Membranotropic Peptide-Functionalized Poly(lactide)-graft-poly

Feb 13, 2015 - Biorthogonal click chemistry on poly(lactic-co-glycolic acid)-polymeric particles. Jason Olejniczak , Guillaume Collet , Viet Anh Nguye...
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Membranotropic Peptide-Functionalized Poly(lactide)-graf tpoly(ethylene glycol) Brush Copolymers for Intracellular Delivery Dorothee E. Borchmann,† Rossella Tarallo,† Sarha Avendano,† Annarita Falanga,‡ Tom P. Carberry,† Stefania Galdiero,*,‡ and Marcus Weck*,† †

Molecular Design Institute and Department of Chemistry, New York University, New York, New York 10003, United States Department of Pharmacy, University of Naples “Federico II”, Via Mezzocannone 16, Naples 80134, Italy

‡

S Supporting Information *

ABSTRACT: A biodegradable delivery scaffold based on poly(lactide)-graf t-poly(ethylene glycol) (PLA-g-PEG) is introduced and tested in vitro. The use of a functional ringopening polymerization initiator (containing a masked aldehyde) on an azido PEG-lactide monomer combines two known methodologies to afford functionalized PLA. The resulting copolymer features two compatible functional groups and decreased hydrophobicity due to the PEG. The functional groups are capable of performing high-yielding orthogonal postpolymerization reactions, namely strain-promoted azide− alkyne click chemistry and reductive amination. PLA-g-PEG was functionalized with a fluorescent dye (7-nitrobenzoxadiazole, NBD) and a cell internalization peptide, gH625. The resulting delivery vehicle was investigated for cell uptake with HeLa cells, showing that the gH625 conjugation exhibited enhanced cellular uptake and localization in close proximity to the nuclei. The presented methodology is a new approach toward targeted delivery.

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reactive 2-bromopropionyl bromide.14 Despite the synthetic progress in the field, poly(ester)s have mainly been used as nanoparticles15 or as sacrificial units of block copolymers16−19 rather than the drug-bearing unit. Uhrich and co-workers conjugated ibuprofen and naproxen to diacid monomers that were reacted with diols at elevated temperatures.20 The strategy reported effectively generates a scaffold with the drug attached to the biodegradable unit. This approach, however, does not allow for easy incorporation of other drug molecules and/or targeting groups. Wang and co-workers suggested a postpolymerization functionalization on poly(phosphonate) by thiol−Michael addition to afford doxorubicin-bearing poly(ester)s.21 While these poly(ester)s are drug bearing, targeting moieties in agreement with the Ringsdorf model have never been incorporated. In this contribution, we seek to introduce a scaffold based on poly(lactide), a biodegradable polymer derived from a biorenewable monomer, as delivery vehicle that also contains a targeting moiety. The Weck group has reported a number of strategies toward functional lactides.22−25 Recently, a poly(lactide)-graf t-poly(ethylene glycol) (PLA-g-PEG) copolymer scaffold studded with azides could be generated by subjecting 3-(3-((2-(2-(2azidoethoxy)ethoxy)ethyl)thio)propyl)-6-methyl-1,4-dioxane2,5-dione (N3-TEG-lactide) (2, Scheme 1) to a ring-opening polymerization (ROP).25 This material provides functional

INTRODUCTION Cancer therapy is often hampered by the hydrophobicity and lack of specificity of common small-molecule drugs such as anthracyclines or paclitaxel.1,2 One approach to increase efficacy is by anchoring the drug of interest to a macromolecular scaffold.3 In 1975, Ringsdorf postulated a model for polymer scaffolds for drug delivery.4,5 It states that linear biocompatible polymers should contain drugs, solubilizing groups, and targeting moieties as essential components. Based on this model, in the 1980s and 1990s poly(2-hydroxypropyl methacrylate)s (PHPMA)s have been investigated as carriers for doxorubicin.6−8 PHPMA, however, features an all-carbon backbone that lacks biodegradability. The long-term fate of such nondegradable constructs remains elusive and has scarcely been investigated.7 Although biodegradable systems could address this shortcoming, only few such systems have been reported. Biodegradable poly(ester)s have found prevailing applications as pharmaceutical adjuvants or tissue engineering scaffolds.9−11 Their inherent hydrophobicity and functional group paucity, however, diminish the application of such homopolymers in compliance with the Ringsdorf model. Researchers have strived to synthesize functional poly(ester)s to address some of these shortcomings.12 Baker and co-workers have reported alkynecontaining glycolides by self-condensation of two α-hydroxyacids.13 Polymer-analogous reactions were performed successfully on the resulting polymer. An elegant strategy that yielded allyl lactide was reported by Cheng and co-workers and circumvents the low-yielding self-condensation step by using a © XXXX American Chemical Society

