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Article
Systemic mRNA Delivery to the Lungs by Functional Polyester-based Carriers
Yunfeng Yan, Hu Xiong, Xinyi Zhang, QIANG CHENG, and Daniel John Siegwart
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01356 • Publication Date (Web): 15 Nov 2017
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4 Systemic mRNA Delivery to the Lungs by Functional Polyester-based Carriers
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6 Yunfeng Yan a,b,*, Hu Xiong b, Xinyi Zhang b, Qiang Cheng b, and Daniel J. Siegwart b,*
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9 College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou,
10 Zhejiang 310014, China
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12 b
Simmons Comprehensive Cancer Center, Department of Biochemistry, The University of Texas
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Southwestern Medical Center, Dallas, TX 75390, United States
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15 *
Corresponding authors. Email address: Daniel.Siegwart@UTSouthwestern.edu (D.J. Siegwart),
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17 yfyan@zjut.edu.cn (Y. Yan).
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21 Abstract
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24 Messenger RNA (mRNA) has recently come into focus as an emerging therapeutic class with great
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26 potential for protein replacement therapy, cancer immunotherapy, regenerative medicine, vaccines, and
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gene editing. However, the lack of effective and safe delivery methods impedes the broad application of
29 mRNA-based therapeutics. Here, we report a robust approach to develop efficient polymeric delivery
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31 carriers for mRNA. Lead polyesters were identified by in vitro screening of a 480-member
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33 combinatorially modified poly(trimethylolpropane allyl ether-co-suberoyl chloride) library for the
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35 delivery of luciferase encoding mRNA (Luc mRNA) to IGROV1 cells. Formulation of mRNA polyplex
36 nanoparticles (NPs) with Pluronic F127 decreased the surface charge. Although this improved the
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38 stability of mRNA nanoparticles, the delivery potency decreased with increased F127 content. Thus, we
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40 determined that NP stabilization with 5% F127 could balance the protective effects and delivery potency.
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42 5% F127 formulated PE4K-A17-0.33C12 mRNA NPs enabled luciferase expression predominantly in
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the lungs after intravenous injection into mice. The efficient mRNA delivery specifically to lungs by
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45 degradable carriers suggests potential for the treatment of pulmonary diseases.
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48 Keywords: Polyesters, mRNA, nanoparticles, drug delivery
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53 Introduction
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56 Messenger RNA (mRNA)-based therapeutics have recently generated great attention because of the
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broad range of potential applications.1-3 Synthetically modified and codon-optimized mRNAs are
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capable of expressing encoded proteins in cells, which enables wide utilization in protein replacement
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5 therapy, immunotherapy, regenerative medicine, and infectious disease vaccines.4-12 In particular, the
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7 effective and controllable expression of target proteins by delivered mRNA provides a reliable approach
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9 for the production of the key nuclease for the emerging genome editing technologies, e.g. zinc finger
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nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly
12 interspaced short palindromic repeat (CRISPR)/Cas.13-18 Due to the great potential in clinical
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14 applications, there has been significant investment in a wide array of biotechnology enterprises.1-3
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17 mRNA-based therapeutics hold some conceptual advantages over other gene therapy methods.
