The effect of Al and Cr elements on the oxidation resistance of MoSi2 via first-principles calculation

Article Cite This: Ind. Eng. Chem. Res. 2019, 58, 6982−6991

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Postfunctionalization of Noncationic RNA−Polymer Complexes for RNA Delivery Ziwen Jiang,† Wei Cui,‡,∥ Jesse Mager,*,‡,∥ and S. Thayumanavan*,‡,§,∥ †

Department of Chemistry, ‡Department of Veterinary and Animal Sciences, §Molecular and Cellular Biology Program, and ∥Center for Bioactive Delivery at the Institute for Applied Life Sciences, University of Massachusetts, Amherst, Massachusetts 01003, United States

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ABSTRACT: We present a postfunctionalization strategy to formulate noncationic RNA−polymer complexes for RNA delivery. Utilizing the methylated pyridinium moieties on the polymer, RNA−polymer polyelectrolytes were first formed via electrostatic interaction. Next, the polyelectrolytes were partially cross-linked with dithiothreitol, “locking” the RNA inside the complex and leaving the rest of the N-methylated pyridyl disulfide groups to subsequently react with thiols. We carried out the postfunctionalization using thiolated oligoethylene glycol and removed a majority of the cationic moieties among the RNA− polymer complexes. The strategy facilitates the noncationic RNA−polymer complexes with tunable RNA release rates in the presence of intracellularly abundant glutathione. With significantly reduced cytotoxicity, the resultant complexes protect RNA against the enzymatic degradation by ribonuclease A and fetal bovine serum. Moreover, the RNA−polymer complexes demonstrated successful siRNA delivery and gene specific knockdown in HeLa cells.

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INTRODUCTION Cytotoxicity of cationic materials has been a concern for the development and translation of therapeutic delivery.1−5 Cationic materials are traditionally preferred because of their high cellular uptake efficiency.6,7 Because cell membrane generally has net negative surface charge,8 positively charged delivery vehicles can interact with cell membrane through electrostatic interaction. Meanwhile, nucleic-acid-based cargos can benefit from the electrostatic interaction to improve the loading capacity of cationic vehicles.9−11 However, cationic moieties at high dosage could hamper the integrity of cell membrane and mitochondrial membrane,12 inducing a significant level of toxicity toward cells. Thus, the biocompatibility of cationic materials needs to be carefully evaluated in vitro and in vivo. RNA therapeutics have been widely facilitated by cationic materials, including cationic lipids13−15 and polymers,16−18 as their electrostatic complementarity can result in effective complexation.Among RNA-based technologies, RNA interference (RNAi) utilizes double-stranded RNA (dsRNA) and is a robust tool for gene silencing.19 Once entering the cytosol, the dsRNA of interest can initiate the endogenous RNAi mechanism, leading to the degradation of target mRNA and subsequent gene specific knockdown.20 Cationic materials can complex with RNA and protect RNA from hydrolysis and enzymatic degradation,21,22 offering a suitable scaffold to load © 2019 American Chemical Society

RNA molecules. The overall positive charge of such an RNA− polymer complex can further facilitate an efficient intracellular delivery of RNA. We designed experiments to utilize the capability of cationic polymers to complex with RNA and subsequently remove the positive charges among the complex, reducing the cytotoxicity of the complex. Previously, we have reported such a “bait-andswitch” supramolecular strategy to deliver dsRNA into preimplantation mammalian embryos.23 Herein we developed a postfunctionalization strategy for noncationic RNA−polymer complexes to achieve RNAi. In detail, a series of cationic polymers are synthesized to complex with dsRNA. The positive charges within these polymers are contributed by methylated pyridyl disulfide (MPDS) side chains. Next, the RNA−polymer polyelectrolytes are partially cross-linked by the addition of dithiothreitol (DTT), leaving the rest of the MPDS side chain to be further functionalized with thiols (Scheme 1). The majority of cationic moieties in the complex are expected to be removed during these two steps. Thus, a postfunctionalization strategy provides a simple approach to varying the cross-linking degree of the complex and tuning the release rate of the RNA cargos. Received: Revised: Accepted: Published: 6982

February 4, 2019 April 4, 2019 April 4, 2019 April 4, 2019 DOI: 10.1021/acs.iecr.9b00666 Ind. Eng. Chem. Res. 2019, 58, 6982−6991

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Industrial & Engineering Chemistry Research Scheme 1. Formation and Postfunctionalization Process of the Noncationic RNA−Polymer Complex

Meanwhile, it allows the RNA−polymer complexes to be postdecorated with functional molecules. The formulation via postfunctionalization protected RNA against enzymatic degradation and demonstrated gene specific knockdown in HeLa cells.

