Facile Synthesis of a Degradable Poly(ethylene glycol) Platform with

Aug 20, 2018 - We report a new synthetic strategy that allows for the facile synthesis of acid-degradable poly(ethylene glycol) (PEG) with tunable aci...
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Facile Synthesis of a Degradable Poly(ethylene glycol) Platform with Tunable Acid Sensitivity at Physiologically Relevant pH Shan Su, Fu-Sheng Du,* and Zi-Chen Li* Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Polymer Chemistry & Physics of Ministry of Education, Department of Polymer Science & Engineering, College of Chemistry and Molecular Engineering, Center for Soft Matter Science and Engineering, Peking University, Beijing 100871, China

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S Supporting Information *

ABSTRACT: We report a new synthetic strategy that allows for the facile synthesis of acid-degradable poly(ethylene glycol) (PEG) with tunable acid sensitivity at physiologically relevant pH. This approach relies on the step-growth polymerization of telechelic OEG/PEG diamine and bis(maleic anhydride)s with the acid-cleavable maleamic acid derivatives formed in situ during the polymerization. These polymers are stable and negatively charged in neutral and basic aqueous solution, but in physiologically acidic media, they can be degraded selectively and completely into metabolizable bis(maleic acid)s and OEG/PEG diamines. The polymers derived from the short OEG diamine have been used as acid-responsive polyanions to coat the positively charged DNA/PEI polyplex, thus enhancing the stability of the coated polyplex against bovine serum albumin at pH 7.4. Upon exposure to a weakly acidic medium, the positively charged DNA/PEI polyplex can be quickly regenerated.

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conditions.33 Among these cleavable linkages, those sensitive to mildly acidic pH are more appealing to degrade the PEGs in tumor tissues (pH = 6.5) and endosamal/lysomal compartments (pH = 5.5).34−38 Accordingly, pH-sensitive acetals,39−41 cis-aconitic acids,42 esters,43 hemiacetals,44,45 and vinyl esters,46−48 have been incorporated into PEG backbones. Despite these important progresses, it remains a challenge to design degradable PEGs that are susceptible to a pH gradient existed in the tumor tissues and endosamal/lysomal compartments. Given their adjustable rates of hydrolysis achieved by varying the substituted groups, we envision that maleamic acid derivatives can be used to design a degradable PEG platform with tunable acid sensitivity at physiologically relevant pH. Previously, Brocchini and co-workers reported the synthesis and acid-triggered degradation of PEG analogue containing maleamic acid repeating units in the polymer backbone.42 However, the polymers possessed ambiguous structures due to the poorly controlled synthetic procedure, thus leading to an incomplete degradation of the polymer with complex degradation kinetics. Recently, we optimized the reaction conditions between aliphatic primary amines and substituted maleic anhydride derivatives. Thus, we could accurately control the structures of the resulting mono and dialkyl substituted maleamic acid derivatives and elucidate the substituent effects on the acid-sensitivity and pH-related

INTRODUCTION Maleamic acid derivatives are one of the important acidsensitive linkers due to the amide hydrolysis catalyzed by the intramolecular adjacent carboxylic group under mildly acidic conditions.1−3 The acid-sensitivity of maleamic acid derivatives can be easily tuned by varying the substituents on the cisdouble bonds, and the dialkyl-substituted derivatives can even be susceptible toward physiological relevant pH.4,5 Moreover, the N-alkyl substituted maleamic acid derivative has a negatively charged carboxylate moiety at neutral pH, but it decomposes upon acid triggering to expose a positively charged amine. These unique features of tunable acid sensitivity and charge conversion make maleamic acid derivatives good caging moieties to modify small molecule drugs6,7 and antibodies.8 Recently, maleamic acid derivatives have been introduced into polymer systems as acid-sensitive linkers for the delivery of nucleic acids,9−11 proteins,12−14 drugs,15−17 and imaging agents.18,19 These linkers can be located at the side chain or main chain of linear polymers,20−25 or used as acid-labile cross-linking points in polymer networks.26,27 Poly(ethylene glycol) (PEG) is a widely used nonionic, hydrophilic, and biocompatible platform polymer in pharmaceuticals for drug and protein delivery.28−30 To overcome the limitation of nondegradability of high-molecular weight PEG which cannot be effectively renal excreted and may accumulate in organs such as liver and kidney,31,32 one of the most widely used approach is to introduce cleavable moieties into the PEG structures to permit their degradation at physiological © XXXX American Chemical Society

