Letter pubs.acs.org/macroletters
Chemoenzymatic Synthesis of Cholesterol‑g‑Poly(amine-co-ester) Amphiphilic Copolymer as a Carrier for miR-23b Delivery Jiawen Chen,†,§ Wei Jiang,‡,§ Haobo Han,† Jiebing Yang,† Wenqi Chen,† Yudi Wang,† Jun Tang,*,‡ and Quanshun Li*,† †
Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Sciences, and ‡Department of Polymer Science, College of Chemistry, Jilin University, Changchun 130012, China S Supporting Information *
ABSTRACT: The lipase-catalyzed polymerization of Nmethyldiethanolamine, diethyl sebacate and ω-pentadecanolide was performed to construct a cationic poly(amine-coester), and a hydrophobic N-(2-bromoethyl)carbamoyl cholesterol was then grafted onto its main chain through a quaternization reaction to prepare the amphiphilic copolymer Chol-g-PMSC-PPDL. The copolymer efficiently bound and condensed miR-23b to form stable nanocomplexes, which showed favorable cellular uptake and miR-23b transfection efficacy due to the introduction of the hydrophobic segment. After miR-23b delivery, an obvious inhibition of cell proliferation could be induced, which was attributed to the induction of cell apoptosis and cell cycle arrest. Moreover, the carrier-mediated miR-23b delivery could inhibit the migration and invasion of tumor cells. Overall, the work provides a novel chemoenzymatic strategy for constructing biodegradable and biocompatible poly(amine-co-ester) derivatives, which are promising carriers for oligonucleotide delivery to achieve tumor gene therapy.
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synthesized through chemical polymerization. Enzymatic polymerization has developed rapidly in the past two decades and become an alternative technique for constructing biomedical polymers due to the high activity and selectivity of enzymes in mild reaction conditions and their outstanding tolerance against organic functional groups.18,19 Recently, several poly(amine-co-esters) have been synthesized through the enzymatic polymerization of diesters and amino-substituted diols with a high transfection efficiency of plasmid DNA and low cytotoxicity.20−24 Moreover, combining with the ringopening polymerization of lactones could further adjust the carriers’ molecular weight and hydrophobicity, which could be used to reduce the density of positive charges to decrease the carriers’ cytotoxicity and strengthen the interaction with cell membrane to promote endocytosis, respectively.21 In addition, the enhanced hydrophobicity could facilitate the gene release from polycation carriers, which is an important influencing factor in miRNA delivery. To date, the transfection efficacy of these poly(amine-co-esters) has not been evaluated using miRNA as cargo. Herein, a lipase-catalyzed combination of polycondensation of N-methyldiethanolamine (MDEA) and diethyl sebacate (DES) with ring-opening polymerization of ω-pentadecanolide
icroRNAs (miRNAs) are a group of endogenous noncoding RNAs with 19−22 nucleotides in length that can interact with many physiologically essential genes and regulate their expression at the post-transcriptional level.1 Thus, they play critical roles in various biological processes, including cell proliferation, differentiation, and migration.1 Among them, miR-23b has been identified as a key tumor suppressor that could inhibit the cell proliferation, migration and invasion through regulating the expression of a series of genes such as survivin, PTEN, MMP-9, E-cadherin, and Vimentin. 2−6 Recently, miR-23b has been found to depress the epithelialmesenchymal transition or increase the cisplatin sensitivity by directly targeting Pyk2 or Src, which would achieve the inhibition of cell migration and invasion and enhance the antitumor efficacy of chemotherapeutics.7,8 Since miR-23b is usually down-regulated in many human tumors, carriermediated miR-23b transfection is a potential route to achieve tumor gene therapy. To date, various cationic polymers have been developed to serve as carriers for gene delivery, such as poly(ethylenimine) (PEI),9,10 poly(L-lysine),11 poly(β-amino esters),12 polyamidoamine dendrimers,13 and other cationic copolymers.14−16 Compared to these carriers, copolyesters containing tertiary amino substituents, namely, poly(amine-co-ester), exhibit favorable characteristics as superior gene carriers such as excellent transfection efficiency, low cytotoxicity, and good biodegradability.17 These cationic polymers were mainly © XXXX American Chemical Society