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SIRNA REQUIRES HIGH POSITIVE CHARGE DENSITY ON POLYMERS FOR COMPLETE COMPLEXATION The cationic polymer for the “bait-and-switch” supramolecular strategy is mainly composed of MPDS side chains as the cationic moieties, with charge-neutral oligoethylene glycol (OEG) side chains to modulate the overall charge density of the cationic polymer. We previously demonstrated that the polymer can be efficiently complexed with long dsRNA.23 To extend the applicability of our system, we hypothesized that small interfering RNA (siRNA), the most widely used RNA interference tool, could be compatible with this bait-and-switch supramolecular strategy. Therefore, we used an siRNA designed against GFP (siGFP) to test the efficacy of our strategy. First, we attempted this with P1 (OEG:PDS molar ratio = 12:88; PDS, pyridyl disulfide ethyl methacrylate), the optimal candidate that we used for long dsRNA. The binding between polymer and RNA was evaluated by tuning the ratio between polymer and RNA, denoted as N/P ratio. However, the P1−siGFP binding requires a much higher polymer dosage to completely bind with siRNA compared to P1 with long dsRNA (Figure 1b). To minimize the dosage of polymer for the complexation, we synthesized P2, a polymer with the same OEG:PDS ratio as P1 but with lower molecular weight. The hypothesis of using polymers with lower molecular weight was based on the molecular weight difference between P1 and dsRNA. During the long dsRNA−polymer complexation, the molecular weight of dsTuba1a is ∼25 times more than that of P1. Since the molecular weight of siRNA is ∼20 times lower than that of dsTuba1a, we hypothesize that decreasing the molecular weight of polymer could improve the complexation performance with siRNA in a similar fashion. We also prepared a methylated poly(pyridyl disulfide) homopolymer (P3) to test binding with siRNA. Through agarose gel electrophoresis assays, we observed

Figure 1. (a) N-Methylation reaction of PDS-based random copolymer (P1′ and P2′) and PDS homopolymer (P3′). Optimization of the complexation between siGFP and (b) P1, (c) P2, and (d) P3. The ratio in each these figures represents the N/P ratio: the molar ratio between the positive charge in polymers and the negative charge in RNA. Even at N/P = 70/1, P1 and P2 did not completely complex with siGFP. When N/P is higher than 15/1, P3 completely complexed with siGFP.

that the P2−siGFP binding behavior is similar to that of P1− siGFP (Figure 1c). However, the optimal N/P ratio for P3− siGFP binding was significantly reduced compared to other polymers (Figure 1d). These results demonstrate the importance of positive charge density in polymers for smaller RNA binding, rather than the molecular weight of polymers. 6983

DOI: 10.1021/acs.iecr.9b00666 Ind. Eng. Chem. Res. 2019, 58, 6982−6991

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Industrial & Engineering Chemistry Research

Figure 2. (a) Dynamic light scattering and (b) ζ potential results during the formulation process of siGFP−P3 complexes. The inset of (a) shows the TEM image of the P3−siGFP post-PEGylated complex. The scale bar stands for 100 nm.

Figure 3. (a) Time-dependent dsRNA release after the addition of 2 mM glutathione (GSH) at 37 °C. Lane 1, dsRNA (dsTuba1a). Lane 2, dsRNA− P1 polyelectrolyte. Lanes 3−8, dsRNA−P1 complex that aimed at different cross-linking degrees: 3, 10%; 4, 20%; 5, 30%; 6, 50%; 7, 80%; 8, 100%. (b) The release profile of dsTuba1a with different cross-linking degrees by analyzing RNA band intensity. The band intensity of lane 1 was normalized as 100% at each time point.

