Article pubs.acs.org/Biomac
A pH-Responsive Detachable PEG Shielding Strategy for Gene Delivery System in Cancer Therapy Xiuwen Guan,†,‡ Zhaopei Guo,† Tinghong Wang,§ Lin Lin,† Jie Chen,† Huayu Tian,*,† and Xuesi Chen*,† †
Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China ‡ University of Chinese Academy of Sciences, Beijing 100049, P. R. China § Changchun Chaoyang People’s Hospital, Changchun 130022, P. R. China S Supporting Information *
ABSTRACT: In this study, a pH-responsive detachable polyethylene glycol (PEG) shielding strategy was designed for gene delivery in cancer therapy. Polyethylenimine/DNA complex (PEI/ DNA) was in situ shielded by aldehyde group-modified PEG derivatives. The aldehyde groups of PEG could react with the amino groups of PEI by Schiff base reaction. The Schiff base bond was stable in neutral pH but labile in slightly acidic pH, which made the PEG sheddable in tumors. PEG-coated nanoparticles (NPs) had distinct advantages compared to their mPEG counterpart, possessing decreased zeta potential, more compressed size, and enhanced stability. PEG/PEI/DNA NPs showed not only high tumor cell uptake and transfection efficiency in vitro but also efficient accumulation and gene expression in solid tumors in vivo. This pH-responsive detachable PEG shielding system has the potential to be applied to other polycationic nanoparticles that contain amino groups on their surfaces, which will have broad prospects in cancer therapy.
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INTRODUCTION Cationic polymers are widely utilized as gene carriers to effectively load negatively charged genes through electrostatic interactions to accomplish successful gene delivery.1−4 As a typical polycationic carrier, polyethylenimine (PEI) is widely used for gene delivery due to its efficient DNA condensation, capable endosome escape, and excellent transfection efficiency.5 PEI with a molecular weight of 25 kDa (PEI25k) has been regarded as the “gold standard” for gene carriers.6,7 However, the positively charged PEI/gene complex may interact with negatively charged macromolecules or normal cell membranes during the transport process in bodily fluid, which is not only unfavorable for the PEI/gene to arrive in target tissues but also toxic to normal cells.8 Given all this, rational modification of PEI is indispensable for resolving these disadvantages. PEGylation is one of the most effective methods of biomaterial modification.9,10 It can decrease cytotoxicity and immunogenicity, improve stability, prevent aggregation, and assist long circulation.11,12 In early studies, PEGylation was achieved by irreversibly conjugating PEG on the polycations.13,14 Although some improvements had been realized, these studies also showed that the inseparable PEG might disturb cellular uptake and severely impact the transfection efficiency of polycations.15,16 To circumvent these unfavorable © 2017 American Chemical Society
impacts, reversible PEG modification was extensively designed, utilizing sensitive chemical bonds that could respond to physical factors (optical, thermal, acoustic, etc.) or chemical factors (pH, reductive, enzymatic, etc.).17−25 When the PEGmodified system was applied in vivo, some specific environments could trigger the detachment of PEG, which would benefit the delivery process. Therefore, this kind of strategy has been, and continues to be, extensively designed and applied in delivery systems. However, it has not always been easy to obtain truly sheddable PEGylation systems as expected. A series of parameters had to be considered to synthesize and screen the proper PEGylation to meet the requirements of various applications. The optimization process required repeated synthesis/screening cycles, which was complicated and timeconsuming. Therefore, a simple synthesis process and convenient PEGylation was highly desired to construct practical delivery systems to make the screening and optimizing process easy and timesaving; this kind of perspicuous and easily Received: January 17, 2017 Revised: March 6, 2017 Published: March 8, 2017 1342
