Real-Time Fluorescence Tracking of Gene Delivery via Multifunctional

Oct 21, 2014 - Real-Time Fluorescence Tracking of Gene Delivery via ... minimize clearance by the reticuloendothelial system after intravenous adminis...
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Real-Time Fluorescence Tracking of Gene Delivery via Multifunctional Nanocomposites Min Bai, Xilin Bai, and Leyu Wang* State Key Laboratory of Chemical Resource Engineering, Beijing Key Laboratory of Environmentally Harmful Chemical Analysis, Beijing University of Chemical Technology, Beijing 100029, P.R. China S Supporting Information *

ABSTRACT: Fluorescence imaging of transduced cells and tissues is valuable in the development of gene vectors and the evaluation of gene therapy efficacy. We report here the simple and rational design of multifunctional nanocomposites (NCs) for simultaneous gene delivery and fluorescence tracking based on ZnS:Mn2+ quantum dots (QDs) and positively charged polymer coating. The positively charged imidazole in the as-synthesized amphiphilic copolymer can be used for gene loading via electrostatic interaction. While the introduced poly(ethylene glycol) (PEG) can be used to reduce the binding of plasma proteins to nanovectors and minimize clearance by the reticuloendothelial system after intravenous administration. Most importantly, these multifunctional nanovectors showed much lower cellular toxicity than the commercial polyethylenimine (PEI) transfection vectors. On the basis of the red fluorescence of QDs, we can realtime track the gene delivery in cells, and the transfection efficacy of pDNA encoding enhanced green fluorescence protein (pEGFP) was monitored via the green fluorescence of the GFP expressed by the pDNA delivered into the nuclei. Fluorescence imaging analysis confirmed that the QDs-based nanovectors delivered pDNA into HepG2 cells efficiently. These new insights and capabilities pave a new way toward nanocomposite engineering for fluorescence imaging tracking of gene therapy.

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ver the past decades, gene detection1−4 and therapy have demonstrated attractive potentials5−7 and an enormous amount of research in the field of gene delivery has been conducted worldwide.8−10 Despite viral vectors offering greater transfection efficiency, nonviral vectors, which are typically based on cationic lipids or polymers, are preferred because of safety concerns with viral vectors. So far, significant efforts have been focused on the gene carrier design toward achieving safe, efficient, and specific delivery and high gene expression in target organs and tissues.8−13 It should be mentioned that it is very important not only to figure out how a vector enters a cell but also to follow its fate within the cell interior. Therefore, the real-time fluorescence imaging may be a useful tool and is highly desirable for tracking of gene delivery, especially for siRNAs and miRNAs that cannot express any fluorescence proteins but are essential for normal cellular processes.14,15 Recently, a series of multifunctional nanomaterials has been developed that can be used in biomedical and pharmaceutical fields for diagnosis and therapy.16−19 These nanomaterials not only can serve as carriers for the drug delivery to target tissues but also can act as tools for imaging.19,20 Although a lot of attention has been paid to the drug delivery with fluorescence imaging guidance,21−25 not much research includes the fluorescence tracking of gene delivery. For the sake of real-time tracking of the gene delivery and transfection, fluorescence tags including fluorescent proteins, organic dyes, and nanoparticles (NPs) can be covalently labeled to the gene. © 2014 American Chemical Society

