Fentanyl Initiated Polymers Prepared by ATRP for Targeted Delivery

Article pubs.acs.org/bc

Fentanyl Initiated Polymers Prepared by ATRP for Targeted Delivery Devora Cohen-Karni,†,‡,# Marina Kovaliov,†,‡,# Shaohua Li,†,‡ Stephen Jaffee,‡ Nestor D. Tomycz,‡ and Saadyah Averick*,†,‡ †

Neuroscience Disruptive Research Lab, Allegheny Health Network Research Institute, Allegheny General Hospital, Pittsburgh, Pennsylvania 15212, United States ‡ Neuroscience Institute, Allegheny Health Network, Allegheny General Hospital, Pittsburgh, Pennsylvania 15212, United States S Supporting Information *

ABSTRACT: The targeted delivery of polymers to neurons is a challenging yet important goal for polymer based drug delivery. We prepared a fentanyl based atom transfer radical polymerization (ATRP) initiator to target the Mu opioid receptor (MOR) for neuronal targeting. We incorporated our recently discovered rigid acrylate linking group into the initiator to retain a high degree of binding to the MOR and grafted random or block copolymers of poly(oligo(ethylene oxide) methacrylate)-block-(glycidyl methacrylate). Trifluoroethanol promoted amine ring opening of the glycidyl methacrylate was used for post-polymerization modification of the fentanyl initiated polymers to attach a near-infrared fluorescent dye (ADS790WS) or to build a targeted siRNA delivery system via modification with secondary amines. We examined the biocompatibility, cellular internalization, and siRNA binding properties of our polymer library in a green fluorescent protein expressing SY SH5Y neuroblastoma cell-line.

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INTRODUCTION Targeted delivery to neurons is a potential tool for the next generation delivery of anti-cancer and anti-addiction therapeutics.1−9 Selective delivery has the unique potential to alleviate symptoms and minimally impact normal physiological functions due to the neuronal specificity of many diseases. The targeted delivery to neurons is challenging, and novel targets are required for delivery to these cells.10−14 The Mu opioid receptor (MOR) presents an attractive target for neuronal delivery due to the ubiquitous nature of its expression and presence in both the central and peripheral nervous systems.15,16 In addition, usurping the endocytotic pathway exhibited by the MOR is an attractive pathway for delivery into the cells.17,18 Furthermore, the MOR has a diverse range of known agonists/antagonists that can potentially be incorporated into polymer chain-ends for targeted delivery.12,14 Fentanyl is a potent MOR agonist.19,20 It is used both therapeutically and in basic research. Tailormade fentanyl derivatives, unlike other MOR ligand derivatives, can be made synthetically with high yields and high purity level from readily available starting materials. Our recent discovery that highmolecular-weight moieties can be incorporated onto fentanyl, while still retaining high affinity to the MOR by utilizing a “rigid” acrylate linking group, enables its’ addition as a polymerchain end as a neuronal targeting agent.21 In this manuscript we report the preparation of atom transfer radical polymerization initiators (ATRP) that incorporate the rigid fentanyl moiety for the targeted delivery to MOR expressing cells. The use of activators generated by electron transfer (AGET) or initiators for continuous activator regeneration (ICAR) ATRP allows for exacting control over polymer architecture © XXXX American Chemical Society

and composition with the distinct advantage of utilizing an air stable precatalyst (Cu(II)) and (re)generation of the activator species (i.e., Cu(I)) in situ; to generate the active species in AGET ATRP, a reducing agent such as ascorbic acid is introduced, and in ICAR ATRP, a free radical initiator such as azobis(isobutyronitrile) generates a radical to reduce the Cu(II) species.22−29 The ability to silence gene expression using short interfering RNA (siRNA) makes this tool an attractive mechanism to control the expression of proteins that are implicated in a variety of disease states.30−32 siRNA delivery has been challenged by the poor drug-like properties of this moleculesiRNA is a negatively charged biomolecule with an average molecular weight of approximately 13.5k, that is readily degraded by both pH and enzymes leading to rapid clearance and poor cell permeability.6,33−36 To overcome these challenges cationic lipids and polymers have been used to complex and deliver siRNA.37 Reversible deactivation radical polymerization (RDRP) methods are powerful tools for preparing biocompatible cationic polymers with functional chain-ends for the targeted delivery of siRNA.24,28,38−41 Here we present our efforts to prepare fentanyl labeled polymers for the targeted delivery to neurons and the initial in vitro characterization of these biohybrids. Random and block copolymers comprised of oligo((ethylene oxide)methacrylate) (OEOMA) and glycidyl methacrylate (GMA) were grown from the fentanyl initiator. The polymers were designed to Received: February 13, 2017 Revised: March 17, 2017 Published: March 22, 2017 A