of the two-step process, the post-PEGylated siRNA−P3 complex was formulated and characterized (Figure 2, Figures S2 and S3). From the dynamic light scattering (DLS) measurement, the size of the complex between P3 and siGFP was slightly larger after DTT-induced cross-linking (20% feed ratio) and post-PEGylation, resulting in ∼60 nm complexes (Figure 2a). ζ potential results demonstrated the charge conversion throughout the formulation process. P3 is cationic in water. The polyelectrolyte complex between P3 and siGFP turned negative at the optimal N/P ratio (20/1 was selected), suggesting that the polyelectrolyte may have partial siRNA strands exposed on the complex surface (Figure 2b). Likewise, we formulated and characterized post-PEGylated long dsRNA− polymer complex. The post-PEGylated long dsRNA−polymer complex has similar size and charge properties compared to the post-PEGylated siRNA−polymer complex (Figure S4). The similar physiochemical properties (hydrodynamic diameter and surface ζ potential) among different RNA−polymer complexes allow us to study their biological activities with fewer parametrical variations.7,25

Indeed, multivalency has been shown to be an important factor to achieve a stable polymer−nucleic acid complex.24 siRNA is less multivalent than long dsRNA, thus requiring higher positive charge density within polymers to achieve complete binding.

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RNA RELEASE RATE CAN BE TUNED BY VARYING THE CROSS-LINKING PERCENTAGE WITHIN THE RNA−POLYMER COMPLEX We next developed our RNA−polymer complex with a postPEGylation step. Such modification is expected to improve the stability of the complex against enzymatic degradation, due to increased steric protection by the polymer attachment. We varied the DTT dosage to tune the cross-linking percentage within the RNA−polymer complex, followed by the addition of thiolated oligoethylene glycol (m-PEG-thiol) to cleave the rest of the methylated PDS moieties. We have demonstrated that the thiol−disulfide exchange reaction on MPDS units is quantitative by analyzing the concentration of N-methyl-2-pyridinethione, the byproduct of the reaction.23 Additionally, we confirmed that the subsequent PEGylation is efficient (Figure S1). On the basis 6984

DOI: 10.1021/acs.iecr.9b00666 Ind. Eng. Chem. Res. 2019, 58, 6982−6991

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Industrial & Engineering Chemistry Research

Figure 4. (a) Schematic illustration of the metabolic stability test for the post-PEGylated RNA−P1 complex. Time-dependent dsRNA release after the addition of glutathione (GSH), evaluating the stability of dsRNA within the complex against (b) RNase A or (c) FBS treatment. “+” denotes the presence of the substance; “−” denotes the absence of the substance. RNA, dsTuba1a; Polymer, P1; RNase A, ribonuclease A, 20 pg μL−1; FBS, fetal bovine serum.

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NONCATIONIC RNA−POLYMER COMPLEXES PROTECT RNA FROM ENZYMATIC DEGRADATION One goal of encapsulating RNA molecules within a delivery vehicle is to enhance their stability in a serum-containing environment as well as to protect against nucleases. To evaluate the metabolic stability of dsRNA within the RNA−polymer complex, we tested the complex with ribonuclease A (RNase A) or fetal bovine serum (FBS). After incubating the enzyme of interest with our complex for 30 min at 37 °C, we inhibited the enzymatic activity of RNase A or FBS and applied GSH to the complex (Figures S7 and S8). If the RNA is protected from enzymatic degradation through the polymer complexation, we expect that the released RNA will have the same shift as the RNA control on agarose gel. As shown in Figure 4a, a similar RNA release profile was observed with or without the RNase A

Next, we hypothesized that the DTT dosage can affect the RNA release rate from the cross-linked RNA−polymer complex. Increasing the DTT dosage has been demonstrated to cause an increased cross-linking degree among the polymers with PDS moieties, resulting in a decreased cargo release rate.26 The intracellularly abundant glutathione (GSH) was utilized as the trigger to induce RNA release by disrupting the disulfide bond among the complex.27,28 We observed an increased rate of GSHtriggered RNA release when reducing the cross-linking density within the RNA−polymer complex (Figure 3). The phenomenon was also confirmed with a 900 bp double-stranded DNA and siRNA (Figures S5 and S6). The formulation with the postPEGylation step thus offers an additional control element, where the GSH-triggered release rate of RNA from the complex can be fine-tuned for experimental design. 6985

