Fluorine-Doped Cationic Carbon Dots for Efficient Gene Delivery

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Fluorine-doped Cationic Carbon Dots for Efficient Gene Delivery Gancheng Zuo, Aming Xie, Xihao Pan, Ting Su, Junjian Li, and Wei Dong ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00521 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on May 1, 2018

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ACS Applied Nano Materials

Fluorine-doped Cationic Carbon Dots for Efficient Gene Delivery Gancheng Zuo†, Aming Xie*, §, Xihao Pan†, Ting Su†, Junjian Li†, Wei Dong*, † †

School of Chemical Engineering, Nanjing University of Science & Technology,

Nanjing 210094, P. R. China §

School of Mechanical Engineering, Nanjing University of Science & Technology,

Nanjing 210094, P. R. China ABSTRACT: Carbon dots (CDs) focus great attention in a broad range of adhibitions due to its excellent optical property, high biocompatibility and property adjustability. However, the developed CDs can be used rarely as effective gene vectors up to now. In this work, we devised and synthesized a novel fluorine-doped cationic CDs (FCDs) using tetrafluoroterephthalic acid as fluorine source and using branched polyethyleneimine (b-PEI) to furnish positive charge sites. The FCDs achieves dramatic pEGFP and luciferase gene transfection efficiency as well as low cytotoxicity in commonly used cell lines at low weight ratio, even in primary cells and stem cells. It is worthy to point out that the FCDs possess superior efficiency and biocompatibility, compared to some widely used commercial reagents such as 25 kDa polyethyleneimine (25k PEI) and Lipofectamine 2000. In addition, the FCDs show excellent efficient transfection even at high serum concentration and low DNA dose, indicating potential practical applications. Keywords: fluorine doping, cationic carbon dots, gene delivery, PEI, serum resistance

1. INTRODUCTION The high-efficiency delivering of exogenous nucleic acid into cells is extremely importance in gene therapy and has attracted tremendous interest during the past few decades. 1-3 Comparing with viral gene vectors, non-viral vehicles are considered some advantages, such as structural and functional designability, facile synthesis, high transfection efficiency and low immunogenicity.4,5 Current non-viral gene vehicles mainly focus on cationic lipids and cationic polymers. Numerous cationic polymers, including polyethyleneimine (PEI)6-8 and poly (β-amino ester), 9-12 are widely used vehicles for gene delivery. Lipofectamine 2000 (Lipo 2000) is a cationic lipid-based gene delivery reagent that shows high transfection efficiency in most cell lines.13,14 However, all these developed cationic non-viral vectors suffer the major hurdle of successful clinic applications.15 Therefore, the design of non-viral vehicles with high-efficiency of nucleic delivery is still highly

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desirable. Emerging as a fascinating carbonaceous nanomaterial, carbon quantum dots (CDs) have received significant focus in recent years,16-19 and been used in a wide range of adhibitions, such as bioimaging,20-23 optoelectronic devices,24,25 theranostics,26 chemical sensing,27,28 rely on their excellent biocompatibility, chemical inertness, tunable photoluminescence, hypotoxicity and excellent light-stability. In the past several years, quite a few CDs have been reported as gene vectors. However, these unprocessed CDs cannot achieve such high gene delivery efficiency that is higher than the efficiency of commercial gene delivery reagents, such as Lipofectamine 2000.29-34 The delivery process is in fact very complicated and composed of several parts including package and delivery of gene, cellular uptake, endosomal escape and genetic expression.35 Therefore, any factor that can optimize one of these steps should be beneficial to gene delivery. Fluorine, which is lacked in biological body, but is widely employed to modify the natures of drugs, biomaterials and cells.36-40 The introduction of fluorine can increase the therapeutic effect or pharmacokinetics of many medicines,36,37 improve protein stability,39 permit the detection or imaging of the marked compounds41, 42 and enhance phase-separation tendency in both non-polar and polar environments.43,44 Fluorinated biomaterials have high affinity to endosome/lysosome and cell membrane,44,45 and can through the lipid bilayer of these membranes easily. Fluorination can decrease surface energy of cationic polymer, and thus make these fluorine-contained cationic polymers can associate with each other at low concentrations, boosting synergistic improving effect.45-48 Hence, fluorine-embellished may be an outstanding strategy to enhance the delivery efficiency of polymeric vehicles through the improvement of cellular uptake process. 49-53 Recently, our group demonstrated that F-doping can prolong the emission wavelength of CDs.54 Then, considering the effect of fluorine,35 we had tried to evaluate the gene delivery efficiency of this F-doping CDs. However, this F-doping CDs cannot achieve the effect of gene delivery, just due to the lack of sufficient positive charge that can package target gene and protect it from enzymatic degradation. Herein, we design a fluorine-doped cationic CDs (FCDs) for gene delivery. The FCDs was prepared by a solvothermal process using tetrafluoroterephthalic acid as fluorine source and branched polyethyleneimine (b-PEI) to furnish positive charge sites. For the FCDs, abundant


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positive nitrogen sites of b-PEI furnish the binding, transport of negative DNA. On the other hand, the residual fluorine bearing moieties facilitate enhanced affinity of encapsulated DNA to cytomembrane as well as karyotheca and endosome/lysosome membrane, thus may greatly improve the efficiency of gene delivery. Therefore, it is believed that the FCDs possess huge potential in application as efficient gene vectors. In addition, we systematically explore chemical structures of the FCDs and thoroughly analyze the enhancing mechanism of nucleic acid delivery.

2. EXPERIMENTAL SECTION 2.1. Materials Tetrafluoroterephthalic acid (TFTA), terephthalic acid, 1.8 kDa branched-polyethyleneimine (1.8k b-PEI), 25 kDa branched-polyethyleneimine (25k PEI) and Methyl tetrazolium (MTT) were purchased from Sigma-Aldrich. DMEM and RPMI 1640 Medium, DMEM/F12, penicillin, streptomycin and fetal bovine serum (FBS) were obtained from GIBCO. Lipofectamine 2000 (Lipo 2000), Lyso-Tracker Red and YOYO-1 were supplied by Invitrogen (Carlsbad, CA, USA). Glo Lysis Buffer and luciferase assay kit (Bright-Glo

TM

) were purchased from Promega (USA).