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For example, mRNA does not integrate into the host genome. Thus, there is limited risk of mutagenesis
20 or persistent expression of gene editing related proteins. mRNA is translated to functional proteins in
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22 cytoplasm and does not need to enter the nucleus, which aids utility in non-dividing or slowly dividing
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24 cells, e.g. dendritic cells.1, 19 Furthermore, comparing with recombinant proteins, the chemical synthesis
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26 of mRNA is easier, cheaper, and carries a lower biosafety risk.1, 20
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28 There are some ongoing challenges to realize the broad potential of mRNA-based therapeutics,
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30 including improving their translatability and stability, reducing the immunogenicity of mRNAs, and
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32 efficiently delivering mRNA into targeted cells.1, 3, 20-21 Translatability and stability has been increased
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by optimal modification of mRNAs. Some inherent immunogenicity has been suppressed through
35 removal of double stranded mRNA by HPLC purification.21-22 Like other nucleic acids, the safe and
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20-21, 23
37 efficient delivery of mRNA is one of the main unmet challenges for mRNA drugs. Single
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39 stranded mRNAs are less stable than double stranded DNA or siRNA;24-25 thus, there are more stringent
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41 requirements of suitable carriers to protect mRNA from degradation by endonucleases and facilitate its
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uptake into target cells. There has been significant progress in both fundamental and translation research
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44 of DNA and siRNA delivery in the past decade.13, 26-27 However, studies on systemic delivery of mRNA
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46 are still limited.2-3, 24, 28
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49 A variety of carrier types, most prominently cationic polymers and cationic lipid-based
50 nanoparticles (LNPs), have found utility in gene delivery. LNPs represent an advanced delivery platform
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52 for siRNA therapeutics and are currently in human clinical trials. LNPs have been recently adapted for
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54 mRNA delivery in vivo.4, 8, 12, 21, 28-35
Adapting LNPs for longer RNA cargo has required a new
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56 understanding of LNPs.17, 30-31 Although cationic polymers have been used widely for decades as carriers
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for long plasmid DNA (pDNA), research as mRNA carriers has only recently been investigated. Given
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that they are already optimized for long nucleic acids, polymers have great potential for mRNA
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5 delivery.36-37 Positively charged polymers possess controllable structures and diverse functionalities.
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7 Therefore, cationic polymers have been investigated as another type of candidate for mRNA delivery.25,
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9 For example, polyethyleneimine (PEI) or poly(2-dimethylaminoethyl methacrylate)
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(PDMAEMA) based polymers were able to encapsulate mRNA and there is efficient expression of
12 luciferase or GFP in cells after treatment with polymer-mRNA polyplex nanoparticles.38-39 But the
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14 clinical translation of these polymers may be limited by their poor degradability. A series of
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16 polyaspartaminde carriers modified with PEI, PEG, or 1,2-diaminoethane was also recently employed
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18 for the efficient mRNA delivery on a variety of disease models including neurological disorders,
19 osteoarthritis, Alzheimer’s disease, and pancreatic cancer.40-41 mRNA delivery using
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21 poly(glycoamidoamine) brush polymers was also reported.43 Recent research showed that charge-
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23 altering releasable transporters are capable of efficacious luciferase mRNA delivery to cultured cells and
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25 animals.44 Current progress suggested the feasibility of delivering synthetic mRNA for the target protein
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expression in cells. Nevertheless, investigation of mRNA delivery carriers is still limited compared to
28 siRNA and pDNA. Delivery carriers must be further improved according to the unique physiochemical
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30 properties of single stranded mRNA. In particular, degradability and functionality of polymeric vehicles
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32 need to be comprehensively considered to meet the requirements in safety, in vivo stabilization, and
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34 efficacy.
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36 We recently reported a facile approach for the scalable preparation of functional polyesters with
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38 tunable molecular weight by the polycondensation of trimethylolpropane allyl ether and diacyl
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40 chlorides.45-47 Combinatorial modification of the side chain –ene group allows for construction of
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42 polyesters with diverse chemical functionality and physical properties. Previous studies have suggested
43 that the versatile platform was efficacious for short RNA delivery both in vitro and in vivo.46-47
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45 Remarkably, high throughput screening of these functional polyesters enabled the discovery of cancer
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47 selective siRNA nanoparticles that do not require any targeting modification, replying only on
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49 physicochemical properties of the polymers themselves to mediate cancer selective uptake.46
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51 In this paper, we aimed to tackle the delivery challenge of emerging mRNA therapeutics using
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53 the unique combination of functionality and degradability of this polymer platform (Figures 1 and S1).