Nonetheless, the involvement of these foreign tags via a covalent bond may result in low efficacy of gene transfection and therapy. Electrostatic interaction may be a good alternative strategy for gene loading, which may enable the good maintenance of the gene native properties. Gao and coworkers26 reported novel CdSe@proton-sponge multifunctional NPs for successful siRNA delivery with significantly reduced cytotoxicity and real-time fluorescence tracking of siRNA delivery in live cells. They also developed a new generation of nanoparticle carrier that allows efficient delivery and real-time imaging of siRNA in live cells. This innovative nanovector combined two distinct types of nanomaterials, semiconductor quantum dots and amphipols.27 By encapsulating the quantum dots (QDs) in acetone with poly(ethylene glycol) (PEG)−poly-ε-caprolactone (PCL)−polyethylenimine (PEI) triblock copolymers, a multifunctional gene vector was successfully developed for simultaneous gene delivery and fluorescence resonance energy transfer (FRET) tracking of organic dye bioconjugated siRNA,28 but the hydrophobic QDs have to be transferred from oil into acetone before the fabrication of nanovectors. Via ligand-exchange, water-dispersible CdSe/ZnSe QDs were conjugated with L-arginine and Received: July 17, 2014 Accepted: October 21, 2014 Published: October 21, 2014 11196

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used for siRNA delivery and fluorescence imaging.29,30 After bioconjugation with peptides, the amine-functionalized QDs were successfully applied for the fluorescence tracking and delivery of Cy-3-labeled RNA.31 The current fluorescence gene nanovectors are mainly based on the hydrophilic NPs. However, the surface coating of hydrophobic NPs is timeconsuming, and the ligand-exchange modification will further result in low fluorescence efficiency. Therefore, it is highly desirable to develop a facile and versatile one-pot strategy for the fabrication of composite nanovectors by encapsulating hydrophobic fluorescence NPs with amphiphilic and positively charged polymer for simultaneous gene delivery and fluorescence tracking. Herein, we developed the multifunctional nanocomposites (NCs) that can be used simultaneously for gene delivery and real-time fluorescence tracking of delivery efficiency and therapeutic effects. Moreover, these NCs can also be easily extended to coencapsulation of hydrophobic drugs for the drug/gene cocktail nanodrugs. As shown in Scheme 1, to fabricate the inorganic−organic multifunctional NCs, we first synthesized the amphiphilic and positively charged copolymer [poly−styrene−methacrylic acid−1-allyl-3-methylimidazolium chloride, P-St-MAA-AMIM] (Scheme 1B), and then, via a facile ultrasonication emulsion strategy (Scheme 1A),32 the mixture chloroform solution containing hydrophobic ZnS:Mn2+ QDs, P-St-MAA-AMIM, and polyethylene glycol−poly(lacticco-glycolic acid) (PEG−PLGA) was successfully transferred into the oil-in-water (O/W) micelle. The red fluorescent and positively charged spheric NCs (termed as NCs-PEG) with QDs embedded in the polymer matrixes were successfully fabricated after the evaporation of chloroform. To the best of our knowledge, the surface modification with poly(ethylene glycol), named PEGylation, is the predominant method used to reduce the binding of plasma proteins33,34 to nonviral vectors during the in vivo circulation, minimize clearance by the reticuloendothelial system,28 and thus prolong the circulation time28 and facilitate accumulation in targeted tissues via enhanced permeation and retention effects.35,36 Thus, the PEG−PLGA was also introduced into the nanocomposite fabrication. Moreover, the hydrophobic PLGA moieties are helpful for the encapsulation of hydrophobic QDs (and hydrophobic drugs), and then, it is easy to get the uniform composite nanospheres or nanocarriers for hydrophobic drugs. As a proof of concept, these NCs-PEG nanospheres were condensed with pDNA encoding enhanced green fluorescence protein (pEGFP) for the gene delivery via electrostatic interaction. As shown in Scheme 1A, real-time visualization of the gene delivery is possible through the red fluorescence of ZnS:Mn2+ QDs encapsulated in the NCs-PEG. Meanwhile, the transfection efficacy can be determined via the green fluorescence from the green fluorescence proteins (GFP) expressed by the pEGFP entered the nuclei. Most importantly, these multifunctional nanovectors showed much lower cellular toxicity than the commercial PEI-6000 transfection agents.