BCA protein assay was purchased from Solarbio (Beijing, China). All chemicals were analytical-grade and used as received without further purification unless indicated. 2.2. Instrumentations FCDs were observed on a high-resolution transmission electron microscope (HR-TEM, Tecnai G2 F20, FEI). The average particle size and zeta potential were assessed using Zetasizer 3000 HAS (Malvern Instrument, Inc, Worcestershire, UK). The molecular weight and the molecular weight distribution of FCDs were determined by gel permeation chromatography using a Sodex OHpack SB-803 HQ (Phenomenox, USA). Fourier transform infrared (FT-IR) spectra were recorded on a Bruker TENSOR-27 spectrometer. X-ray photoelectron spectrum (XPS) was recorded using a Thermo Scientific ESCALAB 250Xi. The

19

F nuclear magnetic resonance (19F

NMR) spectra of FCDs was carried out on a 500 MHz Avance-Ⅱ spectrometer from Bruker using D2O as the solvent. The images of cells were visualized and photographed by a fluorescence microscope (Nikon, Japan). The luminescence of sample was assessed by 1420 multilabel counter (Wallac, USA). 2.3. Synthesis of FCDs. Firstly, TFTA, terephthalic acid and 1.8k b-PEI were put in 15 mL of ethanol, respectively.



Then, the TFTA or terephthalic and 1.8k b-PEI solutions were transferred to a teflon-lined stainless-steel autoclave. Before heating for 6 h at 180 oC, two solutions were stirred well. Then let autoclave naturally cooled to room temperature. The products were dialyzed at 3.5 kDa dialysis tube. The fluid after dialysis was further lyophilized. FCDs or undoped CDs (UCDs), a brown gel, was obtained. 2.4. Characterization of FCDs/pDNA Polyplexes Complexes, containing a moderate amount of pDNA (1 µg), were freshly prepared at various weight ratios. After incubation for 20 min at room temperature, the samples were electrophoresed on 1% agarose gels and stained by ethidium bromide at 100 V for 30 min. The gel was photographed using an UV illuminator. Size and zeta-potential of the complexes were further characterized at 25 oC using dynamic light scattering. 2.5. Cell Culture, Gene Transfection and Cellular Imaging HEK 293T, NIH 3T3, COS-7, and HepG2 cells were obtained from ATCC and incubated in complete DMEM (10% FBS) at 37 oC under humidified 5% CO2 atmosphere. B16F10 and A549 cells were obtained from ATCC and incubated at same environment with complete RPMI 1640 (10% FBS). Primary 3T3-L1 and mESCs cells were extracted from mouse and incubated at same environment with complete DMEM/F12 (15% FBS). These complete culture mediums all contain penicillin (100 units mL-1) and streptomycin (100 µg mL-1). The cells were seeded into 24-well plates for 18-24 h and followed by the cultured with FCDs/pDNA complexes (1 µg pDNA) with the cells for another 4h. The density of cells was 1 × 105 cells per well. The complexes were prepared in advance at different weight ratios and dissolved in 200 µL medium. Next, the mediums were replaced by 900 µL fresh medium containing 10% FBS were added. Then the cells were cultured for another 48 h except HEK 293T cells. After removal of the old medium, HEK 293T cells were cultured for another 24 h. Lipo 2000 was used as controls, and gene transfection was conducted according to the product’s protocols. The optimal weight ratio of FCDs/DNA, UCDs/DNA, 1.8k b-PEI/DNA, 25k PEI/DNA and Lipo 2000/DNA is 2, 2, 3, 3, 3, respectively. The effect of EGFP plasmid delivery in the cells was directly visualized and photographed with a fluorescence microscope, and the transfection efficacy was quantitatively measured using flow cytometry. The luciferase activities were given as relative light units (RLU) per mg of cell protein. The extent of pEGFP mRNA expression level upon


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transfection was measured by quantitative real-time PCR technique. In mechanistic studies of fluorine-doped effect, YOYO-1, Lyso-Tracker Red and DAPI were used to label plasmid (green), endosomes (red) and cell nuclei (blue), respectively. The cells were seeded into 6-well plates for 18-24 h and followed by the cultured with FCDs (100 µg mL-1) with the cells for 4 h. The density of cells was 2 × 105 cells per well. 2.6. Cell Viabilities The cells were seeded into 96-well plates for 18-24 h and followed by the cultured with samples (the concentrations of MTT test correspond to the concentrations of gene transfection experiment) with the cells for 24 h. The density of cells was 1 × 104 cells per well. Then, cells were incubated with 50 µL of 1 × MTT solution for 4 h. Before adding 150 µL of DMSO to each well and shaking for 15 min, the culture medium was removed all. Then, cell viability was measured at 550 nm and estimated according to the equation: Cell viability % = / Where ODs is obtained in the presence of samples and ODu is obtained in the absence of samples. 2.7. Statistical Analysis All experiments were repeated at least three times (n ≥ 3). The results are expressed as mean ± standard deviation (SD). Statistical analysis was evaluated by using two population Student’s t-test. P values < 0.05 were considered statistically significant (*p < 0.05, **p < 0.01 and ***p < 0.005).

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of FCDs



Figure 1. (a) Synthetic Route for FCDs. (b) HR-TEM image, (c) Size distribution of FCDs. As is well known, surficial positive charge of vectors is extremely crucial to effective delivery of gene into cells. Since branched polyethyleneimine bears large amount of primary, secondary and tertiary amino groups and thus may furnish enhanced water-solubility and surficial positive charge, we chose 1.8k b-PEI as nitrogen source to synthesize undoped CDs (UCDs) via a solvothermal process at 180 oC (Figure 1a). To introduce fluorine moiety, TFTA was chosen as the aromatic fluorine source to prepare FCDs. Figure 1b displays HR-TEM image of the prepared FCDs. The magnified image shows clear crystal lattice and the lattice spacing value is measured to be about 0.3 nm, indicating formation of crystal CDs under solvothermal condition. It is determined from the HR-TEM image that the average diameter of FCDs is 4.8 ± 0.5 nm (Figure 1c), demonstrating that FCDs belongs to normal CDs. The equivalent molecular weight of FCDs is about 66 kDa, obtained from the GPC test (Table 1). Polymer dispersity index (PDI) is 1.34, displaying typical characteristic of polymer. It is hard to imagine CDs with an average diameter of 4.8 nm can reach such an equivalent molecular weight. We infer that the FCDs are constructed by a carbonaceous nucleus and positive polymer film outside (nuclear@polymer, Figure 1a). The hydrodynamic particle size of FCDs was evaluated with dynamic light scattering (DLS) test, and


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the average hydrodynamic particle size of FCDs in water is 136 ± 5 nm (Table 1 and Figure S1) and much larger than that observed by HR-TEM, further demonstrate nuclear@polymer structure. Before investigating gene delivery, we measured the zeta potential of FCDs in neutral water (Table 1). The positive zeta potential indicates that partial protonation of N sites take place under this condition. It also demonstrates that the FCDs have potential in the application of gene delivery. Table 1. Characterization of FCDs. Mwa (kDa)

Sample FCDs

66 a

PDI

Average size

Zeta potential

Mw/Mna

(nm)

(mV)

1.34

136 ± 5

12.6 ± 0.3

Determined by gel permeation chromatography (GPC).

