higher lactic content after the initial glycolic unit loss, which is expected to erode slower. Besides the increased block length or blockiness values, both the Expansorb® and Purasorb® polymers had higher residual monomers, as reported from their certificates of analysis, than the Resomer® polymers. Expansorb® DLG 50-2A and DLG 50-5A both had 0.5% total residual monomer and Purasorb® PDLG 5004A had 1.3%, while Resomer® RG 502H and 504H had 0.2% and 0.3% total residual monomer, respectively. The presence of residual monomer could theoretically contribute to faster degradation due to the increased presence of carboxylic acids and increased plasticization, resulting in enhanced acid-catalyzed hydrolysis, but may not be as influential in smaller geometries (e.g., thin films) since the residual monomer would be expected to efficiently escape the polymer matrix upon hydration and thus, likely does not contribute to the differences in behavior of these films [37, 38].
Microspheres and implants of Wako® 7515, a PLGA synthesized by polycondensation , consistently degraded faster than the comparable formulations from Expansorb® DLG 75-2A, Resomer® RG 752H and Purasorb® PDLG 7502A, PLGAs synthesized by ring-opening polymerization (ROP) (Fig. 3, Table 2). The initial apparent first order degradation rate constant, determined from the first 7 days of incubation, of Wako® 7515 microspheres was 0.068 days− 1 compared to 0.061, 0.060 and 0.052 days− 1 of Expansorb® DLG 75-2A, Resomer® RG 752H and Purasorb® PDLG 7502A microspheres, respectively. The initial apparent first order degradation rate constant of Wako® 7515 implants was 0.106 days− 1 compared to 0.075, 0.086 and 0.082 days− 1 of Expansorb® DLG 75-2A, Resomer® RG 752H and Purasorb® PDLG 7502A implants, respectively. Wako® 7515 microspheres also had a significantly faster apparent rate of erosion compared to the other three manufacturers, and Wako® 7515 implants showed significantly faster kero values than Resomer® RG 752H and Purasorb® PDLG 7502A, which is consistent with previous data from our group studying leuprolide-loaded microspheres, where Wako® 7515 microspheres showed faster drug release compared to Resomer® RG 752H microspheres . The glycolic blockiness and block length values previously determined  from these four polymers alone do not completely explain the patterns observed since Wako® 7515 had a drastically lower glycolic block length but only a slightly higher blockiness value compared to the others. There may be multiple variables in their manufacturing processes consistently causing differences in performance, not only the different synthesis methods, polycondensation vs. ROP, but the different types and levels of catalysts and initiators, the reaction temperature and timing conditions, or the purification process, that would require more information and further investigation, ideally with more batches.
High molecular weight, 75/25 ester end-terminated PLGA films across three brands, Expansorb® DLG 75-9E, Lactel® DL-PLG B6007-2, and Resomer® RG 756S were compared (Fig. 5). Resomer® RG 756S films had a lower overall water uptake while Expansorb® DLG 75-9E and Lactel® DL-PLG B6007-2 had similar water uptake profiles (Fig. 5a). Subsequently, Resomer® RG 756S films had a slower and steadier overall degradation and minimal mass loss after 56 days compared to Expansorb® DLG 75-9E and Lactel® DL-PLG B6007-2 (Fig. 5b,c). These trends were apparent in overall degradation rates and half-lives. Resomer® RG 756S degradation rate constant was 0.022 days− 1 and t1/2 was 31.1 days compared to Expansorb® DLG 75-9E and Lactel® DL-PLG B6007-2 which both had higher degradation rates, 0.038 and 0.035 days− 1, respectively, and shorter half-lives, 18.5 and 20 days, respectively (Table 2). All three polymer films eroded slowly for the first 42 days (Fig. 5c), Resomer® RG 756S films maintained this minimal erosion by the end of the 60 days incubation while Expansorb® DLG 75-9E and Lactel® DL-PLG B6007-2 films started to show erosion after 42 days and had similar ton values, 20.8 and 21.8 days, respectively, and similar keros values, 0.010 days− 1 (Table 3). Based on our previous analyses, Resomer® RG 756S polymer had a higher blockiness value, Rc=1.7, but a lower glycolic block length, LG=1.7, compared to both Expansorb® DLG 75-9E polymer, Rc=0.5 and LG=2.4, and Lactel® DL-PLG B6007-2 polymer Rc=0.6 and LG=2.1 . This lower glycolic block length in Resomer® RG 756S may have more influential than its higher blockiness value leading to the lower water uptake and slower degradation and erosion. Overall, the higher molecular weight, higher L/G ratio, and ester end-capping demonstrate how a combination of PLGA properties can be used to dramatically reduce degradation and erosion and would be expected to lead to a slower drug release.