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Figure 1. Schematic of the pH-responsive detachable PEG shielding system. PEI/DNA was in situ shielded by aldehyde-modified mPEG or PEG via rapid and efficient Schiff base reaction between aldehyde-modified PEGs and amino-containing PEI. The ultrasensitive pH-responsive Schiff base bond made the PEG-shielding stable during circulation but detachable as soon as the NPs arrived at the slightly acidic tumor areas. (mPEG2k and PEG2k), and calf thymus DNA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Luciferase plasmid DNA (pGL3control), cell lysate, and the luciferase reporter gene assay kit were purchased from Promega (Mannheim, Germany). Methyl thiazolyl tetrazolium (MTT) was purchased from Amresco (Solon, Ohio, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, USA). Cy5labeled DNA (Cy5-DNA) and the plasmid DNA, which expressed RFP, were purchased from Invitrogen (Carlsbad, CA, USA). The other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Synthesis of Aldehyde Group-Modified PEGs and Characterizations. The aldehyde group-modified mPEG (mPEG-CHO) and PEG (OHC-PEG-CHO) were prepared according to a simple onestep method. Briefly, mPEG2k (10 g) or PEG2k (5 g), 4carboxybenzaldehyde (1.125 g), EDC·HCl (4.793 g), and DMAP (0.122 g) were dissolved in 150 mL of dichloromethane (DCM) and stirred for 48 h at 25 °C. After the reaction was completed, the solution was concentrated by a rotary evaporator, and then, the mixture was washed five times with saturated NaCl solution and another three times with 5% NaCl solution. Subsequently, the organic layer was collected, and 50−100 g of anhydrous magnesium sulfate as dehydration agent was added and left to stand for ∼12 h. After filtration, the filtrate was concentrated and deposited twice with excess diethyl ether. The product was dried under a vacuum at room temperature overnight. Then, the products were dialyzed (MWCO = 1000 Da) with deionized water for 3 d. After lyophilization, the final products mPEG-CHO (78.7% yield) and OHC-PEG-CHO (75.2% yield) were obtained, and the products were characterized. The 1H NMR spectra were studied in D2O with a Bruker AV-300 NMR spectrometer. 13C NMR spectra were studied in D2O with a Bruker AV-500 NMR spectrometer. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was detected by a Bruker Autoflex III mass spectrometer. For the pH sensitivity of the Schiff base reaction to be further verified, mPEGCHO (or OHC-PEG-CHO) and PEI were dissolved in D2O and then mixed together (1:1 mass ratio in 0.5 mL of D2O). The solutions were adjusted to different pH values, and after 5 min, they were analyzed by 1 H NMR through a Bruker AV-300 NMR spectrometer. Preparation of the NPs. The mPEG/PEI/DNA and PEG/PEI/ DNA NPs (for simplicity, mPEG-CHO and OHC-PEG-CHO are abbreviated as mPEG and PEG) were prepared at different mass ratios. First, PEI, DNA, mPEG, and PEG were dissolved in ddH2O. PEI aqueous solution (0.25 mg/mL) was mixed with DNA aqueous solution (0.1 mg/mL) in an equal volume and vortexed for 15 s. After
operated strategy would also provide more possibilities to better cognize, balance, and employ PEGylation. In this study, a facile pH-responsive detachable PEG shielding strategy was designed for the gene delivery system in cancer therapy (Figure 1). A PEI/DNA complex was used as the model polycationic gene delivery system and was in situ shielded by the reaction of aldehyde-modified mPEG and PEG with amino groups from complex. The reaction could be accomplished rapidly and efficiently, and the generated Schiff base bond was stable in neutral or alkaline pH but labile in slightly acidic pH. The PEG shielded NPs were stable in normal tissue during the transport process. Once accumulated at a tumor area, the slightly acidic tumor extracellular pH could trigger the cleavage of the Schiff base bonds and the detachment of PEGs within a short time period; the PEI/ DNA was exposed and further contacted with tumor cell membranes, resulting in cellular uptake to accomplish the final gene expression. This pH-responsive detachable PEG shielding system was expected to possess the following distinguished merits. First, the aldehyde-modified PEGs were easy to synthesize. Second, the shielding process was organic solventfree, and the Schiff base reaction was fast and efficient. Third, PEGs could effectively shield the positive charges, reduce cytotoxicity, and prolong circulation time. The dialdehydeterminated PEG could also tighten the PEI/DNA complex to decrease size and increase stability. Lastly, for in situ PEG shielding on PEI/DNA, cellular uptake was expected to be low in a normal physiological environment (pH 7.4) but increase sharply after PEG deshielding in the slightly acidic tumor extracellular pH (pH 6.8). The zeta potential and particle size, stability, gene transfection, cytotoxicity, and cellular uptake of the delivery system were well characterized. Biodistribution was investigated after the NPs were intravenously injected into tumor-bearing mice, and a reporter gene was used to further explore the gene expression in solid tumors in vivo. This strategy was expected to have broad prospects for gene and drug delivery in cancer therapy.