Then, we explored the chemical structure of FCDs by FT-IR spectrometer, XPS and 19F NMR. Generally, under solvothermal process even at high temperature, it is hard to convert all alkyl amines into aromatic nitrogen atoms. As a result, some of branched PEI chain can be retained, evidenced by the broad absorption band from 3000 to 3600 cm-1 (stands for N-H and O-H bonds), stronger absorption bands than 1.8k b-PEI at 1500-1700 cm-1 and 1200-1400 cm-1(stands for -CONH-, -C=C- and –COOH moieties) in FT-IR spectra (Figure 2a). The XPS signal at binding energy of 284.8, 398.4, 531.6 and 686.8 eV are attributed to C 1s, N 1s, O 1s and F 1s, respectively (Figure 2b), indicating successful retention of fluorine atoms in the prepared CDs. The XPS elemental analysis of FCDs shows the elemental composition: C (60.6%), N (28.0%), O (8.5%), F (2.9%). C 1s high-resolution spectrum exhibits C-H (284.4 eV), C-C/C-N (284.8 eV), C-O (285.4 eV) and C-F/C=O (287.2 eV) bonds on the surface of FCDs (Figure S2).55, 56 N 1s high-resolution spectrum exhibits N-H (398.3 eV) and N-C (398.8 eV) bonds (Figure S3). F 1s high-resolution spectrum exhibits covalent C-F bonding pattern (Figure S4).55 In addition, NMR spectrum of 19F is used to determine the existence form of fluorine in FCDs. Figure 2c shows that all peaks are at the range of chemical shift (-160 to -100), which can put down to the signals of fluorine atom binding to aromatic ring. Based on the characterization and analysis above, we infer that FCDs is constructed a carbonaceous nuclear and several branched chains which contain aromatic fluorine moieties and branched PEI fragments, as depicted in Figure 1a.



Figure 2. (a) FTIR spectra, (b) XPS spectra, (c) 19F NMR spectra of FCDs. UV-vis absorption and FL spectra were used to evaluate the optical properties of FCDs. As shown in Figure S5 (black line), FCDs exhibit two characteristic absorption peaks at ca. 290 nm and 350 nm, which can be assigned to π-π* (aromatic C=C) and n-π* (carboxyl C=O) transitions, respectively. 350 nm and 465 nm are FCDs’ optimal excitation and emission wavelength, respectively (Figure S5: red line and blue line). 3.2. Packing Capacity of DNA by FCDs As is well known, there are abundant -PO3- moieties exposing outside of DNA, thus making the surface of DNA extra negative. Due to the containing of PEI fragments on the surface of FCDs thus furnishes highly positive surficial charge, FCDs tends to combine with DNA to form complex through the electrostatic attraction. The packing capacity of DNA by FCDs and UCDs were evaluated by binding ratio obtained from agarose gel electrophoresis, dynamic light scattering and zeta potential tests. It is found from Figure 3a,b that FCDs have a similar DNA packing capacity to that of UCDs, implying a negligible influence by F-doping on the electrostatic binding behavior. The binding ratio of 1.8k b-PEI to DNA is obviously higher than that of FCDs to DNA (Figure 3c). This is probably attributed to the increased positive charge/molecular weight ratio of FCDs. The weight ratio of 25k PEI to DNA is nearly equal to 0.5, indicating an equivalent binding capacity to that of FCDs (Figure 3d). Both FCDs and UCDs can effectively condense DNA into gradually


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size-reduced nanoparticles with the increasing weight ratio (Figure 3e). The FCDs/DNA complexes can form uniform nanoparticles, and the diameters of these nanoparticles reach 150-200 nm at weight ratio of 2 (Figure 3f). In addition, zeta potential shows a change of negative value–zero–positive value (Figure 3g), indicating gradually increased positive charge in FCDs or UCDs. This packing capacity and the property of condensing DNA into complex nanoparticles are indispensable for efficient cellular uptake of DNA, protecting DNA from nucleic acid enzymatic degradation, and thus successful transportation of DNA into target cells.57

Figure 3. Agarose gel electrophoresis retardation assay of (a) FCDs, (b) UCDs, (c) 1.8k b-PEI, and (d) 25k PEI with EGFP plasmid at different weight ratio. (e) Hydrodynamic size and (g) zeta potential of FCDs/DNA and UCDs/DNA complexes at different weight ratio. (f) TEM image of FCDs/DNA complexes at weigh ratio of 2. ***p < 0.005 in (e) indicates that polyplex size of UCDs and FCDs (w/w=2, n=3) are smaller than that of UCDs and FCDs (w/w=0.1, n=3) *p < 0.05 in (g) indicates higher positive zeta potential compared to FCDs (w/w=2, n=3). Error bars represent the standard error (n=3). 3.3. The Delivery Efficiency of FCDs The transfection efficiency of a vector reflects its gene delivery capacity. It was first evaluated by HEK 293T cells. As comparison, commercial reagent PEI with average molecular weight of 25k PEI and Lipo 2000 were used instead of FCDs under the same condition. As shown in Figure 4a, the optimal gene transfection weight ratio of FCDs to DNA is 2, observed from the fluorescent transfection data. Naked FCDs without DNA was used to determine the fluorescence



