Galactose-Based Amphiphilic Block Copolymers: Synthesis

Apr 4, 2013 - Redox-responsive amphiphilic diblock copolymers, poly(6-O-methacryloyl-d-galactopyranose-co-2-(N,N-dimethylaminoethyl) ...
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Galactose-Based Amphiphilic Block Copolymers: Synthesis, Micellization, and Bioapplication Ying Wang, Chun-Yan Hong,* and Cai-Yuan Pan* CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, 230026, Anhui, People’s Republic of China ABSTRACT: Redox-responsive amphiphilic diblock copolymers, poly(6-O-methacryloyl-D-galactopyranose-co-2-(N,N-dimethylaminoethyl) methacrylate)-b-poly(pyridyl disulfide ethyl methylacrylate) (P(MAGP-co-DMAEMA)-b-PPDSMA) were obtained by deprotection of poly((6-O-methacryloyl1,2:3,4-di-O-isopropylidene- D -galactopyranose)-co-DMAEMA)-b-PPDSMA [P(MAlpGP-co-DMAEMA)-b-PPDSMA], which were prepared via reversible addition−fragmentation chain transfer (RAFT) polymerization of PDSMA using P(MAlpGP-co-DMAEMA) as macro-RAFT agent. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) studies showed that diblock copolymers P(MAGP-co-DMAEMA)-b-PPDSMA can self-assemble into micelles. Doxorubicin (DOX) could be encapsulated by P(MAGP-co-DMAEMA)-b-PPDSMA upon micellization and released upon adding glutathione (GSH) into the micelle solution. The galactose functional groups in the PMAGP block had specific interaction with HepG2 cells, and P(MAGP-co-DMAEMA)-b-PPDSMA can act as gene delivery vehicle. So, this kind of polymer has potential applications in hepatoma-targeting drug and gene delivery and biodetection.

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INTRODUCTION Very recently, drug and gene delivery have attracted great attention for their potential application in the clinic.1−4 Various polymeric micelles have been used as delivery vehicles of the drugs,5−7 and they are generally formed by self-assembly of amphiphilic copolymers in aqueous solution while the hydrophobic drug is loaded in the hydrophobic core of micelles.8−11 Recently, much effort has been directed toward development of the responsive micelles which are disassembled under an external stimuli (pH,12,13 temperature,14,15 redox potential,16−29 specific molecules,20 etc.). These stimuli-responsive micelles have emerged as novel programmable delivery system in which the release of encapsulated contents can be readily modulated by the stimulus, resulting in significantly enhanced therapeutic efficacy and minimal side effects.21,22 Pyridyl disulfide methylacrylate (PDSMA) based copolymers have attracted great attention due to their avirulence, excellent biocompatibility, and redox-responsiveness.16,23−26 The polymerization of pyridyldisulfide-based monomer via the reversible addition− fragmentation chain transfer polymerization (RAFT) was reported in previous publications.27−30 In these earlier studies, modification of hydrophobic pyridyldisulfide-based polymer with hydrophilic moieties such as a tripeptide, doxorubicin, and PEG chains via thiol-disulfide exchange and thiol−ene/thiol− acrylate addition reactions led to the formation of spherical nanoparticles in water, which can act as a potential drug delivery vehicle.26,29,30 In other words, after adding some specific hydrophilic small molecules, the hydrophobic PDSMA blocks can be transferred to the hydrophilic moieties via thioldisulfide exchange and thiol−ene/thiol−acrylate addition reactions. Therefore, for the micelles with PDSMA as the © XXXX American Chemical Society

core, they can be disassembled via adding some specific hydrophilic small molecules. Bulmus groups29 have investigated the water-insoluble/water-soluble exchange of the PDSMA homopolymer by adding different mercapto-compounds. However, to the best of our knowledge, no published paper investigated the control release of drug using the waterinsoluble/water-soluble exchange of the PDSMA. Gene therapy is an attractive approach to treat diseases caused by genetic disorders, mutation, or genetic defects such as leukemia and tumors. Since cationic polymers were employed as the vector for gene delivery in the 1970s,31 a variety of synthetic and natural cationic polymers have been investigated as the gene vectors.32,33 Among these gene vectors, PDMAEMA has received a lot of attention for gene delivery due to its relatively low toxicity and high buffer capacity.34−38 PDMAEMA is a synthetic polycation, which can be protonated at physiological pH. In addition, DNA can be condensed and protected by the interaction between positively charged PDMAEMA and negatively charged DNA.39,40 Challenge is remained in development of cellular specific polymeric micellar system for targeted drug and gene delivery. Among the reported targeting ligands, galactose is a specific liver-targeting ligand, which can mediate the delivery of drugs to liver parenchymal cells through its strong binding with asialoglycoprotein receptors (ASGP-R) on the surface of hepG2 human hepatoma cells,41−43 and it has attracted broad attention because of its low cost, ready availability. Therefore, Received: January 23, 2013 Revised: March 25, 2013