DOI: 10.1021/acs.iecr.9b00666 Ind. Eng. Chem. Res. 2019, 58, 6982−6991

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Figure 5. Delivery of Cy3−siRNA to HeLa cells after RNA−polymer complexation. Confocal microscopy images showing Cy3−siRNA delivery to HeLa cells at (a) 24 h and (b) 48 h. (c) Z-Stack image of Cy3−siRNA delivery at 24 h. The scale bar in each figure represents 20 μm. (d) Flow cytometry results of Cy3−siRNA delivery by P3 complexation.

increased from ∼3% after incubating with the complex for 1 h to ∼60% after 4 h (Figure 5d, Figure S11). These results suggest that, although being negatively charged, the siRNA−polymer complex still interacts with cells and is indeed internalized.

treatment for the post-PEGylated dsRNA−P1 complex, demonstrating protection from nuclease degradation provided by the complex. Meanwhile, the free dsRNA band was completely shifted after incubation with RNase A, indicating the degradation of dsRNA. We also evaluated the RNA stability in the presence of FBS, the most widely used supplement for cell culture media. Previous studies have shown hydrolysis of dsRNA due to the presence of nuclease in FBS.29 As shown in Figure 4b, the RNA−polymer complex provides significant protection for the encapsulated RNA molecules in both 5% and 10% FBS.

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METHYL-β-CYCLODEXTRIN TREATMENT REDUCED THE CELLULAR UPTAKE OF RNA−POLYMER COMPLEXES We next examined cellular uptake of the noncationic RNA− polymer complexes in the presence of pharmacological inhibitors of distinct cellular mechanisms.31,32 Chlorpromazine (CPZ) induces a loss of clathrin and adapter protein complex 2 on the cell surface.33 Amiloride (AMI) is widely used as an inhibitor for macropinocytosis via the inhibition of Na+/H+ exchange.34 Methyl-β-cyclodextrin (MβCD) was chosen as the inhibitor for lipid-raft-mediated endocytosis by depleting plasma membrane cholesterol, an essential molecule for lipid raft formation.35 Nystatin (NYS) disassembles caveolae and cholesterol assemblies in the membrane and hence was utilized as an inhibitor for caveolae-mediated endocytosis.36 Additionally, fucoidan (FCD) was used as an inhibitor for scavengerreceptor-mediated endocytosis, as scavenger receptors typically recognize negatively charged macromolecules.37 After treating HeLa cells with each different inhibitor, we applied the postPEGylated Cy3−siRNA−P3 complex to the pretreated HeLa cells for 3 h and measured the uptake of the complex. As shown in Figure 6, multiple endocytic pathways were involved in the

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CELLULAR UPTAKE OF THE RNA−POLYMER COMPLEXES IS TIME-DEPENDENT The cellular uptake of the RNA−polymer complex was evaluated by confocal microscopy and flow cytometry. On the basis of the formulation between siGFP and P3, a Cy3-labeled negative control siRNA (Cy3−siRNA) was formulated with P3 and incubated with growing HeLa cells. The Cy3 fluorescence signal was used as an indication of the localization of the RNA− polymer complex. The cellular uptake of the Cy3−siRNA−P3 complex showed a time-dependent increase primarily during the initial 48 h incubation with HeLa cells (Figure 5, Figure S10). The fluorescence of Cy3 decreased after 72 h of incubation (Figure S10), possibly due to the increased proliferation of cells.30 The time-dependent increase of the Cy3−siRNA−P3 complex within HeLa cells was further confirmed by flow cytometry. The percentage of cells with Cy3 fluorescence 6986

DOI: 10.1021/acs.iecr.9b00666 Ind. Eng. Chem. Res. 2019, 58, 6982−6991

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(Figure 7a), demonstrating high biocompatibility of the formulation. After deGFP−HeLa cells were treated with postPEGylated P3−siGFP complex, post-PEGylated P3−siScram (scrambled siRNA, negative control) complex, and siGFP, separately, the cells treated with the P3−siGFP complex showed a ∼35% decrease in deGFP fluorescence (Figure 7b). As expected, no decrease in deGFP fluorescence was observed in the siRNA only group. Moreover, no knockdown was observed in the negative control group that is based on a scrambled siRNA and P3 complex, further confirming the in vitro knockdown capability of our RNA−polymer complex.