interference by green emissive FCDs. And no green fluorescence was observed under the fluorescence microscope. UCDs, 1.8k b-PEI, even commercial reagents 25k PEI and Lipo 2000 show lower pEGFP and luciferase gene transfection efficiencies than that of FCDs. It is demonstrated that doping by F atom caused significantly facilitating effect on the pEGFP transfection of CDs in HEK 293T cells, comparing to the transfection efficiency of UCDs or 1.8k b-PEI (Figure 4b). At an extremely low weight ratio of 2, the transfection efficiency achieves near to 92% in HEK 293T cells, which is much higher than that of 25k PEI (56% in HEK 293T cells) and Lipo 2000 (65% in HEK 293T cells) (Figure 4b), demonstrating the great potential of FCDs as highly efficient gene vector. As can be seen, the mean fluorescence intensity (MFI) of FCDs is also much higher than that of UCDs, 25k PEI and Lipo 2000 in HEK 293T cells (Figure 4b). And FCDs mediated 47.4-, 5.6- and 3.8- fold enhancements of luciferase activity compared to UCDs, 25k PEI and Lipo 2000, respectively (Figure 4c). Furthermore, the transfection efficiency of FCDs and UCDs was further evaluated with quantitative RT-PCR (Figure 4d). The pEGFP mRNA expression levels of FCDs-treated cells were 6.1 times, 3.3 times and 1.9 times higher than in UCDs, 25k PEI and Lipo 2000 transfected cells, respectively. In addition, low dose of FCDs used provides the foundation for high cell viability of the cells during gene transfection. Meanwhile, high cell viability was observed using FCDs or UCDs as the gene vector, indicating low cytotoxicity of CDs. Such cytotoxicity on HEK 293T cells is even lower than that of widely used non-viral nucleic acid vehicles, such as 25k PEI, Lipofectamine 2000 (Figure 4e).


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Figure 4. The gene delivery efficiency and cytotoxicity of FCDs in HEK 293T cells. (a, b) pEGFP and (c) luciferase gene transfection efficiency of FCDs, UCDs, 1.8k b-PEI, 25k PEI and Lipo 2000 at their optimal weight ratio for 24 h. (d) EGFP mRNA expression levels of cells treated with FCDs/pEGFP, UCDs/pEGFP, 25k PEI/pEGFP and Lipo 2000/pEGFP. UCDs/pEGFP treated cells serve as control. (e) Cytotoxicity of FCDs, UCDs, 1.8k b-PEI, 25k PEI and Lipo 2000 at their optimal weight ratio for 24 h. (b) Determine the percent of positive EGFP cells (columns) and MFI (red diamonds) of the transfected cells using flow cytometry. *p < 0.05, **p < 0.01 and ***p < 0.005 in (b, c, d) indicates lower EGFP transfection efficiency, luciferase gene transfection efficiency and EGFP mRNA expression levels compared to FCDs (w/w=2, n=4). (e) The



concentrations of MTT test correspond to the concentrations in (a, b). *p < 0.05 and **p < 0.01 in (e) indicates higher cytotoxicity compared to FCDs (w/w=2, n=6). Scale bar, 200 µm. To investigate the universality, a series of cells were employed. High transfection efficiency of FCDs is further confirmed in COS-7 (85%), A549 (81%), B16F10 (73%), NIH3T3 (58%) and HepG2 (45%) cells (Figure 5, S6-S10), demonstrating its superiority in gene delivery. Compared to UCDs, up to 7-, 44-, 8-, 7-, and 4-fold positive EGFP cells were observed in COS-7, A549, B16F0, NIH3T3 and HepG2 cells at the optimal weight ratio. Furthermore, we also evaluated the transfection efficiency of FCDs in difficulty transfect cell lines, such as primary 3T3-L1 and mESCs. As can be seen, FCDs shows higher gene transfection efficiency than UCDs, 25k PEI and Lipo2000 in primary 3T3-L1 and mESCs cells (Figure S11 and S12). FCDs mediated 273.8-, 32.3- and 4.17- fold enhancements of luciferase activity compared to UCDs, 25k PEI and Lipo 2000 in primary 3T3-L1 cells. Similarly, the MFI of FCDs is also higher than the MFI of UCDs, 25k PEI and Lipo 2000 in these cell lines. Especially, fluorine-doping leads to about 2-fold enhancements of EGFP transfection efficiency compared to Lipo 2000 in these cell lines. In addition, FCDs and UCDs both show low cell cytotoxicity (> 90% cell viability), indicating excellent biocompatibility (Figure 4e, S6-S12). These results demonstrate that fluorine-doping route should be a hopeful strategy in the synthesis of high-efficiency CDs based nucleic acid vehicles.


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Figure 5. Comparison of FCDs with UCDs and Lipo 2000 on pEGFP transfection efficiency in cells lines. (a) EGFP expression (columns), mean fluorescence intensity (red diamonds) in eight cells lines transfected by FCDs, UCDs and Lipo 2000 at their optimal weight ratio for 24h or 48 h. *p < 0.05 and ***p < 0.005 in (a) indicates superior pEGFP transfection efficiency compared to UCDs and Lipo 2000 (n=4). 3.4. Mechanistic Studies of Fluorine-Doped Effect To study the mechanism behind high transfection efficiency of FCDs, the cellular uptake property and endosomal escape ability of FCDs/pDNA and UCDs/pDNA complexes were compared. As can be seen in Figure 6, the fluorescence of pDNA and endosomes in FCDs-treated cells for 2 h were brighter than the fluorescence in UCDs-treated cells, demonstrating that fluorine-doped effect can significantly improve the cellular uptake efficiency of CDs/pDNA complexes. Furthermore, numerous isolated pDNA accumulated inside cytoplasm and cell nuclei of FCDs-treated cells for 4 h, confirming that fluorine-doped effect also can obviously enhance endosomal escape abilities of CDs/DNA complexes. On the basis of above analysis and previous research,35,44,45,49,63,64 the improved mechanism of transfection efficiency by FCDs can be concluded as three parts. Firstly, fluorination in



aromatic rings can be partially retained under the synthetic process, and thus dramatically improves cellular uptake of vector/pDNA polyplexes, promotes endosomal escape of the vector/pDNA polyplexes and modulates the DNA packing/unpacking ability of the vector. Secondly, the high electronegativity of fluorine atom reduces the protonation tendency of adjacent amines as well as conjugate amines in FCDs, and thus weaken density of adjacent positive charges. It is beneficial to elevate pDNA release rate. Finally, fluorine-doping of CDs improves its chemical affinity to cell membrane and endosome/lysosome membrane, and thus facilitates endosomal/lysosome escape of pDNA. In a word, fluorine-doping of CDs can dramatically improve its transfection efficiency.