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EXPERIMENTAL SECTION
Materials. Hyperbranched PEI with molecular weight of 25000 Da (PEI25k), mPEG and PEG with molecular weight of 2000 Da 1343
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respectively. Data were shown as mean ± SD based on triplicate independent experiments. Flow Cytometry Assay. HeLa cells were seeded in 6-well plates at a density of 2.0 × 105 cells/well, and the cells were cultured overnight. After that, the growth medium was replaced by 2 mL of fresh medium at pH 7.4 and 6.8, and then, PEG/PEI/DNA NPs (10:2.5:1 mass ratio) were prepared and added to each well (Cy5-DNA was used to prepare the NPs, and the final DNA concentration in the well was 1 μg/mL). After a 2 h incubation, the cells were collected and washed twice with cold PBS. The cells were analyzed by Guava EasyCyte flow cytometer (Guava Technologies). Confocal Laser Scanning Microscopy. HeLa cells were seeded on coverslips in 6-well plates at a density of 1.0 × 105 cells/well and cultured overnight. After that, the growth medium were replaced by 2 mL of fresh medium at pH 7.4 and 6.8, and then, PEG/PEI/DNA NPs (10:2.5:1 mass ratio) were prepared and added to each well (Cy5DNA was used to prepare the NPs, and the final DNA concentration in the well was 1 μg/mL). After a 2 h incubation, the cells were washed with PBS three times and fixed with 3.7% paraformaldehyde for 15 min at room temperature. After immobilization, the cells were washed three times with PBS, and 1 μL of DAPI (1 mg/mL) was added to each well to stain the cell nuclei for 15 min. After that, the cells were washed five times with PBS, and the coverslips were carefully taken out and placed on the slides enclosed with glycerol. The samples were observed by confocal laser scanning microscopy (CLSM) (ZEISS LSM 780, Germany). Biodistribution. BALB/C nude mice (female, 5−6 weeks) were bought from Beijing Huafukang Biological Technology Co. Ltd. (HFK Bioscience, Beijing). All experimental procedures were in accordance with the guidelines for laboratory animals established by the Animal Care and Use Committee of Northeast Normal University. The biodistribution of the PEG/PEI/DNA NPs was evaluated by ex vivo imaging experiments in tumor-bearing mice. Cy5-DNA was used for NP preparation and in vivo tracking. A subcutaneous xenograft tumor model was generated by injecting HeLa cells (2 × 106 cells/100 μL) into the left flank of the nude mice. After 3−4 weeks, the solid tumors formed. Then, the mice were injected with 0.2 mL of PEG/PEI/DNA NPs (1 mg/kg body weight on a Cy5-DNA basis) via tail vein. After 24 h, the mice were anaesthetized and sacrificed, and the major organs (heart, liver, spleen, lung, and kidney) and tumors were excised and imaged by a Maestro In Vivo Imaging System (Cambridge Research & Instrumentation, Inc., USA). Cy5-DNA was excited by a yellow excitation filter; the fluorescence was detected through an emission filter (645 nm), and the exposure time was 2000 ms. The image data was analyzed by commercial software (Maestro 2.4), and the total or average signals were quantitatively analyzed. In Vivo Gene Expression. The in vivo gene expression was carried out by detecting the fluorescence of RFP. The pDNA that expressed RFP was loaded in the PEG/PEI/DNA system as the reporter gene. The mice were injected with 0.2 mL of PEG/PEI/DNA NPs (1 mg/kg body weight on an RFP pDNA basis) via tail vein. After 48 h, the mice were anaesthetized and sacrificed, and the major organs (heart, liver, spleen, lung and kidney) and tumors were excised and imaged by a Maestro In Vivo Imaging System (Cambridge Research & Instrumentation, Inc., USA). RFP was excited by a yellow excitation filter; fluorescence was detected through an emission filter (625 nm), and the exposure time was 2000 ms. The image data was analyzed by commercial software (Maestro 2.4), and the total or average signals were quantitatively analyzed. Statistical Analysis. All measurements were conducted in triplicate and expressed as mean ± standard deviation. Student’s t test was performed to compare the statistical significance. *p < 0.05 was considered statistically significant. **p < 0.01 and ***p < 0.001 were considered extremely significant.
