Lipase-Catalyzed Ring-Opening Polymerization of Benzyl Malolactonate: An Unusual Mechanism?

The use of safe natural catalyst such as enzymes for ring opening polymerization (ROP) of ß-substituted ß-lactones such as benzyl malolactonate (MLABe) is an important objective considering the biomedical applications of the resulting (co)polymers. However, the preparation of well-defined polymeric materials using such systems requires an understanding of enzyme-substrate interactions. In this context, we investigated the mechanism of lipase-catalyzed ROP of MLABe, because it appears that it is probably not the same as the one widely described for other lactones such ε-caprolactone, propiolactone. and lactide. Enzymatic-catalyzed ROPs of MLABe in the presence of the lipase/acyltransferase CpLip2 and its serine knockout (serine KO) mutant (CpLip2_180A) have led to poly(benzyl malate) (PMLABe) terminated by a monobenzyl fumarate group with monomer conversion higher than 70% and weight-average molar mass of about 3600 g/mol (D = 1.42). On the other hand, only less than 7% of MLABe conversion and no polymer formation were observed when the polymerization reaction was conducted in the presence of inactivated CpLip2 (heated at 100 °C). Moreover, the ROP of MLABe in the presence of imidazole, a synthetic mimic of the catalytic histidine, led to a PMLABe terminated by a monobenzyl fumarate group. On the contrary, neither the enzymatic-catalyzed ROP of benzyl dimethylmalolactonate (diMeMLABe), a MLABe with two methyl groups instead of the two "acidic" protons on the lactone's ring, in the presence of CpLip2 and CpLip2_180A nor its chemical ROP in the presence of imidazole were successful. Together, all these results suggested that the lipase-catalyzed polymerization of malolactonates occurred through the abstraction of one of the two "acidic" protons of the lactone's ring by the histidine of the catalytic triad leading to the corresponding monobenzyl fumarate responsible for the polymerization of the remaining monomer. Finally, molecular modeling of the positioning of the monomer into the catalytic site of the CpLip2 and DFT quantum-chemical calculations highlighted an interaction of (R)- and (S)-MLABe with the catalytic histidine of the enzyme preferentially to serine, in the form of a strong hydrogen bond with one of the "acidic" protons of MLABe, thus, supporting the important role of the catalytic histidine in the polymerization of such cyclic lactones.

Cell Uptake and Biocompatibility of Nanoparticles Prepared from Poly(benzyl malate) (Co)polymers Obtained through Chemical and Enzymatic Polymerization in Human HepaRG Cells and Primary Macrophages.

The design of drug-loaded nanoparticles (NPs) appears to be a suitable strategy for the prolonged plasma concentration of therapeutic payloads, higher bioavailability, and the reduction of side effects compared with classical chemotherapies. In most cases, NPs are prepared from (co)polymers obtained through chemical polymerization. However, procedures have been developed to synthesize some polymers via enzymatic polymerization in the absence of chemical initiators. The aim of this work was to compare the acute in vitro cytotoxicities and cell uptake of NPs prepared from poly(benzyl malate) (PMLABe) synthesized by chemical and enzymatic polymerization. Herein, we report the synthesis and characterization of eight PMLABe-based polymers. Corresponding NPs were produced, their cytotoxicity was studied in hepatoma HepaRG cells, and their uptake by primary macrophages and HepaRG cells was measured. In vitro cell viability evidenced a mild toxicity of the NPs only at high concentrations/densities of NPs in culture media. These data did not evidence a higher biocompatibility of the NPs prepared from enzymatic polymerization, and further demonstrated that chemical polymerization and the nanoprecipitation procedure led to biocompatible PMLABe-based NPs. In contrast, NPs produced from enzymatically synthesized polymers were more efficiently internalized than NPs produced from chemically synthesized polymers. The efficient uptake, combined with low cytotoxicity, indicate that PMLABe-based NPs are suitable nanovectors for drug delivery, deserving further evaluation in vivo to target either hepatocytes or resident liver macrophages.

Poly-l-lactic acid-co-poly(pentadecalactone) Electrospun Fibers Result in Greater Neurite Outgrowth of Chick Dorsal Root Ganglia in Vitro Compared to Poly-l-lactic Acid Fibers.

Electrospun poly-l-lactic acid (PLLA) fiber scaffolds are used to direct axonal extension in neural engineering models. We aimed to improve the efficacy of these fibers in promoting neurite outgrowth by altering surface topography and reducing fiber elastic modulus through the incorporation of a compatibilized blend, poly-l-lactic acid-poly(pentadecalactone) (PLLA-PPDL) into the solution prior to electrospinning. PLLA+PLLA-PPDL fibers had a larger diameter, increased surface nanotopography, and lower glass transition temperature than PLLA fibers but had similar mechanical properties. Increases in neurite outgrowth on PLLA+PLLA-PPDL fibers were observed, potentially due to the significantly increased diameter and surface coverage with nanotopography. Ultimately, these results suggest that greater electrospun fiber diameter and surface topography may contribute to increases in neurite outgrowth.