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55 We discovered efficient delivery carriers by in vitro screening of the polymer library for mRNA delivery
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57 to IGROV1 ovarian cancer cells. Candidate carriers were further formulated with Pluronic F-127 to
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enhance the stability of mRNA polyplex nanoparticles for in vivo use. Formulated mRNA nanoparticles
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5 mediated efficacious luciferase expression in the lungs after intravenous administration to mice. Due to
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7 the facile preparation, tunable molecular weight, degradability and diverse functionality, these functional
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9 polyesters are suitable carriers for in vitro and in vivo delivery of mRNA therapeutics.
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35 Figure 1. (a) A series of degradable functional polyesters was created by combinatorial modification of
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37 –ene containing polyesters with 20 amino thiols and 3 alkyl thiols. See Figure S1 for all chemical
38 structures. (b) Functional polyesters enable mRNA delivery to cells, mediating efficient expression of
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40 target proteins in cytoplasm.
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Materials and Methods
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48 Materials. All chemicals for polymer synthesis and modification were purchased from Sigma-Aldrich
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50 or Fisher Scientific and used as received. Luciferase and mCherry mRNA were purchased from Tri-Link
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52 Biotechnologies. RPMI-1640, fetal bovine serum (FBS), Dulbecco’s modified phosphate buffered saline
53 (PBS), Trypsin-EDTA (0.25%), and PEO101-PPO56-PEO101 (Pluronic F127, Mw = 12,600, PDI = 1.05)
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55 were purchased from Sigma-Aldrich. PEG 2000 DMG lipid (Sunbright GM-020, 1,2-dimyristoyl-sn-
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57 glycerol, methoxypolyethylene glycol) was purchased from NOF America. The Quant-iT RiboGreen
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RNA assay kit and RNAiMax were purchased from Life Technologies and used following the
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5 manufacturer’s protocols. The ONE-Glo + Tox luciferase assay kit was purchased from Promega. D-
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7 Luciferin firefly, sodium salt monohydrate was purchased from Gold Biotechnology.
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10 Preparation and characterization of mRNA polyplex nanoparticles. The functional polyesters were
11 synthesized according to our previous papers.45-47 Unmodified polyesters were prepared via the
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13 polycondensation of trimethylolpropane allyl ether (TPAE) and suberoyl chloride (diACl-C8) with
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15 pyridine as catalyst in dichloromethane (DCM).45 The polyesters were functionalized through the thiol-
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17 ene reaction with different amino and alkyl thiols at varying amino/alkyl molar ratios in DMF or DMSO
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using previously reported conditions.46 mRNA polyplex nanoparticles for in vitro studies were prepared
20 by mixing functional polymers (3 g/L in DMSO) with mRNA (citric acid/trisodium citrate buffer, pH
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22 4.2, 10 mM) in 96-well plates at a polymer/mRNA ratio of 30:1 (wt/wt) and final mRNA concentration
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24 of 1.25 ng/µL. The polyplex nanoparticles were incubated on a shaking plate (30 rpm) for 30 min prior
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26 to characterization or addition to cells. To prepare mRNA nanoparticles for in vivo studies, 5 wt% F-127
27 was added to the polymer DMSO solution. Then, the mixture was added into mRNA solution followed
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29 by homogenization and dialysis (cut-off MW of 3,500 Da) against PBS for 4 h prior to injection to mice.
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31 The Quant-iT RiboGreen RNA assay kit was used to quantify mRNA binding according to the
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33 manufacture’s protocols. The size and surface charge of the nanoparticles were measured using a
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Zetasizer Nano ZS (Malvern, He-Ne, λ = 632 nm). All measurements were performed in quadruplicate
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36 and the average with standard deviation was reported.