DOI: 10.1021/acs.bioconjchem.7b00078 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry Scheme 1. Synthesis of Fen-Acry-EtBPA Fentanyl Initiator

then reacted with 2-hydroxyethyl acrylate and converted to Fen-Acry-EtOH by Pd-catalyzed cross coupling reaction. FenAcry-EtBPA was prepared from Fen-Acry-EtOH by EDC/ DMAP esterification with α-bromophenylacetic acid (BPA), and the structure and purity of the compound were confirmed by 1H NMR (Figure S3 in Supporting Information). This novel derivative of fentanyl allows for polymers to be grafted directly from it. The resulting Fen-Acrylate ATRP initiator, Fen-Acry-EtBPA, was then used to form both the dye labeled polymer as well as the charged polymer for siRNA delivery, as described below. Synthesis of Dye Labeled Fentanyl Chain-End Random Copolymer. In order to visualize the polymer in live cell cultures, we prepared dye labeled fentanyl chain-end random copolymer. To prepare dye labeled fentanyl terminated polymers, we polymerized OEOMA with GMA. GMA was incorporated to facilitate post-polymerization modification with an amine functionalized near-infrared fluorescent (NIR) dye. This strategy was chosen to avoid the polymerization of a bulky dye monomer. As a proof-of-concept of visualization, we prepared random copolymer in one step for dye incorporation (Figure 1). Using a novel method to increase yields of amine

incorporate OEOMA to grant the polymer hydrophilic properties and enhance biocompatibility,42−44 while GMA facilitated the post-polymerization modification and functionalization of the polymers45−47 utilizing our recently reported enhanced ring opening conditions.45 The GMA was reacted with an amine functionalized near-infrared fluorescent dye for cell labeling assays or with several amines that is bearing tertiary amine (cationic) groups for siRNA binding. In summary, our manuscript reports the synthesis and characterization of the fentanyl ATRP initiator and fentanyl chain-end polymers and their biocompatibility, ability to bind cells expressing the MOR, and form polyplexes with siRNA.

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RESULTS AND DISCUSSION Tailormade fentanyl derivatives, unlike other MOR ligand derivatives, can be made synthetically with high yields and high purity level from readily available starting materials. Fentanyl derivatives with novel rigid linking groups such as acrylate (Fen-Acry) retain a relatively high affinity to the MOR (The Fen-Acryl-EtOH was found to have an EC50 of 22.8 nM).21 We have found that the incorporation of the “rigid” acrylate linking group as opposed to a flexible ester or ether linking group retains a higher affinity to the MOR.20 ATRP is an RDRP method that allows for the precise control over polymer composition and yields a “living” chain-end, allowing for the preparation of block copolymers and other complex architectures.28,39 Utilizing this property, fentanyl with a “rigid” linked ATRP initiator was prepared for the targeted delivery of labeled polymers to cells expressing the MOR. In order to create a polymer with a fentanyl at the chain-end we have initially synthesized a Fen-Acrylate ATRP initiator and then continued to graft two different types of polymer from it. First, we grafted a random copolymer for dye incorporation (Fen-Acryp(OEOMA-co-GMA)-S/L. We then verified the biocompatibility of those polymers and used them in cell labeling assay to verify the ability of the fentanyl chain-end polymer to bind and enter into cells expressing the MOR. We then synthesized a block copolymer for the incorporation of charged amine groups for the Fen-Acry-p(OEOMA-b-GMA)-S/L, and assessed their biocompatibility as well as their ability to bind and complex siRNA. Synthesis of Fen-Acrylate ATRP Initiator. As shown in Scheme 1, the initiator Fen-Acry-EtBPA was prepared from the precursor compound Fen-Br, which was synthesized from commercially available reagents by a substitution reaction of norfentanyl and 4-bromophenethyl bromide. The Fen-Br was

Figure 1. Schematic synthesis of dye labeled fentanyl chain-end random copolymers: Fen-Acry-p(OEOMA-co-GMA)-Dye-S and FenAcry-p(OEOMA-co-GMA)-Dye-L.