Received: January 26, 2015 Revised: February 3, 2015

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Scheme 1. Synthesis of PLA with Two Compatible Functional Groups by ROP (In Cartoon: Red = Aldehyde Initiator; Blue = Azide Functional Groups)

distilled prior to use. (4-(1,3-Dioxolan-2-yl)phenyl)methanol (1),36 azido-tri(ethylene glycol)-containing lactide (2),25 and bicyclo[6.1.0]non-4-yn-9-ylmethyl (4-nitrophenyl) carbonate (BCN-active ester)37 were synthesized according to published procedures. Dialysis membranes (Spectra/Por 6) were purchased from SpectrumLabs and used as received. Membranes were washed first in water and then in the desired solvent. The compound to be dialyzed was dissolved in a minimum amount of solvent and then loaded into the bag. This was placed in a gently stirred solution of 1 L of solvent, and the exterior solution was changed once a day for 3 days. Methods. 1H NMR and 13C NMR spectra were recorded on a Bruker AC 400 MHz or Bruker AV 600 MHz spectrometer at room temperature. Chemical shifts are reported against the deuterated solvent residual peak. Deuterated solvents were purchased from Cambridge Isotope Laboratories and used as received. Mass spectra were obtained on an Agilent 1100 Series capillary LCMSD trap XCT spectrometer using methanol as solvent unless otherwise indicated. High-resolution mass spectra were acquired on an Agilent 6224 accurate-mass TOF/LC/MS with acetonitrile as solvent. IR spectra were obtained on a Nicolet Magna-IR 760 spectrometer. Sizeexclusion chromatography (SEC) was carried out using a Shimadzu pump coupled to Shimadzu UV and RI detectors. Tetrahydrofuran (THF) was used as solvent. MALDI-ToF MS spectra were acquired on a Bruker UltrafleXtreme MALDI tandem mass spectrometer in the positive mode. α-Cyano-4-hydroxycinnamic acid in THF or trans-2-[3(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB) in THF was used as matrix. A saturated sodium trifluoroacetate solution in acetone was then spotted on top of the matrix layer as dopant salt. Lastly, the compound of interest was applied in its respective NMR solvent. Fluorescence microscopy was performed on an inverted microscope, Nikon Eclipse Ti, Photometrics CoolSNAP ES monochrome camera with a 40× objective, controlled by Nikon NIS-Elements AR software. NBD images were obtained with separate excitation (465−495 nm) and emission (515−555 nm) filters. tert-Butyl (6-((7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexyl)carbamate (NBoc-hexylenediamine-NBD). To a stirred solution of mono-Boc hexylenediamine (686 mg, 3.17 mmol, 1 equiv) and 4-chloro-7-nitrobenzofurazan (NBD) (633 mg, 3.17 mmol, 1 equiv) in ethanol (8 mL) was added pyridine (catalytic, 260 μL), and the mixture was allowed to stir for 30 min. The reaction mixture was then concentrated and purified by column chromatography (toluene:ethyl acetate 7:3). The product was obtained as red foam (44 mg, 0.107 mmol, 25%). 1H NMR (400 MHz, DMSO-d6): δ = 9.53 (s, br, 1H); 8.49 (d, 1H, J = 8.9 Hz); 6.75 (s, 1H); 6.39 (d, 1H, J = 8.9 Hz); 3.45 (s, br, 2H); 2.89 (q, 2H, J = 6.8 Hz); 1.66 (m, 2H); 1.35 (m,