Scheme 1. (A) Schematic Illustration for the Fabrication of Composite NCs-PEG Gene Nanovectors, Intracellular Delivery of Plasmid DNA to the Nucleus, and Expression of Green Fluorescence Protein (GFP) in a Cancer Cell.a (B) Scheme for the Synthesis of the Amphiphilic and Positively Charged Functional Copolymer

a The red fluorescence of encapsulated ZnS:Mn2+ QDs can track the gene delivery, and the green fluorescence of GFP is capable of indicating the gene transfection efficacy. After the plasmid DNA/NCsPEG complexes were internalized by the cancer cell and escaped from the endosome, the intracellular release of plasmid DNA from the nanocomposite stimulated by the mildly acidic environments in cancer cells led to the enhanced gene transfection and strong green fluorescence.

ethanol, and methanol were supplied by Beijing Chemical Factory. 2-Amino-2-(hydroxymethyl)-1, 3-propanediol (Tris, (HOCH2)3CNH2), and ethidium bromide (EB) were obtained from Amresco. Agarose was obtained from Spain BIOWEST. PEG1000-PLGA5600 (50/50) was supplied by Jianan Daigang Biomaterial Co. Ltd. The plasmid DNA used in this work was pDNA encoding enhanced green fluorescence protein (pEGFP) and provided as a gift by professor Fujian Xu (College of Materials Science and Engineering, Beijing University of Chemical Technology). All the chemicals were of analytical grade and used as received without further purification. Ultrapure water was obtained with a Milli-Q filtration system. Characterization. The photoluminescence measurements were carried out on an F-4600 spectrophotometer (Hitachi)



EXPERIMENTAL SECTION Reagents and Chemicals. 1-Allyl-3-methylimidazolium chloride was purchased from Shanghai Flute Cypress Chemical Technology Co. Ltd. Methacrylic acid (MAA), styrene (St), and 2,2′-azobis(isobutyronitrile) (AIBN) were obtained from Tianjin Chemical Factory (China) and used as received. Oleic acid was purchased from Alfa Aesar. ZnCl2, MnCl2, NaOH, Na2S, Na2EDTA·2H2O, acetic acid, cyclohexane, chloroform, 11197

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equipped with a plotter unit and a quartz cell (1 cm × 1 cm). The shape and size of the NCs-PEG nanospheres and QDs were examined via the H-800 transmission electron microscope (TEM, JEOL) with a tungsten filament at an accelerating voltage of 100 kV. Dynamic light scattering (DLS) particle size analysis and zeta potentials of the pDNA/NCs-PEG complexes and QDs were measured using a Zetasizer Nano-ZS90 (Malvern) zeta and size analyzer, and the procedures are similar to those described earlier.37 Gene loading ability of NCs-PEG was examined on the agarose gel electrophoresis (DYY-6C, Beijing Liuyi Instrument Factory). The cytotoxicity tests were carried out by an ELISA plate reader (F50, TECAN). Confocal laser scanning microscopy (CLSM) observation was performed using TCS SP5 two-photon confocal microscopes (Leica) equipped with a Mai Tai NIR diode laser. Energy dispersive spectrum (EDS) analysis was conducted on a Veeco DI scanning electron microscope (SEM). FT-IR spectra were performed on Nexus 670 Fourier-transform infrared spectrophotometer (Nicolet, USA). The 1H and 13C spectra of functional copolymer were recorded on a Bruker DRX-600 spectrometer (Bruker, Ettlingen, Germany) at room temperature by using CDCl3 as the solvent. The gel permeation chromatography (GPC) analysis was performed on a Waters 2414 Refractive Index Detector and Waters 1515 Isocratic HPLC Pump, where THF was used as eluent and the commercial narrow-polydispersity polystyrene was used as calibration standard. Flow cytometry analysis was carried out on a BD FACSAria II Flow Cytometer. Synthesis and Characterization of Functional Copolymer. The functional copolymer (P-St-MAA-AMIM) used for gene loading was prepared as follows. In brief, styrene (5.1 mL), MAA (46 μL), 1-allyl-3-methylimidazolium chloride (AMIM, 0.368 mg), and AIBN (96.0 mg) were added into chloroform solution (35.0 mL). Herein, the styrene, MAA, and AMIM were used as monomers and AIBN was the initiator. The mixture solution was then transferred into a 45 mL Teflonlined autoclave and heated at 100 °C for 10 h. Then, 100 mL of methanol was added to precipitate the white products. The copolymer was purified by washing with chloroform (10.0 mL), precipitating with methanol (100 mL), and then centrifuging. This purification cycle was repeated at least twice. The molecular weight (Mw) of the as-prepared functional copolymer was about 3284 through GPC analysis (Figure S1, Supporting Information). The 1H NMR and 13C NMR spectra of the functional copolymer were shown in Figure S2, Supporting Information. The resonance peaks at δ 6.64−7.29 ppm and δ 125.33−128.00 ppm were ascribed to the protons and carbon of the benzene ring, respectively. The peaks at δ 0.95−0.99 and 1.31−1.49 ppm (1H NMR) and δ 26.61−27.90 and 44.24−45.92 ppm (13C NMR) were associated with the methacrylic acid backbone. δ 6.53−6.57, 2.6, and 1.91−2.12 ppm of 1H NMR and δ 32.12, 40.44, and 145.36 ppm of 13C NMR were assigned to AMIM group signals. These results indicated the successful synthesis of the desired functional polymer. Preparation of Nanocomposites (NCs-PEG).32 Before the NCs-PEG fabrication, ZnS:Mn2+ QDs were synthesized according to the reported methods.38,39 The as-prepared QDs (15.0 mg) with average size of 9.1 nm (Figure S3, Supporting Information), functional copolymer (50.0 mg), and PEG1000PLGA5600 (0.2 mg) were dispersed into chloroform to produce 1.0 mL of transparent solution. Thereafter, the mixture solution was injected into 10.0 mL of deionized water containing 0.06