Compared to 75/25, acid-terminated PLGA implants of similar molecular weight, PLA implants typically showed slower degradation and significantly reduced erosion over 56 days, as expected with the 100% racemic lactide polymers. PLA implants, Expansorb® DL 100-2A, Resomer® R 202H, and Purasorb® PDL 02A eroded very slowly, with a small initial erosion phase in the first few weeks followed by a small increase in erosion that resulted in 25-35% mass loss after 56 days of incubation (Fig. 6c). Between the manufacturers, Resomer® R 202H had the slowest water uptake, and slowest degradation and erosion (Fig. 6a-b, Tables 2 and 3). Resomer® R 202H also had the highest dry Tg, ~51°C, indicative of its lower reported residual monomer (0.2%), while Expansorb® DL 100-2A and Purasorb® PDL 02A revealed Tg values of ~45°C and ~46°C, respectively (Table 1) and higher reported residual monomers (1.8 and 2%, respectively). To further characterize these differences, we evaluated the molecular diffusion as a function of incubation time with spherical microspheres of these same PLA polymers incubated with BODIPY fluorescent dye, which is known to diffuse through solid PLGA over reasonable time scales and can be monitored by laser fluorescence confocal microscopy and diffusion coefficients can be calculated using the spherical geometry . Increases in the diffusion coefficient of BODIPY, may be indicative of higher polymer chain mobility and polymer hydrolysis rate. Fig. 6d-g shows the BODIPY diffusion coefficients and representative confocal images of a single microsphere of each of the three polymers. Purasorb® PDL 02A, the fastest degrading polymer, had a significantly greater diffusion coefficient on day 21 of incubation, compared to the other two polymers, and the confocal image further showed how degraded these microspheres became, while Resomer® R 202H, the slowest degrading, was still “intact” by day 21.
We observed differences in sequence distribution, glass transition temperatures, and in reported residual monomer between polymer manufacturers that manifested in differences between their water uptake, degradation, and erosion behaviors when formulated into films, implants and microspheres. These differences may arise from the polymer synthesis process and manufacturing parameters. For example, the ROP method has several variables involved such as the reaction temperature, time, amount of catalyst present, amount of co-initiator present, timing of monomer addition, and the extent of purification. Differences in optimized conditions between manufacturers, or inherent variations between batches within the same manufacturer can lead to deviations in polymer properties that may not be monitored or reported. The amount of catalyst present can help to increase the molecular weight quickly, but can also result in residual monomer, or higher chain dispersity. The addition of monomer to the growing chain can be affected by the catalyst used and by the reactivity of each monomer. Glycolide/glycolic acid has a slightly higher reactivity of addition to the growing polymer chain end, both when adding to a lactic or glycolic unit, which can result in “blockiness” of lactic or glycolic units [39, 40]. Over time, side reactions including chain back-biting and chain rearrangement can lead to the formation of cyclic dimers and more chain polydispersity or residual monomers . In general, ROP offers better control over the sequence distribution and molecular weight than polycondensation, especially when no catalyst is used in polycondensation . The effects of the monomer sequence have been extensively studied by Meyer et al. and it is clear that the heterogeneity of the sequence, the increased number or lengths of the glycolic linkages, leads to significantly faster swelling and hydrolysis and, thus, polymer erosion [14, 16, 43]. These studies were done comparing a typical polymer synthesized by ROP to a sequence-controlled polymer, so the differences were very evident. Here, we compared random (co-)polymers, synthesized by ROP, except for Wako® 7515, and their differences in glycolic blockiness values and block lengths are less evident. The differences in glycolic sequence that we observed between manufacturers appeared to result in increased initial degradation and generally resulted in heterogenous degradation of the glycolic and lactic monomers. This faster initial hydrolysis could potentially lead to a faster initial release of drug which could result in a non-ideal release profile, or dose dumping. We also observed apparent effects of increased residual monomer and lower glass transition temperature on water uptake, hydrolysis, and erosion. Although the residual monomers would be expected to diffuse out of smaller geometries quickly, it is possible they are more influential in a larger geometry as seen in the acid-terminated PLA implants. To gain more insight into the effects of manufacturing variables, it would be interesting to investigate the differences between multiple product batches within the same manufacturer. The data here is a limited example but begins to establish the effects of variations between manufacturers or polymer synthesis methods on PLGA degradation and erosion behavior.