20 min of incubation at room temperature, the PEI/DNA complexes (2.5:1 mass ratio) were formed. Then, different concentrations of mPEG or PEG aqueous solution were added to the PEI/DNA complexes, and the pH of the solutions were adjusted to 7.4. After 5 min incubation at room temperature, the mPEG/PEI/DNA or PEG/ PEI/DNA NPs with various mass ratios of (0, 2.5, 5, 7.5, 10, 15, 20):2.5:1 were obtained. Zeta Potential and Particle Size. The zeta potential and particle size of the freshly prepared mPEG/PEI/DNA and PEG/PEI/DNA NPs ((0, 2.5, 5, 7.5, 10, 15, 20):2.5:1 mass ratio; the final concentration of DNA was 0.025 mg/mL) were measured by zeta potential/BI-90Plus particle size analyzer (Brookhaven, USA) at room temperature (n = 5). Data were shown as mean ± standard deviation (SD) based on triplicate independent experiments. Stability Studies. PEI/DNA and PEG/PEI/DNA NPs were prepared according to the above-mentioned method at mass ratios of 2.5:1 and 10:2.5:1, respectively. Then, the NPs were incubated in PBS (pH 7.4) at 37 °C under gentle stirring (the final concentration of DNA was 0.025 mg/mL). At different time points, the mean diameters of the NPs were measured by the zeta potential/BI-90Plus particle size analyzer. For the antiaggregation ability of the NPs under protein conditions to be further investigated, the size of the NPs was measured under different amounts of BSA solution (0, 0.1, 0.3, and 0.5%). Gel Retardation Assay. The DNA binding ability of the PEG/ PEI/DNA NPs was tested by gel retardation assay. Agarose gel (1% w/v) was used for electrophoresis. The PEG/PEI/DNA NPs with different mass ratios were prepared and mixed with loading buffer. Electrophoresis (DY-4C, Liuyi, Beijing, China) proceeded in TAE running buffer at 85−100 V for 40 min. The gel was visualized by UV gel imaging system (UVP EC3, UVP Inc., Upland, USA) to evaluate the DNA binding ability of the PEG/PEI/DNA NPs. In Vitro DNA Transfection. The DNA transfection experiment of the NPs was carried out in HeLa cells. The luciferase plasmid DNA (pGL3-control) was used as the reporter gene. HeLa cells were seeded in a 96-well plate at a density of 8000 cells/well and then cultured at 37 °C in 5% CO2 overnight. Before transfection, the culture medium (DMEM) was replaced with 180 μL/well of fresh DMEM with different pH values (the fresh DMEM was adjusted to pH 7.4 and 6.8 by sterile dilute HCl aqueous solution before the experiment). The PEG/PEI/DNA NPs with various mass ratios were prepared and added to each well ((0, 2.5, 5, 7.5, 10, 15, 20):2.5:1 mass ratio; the final DNA concentration in the well was 1 μg/mL). The cells were incubated with the NPs for 2 h. Then, the culture medium was replaced with 200 μL/well of fresh DMEM, and the cells were cultured in an incubator for 48 h. Then, the cells were lysed with 50 μL of cell lysate and frozen at −80 °C. After thawing, 20 μL of supernatant of the cell lysate was added to 100 μL of luciferase substrate. The relative light units (RLU) were tested by luminometer (Turner Biosystems and Promega) and normalized to total protein content (BCA protein assay kit, Sigma). Luciferase activity was expressed as RLU/mg protein. Cytotoxicity Assay. The cytotoxicity of the PEG/PEI/DNA NPs was assessed with methyl thiazolyl tetrazolium (MTT) assay. HeLa cells were seeded in 96-well plates at 1.0 × 104 cells/well and cultured at 37 °C in 5% CO2. Before the experiment, the culture medium (DMEM) was replaced with 180 μL/well of fresh DMEM with different pH values. PEG/PEI/DNA NPs with different mass ratios were prepared and added to each well ((0, 2.5, 5, 7.5, 10, 15, 20):2.5:1 mass ratio; the final DNA concentration in the well was 1 μg/mL). The cells were incubated with the NPs for 2 h. Then, the culture medium was replaced with 200 μL/well of fresh DMEM, and the cells were cultured in an incubator for 48 h. The cytotoxicity of the OHCPEG-CHO was also studied, and the final concentrations of OHCPEG-CHO were 3.125−1000 μg/mL. After 48 h, 20 μL of MTT (5 mg/mL) was added to each well and incubated for 4 h; then, the MTT solution was carefully removed, and 200 μL of DMSO was added to dissolve the MTT formazan crystals. The absorbency was measured on a Bio-Rad 680 microplate reader at 492 nm. The cell viability (%) was calculated as cell viability (%) = (Asample/Acontrol) × 100%, where Asample and Acontrol mean the absorbencies of the sample and control wells,
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RESULTS AND DISCUSSION Synthesis of Aldehyde-Modified PEGs and Characterizations. Aldehyde group-modified mPEG or PEG was synthesized through a simple one-step method as shown in 1344
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Figure 2. (A) Synthesis route of aldehyde group-modified mPEG and PEG and their further reaction with PEI. (B) 1H NMR spectra of aldehyde group-modified mPEG and PEG. (C, D) 1H NMR spectra of reactions between aldehyde group-modified mPEG (C) or PEG (D) with PEI at pH 7.4 and 6.8. The peaks of the aldehyde groups are marked by a dotted box to show the pH sensitivity of the Schiff base bonds.