mg of NaOH with ultrasonic treatment (300 W) and magnetic stirring for 6 min. The NCs-PEG was obtained via removing the chloroform by evaporating at 60 °C for 2 h. The obtained NCs-PEG product was centrifuged at 12 000 rpm for 15 min. Finally, the functional NCs-PEG was redispersed into DI water (6.0 mL) and stored for later use. The final concentration of the colloidal solution is ca. 16.8 mg/mL. As a control, the NCs without PEG were prepared using the same protocol in the absence of PEG−PLGA. It should be mentioned that the shape and size of the as-prepared QDs were characterized via transmission electron microscope (TEM) and dynamic light scattering (DLS) technology (Figure S3, Supporting Information). In addition, the coexistence of Zn, S, and Mn elements in the QDs was unambiguously confirmed via the results of energy dispersive spectroscopy (EDS) analysis (Figure S4, Supporting Information). DNA Loading Test. As a proof of concept, NCs-PEGs were examined for their ability to bind pDNA through agarose gel electrophoresis. In brief, NCs-PEGs were mixed with 1.0 μg of pDNA at various mass ratios of pDNA to NCs-PEG (1:0, 1:1, 1:5, 1:10, 1:15, 1:20, and 1:50). Each mixture was vortexed and incubated at 37 °C for 3 h. The pDNA/NCs-PEG complexes were loaded onto 1% agarose gel stained with 2.5 μL of ethidium bromide (10 mg/mL) per 50 mL of agarose solution by mixing with the loading buffer. Gel electrophoresis was carried out in 0.5× TAE buffer at 80 mV for 90 min. The gel was then analyzed on a gene genius bioimaging system to show the position of the pDNA/NCs-PEG complex relative to that of the naked pDNA. Cell Viability. The cytotoxicity of the nonviral nanocomposite vectors was evaluated using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay40−42 in the HepG2 (Hepatocellular carcinoma, human) cell line. Yellow MTT is reduced to purple formazan in the mitochondria of living cells. The absorbance of this colored solution can be quantified by measuring the absorbance at a certain wavelength (490 nm) by a spectrophotometer or a plate reader. This reduction takes place only when the mitochondrial reductases are active, and therefore, conversion can be directly related to the number of viable cells.43 The HepG2 cells were cultured in DMEM, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/ mL streptomycin at 37 °C, under 5% CO2 and a 95% relative humidity atmosphere. The cells were seeded in a 96-well microtiter plate, and then, different amounts (0−48.0 μg/mL) of NCs-PEG were added and cultured for 24 or 48 h, respectively. Thereafter, 10 μL of sterile-filtered MTT stock solution in PBS (4.0 mg/mL) was added to each well. The 96well microtiter plate was incubated at 37 °C for another 3 h, under 5% CO2 and a 95% relative humidity atmosphere. The absorbance of soluble formazan produced by cellular reduction of MTT in each well was measured at 490 nm using an ELISA plate reader (F50, TECAN). The increase in viable cell number results in the increase in the amount of MTT formazan and the increase in absorbance. Transfection Assay. The HepG2 cells were seeded in 6well plates in 1.0 mL of complete DMEM culture media per well (10% FBS) and incubated for 24 h under standard incubation conditions. Then, the original cell culture media were replaced with the pDNA/NCs-PEG complex (20.0 μg of NCs-PEG + 1.0 μg of pDNA) in 1.0 mL of serum-free DMEM culture media for each well. After incubation for 4 h, the transfection media were replaced with 2.0 mL of complete 11198