handles for postpolymerization reactions and features triethylene glycol (TEG) chains dangling off the PLA backbone in a brush-like fashion. We have demonstrated reduced nonspecific biomacromolecule adhesion using these materials.24 We suggest that the use of this copolymer as drug delivery vehicle could exert a similar effect as protein and drug PEGylation: longer circulation times and enhanced hydrophilicity.26 In accordance with the Ringsdorf model, it features both solubilizing groups and functional handles for drug attachment. Herein, we add a handle for attachment of uptake enhancement using an aldehyde-containing ROP initiator, effectively generating a telechelic PLA-g-PEG that contains one aldehyde at the polymer chain end and azides along each repeat unit. The pendant azides are exploited for attachment of fluorescent dyes that monitor cell uptake, serving as models for hydrophobic cargo such as anticancer drugs. The aldehyde-containing initiator enables the installation of a virus-derived, membranotropic peptide, gH625,27−29 which has successfully aided internalization of various macromolecular cargoes.30−33 gH625 is a 20 amino acid fragment derived from the envelope protein of the Herpes simplex virus.27,34 The presence of both hydrophilic and hydrophobic residues results in the formation of an amphiphatic helix, as shown by circular dichroism in a membrane-mimicking environment, and allows for interactions with the cellular membrane.28 A recent study demonstrated that the peptide when attached to a dendritic scaffold has a high affinity for the phospolipid bilayer.35 Uptake studies with quantum dots,30 liposomes,31 dendrimers,32 and nanoparticles33 suggested a nonactive translocation mechanism to cross the lipid bilayer but may vary depending on the cargo. This contribution describes the synthesis of an elaborate gH625-PLA conjugate containing fluorescent dyes, its detailed characterization, and application for uptake studies with living HeLa cells. Preliminary mechanistic studies suggest a mixed mechanism of cellular uptake.

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EXPERIMENTAL SECTION

Materials. All chemicals were purchased from Sigma-Aldrich and used as received unless otherwise noted. The polymerization solvent dichloromethane (DCM) was stirred over calcium hydride and B