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Preparation of P(MAlpGP-co-DMAEMA). Into a 10 mL polymerization tube, MAlpGP (1.0 g, 3.05 mmol), DMAEMA (2.0 g, 12.74 mmol), AIBN (0.74 mg, 0.045 mmol), CPDB (35 mg, 0.158 mmol), and THF (4 mL) were added. The mixture was degassed through three freeze−pump−thaw cycles. The polymerization tube was then flame-sealed under vacuum, and the sealed tube was immersed into an oil bath thermostatted at 70 °C. After 10 h, the polymerization tube was cooled to room temperature rapidly, then the reaction mixture was precipitated into an excess of n-hexane. The obtained product was dried overnight in a vacuum oven at room temperature. Preparation of P(MAlpGP-co-DMAEMA)-b-PPDSMA. A typical polymerization procedure is as follows. Into a 5 mL polymerization tube, P(MAlpGP-co-DMAEMA) (Mn,NMR = 12000 g/mol, 0.2 g, 1.67 × 10−5 mol), PDSMA (0.5 g, 1.976 × 10−3 mol), AIBN (0.6 mg, 3.66 × 10−6 mol), and THF (2.2 mL) were added. After three freeze− vacuum−thaw cycles, the polymerization tube was sealed under vacuum and placed in an oil bath thermostatted at 70 °C. After the prescribed time, the tube was cooled to room temperature rapidly and the polymer was obtained by precipitation from n-hexane. The obtained product was dried overnight in a vacuum oven at room temperature. Preparation of P(MAGP-co-DMAEMA)-b-PPDSMA. The block copolymer P(MAlpGP-co-DMAEMA)-b-PPDSMA2 (Mn = 20000 g/ mol, 200 mg) was stirred in 80% formic acid (20 mL) for 48 h at room temperature, then deionized water (3 mL) was added and the mixture was stirred for additional 3 h. The final solution was dialyzed against deionized water for 3 days to remove the formic acid. The product was obtained in 96% yield as a white cotton-like solid by freeze-drying. Preparation of Aqueous Micellar Solution. Amphiphilic diblock copolymer P(MAGP-co-DMAEMA)-b-PPDSMA2 (Mn = 20000 g/mol, 10 mg) was dissolved in DMF (1 mL). Under vigorously stirring, deionized water (9 mL) was added slowly. After the addition was completed, the dispersion was stirred for another 5 h, then DMF was removed by dialysis [molecular weight (Mw) cutoff: 3.5 kDa] against deionized water for 24 h. Drug Loading and Release. P(MAGP-co-DMAEMA)-bPPDSMA2 (80 mg) and 20 mg DOX·HCl (4 mL triethylamine should be added) or 20 mg Nile red were dissolved in 2 mL DMF. Into the solution, 10 mL of deionized water was slowly added under vigorously stirring. After stirring for another 5 h, the mixture was subjected to dialyze against PBS buffer (pH 7.4, 10.0 mM) at room temperature for 24 h, and the external buffer solution was refreshed every 6 h during this period. After this process is completed, the polymer concentration of the drug-encapsulated micellar solution was adjusted to 1.0 mg/mL for subsequent in vitro drug release experiments. The content of drug loaded (DLC) and the drug loading efficiency (DLE) were determined by the following formulas:

