Cationic Polythiophenes as Gene Delivery Enhancer - ACS Publications

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Cationic Polythiophenes as Gene Delivery Enhancer Yajie Zhang, Xiao Li, Tiantian Wu, Jian Sun, Xuewei Wang, Leilei Cao, and Fude Feng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 11 May 2017 Downloaded from http://pubs.acs.org on May 15, 2017

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ACS Applied Materials & Interfaces

Cationic Polythiophenes as Gene Delivery Enhancer Yajie Zhang, Xiao Li, Tiantian Wu, Jian Sun, Xuewei Wang, Leilei Cao and Fude Feng* Department of Polymer Science & Engineering, School of Chemistry & Chemical Engineering, Nanjing University, Nanjing 210023, P. R. China

Email: [email protected]

KEYWORDS: polythiophene, conjugated polyelectrolyte, gene delivery, endosomal escape, photochemical internalization

ACS Paragon Plus Environment

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ABSTRACT: There is urgent demand of easily available and highly effective method to improve transgene performance of polymeric gene carriers at low consumption of delivery materials. We developed biocompatible multicomponent nanocomposites in which small quantities of cationic polythiophenes were engineered into the outer shell of polypeptide/DNA polyplexes without covalent linkages. We revealed the introduction of polythiophenes in small quantities led to multiple outcomes including modulation of polyplex size and zeta potential, increase of polyplex stability, promotion of endo-lysosome membrane disruption, light-induced generation of reactive oxygen species (ROS), and significant enhancement of gene delivery to tumor cells. The factors such as structural architectures, molecular weights, photosensitizing capability, and percentage composition of polythiophenes were investigated.

During the past decade, gene therapy has attracted worldwide research interests particularly in the field of cancer therapeutics.1 Gene delivery vehicles play a key role in achieving an effective gene therapy, by successful delivery of therapeutic gene to target cells.2 A variety of parameters are required for an ideal gene carrier with respect to availability, safety and efficiency. Viral vectors have found wide use in biological applications for their high delivery efficiency.3 However, concerns associated with the safety issue of virus urge researchers to seek efficient nonviral gene carriers such as liposomes and polymers.4 Cationic polymers are capable of compacting and condensing large-sized DNA into nanoparticles, with polyethylenimine (PEI) being recognized as a gold standard gene carrier.5 However, the significant cytotoxicity and nondegradability of PEI impede its clinic applications. To reduce the cytotoxicity and improve the transfection efficiency,


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many intelligent strategies have been exploited on the design of the polymer structures featured by biodegradable backbones, functionalized side-chains, and stimuli-responsiveness (pH, heat, light), etc.6-8 Photochemical internalization (PCI) is a method based on the principle of photodynamic therapy (PDT) for endo-lysosomal escape of macromoleclues and nanoparticles by the use of light, oxygen and photosensitizer.9-13 Transgene systems

using cationic polymers,

such as

PEI,

oligoethylenimine, poly(L-lysine), polyamidoamine (PAMAM) dendrimer and PEG-PAsp(DET)PLys triblock copolymer, have been reported with enhanced gene transfer by exciting photosensitizer agents which are electrostatically bound or covalently conjugated to the polymers.14-17 Ideally, separation of photosensitizer from polyplex, attaching of photosensitizer to the endo-lysosome inner membrane and avoidance of photosensitizer relocation to other organelles are preferred while maintaining the integrity of nucleic acids. However, this would raise the bar on the controllability and reliability of photoactivation that relies not only on the property of photosensitizer but also on the tunability of distance between photosensitizer and other objects including nucleic acid and endo-lysosome inner membrane. Undoubtedly, the location or distribution of photosensitizer has profound influence on the gene delivery performance. Conjugated polyelectrolytes (CPEs) have been widely used in biosensing, cell imaging, antibiosis, and drug delivery applications due to their unique optical properties. 11, 18-27 Compared to other polymers, CPEs functionalized with positive charges have DNA binding affinity as well as capability of oxygen photosensitization, which allows them to be used in gene delivery studies.28-30 Among water soluble CPEs, polythiophenes (PTs) have received increasing research interests in cell studies including cell adherence, differentiation and imaging, to tissue engineering