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Macromolecules 17H). 13C NMR (150 MHz, DMSO-d6): δ = 155.6, 145.1, 144.1, 137.8, 120.5, 99.0, 72.3, 43.3, 29.4, 28.2, 27.6, 26.1, 25.9. IR (ATR) ν (cm−1) = 3323, 2934, 1688, 1621, 1585, 1529, 1507, 1445, 1403, 1365, 1298, 1273, 1173, 999, 668, 613. MS-ESI (M + H)+: cleavage of Bocprotecting group during ionization; only amine observed (see below). N 1-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)hexane-1,6-diamine (Hexylenediamine-NBD). The Boc-protected dye (643 mg, 1.69 mmol) was dissolved in a 1:1 solution of TFA:DCM and stirred at room temperature for 1 h. The mixture was concentrated, resuspended in acetonitrile, and precipitated into cold diethyl ether. The product was isolated as golden crystals (442 mg, 1.58 mmol, 94%). 1H NMR (400 MHz, DMSO-d6): δ = 9.56 (s, br, 1H); 8.50 (d, J = 8.70 Hz, 1H); 7.77 (s, br, 1H); 6.40 (d, J = 8.70 Hz, 1H); 3.53−3.38 (m, 2H); 2.84−2.70 (m, 2H); 1.73−1.61 (m, 2H); 1.60−1.47 (m, 2H); 1.43−1.30 (m, 4H). 13C NMR (150 MHz, DMSO-d6): δ = 158.7, 145.7, 144.6, 138.4, 121.0, 99.5, 43.7, 39.2, 27.9, 27.4, 26.3, 25.9. IR (ATR) ν (cm−1) = 3209, 3145, 3060, 2938, 2858, 1683, 1619, 1585, 1529, 1489, 1443, 1404, 1350, 1295, 1257, 1198, 1171, 1123, 997, 823, 719, 595. MS-ESI (M + H)+ m/z: calcd for C12H17N5O3, 279.30; found, 279.13. BCN-Hexylenediamine-NBD (5). A previously reported BCN active ester37 (200 mg, 0.634 mmol, 1 equiv) hexylenediamine-NBD (195 mg, 0.698 mmol, 1.1 equiv) and triethylamine (265 μL, 1.90 mmol, 3 equiv) were dissolved in acetonitrile (20 mL) and stirred for 16 h. The crude mixture was concentrated under reduced pressure and purified by column chromatography (toluene:ethyl acetate 4:1, gradient to 1:1). An orange foam was isolated (261 mg, 0.546 mmol, 90%). 1H NMR (600 MHz, MeCN-d3): δ = 9.58 (s, br, 1H); 8.70 (d, J = 8.70 Hz, 1H); 7.09 (t, J = 5.5 Hz, 1H); 6.65 (d, J = 8.70 Hz, 1H); 4.03 (d, J = 6.83 Hz, 2H); 3.74 (s, br, 2H); 3.22 (q, J = 6.04 Hz, 2H); 2.45 (d, J = 12.94 Hz, 2H); 2.40−2.33 (m, 2H); 2.24−2.19 (m, 2H); 1.69−1.41 (m, 10H); 0.86−0.70 (m, 3H). 13C NMR (150 MHz, DMF-d7): δ = 157.2, 145.7, 144.8, 121.7, 99.3, 98.9, 68.0, 44.0, 40.8, 33.4, 28.2, 26.8, 26.6, 24.2, 22.9, 21.1. IR (ATR) ν (cm−1) = 3327, 2929, 2857, 1661, 1620, 1583, 1529, 1495, 1442, 1407, 1386, 1349, 1293, 1252, 1178, 1132, 1096, 1035, 997, 901, 843, 810, 779, 737, 661, 595. MS-ESI (M + H)+ m/z: calcd for C23H29N5O5, 455.51; found, 455.21. Dioxolane-PLA-g-PEG3 (3). 125 mg (0.349 mmol, 15 equiv) of N3-TEG-lactide25 was transferred to a Schlenk reactor and dried under vacuum for 1 day. The Schlenk tube was transferred to a nitrogen-filled glovebox. A stock solution of 18 mg of 1,5,7-triazabicyclo[4.4.0]decene (TBD) in 25 mL of dichloromethane was prepared and 1 mL of this solution (0.73 mg of TBD; 1.5 mol %) was added to the initiator (4 mg, 0.023 mmol, 1 equiv) that was dissolved in 300 μL of dichloromethane. This solution was added to the monomer (c = 0.25 M). After 2 min polymerization time, the polymerization was quenched by addition of excess benzoic acid in dichloromethane. The crude polymer was dialyzed against acetone for 3 days with solvent changes after 12, 24, and 48 h. 103 mg of polymer was obtained as a yellow viscous oil (82%). 1H NMR (400 MHz, CDCl3): δ = 7.47 (d, 0.2H, J = 8.3 Hz); 7.34 (t, 0.2H, J = 5.0 Hz); 5.8 (s, 0.1 H); 5.15 (s, br, 2H); 4.51−4.18 (m, 0.1 H); 4.15−3.95 (m, 4H); 3.69−3.57 (m, 8H); 3.38 (t, 2H, J = 4.5 Hz); 2.70 (s, br, 2H); 2.59 (s, br, 2H); 2.00 (s, br, 2H); 1.73 (s, br, 2H); 1.56 (s, br, 3H). 13C NMR (150 MHz, CDCl3): δ = 169.7, 128.4, 126.9, 103.5, 71.2, 70.8, 70.5, 70.2, 65.5, 50.8, 41.1, 31.5, 25.1, 16.9. Deprotection of Dioxolane-PLA-g-PEG3 (3) To Yield Aldehyde-PLA-g-PEG 3 (4). Dioxolane-PLA-g-PEG3 (98 mg) was dissolved in acetone (1 mL) and exposed to one spatula tip of ptoluenesulfonic acid monohydrate for 1 h. The reaction mixture was then dialyzed for 3 days against acetone with solvent changes after 12, 24, and 48 h. 71 mg of a viscous yellow oil was isolated. 1H NMR (600 MHz, CDCl3): δ = 10.02 (s, 0.1H); 7.88 (d, J = 7.85 Hz, 0.2H); 7.48 (d, J = 7.85 Hz, 0.2H); 4.45−4.18 (m, 0.2H); 5.29−5.00 (m, 2H); 3.77−3.51 (m, 8H); 3.49−3.15 (s, br, 2H); 2.80−2.43 (m, 4H); 2.17− 1.86 (s, br, 2H); 1.86−1.63 (s, br, 2H); 1.63−1.40 (s, br, 3H). 13C NMR (150 MHz, CDCl3): δ = 191.7, 169.3, 168.8, 130.1, 128.4, 128.3, 72.3, 71.0, 70.7, 70.4, 70.1, 69.1, 50.7, 31.9, 31.4, 29.9, 22.7, 16.8.