ring opening with epoxides, namely, using trifluoroethanol as solvent,45 we were able to achieve efficient incorporation of the dye into the polymer backbone. Fen-Acry-p(OEOMA-coGMA) was synthesized using activators generated by electron transfer (AGET) ATRP conditions with OEOMA:GMA ratio of 90:10 using the Fen-Acry-EtBPA initiator (Scheme 2). In AGET ATRP, an air stable polymerization mixture using a Cu(II) halide salt precatalyst is degassed and activators are generated in situ via introduction of a reducing agent such as ascorbic acid. Two polymerization periods (1 and 2 h) were used, to form a short (S) and a long (L) version of the polymer, labeled Fen-Acry-p(OEOMA-co-GMA)-S and Fen-Acry-p(OEOMA-co-GMA)-L. 1H NMR analysis was used to B


Article

Bioconjugate Chemistry Scheme 2. Synthesis of Dye-Containing Fentanyl Chain-End Polymer

Table 1. AGET ATRP of Fen-Acry-p(OEOMA-co-GMA) Using Fen Initiator polymer Fen-Acry-p(OEOMA-coGMA)-S Fen-Acry-p(OEOMA-coGMA)-L

monomer OEOMA500/ GMA OEOMA500/ GMA

initiator Fen-AcryEtBPA Fen-AcryEtBPA

[OEOMA]/[GMA]/[I]/ [CuBr2]/ [TPMA]/[AscA]

time (min)

conv %

Mn,theo kDa

Mn,GPCa kDa

Mw/ Mn

90/10/1/0.09/0.27/0.08

60

49

22.7

5.9

1.26

90/10/1/0.09/0.27/0.08

120

63

29.2

7.0

1.27

a Mn was measured by aqueous GPC using 100 mM sodium phosphate buffer with 0.2 vol % trifluoroacetic acid (pH = 2.5), calibrated using linear poly(ethylene oxide) standards.

Figure 2. GPC traces of (A) absorbance at 790 nm and (B) UV−vis spectra of fentanyl chain-end polymer before and after reaction with the NIR dye, both of which confirm dye incorporation.

the amine-containing dye.45 This functionalization method facilitated high yields and purities. Molecular weight and distribution was measured by GPC (Figures S17 and S18 in Supporting Information). Dye incorporation was confirmed by GPC and UV−vis spectra. Both of the Fen-Acry-p(OEOMA-co-GMA)-Dye polymers had a distinct absorption peak at 790 nm while Fen-Acryp(OEOMA-co-GMA) had no absorption response in the same wavelength (Figure 2 and Figure S19 in Supporting Information). UV−vis spectrum of the Fen-Acry-p(OEOMAco-GMA)-Dye (Figure 2) also showed a strong absorption band between 600 and 850 nm with a peak at 800 nm which corresponds to the absorption of the dye. Synthesis of Cationic Fentanyl Terminated Block Copolymers. To allow for siRNA binding, we prepared block copolymers. Block copolymers have been previously used

characterize the polymers. Proton peaks from both the ethylene oxide and epoxy groups were observed in their 1H NMR spectra (Figures S4 and S5 in Supporting Information), indicating polymer formation. The molecular weight of both polymers was measured by aqueous GPC (Table 1). A similar discrepancy of theoretical and measured Mn was observed in our previous measurements. It is explained by the use of linear poly(ethylene oxide) as standards for GPC which significantly decreases the apparent molecular weight for OEOMA containing polymers.25,48,49 The post-polymerization incorporation of an amine-containing NIR dye (ADS790WS) was accomplished by reacting the dye with either polymer in trifluoroethanol (TFE) at 50 °C for 48 h. TFE was found to accelerate the ring-opening reaction of epoxides and was applied here for the conjugation reaction with C


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Bioconjugate Chemistry

Figure 3. Schematic synthesis of amine-containing fentanyl chain-end block copolymers Fen-Acry-p(OEOMA-b-amine)-S-1/2/3 and Acryp(OEOMA-b-amine)-L-1/2/3 and siRNA polyplex formation.