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applications, due to their excellent biocompatibility, easy availability and remarkable opti-electric properties.30-32 The performance of cationic polythiophenes in gene delivery is to be investigated. In our previous work on in vitro gene delivery to macrophage cells using a star-shaped degradable polyaspartate (SP, Figure 1), SP showed strong transfection capability and low cytotoxicity.[20] However, one of the drawbacks of polymeric vectors such as SP and PEI is that it requires relatively high DNA loading for successful gene delivery to tumor cells. It is necessary to reduce the consumption of DNA and gene carriers without compromising transfection efficiency, in order to minimize the risk of cytotoxicity and expand the carriers to more cell lines and animal models. Wu and co-workers have explored systemic studies on the free polymer effect and demonstrated that DNA-unbound free polymers in solution in large amount increase transfection efficiency.33 This observation was complementary with our finding that cationic polymers form core-shell structure by assembling with DNA to result in a polymeric shell where a population of free polymers are confined via secondary interactions.34 The polymeric shell of polyplex allows anchor of cationic polythiophene (cPT) chains in small quantities to the outer coating of polyplex and necessities a close insight into the role of confined cPT chains in in vitro gene transfer process. In the present study, as shown in Figure 1a, cPTs are introduced to the SP/plasmid polyplex shell as endo-lysosomal membrane destabilizer in dark or by PCI to affect transgene process. The goal is to enhance transgene efficiency, save consumption of delivery materials, minimize cytotoxicity, and more importantly add understanding to the relationship between cPT structures/properties and transfection outcomes. We prepared a series of linear and hyperbranched polythiophene esters by widely-used FeCl3 oxidative polymerizations (Figure S1 and Figure 1b). Linear polythiophene esters were obtained using ethyl thiophene-3-acetate (TEt) and ethyl thiophene-3-manolate (TMEt) as monomers,


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respectively. After separation by size exclusion chromatography (SEC), linear polymers were collected in fractions including TEt-based LPT-1~2, and TMEt-based LPTM. The same procedures were applied to the preparation of hyperbranched polythiophene esters except that a synthetic three-arm branch unit (T3) was co-polymerized with TEt or TMEt to afford TEt-based BPT-1~3 and TMEt-based BPTM-1~2. By 1HNMR, successful integration of T3 into polymer structures for BPT(Et) series and BPTM(Et) series was clearly confirmed by the proton resonances peaked at 2.51, 3.21, and 3.73 ppm (Figure S2-3). All of the purified polymers were analyzed by GPC in THF using polystyrene as standard for estimation of molecular weights (Figure 1c), showing unimodal distributions in GPC traces (Figure S4) with narrow polydispersity indexes (PDIs). cPTs were yielded by aminolysis of polythiophene esters with excess diethylenetriamine (DET) followed by acidification and purification treatments. The primary amines of cPTs were presumably protonated, which ensured good water solubility.

Figure 1. (a) Schematic illustrations of the mechanism for cPT-enhanced endo-lysosome escape in gene delivery to cells. (b) Chemical structures of linear and hyperbranched polythiophenes, and SP. (c) The GPC-based molecular weights and PDIs for PTs and SP. (d) The fluorescence quantum yields of PTs and singlet oxygen quantum yields of cPTs.