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CONCLUSIONS In summary, we describe a strategy to postfunctionalize noncationic RNA−polymer complexes with negligible cytotoxicity. The key component of our strategy is the methylated pyridyl disulfide moieties among the polymeric scaffold. The cationic moieties initiate complexation with RNA molecules, and the density of these positive charges significantly affects the binding between cationic polymers and RNA. Moreover, the methylated pyridyl disulfide moieties on the polymer side chain can undergo an efficient thiol−disulfide exchange reaction, enabling the polymer to be either functionalized with a monothiol or cross-linked by a dithiol (or multithiol). During the exchange reaction, the cationic moieties are cleaved in situ, significantly reducing the toxicity concern of polymeric materials. As a result, the chemical design allows us to tune the RNA release rate of the complex by varying the cross-linker concentration. We utilized dithiothreitol as the cross-linker to build in disulfide bonds among the polymeric scaffold, allowing the complex to be responsive to glutathione, a potent and abundant intracellular redox signaling tripeptide. Here we show that the postfunctionalization step with PEG-thiol protects the complexed RNA from enzymatic degradation. We envisage that the postfunctionalization strategy can be further applied to the incorporation of small molecule targeting ligands or biomacromolecules that contain thiol as well as the construction of supramolecular clustering in a broader context. Importantly, we demonstrate gene specific knockdown with long dsRNA in preimplantation mammalian embryos23 and now siRNA-

Figure 6. Relative cellular uptake efficiencies of Cy3−siRNA to HeLa cells after P3 complexation, in the presence of different inhibitors as compared to those without inhibitors. FCD, fucoidan; NYS, nystatin; AMI, amiloride; MβCD, methyl-β-cyclodextrin; CPZ, chlorpromazine. Error bars denote the standard deviation of replicates. *, P < 0.01, n = 4.

uptake of the RNA−polymer complexall of the inhibitors besides the micropinocytosis inhibitor resulted in a decrease in uptake of Cy3−siRNA−P3. Cells treated with MβCD showed the most significant loss of complex uptake, suggesting that lipidraft-mediated endocytosis is a major endocytic pathway required for cellular uptake of P3−siRNA complexes.

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POSTFUNCTIONALIZED RNA−POLYMER COMPLEXES ARE HIGHLY BIOCOMPATIBLE AND CAPABLE OF GENE SPECIFIC KNOCKDOWN Finally, we employed HeLa cells that stably express destabilized green fluorescent protein (deGFP) to evaluate the RNAi capability of siRNA delivery with our post-PEGylated siRNA− polymer complex. We chose to target deGFP38−40 as a reporter to quantify the knockdown efficiency. Before assessment of the knockdown efficiency of the complex, the cytotoxicity of postPEGylated siRNA−P1 complexes was evaluated at different concentrations. The RNA−polymer complex exhibited negligible cytotoxicity after incubating with HeLa cells for 24 h

Figure 7. (a) Cytotoxicity profile of the siRNA−P3 post-PEGylated complex on HeLa cells after 24 h of incubation. (b) Percentage of deGFP expression in deGFP−HeLa cells after incubating with siGFP−P3 complex, siScram-P3 complex, or siGFP for 24 h. Error bars denote the standard deviation of replicates in each figure. n = 3. *, P < 0.005. 6987

DOI: 10.1021/acs.iecr.9b00666 Ind. Eng. Chem. Res. 2019, 58, 6982−6991

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Industrial & Engineering Chemistry Research