Figure 2A. The 1H NMR spectra of the modified mPEG and PEG are shown in Figure 2B; the characteristic peak of the aldehyde groups was at ∼10 ppm. The signal assignment for 13 C NMR is presented in Figure S1. The MALDI-TOF-MS measurement of the PEG molecular weight is shown in Figure S2. These results demonstrate that the aldehyde groupmodified PEGs were successfully prepared. When PEGs were mixed with PEI solution at pH 7.4, the aldehyde peak fully disappeared, but when the solutions were adjusted to pH 6.8, the aldehyde peak appeared again (Figure 2C, D). These results verified that the aldehyde groups of PEGs could react with the amino groups of PEI by Schiff base reaction in neutral pH, which could be applied to realize PEG shielding on the surface of PEI/DNA complexes. Moreover, the Schiff base bond was
cleavable in slightly acidic pH, which was conducive to PEG detachment in the tumor extracellular area (pH ∼6.8). Zeta Potential and Particle Size. The zeta potential and particle size of the mPEG/PEI/DNA and PEG/PEI/DNA NPs with different mass ratios are shown in Figure 3. The zeta potential gradually decreased with increasing PEG mass ratio (Figure 3A, C), which demonstrates that both mPEG and PEG could effectively shield the surplus positive charges of PEI/ DNA complexes. It was interesting to find that mPEG and PEG had distinct effects on PEI/DNA particle size regulation. For mPEG/PEI/DNA (Figure 3B), the particle size markedly increased at a mass ratio of 2.5:2.5:1, the reason for this might be due to the fact that one terminal of the hydrophilic mPEG tended to stretch in the water, the force of which drove PEI/ 1345
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Figure 3. Zeta potential and particle size of mPEG/PEI/DNA NPs (A, B) and PEG/PEI/DNA NPs (C, D) with different mass ratios; the values of the x-axis represent the mass ratios of mPEG or PEG to PEI/DNA.
Figure 4. (A) The particle sizes of PEI/DNA and PEG/PEI/DNA NPs incubated in PBS (pH 7.4) at 37 °C under gentle stirring. (B) Particle sizes of PEI/DNA and PEG/PEI/DNA NPs in different concentrations of BSA solutions.
them to attach on the PEI/DNA surface with only one terminal as the other terminal was stretched in water, which drove PEI/ DNA expansion. Even so, PEG/PEI/DNA assuredly showed much smaller size and a lower polydispersity index (PDI; Table S1) than those of mPEG/PEI/DNA. The smaller size was more favorable for long-circulation behaviors in physiological environments.26,27 Considering this, we chose PEG/PEI/ DNA to further explore the practicability of this strategy. As PEG/PEI/DNA with a mass ratio of 10:2.5:1 showed the smallest size while retaining an acceptable zeta potential, this ratio was selected for further studies. Stability Studies. For the effects of PEG shielding/ compression on the stability of NPs to be explored, PEI/ DNA and PEG/PEI/DNA NPs were incubated in PBS (pH 7.4) at 37 °C under gentle stirring for 96 h (Figure 4A). The size of the PEI/DNA gradually increased over time during the first 12 h and severely increased after 24 h incubation in PBS. However, the size of PEG/PEI/DNA NPs was almost unaltered over the 96 h test, which verifies the superior stability of PEGshielded NPs. For the antiaggregation ability of the NPs in protein-containing solution to be investigated further, the size
DNA expansion, leading to increased particle size. Further adding mPEG decreased the particle sizes; the hydrophobicity and π−π stacking interaction of the benzene rings of mPEG could condense the PEI/DNA complexes. The particle size tended to grow slightly when further increasing the mPEG mass ratio to 15 and 20. As excess aldehyde mPEG might occupy the amino of PEI, which might in turn decreases the electrostatic force between PEI and DNA, the PEI/DNA slightly swells. For PEG/PEI/DNA (Figure 3D), it was exciting to find that the particle size was greatly reduced after the PEI/ DNA complexes were shielded by PEG, the dialdehydeterminated PEG could effectively compress the PEI/DNA complexes. With the PEG mass ratio increased, the hydrophobicity and π−π stacking interaction of benzene rings of PEG also made the NPs smaller. The NPs at a mass ratio of 10:2.5:1 showed the most significant size compression. The particle size increased upon adding more PEG. Excess aldehyde PEG would consume the amino of PEI, which might decrease the binding force between PEI and DNA; thus, the PEI/DNA complexes became loose. Furthermore, there was competition between the excess PEG molecules, which might cause some of 1346