Figure 6. Mechanistic studies of the fluorine effect of FCDs on cellular uptake property and endosomal escape ability. Confocal laser scanning microscopy images of COS-7 cells treated with FCDs/pDNA and UCDs/pDNA complexes for 2h and 4h, respectively. YOYO-1, Lyso-Tracker Red and DAPI were used to label plasmid (green), endosomes (red) and cell nuclei (blue),


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respectively. The arrows indicate co-localization of YOYO-1-labelled pDNA and Lyso-Tracker Red-stained endosomes. The triangles indicate the YOYO-1-labelled pDNA were released from complexes and accumulated inside cytoplasm and cell nuclei. Scale bar, 200 µm. 3.5. Gene Delivery by FCDs at High Serum Concentration and Low DNA Dose For clinical applications, an ideal vector should not only exhibit high transfection efficiency and excellent biocompatibility, but also allow to effectively transfect at high serum concentration as well as low DNA dose. Firstly, we tested the transfection efficiency of FCDs and Lipo 2000 in medium containing 0-50% FBS. As shown in Figure 7a,b, higher than 85% of transfection efficiency is maintained for FCDs in the presence of 30% FBS. Even with 50% FBS, FCDs still shows transfection efficiency of 56 % in HEK 293T cells. However, for Lipo 2000, the capacity of serum resistance is very low, evidenced by the dramatically decreased transfection efficiency (only 13% in HEK 293T with 50% serum). The outstanding serum resistance of FCDs/DNA complexes is mainly owing to the inert property of FCDs against serum proteins and low zeta potential (Figure 3f). Then, we evaluated the transfection efficiency of FCDs at low DNA dose in COS-7 cells (Figure 7c and d). Figure 7c shows a positive correlation between gene transfection efficiency and DNA dosage with DNA dosage from 0.1-1 µg. At this concentration range, the cytotoxicity of FCDs can be completely ignored. With the increasing of DNA dosage to 1 – 2 µg accompanying with increasing FCDs dosage, the cell cytotoxicity slightly increases and leads the slight decline of gene transfection efficiency. In a word, the excess DNA is not beneficial to gene transfection. The variation tendency is further demonstrated by the fluorescence signals (Figure 7d). In a word, lower DNA dosage means lower dosage of gene vectors, accompanying with lower cytotoxicity and fewer side effects. FCDs shows moderate transfection efficiency (about 40%) at only low DNA doses of 0.2 µg in COS-7 cells, intensely indicating its huge potential in practical applications. It is attributed to the high affinity of FCDs to phospholipid membrane, such as cell membranes, endosome/lysosome membrane.44, 45



Figure 7. FCDs outperforms commercial Lipo 2000 at high serum concentration and low DNA doses. (a, b) Transfection efficiency of FCDs (weight ratio at 2) and Lipofectamine 2000 at different FBS concentrations in HEK 293T cells. (c, d) Transfection efficiency of FCDs (weight ratio at 2) and Lipo 2000 at different DNA dosage in COS-7 cells. (b, c) The nucleic acid delivery efficiency was quantified by flow cytometry. Red diamonds in (b, c) represent mean fluorescence intensity. *p < 0.05, **p < 0.01 and ***p < 0.005 indicate lower EGFP transfection efficiency compared to FCDs group (n=4). Scale bar, 200 µm. 3.6. Cellular Imaging of FCDs Since FCDs has been demonstrated excellent fluorescence properties and low cytotoxicity,


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FCDs is expected to potentially serve as bioimaging reagent. We chose HEK 293T cells and A549 cells to evaluate the cellular imaging property of FCDs (Figure S13). It is found from these images that cytoblast regions were stained with brighter fluorescence, which implies FCDs can across cytomembrane/karyotheca and enter into cells, even enter into cell nucleus. Noteworthy, this result further demonstrates high-efficiency cellular uptake of FCDs.

4. CONCLUSIONS In summary, we have designed and prepared a fluorine-doped cationic CDs by a solvothermal process using TFTA as fluorine source and b-PEI to furnish positive charge sites. The FCDs shows a nuclear/polymer nanostructure, including a carbonaceous nuclear and a fluorine-bearing cationic polymer film outside. The gene delivery efficiency of FCDs is very high in commonly used cell lines, primary cells and stem cells, even superior to some common used commercial reagents, such as 25k PEI and Lipo 2000. This is mainly attributed to the fluorine-doping effect, because fluorine-doping dramatically enhances affinity of encapsulated DNA to cytomembrane as well as endosome/lysosome membrane of DNA. Furthermore, the FCDs also show high-efficiency gene delivery at high concentration of serum and low DNA dose, further demonstrating its huge potential in practical applications. Although FCDs show high gene delivery efficiency and excellent biocompatibility in vitro, lacking of degradable chemical structure may limit its in vivo application. We will consider this issue in our further research on FCDs based gene delivery system.

ASSOCIATED CONTENT Supporting Information Supporting Information displays Figures S1−S13, including hydrodynamic size of FCDs; high resolution C 1s, N 1s and F 1s XPS spectra of FCDs; UV-vis absorption, FL excitation and FL emission spectra of FCDs; gene delivery efficiency and cytotoxicity of FCDs in COS-7, A549, B16F10, NIH3T3, HepG2, primary 3T3-L1 and mESCs cells; cellular imaging of HEK 293T and A549 cells by FCDs.

AUTHOR INFROMATION Corresponding Author * E-mail: [email protected] (Aming Xie)



* E-mail: weidong@ njust.edu.cn (Wei Dong) ORCID Aming Xie: 0000-0001-5381-0689 Wei Dong: 0000-0002-6712-8960 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The financial supported by the National Natural Science Foundation of China (NSFC: 51573078) is gratefully acknowledged.