Received: May 17, 2018 Revised: August 3, 2018

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DOI: 10.1021/acs.macromol.8b01053 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules Scheme 1. Syntheses of Acid-Cleavable PEGs from Bis(maleic anhydride)s and Diamines

hydrolysis.3 Herein, we demonstrate a straightforward, onestep procedure toward acid-sensitive PEGs with defined structures via the step-growth polymerization of bis(maleic anhydride)s and diamines (Scheme 1). Two bis(maleic anhydride)s (M1 and M2) and two OEG and PEG primary diamines (M4 and M5) were used to afford two series of PEGs with pendent negatively charged carboxylic acid groups (P1aP3a and P1b-P3b). They are both acid-degradable at physiologically relevant pH, and P1b-P3b have high density of negative charges, enabling them as acid-sensitive coatings for positively charged nanoparticle surface. Compared to other maleamic acid-modified polyanions, which underwent a pHsensitive charge conversion to produce polycations that might be cytotoxic,13,49,50 the main-chain degradable PEGs would degrade into small molecule products that are easily metabolized. Additionally, the end groups of these macromolecular scaffolds should be either maleic anhydride or amine. The terminal maleic anhydride can easily react with the amino groups of protein (lysine group) or small molecule drugs (such as doxorubicin), generating prodrug and polymer−protein conjugate. Alternatively, the terminal amino groups can react with activated ester modified fluorescent probes for bioimaging. Therefore, these polymers can be further modified to deliver drugs or proteins.

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degradation products were measured with a quadrupole rods SQ Detector 2 mass spectrometer (Waters Corporation) in the positive ion mode and negative ion mode, respectively. Gel permeation chromatography (GPC) analysis was used to determine the numberaverage molecular weights (Mn) and the dispersity (Đ) of the polymers using a Waters GPC equipped with a Waters 2414 refractive index detector, a Waters 1525 binary HPLC pump, and three Waters Styragel columns (1 × 104, 1 × 103, and 500 Å pore sizes). THF was used as the mobile phase at a flow rate of 1 mL/min, and the column oven was set at 35 °C. The calibration was performed using polystyrene standards, and the data were calculated with a Breeze 3.30 software. For GPC analyses, the in situ formed polymer solutions were used prior to the addition of NaHCO3 aqueous solution. Particle size and zeta potential of the polyplexes were evaluated on a PALS analyzer (Brookhaven Instruments Corporation, BIC) equipped with a 35 mW solid state laser (660 nm). The intensity-averaged particle size of each sample was measured by dynamic light scattering (DLS, detection angle 90°) and analyzed with BIC particle sizing software (9kpsdw32, ver. 2.3). Zeta potentials were measured by a zeta potential analyzer (ZetaPALS, Brookhaven Instruments, Holtsville, NY) and analyzed with BIC PALS potential analyzer software (palsw32, ver. 3.43). Synthesis of N,N′-(Hexane-1,6-diyl)bis(3-(4-methyl-2,5dioxo-2,5-dihydrofuran-3-yl) Propanamide) (Monomer M1). Oxalyl chloride (5.5 mL, 64.0 mmol) and DMF (400 μL) were added to a solution of CDM (2.95 g, 16.0 mmol) in DCM (40 mL). The reaction mixture was stirred at 0 °C for 10 min and at room temperature for additional 1 h. Then, the unreacted oxalyl chloride and DCM was removed by rotary evaporation. The acyl chlorideactivated CDM (dissolved in 5 mL of THF) was added dropwise to the solution of 1,6-hexanedimine (0.93 g, 8.0 mmol) and pyridine (1.27 mg, 16.0 mmol) in anhydrous THF (24 mL) at 0 °C within 2 h. The mixture was stirred at 0 °C for 10 h and stirred at room temperature for another 12 h. Subsequently, the concentrated reaction mixture was precipitated into 0.1 N HCl (100 mL) three times. The precipitate was collected and dried under high vacuum at 40 °C overnight to give M1 as a light brown powder in 38%. 1H NMR (400 MHz, CDCl3, ppm): δ 5.69 (s, 2H), 3.21 (dt, J = 6.4, 6.4 Hz, 4H), 2.80 (t, J = 7.0 Hz, 4H), 2.55 (t, J = 7.0 Hz, 4H), 2.13 (s, 6H), 1.51−1.39 (m, 4H), 1.32−1.24 (m, 4H). 13C NMR (101 MHz, CDCl3, ppm): δ170.5, 165.9, 142.7, 142.3, 39.2, 32.9, 29.3, 25.9, 20.5, 9.7. ESI-MS: calcd. for C22H28N2O8: 448.473; found: 449.19164 [M + H]+, 471.17379 [M + Na]+, 487.14772 [M+K]+. Synthesis of 3,3′-(Pentane-1,5-diyl)bis(furan-2,5-dione) (Monomer M2). (1Z,8Z)-Tetramethyl-nona-1,8-diene-1,2,8,9-tetracarboxylate (Compound 1). In a flame-dried, nitrogen-flushed flask were added freshly polished magnesium ribbon (2.0 g) and anhydrous THF (3 mL). A solution of 1,5-dibromopentane (2.59 g, 11.25 mmol) in THF (15 mL) and a crystal of iodine were added to the above mixture. The mixture was refluxed for 2 h, and cooled down to room temperature. The above generated Grignard reagent was added dropwise to a suspension of CuBr·Me2S (5.55 g, 27 mmol) in THF (35 mL) at −40 °C for 2 h and then cooled to −78 °C, and DMAD (3.84 g, 27 mmol) was added dropwise to give a dark red-brown mixture. After being stirred for 1 h, the reaction mixture was quenched with a saturated solution of ammonium chloride (100 mL, adjusted to pH 8 with ammonia) and allowed to warm to room