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39 In vitro delivery of mRNA. IGROV1 ovarian cancer cells were cultured in RPMI-1640 medium
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41 supplemented with 5% FBS (37 oC, 5% CO2). For in vitro screening assays, IGROV1 cells were seeded
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into opaque white 96-well plates (10,000 cells/well) and incubated for 24 h. The medium was replaced
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44 with the fresh RPMI-1640 medium with 5% FBS (200 µL/well) and 20 µL luciferase mRNA (Luc
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46 mRNA) polyplex nanoparticles (25 ng mRNA/well, 181 pM) were added to cells. After incubation for
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48 24 h, the media was gently removed. The cell viability and luciferase expression were measured using
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ONE-Glo + Tox luciferase assay kits and normalized to untreated cells. All transfection assays were
51 performed in triplicate and the average with standard deviation was reported. RNAiMax positive control
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53 experiments were conducted following the manufacturer’s protocol. For in vitro fluorescence and
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55 luminance imaging, IGROV1 cells were seeded into transparent 24-well plates (40,000 cells/well) and
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57 incubated overnight. Similarly, nanoparticles with 100-800 ng mRNA were added into cells with 800 µL
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fresh RPMI-1640 medium (5% FBS). After 24 h transfection, the mCherry or luciferase signal was
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5 evaluated using an EVOS FL microscope (AMG) or IVIS Lumina imaging system (Caliper Life
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7 Science). Luc mRNA NPs treated cells were collected and the luciferase expression were quantitated
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9 using a Luciferase Assay System kit (Promega) according to the manufacture’s protocols.
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11 In vivo delivery of luciferase mRNA. All animal experiments were approved by the Institutional
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13 Animal Care and Use Committee (IACUC) of the University of Texas Southwestern Medical Center and
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15 were consistent with local, state and federal regulations as applicable. NOD SCID mice were purchased
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17 from the UT Southwestern animal breeding core. Luc mRNA loaded nanoparticles with selected
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functional polyesters and F127 were prepared as described above. 150 µL of mRNA NPs (20 µg Luc
20 mRNA) were administered to NOD SCID mice via tail vein injection (n=4). After 24 h, 80 µL of D-
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22 luciferin PBS solution (40 g/L) was injected subcutaneously in the neck scruff 10 min prior to
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24 bioluminescence imaging on an IVIS Lumina imaging system. Ex vivo imaging on systemic organs was
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26 performed immediately after live animal imaging.
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31 Results and Discussion
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In vitro screening of functional polyesters for mRNA delivery. Nucleic acid delivery involves various
35 steps including the formation of nanoparticles, endocytosis, intracellular trafficking and release, which
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37 requires a subtle balance among physicochemical properties of carriers, particularly electrostatic binding
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39 and stabilizing hydrophobicity.3, 17, 46-56
Due to the challenges of identifying efficacious carriers,
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41 combinatorial high throughput screening has proven to be an efficient way to identify materials for
42 siRNA and plasmid DNA delivery.57-59 Therefore, we constructed a robust library containing 480
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44 functional polyesters with degradability and diverse chemical functional groups, and used a similar
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46 screening strategy for the discovery of carriers for mRNA delivery. In vitro studies (Figures 2 and S2)
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48 show that some mRNA polyplex nanoparticles enable high luciferase expression in IGROV1 cells,
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indicating efficient luciferase mRNA delivery by these polymers. Particularly, A17 (cysteamine)
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51 modified polyesters had promising delivery potency. This functional group was also active in our prior
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53 study of siRNA carriers, suggesting that the same pKa may be suitable for siRNA delivery and mRNA
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55 delivery. Comparing the overall results to our prior siRNA delivery screen,46 we identified a smaller
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number of effective polymeric carriers for mRNA delivery, implying that the RNAs with different
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37 Figure 2. In vitro screening of functional polyesters for luciferase mRNA (Luc mRNA) delivery to
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39 IGROV1 cells enabled identification of efficacious mRNA carriers. Functional polyesters with
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41 molecular weight of 4,200 g/mol (PE4K) and 8,300 g/mol (PE8K) were functionalized with amino thiols
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43 (A1-A20) and alkyl thiols (C6-C12) at different molar feed ratios (alkyl thiol : amino thiol) of 1:4, 1:2,
44 1:1, and 2:1. See Figure S1. The functional polyesters were named by the molecular weight of polyester
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46 followed by the amino thiol, the molar fraction of alkyl thiols, and the alkyl thiol in the modification.
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48 The Luc mRNA dose was 25 ng mRNA/well (181 pM). The luciferase signal was normalized to
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50 untreated cells.
