Tumor Acidity-Induced Sheddable Polyethylenimine-Poly(trimethylene

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Tumor Acidity-Induced Sheddable PolyethyleniminePoly(trimethylene carbonate)/DNA/Polyethylene Glycol-2,3Dimethylmaleicanhydride Ternary Complex for Efficient and Safe Gene Delivery Caiyan Zhao,† Leihou Shao,† Jianqing Lu, Xiongwei Deng, and Yan Wu* CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, No. 11 Beiyitiao, Zhongguancun, Beijing 100190, China S Supporting Information *

ABSTRACT: Amphiphilic PEI derivatives/DNA complexes are widely used for DNA delivery, but they are unstable in vivo and have cytotoxicity due to the excess cationic charge. PEGylation of cationic complexes can improve sterical stability and biocompatibility. However, PEGylation significantly inhibits cellular uptake and endosomal escape. In this work, sheddable ternary complexes were developed by coating a tumor acidity-sensitive β-carboxylic amide functionalized PEG layer on the binary complexes of amphiphilic cationic polyethylenimine-poly(trimethylene carbonate) nanoparticles/DNA (PEI-PTMC/DNA). Such sheddable ternary complexes markedly reduced their nonspecific interactions with serum protein in the bloodstream and obtained minimal cytotoxicity due to the protection of the PEG shell. At the tumor site, the PEG layer was deshielded by responding to the tumor acidic microenvironment and the positively charged complexes re-exposed that had higher affinity with negatively charged cell membranes. Meanwhile the positively charged complexes facilitated endosomal escape. Accordingly, this delivery system improved the biocompatibility of gene-loaded complexes and enhanced the gene transfection efficiency. Such PEGylated complexes with the ability to deshield the PEG layer at the target tissues hold great promise for efficient and safe gene delivery in vivo. KEYWORDS: sheddable, tumor acidity, PEGylation, polyethylenimine-poly(trimethylene carbonate), gene delivery

1. INTRODUCTION

Hydrophobic modification on polycation can improve stability and promote cellular uptake by self-assembly into compact micelle architecture.15−17 Most of the amphiphilic cationic copolymers have shown effective transfection and alleviative cytotoxicity. Previous research showed that poly(trimethylene carbonate) (PTMC), a hydrophobic biomaterial approved by the FDA, possesses high biocompatibility, nontoxicity, and good biodegradability.18−21 Unlike other hydrophobic polymers, the degradation of PTMC cannot release detrimental acidic compounds that would cause inflammation and other unwanted damage in the body.22−24 So incorporation of the PTMC chain on PEI may be able to improve the biocompatibility of PEI. Whereas, amphiphilic cationic carriers still cannot simultaneously meet the high stability in plasma and efficient gene transfection. The excess cationic charge of the cationic copolymer/DNA complexes leads to the high toxicity and unstability in vivo due to serum

Gene therapy technology has exhibited promising potential as a biological tool for the treatment of cancer.1,2 However, its therapeutic efficacy is hampered due to some intractable problems including the nuclease degradation, inability to transport into cells and other biological barriers.3,4 To date, cationic polymers, one of the most important nonviral vectors, have shown great potential for gene delivery.5 Among them, polyethylenimine (PEI, refers to branched PEI 25 kDa) is regarded as the gold standard for gene transfection with outstanding properties such as chain flexibility and facile manufacture.6−8 Moreover, PEI can exert a unique “proton sponge effect” for endosomal escape and exhibit remarkable transfection efficiency.9 However, it fails to achieve satisfactory efficacy for systemic gene delivery. One main reason is that the nonspecific interaction between positively charged complexes and serum components leads to severe aggregation and rapid clearance from the circulation by the reticuloendothelial system (RES).10 Another reason is that the cationic causes devastating interaction with cellular membrane, leading to inevitable cytotoxicity.11−14 © XXXX American Chemical Society

Received: January 21, 2016 Accepted: February 23, 2016

A

DOI: 10.1021/acsami.6b00825 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Scheme 1. Schematic Illustration of (A) the Formation of PEI-PTMC/DNA/PEG-DA Ternary Complex and (B) Deshielding of PEG Shell at Tumor Aciditya

a

The cationic PEI-PTMC copolymers were self-assembled into nanoparticles and complexed with DNA to form a binary complex. Then the PEIPTMC/DNA complex was coated with PEG-DA to form ternary complex. In a tumor acidic environment, PEG-DA degraded and generated a positively charged amino group, triggering PEG shell deshielded via electrostatic repulsion and re-exposing positive complex.

or negative charge in the physiological conditions but reverse positive charged at lower pH (6.5−6.8) of the tumor extracellular space. Inspired by the pH-triggered charge-reversal characteristic of β-carboxylic amide, we introduce a pHsensitive charge-conversional function to PEG by conjugating 2,3-dimethylmaleicanhydride (DA) to mPEG-NH2. In this study, a ternary tumor acidity-induced charge-reversal PEG corona coated PEI-PTMC/DNA complex was fabricated. The formation and the “deshielding strategy” of the ternary complex are illustrated in Scheme 1. A stable core was prepared by cationic amphiphilic PEI-PTMC nanoparticles (NPs) complexed with plasmid DNA (pDNA). A functionalized PEG layer was decorated on the surface of the binary complexes to form noncovalent post-PEGylated ternary complexes (PEI-PTMC/DNA/PEG-DA). The functionalized PEG layer shielded the DNA-loaded complexes from aggregation and recognition by the body immune system during systemic circulation and the positively charged complexes re-exposed only at the tumor sites via electrostatic repulsion between charge-reversal PEG and positive complexes to self-remove the PEG layer. The re-exposed positively charged complexes could obtain higher affinity with negatively charged cell membranes. After endocytosis, the complexes would efficiently escape from endosomal entrapment and migrate into cytosol benefiting from the abundant amine in PEI. So the PEI-PTMC/DNA/PEG-DA ternary complexes would be an ideal option for efficient and safe gene delivery.