Scheme 3. Synthesis of Fentanyl Chain-End Block Copolymer with Variable Block Lengths and Tertiary Amine Groups

Table 2. AGET ATRP of polyOEOMA Using Fen Initiator and Growth of a Second Block of polyGMA Using ICAR ATRP polymer a

Fen-Acry-pOEOMA Fen-Acry-p(OEOMA-b-GMA)-Lb Fen-Acry-p(OEOMA-b-GMA)-Sc

monomer

initiator

time (min)

conv %

Mn,theo kDa

Mn,GPCd kDa

Mw/Mn

OEOMA500 GMA GMA

Fen-Acry-EtBPA Fen-Acry-pOEOMA Fen-Acry-pOEOMA

60 120 20

20 38 6

10.6 34.9 11.2

9.9 41.8 11.8

1.15 1.18 1.11

AGET ATRP conditions: [OEOMA]0/[I]0/[CuBr2]0/[TPMA]0/[AscA]0 = 50/1/0.1/0.1/0.09, T = 40 °C. bICAR ATRP conditions: [GMA]0/ [I]0/[CuBr2]0/[TPMA]0/[AIBN]0 = 470/1/0.05/0.05/0.2, T = 60 °C. cICAR ATRP conditions: [GMA]0/[I]0/[CuBr2]0/[TPMA]0/[AIBN]0 = 100/1/0.05/0.05/0.2, T = 60 °C. dMn was measured by DMF GPC, calibrated using linear polystyrene standards. a

(Scheme 3, Table 2). The Fen-Acry-pOEOMA was purified by dialysis and the structure of the polymer was confirmed by 1H NMR (Figure S8 in Supporting Information). The polymer Mn of 9900 g/mol was determined by GPC and matches the theoretical Mn (10 600 g/mol) calculated by 1H NMR proton peak integration. The average number of repeating units of OEOMA was calculated based on Mn to be ∼20. ICAR ATRP48 was used to grow the second block-pGMA from the Fen-AcrypOEOMA. The pGMA block was used as a reactive handle to incorporate tertiary amines. The reaction was conducted using TFE as a solvent due to the enhanced rate of the ring opening reaction in TFE.45 To study the effects of the amine-containing block length, two types of block copolymers with different pGMA block lengths were prepared. The ratio of [mGMA] to [mOEOMA] was calculated by 1H NMR to be 0.3:1 for the Fen-Acryp(OEOMA-b-GMA)-S, and 9:1 for Fen-Acry-p(OEOMA-bGMA)-L. The two Fen-Acry-p(OEOMA-b-GMA) block copolymers were reacted with three different secondary amines to afford a total of six block copolymers with variable second block length and tertiary amines: Fen-Acry-p(OEOMA-bamine)-S/L-1,2,3 (Scheme 3). 1H NMR was used to character-

for delivery of complex biological molecules including nucleic acid therapeutics such as plasmid DNA and short interfering RNA.34,50,51 In those cases, block copolymer architecture is selected to form a biocompatible first block outer shell and a cationic second block capable of complexing anionic biological molecules forming the core.50,52,53 The structure of the cationic group is critical for efficient siRNA binding. Therefore, a library of block copolymers was prepared with two lengthsshort and long (S/L)and one of three different cationic amines (N,N,N′-trimethylethylenediamine (1), 1-methylpiperazine (2), or 3,3′-iminobis(N,N-dimethylpropylamine)) (3). Inspired by Seigwart and co-workers,43 a polyGMA block was grown from a biocompatible first block and reacted with amines postpolymerization. For the purpose of siRNA delivery to cells expressing the MOR we synthesized Fen chain-end block copolymers (Figure 3). The Fen-Acry-EtBPA initiator was used to prepare the block copolymers. The block design included a pOEOMA block to provide biocompatibility and a pGMA block to react with the tertiary amine to form complex with the siRNA. In order to synthesize the block copolymers, a single block pOEOMA was first grafted from Fen-Acry-EtBPA initiator by AGET ATRP D


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Figure 4. Biocompatibility of fentanyl terminated polymers: Biocompatibility assays were performed for the chain-end fentanyl polymers Fen-Acryp(OEOMA-co-GMA)-Dye-S/L and Fen-Acry-p(OEOMA-b-amine)-S/L-1,2,3 by incubating SH-SY5Y cells with the compounds for 48 h (as described in the Experimental Section). Each point represents the ATP level as a percent of the control (untreated) cells, the average of three replicates. Error bars represent triplicate variability. Fen-Acry-p(OEOMA-co-GMA)-Dye and Fen-Acry-p(OEOMA-b-amine)-S-1,2,3 polymers are fully biocompatible in the entire tested range (0.5 μg/mL to 1 mg/mL). However, Fen-Acry-p(OEOMA-b-amine)-L-1,2,3 appear toxic at concentrations higher than 10 μg/mL.

