Introduction: poly(lactide-co-glycolide) (PLGA) nanoparticles due to their wide range of resorption times are used extensively for immobilization and timed-release of proteins in drug delivery and regenerative medicine. However, protein denaturation by surface adsorption and acidic degradation products of PLGA significantly reduces protein bioactivity in vitro and in vivo. Due to chain flexibility and electrical neutrality of polyethylene glycol (PEG) macromers, PEGylation is used to increase stability of aqueous nanoparticle suspensions. The objective of this work was to investigate the effect of sequential chain-extension of PEG with short segments of lactide (L) and glycolide (G) monomers by ring-opening polymerization on self-assembly and stability in aqueous solution, and bioactivity of proteins grafted to the self-assembled nanogels (NGs).
Materials and method: The PEG-LG block copolymers were synthesized by melt ring-opening polymerization with sequential addition of L and G monomers to the reaction using PEG as the initiator. The synthesized copolymer was functionalized with succinimide groups by reacting hydroxyl end-groups of the copolymer with N,N/-disuccinimidyl carbonate. The maromers were dissolved in DMSO and self-assembled to form NGs by dialysis (3.5 kDa MW cutoff) against PBS for 8 h. For protein grafting, NGs were suspended in 0.5 mL PBS by sonication for 1 min. Next, 0.5 mL of the protein in PBS (800 ng/mL) was added to the NG suspension, and the amine group of the protein was allowed to react with succinimide end-groups of the chain extended macromer in NGs under ambient conditions for 12 h. Size distribution of the NGs was measured by dynamic light scattering. Zeta potential of the NGs was measured with a ZetaPlus analyzer. NGs degradation kinetic was measured by incubation in PBS at 37°C as we described previously. Release kinetics of the grafted protein was measured by incubating 1 mg protein-loaded NGs in 1 mL PBS at 37°C as we previously described. At each time point, the suspension was centrifuged at 18350 rcf for 10 min, the supernatant was removed, the NGs were resuspended in 1 mL fresh PBS and incubated until the next time point. The amount of bovine serum albumin (BSA) in the supernatant was measured with the ninhydrin reagent. For BMP2-grafted NGs, the protein concentration in the supernatant was measured by ELISA. The abbreviation P4 is for 4.6 kDa PEG MW and 10 LG:PEG molar ratio; P8 is for 8 kDa PEG MW and 20 LG:PEG molar ratio; P12 is for 12 kDa PEG MW and 30 LG:PEG molar ratio. Abbreviation I, II, and III are for L/G ratio of 75/25, 60/40, and 50/50, respectively.
Results: Figure 1a to 1c show the effect of PEG molecular weight, LG segment length, and L/G ratio in the chain-extended macromer on NG size distribution. The morphology of the dried NGs is shown in the inset SEM images in (a-c). NGs size was <200 nm. The average size ranged from 120±6 to 140±13 nm for P4 NGs, 150±11 to 160±17 nm for P8 NGs, and 170±12 to 190±15 nm for P12 NGs, respectively. The NGs were relatively monodisperse with PD<0.2 and the mean NG size increased with decreasing lactide fraction. All NGs were negatively charged with zeta potential between -23 and -15 mV. The absolute value of the zeta potential increased with increasing PEG molecular weight while the lactide fraction did not significantly affect zeta potential of the NGs. The effect of concurrent (random, c) and sequential (block, s) addition of L and G monomers to the polymerization reaction on colloidal stability of the NGs was also investigated. The NGs generated from P4-I macromers with concurrent addition of LG monomers (P4-Ic) had a significantly wider distribution (PD=0.31±0.05) than the sequential addition (0.07±0.01). P4-Is NGs was more stable in aqueous solution than P4-Ic NGs because their mean size did not change with incubation time and their precipitation from solution occurred over a narrower centrifugation speeds. Grafting BMP2 and VEGF to the NGs from sequential chain-extension led to only 30% and 20% denaturation of the proteins, respectively. Results of this work indicate that nanogels self-assembled from PEG macromers chain-extended with sequential polymerization of lactide and glycolide are a more efficient platform for delivery of proteins compared to PEGylated PLGA nanoparticles.
Figure 1. Effect of PEG molecular weight, LG segment length, and L/G ratio in PEG macromers chain-extended with sequential polymerization of L and G on NG assembly and size distribution; Morphology of the dried NGs is shown in the inset SEM images in (a-c).