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The neutral polymers were then characterized by UV-vis absorptionand steady-state fluorescence in chloroform (Figure S5a). For the same polymer series, the maximum absorption wavelength (max) red-shifted with the increase of molecular weight, due to the extension of πconjugation length. For the same reason, linear polymers LPT-1~2 showed red-shifted max compared to branched polymers BPT-1~3. It seems that malonate-modified polymers BPTM-1~2 led to red-shift of max relative to acetate modified polymers BPT-1~3, likely due to the ketone to enol isomerization of malonate which brings side chains into π-conjugation with polymer backbones. The relationship between maximum fluorescence wavelength (em) and polymer structure follows the similar laws (Figure S5a). The fluorescence quantum yields (ФF) of polythiophene esters in chloroform were determined using quinine sulfate in 1.0 M H2SO4 as standard (ФF 0.545) 23 and shown in Figure 1d. Photophysical characterizations of cPTs were carried out in water, including UV-vis absorption, steady-state fluorescence and oxygen sensitization. The fluorescence spectra of cPTs were similar to their neutral precursors in chloroform without visible red shifts in em (Figure S5b). Malonate conjugation led to low ФF values, but other cPTs except the smallest polymer cBPT-3 displayed moderate ФF values (>0.20) (Figure 1d). The cPT solutions emitted bright yellow light under UV lamp (Figure S6). These spectroscopic properties are in consistency with the good water-solubility of cPTs. The largest cPTs, including cBPT-1, cBPTM-1 and cLPT-1, indicated the strongest capabilities of oxygen sensitization which were characterized by 1O2 quantum yields (ΦΔ) in airequilibrated water (Figure 1d). The ΦΔ values, determined using 9,10-Anthracenediylbis(methylene) dimalonic acid (ABDA) as 1O2 trapping agent and rose bengal as standard (ΦΔ 0.75 in water),12 fell as the molecular weights of cPTs decreased. As shown in Figure S7, the effects


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of light irradiation on the smallest polymers toward ABDA absorption were very minor. The difference of cPTs in ROS generation capability provides a chance to investigate the role of ROS in transgene process. DNA binding and condensing capability of cPTs were examined by transmission electronic microscope (TEM) and electrophoresis analysis. Unlike SP, the cPTs alone were unable to condense plasmid pGFP into globular particles as visualized by TEM (Figure S8). Additionally, DNA leakage occurred during electrophoresis of cPT/pGFP polyplexes prepared at N/P 5, more significant for the use of small-sized cPTs (Figure S9). Accordingly, cPTs alone were not suitable for gene delivery due to the poor DNA binding and condensing capability, in sharp contrast to SP.


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Figure 2. (a) EB exclusion results for estimation of DNA binding affinity. (b) Hydrodynamic diameters and zeta potentials of polyplexes in aqueous solution. (c) Cell viability of HepG2 cells by MTT assay. (d) Chemiluminescence intensity in HeLa cells after transfection of pLuc by polyplexes for 40 h.

Stability of polyplex formed between gene carrier and DNA has been recognized to be one of key factors in gene delivery system. Interestingly, the introduce of small quantity of cPTs in the SP/pGFP polyplex was found to enhance the polyplex stability as revealed by the EB exclusion assay. Higher cPTs/SP/pGFP polyplex stability was correlated to the strongerfluorescence quenching of EB/DNA complex as DNA-bound EB was displaced by the added polymer. As shown in Figure 2a, the addition of SP/cPTs where cPTs accounted for only 2.5% or 5% total N/P ratios exhibited greater extent of EB fluorescence quenching than SP or branched PEI (25 KDa) alone at each tested N/P ratio. In most cases the presence of 2.5% cPTs was more effective in EB displacement. To check whether it is common to other cationic polymers such as PEI, we also tested PEI/SP blends using a variety of commercial PEI materials, and found that the PEIs had slight impact on EB fluorescence quenching (Figure S10). DNA binding ability was then evaluated in terms of CE 50 values which were defined as the charge excess required to gain a 50% reduction of EB fluorescence.