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29 Figure 3. Fluorescence images of IGROV1 cells after 24 h-treatment with mCherry mRNA polyplex
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31 nanoparticles verified the successful mRNA delivery by selected polymers with varying mRNA dose
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33 (100, 200, 400, and 800 ng/well). Scale bars = 200 µL.
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38 To validate the mRNA delivery indicated by the normalized luciferase expression (Figure 2),
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40 fluorescence imaging of IGROV1 cells was performed after 24 h-treatment with mCherry mRNA NPs.
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42 Figure 3 shows high mCherry signal under a regular measurement setting (60% intensity), proving
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successful mCherry mRNA delivery by selected functional polymers. For PE4K-A17-0.33C6 and
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45 PE4K-A17-0.2C12, the mCherry signal was strong and overall dose responsive (except for a slight
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47 decrease at 2178 pM for PE4K-A17-0.33C6 due to toxicity or possible aggregation at high
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49 concentration). There was very limited mCherry expression at low mRNA dose when the polymer chain
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51 length doubled (PE8K-A17-0.33C6). But mCherry protein expression increased after the mRNA dose
52 was increased to 1272 pM (400 ng/well). It is worth noting that the mass concentration of mRNA in
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54 screening studies (Figure 2) is identical to the lowest does in 24-well plates. We speculate that
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1-(4-(2-hydroxyethyl)piperazin-1-yl)-4-mercaptobutan-1-one (A5), the mCherry expression was
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5 relatively low for all doses, indicating the predominant role of the side chain amine for cellular uptake
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7 and endosomal release of mRNA. Overall, this approach allowed identification of efficient mRNA
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9 delivery carriers via high throughput screening of the versatile polymer library. Focusing on these potent
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polymers, we next evaluated effects of Pluronic F127 formulation on physicochemical properties of
12 mRNA polyplex NPs and the efficacy of mRNA delivery because the formulation with amphiphilic
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14 stabilizers has been shown to improve the in vivo stability of functional polyester nucleic acid NPs.32, 47
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17 Effects of Pluronic F127 on mRNA delivery. PPO segments of Pluronic F127 could insert into the
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hydrophobic domain and leave hydrophilic PEG on the surface of formulated mRNA polyplex NPs
20 which benefits nucleic acid delivery in vivo by protecting NPs from protein adsorption and aggregation
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22 at high concentration. On the other hand, too much PEG on the surface may reduce NP-cell interactions
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24 and cellular uptake.47 To find optimal formulation conditions for mRNA delivery, we evaluated Luc
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26 mRNA delivery efficacy of NPs formulated with 5 or 10% F127 or PEG2000 DMG lipid (Figures 4 and
27 S3). The addition of F127 did not alter the cell viability, but did lead to a decrease in luciferase
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29 expression of IGROV1 cell with increased F127 content for some formulations. Without addition of
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31 F127, some functional polyesters showed comparable mRNA delivery capability to the commercial
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33 reagent RNAiMax. In contrast to our prior work with F127-stabilized siRNA NPs, where activity was
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maintained, there was drop in delivery efficacy of the formulated NPs with 10% F127 for most of the
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36 lead polyesters. Our previous study showed that 5% PEG2000 DMG lipid provided efficient shielding
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38 for siRNA polyplex nanoparticles without compromising delivery efficacy.47 However, formulation of
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40 mRNA NPs with a PEG2000 DMG lipid induced a decrease over 2 orders of magnitude (Figure S3),
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42 which illustrates the challenge of delivering much longer RNAs that are more challenging to load and
43 stabilize. Due to the potential retention or loss of activity,32, 47 the high-throughput screen of polyplexes
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45 was conducted without stabilizers. Nevertheless, the addition of F127 is essential for in vivo delivery