REFERENCES (1) Kanasty, R.; Dorkin, J. R.; Vegas, A.; Anderson, D. Delivery Materials for siRNA Therapeutics. Nat. Mater. 2013, 12, 967-977. (2) Akinc, A.; Zumbuehl, A.; Goldberg, M.; Leshchiner, E. S.; Busini, V.; Hossain, N.; Bacallado, S. A.; Nguyen, D. N.; Fuller, J.; Alvarez, R.; Borodovsky, A.; Borland, T.; Constien, R.; de Fougerolles, A.; Dorkin, J. R.; Narayanannair Jayaprakash, K.; Jayaraman, M.; John, M.; Koteliansky, V.; Manoharan, M.; Nechev, L.; Qin, J.; Racie, T.; Raitcheva, D.; Rajeev, K. G.; Sah, D. W.; Soutschek, J.; Toudjarska, I.; Vornlocher, H. P.; Zimmermann, T. S.; Langer, R.; Anderson, D. G. A Combinatorial Library of Lipid-Like Materials for Delivery of RNAi Therapeutics. Nat. Biotechnol. 2008, 26, 561-569. (3) Yang, J.; Zhang, Q.; Chang, H.; Cheng, Y. Surface-Engineered Dendrimers in Gene Delivery. Chem. Rev. 2015, 115, 5274-5300. (4) Zhou, J.; Liu, J.; Cheng, C. J.; Patel, T. R.; Weller, C. E.; Piepmeier, J. M.; Jiang, Z.; Saltzman, W. M. Biodegradable Poly(amine-co-ester) Terpolymers for Targeted Gene Delivery. Nat. Mater. 2012, 11, 82-90. (5) Samal, S. K.; Dash, M.; Van Vlierberghe, S.; Kaplan, D. L.; Chiellini, E.; van Blitterswijk, C.; Moroni, L.; Dubruel, P. Cationic Polymers and Their Therapeutic Potential. Chem. Soc. Rev. 2012, 41, 7147-7194. (6) Thomas, M.; Lu, J. J.; Ge, Q.; Zhang, C.; Chen, J.; Klibanov, A. M. Full Deacylation of Polyethylenimine Dramatically Boosts Its Gene Delivery Efficiency and Specificity to Mouse Lung. Proc. Natl. Acad. Sci. U S A 2005, 102, 5679-5684. (7) Bryson, J. M.; Fichter, K. M.; Chu, W. J.; Lee, J. H.; Li, J.; Madsen, L. A.; McLendon, P. M.; Reineke, T. M. Polymer Beacons for Luminescence and Magnetic Resonance Imaging of DNA Delivery. Proc. Natl. Acad. Sci. U S A 2009, 106, 16913-16918. (8) Yang, J.; Hendricks, W.; Liu, G.; McCaffery, J. M.; Kinzler, K. W.; Huso, D. L.; Vogelstein, B.; Zhou, S. A Nanoparticle Formulation That Selectively Transfects Metastatic Tumors in Mice. Proc. Natl. Acad. Sci. U S A 2013, 110, 14717-14722. (9) Breunig, M.; Lungwitz, U.; Liebl, R.; Goepferich, A. Breaking Up the Correlation Between Efficacy and Toxicity for Nonviral Gene Delivery. Proc. Natl. Acad. Sci. U S A 2007, 104, 14454-14459.


Page 18 of 23

Page 19 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(10) Huang, J. Y.; Gao, Y.; Cutlar, L.; O'Keeffe-Ahern, J.; Zhao, T.; Lin, F. H.; Zhou, D.; McMahon, S.; Greiser, U.; Wang, W.; Wang, W. Tailoring Highly Branched Poly(beta-amino ester)s: a Synthetic Platform for Epidermal Gene Therapy. Chem. Commun. 2015, 51, 8473-8476. (11) Cutlar, L.; Zhou, D.; Gao, Y.; Zhao, T.; Greiser, U.; Wang, W.; Wang, W. Highly Branched Poly(beta-Amino Esters): Synthesis and Application in Gene Delivery. Biomacromolecules 2015, 16, 2609-2617. (12) Gao, Y.; Huang, J. Y.; O'Keeffe Ahern, J.; Cutlar, L.; Zhou, D.; Lin, F. H.; Wang, W. Highly Branched Poly(beta-Amino Esters) for Non-Viral Gene Delivery: High Transfection Efficiency and Low Toxicity Achieved by Increasing Molecular Weight. Biomacromolecules 2016, 17, 3640-3647. (13) Dalby, B.; Cates, S.; Harris, A.; Ohki, E. C.; Tilkins, M. L.; Price, P. J.; Ciccarone, V. C. Advanced Transfection with Lipofectamine 2000 Reagent: Primary Neurons, siRNA, and High-Throughput Applications. Methods 2004, 33, 95-103. (14) Clements, B. A.; Incani, V.; Kucharski, C.; Lavasanifar, A.; Ritchie, B.; Uludag, H. A Comparative Evaluation of Poly-L-Lysine-Palmitic Acid and Lipofectamine 2000 for Plasmid Delivery to Bone Marrow Stromal Cells. Biomaterials 2007, 28, 4693-4704. (15) Liu, S.; Zhou, D.; Yang, J.; Zhou, H.; Chen, J.; Guo, T. Bioreducible Zinc(II)-Coordinative Polyethylenimine with Low Molecular Weight for Robust Gene Delivery of Primary and Stem Cells. J. Am. Chem. Soc. 2017, 139, 5102-5109. (16) Pan, L.; Sun, S.; Zhang, A.; Jiang, K.; Zhang, L.; Dong, C.; Huang, Q.; Wu, A.; Lin, H. Truly Fluorescent Excitation-Dependent Carbon Dots and Their Applications in Multicolor Cellular Imaging and Multidimensional Sensing. Adv. Mater. 2015, 27, 7782-7787. (17) Xie, Z.; Wang, F.; Liu, C. Y. Organic-Inorganic Hybrid Functional Carbon Dot Gel Glasses. Adv. Mater. 2012, 24, 1716-1721. (18) Liu, J.; Liu, Y.; Liu, N.; Han, Y.; Zhang, X.; Huang, H.; Yeshayahu Lifshitz; Shuit-Tong Lee; Jun Zhong; Kang, Z. Metal-Free Efficient Photocatalyst for Stable Bisible Water Splitting via a Two-Electron Pathway. Science 2015, 347, 970-974. (19) Tang, Q.; Zhu, W.; He, B.; Yang, P. Rapid Conversion from Carbohydrates to Large-Scale Carbon Quantum Dots for All-Weather Solar Cells. ACS Nano 2017, 11, 1540-1547. (20) Wang, F.; Xie, Z.; Zhang, H.; Liu, C.; Zhang, Y. Highly Luminescent Organosilane-Functionalized Carbon Dots. Adv. Funct. Mater. 2011, 21, 1027-1031. (21) Zheng, M.; Liu, S.; Li, J.; Qu, D.; Zhao, H.; Guan, X.; Hu, X.; Xie, Z.; Jing, X.; Sun, Z. Integrating Oxaliplatin with Highly Luminescent Carbon Dots: an Unprecedented Theranostic Agent for Personalized Medicine. Adv. Mater. 2014, 26, 3554-3560. (22) Jiang, K.; Sun, S.; Zhang, L.; Lu, Y.; Wu, A.; Cai, C.; Lin, H. Red, Green, and Blue Luminescence by Carbon Dots: Full-Color Emission Tuning and Multicolor Cellular Imaging. Angew. Chem. Int. Ed. Engl. 2015, 54, 5360-5363. (23) Baker, S. N.; Baker, G. A. Luminescent Carbon Nanodots: Emergent Nanolights. Angew. Chem. Int. Ed. Engl. 2010, 49, 6726-6744. (24) Lou, Q.; Qu, S.; Jing, P.; Ji, W.; Li, D.; Cao, J.; Zhang, H.; Liu, L.; Zhao, J.; Shen, D. Water-Triggered Luminescent "Nano-Bombs" Based on Supra-(Carbon Nanodots). Adv. Mater. 2015, 27, 1389-1394. (25) Cao, L.; Meziani, M. J.; Sahu, S.; Sun, Y. Photoluminescence Properties of Graphene versus