protein adhesion, although the high cationic charge is essential to achieve high transfection efficiency.25 Polyethylene glycol (PEG) has been widely used to modify polycations to reduce the cytotoxicity and prolong the circulation time via shielding cationic charge of cationic complexes during systemic circulation.26−28 However, PEGylation causes the risk of overexposure the complexes to other tissues and markedly hinder the cellular uptake and endosomal escape.29−31 Hence, PEGylation limits the gene transfection efficiency. Recently, scientists have proposed a smart “sheddable” strategy in which the shielding PEG or other polymers can be fully or partially removed from the nanoparticle in tumor tissue by responding to local stimuli to reveal an active group for improved intracellular delivery efficiency.32−36 Such “sheddable” nanocarrier exhibits superior efficacy in delivering therapeutic agents. Various enzyme overexpressed in tumors at different stages has been utilized to trigger the protective PEG corona deshielding in delivery systems to promote cellular uptake.37,38 Meanwhile, it is known that the extracellular pH of tumor tissue (6.5−6.8) is often lower than that of normal physiological conditions (∼pH 7.4) due to the increased glucose uptake and metabolism in tumor microenvironments,39−42 which is a more practical trigger for designing such a nanoparticle. A tumor acidity-activated sheddable sulfonamide/poly(ethylenimine)/pDNA delivery system has been reported to deliver DNA that had shown enhanced transfection efficiency in vitro and reduced cytotoxicity.43 However, the in vivo delivery efficacy has not been verified. An exciting aspect is that β-carboxylic amide has a pHresponsive hydrolysis characteristic.44−46 It is stable and negatively charged at neutral pH but quickly hydrolyzes to expose a positively charged amino group at a slightly acidic environment, such as tumor tissues. Therefore, the polymers bearing acid-sensitive β-carboxylic amide can maintain neutral

2. EXPERIMENTAL SECTION 2.1. Materials. Branch poly(ether imide) (PEI, 25 kDa) and polyethylene glycol (mPEG2k-NH2, mPEG2k-COOH) were purchased from Sigma-Aldrich (Milwaukee); poly(trimethylene carbonate) (PTMC-COOH, 20 kDa) was purchased from Jinan Daigang Biotechnology Co. Ltd. (Shandong, China); 2,3-dimethylmaleic anhydride (DA), dicyclohexylcarbodiimide (DCC), and 4-dimethylaB