Received: March 18, 2017 Accepted: April 24, 2017
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DOI: 10.1021/acsmacrolett.7b00211 ACS Macro Lett. 2017, 6, 523−528
Letter
ACS Macro Letters Scheme 1. Chemoenzymatic Synthesis of Cholesterol-g-Poly(amine-co-ester) Copolymer Chol-g-PMSC-PPDL
elucidated by FT-IR and 1H and 13C NMR characterization (Figures 1, S6, and S7). The grafting ratio of cholesterol was
(PDL) was carried out to construct the copolyester PMSC− PPDL, and hydrophobic N-(2-bromoethyl)carbamoyl cholesterol (Be-chol) was then grafted onto its main chain through a quaternization reaction to obtain the cationic amphiphilic copolymer Chol-g-PMSC-PPDL (Scheme 1). The copolymer was used as a carrier for miR-23b delivery to systematically evaluate the inhibition of tumor proliferation and migration using the human lung adenocarcinoma cell line A549 as a model. The poly(amine-co-ester) PMSC−PPDL was successfully synthesized through the combination of polycondensation of MDEA and DES with a ring-opening polymerization of PDL using Novozym 435 as the catalyst in a two-stage manner. During the first stage, monomers were converted to nonvolatile oligomers, and the polymer chain growth could be accelerated under high vacuum to remove the byproduct ethanol at the second stage.21 The structure was characterized by FT-IR and 1 H and 13C NMR (Figures S1−S3). Through the integration of peaks at 4.04−4.07 ppm from lactone units and 4.15−4.18 ppm from MDEA units, the ratio of PPDL in the copolymer was calculated to be 14.5%. Then, Be-chol was synthesized through the reaction of cholesteryl chloroformate and 2-bromoethylamine hydrobromide according to previous reports,25−27 and its structure was confirmed by FT-IR and 1H NMR (Figures S4 and S5). The ratio of the H1, H2, HN, H4, and H5 peak areas was determined to be 1:1:1:2:2, confirming the successful synthesis of Be-chol. Finally, the hydrophobic Be-chol was grafted onto the main chain of PMSC−PPDL through a quaternization reaction to obtain the amphiphilic copolymer Chol-g-PMSC-PPDL. The introduction of hydrophobic cholesterol will be beneficial for decreasing the carrier’s cytotoxicity and further improving the cellular uptake of carrier/miR-23b nanoparticles. The structure of Chol-g-PMSC-PPDL was
Figure 1. 1H NMR spectrum of Chol-g-PMSC-PPDL. 1H NMR (CDCl3, ppm): 2.70 (a), 1.57 (b), 1.24−1.28 (c and d), 4.02−4.17 (e and j, −CH2O− group), 2.28−2.35 (f and g), 5.38 (h), and 0.70 (i).
calculated to be 9.7% based on the peak areas of 5.38 ppm (h) and 4.02−4.17 (e and j), as shown in Figure 1, and the grafting ratio could be modulated by changing the ratio of PMSC− PPDL and Be-chol. The number-average molecular weight (Mn) of Chol-g-PMSC-PPDL was determined to be 8300 g/ mol through GPC analysis, lower than that of PMSC−PPDL (19200 g/mol; Figure S8). The reduced Mn value was probably caused by the degradation of ester bonds in PMSC−PPDL at 110 °C during the cholesterol grafting reaction. This phenomenon was consistent with the similar grafting reaction,27 in which the high temperature could cause the 524
DOI: 10.1021/acsmacrolett.7b00211 ACS Macro Lett. 2017, 6, 523−528
Letter
ACS Macro Letters degradation of main chain and result in a lower molecular weight. The cytotoxicity of Chol-g-PMSC-PPDL was evaluated by MTT, indicating that the carrier was of excellent biocompatibility with a cell viability of >92% at a carrier concentration of 150 μg/mL (Figure S9). In contrast, lower cell viability (