(26)

(27)

(28) (29)

(30)

(31)

(32)

(33) (34)

(35)

(36) (37) (38)

(39) (40)

(41)

Other Carbon Nanomaterials. Acc. Chem. Res. 2012, 46, 171-180. Huang, P.; Lin, J.; Wang, X.; Wang, Z.; Zhang, C.; He, M.; Wang, K.; Chen, F.; Li, Z.; Shen, G.; Cui, D.; Chen, X. Light-Triggered Theranostics Based on Photosensitizer-Conjugated Carbon Dots for Simultaneous Enhanced-Fluorescence Imaging and Photodynamic Therapy. Adv. Mater. 2012, 24, 5104-5110. Zhu, A.; Qu, Q.; Shao, X.; Kong, B.; Tian, Y. Carbon-Dot-Based Dual-Emission Nanohybrid Produces a Ratiometric Fluorescent Sensor for in Vivo Imaging of Cellular Copper Ions. Angew. Chem. Int. Ed. Engl. 2012, 51, 7185-7189. Ding, C.; Zhu, A.; Tian, Y. Functional Surface Engineering of C‑Dots for Fluorescent Biosensing and in Vivo Bioimaging. Acc. Chem. Res. 2013, 47, 20-30. Liu, C.; Zhang, P.; Zhai, X.; Tian, F.; Li, W.; Yang, J.; Liu, Y.; Wang, H.; Wang, W.; Liu, W. Nano-Carrier for Gene Delivery and Bioimaging Based on Carbon Dots with PEI-Passivation Enhanced Fluorescence. Biomaterials 2012, 33, 3604-3613. Hu, L.; Sun, Y.; Li, S.; Wang, X.; Hu, K.; Wang, L.; Liang, X.; Wu, Y. Multifunctional Carbon Dots with High Quantum Yield for Imaging and Gene Delivery. Carbon 2014, 67, 508-513. Wang, L.; Wang, X.; Bhirde, A.; Cao, J.; Zeng, Y.; Huang, X.; Sun, Y.; Liu, G.; Chen, X. Carbon-Dot-Based Two-Photon Visible Nanocarriers for Safe and Highly Efficient Delivery of siRNA and DNA. Adv. Healthc. Mater. 2014, 3, 1203-1209. Yang, X.; Wang, Y.; Shen, X.; Su, C.; Yang, J.; Piao, M.; Jia, F.; Gao, G.; Zhang, L.; Lin, Q. One-Step Synthesis of Photoluminescent Carbon Dots with Excitation-Independent Emission for Selective Bioimaging and Gene Delivery. J. Colloid Interface Sci. 2016, 492, 1-7. Dou, Q.; Fang, X.; Jiang, S.; Chee, P. L.; Lee, T.; Loh, X. J. Multi-Functional Fluorescent Carbon Dots with Antibacterial and Gene Delivery Properties. RSC Adv. 2015, 5, 46817. Zhang, M.; Zhao, X.; Fang, Z.; Niu, Y.; Lou, J.; Wu, Y.; Zou, S.; Xia, S.; Sun, M.; Du, F. Fabrication of HA/PEI-Functionalized Carbon Dots for Tumor Targeting, Intracellular Imaging and Gene Delivery. RSC Adv. 2017, 7, 3369. Wang, M.; Liu, H.; Li, L.; Cheng, Y. A Fluorinated Dendrimer Achieves Excellent Gene Transfection Efficacy at Extremely Low Nitrogen to Phosphorus Ratios. Nat. Commun. 2014, 5, 3053. Müller, K.; Faeh, C.; Diederich, F. Fluorine in Pharmaceuticals: Looking Beyond Intuition. Science 2007, 317, 1881-1886. Purser, S.; Moore, P. R.; Swallow, S.; Gouverneur, V. Fluorine in Medicinal Chemistry. Chem. Soc. Rev. 2008, 37, 320-330. Liu W.; Huang X.; Cheng M.; Robert J. N.; William A. G.; Groves, J. T. Oxidative Aliphatic C-H Fluorination with Fluoride Ion Catalyzed by a Manganese Porphyrin. Science 2012, 337, 1322-1325. Buer, B. C.; Meagher, J. L.; Stuckey, J. A.; Marsh, E. N. G. Structural Basis for the Enhanced Stability of Highly Fluorinated Proteins. Proc. Natl. Acad. Sci. 2012, 109, 4810–4815. Wang, Y.; Lee, W. C.; Manga, K. K.; Ang, P. K.; Lu, J.; Liu, Y. P.; Lim, C. T.; Loh, K. P. Fluorinated Graphene for Promoting Neuro-Induction of Stem Cells. Adv. Mater. 2012, 24, 4285-4290. Takaoka, Y.; Sakamoto, T.; Tsukiji, S.; Narazaki, M.; Matsuda, T.; Tochio, H.; Shirakawa, M.; Hamachi, I. Self-Assembling Nanoprobes That Display off/on 19F Nuclear Magnetic


Page 20 of 23

Page 21 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(42)

(43) (44)

(45) (46)

(47)

(48)

(49) (50)

(51)

(52) (53)

(54)

(55)

(56)

(57)