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ACS Applied Materials & Interfaces minopyridine (DMAP) were purchased form GL Biochem Co. Ltd. (Shanghai, China); ethidium bromide was purchased from SigmaAldrich (Milwaukee, WI). All other regents were analytical grade and used as received. The human embryonic kidney cell line 293T and human cervical cancer cell line Hela was purchased from American Type Culture Collection (ATCC, Manassas, VA). High-glucose Dulbecco’s modified Eagle’s medium (H-DMEM), low-glucose Dulbecco’s modified Eagle’s medium (L-DMEM), and fetal bovine serum (FBS) were purchased from Wisent Inc. (Multicell, Wisent Inc., St. Bruno, Quebec, Canada); 0.25% trypsin-EDTA and antibiotic solutions of penicillin and streptomycin were obtained from Invitrogen (Carlsbad, CA); 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Milwaukee, WI); DiR and Lysotracker Green were purchased from Molecular Probes Inc. (Eugene, OR); agarose was purchased from GENTECH (Shanghai, China); EGFPN1 plasmid (4700 bp) was extracted from Escherichia coli according to the protocol of the plasmid extraction kit of TIANGEN; Texas Redoligodeoxynucleotides (ODN) was purchased from Sangon Biotech Co. Ltd. (Shanghai, China). 2.2. Synthesis of PEI-PTMC. PTMC20k-COOH (4.0 g, 0.2 mmol) and DCC (4.1 g, 20 mmol) were dissolved in 10 mL of dried DMF and stirred at room temperature for 2 h. Then PEI25K (5.0 g, 0.2 mmol) and DMAP (0.27 g, 2 mmol) were added to the above solution. The solution was kept stirring at room temperature for 24 h. The resulting mixture was filtered, and the filtrate was dialyzed (MWCO = 30 000 Da) against distilled water for 48 h then lyophilized to obtain PEI-PTMC. 2.3. Synthesis of PEG-DA. mPEG2k-NH2 (400 mg, 0.2 mmol) and DA (276 mg, 2 mmol) in 20 mL of PBS (pH 8.0) was stirred at room temperature for 24 h. Then the solution was dialyzed (MWCO = 1000 Da) against distilled water at neutral pH for 24 h and lyophilized to obtain PEG-DA. 2.4. Characterization of Copolymer. The chemical structure of PEI-PTMC and PEG-DA were characterized by using nuclear magnetic resonance (1H NMR) and Fourier transform-infrared spectroscopy (FT-IR). 1H NMR spectra were recorded on a Bruker AVANCE 400 NMR spectrometer (Billerica, MA) using DMSO-d6 as solvent. FT-IR was analyzed on a spectrophotometer (Perkin Elmer) using KBr as a reference. The samples were mixed with KBr to make transparent slices for measurement. 2.5. Preparation of Binary and Ternary Complexes. The charge ratio (N/P or N/P/C) of binary or ternary complexes was indicated as the mole ratio of the tertiary amine groups (N) on PEIPTMC to the phosphate groups (P) on DNA and the carboxyl groups (C) on PEG-DA. PEI-PTMC NPs were first prepared as follows: 20 mg of PEI-PTMC copolymer was dissolved in DMF. Then the solution was added dropwise to 5 mL of double distilled water and stirred for 30 min. The mixture was placed in a dialysis bag (MWCO = 3500 Da) and dialyzed against double distilled water to remove DMF at room temperature. The final volume was adjusted to 20 mL, and the final concentration was 1 mg/mL. Then pDNA (290 ng/μL) was added into the above PEI-PTMC NPs at different N/P ratios to obtain binary complexes PEI-PTMC/DNA. After incubation for 15 min, PEG-DA (10 nmol/μL) was added into the preprepared binary complexes to construct ternary complexes PEI-PTMC/DNA/PEGDA. For in vivo fluorescent imaging, DiR, a near-infrared fluorescent probe was loaded into PEI-PTMC NPs. The DiR-loaded complexes were prepared using the procedures similar to as described above. 2.6. Characterization of Particle Size and Zeta Potential. The particle size and zeta potential of complexes were characterized by using a Zeta Sizer Nano series Nano-ZS (Malvern, U.K.). The determinations were performed at 633 nm with a constant angle of 90, respectively. Various N/P ratios of binary complexes and various N/P/ C ratios of ternary complexes containing 3 μg of pDNA were prepared and diluted with PBS to 0.8 mL before measurement. 2.7. Transmission Electron Microscopy (TEM). The morphology of complexes was observed using a Tecnai G2 20 STWIN transmission electron microscope (TEM, Philips, Netherlands) with

200 kV acceleration voltage. The samples were prepared by adding a drop of complex solution onto a copper grid and air-dried. 2.8. Gel Retardation Assay. A volume of 10 μL of well-incubated complexes solution containing 1 μg of pDNA were mixed with 2 μL of 6× loading buffer (Takara Biotechnology, Dalian, China), then the suspensions were loaded onto 1% agarose gel containing 5 μg/mL ethidium bromide and the electrophoresis was carried out at a voltage of 120 V in 1× TAE running buffer for 15 min. The pDNA retardation was analyzed on an image master VDS thermal imaging system (BioRad, CA) at a UV light wavelength of 254 nm. 2.9. Bovine Serum Albumin Absorption. A bovine serum albumin (BSA) absorption assay was used to examine protein adsorption capacity of complexes according to a previous study. In brief, 200 μL of complexes solutions (polycation concentration, 22 μM) was mixed with equivalent volume BSA solution (2 mg/mL). After shaking for 30 min, the adsorption was measured at 280 nm using a UV spectrophotometer TU1810 (Beijing Purkinje General Instrument Co. Ltd., China). Subsequently the sample was centrifuged at 13 000 r/min for 10 min and the adsorption of BSA in the supernatants was measured the same as above. The concentration was calculated using the calibration curve of BSA. The percent of BSA adsorbed onto the complexes was calculated using the following equation:

q=

(C i − Cs)V n

Ci represents the initial concentration of BSA. Cs represents the concentration of BSA in the supernatant. V and n represent the ultimate volume of the solution and the molar amount of the polycations in complexes, respectively. 2.10. Cell Viability. 293T cells were seeded in a 96-well plate at a density of 1 × 104 cells per well and cultured in complete H-DMEM containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C for 24 h. A volume of 10 μL of PEI-PTMC/ pDNA binary complexes at different N/P ratios or PEI/pDNA, PEIPTMC/pDNA, and PEI-PTMC/pDNA/PEG-DA complexes at different concentrations were added into each well. The cells were incubated for 24 h then the medium was replaced by 100 μL of MTT (0.5 mg/mL) for another 4 h. Afterward the MTT solution was replaced by 150 μL of DMSO to dissolve the formazan crystals formed by viable cells. The absorbance of the solutions was measured using an Infinite M200 microplate reader (Tecan, Durham) at 570 nm, with 630 nm as the reference wavelength. 2.11. In Vitro Transfection. EGFP-N1 plasmid was used as a model gene to investigate the gene transfection efficiency. 293T cells were seeded into 24-well plates (0.5 mL, 5 × 104 cells per well) and incubated in complete H-DMEM containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C for 24 h. One hour before transfection, the culture medium was replaced by fresh DMEM without FBS. Complexes containing 1 μg of EGFP-N1 plasmid were added into each well. The cells were transfected for 4 h. Then the transfection solution was replaced by 500 μL of complete H-DMEM for an additional 48 h. The expression of EGFP-N1 plasmid in the cells was directly observed by using an inverted fluorescence microscope (Olympus IX 70, Olympus, Tokyo, Japan). The transfection efficiency was quantified by using flow cytometry (Applied Biosystems, Life Technologies, Carlsbad, CA). 2.12. Cellular Distribution of Complexes. 293T cells were seeded in each of the 35 mm glass dishes at a density of 1 × 105 cells and cultured for 24 h. The media were substituted by with complexes suspensions containing 1 μg of Texas Red-ODN in fresh DMEM without FBS. After incubation for 4 h, the cells were washed three times with PBS and the lyso/endosome was stained with LysoTracker Green. The cellular distribution of complexes was observed using a confocal laser scanning microscope (CLSM) (LSM 710, Carl Zeiss Microscope Co. Ltd., Germany). 2.13. Hemolysis Assay. Red blood cells (RBCs) were isolated from freshly whole blood by centrifuging at 1500g for 5 min and washed five times with sterile saline. Then RBCs were suspended in C