EXPERIMENTAL SECTION

Materials. 2-Propionic-3-methylmaleic anhydride (carboxy-dimethylmaleic anhydride, CDM, 97%, TCI), NH2-PEG77-NH2 (M4, >95%, Mw ≈ 3400, Shanghai Peng Sheng Biological Technology Co., Ltd.), 1,8-diamino-3,6-dioxaoctane (NH2-OEG2-NH2, M5, 98%, Aldrich), dimethyl acetylenedicarboxylate (DMAD, 96%, Tianjin Heowns Technology Co., Ltd.), branched polyethylenimine (PEI, Mw ≈ 25 000, Aldrich), salmon sperm DNA (ssDNA,Aldrich) were used as-received. 1,6-Hexanediamine (99.5%), tetrahydrofuran (THF, 99.9%, SuperDry), N,N-dimethylformamide (DMF, 99.8%, SuperDry) dichloromethane (DCM, 99.9%, SuperDry), and copper(I) bromide-dimethyl sulfide (CuBr·Me2S, 98%) were purchased from J&K Chemical, Ltd. Deuterated phosphate buffers (PB) with pH 7.4, 6.5, and 5.5 were prepared from NaOD (40 wt % in D2O, Alfa) and deuterated phosphoric acid (85 wt % in D2O, Alfa). Other reagents were purchased from Beijing Chemical Reagent Co. and used without further purification unless otherwise specified. 1,5-Dibromopentane (97%, Aldrich) and triethylamine (TEA, AR, Beijing Chemical Reagent Co.) were dried over CaH2 and distilled into flame-dried bottles immediately prior to use. Pyromellitic dianhydride (M3, J&K Chemical, Ltd. 99.5%) was purified by dehydration in trifluoroacetic anhydride prior to use. Measurements. 1H NMR and 13C NMR spectra were recorded on a Bruker ARX 400 MHz spectrometer using MeOD, D2O or CDCl3 as the solvent. Tetramethylsilane was used as an internal standard. Electrospray ionization mass spectroscopy (ESI-MS) of monomers M1 or M2 was performed on a Bruker APEX-IV Fourier transform mass spectrometer in the positive ion mode and the negative ion mode, respectively. Mass spectra (MS) of the polymer B

