A self-assembled coumarin-anchored dendrimer for efficient gene

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A self-assembled coumarin-anchored dendrimer for efficient gene delivery and light-responsive drug delivery Hui Wang, Wujun Miao, Fei Wang, and Yiyun Cheng Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00246 • Publication Date (Web): 23 Apr 2018 Downloaded from http://pubs.acs.org on April 23, 2018

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Biomacromolecules

A self-assembled coumarin-anchored dendrimer for efficient gene delivery and light-responsive drug delivery Hui Wang,1 Wujun Miao,2 Fei Wang,3 Yiyun Cheng*,1 1

Shanghai Key Laboratory of Regulatory Biology, East China Normal University, Shanghai,

200241, P. R. China. 2

Changzheng Hospital, Department of Orthopedic Oncology, Shanghai, P.R. China.

3

Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases with

Integrated Chinese-Western Medicine, Shanghai Institute of Traumatology and Orthopedics, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, P. R. China. *Correspondence should be addressed to Yiyun Cheng. E-mail: [email protected]

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ABSTRACT The assembly of low molecular weight polymers into highly efficient and non-toxic nanostructures has broad applicability in gene delivery. In this study, we reported the assembly of coumarin-anchored low generation dendrimers in aqueous solution via hydrophobic interactions. The synthesized material showed significantly improved DNA binding and gene delivery, and minimal toxicity on the transfected cells. Moreover, the coumarin moieties in the assembled nanostructures endow the materials with light-responsive drug delivery behaviors. The coumarin substitutes in the assembled nanostructures were cross-linked with each other upon irradiation at 365 nm, and the cross-linked assemblies were degraded upon further irradiation at 254 nm. As a result, the drug-loaded nanoparticle showed a light-responsive drug release behavior and light-enhanced anticancer activity. The assembled nanoparticle also exhibited a complementary anticancer activity through the co-delivery of 5-fluorouracil and a therapeutic gene encoding tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This study provided a facile strategy to develop light-responsive polymers for the co-delivery of therapeutic genes and anticancer drugs. KEYWORDS: dendrimer; gene delivery; self-assembly; coumarin; light-responsive.


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INTRODUCTION Nucleic acid-based therapeutics has been considered as one of the most promising strategies for the treatment of various diseases

1, 2

. The key to success in gene delivery is the development of

efficient and non-toxic gene carriers

3-7

. Cationic polymers were widely used as gene carriers,

and these materials complex with nucleic acids via electrostatic interactions

8, 9

. The formed

positively charged nanoparticles avoid enzymatic degradation and are beneficial for efficient endocytosis

10

. However, the nanoparticles are prone to destabilization by salts and polyanionic

biomolecules abundant in cell culture media and physiological environments

11

. Generally,

polymers with high molecular weight and/or high charge density are essential for complex stabilization, but result in serious toxicity on the transfected cells. On the other hand, low molecular weight polymers with relatively low charge density are generally non-toxic, but they are not capable of forming condensed polyplexes with nucleic acids, and thus fail to achieve efficient gene delivery

12

. Breaking down the transfection efficacy-toxicity correlation was

considered as one of major challenges in cationic polymer-mediated gene delivery systems 13-15. Recently, supramolecular strategies were used to dissolve the dilemma of balancing transfection efficiency and cytotoxicity for cationic polymers 16-18. The low molecular weight polymers were assembled into various nanostructures via hydrophobic, hydrogen-bonding, ionic, or fluorophilic interactions

19-24

. Alternatively, the polymers were conjugated to biocompatible nanoparticles or

biomacromolecules to generate hybrid nanostructures, or crosslinked into nanoclusters with stimuli-responsive property

25

. These assembled nanostructures showed much improved DNA

binding affinity and transfection efficacy, and could be degraded into low molecular weight species after cell internalization, which minimizes the damage of cationic polymers to transfected cells and ensures minimal cytotoxicity 18, 26. Among these strategies, the assembly of amphiphilic materials consisted of low molecular weight polymers and hydrophobic substitutes via hydrophobic interactions is the most investigated strategy 27-29. Hydrophobic ligands such as aliphatic chains, perfluoroalkane chains, and cholesterols were anchored to low molecular weight polymers via irreversible covalent linkages, dynamic covalent bonds or host-guest interactions 3032