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Figure 1. (A) Hydrodynamic diameter and (B) transmission electronic microscopic image of PEI-PTMC NPs. saline and 0.5 mL was added to 0.5 mL of PEI-PTMC/pDNA binary complexes, PEI-PTMC/pDNA/PEG-DA ternary complexes, or PEI/ pDNA complexes in saline at a final polycation concentration of 0.2, 0.5, 1, 2, 5, or 10 μM. The samples were incubated for 3 h and centrifuged at 1500g for 5 min. The optical density (OD) of the supernatant was read at 545 nm using a UV−vis spectrophotometer. The positive reference (100% lysis) was a blood/water mixture, and the negative reference (0% lysis) was a blood/saline mixture. The hemolytic ratios of the samples were calculated as follows:

hemolytic ratio(%) =

PEI-PTMC copolymer was synthesized by grafting hydrophobic PTMC with hydrophilic PEI. The resulting PEI-PTMC conjugates were characterized by 1H NMR and FT-IR spectroscopy. In Figure S1A, a broad peak at δ ∼2.58 ppm corresponded to the methylene protons of PEI. 1H NMR of PEI-PTMC conjugates displayed new absorption peaks. The signals at δ 4.15 ppm and δ 1.96 ppm were assigned to the protons of methylene (2H, −CH2−CH2−CH2−OCO−) and (2H, −CH2−CH2−CH2−OCO−) repeat units in the PTMC, respectively. In FT-IR spectra analysis (Figure S1B), the PEI polymer showed the characteristic absorptions of amino groups at around 3300 cm−1. After modified with PTMC, the symmetric (CO) band of the poly(alkyl carbonate)s at 1743 cm−1 appeared, and meanwhile the absorption peaks of amino groups at 3300 cm−1 are still observed, indicating that the PEI-PTMC copolymer had been successfully prepared. The amino-groups in mPEG-NH2 were amidated with DA to produce pH-dependent charge-conversional PEG-DA. It could be seen from Figure S2A that the mPEG-NH2 showed the characteristic methylene peak at δ 3.51 ppm. Compared with the 1H NMR spectra of mPEG-NH2, the 1H NMR spectrum of mPEG-DA exhibited a single peak at δ 1.88 ppm that was attributed to the protons of −CH3 (3H, −C(CH3) C(CH3)−) in DA. Further evidence for the functionalization of PEG was offered by FT-IR spectra. As shown in Figure S2B, mPEG-NH2 showed the characteristic peaks of −C−O−C− at 1107 cm−1. However, in the spectra of PEG-DA, a very broad absorption peak of −COOH at 3700−3100 cm−1 was observed and the new slight absorption at 1706 cm−1 is assigned to the stretching vibration of −CO. Together, these results demonstrated the successful synthesis of functionalized derivatives PEG-DA. 3.2. Preparation and Characterization of PEI-PTMC NPs. First, the biodegradable, amphiphilic, cationic PEI-PTMC self-assembled into nanoparticles in aqueous media. The size distribution and zeta potential were determined by DLS. As shown in Figure 1A, the hydrodynamic diameter of PEI-PTMC NPs was 112.5 nm (PDI = 0.229). The zeta potential of PEIPTMC NPs was 46.1 mV. The high zeta potential laid the foundation for electrostatic interactions with negatively charged nuclei acids. TEM (Figure 1B) showed that the PEI-PTMC copolymer self-assembled into homogeneous spherical structure. These data indicated that the hydrophobic interaction among the PTMC moiety in an amphiphilic PEI-PTMC