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47 because it can improve tolerability and more importantly, greatly improves NP stability in vivo. Among
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49 the three conditions tested (5% F127, 10% F127, and 5% PEG2000 DMG), 5% F127 was selected for
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use in studies in vivo because it most optimally balanced delivery efficacy and NP stability. The dose
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52 response of selected NPs with and without F127 formulation (Figure 4b) further confirmed the mRNA
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Figure 5. Physicochemical properties of mRNA polyplex nanoparticles with selected polyesters. (a)
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54 Next, the binding capability of the lead polyesters with mRNA and the resulting properties of
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56 polyplex NPs were measured. Figure 5a shows that all selected A17 modified polyesters completely
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bound mRNA under the preparation conditions (pH 4.2, 10 mM, and polymer/mRNA weight ratio of
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30:1). The flexibility of single stranded mRNA may benefit the charge neutralization between cationic
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5 polymers and negatively charged nucleic acids. Most of the selected NPs were smaller than 200 nm. The
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7 formulation with F127 did not change NP size of C6 and C12 modified polymers, but did decrease the
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9 size of NPs of C10 polymers (Figure 5b). The diameter of NPs with or without F127 remained constant
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within 0.5 to 2 hours of monitoring prior to addition to cells. Furthermore, the surface charge of mRNA
12 NPs with selected polyesters dramatically decreased after addition of F127, indicating the formation of
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14 the protective PEG layer on the NP surface (Figure 5c). Meanwhile, the formulation-induced charge
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16 decrease weakened NP interactions with cell surfaces, resulting in decrease in mRNA delivery in vitro
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Figure 6. Luminescence images of IGROV1 cells after 24 h-treatment with Luc mRNA polyplex
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Luminescence imaging of cells treated with Luc mRNA NPs. Luciferase encoded mRNA is powerful
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5 for mRNA delivery evaluation because of the convenient luminescence measurement both in vitro and
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7 in vivo. Prior to the administration of mRNA NPs to mice, in vitro luminescence imaging was performed
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9 on IGROV1 cells for the selected formulations (Figure 6). For cases of NPs without F127, the
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luminescence signal decreased at highest mRNA dose due to the cytotoxicity and possible aggregation.
12 After formulation with 5% F127, the luminescence of cells was lower than that without formulation at
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14 low mRNA dose. But the luciferase expression was stronger at high dose due to the improvement in NP
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16 stability. Further increase of F127 content to 10% led to a decrease in luminescence, suggesting
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18 inefficient Luc mRNA delivery by polyesters with 10% F127.
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Figure 7. In vivo efficacy of mRNA NPs delivered by tail vein injection to mice at a dose of 1 mg/kg
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56 mRNA. (a) Lead Luc mRNA NPs enable efficient mRNA delivery to mice. (b) Representative ex vivo
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images show luciferase expression mainly in the lungs (minor spleen expression) after systemic
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In vivo evaluation of mRNA delivery. Based on the promising in vitro luminescence imaging (Figure
12 6), we focused on 5% F127 formulated NPs in studies in vivo. There was clear luciferase expression in
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14 mice after i.v. administration of Luc mRNA NPs with 5% F127. When F127 content increased to 10%,
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16 there was almost no luciferase detected under the same measurement setting (Figure 7a), in agreement
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18 with reduced in vitro delivery efficacy observed with higher F127 content. Luminescence images of
19 excised organs following i.v. injection of Luc mRNA NPs with PE4K-A17-0.33C12 (Figure S4) and 5%
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21 F127 demonstrate luciferase expression predominantly in the lungs, with less delivery to the spleen