DOI: 10.1021/acs.macromol.8b01053 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules Scheme 2. Synthesis of Monomer M1

temperature. After 30 min and removal of most of THF by rotary evaporation, the aqueous phase was extracted with ethyl acetate (3 × 200 mL), and the combined organic extract was washed with brine (200 mL), dried and concentrated by rotary evaporation. The obtained deep brown crude product was purified by silica column chromatography (petroleum ether/ethyl acetate, 4:1, v/v) to afford a light yellow solid (2.0 g, yield: 15%).1H NMR (400 MHz, CDCl3, ppm): δ 5.81 (s, 2H), 3.83 (s, 6H), 3.72 (s, 6H), 2.35 (t, J = 7.4 Hz, 4H), 1.56−1.45 (m, 4H), 1.43−1.32 (m, 2H). 13 C NMR (101 MHz, CDCl3, ppm): δ 169.3, 165.4, 150.4, 119.4, 52.4, 51.9, 34.2, 28.1, 26.6. ESI-MS: calcd. for C17H24O8: 356.373; found: 357.154394 [M + H]+, 379.136338 [M + Na]+, 395.110276 [M + K]+. (1Z,8Z)-Nona-1,8-diene-1,2,8,9-tetracarboxylic Acid (Compound 2). An aqueous solution of 1 N LiOH (114 mmol, 114 mL) was add to a solution of compound 1 (2.0 g, 5.7 mmol) in THF (100 mL), and the mixture was stirred at 50 °C for 5 h. The reaction mixture was acidified with 1 N HCl to pH 2, and the solvent was removed under reduced pressure. The obtained solid was washed by acetone (3 × 200 mL) to afford a white powder (1.7 g, yield: 98%). 1H NMR (400 MHz, CD3OD, ppm): δ 6.16 (s, 2H), 2.37 (t, J = 7.4 Hz, 4H), 1.57− 1.45 (m, 4H), 1.44−1.34 (m, 2H). 13C NMR (101 MHz, CD3OD, ppm): δ 170.0, 169.9, 149.6, 129.0, 36.1, 28.9, 28.8. ESI-MS: calcd. for C13H16O8: 300.265; found: 299.076144 [M-H]−. 3,3′-(Pentane-1,5-diyl)bis(furan-2,5-dione) (Monomer M2). The above white powder (compound 2, 0.50 g, 1.67 mmol) was added to trifluoroacetic anhydride (TFAA, 5.0 mL), and the suspension was stirred for 2 h. at ambient temperature. TFAA was removed by rotary evaporation to afford a light yellow powder M2 (0.44 g, quantitative yield). 1H NMR (400 MHz, CDCl3, ppm): δ 6.62 (s, 2H), 2.55 (t, J = 7.4 Hz, 4H), 1.77−1.66 (m, 4H), 1.56−1.42 (m, 2H). 13C NMR (101 MHz, CDCl3, ppm): δ 165.8, 163.8, 153.0, 128.8, 28.7, 26.6, 25.8. ESI-MS: calcd. for C13H12O6: 264.235; found: 265.070665 [M + H]+. Synthesis of Polymers. These polymers were prepared by the nucleophilic polyaddition of the diamines (M4 and M5) to the bisanhydrides (M1, M2, and M3). P1a. Monomer M1 (36.0 mg, 0.08 mmol, 1 equiv), M4 (263.9 mg, 0.08 mmol, 1 equiv), anhydrous DMF (0.5 mL), and TEA (4.8 equiv to M1) were sequentially transferred into a 10 mL Schlenk flask. The mixture was degassed by three freeze−pump−thaw cycles and stirred under N2 atmosphere at 0 °C for 48 h. After 0.1 N NaHCO3 (2.4 mL, 3 equiv) was added to the polymerization solution, DMF was removed by rotary evaporation under reduced pressure. The residue was lyophilized to afford P1a as a light brown solid (quantitative yield). This sample was used without further purification. P2a-α/β with various α/β ratios of the substituent structure: The similar protocol was used starting from M4 and M2. The polymerizations were carried out in DCM for 48 h at different temperatures: −20 °C for P2a-55/45, 0 °C for P2a-70/30, and 30 °C for P2a-90/10. The ratio of α- to β-configuration regarding the position of the substituent on the cis-double bond of P2a series polymers was measured by comparing the peak intensities (Ia1/Ia2) of protons a1 and a2 in the 1H NMR spectrum (Figure S3 of the Supporting Information, SI). All these P2a-α/β polymers are white solids and obtained in quantitative yields. P3a. This polymer was obtained as a white solid from M4 and M3 following the same protocol as for P2a (0 °C, quantitative yield). For the Pnb series polymers (P1b, P2b, and P3b), the same synthetic procedure as for P1a was applied, except using anhydrous DMF as the solvent. The polymerization temperatures were 0 °C for P1b and P3b, and −20 °C for P2b. The yields of all these polymers