PLA-g-PEG Hexylenediamine−Dye Conjugate (6). AldehydePLA-g-PEG3 (23 mg; 0.063 mmol based on azide groups) and NBDhexylenediamine-BCN (43 mg; 0.094 mmol; 1.5 equiv per azide) were stirred in N,N-dimethylformamide (DMF) (24 mL) at room temperature for 16 h. The crude reaction mixture was dialyzed against DMSO for 3 days with three solvent changes. The dye−polymer conjugate was isolated as a red thin film (53 mg, 0.008 mmol, 89%). 1 H NMR (600 MHz, DMF-d7): δ = 10.08 (end-group); 9.43 (s, br, 0.9H); 8.55 (s, br, 0.9H); 7.65 (end-group); 7.34 (end-group); 6.92 (s, br, 0.9H); 6.50 (s, br, 0.9H); 5.44−5.10 (m, br, 2H, PLA-backbone used for calibration); 4.45 (s, br, 1.7H); 3.98−3.70 (m, 5H); 3.67− 3.49 (m, 8H), 3.46 (s, br, 1.5H) (signals overlapping with DMF solvent peak not picked/integrated); 2.38−2.20 (d, br, 2H); 2.16− 1.92 (m, br, 3H); 1.85−1.63 (m, br, 4.4H); 1.61−1.22 (m, 14H); 0.92−0.62 (m, 3.7H). 13C NMR (150 MHz, DMF-d7): δ = 157.1, 145.1, 144.2, 138.0, 134.5, 121.7, 99.4, 71.0, 70.5, 70.2, 67.7, 47.8, 42.1, 41.0, 40.9, 40.8, 40.6, 31.3, 28.2, 27.6, 26.8, 25.9, 24.7, 22.9, 22.7, 16.9. gH625−Polymer Conjugate (7). One equivalent of dye− polymer was stirred with 3 equiv of a modified gH625 in DMF for 16 h. The gH625 featured a terminal lysine to provide a primary amine for reductive amination. Schiff base formation was confirmed on the next morning via a reduction of intensity of the aldehyde peak belonging to the polymer at 10.5 ppm (1H NMR). Five equivalents of the reducing agent sodium cyanoborohydride in DMF was added to the mixture. After stirring for 2 h at room temperature, the reaction mixture was purified by dialysis (MWCO = 8 kDa; solvent: MeCN/ H2O 3:2 two cycles, DMSO two cycles). Cell Uptake Studies. HeLa cells were cultured in low glucose DMEM (10% FBS, 1% Pen/Strep, 1% L-Glu) and maintained at 37 °C and 5% CO2. For the cellular uptake studies polymer−NBD and gH625−polymer−NBD were diluted in the cell culture medium (with FBS) with 0.6% of DMSO at the final concentration of 15 μM (concentration of NBD dye). 70% confluent HeLa cells, seeded on 35 mm dish with a 10 mm glass bottom well, were incubated with 400 μL of compound for 4 h at 37 °C. After incubation, the cells were rinsed twice with PBS to remove noninternalized compounds, and 400 μL of HBSS was added for imaging. For experiments with the metabolic inhibitor, HeLa cells were preincubated with NaN3 for 30 min at 37 °C. Afterward, the cells were washed twice with PBS and then incubated with the polymers as described above. All experiments were run in triplicates.