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Biomacromolecules of the NPs was measured under different concentrations of BSA solutions (Figure 4B). PEI/DNA complexes were severely affected by the BSA proteins; the size varied sharply in different BSA concentrations. In 0.1% BSA, the BSA molecules were absorbed on the surfaces of positively charged PEI/DNA complexes to form a protein corona, which increased the size to 900 nm. Upon further increasing the BSA concentration, the particle size became smaller. Size decreasing phenomena for complexes in high BSA concentration were also reported in another study, where it was reported that high BSA concentration might condense the complexes to some extent.28 Encouragingly, the size of PEG/PEI/DNA NPs remained almost unchanged; the NPs remained uncorrupted in the BSA solutions. The enhanced stability and antiaggregation ability of PEG/PEI/DNA NPs were attributed to the neutral and hydrophilic PEG shielding, which had created a hydrophilic corona on the PEI/DNA complexes to shield the surface positive charges and sterically impede nonspecific protein binding.29 Gel Retardation Assay. The DNA binding ability of the PEG/PEI/DNA NPs was verified by gel retardation assay. Different mass ratios of PEG/PEI/DNA NPs were prepared and tested (0, 2.5, 5, 7.5, 10, 15, and 20:2.5:1). Considering the aldehyde groups of PEG would react with PEI, the positively charged groups of PEI would be partly consumed. Therefore, it was important to certify the DNA binding ability of PEG/PEI/ DNA NPs. The results are shown in Figure 5; bright bands
Figure 6. Transfection efficiency of the in situ shielding PEG/PEI/ DNA NPs at different pH values (pH 7.4 and 6.8); the values of the xaxis represent the mass ratios of PEG to PEI/DNA.
90% even when the concentration was up to 1000 μg/mL, which showed a low cytotoxicity. The cell viability of PEG/ PEI/DNA NPs is shown in Figure 7B. At pH 7.4, the cell viability of PEI/DNA complexes was 83.2%, when they were further shielded by PEG, the cytotoxicity was notably alleviated. The result verified that PEG could effectively shield the positive charges and decrease the cytotoxicity of PEI/DNA. When the pH decreased to 6.8, PEG was detached from the NPs, and the positively charged PEI/DNA was exposed; thus, the cell viability was lower than that at pH 7.4. Flow Cytometry Assay. A cellular uptake study was carried out by flow cytometry, and the results are shown in Figure 8. PEI/DNA had a slightly higher uptake at pH 6.8 than at 7.4, which is consistent with the transfection results. For PEG/PEI/ DNA NPs, PEG shielding induced much lower fluorescence intensity at pH 7.4 than 6.8, which reflected that PEG effectively shielded the positive charges of the NPs and prevented the interaction between the cells and the PEGshielded NPs. Whereas at pH 6.8, the cellular uptake was markedly increased; as the PEG layer was detached away from the NPs, the exposed positively charged PEI/DNA complexes interacted with the negatively charged cell membranes and resulted in further internalization. Confocal Laser Scanning Microscopy. The cellular internalization of the NPs was monitored by CLSM (Figure 9). The cell nucleus was stained in blue by DAPI, and red fluorescence was from Cy5-DNA. PEI/DNA could enter into the cells, and little difference was found between pH 7.4 and 6.8. However, it was remarkable that less PEG/PEI/DNA NPs were found in the tumor cells at pH 7.4, but internalized NPs were markedly increased at pH 6.8. This result was consistent with the cell uptake study, which was tested by flow cytometry and further confirmed that a slightly acidic environment could remove the PEG shielding from the NPs. Biodistribution. The biodistributions of the PEI/DNA and PEG/PEI/DNA NPs were evaluated by ex vivo imaging on a subcutaneous tumor model. Cy5-DNA was used for NP preparation and tracking. The tumor-bearing mice were injected with the NPs via tail vein. After 24 h, the major organs (heart, liver, spleen, lung, and kidney) and tumors were excised and imaged; the results are shown in Figure 10 and Figure S3. PEG/PEI/DNA NPs showed more effective accumulation in tumor tissue than that of PEI/DNA. This result was mainly attributed to the following two reasons: First, PEG effectively shielded the positive charges of PEI/DNA, which was helpful for enhanced stability and long circulation.