sample absorbance − negative control positive control − negative control

2.14. Red Blood Cells Aggregation Study. The RBCs were mixed with PEI-PTMC/pDNA binary complexes or PEI-PTMC/ pDNA/PEG-DA ternary complexes in saline at a final polycation concentration of 2 μM. Saline was used as a negative control and modified fluid gelatin (Gel) as a positive control. The cellular morphology of the RBCs was observed with CLSM. 2.15. In Vivo Distribution of Complexes. Female BALB/c nude mice (4−6 weeks-old) were purchased from Vital River Laboratory Animal Center (Beijing, China). All animal procedures were performed in accordance with protocols approved by the ethics committee of Peking University. The tumor model was established by inoculating 1 × 107 Hela cells in the armpit of mice. When the tumor volume grew to a diameter of about 100 mm3, the mice were randomly divided into five groups (n = 3 per group) and injected in the caudal vein with physiological saline, DiR, DiR-loaded PEI-PTMC/pDNA complexes, DiR-loaded PEIPTMC/pDNA/PEG-COOH, or DiR-loaded PEI-PTMC/pDNA/ PEG-DA complexes (DiR 500 ng/mL). After 24 h, near-infrared imaging was carried out using a Maestro in vivo imaging system (Cambridge Research & Instrumentation, Woburn, MA). 2.16. In Vivo Transfection. Hela tumor-bearing BALB/c mice were used to assay the in vivo gene expression efficacy of DNA-loaded complexes. The mice were treated with naked DNA, PEI-PTMC/ DNA binary complexes, PEI-PTMC/DNA/PEG-COOH, or PEIPTMC/DNA/PEG-DA ternary complexes. All of the complexes contained 30 mg of EGFP plasmid. After tail-vein injection of 48 h, the mice were sacrificed, and tumors were dislodged and photographed using an in vivo imaging system. 2.17. Statistical Analysis. Data were expressed as mean standard deviation (S.D.). Statistical analysis was determined using Student’s t test and *p < 0.05, **p < 0.01, and ***p < 0.001 were used to show statistical significance.

3. RESULTS AND DISCUSSION 3.1. Synthesis and characterization of PEI-PTMC and PEG-DA. We here describe the successful synthesis of amphiphilic PEI-PTMC and functionalization of PEG. The D


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Figure 2. (A) Agarose gel electrophoresis assay of PEI-PTMC/pDNA binary complexes; (B) particle size and zeta potential of PEI-PTMC/pDNA binary complexes at different N/P ratios; (C) fluorescence microscope images of 293T cells transfected with PEI-PTMC/pDNA binary complexes at different N/P ratios in vitro.

Figure 3. (A) Particle size and zeta potential of PEI-PTMC/pDNA/PEG-DA ternary complexes in different N/P/C ratios at pH 7.4; (B) changes of PEI-PTMC/pDNA/PEG-DA ternary complexes in size and zeta potential after incubation at pH 6.5 for 2 h; (C) agarose gel electrophoresis assay of ternary complexes at various N/P/C ratios after incubation at pH 7.4 and pH 6.5; (D) transmission electronic microscopic image of PEI-PTMC/ pDNA/PEG-DA complexes at N/P/C ratio of 12:1:20.

E


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the positively charged PEI-PTMC/pDNA complexes were reexposed, demonstrating the slightly acidic environment could degrade the amide bond of PEG-DA and further trigger the PEG chain detached from the complexes. The pDNA retardation capacity of the ternary complexes after exposure at pH 6.5 was also tested. As shown in Figure 3C (lower), no pDNA release was observed, indicating the pH of PBS buffer did not interfere with DNA binding. In the TEM measurement (Figure 3D), the ternary complexes (the N/P/C ratio of 12:1:20) were uniformly dispersed as individual nanoparticles. In view of the best in charge reverse ability and particle size, the complexes formed at N/P/C ratio of 12:1:20 were chosen as a representative for further investigation. 3.5. Stability of the Complexes. The serum stability is important for the complexes to be applied in vivo. If the particles are unstable, they may aggregate to form large particles due to potential nonspecific interactions with serum components and be cleared from the blood circulation rapidly following systemic administration. Figure 4A and Figure S5 presented the size and zeta potential changes of PEI-PTMC/ pDNA binary complexes and PEI-PTMC/pDNA/PEG-DA ternary complexes after incubation with 10% FBS. The PEI-

copolymer hold it together to form tight and spherical particles with small size. 3.3. Assessment of Binary Complexes. To investigate the potential of PEI-PTMC NPs in gene delivery, the physiochemical characterization and in vitro experiments were performed. pDNA encoding green fluorescent protein (GFP) was chose as a model nucleic acid to complex with PEI-PTMC NPs. A vital factor of the carrier for gene delivery is the ability of binding DNA to form complexes. So, the agarose gel electrophoresis was carried out to evaluate the binding ability of PEI-PTMC and pDNA. Figure 2A showed that the PEI-PTMC displayed excellent pDNA retarding abilities at the low N/P ratio of 3:1 and demonstrated that PEI-PTMC NPs can effectively form complexes with DNA. Figure 2B showed the particle size and zeta potential of the binary complexes. The particle sizes decreased with the increasing of N/P ratio while the zeta potentials increased as the increasing of N/P ratio. The PEI-PTMC/pDNA complexes could form nanoparticles within 200 nm at the N/P ratio of 12 or higher. In addition, the TEM imaging (Figure S3) showed that the binary complexes (at the N/P ratio of 12:1) still maintained a spherical structure. The in vitro cytotoxicity and gene transfection efficiency of PEI-PTMC/pDNA were performed with 293T cells at N/P ratios from 7 to 20. As shown in Figure S4, as the N/P ratio increased, the cell viability decreased, indicating that the toxicity of the binary complexes increased at high N/P ratios, especially at N/P above 15. Figure 2C showed that the cells treated with PEI-PTMC/pDNA complexes exhibited good transfection efficiency. The complexes at a N/P ratio above 12/1 exhibited brighter green fluorescence due to a large amount of GFP expression. Whereas, the gene transfection efficiency was slightly reduced at the N/P ratio of 20:1 that might be attributed to the high toxicity. 3.4. Characterization of Ternary Complexes. On the basis of the preferable transfection efficiency of cationic amphiphilic PEI-PTMC, a tumor acidity-sensitive chargereversal PEG-DA was synthesized for coating PEI-PTMC/ DNA binary complexes with an optimal N/P ratio of 12:1 to improve the biocompatibility of the gene delivery system. The agarose gel electrophoresis assay, particle size, zeta potential, and TEM imaging were first carried out to characterize the ternary complexes. As shown in Figure 3C (upper), the agarose gel electrophoresis assay indicated that pDNA detachment was not observed when the PEG-DA was coated onto the PEIPTMC/pDNA binary complexes to form a PEI-PTMC/ pDNA/PEG-DA ternary structure even with a ratio of N/P/ C up to 10:1:30, indicating that the introduction of functionalized PEG layer did not interfere with DNA retardation.47 The particle size and zeta potential of ternary complexes were measured by DLS. As shown in Figure 3A, for all the four investigated N/P/C ratios, the size of PEI-PTMC/ pDNA/PEG-DA ternary complexes are larger than that of PEIPTMC/pDNA binary complexes at a N/P ratio of 12:1. In addition, as the ratio of PEG increased, zeta potentials of ternary complexes decreased and switched to negative when the N/P/C ratio reached to 12:1:20, implying that the formation of PEG corona shielded the positive charge of binary complexes. However, after incubation at pH 6.5 for another 2 h (Figure 3B), all of the complexes obviously exhibited a positive surface and the zeta potential of complexes was higher than that at pH 7.4. Meanwhile, the size of the complexes was slightly smaller than that at pH 7.4 for all groups. These findings might be because the PEG shell was deshielded at acidic conditions and