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RESULTS AND DISCUSSION Synthesis of Bifunctional PLA-g-PEG. To incorporate both oligo(ethylene glycol) chains and azide moieties in a PLA scaffold, we started with the previously reported N3-TEGlactide monomer 2.25 We used thiol−ene chemistry as key step to install the bifunctional TEG on the allyl lactide. For a second functional moiety that could be functionalized in an orthogonal manner to azides on the PLA-g-PEG scaffold, a functional initiator was applied in the ROP of lactide.38,39 Previously reported functional initiators include anthracene,40 pyridyl disulfide,41 protected maleimide,42 and protected aminecontaining molecules.43 Since peptides are rich in functional group-containing amino acids, the desired end-group should be thiol- or amine-reactive. Maleimides and azides, however, are not fully compatible,44 and pyridyl disulfide chemistry creates only transient bonds. Therefore, we chose reductive amination via a terminal lysine as postpolymerization functionalization strategy and an aldehyde moiety as functional group on the ROP initiator. Kataoka and co-workers reported commercially available 3,3diethoxypropanolate as initiator for the anionic ROP of ethylene oxide and lactide to generate block copolymers.45,46 After exposure to strong acid (pH 2), the protected aldehyde could be liberated without notable polymer hydrolysis, presumably due to the micellar architecture of the block C

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Figure 1. 1H NMR spectroscopic overlay of dioxolane-PLA-g-PEG 3 (top) and aldehyde-PLA-g-PEG 4 (bottom). Inset: SEC overlay of 3 (blue) and 4 (red).

Scheme 2. Synthesis of PLA-g-PEG Hexylenediamine−Dye Conjugate 6 (by SPAAC) and gH625−Dye−Polymer Conjugate 7 (by Reductive Amination)

D

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Figure 2. 1H NMR spectroscopic overlay of 4, 6, and 7, highlighting the signals of interest.

ppm was observed, indicating the presence of the sp-hybridized alkyne carbons (Figure S6). Synthesis of NBD-PLA-g-PEG. Initially, we carried out screening reactions with a sterically less-demanding BCN alcohol to optimize functionalization conditions (temperature, time) using only 1 equiv of BCN per azide. The polymer was reacted with 1.5 equiv of the NBD dye per azide group (due to the higher steric demand) for 12 h at room temperature. After purification, 1H NMR spectroscopic analysis indicated 85% functionalization by integration of the new methylene signal at δ 4.5 ppm in relation to PLA backbone protons at δ 5.4−5.1 ppm (Figure 2). Since the dye−polymer conjugate does not ionize well during MALDI-ToF analysis (Figure S16), we performed UV−Vis spectrometric analysis to further quantify the functionalization yield. A calibration curve at λ = 330 nm was recorded (Figure 3) and used to determine the degree of functionalization as 83.3%, which corroborates the 1H NMR spectroscopic result. By SEC analysis, only broadening of Đ was observed. A molecular weight increase in accordance with the 1 H NMR spectroscopic and UV−vis analyses could not be confirmed. This can be attributed to the brush architecture of the polymer: the hydrodynamic radius might not change significantly by increasing the brush lengths instead of the backbone length.51 We confirmed the purity of the dye− polymer conjugate after dialysis using 13C NMR spectroscopy, by following the disappearance of the signal at δ 99.7 ppm attributed to the alkyne sp-carbons. Conjugation of gH625. To install the gH625 peptide onto the scaffold, a modified derivative featuring a terminal lysine was synthesized. This peptide and the NBD-PLA-g-PEG conjugate were stirred in DMF overnight at ambient temperatures. An excess of gH625 peptide (3 equiv compared to the NBD-PLA-g-PEG aldehyde) was added to the reaction mixture