were quantitative. P1b is a light brown solid, P1b and P2b (α/β ratio: 52/48) are white solids. pH-Dependent Degradation of Polymers. All the hydrolysis experiments of polymers were performed in deuterated phosphate buffer (PB). For P1a, P2a-55/45, P2a-90/10, and P3a, 5 mg of polymer was dissolved in 500 μL of PB solution (50 mM, pH 5.5, 6.5 or 7.4) in a NMR tube. The tube was incubated at 37 °C, and the 1H NMR spectra were recorded at specific time points. After complete degradation as indicated by 1H NMR, the solution was alkalified with 1 N Na2CO3 to pH 9 and lyophilized. The degradation products were analyzed by GPC and MS, respectively. For P1b, P2b, and P3a, the similar procedure was followed but the polymer concentration was 2 mg/mL. Formation and Characterization of DNA/PEI/Pnb Ternary Polyplex. Formation of Polyplex. The solutions of ssDNA (25 μg/ mL), PEI (1.0 mg/mL), and Pnb (5.0 mg/mL) were first prepared in ultrapure water and filtered through a 0.45 μm membrane (Millipore). The following is the general procedure for polyplex formation. A known amount of PEI solution was quickly added to a DNA solution (final N/P ratio = 10). The mixed solution was vortexed for 10 s and incubated for another 15 min at room temperature to afford the DNA/PEI polyplex. Then, a Pnb solution was quickly added to the DNA/PEI polyplex (final CO2H/N ratio = 2, represents the molar ratio of carboxylic acid groups in Pnb to the nitrogen atoms of PEI). The ternary polyplex was obtained after vortexing for 10 s and incubation at room temperature for additional 15 min. Size and Zeta Potential Measurements. The stock polyplex solution was diluted in phosphate buffers (10 mM) with varied pH (5.5, 6.5 or 7.4). The final DNA concentration was 12 μg/mL. Each sample was incubated at 37 °C prior to the measurements of particle size and zeta potential. Experiments were carried out in triplicate. Stability of the Polyplexes in Albumin Solution. BSA in deionized water (26 μL, 40 mg/mL) was added into 2 mL of polyplex solution in PB (pH 7.4, 10 mM). The final concentration of ssDNA was 12 μg/mL. Then, the particle size of the polyplexes was measured by DLS at 37 °C against time. Experiments were carried out in triplicate. Cytotoxicity Assay. Cell viability was measured by CCK-8 assay. P1a, P2a-55/45, P3a, P1b, P2b, and P3b were dissolved in PB solution (10 mM, pH 7.4) with various polymer concentrations. The polymer solutions were incubated at 37 °C prior to use. HeLa cells were seeded in 96-well plates and incubated at 37 °C under a 5% CO2 humidified atmosphere for 24 h. Then, 10 μL of the polymer solution was added to each well, and the cells were cultured for another 24 h before CCK-8 assay. The absorbance of the solution was measured at 450 nm by a PerkinElmer EnSpire multimode microplate reader. The cell viability was calculated using the following equation: Cell viability (%) = (Abssample/Abscontrol) × 100. Experiments were performed in triplicate.

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RESULTS AND DISCUSSION Synthesis of Bis(maleic Anhydride) Monomers M1 and M2. M1 was prepared in a one-pot two step procedure starting from 2-propionic-3-methylmaleic anhydride (Scheme 2). Acyl chloride-activated CDM was obtained in quantitative yield by the reaction of 2-propionic-3-methylmaleic anhydride with oxalyl chloride in DCM. Then 1,6-hexanediamine and pyridine were added to the solution, and the mixture was allowed to react for 12 h. The overall yield of M1 was 38%. M2 was synthesized by a three-step approach starting from 1,5dibromopentane and dimethyl acetylenedicarboxylate (Scheme C

DOI: 10.1021/acs.macromol.8b01053 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules Scheme 3. Synthesis of Monomer M2