Figure 4. (A) Changes in particle size of PEI-PTMC/pDNA binary complexes and PEI-PTMC/pDNA/PEG-DA ternary complexes after incubation with 10% fetal bovine serum; (B) protein adsorption of PEI/pDNA, PEI-PTMC/pDNA binary complexes, and PEI-PTMC/ pDNA/PEG-DA ternary complexes with BSA. F


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The result further confirmed the improved biocompatibility and safety of PEI-PTMC/DNA/PEG-DA ternary complexes. 3.7. In Vitro Gene Transfection. It is well-known that efficient gene transfection of complexes is necessary for gene delivery. In order to investigate the transfection efficiency of PEI-PTMC/DNA/PEG-DA ternary complexes, PEI/DNA complexes and PEI-PTMC/DNA binary complexes were used as positive controls and the unsensitive PEG-COOH was used to coat the PEI-PTMC/DNA binary complexes to form unsheddable ternary complexes as a negative control. Figure 6 and Figure S6 showed the expression of GFP in the transfected 293T cells after incubation with the following four DNA complexes formulations: PEI/pDNA (N/P = 12:1), sheddable PEI-PTMC/pDNA/PEG-DA ternary complexes (N/P/C = 12:1:20), unsheddable PEI-PTMC/pDNA/PEGCOOH ternary complexes (N/P/C = 12:1:20), and PEIPTMC/pDNA binary complexes (N/P = 12:1) at pH 7.4 or 6.5, respectively. The cells treated with both sheddable PEIPTMC/pDNA/PEG-DA ternary complexes and unsheddable PEI-PTMC/pDNA/PEG-COOH ternary complexes exhibited weaker green fluorescence compared with PEI/pDNA and PEIPTMC/pDNA complexes at pH 7.4, indicating that coating with the PEG shell significantly lowered the transfection efficiency. After incubation at pH 6.5, the cells treated with sheddable PEG-DA coated ternary complexes showed comparable green fluorescence intensity to PEI/pDNA and PEIPTMC/pDNA complexes. In contrast, unsheddable PEGCOOH coated ternary complexes still showed dim green fluorescence. The transfection efficiency was further quantified by flow cytometry (Figure 6D). The results exhibited the same tendency with fluorescence microscope images. These results demonstrated that the transfection efficiency of DNA-loaded cationic complexes could not be inhibited by coating a pHinduced charge-reversal PEG shell. The deshielding of PEG layer at slightly acidic tumor extracellular microenvironment would re-expose the positively charged surface of complexes which was beneficial for cellular uptake and endsomal escape. 3.8. Intracellular Distribution. The successful endosomal escape is a key step for the gene carrier to obtain efficient gene transfection,6,48 so it is necessary to illustrate the intracellular trafficking of PEI-PTMC/DNA binary complexes and PEIPTMC/DNA/PEG-DA ternary complexes. Here, the intracellular distribution of the complexes after cellular internalization was observed using confocal laser scanning microscopy (CLSM). As shown in Figure 7, Lysotracker Green was used to identify lyso/endosome. For PEI/Texas Red-ODN complexes, most of the red fluorescence was localized in the cytoplasm and nucleus, indicating that PEI had good buffering capacity, which facilitated it’s escape from the endosome. However, some of the red fluorescence localized on the cell surface that might be because the PEI/Texas Red-ODN complexes had low stability and aggregation occurred. Similar to PEI, the cells treated with binary and ternary complexes showed obvious red fluorescence in the cytoplasm and nucleus, indicating that the binary and ternary complexes had strong endosomal escape ability. These results suggested that the functionalized PEG layer could shed from the cationic complexes in acid tumor sites and not cause the inhibition to endosomal escape. So, the ternary complexes exhibited comparable endosomal escape and transfection efficiency to PEI-PTMC/DNA binary complexes. 3.9. In Vivo Distribution. It is well-known that hemolysis (destruction of RBCs) in vivo can lead to anemia, jaundice, and other pathological conditions,49 so the hemolytic potential