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Relative

to PEIs/SP, SP or PEI alone, cPTs/SP were examined with CE50 values which were correlated to enhanced DNA stability by cPTs, small-sized cPTs such as cBPTM-2, cLPT-2 and cBPT3 in particular (Figure S11). Considering the possible photodamage of DNA from cPTs/SP upon PCI treatment, we formulated cPTs-decorated SP/pGFP polyplexes by mixing cPT chains with pre-formed SP/DNA particles instead of blending cPTs and SP in order to keep cPT chains away from DNA. The new polyplexes were quite stable at pH 5 or pH 7 during incubation at 37 ℃ in dark or treated by white


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light irradiation, with invisible DNA migration as indicated by electrophoresis (Figure S12). Reduced hydrodynamic diameters, determined by dynamic light scattering (DLS) technique, were observed with short cPTs and low cPT population. This trend is in good consistency with the DNA stability results discussed above, implying that 2.5% short cPTs such as cBPTM-2, cLPT-2 and cBPT-3 are more effective in constructing stable nanocomposites composed of rigid polythiophenes and soft polypeptides, despite of lowered zeta potentials relative to the 5% population counterparts (Figure 2b). These results reveal that the cPT chains interacted with SP/DNA polyplexes and were likely enriched in the coating shell of polyplex particles. To evaluate the cytotoxicity of cPT-decorated polyplexes, cell viabilities were determined by MTT assay with the polyplexes-treated HepG2 cells. Compared to SP/pGFP, the multicomponent nanocomposites indicated similar cell viabilities under the same treatment (Figure 2c). However, long-chained cPTs like cLPT-1 and cBPT-1 led to increased photocytotoxicity, correlated to their high ROS generation capability. For the same reason, lesser photocytotoxicity was observed with the cells treated by 2.5% cPTs-polyplexes under reduced light exposure (1 min, 500 mW/cm2). To investigate in vitro ROS generation capability of 2.5 or 5% cPTs-polyplexes upon light illumination, dichlorofluorescein diacetate (DCFH-DA) was used as an ROS indicator on a basis of that the fluorescence intensity of highly fluorescent dichlorofluorescein (DCF), the oxidized product of DCFH-FA, is generally proportional to the amount of intracellular ROS.15 As shown in Figure S13, the images for nanocomposites–treated HeLa cells indicated bright green emission, with much stronger brightness than those for HepG2 cells by the same treatment. This difference suggests that HeLa cells have much greater oxidative stress level. The quantitative in vitro ROS level for polyplex-treated cells growing in 96-well plates was examined in terms of IROS, defined as the ratio between fluorescence intensity of DCF at 525


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nm and total protein amount (Figure S14a). Generally, treatment of the cells by short cPTspolyplexes led to small IROS values. The ROS generation could be abolished by the cotreatment of cells with ROS scavenger ascorbate (Figure S14b). These results demonstrate potential PCI machanism-based application of cPTs-polyplexes in in vitro transfection studies. Transfection of HepG2 and HeLa cells with polyplexes prepared at total N/P 5 was carried out at a pGFP loading of 0.2 g/well in 96-well plates. With the given pGFP loading, pGFP delivery by PEI and SP alone led to low level of GFP expression. In contrast, GFP expression on PCI or non-PCI treated HeLa cells was greatly improved after transfection by 2.5 or 5% cPTs-polyplexes (Figure S15). Quantitative transgene efficacy for delivery of luciferaseencoding plasmid (pLuc) was achieved by chemiluminescence assay using the same transfection protocol except that half of cells were subjected to BCA assay for total protein quantification. Transfection efficacy, termed ITE, was estimated by the ratio between luminescence intensity and total protein amount (Figure 2d). The transfection enhancement by the small quantity of cPTs was very significant for HeLa cells relative to HepG2 cells, likely due to the strong intrinsic suppression of ROS in HepG2 cells even under PCI treatment (Figure S16). Transfection was blocked if ROS was depleted by co-treatment with ascorbate (Figure S17). The shortest polymers cLPT-2 and cBPT-3 in 2.5% population exhibited 5-fold enhancement of ITE in non-PCI treated samples. In despite of the lowest IROS, however, cBPT3 in 2.5% population led to 10-fold enhancement of ITE in PCI-treated samples. It is not necessary for one to pursue strong photosensitizers for PCI, since they are not utilized to induce cell death.