many galactose-based glycopolymers are applied in biorecognition systems.44 Wu et al.45 synthesized galactosylated and fluorescein isothiocyanate (FITC) labeled polycaprolactone-gdextran polymers, and the galactosylated micelles could be selectively recognized by ASGP-R on the surfaces of HepG2 cells. Wang et al.46 developed reactive micelles based on diblock copolymer of poly(ethyl ethylene phosphate) and poly(εcaprolactone), and the micelles were further surface conjugated with galactosamine to target ASGP-R of HepG2 cells. Galactose-functionalized azacitidine-conjugating amphiphilic random copolymers were prepared and their hepatomatargeting function was investigated.47 Therefore, the micelles not only act as drug and gene delivery vehicles but also have the hepatoma-targeting function, thus they have broad application in biosystem. In our study, redox-responsive copolymers P(MAGP-coDMAEMA)-b-PPDSMA were synthesized. Introduction of the biocompatible PMAGP and PPDSMA not only endues the bock copolymers with the redox-responsive and target character but also reduces the cytotoxicity of PDMAEMA. Self-assembly of the block copolymers, P(MAGP-co-DMAEMA)-b-PPDSMA can form redox-sensitive micelles with disulfide bonds in their hydrophobic part, and then DOX is loaded in the micelles. The DOX-loaded micelles can be disassembled in response to GSH, with a concomitant release of the loaded DOX. So, it is expected that the micelles can act as gene delivery vehicles, also can be selectively recognized by HepG2 cells. Thus, this kind of polymers can act as hepatoma-targeting drug and gene delivery vehicles.

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EXPERIMENTAL SECTION

Materials. 6-O-Methacryloyl-1,2;3,4-di-O-isopropylidene-D-galactopyranose (MAlpGP) was prepared as reported,48 and its 1H NMR (300 MHz, CDCl3, δ, ppm): 1.21−1.60 (m, 12H, -CH3), 1.95 (s, 3H, -CH3), 4.08, 4.19−4.42, 4.63, and 5.54 (7H, sugar moiety), 5.57 and 6.14 (s, 2H, =CH2). Pyridyl disulfide methylacrylate (PDSMA) was synthesized according to literature.16 2-(N,N-Dimethylaminoethyl) methacrylate (DMAEMA, 97%, Alfa) was purified by passing through basic alumina columns, then vacuum-distilled, and stored at −20 °C prior to use. Toluene and THF were refluxed over sodium for 24 h and distilled prior to use. Azobis(isobutyronitrile) (AIBN, Aldrich) was recrystallized from ethanol. Cyanoisopropyl dithiobenzoate (CPDB) was prepared according to the literature.49,50 1H NMR (300 MHz, CDCl3, δ, ppm): 1.94 (6H, m, −CH3), 7.31−7.97 (5H, m, aryl H). DOX·HCl was purchased from Aladdin and used as received. Poly(ethylenimine) (PEI, branched, Mw = 25 kDa, 99%) was purchased from Aldrich. All other reagents with analytical grade were purchased from Shanghai Chemical Reagent Co. and used without further purification. Characterization. 1H NMR measurements were carried out on a Bruker AV300 NMR spectrometer using CDCl3 or D2O or DMSO-d6 as solvent. Molecular weight and molecular weight distribution (Mw/ M n ) were determined on a Waters 150C gel permeation chromatography (GPC) equipped with microstyragel columns (500, 103 and 104 Å) and RI 2414 detector at 30 °C, monodispersed polystyrene standards were used in the calibration of Mn, Mw, and Mw/ Mn, and tetrahydrofuran (THF) was used as eluent at a flow rate of 1.0 mL/min. UV/vis measurement was performed on a Unico UV/vis 2802PCS spectrophotometer. Transmission electron microscopy (TEM) observations were conducted on a JEM-100SX electron microscope at an acceleration voltage of 100 kV. The sample for TEM observation was prepared by placing 10 μL of micellar solution on a copper grid coated with thin films of Formvar and carbon successively. Dynamic light scattering (DLS) measurements were carried out on a DynaPro light scattering instrument (DynaPro-99E) at 25 °C with 824.3 nm laser, and data was analyzed with DYNAMICS V6 software.

DLC = (weight of loaded drug/weight of polymer) × 100% DLE = (weight of loaded drug/weight of drug in feed) × 100% The drug release was performed under both reduction-insensitive and reduction-sensitive conditions. For the reduction-sensitive experiment, the drug release was performed under various pHs (pH = 5, 7.4, and 9). A typical procedure is as follows: 2.0 mL of an aqueous dispersion of DOX-loaded micelles (1.0 mg/mL) was transferred to a dialysis bag with a molecular weight cutoff of 3.5 kDa and then immersed in 50 mL of various buffer solutions (pH = 5, 7.4, and 9) containing 10 mM GSH, which was purged with pure nitrogen for 30 min. The samples (2 mL) were taken at predetermined time intervals for estimating the amount of drug released, and the same method was employed for the reduction-insensitive experiment except use of pure PBS solution to replace the buffer solution. UV absorbance of the dialysis solution at 497 nm was monitored to determine the DOX releasing profile. A series of parallel experiments were conducted. In Vitro Cytotoxicity Measurement. Cell viability was examined by the MTT assay. HepG2 cells were seeded in a 96-well plate at an initial density of 5000 cells/well in 100 μL of DMEM complete B