PTMC/pDNA complexes became large aggregates over the time, while the size of PEGylated PEI-PTMC/pDNA/PEG-DA complexes only increased slightly. In addition, the zetapotential of unPEGylated PEI-PTMC/pDNA complexes was significantly decreased to about −17 mV. In contrast, the PEGylated PEI-PTMC/pDNA/PEG-DA complexes retained the zeta potential of nearly −3 mV, indicating the PEG layer minimized nonspecific interactions of nanocomplexes with serum proteins. Furthermore, BSA was used as a model protein to test protein adsorption capacity of binary and ternary complexes due to albumin constitutes the largest fraction of proteins in blood plasma. The PEI-PTMC/pDNA binary complexes, PEI-PTMC/pDNA/PEG-DA ternary complexes, or the parental cationic polymer PEI/pDNA complexes were incubated with excess BSA. As shown in Figure 4B, the resistance against the BSA adsorption of PEI-PTMC/pDNA complexes was better than that of PEI/pDNA complexes, which might be due to the presence of the hydrophobic segment. After coating with PEG-DA, the BSA absorption of ternary complexes further reduced nearly 2-fold compared to that of PEI-PTMC/pDNA binary complexes, indicating that the PEI-PTMC/pDNA/PEG-DA ternary complexes became more stable than the PEI-PTMC/pDNA binary complexes. This phenomenon suggested that the stability of the positively charged polycation/DNA complexes against serum protein could be enhanced by coating a PEG-DA layer to its surface. So, the PEGylation of DNA-loaded complexes might be suitable for the gene delivery in vivo. 3.6. In Vitro Cytotoxicity. The cytotoxicity of PEI-PTMC/ pDNA binary complexes, PEI-PTMC/pDNA/PEG-DA ternary complexes, and PEI/pDNA complexes were evaluated by MTT assay. As shown in Figure 5, for PEI/pDNA complexes, the cell

Figure 5. In vitro cytotoxicity of 293T cells after treated with PEI/ pDNA, PEI-PTMC/pDNA binary complexes, and PEI-PTMC/ pDNA/PEG-DA ternary complexes.

viabilities dropped sharply as the complexes concentration increased. This is mainly because the strong interaction between the positively charged PEI and the negatively charged cell membrane led to the damage of the cell membrane and high toxicity in cell lines. The binary and ternary complexes exhibited much better biocompatibility compared to PEI. Especially, PEI-PTMC/pDNA/PEG-DA ternary complexes showed highest cell viability of over 90% even at high concentrations, indicating that PEGylated cationic copolymer/DNA complexes not only reduced the interaction with serum components but also minimized the cell cytotoxicity. G


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Figure 6. Fluorescence microscope images of 293T cells transfected with sheddable PEI-PTMC/pDNA/PEG-DA ternary complexes (N/P/C = 12:1:20) at (A) pH 7.4 and (B) pH 6.5; and (C) PEI/pDNA (N/P = 12:1) at pH 7.4 in vitro. (D) The expression of GFP in 293T cells quantified by flow cytometry.

Figure 7. In vitro intracellular distribution of PEI-PTMC/Texas Red-ODN binary complexes and PEI-PTMC/Texas Red-ODN/PEG-DA ternary complexes in 293T cells with PEI/Texas Red-ODN complexes as a positive control. Lyso/endosome was stained with Lysotracker Green (green).

hemolysis even the polycation concentration as low as to 2 μM, indicating the PEGylation could effectively improve the hemocompatibility of PEI-PTMC/DNA/PEG-DA complexes. The dispersion states of the RBCs when mixed with PEIPTMC/pDNA complexes, PEI-PTMC/pDNA/PEG-DA complexes, normal saline (negative control), or Gel (positive control50) were observed by confocal microscopy (Figure S8). The RBCs treated with PEI-PTMC/pDNA complexes, PEIPTMC/pDNA/PEG-DA complexes, and normal saline showed no obvious aggregation or morphological changes, but obvious

must be evaluated before investigating the in vivo distribution and transfection efficiency of DNA-loaded complexes. According to the ISO/TR 7405-1984 (f), the samples were considered hemolytic if the percent of hemolysis was >5% and the release of hemoglobin resulted in the supernatant appearing red. As shown in Figure 8A,B and Figure S7, no visible hemolytic effects were seen for ternary complexes even at the high concentration. However, the binary complexes induced hemolysis when the polycation concentration reached to 5 μM. In addition, the PEI/pDNA complexes cause more serious H


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Figure 8. Hemolysis assay of (A) PEI-PTMC/pDNA/PEG-DA ternary complexes and (B) PEI-PTMC/pDNA binary complexes, using water as a positive control and saline as a negative control; (C) in vivo distribution of (a) saline, (b) DiR, (c) DiR-loaded PEI-PTMC/pDNA binary complexes, (d) DiR-loaded PEI-PTMC/pDNA/PEG-COOH ternary complexes, and (e) DiR-loaded PEI-PTMC/pDNA/PEG-DA ternary complexes after intravenous injection 24 h to mice.