copolymer with the PLA block buried in the core. Previous studies on PLA-g-PEG, however, had shown brush copolymer degradation under these harsh deprotection conditions;25 thus, we investigated the 1,3-dioxolane protecting group on p(hydroxymethyl)benzaldehyde as the initiator instead. ROP using 1,5,7-triazabicyclodecene (TBD) as catalyst47 proceeded with reliable initiator incorporation, as evidenced by 1H NMR spectroscopy (Figure 1) and MALDI-ToF spectrometry (Supporting Information Figure S13). The 1,3-dioxolane group was removed quantitatively after 1 h of exposure to ptoluenesulfonic acid. No backbone degradation was observed by size-exclusion chromatography (SEC) (Figure 1). By 1H NMR spectroscopy, the signals attributed to the 1,3-dioxolane group at δ 5.80 and 4.03−4.12 ppm disappeared, while a new signal at δ 10.01 ppm attributed to the aldehyde proton emerged (Figure 1). Synthesis of the NBD Dye. The NBD derivatives were designed to feature both an acid-cleavable carbamate bond and a literature-known bicyclononyne (BCN)37 for anchoring them to the polymeric carrier via strain-promoted azide−alkyne click chemistry (SPAAC).48−50 This modular approach allows for exchanging the carbamate bond with other stimuli-responsive spacers to ensure drug release. We previously reported that copper-catalyzed 1,3-dipolar cycloadditions are prohibitive on PLA-g-PEG scaffolds, owing to the ability of PEG-chains to complex Cu(I).25 The BCN was attached to the NBD-amine via an active ester (Supporting Information, Scheme S1).37 This method creates a carbamate bond simultaneously, installing both a functional handle for polymer attachment and a cleavable spacer, in one step. The identity of the product could be confirmed by conventional analytical methods. A characteristic signal in the 13C NMR spectroscopy at δ 99.7 E

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Gratifyingly, the dye−polymer−peptide conjugate ionized more easily than the dye−polymer precursor, and a distinct molecular weight increase after conjugation was observed (Figure S16). By the crude 1H NMR spectrum, 62% of the aldehyde groups had been functionalized with gH625 peptide (Figure 2 and Figure S15). Dialysis membranes of 8000 g/mol molecular weight cutoff were used for dialysis, suggesting that some unreacted polymer chains (MW = 6200 g/mol) remained in the mixture. Nevertheless, cell uptake studies were carried out to gain preliminary insights into the applicability of the novel scaffold as delivery vehicle. Cell Uptake Studies. In order to ensure the integrity of the scaffold for the duration of the cell studies, we tested the strength of the carbamate bond on NBD-PLA-g-PEG in DMF solution with acetic acid. No carbamate bond cleavage could be observed by 1H NMR spectroscopy (until up to 12 h observation time, Figure S18) upon the addition of one drop of acetic acid to a DMF solution of the scaffold. These suggest that all fluorescence signals in the cell studies originate from the macromolecular scaffold within the cells and not from small molecule dyes released from the scaffold during the cell study (5 h observation time). In the future, the dye concept can be expanded to feature a disulfide bond (redox trigger) instead of the carbamate bond to ensure triggered release. gH625-aided cell uptake of macromolecular cargo had been studied in the past,30−33 and mechanistic studies indicate that gH625-decorated cargo tends to undergo passive cell uptake due to lipid mixing between gH625 and the extracellular membrane.35 To gain insights into the cellular uptake of our novel delivery scaffold, we carried out internalization experiments on nonfixed living cells. HeLa cells were incubated with NBD-PLA-g-PEG and gH625-NBD-PLA-g-PEG in a 15 μM concentration at 37 °C for 4 h. After the incubation time, the cells were rinsed three times with PBS buffer, and HBSS buffer was added to perform microscope imaging of the living cells. As shown in Figure 4, the covalent attachment of the cellpenetrating peptide gH625 to NBD-PLA-g-PEG not only increases the polymer translocation across the plasmatic membrane but also leads to a different intracellular distribution