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Scheme 1. Synthesis of P(MAGP-co-DMAEMA)-b-PPDSMA

Table 1. Preparation of P(MAlpGP-co-DMAEMA)-b-PPDSMA sample P(MAlpGP-co-DMAEMA) P(MAlpGP-co-DMAEMA)-bPPDSMA 1 P(MAlpGP-co-DMAEMA)-bPPDSMA2 P(MAlpGP-co-DMAEMA)-bPPDSMA3

RAFT agent CPDB P(MAlpGP-coDMAEMA) P(MAlpGP-coDMAEMA) P(MAlpGP-coDMAEMA)

time (h)

conv.a (%)

Mn,thb (g/mol)

Mn,GPCc (g/mol)

10 8

64 17

12400 17200

9000 14000

1.11 1.24

12000 17000

1/5/0 1/5/1.65

12

40

24100

20000

1.25

24000

1/5/4

16

64

31400

24000

1.28

31200

1/5/7

Mw/Mnc Mn,NMRd

[M]/[D]/[P]e (molar ratio)

Determined by gravimetry. bTheoretical molecular weight: Mn,th = [PDSMA]0/[P(MAlpGP-co-DMAEMA)]0 × MPDSMA × conversion + Mn,P(MAlpGP‑co‑DMAEMA), where [PDSMA]0/[P(MAlpGP-co-DMAEMA)]0 is the molar ratio of PDSMA and macro-RAFT agent in feed, MPDSMA, and Mn,P(MAlpGP‑co‑DMAEMA) are the molecular weights of PDSMA and P(MAlpGP-co-DMAEMA), respectively. cDetermined by GPC with calculation based on polystyrene standards. dDetermined by 1H NMR data. eDetermined by 1H NMR, [M]/[D]/[P] represents the molar ratio of MAlpGP, DMAEMA, and PPDSMA in the resultant polymers. a

deionized water), respectively, for 2 h at 37 °C. After removing the medium, the HepG2 and A549 cells were washed three times using phosphate-buffered saline (PBS) and fixed with 4% formaldehyde, then the slides were mounted and observed with a Nikon Eclipse TE2000U Inverted Microscope with a 10× objective. Transfection Activity In Vitro. Transfection experiments were performed with COS-7 cells using gWiz-Luc plasmid. All transfection studies were performed in 48-well plates with cells plated 24 h before transfection at a seeding density of 40000 cells per well. The polyplexes were prepared by adding the polymer solution into plasmid solution at desired N/P ratios and incubated for 30 min. On the day of transfection, the medium was removed and 25 μL polyplexes containing 0.04 μg DNA mix with 150 μL of FBS-free DMWM was added. After 4 h of incubation, the transfection mixture was removed and the cells were cultured for additional 24 h in 150 μL of fresh full DMEM media. To determine levels of luciferase expression, the culture medium was discarded and the cell lysates were harvested after incubation of cells for 30 min at room temperature in 100 μL of cell lysis reagent buffer (Promega). To measure the luciferase content, 100 μL of luciferase assay buffer (20 mM glycylglycine (pH 8), 1 mM MgCl2, 0.1 mM EDTA, 3.5 mM DTT, 0.5 mM ATP, and 0.27 mM coenzyme A) was automatically injected into 20 μL of cell lysate, and the luminescence was integrated over 10 s using single tube Sirius

medium. Micellar solution of P(MAGP-co-DMAEMA)-b-PPDSMA was then added to achieve varying final polymer concentrations. After incubation for 24 h, MTT reagent (in 20 μL PBS buffer, 5 mg/mL) was added to each well, and the cells were further incubated with 5% CO2 for 4 h at 37 °C. The culture medium in each well was removed and replaced by 100 μL of DMSO. The plate was gently agitated for 15 min and the absorbance values were recorded at a wavelength of 490 nm upon using a Thermo Electron MK3 μm. The cell viability is calculated as A490,treated/A490,control × 100%, where A490,treated and A490,control are the absorbance values with or without the addition of micelles, respectively. Each experiment was done in quadruple. The data are shown as the mean value plus a standard deviation (±SD). Cellular Uptake Assay. A cellular uptake assay of the Nile Redloaded micelles prepared from P(MAGP-co-DMAEMA)-b-PPDSMA was carried out. The human hepatoma cells HepG2 overexpressing ASGP-R were used for experimental group, while A549 cells bearing no ASGP-R were used as negative control group. The prepared HepG2 cells and A549 cells (5 × 104) were seeded on coverslip in 24-well plate and then incubated in 500 μL of DMEM (Dulbecco’s Modified Eagle’s Medium, containing 10% hyclone fetal bovine serum, 50 units mL −1 penicillin, and 50 units mL −1 streptomycin) medium containing 100 μL of Nile Red-loaded P(MAGP-co-DMAEMA)-b-PPDSMA micellar solution (3 mg/mL in C