3.10. In Vivo Gene Transfection. Gene transfection in vivo was examined using tail-vein injection of ternary PEI-PTMC/ DNA/PEG-DA complexes at a dose of 30 mg of EGFP plasmid DNA. The naked DNA, PEI-PTMC/DNA binary complexes and PEI-PTMC/DNA/PEG-COOH complexes were also studied for comparison (Figure 9). The administration of naked plasmid and PEI-PTMC/pDNA binary complexes resulted in neglectable gene expression, but the PEGylated ternary complexes showed transfection ability in vivo. The tumor fluorescence intensity in PEI-PTMC/pDNA/PEG-DA complexes treated mice was notably higher than that in unsheddable PEI-PTMC/pDNA/PEG-COOH complexes treated group. As discussed previously, the detachment of PEG could significantly improve the tumor uptake and endosomal escape, facilitating the pDNA release and GFP expression in tumor. These promising results provide fundamental evidence for the use of PEI-PTMC/DNA/PEG-DA as a potential vector for the tumor-targeted gene delivery in the gene therapy.

aggregates were observed from the RBC treated with Gel. These results demonstrated that the PEI-PTMC/DNA/PEGDA ternary complexes had good blood compatibility. The in vivo distributions were then evaluated to investigate whether the DNA-loaded complexes have the preferential systemic genes delivery potency. The naked fluorescent dye DiR or DNA complexes loaded with DiR were intravenously administered into tumor-bearing nude mice, and fluorescence distribution in vivo were examined at 24 h postinjection. As shown in Figure 8C, strong fluorescence in naked DiR and DiR-loaded PEI-PTMC/pDNA binary complexes treated mice was observed in the abdomen that may be due to the high macrophage uptake nature of livers. Compare with the DiR and DiR-loaded PEI-PTMC/pDNA mediated in vivo distribution, the fluorescence of PEGylated nanocomplexes treated animals could accumulate in the tumor tissues, indicating that the presence of the hydrophilic PEG chain shielded the surface charge density of the complexes, extended the blood circulation and improved accumulation in the tumor tissue through enhanced permeability and retention (EPR) effect. However, large amounts of unsheddable PEI-PTMC/pDNA/PEGCOOH complexes mainly accumulated in the abdomen, which might be because the PEGylation hindered the uptake of the nanocomplexes by the tumor and the prolonged systemic circulation provided the particles an increased chance to exposure to other tissues and caused body immune system capture. The fluorescence in the PEI-PTMC/pDNA/PEG-DA complexes treated mice was primarily located in the tumor, indicating that the sheddable PEI-PTMC/DNA/PEG-DA has excellent targeting efficiency in tumors.

4. CONCLUSIONS In summary, on the basis of the polycation PEI, we developed a sheddable ternary nanocomplex system PEI-PTMC/DNA/ PEG-DA to achieve a highly effective and meanwhile safe gene delivery. NPs formed by amphiphilic PEI derivatives PEIPTMC could complex with DNA to obtain high gene transfection efficiency. A tumor acid-responsive β-carboxylic amide functionalized PEG was successfully decorated on the surface of the PEI-PTMC/DNA core. Excitingly, the PEGylation of polycation complexes possessed good serum stability and significantly reduced cytotoxicity compared with PEI and PEI-PTMC/DNA binary complexes. Moreover, the I



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ACKNOWLEDGMENTS This study was supported by National Natural Science Foundation of China (Grants 81272453 and 81472850), supported by the Key Research Program of the Chinese Academy of Sciences (Grant No. KGZD-EW-T06), and supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA09030301).

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Figure 9. (A) In vivo EGFP gene expression in tumor after intravenous injection 48 h to mice; (B) relative mean fluorescence intensity of EGFP gene expression at 48 h: (a) naked DNA group, (b) PEIPTMC/DNA binary complexes group, (c) represents PEI-PTMC/ DNA/PEG-COOH ternary complexes group, and (d) PEI-PTMC/ DNA/PEG-DA ternary complexes group.

ternary PEI-PTMC/DNA/PEG-DA complexes still had strong buffering capacity enough to escape from the endosome and kept high transfection efficiency at tumor extracellular pH. Namely, introduction of the β-carboxylic amide functionalized PEG shell to cationic complexes dramatically improved the biocompatibility and safety but did not sacrifice the high transfection efficiency. In addition, systemic administration of PEI-PTMC/DNA/PEG-DA complexes achieved simultaneous enhanced tumor targeting and gene transfection efficiency in tumor tissues. These excellent characteristics suggested that the PEI-PTMC/DNA/PEG-DA ternary carrier represented major progress for gene delivery in vivo.

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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/acsami.6b00825. Synthetic route of copolymer, 1HNMR and FT-IR spectra, TEM image, cell viability assay, zeta potential analysis, fluorescence microscope images, and blood compatibility experiments (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86 10 82545614. Fax: +86 10 62656765. E-mail: [email protected]. Author Contributions †

Caiyan Zhao and Leihou Shao contributed equally.

Notes

The authors declare no competing financial interest. J


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Tumor Acidity-Induced Sheddable Polyethylenimine-Poly(trimethylene

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