trations higher than 10 μg/mL. These results indicate that the block copolymer biocompatibility depends on the block length ratio. Since the OEOMA block lengths are equal, the longer polymers contain a longer GMA block: [mOEOMA]:[mGMA] 1:0.3 for Fen-Acry-p(OEOMA-b-amine)-S-1,2,3 vs [mOEOMA]:[mGMA] 1:9 in Fen-Acry-p(OEOMA-b-amine)-L-1,2,3. When further reacted the higher cationic charge contributed by the charged amines is toxic to the cells, in accordance with previous reports.54−56 This difference is an indication of our ability to control the properties and the flexibility allowed in optimization of this delivery system. Cellular Internalization of Dye Labeled Fentanyl Terminated Polymers. For the purpose of assessing the ability of MOR expressing cells to bind and internalize the fentanyl chain-end polymers, the dye containing polymers were used, to allow for fluorescent imaging. Cellular internalization

ize all polymers, and determine the quantitative consumption of the proton peaks from the epoxide upon reaction with the amine. The Mn of all polymers was determined by GPC (see Supporting Information). Biocompatibility of Fentanyl Terminated Polymers. Since all the polymers in this study are intended for future use with live cells, in vitro biocompatibility assays were performed to assess the toxicity of the polymers for use in a physiological context. Chain-end fentanyl polymers Fen-Acry-p(OEOMA-coGMA)-Dye and Fen-Acry-p(OEOMA-b-amine)-S/L-1,2,3 were incubated with SH-SY5Y cells for 48 h (as described in the Experimental Section). As presented in Figure 4, both FenAcry-p(OEOMA-co-GMA)-Dye and Fen-Acry-p(OEOMA-bamine)-S-1,2,3 polymers were found fully biocompatible in the entire tested range (0.5 μg/mL to 1 mg/mL). However, the Fen-Acry-p(OEOMA-b-amine)-L-1,2,3 were toxic at concenE


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Bioconjugate Chemistry of targeted polymers was studied using a GFP expressing SHSY5Y neuroblastoma cell line that endogenously expresses the MOR. Using this combination the cells are imaged in green and the polymer is red. To verify cell binding and internalization, the fentanyl chain-end dye containing polymers were added to cells and the dye incorporated polymer was used for imaging. SH-SY5Y cells were incubated with Fen-Acry-p(OEOMA-coGMA)-Dye for 1 h in culture prior to fixing and staining. Fluorescent imaging of the cells revealed colocalization of the NIR modified polymer (red) with the GFP (green) expressing SH-SY5Y cells as presented in Figure 5. (Full size images, as

Figure 5. Localization of the fentanyl chain-end polymers in endogenously expressing MOR cells. As described in Experimental Section, SH-SY5Y cells were incubated with Fen-Acry-p(OEOMA-coGMA)-Dye-S or -L (as indicated) for 1 h in culture prior to fixing and staining with the nuclear counter stain DAPI (blue). The EVOS imaging system was used for imaging: of the cells with the NIR modified polymer (Cy7, red) and GFP expressing SH-SY5Y cells (green) and DAPI (blue). The merged images indicate the localization of the polymer around the nucleus of the SH-SY5Y cells. (Full size images, as well as images of cells incubated in media only (without polymer) are presented in Figures S29−31 in Supporting Information).

Figure 6. Polyplex formation assays were performed by mixing siRNA and polymer at a weight/weight (w/w) ratio ranging from 1:0.06 to 1:40 (siRNA:Polymer), as indicated. The formed polyplexes were evaluated using agarose gel electrophoresis, stained with ethidium bromide and UV visualized.