achieve high conversion, and get polymers with defined structures, we followed our established reaction conditions of small molecules.3 P1a was obtained in DMF with TEA as the catalysis at 0 °C, while P2a and P3a were prepared in DCM at 0 °C or −20 °C. Similar as in the small molecule reactions, polymer P2a also has two isomers, thus three P2a-α/β polymers with different α/β ratios were obtained by varying the reaction temperature. Because of the high density of the pendent carboxylic acid groups of P1b-P3b, they are not soluble in DCM even in the acid forms; these polymers were thus prepared in DMF. All these polymers were obtained in the salt forms to avoid plausible prehydrolysis during storage. The polymer structures were characterized by 1H and 13C NMR. As shown in Figures 2A and S2, the two characteristic methyl proton signals at 1.87 ppm (proton a1) and 1.82 ppm(proton a2) assigned to two isomeric maleamic acid units of P1a were clearly resolved. Of importance, we only found negligible amount of undesired imide unit (1.94 ppm, proton a3), confirming the clean and controllable polymerization reaction. For the P2a-α/β polymers, the α/β ratio could be determined by comparing the peak intensities of proton a1 (5.56 ppm) and a2 (5.83 ppm) (Figures 2B, S3, and S4). In the 1 H NMR spectrum of P3a, it possesses two types of symmetric units in the backbone, central symmetry (proton a) and axial symmetry (proton e and f) (Figures 2C and S5). The structures of P1b-P3b were also confirmed by NMR (Figures S6−S8). P2b possesses an α/β ratio of 48/52. Interestingly, P2a-55/45 (in DCM, −20 °C) and P2b-48/52 (in DMF, −20 °C) show similar α/β ratios, implying that reaction temperature is the key parameter influencing the ratio of α-unit to βunit. The degree of polymerization (DP) and number-average molecular weight of these polymers were estimated by terminal group analysis method, the detailed analysis was provided in

3). Compound 1 was obtained in a relatively low yield of 15%. Then, this compound was hydrolyzed to the tetracarboxylic acid in 98% yield. Finally, M2 was obtained in the presence of trifluoroacetic anhydride in quantitative yield. Their structures were confirmed by NMR and mass spectroscopy (Figures 1 and S1).

Figure 1. 1H NMR spectra of monomer M1 (A) and M2 (B).

Polymer Synthesis and Characterization. The one-pot “A2 + B2” polymerizations of M1/M2 and M4/M5 were carried out to afford the desired two series of polymers P1 and P2. For comparison, pyromellitic dianhydride (M3) was also used to get polymer P3 (Table 1). To minimize side reactions, Table 1. Characterization of Acid-Cleavable PEGsa monomers sample

α/β ratio

P1a P2a

− 55/45 70/30 90/10 − − 48/52 −

P3a P1b P2b P3b

A2 M1 M2 M2 M2 M3 M1 M2 M3

B2 M4 M4 M4 M4 M4 M5 M5 M5

solvent DMF DCM DCM DCM DCM DMF DMF DMF

T ( °C)

DPb

0 −20 0 30 0 0 −20 0

19 19 18 17 16 24 24 36

Mnb (kDa) 71.8 69.6 66.0 62.3 57.9 14.3 9.9 13.2

Mnc (kDa)

Mnd (kDa)

Đd

76.9 72.0 70.6 74.2 70.1 15.8 12.1 14.1

− 64.3 49.0 42.0 55.4 −e −e −e

−e 1.90 1.61 1.55 1.71 −e −e −e

e

a

The yields of all polymers are quantitative. bDetermined by 1H NMR spectra via terminal group analysis (Figure S9). cMeasured by Fluorescamine-labeling assay of terminal maino groups (Figure S10). dMeasured by GPC using THF as eluent (Figure S10). eThese polymers were insoluble in THF, and no signals were detected in GPC analysis regardless of using DMF and or H2O as the mobile phase. D

DOI: 10.1021/acs.macromol.8b01053 Macromolecules XXXX, XXX, XXX−XXX

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the SI (Figures S9 and S10). P2a and P3a in their acid forms are soluble in THF, and they were also characterized by GPC (Figure S11). Collectively, this one-pot synthetic approach is feasible for the preparation of main chain acid-degradable PEGs with maleamic acid linkages. pH-Dependent Degradation of Polymers. The acidsensitivity and pH-dependent degradation are the critical parameters for the design of pH-responsive polymers that may be used for programmable delivery systems.3,12,51 Herein, we first studied the pH-dependent degradation of P1a-P3a in phosphate buffer (PB) at 37 °C by 1H NMR and GPC (Figures 3 and 4). P1a and P2a could degrade in weakly acidic PBs. For P1a, 90% of the acid-labile Units were hydrolyzed in 4 h at pH 5.5, producing the dialkyl substituted bis(maleic acid) and the PEG diamine (M4), which could be detected by both GPC and MS (Figures 3A, 4A, and S12). Besides, a weak but clearly detectable methyl signal of the substituted maleimide was also observed in the final degradation products. With increasing pH, the fraction of the imide byproduct increased form 6% at pH 5.5 to 40% at pH 7.4 (Figures S13). The formation of the imide linkage inhibited the complete degradation of P1a and resulted in the production of a PEG dimer (Figure 4A). Figure 3B shows the time-dependent 1H NMR spectra of P2a-55/45 at pH 5.5. A continuous decrease in the signal intensity of the maleamic acid units with concurrent increase of the degradation products (monoalkyl substituted bis(maleic acid)) and M4) were detected. P2a-55/45 degraded completely and cleanly in 72 h at pH 5.5 (Figures 4A and