compensated by adding 5 mM sodium phosphate buffer (pH 7.4). The byproducts from the cross-linking and PEGylation reactions were purified by Amicon centrifugal filters with 3K molecular weight cutoff (MWCO). The optimal dosage for the siRNA−polymer (P1, P2, or P3) complexation was obtained via the optimization of the N/P ratio. For the calculation of the N/P ratios, the average mass per charge for siRNA was 325 Da. The average mass per positive charge for P1, P2, and P3 was 429, 429, and 419 Da, respectively. In each well, 100 ng of siRNA was first added into nuclease-free water. Next, an increasing amount of polymer (P1, P2, or P3, 2 mg mL−1 stock solution) was added into the RNA aqueous solution to elevate the N/P ratio. The final volume of each siRNA−polymer mixture solution was maintained at 20 μL. The optimal N/P ratio for P3 is determined as 20/1. The mixture was further formulated with DTT-induced cross-linking and post-PEGylation reactions. During the post-PEGylation step, m-PEG4-thiol (BroadPharm) was used for the siRNA− polymer formulation. Nuclease-free water was used throughout the formulation process. The solution (water) was replaced with 5 mM sodium phosphate buffer (pH 7.4) during the complex purification using Amicon centrifugal filters with 3K MWCO. The concentration of siRNA within the post-PEGylated complex is calculated using the feed dosage of siRNA and the final volume of phosphate buffer. Glutathione-Triggered (GSH-Triggered) dsRNA Release. The post-PEGylated RNA−polymer complexes were formulated, and the overall sample volume was adjusted to 40 μL with 5 mM phosphate buffer (pH = 7.4). For the evaluation of the RNA release, GSH stock solution (10 μL) was added into the complex-containing solution to obtain the final volume at 50 μL, with the final GSH concentration at 2 mM (Figure S9). Each sample was incubated at 37 °C. At each time point, the solution was well mixed with a pipet, and 7 μL of solution was collected to check the RNA release using similar electrophoresis procedures as above. Metabolic Stability Test of dsRNA within the Complex against RNase A and FBS. The experiments were all based on the post-PEGylated dsTuba1a−P1 complex. The preformed complex was started by complexing 100 ng of dsTuba1a and 5.32 μg of P1, with the additional corresponding amount of DTT and m-PEG8-thiol to aim at a 20% cross-linking percentage. The volume containing the post-PEGylated dsTuba1a−P1 complex was adjusted to 30 μL using 5 mM sodium phosphate buffer (pH 7.4). RNase A (20 pg μL−1 as the final concentration) or FBS (5% or 10% as the final concentration) was added afterward and incubated at 37 °C for 30 min, followed by the addition of the corresponding inhibitor. For RNase A inactivation, SUPERase·In (8 U μL−1) was added and incubated for 30 min at 37 °C. For FBS inactivation, SDS was added to obtain its final concentration at 0.3% and incubated at room temperature for 5 min. After the inactivation of the enzyme, GSH was added into the solution at a final concentration of 2 mM and incubated at 37 °C. Subsequently, the dsTuba1a release was monitored at different time points using agarose gel electrophoresis. General Procedure for Cell Culture and Viability Assay. Cell Culture. HeLa cells with or without destabilized GFP expression were grown and passaged in Dulbecco’s modified Eagle’s medium:nutrient mixture F-12 (DMEM/F12, ThermoFisher, 10565018) supplemented with 10% fetal bovine serum (FBS, ThermoFisher, 10437028) and 1% antibiotics (100 U mL−1 penicillin−streptomycin, ThermoFisher,#15140122).

mediated silencing with this novel design, providing a highly biocompatible and nontoxic polymeric tool for RNA delivery.

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EXPERIMENTAL SECTION Reagents were acquired from commercial sources without further purification. NMR spectra were acquired from a Bruker AdvanceIII 400 (or 500) NMR spectrometer. Gel permeation chromatography (GPC) was conducted using tetrahydrofuran as the eluent on an Agilent 1260 LC instrument. Calculations of molecular weights were based on polystyrene standards. Dynamic light scattering and ζ potential results were obtained on a Malvern Zetasizer Nano ZS instrument. The transmission electron microscopy (TEM) image was obtained from a JEOL 2000FX electron microscope. Absorption spectra were acquired with a PerkinElmer Lambda 35 UV/vis spectrophotometer. Confocal microscopy images were captured from a Nikon fluorescence microscope equipped with a Yokogawa spinning disk. Flow cytometry experiments were performed on a ThermoFisher Attune NxT flow cytometer. The infrared spectra were obtained on a Bruker Alpha FT-IR spectrometer (3500− 400 cm−1). The thermogravimetric analysis (TGA) was carried out under N2 flow using a TA Instrument Q50 thermogravimetric analyzer (25−600 °C). The statistical analysis was performed with GraphPad Prism 7.00 software using the unpaired t test. The significant difference was qualified when P < 0.01. General Procedure for the N-Methylation Reaction of Polymers. The synthesis of pyridyl disulfide ethyl methacrylate (PDS) monomer was based on our previous report.41 PEG− PDS random copolymers (P1′ and P2′) or PDS homopolymers (P3′) were obtained following a previous report via reversible addition−fragmentation chain-transfer polymerization.26 The average molecular weight of poly(ethylene glycol) methyl ether methacrylate monomer is 500 g mol−1 for the synthesis of P1′ and P2′. The N-methylation reaction of P1′, P2′, and P3′ was conducted following our previous report,23 respectively resulting in the N-methylated polymers: P1, P2, and P3. Preparation of dsRNA and siRNA Sequence. Long double-stranded RNA of Tuba1a (dsTuba1a42) was prepared following a previous report and used as the model long dsRNA. Cy3-labeled negative control siRNA (Cy3−siRNA, Sigma SIC003), siRNA negative control (scrambled siRNA, siScram, Sigma SIC001), and siGFP (Dharmacon P-002048-01-20) were purchased from commercial sources. The GFP siRNA (siGFP) sequence is 5′-GCAAGCTGACCCTGAAGTTC. General Procedure for Polymer−RNA Complexation and Cross-Linking. The complexation between RNA and polymer was assessed using electrophoresis on agarose gel (2%) for 25 min at 72 V (Thermo Electron Corporation EC-105 Compact Power Supply) in Tris-acetate-EDTA (TAE) running buffer. RNA was visualized by ethidium bromide staining. The complexation and cross-linking between polymer (P1) and dsRNA were carried out on the basis of a previous report.23 For the formulation with post-PEGylation step, DTT stock solution was spiked into the solution containing P1−dsRNA polyelectrolyte (when N/P = 40/1) and incubated for 30 min at room temperature. The volume of DTT solution was varied to tune the feed dosage to aim at different cross-linking percentages within the complex. The rest of the PDS units were replaced by adding a quantitative amount of m-PEG-thiol and incubating at room temperature for 1 h. During the post-PEGylation step, mPEG8-thiol (BroadPharm) was used for the dsRNA−polymer formulation. The sample volume difference in each sample was 6988