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23 (Figure 7b). The mixture of mRNA and functional polyesters without F127 became turbid immediately
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25 under the in vivo formulation concentration (0.13 g mRNA/L) and there was a precipitate on the bottom
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of the dialysis cup after 24 hour dialysis against PBS. Therefore, un-formulated NPs (i.e. NPs without
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28 the F127 stabilizer) were not included in the in vivo studies. A large number of studies showed that
29
30 nucleic acid NPs readily accumulate in the liver after i.v. administration due to the size of nanoparticles
31
32 and possible adsorption of hepatocyte targeting proteins in serum (e.g. ApoE).60 It has been shown that
33
34
altering administration method to aerosol inhalation enables specific delivery of siRNA or mRNA in the
35 lungs.24, 46 Our results indicate that functional polyesters enable efficient mRNA delivery to the lungs
36
37 but not the liver via i.v. injection, which is consistent with data on poly(β-amino ester) (PBAE)
38
39 polymers in a recent report.32 It was shown that PEGylated mRNA polyplex NPs accumulated in
40
41 multiple organs (including liver) but that the mRNA was only translated to protein in the lungs.32 It was
42
further speculated that specific protein adsorption or physiology may play a role in lung delivery.32, 60-63
43
44 Moreover, a report on a degradable LNP indicated preferential delivery to the spleen, hypothesized to be
45
46 due to the degradable bonds.64 Thus, factors including surface charge, size, and degradability might
47
48 contribute to lung mRNA delivery of degradable mRNA NPs. As shown in luminescence images,
49
50
formulation with additional F127 (10%) diminished luciferase activity. Ex vivo organ images from mice
51 injected with PE4K-A17-0.33C12 - 5% F127 NPs show more than 30-fold higher luciferase expression
52
53 in the lungs that mRNA NPs with PE4K-A17-0.2C12 -10% F127 (Figure S5). Inclusion of additional
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55 F127 may provide better protection from the aggregation or precipitation, but it also sacrifices the
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mRNA delivery potency. The balance between these two factors should be carefully considered in the
4
5 design and formulation of nucleic acid NPs.
6
7
8 Conclusions
9
10
11
In this paper, we report a robust functional polyester library for the discovery of efficient delivery
12 carriers for emerging mRNA therapeutics. In vitro screening of the polyester library enabled
13
14 identification of amine A17 modified polyester series as being potent carriers for Luc mRNA delivery to
15
16 IGROV1 cells. Successful delivery was validated by fluorescence imaging of cells treated with polyplex
17
18 NPs of mCherry mRNA and selected polyesters. To improve the stability of mRNA NPs at high
19 concentration and in serum, formulated mRNA NPs were prepared by adding 5 or 10% triblock
20
21 copolymer PEO101-PPO56-PEO101 (F127) into the stock solution of the lead functional polyesters prior to
22
23 mixing with mRNA. Formulation with F127 led to a slight decrease in the size and a remarkable
24
25 decrease in surface charge of mRNA NPs depending on the polyester and F127 content. It did not
26
sacrifice the encapsulation of mRNA into polyplex NPs, but the delivery efficacy decreased with higher
27
28 F127 content. Luminescence imaging showed that the formulation with 5% F127 enabled efficient
29
30 delivery of Luc mRNA at high dose whereas addition of 10% F127 significantly decreased the potency
31
32 across the entire dose range. To balance the protective effect and delivery potency, formulated mRNA
33
34
NPs with moderate F127 coating (5%) were examined in vivo. Bioluminescence imaging demonstrated
35 efficient Luc mRNA delivery to mice after intravenous injection of NPs, where the luciferase expression
36
37 was mainly in the lungs. The safe and efficient mRNA delivery specifically to lungs by functional
38
39 polyesters may hold potential for the treatment of pulmonary diseases.
40
41
42
Acknowledgements
43
44 D.J.S. gratefully acknowledges financial support from the Cancer Prevention and Research Institute of
45
46 Texas (CPRIT) (R1212), the Welch Foundation (I-1855), the American Cancer Society (RSG-17-012-
47
48 01), and the Mary Kay Foundation (049-15). Y.Y. gratefully acknowledges financial support from
49
Zhejiang University of Technology (105008529).
50
51
52 Supporting Information
53
54
55 The Supporting Information is available free of charge on the ACS Publications website at DOI:
56
57
10.1021/acs.biomac.
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This information includes the chemical structures of functional polyesters modified with amino- and
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5 alkyl-thiols used in the study, cell viability after treatment with mRNA polyplex NPs, effects of
6
7 PEG2000 DMG lipid on mRNA delivery, and ex vivo quantification of luminescence intensity in organs
8
9 after administration of mRNA polyplex NPs
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11
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