Figure 2. 1H NMR spectra of P1a (A), P2a-55/45 (B), and P3a (C) in D2O.

Figure 3. (A, B) Time-dependent 1H NMR spectra during the hydrolysis of (A) P1a and (B) P2a-55/45 in deuterated PB (pH = 5.5) at 37 °C. (C) Hydrolysis kinetic curves of P1a, P2a-55/45, P2a-90/10, and P3a at various pHs (pH 5.5, 6.5, and 7.4). The extent of hydrolysis (Ed) of P1a was estimated by comparing the integrals of peaks yellow, blue and asterisk (Ed = Iblue/(Iyellow + Iblue + Iasterisk)) and Ed of P2a was estimated by comparing the integrals of peaks red and green (Ed = Igreen/(Ired + Igreen)). E

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degradation kinetic profiles of P1a-P3a at three pHs (7.4, 6.5, and 5.5), which are approximately encountered in blood, extracelluar matrix of tumor, and endo/lysosome, respectively (Figure 3C).52,53 At the same pH, the degradation rate of these polymers followed the order of P1a ≫ P2a-55/45 > P2a-90/ 10 ≫ P3a, consistent with that of the small molecule analogues.3 P2a-55/45 shows the best hydrolysis selectivity (the ratio of t1/2‑pH7.4 to t1/2‑pH5.5 was 766). We also investigated the acid-assisted degradation profiles of P1b-P3b by 1H NMR and MS (Figures S18−S26). A similar trend as those of P1a-P3a was observed. However, at the same pH, the degradation rates of P1b or P2b-48/52 were slower than those of P1a and P2a-55/45. The detailed reason is currently under investigation, but is probably due to the more flexible chain conformation of P1a and P2a. Shielding Effect of P2b on DNA/PEI Polyplex. In genedelivery field, synthetic nonviral vectors with a positively charged surfaces are beneficial for their cellular uptake and endo/lysosomal escaping, but undesired for their circulation in blood or diffusion in the extracellular matrices.54,55 To solve this dilemma, much effort has directed toward temporally shielding the positive charges by coating the nanoparticles with polyanions and then deshielding at a specific stage. Negatively or neutrally charged nanoparticles would be relatively stable during circulation due to the reduced interaction with blood components such as serum proteins, but could recover the surface positive changes upon approaching the weakly acidic environments of tumor tissues or endo/lysosome.56,57 Herein, we explored the feasibility of using P1b-P3b as the acidsensitive polyanions by making a ternary polyplex for potential gene delivery (Figure 5A). Salmon sperm DNA (ssDNA) and

Figure 4. (A, B) GPC traces of polymers before (red) and after (black) acidic degradation, and monomer NH2-PEG77-NH2 (M4) (blue). The degradation experiments were performed in 50 mM PB (37 °C) at varied pHs (5.5, 6.5, and 7.4) for P1a (A) and at pH 5.5 for P1a, P2a-55/45, and P2a-90/10 (B). dPna denotes the completely degraded sample.

S14). P2a-90/10 also degraded into the same products but in a much slower rate, because the β-isomer of the maleamic acid units was hydrolyzed faster than the α-isomer (Figures S15 and S16).3 As expected, P3a was very stable in neutral or weakly acidic media, only negligible amount of the degradation products could be detected even at pH 5.5 for 3 days (Figure S17). On the basis of the 1H NMR spectra, we calculated the

Figure 5. (A) Formation and acid-sensitive deshielding of the ternary polyplex. (B−D) The change of particle size (above) and zeta potential (below) of polyplexes coated with (B) P1b, (C) P2b, and (D) P3b after incubation for different times at various pHs. For P3b polyplexes at all pHs or P2b polyplex at pH 5.5, the deshielded polyplex nanoparticles aggregated rapidly into large clusters more than 10 μm. F