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Figure 3. (a) CLSM images for HeLa cells incubated with polyplexes for 40 h with or without PCI treatment. The green channel denotes expression of GFP protein, red channel for Rho-pGFP, and blue channel for Hoechst 33258. (b) CLSM images for HeLa cells immediately after PCI treatment at 5 h post incubation with polyplexes, using non-PCI treated cells for comparison. Scale bar: 20 μm.

To look into the role of cPTs in transfection process, intracellular distribution of rhodamine-labeled plasmid DNA (Rho-pGFP) and expressed GFP protein in HeLa cells were imaged after 40 h of transfection by 2.5% or 5% cLPT-2-polyplexes using confocal laser scanning microscopy (CLSM) (Figure 3a). For the control using PEI and SP along as delivery materials, independent on light treatment, Rho-pGFP was visualized as numerous red punctate clusters in the perinuclear region, which is characteristic for lysosomal entrapment. Very weak dispersed red fluorescence was observed with SP/pGFP-treated cells. These findings indicated efficient cell uptake of PEI or SP polyplexes, but very limited Rho -pGFP escape


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from lysosomes (Figure 3a). For transfection of non-PCI treated cells by cLPT-2 decorated polyplexes, highly dispersed red fluorescence of Rho-pGFP was spread over the cells. Compared to non-PCI treated cells, the PCI-treated cells showed brighter Rho-pGFP fluorescence all over the cells, which was correlated to successful escape of Rho -pGFP from lysosomes. Similar results were obtained with cLPTM (Figure S18a). Efficient cell uptake of polyplexes by HepG2 cells was comparable to that by He La cells (Figure S18b), as indicated by the red punctate clusters around nucleus membranes. However, dispersed red fluorescence throughout the cells was rare. It seems the low ITE values for transfection of HepG2 cells were not linked to cell uptake, but rather correlated to the unsuccessful escape of Rho-pGFP from endo-lysosomes. Unlike HeLa cells, PCI treatment on HepG2 cells was not effective in elevating intracellular ROS level, and led to slight improvement of endosomal escape. To investigate the role of cPTs in endo-lysosomal escape, rhodamine-labeled pLuc (RhopLuc) in nanocomposites was imaged under confocal microscope after 5 h of incubation with HeLa cells. Lysosomes were visualized in green channel by exciting Lysotracker green that emitted green fluorescence at acidic pH. cPTs exhibited negligible emission and did not interfere with imaging experiments (Figure S19). As shown in Figure 3b, after cellular internalization of PEI and SP polyplexes, Rho-pGFP was well colocalized with Lysotracker, which suggests the proton sponge effect of PEI and SP was not effective enough for endosome membrane rupture. The green fluorescence intensity was unaffected by light illumination. In contrast, for cPTs-polyplexes-treated cells, remarkable changes of colocalization between Rho-pGFP and Lysotracker occurred before and after PCI treatment (Figure 3b and Figure S20-S21). Reduced colocalization for PCI treated cells suggests a separation of Rho-pGFP