Figure 5. DNA gel retardation assay of free DNA and PEG/PEI/DNA NPs with various mass ratios (0, 2.5, 5, 7.5, 10, 15, and 20:2.5:1).
were found for free DNA, but there was no free DNA observed in the lanes of PEG/PEI/DNA NPs. The results meant that the PEG/PEI/DNA NPs could tightly retard the DNA in sampleadding holes under these tested mass ratios. In Vitro DNA Transfection. For the effect of PEG shielding to be investigated, the DNA transfection of PEG/ PEI/DNA NPs was tested at different pH levels. After in situ PEG shielding (Figure 6), the transfection decreased; more PEG led to lower transfection efficiency at pH 7.4. However, the transfection effciency markedly increased at pH 6.8. Furthermore, significant transfection effciency differences were observed for PEG/PEI/DNA NPs between pH 7.4 and 6.8. When PEG/PEI/DNA NPs were in the slightly acidic pH (such as tumor area), the PEG layer would be removed, and the exposure of positively charged PEI/DNA would lead to much higher transfection. Cytotoxicity Assay. The cytotoxicities of the aldehydemodified PEG and PEG/PEI/DNA NPs were also tested in HeLa cells. In Figure 7A, the cytotoxicity of OHC-PEG-CHO was concentration-dependent, and the cell viability was above 1347
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Figure 7. Cytotoxicities of (A) OHC-PEG-CHO at different concentrations and (B) PEG/PEI/DNA NPs with various mass ratios at different pH values (pH 7.4 and 6.8).
Figure 10. Biodistribution of PEI/DNA (P/D) and PEG/PEI/DNA (P/P/D) in tumors and heart, liver, spleen, lung, and kidney. The NPs were injected into the mice via tail vein.
Figure 8. Cellular uptake of PEI/DNA and PEG/PEI/DNA NPs (10:2.5:1 mass ratio) at pH 7.4 and 6.8.
Figure 11. In vivo gene expression of PEI/DNA (P/D) and PEG/ PEI/DNA (P/P/D) in tumors and heart, liver, spleen, lung, and kidney. The NPs were injected into the mice via tail vein.
and kidney) and tumors were excised and imaged, the results of which are shown in Figure 11. The fluorescence of RFP could be observed vividly in the PEG/PEI/DNA group, which meant that the pDNA had been successfully delivered into the tumors and also had realized gene expression. The much stronger fluorescence at the tumor site for PEG/PEI/DNA than for PEI/DNA (Figure S4) confirmed that PEG/PEI/DNA was more effective than PEI/DNA for delivering the genes to the tumor site.
Figure 9. CLSM images of HeLa cells incubated with PEI/DNA and PEG/PEI/DNA NPs (10:2.5:1 mass ratio) at pH 7.4 and 6.8.
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Second, the PEG detachment in slightly acidic tumors caused exposure of positively charged PEI/DNA to tumor cells, which was good for high cellular uptake, leading to efficient accumulation in tumors. In Vivo Gene Expression. In vivo gene expression was carried out by detecting the fluorescence of RFP. The pDNA expressing RFP was loaded in the PEI/DNA and PEG/PEI/ DNA NPs and further injected into the tumor-bearing mice via tail vein. After 24 h, the major organs (heart, liver, spleen, lung,
CONCLUSIONS In this study, a facile pH-responsive detachable PEG shielding strategy was successfully developed for gene delivery system in cancer therapy. Aldehyde-modified mPEG and PEG were shielded on PEI/DNA complexes through pH-responsive Schiff base bonds, which made the PEGs sheddable in slightly acidic tumor areas. Our research showed that PEG/PEI/DNA NPs 1348
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Article
Biomacromolecules