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media (10% FBS) and the cells were further incubated for 12− 48 h. After removing the medium, the cells were washed with PBS for 3 times followed by fixation with 10% formaldehyde solution for 15 min. Fluorescence imaging was performed using a 488 nm light to excite GFP and ZnS:Mn2+ QDs to get the green and red fluorescence, respectively. Flow Cytometry Analysis. The sample preparation was similar to the transfection assay. HepG2 cells were seeded in 6well plates at a density of 2 × 105 cells per well and incubated for 24 h. Then, the original cell culture media was replaced with the pDNA/NCs-PEG complex (80.0 μg of NCs-PEG + 4.0 μg of pDNA) in 1.0 mL of serum-free DMEM culture media for each well. After 4 h, the transfection media was replaced with 2.0 mL of complete media and the cells were further incubated for 12, 24, or 48 h. Then, the cells were trypsinized, washed with PBS, centrifuged, and resuspended in FACS buffer. FACS analysis was carried out on a BD FACSAria II Flow Cytometer with a fixed 488 nm laser. Data from 20 000 events/sample were gated using forward and side scatter parameters to exclude debris and dead cells. Cells without pDNA/NCs-PEG incubation were used as a negative control.



RESULTS AND DISCUSSION Transmission electron microscope (TEM) images (Figure 1a,b) and dynamic light scattering (DLS) (Figure 1c,d) results indicated that our composite NCs-PEG nanovectors are spheres with an average diameter of ∼120 nm. The obtained composite nanospheres were further characterized via Fourier transform infrared spectroscopy (FT-IR) and powder X-ray diffraction (XRD) technologies. The results of FT-IR spectra (Figure S5, Supporting Information) suggest the successful coating of the functional polymer. Meanwhile, the existence of QDs in the composite nanospheres was confirmed via the XRD pattern (Figure S6, Supporting Information). The good stability of pDNA/NCs-PEG complexes was also checked by dispersing the complexes in the DMEM culture media. As shown in Figure S7, Supporting Information, after incubation in DMEM media at 37 °C for 10 h, the shape and size of the nanosphere were well retained, which indicated that the pDNA/NCs-PEG complexes were stable under complex intracellular environments where the pH was 7.2. Due to their good stability, these complexes were applied for gene delivery. Before the gene loading, the surface of the NCs-PEG is pretty smooth (Figure 1a). After the formation of the pDNA/NCs-PEG complexes, the NCs-PEG become sticky (Figure 1b); however, the shape and size of the pDNA/NCsPEG complexes are almost consistent with that of NCs-PEG. Despite the sticky surface, the DLS particle size has no obvious change (Figure 1c,d), suggesting a good stability and no aggregation takes place. It should be mentioned that the encapsulated QDs can be observed as the black dots in the TEM image of a single nanosphere with high magnification (Figure 1e). Even though the QDs were encapsulated and protected by the hydrophobic moieties of the polymer, after the pDNA loading, the fluorescence was still quenched about 50% (Figure 1f), which can be attributed to the high frequency vibration of hydrophilic chemicals including H2O.41,44 However, the red emission of QDs is still naked-eye-visible and strong enough for fluorescence tracking. The pH influence on the DLS size (Figure S8, Supporting Information), fluorescence intensity (Figure S9, Supporting Information), and the zeta potential (Figure S10, Supporting Information) of the NCs-PEG was further investigated before