DOI: 10.1021/acs.iecr.9b00666 Ind. Eng. Chem. Res. 2019, 58, 6982−6991

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Industrial & Engineering Chemistry Research

HeLa cells were cultured in DMEM/F12 for 1 h and incubated with the Cy3−siRNA−polymer complex for another 3 h. For the blank group, HeLa cells were cultured in DMEM/F12 for 4 h. After the fluorescence signal was subtracted from the blank group, the Cy3−siRNA fluorescence of the positive control group was normalized as 100%. General Procedure for the deGFP Knockdown Experiment. A total of 10K deGFP-expressing HeLa cells were mixed with the complex-containing DMEM/F12 and seeded into a 96well plate. For different experimental groups, each well contains 60 nM siGFP within the siRNA−polymer complex, 60 nM siScram within the siRNA−polymer complex, or 60 nM siGFP. On the basis of the initial polymer weight and final sample volume, with the assumption of no sample loss during the formulation process, the final concentration of polymers in each sample is 21.5 ng μL−1. deGFP-expressing HeLa cells cultured in DMEM/F12 were used as 100% expression, and HeLa cells without deGFP expression were used as the blank group. The cells were maintained in a humidified atmosphere (5% CO2) at 37 °C for 24 h. The fluorescence intensity of deGFP within the cells was measured using flow cytometry with an excitation wavelength of 488 nm. After the fluorescence signal was subtracted from the blank sample, the deGFP fluorescence of the cells without any treatment was normalized as 100%.