Resonance Signals for Protein Detection and Imaging. Nat. Chem. 2009, 1, 557-561. Lee, E.; Kamlet, A. S.; Powers, D. C.; Neumann, C. N.; Gregory B. Boursalian; Takeru Furuya; Daniel C. Choi; Jacob M. Hooker; Ritter, T. A Fluoride-Derived Electrophilic Late-Stage Fluorination Reagent for PET Imaging. Science 2011, 334, 639-642. Horvath, I. T.; Rabai, J. Facile Catalyst Separation Without Water: Fluorous Biphase Hydroformylation of Olefins. Science 1994, 266, 72-75. Kasuya, M. C. Z.; Nakano, S.; Katayama, R.; Hatanaka, K. Evaluation of the Hydrophobicity of Perfluoroalkyl Chains in Amphiphilic Compounds That are Incorporated into Cell Membrane. J. Fluorine Chem. 2011, 132, 202-206. Marsh, E. N. G. Towards the Nonstick Egg: Designing Fluorous Proteins. Chem. Biol. 2000, 7, R153-R157. Percec, V.; Imam, M. R.; Peterca, M.; Leowanawat, P. Self-Organizable Vesicular Columns Assembled from Polymers Dendronized with Semifluorinated Janus Dendrimers Act as Reverse Thermal Actuators. J. Am. Chem. Soc. 2012, 134, 4408-4420. V. Percec; M. Glodde; T. K. Bera; Y. Miura; Shiyanovskaya, I.; K. D. Singer; V. S. K. Balagurusamy; P. A. Heiney; I. Schnell; A. Rapp; H. Spiess; Duank, S. D. H. H. Self-Organization of Supramolecular Helical Dendrimers into Complex Electronic Materials. Nature 2001, 419, 384-387. Brittain, S. M.; Ficarro, S. B.; Brock, A.; Peters, E. C. Enrichment and Analysis of Peptide Subsets Using Fluorous Affinity Tags and Mass Spectrometry. Nat. Biotechnol. 2005, 23, 463-468. Wang, M.; Cheng, Y. The Effect of Fluorination on the Transfection Efficacy of Surface-Engineered Dendrimers. Biomaterials 2014, 35, 6603-6613. He, B.; Wang, Y.; Shao, N.; Chang, H.; Cheng, Y. Polymers Modified with Double-Tailed Fluorous Compounds for Efficient DNA and siRNA Delivery. Acta. Biomater. 2015, 22, 111-119. Lv, J.; Chang, H.; Wang, Y.; Wang, M.; Xiao, J.; Zhang, Q.; Cheng, Y. Fluorination on Polyethylenimine Allows Efficient 2D and 3D Cell Culture Gene Delivery. J. Mater. Chem. B 2015, 3, 642-650. Liu, H.; Wang, Y.; Wang, M.; Xiao, J.; Cheng, Y. Fluorinated Poly(propylenimine) Dendrimers as Gene Vectors. Biomaterials 2014, 35, 5407-5413. Wang, H.; Wang, Y.; Wang, Y.; Hu, J.; Li, T.; Liu, H.; Zhang, Q.; Cheng, Y. Self-Assembled Fluorodendrimers Combine the Features of Lipid and Polymeric Vectors in Gene Delivery. Angew. Chem. Int. Ed. Engl. 2015, 54, 11647-11651. Zuo, G.; Xie, A.; Li, J.; Su, T.; Pan, X.; Dong, W. Large Emission Red-Shift of Carbon Dots by Fluorine Doping and Their Applications for Red Cell Imaging and Sensitive Intracellular Ag+ Detection. J. Phys. Chem. C 2017, 121, 26558-26565. Kundu, S.; Yadav, R. M.; Narayanan, T. N.; Shelke, M. V.; Vajtai, R.; Ajayan, P. M.; Pillai, V. K. Synthesis of N, F and S Co-Doped Graphene Quantum Dots. Nanoscale 2015, 7, 11515-11519. Wang, N.; Fan, H.; Sun, J.; Han, Z.; Dong, J.; Ai, S. Fluorine-Doped Carbon Nitride Quantum Dots: Ethylene Glycol-Assisted Synthesis, Fluorescent Properties, and Their Application for Bacterial Imaging. Carbon 2016, 109, 141-148. Jiang, K.; Sun, S.; Zhang, L.; Wang, Y.; Cai, C.; Lin, H. Bright-Yellow-Emissive N-Doped



(58)

(59)

(60)

(61)

(62) (63)

(64)

Carbon Dots: Preparation, Cellular Imaging, and Bifunctional Sensing. ACS Appl. Mater. Interfaces 2015, 7, 23231-23238. Li, J. Y.; Liu, Y.; Shu, Q. W.; Liang, J. M.; Zhang, F.; Chen, X. P.; Deng, X. Y.; Swihart, M. T.; Tan, K. J. One-Pot Hydrothermal Synthesis of Carbon Dots with Efficient Up- and Down-Converted Photoluminescence for the Sensitive Detection of Morin in a Dual-Readout Assay. Langmuir 2017, 33, 1043-1050. Wang, Y.; Kalytchuk, S.; Zhang, Y.; Shi, H.; Kershaw, S. V.; Rogach, A. L. Thickness-Dependent Full-Color Emission Tunability in a Flexible Carbon Dot Ionogel. J. Phys. Chem. Lett. 2014, 5, 1412-1420. Shen, L.; Zhang, L.; Chen, M.; Chen, X.; Wang, J. The Production of pH-Sensitive Photoluminescent Carbon Nanoparticles by the Carbonization of Polyethylenimine and Their Use for Bioimaging. Carbon 2013, 55, 343-349. Tan, J.; Zou, R.; Zhang, J.; Li, W.; Zhang, L.; Yue, D. Large-Scale Synthesis of N-Doped Carbon Quantum Dots and Their Phosphorescence Properties in a Polyurethane Matrix. Nanoscale 2016, 8, 4742-4747. Bao, L.; Liu, C.; Zhang, Z. L.; Pang, D. W. Photoluminescence-Tunable Carbon Nanodots: Surface-State Energy-Gap Tuning. Adv. Mater. 2015, 27, 1663-1667. Maksimenko, A. V.; Mandrouguine, V.; Gottikh, M. B.; Bertrand, J. R.; Majoral, J. P.; Malvy, C. Optimisation of Dendrimer-Mediated Gene Transfer by Anionic Oligomers. J. Gene Med. 2003, 5, 61-71. Santos, J. L.; Oliveira, H.; Pandita, D.; Rodrigues, J.; Pego, A. P.; Granja, P. L.; Tomas, H. Functionalization of Poly(amidoamine) Dendrimers with Hydrophobic Chains for Improved Gene Delivery in Mesenchymal Stem Cells. J. Control Release 2010, 144, 55-64.


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Fluorine-Doped Cationic Carbon Dots for Efficient Gene Delivery

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