Figure 1. TEM images (a, b, and e), DLS size distribution (c and d), and fluorescence spectra (f) of the NCs-PEG before (a, c, and e) and after (b and d) incubation with pDNA (pDNA/NCs-PEG, w/w = 1:40) at 37 °C for 3 h, respectively. Image (e) shows a single nanocomposite with high magnification, and the QDs are clearly shown as black dots in the polymer matrixes.

the gene delivery applications. The DLS average diameter of NCs-PEG was almost unaffected by pH value in the range of pH 4−8 after incubation at 37 °C for 3 h (Figure S8, Supporting Information). The pH value in the range of pH 6−8 had no obvious influence on the fluorescence; however, the fluorescence was slightly quenched under acidic conditions (pH 4−5), which can be attributed to the decomposition of QDs under acidic conditions (Figure S9, Supporting Information). As expected, the NCs-PEG was positively charged at pH 4−7; however, it was negatively charged when the pH was tuned to 8.0, which was attributed to the increasing carboxyl hydrolysis of the functional copolymer (Figure S10, Supporting Information). On one hand, the tumor extracellular environment is relatively acidic (pH ≈ 6.5);45 on the other hand, the positively charged nanovectors are more suitable for the loading of negatively charged DNA, so we chose the weak acid conditions (pH ≈ 6.5) for the gene loading. To the best of our knowledge, the zeta-potential test is a reasonable technique for the rapid characterization of gene loading. Therefore, the zeta-potential of our nanovectors was conducted before and after the gene loading. As shown in Figure 2a, our NCs-PEG nanovectors are positively charged and the zeta potential is about 32 mV at pH 6.5. After the gene loading, the pDNA/NCs-PEG complexes are negatively 11199

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Figure 2. (a) Zeta potential of NCs-PEG (4.2 mg/mL) and pDNA/NCs-PEG complexes (4.2 mg/mL NCs-PEG, 1.0 mg/mL pDNA) at pH 6.5. (b) Agarose gel electrophoresis analysis for NCs-PEG mixed with 1.0 μg of pDNA at various mass ratios of pDNA to NCs-PEG (1:0, 1:1, 1:5, 1:10, 1:15, 1:20, and 1:50).