The cells were cultured in a humidified atmosphere supplied with 5% CO2 at 37 °C. Cell Viability Assay (alamarBlue). A total of 15K HeLa cells per well were seeded in a 96-well plate for 24 h before the evaluation. For the assessment of the biocompatibility of the siRNA−P3 post-PEGylated complex, siScram was used to complex with P3 and formulated as mentioned above. Replicates of the complex from different wells were combined, purified, and concentrated with an Amicon Ultra centrifugal filter unit (MWCO 3 kDa). The concentrated complex was diluted to different concentrations and incubated with cells for 24 h. Next, after washing with phosphate buffer saline (PBS), the cells were supplied with 220 μL of full DMEM/F12 growth medium that contains 10% alamarBlue reagent. After incubation for 70 min, cell viability was calculated by acquiring the fluorescence intensity of alamarBlue at 590 nm (excitation wavelength: 535 nm). General Procedure for Evaluating the Cellular Uptake of the siRNA−Polymer Complex. For the time-dependent Cy3−siRNA uptake evaluation, a total number of 25K HeLa cells per well were seeded in a 48-well plate for 24 h before the experiment. The siRNA−P3 polyelectrolyte was formulated with 0.1 equiv of DTT and 0.8 equiv of m-PEG4-thiol, purified and concentrated. The concentrated complex was diluted using Dulbecco’s modified Eagle’s medium to obtain the siRNA concentration at 60 nM within the complex. The complex in DMEM/F12 was added to HeLa cells and incubated at 37 °C. For the time-dependent experiment, the cellular uptake of the complex was evaluated every hour within the initial 4 h. After the cells were washed with cold PBS, the fluorescence intensity of Cy3−siRNA within the cells was measured using flow cytometry with an excitation wavelength of 488 nm. For the measurement of the cellular distribution of the siRNA−polymer complex, the siRNA−P3 complex was prepared in DMEM/F12 as mentioned above. HeLa cells (20K) in DMEM/F12 were mixed with the complex-containing DMEM/F12 and seeded into a glass bottom dish (Cellvis, D35C4-20-0-N). After incubation for 3 h at 37 °C, FBS was added into the medium to obtain a final concentration of 10%. The cells were maintained in a humidified atmosphere (5% CO2) at 37 °C for different time lengths (12, 18, 24, 36, 48, and 72 h). At each time point, the cells were incubated with 50 nM LysoTracker Green (ThermoFisher, L7526) for 30 min and NucBlue (1 drop in 500 μL of DMEM/F12, ThermoFisher, R37605) for 5 min in FluoroBrite DMEM (ThermoFisher, A1896701) at 37 °C, staining the lysosome and nucleus, respectively. The intracellular distribution of Cy3−siRNA was measured by confocal microscopy with excitation wavelengths of 405, 488, and 561 nm. Procedures for Determining the Endocytic Pathways. A total of 25K HeLa cells were cultured in a 48-well plate for 24 h prior to the experiment. For the inhibition of different endocytic pathways, cells were cultured for 1 h in DMEM/F12 containing fucoidan (0.1 mg mL−1, Carbosynth, YF01606),37 nystatin (25 μg mL−1, Alfa Aesar, J62486),43 amiloride (1 mM, SigmaAldrich, A7410),34 methyl-β-cyclodextrin (3.4 mg mL−1, TCI America, M3156),44 and chlorpromazine (10 μM, TCI America, C2481).45 Subsequently, in the presence of inhibitors, the cells were incubated with Cy3−siRNA at an siRNA concentration of 60 nM within the complex for an additional 3 h. After the cells were washed with cold PBS, the fluorescence intensity of Cy3− siRNA within the cells was measured using flow cytometry with an excitation wavelength of 488 nm. For the positive control,

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.iecr.9b00666.

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Characterization of each polymer, quantification of PEGylation degree, and spectral data (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Ziwen Jiang: 0000-0002-6633-7824 Wei Cui: 0000-0001-7281-3471 S. Thayumanavan: 0000-0002-6475-6726 Author Contributions

All authors planned the project. Z.J. and W.C. conducted the experiments and analyzed the results. Z.J. and S.T. wrote the manuscript with the review of W.C. and J.M. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This contribution was identified by Dr. Chloé Grazon (Boston University) as the Best Presentation in the “Basic Research in Colloids, Surfactants & Nanomaterials” session of the 2018 ACS Fall National Meeting in Boston. We acknowledge the U.S. Army Research Office (W911NF-15-1-0568) for funding support. Z.J. was supported by the Graduate School Travel Grant (UMass Amherst) to attend the 2018 ACS Fall National Meeting. W.C. acknowledges the postdoctoral fellowship from the Lalor Foundation. We thank Dr. James J. Chambers at the Light Microscopy Facility in the Institute for Applied Life Sciences (UMass Amherst) for valuable suggestions, and Dr. Mingxu You (UMass Amherst) for generously sharing the flow 6989

DOI: 10.1021/acs.iecr.9b00666 Ind. Eng. Chem. Res. 2019, 58, 6982−6991

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Industrial & Engineering Chemistry Research

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cytometer. The deGFP-expressing HeLa cells are a generous gift from Dr. Eben Alsberg (University of Illinois at Chicago).

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DOI: 10.1021/acs.iecr.9b00666 Ind. Eng. Chem. Res. 2019, 58, 6982−6991

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DOI: 10.1021/acs.iecr.9b00666 Ind. Eng. Chem. Res. 2019, 58, 6982−6991