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2.56 ppm (d) to the signals at δ = 7.2−8.0 ppm (m′), assuming no loss of the dithiobenzoate functional groups in the polymerization. The results are listed in Table 1. 1 H NMR spectra of the block copolymers are also used to characterize their structure, and a typical 1H NMR spectrum of P(MAlpGP-co-DMAEMA)-b-PPDSMA2 in Figure 1B reveals characteristic signals of both P(MAlpGP-co-DMAEMA) and PPDSMA. All proton signals of the P(MAlpGP-co-DMAEMA) at δ = 3.9−4.5 (i,m,f,g), 4.62 (j), 5.50 (h), 2.08 (e), 2.56 (d), and 4.08 ppm (c) can be seen, and the characteristic signals of PPDSMA appeared at δ = 2.99 (d′), 4.12 (c′), and 7.0−8.6 ppm (e′) are attributed to the methylene protons adjacent to the disulfide group, ester methylene protons, and pyridyl protons. These 1H NMR data support the formation of P(MAlpGP-co-DMAEMA)-b-PPDSMA. In addition, the compositions of P(MAlpGP-co-DMAEMA)-b-PPDSMA copolymers were calculated based on the integral values of proton signals at δ = 5.50 (h), 2.56 (d), and 2.99 ppm (d′), and their Mn,NMRs were calculated based on the integration ratio of δ = 2.56 (d) to 2.99 (d′); the results are listed in Table 1. The molecular weights and molecular weight distributions of P(MAlpGP-co-DMAEMA) and P(MAlpGP-co-DMAEMA)-bPPDSMA were measured by GPC, and the results are shown in Figure 2. All GPC traces of the block copolymers formed are

luminometer (Zylux Corporation). Total cellular protein in the cell lysate was determined by the bicinchoninic acid (BCA) protein assay using a calibration curve constructed with standard bovine serum albumin solutions (Pierce). The luciferase transfection results are expressed as relative light units (RLU) per mg of cellular protein. Unless stated otherwise, the results are expressed as mean RLU/mg of protein (±SD of triplicate experiments).

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RESULTS AND DISCUSSION Synthesis and Characterization of P(MAGP-co-DMAEMA)-b-PPDSMA. Because the RAFT polymerization of DMAEMA with CPDB as RAFT agent exhibited excellent living characteristics in broad polymerization conditions,51 the CPDB was used as RAFT agent in the RAFT copolymerization of MAlpGP and DMAEMA. The feed molar ratio of [CPDB]/ [DSDMA]/[MAlpGP]/[AIBN] was fixed at 1:1.5:20:0.25, and the P(MAlpGP-co-DMAEMA) with Mn,GPC = 9000 g/mol and Mw/Mn = 1.11 was obtained. Then this copolymer was used as macro-RAFT agent in the subsequent RAFT polymerization of PDSMA, forming the diblock copolymers P(MAlpGP-coDMAEMA)-b-PPDSMA, as shown in Scheme 1. The detailed polymerization conditions and results are listed in Table 1. The structures of macro-RAFT agent P(MAlpGP-coDMAEMA) and P(MAlpGP-co-DMAEMA)-b-PPDSMA are verified by their 1H NMR spectra. As shown in Figure 1A, the

Figure 2. GPC traces of (1) P(MAlpGP-co-DMAEMA) (Mn = 9000 g/mol, Mw/Mn = 1.11); (2) P(MAlpGP-co-DMAEMA)-b-PPDSMA1 (Mn = 14000 g/mol, Mw/Mn = 1.24); (3) P(MAlpGP-co-DMAEMA)b-PPDSMA2 (Mn = 20000 g/mol, Mw/Mn = 1.25); and (4) P(MAlpGP-co-DMAEMA)-b-PPDSMA3 (Mn = 24000 g/mol, Mw/ Mn = 1.28).