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from endo-lysosomes. Note that the brightness of green fluorescence was significantly attenuated in PCI-treated cells, which strongly implies that endo-lysosomal pH rise took place due to enhanced permeability of endo-lysosome membrane structures. Meanwhile, in most cases attenuated lysotracker emission already occurred with non-PCI treated cell, which means that endo-lysosomal permeability or preventing endo-lysosomal acidification occurred. Based on the above results, we come to the following conclusions: endo-lysosomal membrane could be destabilized by cPTs that were initially decorated at the surface of polyplexes, and the rupture process was promoted by PCI treatment upon photoactivation of cPTs interacting with endo-lysosome membrane to allow for more efficient endo-lysosomal escape. When it comes to HepG2 cells, it seems cPTs-induced permeability of endo-lysosome membrane took place (Figure S22-S23), which was correlated to the enhanced transgene performance (Figure S16), although the luciferase expression could not reach the level in Hela cells following the same transfection procedure. In summary, we designed and synthesized a series of cPTs. Photophysical studies show cPTs are in well dispersed state in aqueous medium, and activatable to induce ROS generation under white light irradiation. Minimal quantities of cPTs form stable nanocomposites by assembling with SP/plasmid polyplex and enhance in vitro transgene efficacy by raising endolysosomal permeability. Moderate photosensitization of oxygen is correlated to successful gene delivery. Short cPTs such as linear cLPT-2 and hyperbranched cBPT-3 with low photosensitization capability indicate the best performance as transgene enhancer. Our strategy to employ biocompatible polythiophenes as gene delivery enhancer provides a generalized simple, economic and efficient platform for nonviral gene delivery applications.


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ASSOCIATED CONTENT Supporting Information. Experimental details and figures. This material is available free of charge via the Internet at http://pubs.acs.org/.

AUTHOR INFORMATION Correspondent Author *Email: [email protected] (F. F.)

ACKNOWLEDGMENTS We’re grateful to Prof. Wei Wang (Nanjing University) for help with DLS and ζ-potential measurements and Intermediate Chemistry Laboratory (Nanjing University) for help with CLSM. We thank the National Natural Science Foundation of China (Grant No. 21474046), 1000 Young Talent Program, Collaborative Innovation Center of Chemistry for Life Sciences, and the Program for Changjiang Scholars and Innovative Research Team in University for financial support.