charged and the zeta potential is −41.6 mV, suggesting that the pDNA has been uploaded to the nanovectors via electrostatic interactions between the negative phosphates along the DNA backbone and the positive charges displayed on the nanovectors. These results indicate that our nanovectors are suitable for gene loading. To further check the gene loading ability of our nanovectors, the agarose gel electrophoresis analysis was also carried out (Figure 2b). In brief, NCs-PEG were mixed with 1.0 μg of pDNA at various mass ratios of pDNA to NCsPEG (1:0, 1:1, 1:5, 1:10, 1:15, 1:20, and 1:50) and incubated at 37 °C for 3 h before being loaded onto 1% agarose gel. As shown in Figure 2b, if no nanovectors are loaded, that means the ratio of pDNA to NCs-PEG is 1:0 and no pDNA is left in the sample spotting hole on the top of the gel. By increasing the dose of nanovectors step by step, more and more pDNA was trapped in the sample spotting hole. These results further confirmed that the pDNA was successfully combined with NCs-PEG. In other words, our composite nanovectors are applicable for gene loading. Besides the gene loading ability, the cytotoxicity is another major concern with gene nanocarriers. We studied the cytotoxicity of our NCs-PEG nanovectors using the MTT assay.40−42 As a control, the cytotoxicity of NCs without PEG− PLGA and commercial transfection agent polyethylenimine (PEI-6000) was also evaluated. From Figure 3a, with the increase of incubation time and nanovector concentration, the cell vialbility gradually decreases, but the cell survival rate remains basically above 91.4% and 70.5% at the nanovector concentration of 48.0 μg/mL for 24 and 48 h incubation, respectively. Nevertheless, the NCs without PEG demonstrate higher cytotoxicity, which further illustrates that the PEG− PLGA in the process of nanovector circulation plays a key role to stabilize the nanocarriers and decrease the cytotoxicity. It is known that the cancer cells usually have a negatively charged surface. Thus, the positively charged nanocarriers are not only suitable for gene loading but also beneficial for improving cellular uptake. On the other hand, the positively charged surface of the nanocarriers like CTAB-coated gold nanorods46 and polyethylenimine (PEI)47 vectors will result in cytotoxicity to cells. The toxicity is mainly associated with the strong electrostatic interaction of carriers with intracellular molecules and then cuases damage to cells.48−50 Polyethylenimine (PEI), a kind of polycation, has been introduced as nonviral gene carriers with a capability of forming stable complexes by electrostatic interactions with nucleic acids. The polyplex formation leads to improved protection of the genes from

Figure 3. (a) Cytotoxicity of NCs-PEG (square) and NCs without PEG−PLGA (triangle) at different concentrations (3, 6, 12, 24, 48 μg/ mL) in HepG2 cell lines after incubation for 24 h (black) and 48 h (blue), respectively. (b) Cytotoxicity induced by PEI-6000 vectors (10 μg/mL) and NCs-PEG (48.0 μg/mL) after incubation for 24 and 48 h, respectively.

enzyme-mediated digestion and enhanced intracellular delivery. As a control, we also carried out the cytotoxicity tests of PEI6000, a commercially available gene vector. As shown in Figure 3b, when the concentration of PEI-6000 is 10 μg/mL, only 29.1% cells are alive after incubation for 48 h, while the cell viability is over 70.5% for our NCs-PEG nanocomposites even with a much higher concentration of 48.0 μg/mL for 48 h incubation. Their good biocompatibility can be attributed to the PEG−PLGA that helps to block out some positive charge and then lower NCs adhesion to the cell surface. All the results indicate that our nanovectors are powerful and safe agents for gene delivery. For the high efficient gene delivery, in addition to good biocompatibility, high efficiency of cell surface association, and internalization, the safe and quick release of gene from the nanovector is another key issue before the translocation into the nucleus. Generally, once in the endosomal compartment, 11200

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the gene will be cleaved off and released when exposed to the mildly acidic environment such as pH 5.5. Therefore, we conducted the stability tests of our pDNA/NCs-PEG complexes by dispersing them in pH 5.5 Tris buffer solution. As shown in Figure 4, after incubation in Tris buffer (pH 5.5)

Figure 5. Confocal laser scanning microscopy (CLAM) fluorescence images of HepG2 cell lines incubated with pEGFP-condensed NCsPEG at the w/w ratio of 1:15 at 12, 24, and 48 h, respectively. Red: fluorescence of internalized ZnS:Mn2+; green: fluorescence of GFP expressed by pEGFP.