Figure 1. 1H NMR spectra of P(MAlpGP-co-DMAEMA) (A) and P(MAlpGP-co-DMAEMA)-b-PPDSMA (B) recorded in CDCl3.

completely shifted to high molecular weight region and the Mns of block copolymers increase with polymerization time. All the results support also the formation of expected diblock copolymers. The target copolymers, P(MAGP-co-DMAEMA)-bPPDSMAs, were obtained by deprotection reaction of P(MAlpGP-co-DMAEMA)-b-PPDSMA in 80% formic acid,52,53 and the obtained polymer solution was dialyzed against deionized water and lyophilized. The degree of deprotection reaction can be estimated by the 1H NMR spectrum in Figure 3. The almost disappearance of the isopropylidene proton signals at δ = 1.2−1.6 ppm indicates success of the deprotection reaction, and the target block copolymers are successfully synthesized. Aggregation Behavior of the Amphiphilic Diblock Copolymers P(MAGP-co-DMAEMA)-b-PPDSMA in Water. Amphiphilic block copolymer can self-assemble into micelles,

signals of anomeric protons in the MAlpGP units appear at δ = 3.9−4.5 ppm (i,m,f,g), 4.62 ppm (j), and 5.50 ppm (h), and the signals at δ = 2.08 (e), δ = 2.56 (d), and δ = 4.08 (c) are attributed to the methyl protons, the methylene protons adjacent to the nitrogen and ester methylene protons in the dimethylaminoethyl group of DMAEMA units, respectively. The phenyl proton signals of dithiobenzoate units at δ = 7.37− 7.93 ppm (m′) can be clearly seen, and the ascription of other proton signals is marked in the figure. All these facts support that the P(MAlpGP-co-DMAEMA) was successfully synthesized. The molar ratio of MAlpGP and DMAEMA units in the resultant polymers is 1:5, which was calculated based on the integral values of the signals at δ = 5.50 ppm (h) and δ = 2.56 ppm (d). The number-average molecular weight, Mn,NMR of the P(MAlpGP-co-DMAEMA) was also obtained by calculation from the integration ratio of proton signals at δ = 5.50 (h) and D

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reactions.29 Therefore, the micelles self-assembled from P(MAGP-co-DMAEMA)-b-PPDSMA are disassembled in the presence of hydrophilic mercapto-compounds, leading to release of the loaded drug. Glutathione (GSH) is a tripeptide that was found within the cells at millimolar concentrations, and it can be used as a water-soluble reducing agent.58 So, we investigated the drug release behavior of the micelles obtained from P(MAGP-co-DMAEMA)-b-PPDSMA using GSH as the example, and the DOX-loaded micellar solution was utilized for the subsequent in vitro drug release experiments. The drug release behavior of the micelles obtained from P(MAGP-coDMAEMA)-b-PPDSMA2 was investigated at different conditions and the results are shown in Figure 6A. The DLE% and DLC% values are 28.8 and 7.2%, respectively, which are slightly higher than those reported in the literature.59 For the system without GSH, a maximum release of 32% was reached after 48 h, probably, the released drug came from the surface layer of the micelles. However, for the system containing 10 mM GSH, much more DOX was released, which is due to the waterinsoluble/water-soluble exchange of the PDSMA in the presence of hydrophilic mercapto-compounds, GSH, resulting in the disassociation of the micelles. For confirming this explanation, we measured the sizes of P(MAGP-co-DMAEMA)-b-PPDSMA2 micelles before and after control release for 48 h in the presence of 10 mM GSH. As shown in Figure 6B, the micelles after 48 h release was not completely disassociated, but their size decreased significantly (from D = 136 to 12 nm), which is consistent with the control release result in Figure 6A, that is, approximately 25% of the loaded DOX was not released after control release for 48 h. When control release was performed at pH = 7.4 without GSH for 48 h, no obvious change of the micelles’ size was observed (the result is not shown). The solution pH affected the drug release also. As the DOX-loaded micelle solution was placed in pH 7.4 buffer (PBS, 10.0 mM) containing 10 mM GSH, after 50h releasing, ∼75% of the loaded drug was released at pH = 7.4, but at pH 9, only ∼60% of the drug was released, while at pH 5, ∼85% was released. This is understandable because the release rate of DOX decreased in the solution with pH > 8.4, and the PDMAEMA in the shell of P(MAGP-co-DMAEMA)-bPPDSMA micelles is weak base and was collapsed at pH = 9, leading to the release rate decrease of the drug. While at pH = 5, the release rate of DOX increased, and the solubility of PDMAEMA block is better than that at pH = 7.4, leading to the release rate increase of the DOX. Cytotoxicity of the Aggregates. The cytotoxicity of P(MAGP-co-DMAEMA)-b-PPDSMA micelles was evaluated by MTT assay using HepG2 cells. The cells were incubated with various amounts of micelles for 24 h. The PEI with molecular weight of 25 kDa was used as a control. The results in Figure 7 showed that the P(MAGP-co-DMAEMA)-b-PPDSMA micelles were less toxic than 25 kDa PEI, and cytotoxicity of the P(MAGP-co-DMAEMA)-b-PPDSMA micelles slightly decreased with increasing molecular weights of PPDSMA block; perhaps the content of cytotoxic DMAEMA on the surface of the micelles is responsible for the cytotoxicity, and this content decreased with an increase of the biocompatible PPDSMA chain length. In addition, it should be noted that the three micelles maintained over 80% cell viability at concentration of 100 μg/mL, indicating low cytotoxicity of the cationic micelles. Cellular Uptake of Aggregates. It is known that hepatoma cells can recognize galactose- and N-acetylgalactosamine-terminated glycoproteins via the asialoglycoprotein