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Shi, H.; Xiujie, C.; Liu, S.; Xu, H.; An, Z.; Ouyang, L.; Tu, Z.; Zhao, Q.; Fan, Q.; Wang, L.; Huang, W. Hyper-Branched Phosphorescent Conjugated Polyelectrolytes for TimeResolved Heparin Sensing. ACS Appl. Mater. Interfaces 2013, 5, 4562-4568. Li, S.; Chang, K.; Sun, K.; Tang, Y.; Cui, N.; Wang, Y.; Qin, W.; Xu, H.; Wu, C. Amplified Singlet Oxygen Generation in Semiconductor Polymer Dots for Photodynamic Cancer Therapy. ACS Appl. Mater. Interfaces 2016, 8, 3624-3634. Wu, C.; Bull, B.; Szymanski, C.; Christensen, K.; McNeill, J. Multicolor Conjugated Polymer Dots for Biological Fluorescence Imaging. ACS Nano 2008, 2, 2415-2423. Parthasarathy, A.; Pappas, H. C.; Hill, E. H.; Huang, Y.; Whitten, D. G.; Schanze, K. S. Conjugated Polyelectrolytes with Imidazolium Solubilizing Groups. Properties and Application to Photodynamic Inactivation of Bacteria. ACS Appl. Mater. Interfaces 2015, 7, 28027-28034. Achyuthan, K. E.; Bergstedt, T. S.; Chen, L.; Jones, R. M.; Kumaraswamy, S.; Kushon, S. A.; Ley, K. D.; Lu, L.; McBranch, D.; Mukundan, H.; Rininsland, F.; Shi, X.; Xia, W.; Whitten, D. G. Fluorescence Superquenching of Conjugated Polyelectrolytes: Applications for Biosensing and Drug Discovery. J. Mater. Chem. 2005, 15, 2648-2656. Chen, H.; Wang, B.; Zhang, J.; Nie, C.; Lv, F.; Liu, L.; Wang, S. Guanidinium-Pendant Oligofluorene for Rapid and Specific Identification of Antibiotics with MembraneDisrupting Ability. Chem. Commun. 2015, 51, 4036-4039. Li, J.; Zhao, Q.; Shi, F.; Liu, C.; Tang, Y. NIR-Mediated Nanohybrids of Upconversion Nanophosphors and Fluorescent Conjugated Polymers for High-Efficiency Antibacterial Performance Based on Fluorescence Resonance Energy Transfer. Adv. Healthcare Mater. 2016, 5, 2967-2971. Feng, X.; Lv, F.; Liu, L.; Tang, H.; Xing, C.; Yang, Q.; Wang, S. Conjugated Polymer Nanoparticles for Drug Delivery and Imaging. ACS Appl. Mater. Interfaces 2010, 2, 24292435. Feng, X.; Lv, F.; Liu, L.; Yang, Q.; Wang, S.; Bazan, G. C. A Highly Emissive Conjugated Polyelectrolyte Vector for Gene Delivery and Transfection. Adv. Mater. 2012, 24, 54285432. Feng, X.; Tang, Y.; Duan, X.; Liu, L.; Wang, S. Lipid-Modified Conjugated Polymer Nanoparticles for Cell imaging and Transfection. J. Mater. Chem. 2010, 20 (7), 1312-1316. Hu, R.; Li, S.; Bai, H.; Wang, Y.; Liu, L.; Lv, F.; Wang, S. Regulation of Oxidative Stress Inside Living Cells Through Polythiophene Derivatives. Chin. Chem. Lett. 2016, 27, 545549. Yang, G.; Liu, L.; Lv, F.; Wang, S. Conjugated Polyelectrolyte Materials for Promoting Progenitor Cell Growth Without Serum. Sci. Rep. 2013, 3, 1702. Zambianchi, M.; Maria, F. D.; Cazzato, A.; Gigli, G.; Piacenza, M.; Sala, F. D.; Barbarella, G. Microwave-Assisted Synthesis of Thiophene Fluorophores, Labeling and Multilabeling of Monoclonal Antibodies, and Long Lasting Staining of Fixed Cells. J. Am. Chem. Soc. 2009, 131, 10892-10900. Yue, Y.; Jin, F.; Deng, R.; Cai, J.; Chen, Y.; Lin, M. C. M.; Kung, H.; Wu, C. Revisit Complexation between DNA and Polyethylenimine - Effect of Uncomplexed Chains Free in the Solution Mixture on Gene Transfection. J. Controlled Release 2011, 155, 67-76. Zhang, Y.; Wang, Y.; Zhang, C.; Wang, J.; Pan, D.; Liu, J.; Feng, F. Targeted Gene Delivery to Macrophages by Biodegradable Star-Shaped Polymers. ACS Appl. Mater. Interfaces 2016, 8, 3719-3724.


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Wang, L.; Wu, D.; Xu, H.; You, Y. High DNA-Binding Affinity and Gene-Transfection Efficacy of Bioreducible Cationic Nanomicelles with a Fluorinated Core. Angew. Chem., Int. Ed. 2016, 55, 755-759.


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Figure 1. (a) Schematic illustrations of the mechanism for cPT-enhanced endo-lysosome escape in gene delivery to cells. (b) Chemical structures of linear and hyperbranched polythiophenes, and SP. (c) The GPC-based molecular weights and PDIs for PTs and SP. (d) The fluorescence quantum yields of PTs and singlet oxygen quantum yields of cPTs.


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Figure 2. (a) EB exclusion results for estimation of DNA binding affinity. (b) Hydrodynamic diameters and zeta potentials of polyplexes in aqueous solution. (c) Cell viability of HepG2 cells by MTT assay. (d) Chemiluminescence intensity in HeLa cells after transfection of pLuc by polyplexes for 40 h.


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Figure 3. (a) CLSM images for HeLa cells incubated with polyplexes for 40 h with or without PCI treatment. The green channel denotes expression of GFP protein, red channel for Rho -pGFP, and blue channel for Hochest 33258. (b) CLSM images for HeLa cells immediately after PCI treatment at 5 h post incubation with polyplexes, using non-PCI treated cells for comparison. Scale bar: 20 μm.


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Cationic Polythiophenes as Gene Delivery Enhancer - ACS Publications

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