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Macromolecules

and negative charges, good biocompatibility, we envision our acid-degradable PEG to be a platform polymer for future applications in biomedical fields.

polyethylenimine (PEI) were applied as a model DNA and a polycationic carrier, respectively. Initially, PEI compacted ssDNA into binary polyplex nanoparticles with a positive zeta potential of ca. 18 mV and a diameter of 120 nm at the N/ P ratio of 10 (Figure S27A). Upon adding P1b-P3b with a COOH/N ratio of 2, ternary polyplexes were formed with a negatively charged shell (zeta potential of ca. −30 mV) and a slightly increased size (Figure S27B−D). The attempt of coating DNA/PEI polyplex with P1a-P3a was not successful probably owing to the low density of the carboxylate groups along their polymer chains. To investigate the weak acid-responsive deshielding effect, the changes in particle size and zeta potential of the polyplexes were monitored (Figures 5B−D). Since P1b was too acidsensitive, even unstable in PB of pH 7.4 (Figure 5B), both zeta potential and particle size of the ternary polyplex made of P1b increased immediately after incubation for 1 h. Similar phenomenon was reported by Kataoka and co-workers for the poly(amino-acid)-based ternary DNA delivery system.56 The reason for the increase of polyplex size was probably due to the aggregation of the nanoparticles caused by reduction in the repulsive force with partial charge neutralization. In the case of DNA/PEI/P2b polyplexes, the particle size (130 nm) and zeta potential (−27 mV) were almost constant at pH 7.4 and 6.5 for 10 h. However, at pH = 5.5, the zeta potential of the polyplex increased gradually to 5 mV and the particle size increased rapidly to over 10 μm, indicating the formation of large clusters due to the surface charge conversion of the polyplex (Figure 5C). As a control group, for DNA/PEI/P3b polyplexes, both the zeta potential and size remained unchanged for 10 h in the tested pH range, indicating that the polyplexes were stable because of the low acid sensitivity of P3b (Figure 5D). To test the shielding effect of P1b and P2b shells against protein binding, particle sizes of the polyplexes were measured in a PB (pH 7.4) with bovine serum albumin (BSA) (Figure S28). The DNA/PEI binary polyplex showed a drastic increase in size immediately after BSA addition, implying the formation of large aggregates from DNA/PEI polyplex and the negatively changed BSA. For the DNA/PEI/P1b polyplex, however, the addition of BSA only caused a gradual increase in size because of degradation of the P1b coating layer even at pH 7.4. In contrast, the sizes of the ternary polyplexes coated with P2b did not change upon BSA addition, indicating the stabilizing effect of the polyanion shell against protein adsorption. Finally, P1a-P3a and P1b-P3b did not show cytotoxicity in a polymer concentration range of 0−0.5 mg/mL (Figure S29).

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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.macromol.8b01053. Experimental part, additional data for the characterization and hydrolysis of polymers and polyplexes, and the results of cytotoxicity assay (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (F.-S.D.). *E-mail: [email protected] (Z.-C.L.). ORCID

Fu-Sheng Du: 0000-0003-3174-6107 Zi-Chen Li: 0000-0002-0746-9050 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by National Key Research and Development Program of China (No. 2016YFA0201400) and National Natural Science Foundation of China (No. 21534001).

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REFERENCES

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CONCLUSIONS We have developed a facile synthetic method for the preparation of an acid-degradable PEG platform via the reaction between OEG/PEG diamines and bis(maleic anhydride)s. These polymers have well-defined and designable structures, and consistent with small molecules, they exhibited tunable pH-dependent degradation profiles. The acid sensitivity of the PMAs follows the order of dialkyl substituted ≫ monoalkyl substituted (β-isomer > α-isomer). Finally, P2b was demonstrated to be acid-responsive polyanions to coat the positively charged DNA/PEI polyplex, thus enhancing the stability of the coated polyplex against bovine serum albumin at pH 7.4. Upon exposure to a weakly acidic medium, the positively charged DNA/PEI polyplex can be quickly regenerated. With a combination of tunable acid-sensitivity G

DOI: 10.1021/acs.macromol.8b01053 Macromolecules XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.macromol.8b01053 Macromolecules XXXX, XXX, XXX−XXX