Figure 3. UV calibration curves for BCN-hexylenediamine-NBD (5) from the absorbance at λ = 330 nm.

to shift the equilibrium of Schiff base formation. While peptide−polymer hybrids are known to pose challenges for spectroscopic characterization due to the plethora of signals,52−54 we took advantage of the unique location of the aldehyde peak at 10.5 ppm to monitor the reaction. Complete disappearance of the aldehyde peak by 1H NMR spectroscopy was not observed, even at increased reaction times. This can be attributed to the higher steric demand of the peptide compared to small-molecule amines that had been used for the test reactions. The reducing agent sodium cyanoborohyride, chosen for its compatibility to the majority of biologically occurring functional groups, was added after 12 h of reaction time. After 2 h of stirring at room temperature and an additional 2 h at 40 °C, excess peptide and reducing agent were removed by dialysis, and the pure conjugate was analyzed by MALDI-ToF spectrometry to confirm the absence of unreacted peptide.

Figure 4. Cell uptake experiments. NBD-PLA-g-PEG (A−C) and gH625-NBD-PLA-g-PEG (D−F) at 15 μM for 4 h: (A, D) = transmission; (B, E) = fluorescence; (C, F) = overlay. F

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with accumulation of the conjugate in close proximity to the nucleus. To gain insights into the uptake mechanism, an endocytosis inhibition experiment using sodium azide (NaN3) was performed. NaN3 is an oxidative phosphorylation inhibitor commonly used to shut down ATP production within the cellular membrane.55 HeLa cells were first incubated with NaN3for 30 min followed by the addition of NBD-PLA-g-PEG and gH625-NBD-PLA-g-PEG. The microscope images show a significant reduction in intracellular fluorescence intensity for both compounds, suggesting, at least partially, an active internalization mechanism (Figure S17).

CONCLUSION In this contribution, we introduce a novel delivery scaffold based on biodegradable PLA. Using both a functional ROP initiator and a functional monomer featuring TEG allowed us to address known shortcomings of conventional PLA, namely hydrophobicity and functional group paucity, in our PLA-gPEG scaffold. The functionalities on the polymer and brush ends were exploited in an orthogonal fashion to attach NBD dyes to monitor cell uptake and a membranotropic peptide to facilitate cell internalization. NBD dyes were attached by SPAAC while gH625 was attached by reductive amination. Preliminary cell uptake studies with HeLa cells revealed different intracellular distributions for the two conjugates, indicating a random uptake for NBD-PLA-g-PEG and localization in close proximity of the nuclei for gH625-NBD-PLA-gPEG. Inhibition experiments with NaN3 could not completely shut down uptake, suggesting a mixed uptake mechanism. The localization close to the nuclei has the potential to be exploited for the delivery of DNA intercalators such as doxorubicin in cancer therapy. ASSOCIATED CONTENT

S Supporting Information *

Spectral characterization, synthetic scheme for NBD dye, synthetic procedure for modified gH625, and characterization. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (S.G.). *E-mail: [email protected] (M.W.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Tomasz Kurcon, Christopher A. Vaiana, and Prof. Lara K. Mahal for assistance with the cell uptake studies and Dr. Elizabeth Elacqua for proofreading the manuscript. D.E.B. gratefully acknowledges the Herman and Margaret Sokol Doctoral Fellowship. T.P.C. thanks the Margaret Strauss Kramer Foundation. S.A. gratefully acknowledges the Dean of Undergraduate Studies’ Research Fund. M.W. acknowledges a Friedrich Bessel Award from the Humboldt Foundation. The Bruker Avance-400 MHz NMR spectrometer was acquired through support of the National Science Foundation (CHE01162222). The MALDI−ToF MS was acquired through the National Science Foundation under Award CHE-0958457. G

DOI: 10.1021/acs.macromol.5b00173 Macromolecules XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.macromol.5b00173 Macromolecules XXXX, XXX, XXX−XXX