Figure 4. TEM image of pDNA/NCs-PEG complexes after incubation with Tris buffer (pH 5.5) for 24 h.

gene delivery and real-time fluorescence imaging tracking. By encapsulating the hydrophobic ZnS:Mn2+ QDs with PEG− PLGA and amphiphilic and positively charged copolymer, multifunctional nanovectors were easily constructed. With the introduction of PEG, these nanovectors are specifically designed to address longstanding barriers in gene delivery and good biocompatibility. The efficacy of our newly designed nanovectors for gene delivery was highlighted by the successful intracellular transport of plasmid DNA encoding enhanced green fluorescence protein (pEGFP). The red fluorescence of QDs enabled us to ascertain the location, distribution, and long-term viability of the gene. The transfection efficacy of pDNA was evaluated via the green fluorescence of green fluorescence proteins expressed by pEGFP. These results proved that our multifunctional nanoplatforms were versatile tools for diagnostic and therapeutic imaging purposes applicable for biologically active genes. Moreover, our nanovectors are especially suitable for simultaneous delivery of hydrophobic drugs (e.g., paclitaxel)/genes and fluorescent/ magnetic imaging. These new insights and capabilities represent a major step toward nanocomposite engineering for fluorescence imaging and therapeutic applications.

for 24 h, the complexes decomposed totally and no intact composite nanosphere was observed. We can come to the conclusion that the pDNA is released due to the decomposition of nanovectors when exposed to the mildly acidic environment of carcinoma cells. These results further indicate that our nanovectors are suitable for the gene delivery because of their good biocompatibility, novel gene loading ability, mild-acidinduced gene release, and fluorescence imaging tracking. In principle, it is possible to employ the fluorescent NCsPEG for live-cell imaging and monitoring of the intracellular gene transfection. In vitro gene transfection was performed by incubating pDNA/NCs-PEG complexes with HepG2 cells. The uptake and intracellular movement of nanovectors can be visualized via the red fluorescence of encapsulated QDs. Meanwhile, the transfection efficiency of the pDNA in the cell was monitored by the green fluorescence emitted from the green fluorescence protein expressed by the pEGFP. As shown in the fluorescence imaging (Figure 5), the nanovectors were taken up by the cell through endocytosis and distributed in the cytoplasm. No red fluorescence is observed in the nuclei, suggesting that no nanovectors enter the nuclei. In the course of gene transfection, by prolonging the incubation time from 12 to 48 h after gene transfection, green fluorescence was enhanced substantially, indicating that more and more green fluorescence proteins were expressed by pEGFP. These results were further verified by the flow cytometric analysis of the gradually enhanced green fluorescence of GFP in the transfected cells (Figure S11, Supporting Information). Meanwhile, along with the cell division and growth, both the red fluorescence QDs and green fluorescence proteins were divided into the daughter cells (Figure 5). The above observation indicated that pDNA/NCs-PEG was able to escape from the lysosome, have a safe release from the endosome, and demonstrate good transfection efficacy.



ASSOCIATED CONTENT

* Supporting Information S

The GPC analysis (Figure S1), NMR analysis (Figure S2), TEM and DLS results (Figure S3), EDS (Figure S4), FT-IR (Figure S5), XRD (Figure S6), stability tests in culture media (Figure S7), pH influence tests on DLS of the NCs-PEG (Figure S8), pH influence on the fluorescence intensity of the NCs-PEG (Figure S9), pH effects on the zeta potential of the NCs-PEG (Figure S10), and fluorescence flow cytometry analysis (Figure S11). This material is available free of charge via the Internet at http://pubs.acs.org.





CONCLUSION In summary, we proposed an effective and facile strategy for the fabrication of multifunctional nanocomposites for simultaneous

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 11201

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Notes

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The authors declare no competing financial interest.



ACKNOWLEDGMENTS This research was supported in part by the National Natural Science Foundation of China (21475007, 21275015), the State Key Project of Fundamental Research of China (2011CB932403), the Fundamental Research Funds for the Central Universities (ZZ1321, YS1406), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1205). We also thank the support from the “Public Hatching Platform for Recruited Talents of Beijing University of Chemical Technology”.



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dx.doi.org/10.1021/ac5026489 | Anal. Chem. 2014, 86, 11196−11202