Figure 3. 1H NMR spectrum of P(MAGP-co-DMAEMA)-bPPDSMA2 recorded in DMSO-d6.

vesicles, and other morphologies in a selective solvent.54−57 When the water was slowly added into the copolymer solution in DMF, and then DMF was removed by dialysis against deionized water for 24 h, the micelles with PPDSMA core and P(MAGP-co-DMAEMA) corona were formed, which was validated by their 1H NMR spectrum in D2O, as shown in Figure 4. We can find only the characteristic signals of

Figure 4. 1H NMR spectrum of P(MAGP-co-DMAEMA)-bPPDSMA2 recorded in D2O.

P(MAGP-co-DMAEMA), but the signals of PPDSMA are absent. The TEM images and the DLS results are shown in Figure 5. The average diameter of resultant micelles determined by TEM is smaller than that determined by DLS (TEM: A1, 80 nm; B1, 120 nm; C1, 180 nm; DLS: A2, 98 nm; B2, 136 nm; C2, 196 nm); it is reasonable because the size obtained from TEM is in the dry state and the size measured by DLS is in the swollen state. In addition, the average diameters of resultant micelles increase with the increase of the PPDSMA block (Figure 5). Triggered Release of DOX from Micelles. PPDSMA is a hydrophobic polymer with disulfide bonds, but it can be transferred into hydrophilic polymer in the presence of hydrophilic mercapto-compounds by thiol/disulfide exchange E

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Figure 5. TEM images and DLS curves of the micelles formed by self-assembly of P(MAGP-co-DMAEMA)-b-PPDSMA1 (A), P(MAGP-coDMAEMA)-b-PPDSMA2 (B), and P(MAGP-co-DMAEMA)-b-PPDSMA3 (C) in water (2 mg/mL).

Figure 6. (A) DOX release profile of P(MAGP-co-DMAEMA)-b-PPDSMA2 micellar nanocarriers under different conditions; (B) Size change of the P(MAGP-co-DMAEMA)-b-PPDSMA2 micelles before (a) and after control release for 48 h in the presence of 10 mM GSH (b); concentration of the micelles: 2 mg/mL.

receptor (ASGP-R) on the surface of them.60 Once the ligand on the micelles binds to the ASGP-R on the hepatoma cells, the micelles can be rapidly internalized by hepatoma cells.45,61 In order to investigate whether the aggregates of P(MAGP-coDMAEMA)-b-PPDSMA have a targeting function against human hepatoma cells, a cellular uptake assay of the aggregates prepared from P(MAGP-co-DMAEMA)-b-PPDSMA was carried out. The human hepatoma cells HepG2 overexpressing ASGP-R were used for experimental group, while A549 cells bearing no ASGP-R were used as a negative control group. The cellular uptake of Nile Red loaded P(MAGP-co-DMAEMA)-bPPDSMA2 aggregates was studied by fluorescence microscope for investigation on the target interaction between galactose functional groups and HepG2 cells. HepG2 cells having large number galactose-binding asialoglycoprotein receptors and A549 cells were incubated with DMEM medium containing