well as images of cells incubated in media only (without polymer) are presented in Figures S29−S31 in Supporting Information.) These results indicate that the SH-SY5Y cell, which endogenously express MOR, are able to bind and internalize the fentanyl chain-end polymer, suggesting future use of this system for delivery into MOR expressing cells. siRNA Binding of Cationic Fentanyl Terminated Block Copolymers. The capacity of the cationic block copolymers to complex siRNA was evaluated using agarose gel electrophoresis. In order to deliver siRNA, which is negatively charged, several different cationic amine groups were reacted with the GMA containing block copolymer to form the positively charged FenAcry-p(OEOMA-b-amine)-S/L-1,2,3 (Scheme 1). Providing the polymer with a positive charge allows for the interaction and complexation with the negatively charged siRNA. The ability of the polymers to bind and complex with siRNA was investigated using a polyplex formation assay. The results are presented in Figure 6. We have found that the ability and siRNA:polymer ratios at which those polymers form polyplexes depend on the length of the blocks, rather than the specific amine. The longer block copolymers bind siRNA at lower w/w ratios due to the higher amine content of the polymer, which leads to higher positive charge density on the polymer.

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CONCLUSIONS To summarize, we have found that the chain-end fentanyl polymers Fen-Acry-p(OEOMA-co-GMA)-Dye are fully biocompatible, at both lengths, and can be bound and internalized by SH-SY5Y cells which endogenously express the MOR. We have shown that the block copolymers Fen-Acry-p(OEOMA-bamine)-S/L-1,2,3 biocompatibility depends on the block length ratio, due to the amine cationic charge which is toxic to the cells. Not surprisingly, the longer polymers with the higher cationic charge, bind siRNA betterthe ratio of binding correlates directly to length of block copolymer and the increase in positive charge. Therefore, we can tune the properties of the polymer to get the architecture that serves siRNA binding and delivery best. Further studies are required to optimize siRNA delivery. Taken together, our results support the possible use of this system for delivery into MOR expressing cells.

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EXPERIMENTAL SECTION Synthesis and Characterization. Synthesis of Fen-Acryp(OEOMA-GMA) Polymers. Two different approaches were used for polymer synthesis. For the post-polymerization

F


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ascorbic acid solution (2.0 mg in 100 μL DMF) was quickly injected into the reaction mixture via a syringe under N2 protection. The polymerization was continued for 60 min before it was stopped. The crude polymer was purified by repeated precipitation from ethyl ether. 1H NMR (500 MHz, CDCl3): δ 4.09, 3.8−3.5, 3.39, 2.2−1.5, 1.03, 0.88; Fen-AcrypOEOMA20, SEC: Mn = 9900 g/mol, Mw/Mn = 1.15. Synthesis of Fen-Acry-p(OEOMA-b-GMA). To a Schlenk flask equipped with a magnetic stir bar were added GMA (3 mL, 22.7 mmol), Fen-Acry-pOEOMA (480 mg, ∼0.048 mmol), and DMF (2.5 mL). A stock solution of CuBr2 (0.54 mg, 2.4 μmol), TPMA (0.82 mg, 2.4 μmol), and AIBN (1.86 mg, 9.6 μmol) dissolved in 0.5 mL DMF was added, and the flask was tightly sealed with rubber septum. For Fen-Acryp(OEOMA-b-GMA)-S, the ratio of [M0 ]/[I]/[CuBr2 ]/ [TPMA]/[AIBN] was set to be 100/1/0.05/0.05/0.2. The reaction mixture was purged with N2 for 30 min to remove any dissolved oxygen before it was heated at 60 °C for the polymerization. The polymerization continued for the desired time (20 min for Fen-Acry-p(OEOMA-b-GMA)-S or 60 min for Fen-Acry-p(OEOMA-b-GMA)-L, and was stopped by exposure to air. The block copolymer was purified by precipitation in ethyl ether and was collected by filtration: 1H NMR (500 MHz, CDCl3): δ 4.32, 4.10, 3.68, 3.57, 3.40, 3.26, 2.87, 2.66, 2.07−1.93, 1.12−1.04, 0.97; Fen-Acry-p(OEOMA20b-GMA6)-S, SEC: Mn = 11 800 g/mol, Mw/Mn = 1.10; FenAcry-p(OEOMA-b-GMA)-L, SEC: Mn = 41 800 g/mol, Mw/Mn = 1.18. Synthesis of Fen-Acry-p(OEOMA-b-amine) Block Copolymers. Tertiary amine modified block copolymers, Fen-Acryp(OEOMA-b-amine)-S/L-1, Fen-Acry-p(OEOMA-b-amine)S/L-2, Fen-Acry-p(OEOMA-b-amine)-S/L-3, were synthesized using three different secondary amines: (N,N,N′-trimethylethylenediamine (1), 1-methylpiperazine (2), 3,3′-iminobis(N,Ndimethylpropylamine)) (3). Fen-Acry-p(OEOMA-b-GMA) was dissolved in 2,2,2-trifluoroethanol (1 mL) with a polymer concentration of 10 mM. The amines (2 mmol) were added to the solution, and the reaction was stirred at 55 °C under N2 for 24 h. The products were purified by dialysis against water and methanol, and the pure products were characterized by 1H NMR and GPC: FenAcry-p(OEOMA-b-amine)-S-1, SEC: Mn = 10 600 g/mol, Mw/ Mn = 1.21; Fen-Acry-p(OEOMA-b-amine)-L-1, SEC: Mn = 157 000 g/mol, Mw/Mn = 1.16; Fen-Acry-p(OEOMA-bamine)-S-2, SEC: Mn = 11 300 g/mol, Mw/Mn = 1.19; FenAcry-p(OEOMA-b-amine)-L-2, SEC: Mn = 160 700 g/mol, Mw/Mn = 1.21; Fen-Acry-p(OEOMA-b-amine)-S-3, SEC: Mn = 10 100 g/mol, Mw/Mn = 1.28; Fen-Acry-p(OEOMA-b-amine)L-3, SEC: Mn = 202 900 g/mol, Mw/Mn = 1.16. Biocompatibility. SH-SY5Y neuroblalstoma cells were cultured according to manufacturer’s instructions. Cells were plated at 90% confluency in white 96 well plates. Prior to the ATP assay, the tested polymer was added to each well at final concentrations ranging from 0.5 μg/mL to 1 mg/mL, as indicated, in triplicate, and incubated at 37 °C for 72 h. ATP levels were determined using a CellTiter-Glo Luminescent Cell Viability Assay kit from Promega using manufactures instructions, and read using a luminometer (Cytation 3, Biotek). Untreated cells in culturing media (no polymer) were used as control. Cell Binding. SH-SY5Y neuroblalstoma cells (GenTarget Inc., cat# SC042) were cultured according to manufacturer’s instructions. Cells were seeded at 90% confluency on a