(10) Harris, J. M.; Martin, N. E.; Modi, M. Clin. Pharmacokinet. 2001, 40, 539−551. (11) Dufort, S.; Sancey, L.; Coll, J.-L. Adv. Drug Delivery Rev. 2012, 64, 179−189. (12) Veronese, F. M.; Mero, A. BioDrugs 2008, 22, 315−329. (13) Petersen, H.; Fechner, P. M.; Fischer, D.; Kissel, T. Macromolecules 2002, 35, 6867−6874. (14) Petersen, H.; Fechner, P. M.; Martin, A. L.; Kunath, K.; Stolnik, S.; Roberts, C. J.; Fischer, D.; Davies, M. C.; Kissel, T. Bioconjugate Chem. 2002, 13, 845−854. (15) Mishra, S.; Webster, P.; Davis, M. E. Eur. J. Cell Biol. 2004, 83, 97−111. (16) Hatakeyama, H.; Akita, H.; Harashima, H. Adv. Drug Delivery Rev. 2011, 63, 152−160. (17) Romberg, B.; Hennink, W. E.; Storm, G. Pharm. Res. 2008, 25, 55−71. (18) Sun, H.; Guo, B.; Cheng, R.; Meng, F.; Liu, H.; Zhong, Z. Biomaterials 2009, 30, 6358−6366. (19) Wen, H.; Dong, C.; Dong, H.; Shen, A.; Xia, W.; Cai, X.; Song, Y.; Li, X.; Li, Y.; Shi, D. Small 2012, 8, 760−769. (20) Li, J.; Ge, Z.; Liu, S. Chem. Commun. 2013, 49, 6974−6976. (21) Meers, P. Adv. Drug Delivery Rev. 2001, 53, 265−272. (22) Walker, G. F.; Fella, C.; Pelisek, J.; Fahrmeir, J.; Boeckle, S.; Ogris, M.; Wagner, E. Mol. Ther. 2005, 11, 418−425. (23) Knorr, V.; Allmendinger, L.; Walker, G. F.; Paintner, F. F.; Wagner, E. Bioconjugate Chem. 2007, 18, 1218−1225. (24) Nie, Y.; Günther, M.; Gu, Z.; Wagner, E. Biomaterials 2011, 32, 858−869. (25) Xu, S.; Luo, Y.; Haag, R. Macromol. Biosci. 2007, 7, 968−974. (26) Duan, X.; Li, Y. Small 2013, 9, 1521−1532. (27) Liang, S.; Yang, X.; Du, X.; Wang, H.; Li, H.; Liu, W.; Yao, Y.; Zhu, Y.; Ma, Y.; Wang, J. Adv. Funct. Mater. 2015, 25, 4778−4787. (28) Zhou, D.; Li, C.; Hu, Y.; Zhou, H.; Chen, J.; Zhang, Z.; Guo, T. J. Mater. Chem. 2012, 22, 10743−10751. (29) He, H.; Bai, Y.; Wang, J.; Deng, Q.; Zhu, L.; Meng, F.; Zhong, Z.; Yin, L. Biomacromolecules 2015, 16, 1390−1400.
had a prominent advantage in size compression compared with that of its mPEG counterpart due to the dual function of shielding and compression. The results showed that PEG/PEI/ DNA had reduced zeta potential and particle size, enhanced stability, and lower cytotoxicity after PEG shielding, which was beneficial for further in vivo applications. Furthermore, PEG detachment could be triggered by the slightly acidic tumor extracellular pH, leading to high cellular uptake and efficient gene transfection. The system could accumulate at tumor sites and assist in accomplishing effective gene expression in vivo. This pH-responsive, detachable PEG shielding system was a convenient and effective strategy for carrier PEGylation. The aldehyde-modified PEG was also suitable for shielding other kinds of nanoparticles that contained amino groups on surfaces. This strategy could have extensive application prospects in cancer therapy.
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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.biomac.7b00080. 13 C NMR spectra and MALDI-TOF-MS of the materials, PDI data for NP sizes, and quantitative analysis of the total or average signals for biodistribution and in vivo gene expression (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Huayu Tian: 0000-0002-2482-3744 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors are thankful to the National Natural Science Foundation of China (21474104, 51233004, 51403205, 51390484, and 51520105004), National Program for Support of Top-Notch Young Professionals, and Jilin Province Science and Technology Development Program (20160204032GX) for financial support of this work.
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REFERENCES
(1) De Smedt, S. C.; Demeester, J.; Hennink, W. E. Pharm. Res. 2000, 17, 113−126. (2) Tian, H.; Chen, J.; Chen, X. Small 2013, 9, 2034−2044. (3) Liu, X.; Rocchi, P.; Peng, L. New J. Chem. 2012, 36, 256−263. (4) Barnard, A.; Posocco, P.; Pricl, S.; Calderon, M.; Haag, R.; Hwang, M. E.; Shum, V. W.; Pack, D. W.; Smith, D. K. J. Am. Chem. Soc. 2011, 133, 20288−20300. (5) Tian, H.; Tang, Z.; Zhuang, X.; Chen, X.; Jing, X. Prog. Polym. Sci. 2012, 37, 237−280. (6) Boussif, O.; Lezoualc’h, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J.-P. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 7297−7301. (7) Akinc, A.; Thomas, M.; Klibanov, A. M.; Langer, R. J. Gene Med. 2005, 7, 657−663. (8) Guan, X.; Li, Y.; Jiao, Z.; Lin, L.; Chen, J.; Guo, Z.; Tian, H.; Chen, X. ACS Appl. Mater. Interfaces 2015, 7, 3207−3215. (9) Suk, J. S.; Xu, Q.; Kim, N.; Hanes, J.; Ensign, L. M. Adv. Drug Delivery Rev. 2016, 99, 28−51. 1349
DOI: 10.1021/acs.biomac.7b00080 Biomacromolecules 2017, 18, 1342−1349