Nile Red loaded P(MAGP-co-DMAEMA)-b-PPDSMA aggregates for 2 h, respectively. The cell binding and internalization ability were investigated by the fluorescence intensity of cell microphotograph. As shown in Figure 8, after 2 h incubation with Nile Red loaded P(MAGP-co-DMAEMA)-b-PPDSMA2 micellar solution at 37 °C, remarkable fluorescence was observed in HepG2 cells (Figure 8A). As for A549 cells incubated with P(MAGP-co-DMAEMA)-b-PPDSMA2 micellar solution, very weak intracellular fluorescence was observed (Figure 8B). The results reveal that via the ligand−receptormediated recognition, the micelles self-assembled from P(MAGP-co-DMAEMA)-b-PPDSMA have target interaction with HepG2 cells, resulting in a higher cellular uptake quantity of HepG2 cells than that of A549 cells under the same conditions. F

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Figure 9. Luciferase gene expression of P(MAGP-co-DMAEMA)-bPPDSMA1, P(MAGP-co-DMAEMA)-b-PPDSMA2, and P(MAGP-coDMAEMA)-b-PPDSMA3 and bPEI 25 kD in COS-7 cells at different N/P. The luciferase gene expression level is expressed as relative luciferase light units (RLU)/mg of protein (P1, P2, and P3 represent P(MAGP-co-DMAEMA)-b-PPDSMA1, P(MAGP-co-DMAEMA)-bPPDSMA2, and P(MAGP-co-DMAEMA)-b-PPDSMA3, respectively).

Figure 7. Relative cell viability of HepG2 cells evaluated by MTT assay after incubation with micellar solution of P(MAGP-coDMAEMA)-b-PPDSMA1, P(MAGP-co-DMAEMA)-b-PPDSMA2, and P(MAGP-co-DMAEMA)-b-PPDSMA3 at 37 °C and varying polymer concentrations.

explained by that molecular weights of P(MAGP-co-DMAEMA)-b-PPDSMA3 is comparable to PEI 25KD and the cytotoxicity of P(MAGP-co-DMAEMA)-b-PPDSMA3 is much lower than that of PEI 25kD (Figure 7). These results confirm that this kind of polymer micelles can be used as a promising nonviral gene vector.

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CONCLUSION Novel redox-responsive amphiphilic diblock copolymers, P(MAGP-co-DMAEMA)-b-PPDSMA have been successfully synthesized, and the resultant amphiphilic block copolymers can be self-assembled into micelles containing disulfide bonds in their hydrophobic part, then the DOX is loaded in the micelles. In the aqueous solution containing GSH, much more loaded DOX can be released at relatively fast rate owing to hydrophobic PPDSMA becoming hydrophilic via thiol-disulfide exchange reaction. In addition, the micelles not only have binding specifically to the ASGP receptors on hepatoma cells because of the target interaction between galactose functional groups and HepG2 cells but can also act as the gene delivery vehicles; thus, this kind of polymer can act as hepatomatargeting drug and gene delivery vehicles.

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Figure 8. Fluorescence microscopy images of HepG2 cells (A) and A549 cells (B) treated by Nile Red loaded P(MAGP-co-DMAEMA)-bPPDSMA2 micellar solution at 37 °C for 2 h (A1, B1: fluorescence images; A2, B2: bright field images).

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. Notes

Transfection Activity In Vitro. Figure 9 showed the transfection efficacies of P(MAGP-co-DMAEMA)-bPPDSMA1, P(MAGP-co-DMAEMA)-b-PPDSMA2 and P(MAGP-co-DMAEMA)-b-PPDSMA3 and PEI 25kD polyplexes with luciferase gene. For all the samples, luciferase gene expression efficacy increased with the increase of N/P, and at the same N/P, the luciferase gene expression efficacy increased with the molecular weights increase of P(MAGP-co-DMAEMA)-b-PPDSMA, which can be explained by that the complex ability of the polymer and DNA increased with the polymer molecular weights, resulting in the more stable of P(MAGP-coDMAEMA)-b-PPDSMA/DNA polyplexes. In addition, it should be noted that the luciferase gene expression efficacy of P(MAGP-co-DMAEMA)-b-PPDSMA3 at the N/P = 40 and 80 is higher than that PEI 25kD at the N/P = 10, which can be

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support by National Natural Science Foundation of China (Nos. 20974103, 21074121, and 21090354) is greatly acknowledged.

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