reaction with ADS790WS dye two random copolymers (FenAcry-p(OEOMA-co-GMA)-S (S - Short) and Fen-Acry-p(OEOMA-co-GMA)-L (L - Long)) were synthesized using AGET ATRP. For the post-polymerization reaction with various secondary amines (N,N,N′-trimethylethylenediamine, 1-methylpiperazine, 3,3′-iminobis(N,N-dimethylpropylamine)), block copolymers (Fen-Acry-p(OEOMA-b-GMA)-S and FenAcry-p(OEOMA-b-GMA)-L) were synthesized in two steps by AGET and ICAR ATRP. Synthesis of Fen-Acry-p(OEOMA-co-GMA) Random Copolymers. Two copolymers with different lengths were synthesized. The length and molecular weight of the copolymers were controlled by the AGET ATRP reaction time, 1 h for relatively short copolymer, Fen-Acry-p(OEOMAco-GMA)-S, and 2 h for relatively long copolymer, Fen-Acryp(OEOMA-co-GMA)-L. To a Schlenk flask were added CuBr2 (0.8 mg, 3.6 μmol) and TPMA (3.1 mg, 10.8 μmol) dissolved in 0.5 mL DMF. OEOMA500 (1.8 g, 3.6 mmol), GMA (56.8 mg, 0.4 mmol), and Fen-Acry-EtBPA (22.0 mg, 0.04 mmol) were added, and the flask was tightly sealed with a rubber septum. The reaction mixture was then degassed by purging with N2 gas for 30 min. The flask was then heated to 40 °C, and a deoxygenated ascorbic acid solution (100 μL, 36 mM in DMF) was quickly injected into the reaction mixture via a syringe under N2 protection. The polymerization was continued for 1 h for Fen-Acry-p(OEOMA-co-GMA)-S or 2 h for Fen-Acry-p(OEOMA-co-GMA)-L before was stopped. The crude polymer was purified by repeated precipitation from ethyl ether: 1H NMR (500 MHz, CDCl3): δ 4.30, 4.10, 3.88, 3.8−3.5, 3.40, 3.20, 2.85, 2.67, 2.2−1.5, 1.03, 0.87; Fen-Acry-p(OEOMA-coGMA)-S, SEC: Mn = 5900 g/mol, Mw/Mn = 1.26; Fen-Acryp(OEOMA-co-GMA)-L, SEC: Mn = 7000 g/mol, Mw/Mn = 1.27. Synthesis of Fen-Acry-p(OEOMA-co-GMA)-Dye. Fen-Acryp(OEOMA-co-GMA) (100 mg) and ADS790WS dye (8 mg, 0.01 mmol) were dissolved in trifluoroethanol (2 mL). The reaction mixture was kept stirring at 50 °C for 48 h. The polymer was purified by dialysis against water and methanol and dried under high vacuum: Fen-Acry-p(OEOMA-co-GMA)Dye-S, SEC: Mn = 5800 g/mol, Mw/Mn = 1.26; Fen-Acryp(OEOMA-co-GMA)-Dye-L, SEC: Mn = 5900 g/mol, Mw/Mn = 1.26. Synthesis of Fen-Acry-p(OEOMA-b-GMA) Block Copolymers. Block copolymers Fen-Acry-p(OEOMA-b-GMA)-S/L were synthesized it two steps. In the first step, Fen-AcrypOEOMA was synthesized by using AGET ATRP. In the second step, we used Fen-Acry-pOEOMA polymer to initiate the polymerization of GMA to synthesize Fen-Acry-p(OEOMA-b-GMA)-S/L using ICAR ATRP. Two block copolymers with different length were synthesized. The length and molecular weight of the copolymers were controlled by the ICAR ATRP reaction time, 20 min for relatively short block copolymer, Fen-Acry-p(OEOMA-b-GMA)-S, and 60 min for relatively long block copolymer, Fen-Acry-p(OEOMA-bGMA)-L. Synthesis of Fen-Acry-pOEOMA. To a Schlenk flask were added CuBr2 (2.7 mg, 12 μmol) and TPMA (3.5 mg, 12 μmol) dissolved in 0.5 mL DMF. OEOMA500 (3.0 g, 6 mmol) and Fen-Acry-EtBPA (66.0 mg, 0.12 mmol) were added, and the flask was tightly sealed with rubber septum. The reaction mixture was then degassed by purging with N2 gas for 30 min. The flask was then heated to 40 °C, and a deoxygenated G


Article


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chamber slide, and allowed to adhere overnight. The indicated polymer was then added to each well at a final concentration of 0.2 mg/mL. After an hour-long incubation period, the cells were washed and fixed with 4% PFA, and counterstained with DAPI nuclear stain. The slides were then imaged using an Invitrogen EVOS FL Auto Cell Imaging system. Polyplex Formation. The assay was performed as previously described.24 Fen-Acry-p(OEOMA-amine) polymer was mixed with 500 ng of siRNA (IDT) at a weight/weight (w/w) ratio ranging from 1:0.06 to 1:40 (siRNA:Polymer) in a constant volume and incubated for 30 min at room temperature to form polyplexes. The formed polyplexes were evaluated using 2% agarose gel electrophoresis in Tris/Borate/EDTA buffer, stained with ethidium bromide and UV visualized.

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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.bioconjchem.7b00078. List of materials as well as the synthesis of Fen-Br, FenAcry-EtOH, and Fen-Acry-EtBPA, 1H NMR spectra, and GPC traces (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.:+1-412-359-4943. ORCID

Saadyah Averick: 0000-0003-4775-2317 Author Contributions #

D. C.-K. and M. K. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully thank the Allegheny Health Network Research Institute and the Neuroscience Institute for start-up funds to conduct this research.

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ABBREVIATIONS Acry, acrylate; AGET, activators generated by electron transfer; ATRP, atom transfer radical polymerization; BPA, αbromophenylacetic acid; DAPI, 4′,6-diamidino-2-phenylindole; DMAP, 4-dimethylaminopyridine; EDC, 1-ethyl-3-(3(dimethylamino)propyl)carbodiimide; Fen, fentanyl; GFP, green fluorescent protein; GMA, glycidyl methacrylate; GPC, gel permeation chromatography; ICAR, initiators for continuous activator regeneration; L, long; MOR, mu opioid receptor; NIR, near-infrared fluorescent; OEOMA, oligo((ethylene oxide)methacrylate); RDRP, reversible deactivation radical polymerizations; S, short; siRNA, short interfering ribonucleic acid; TFE, trifluoroethanol

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REFERENCES

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