. The hydrophobic substitute in the amphiphilic material controls the diameter, surface charge

density and morphology of the assembled nanostructures, and thus influences the binding affinity with nucleic acids

33

. In addition, the hydrophobic components on the assembled structures are


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beneficial for efficient cellular uptake and endosomal escape via a membrane fusion mechanism 27, 29

. For the perfluoroalkane chain, it also improves the stability of assembled nanostructures

and complexes with nucleic acids to serum proteins, and ensures high tissue penetration ability 23, 34, 35

. Besides gene delivery, supramolecular nanostructures via the assembly of amphiphilic

materials can also be loaded with hydrophobic cargos for sustained drug delivery or co-delivery of therapeutic genes and chemical drugs 36, 37. In this work, we developed a new type of amphiphilic material based on low molecular weight polymers for efficient gene delivery and light-responsive drug delivery. Our design principle relied on the anchoring of a hydrophobic coumarin moiety on the surface of low generation dendrimer to yield an amphiphilic dendrimer for improved gene delivery. Coumarin and its derivatives are a class of hydrophobic olefinic compounds with a variety of therapeutic functions such as anti-coagulant, antibacterial, anti-inflammatory and anticancer activities and fluorescent properties

38-43

. Coumarin undergoes [2 + 2] cyclodimerization upon irradiation with ultraviolet

(UV) lights of wavelength above 300 nm 44. When irradiated with a shorter wavelength UV light, the

cyclobutane

adduct

could

degrade

into

coumarin

monomers.

This

reversible

cyclodimerization reaction was widely used to prepare cross-linked micelles, nanogels, and selfhealing or shape-memory materials

45-48

. Here, coumarin was modified on a generation 1 (G1)

polyamidoamine (PAMAM) dendrimer to construct an amphiphilic G1 dendrimer-coumarin conjugate (G1-CM) for efficient and non-toxic gene delivery. Incorporation of the photoresponsive coumarin group in the amphiphile has the advantage of being able to covalently cross-link and degrade the assembled structure by exposure to different UV light irradiations. Owing to the reversible dimerization of coumarin moieties, the assembled nanocarrier exhibited a light-responsive drug release profile, and showed complementary anticancer activity when codelivering anticancer drugs and therapeutic genes (Scheme 1).


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Scheme 1. Self-assembled coumarin-anchored low generation dendrimer for efficient gene delivery and light-responsive drug delivery.

EXPERIMENTAL SECTION Materials. Ethylenediamine-cored and amine-terminated G1 PAMAM dendrimer (molecular weight: 1430 Da) was purchased from Dendritech, Inc. (Midland, MI). 7-Carboxymethoxy-4methylcoumarin (CM), dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS) and 5fluorouracil (5-Fu) were purchased from Sigma-Aldrich (St Louis, MO). 3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sangon (Shanghai, China). Lipofectamine 2000 (Lipo2000) was purchased from Invitrogen (Thermo Fisher Scientific). The chemicals were used as received without further purification.


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Synthesis and characterization of G1-CM. The coumarin-anchored G1 PAMAM dendrimer was synthesized by reacting G1 polyamidoamine dendrimer with CM. Briefly, CM (0.105 mmol) was activated by NHS (0.126 mmol) and DCC (0.136 mmol) for 4 h in 3 mL N, Ndimethyformamide (DMF) at room temperature. Then, G1 dendrimer (0.070 mmol) and triethylamine (0.157 mmol) dissolved in 3 mL dimethyl sulfoxide (DMSO) were added. The mixture was further stirred at room temperature for 7 days. The product was purified by precipitation in diethyl ether for three times and dried under vacuum at 45 °C for 12 h. The yielding G1-CM was obtained as light yellow gels. The average number of CM conjugated on each G1 dendrimer was determined by 1H NMR (Varian, 699.804 MHz). Characterization of G1-CM assembled structures and G1-CM/DNA polyplexes. G1-CM was dissolved in distilled water at proper concentrations and directly examined by DLS (Zetasizer NanoZS90, Malvern). Also, the materials at proper concentrations was added onto carbon grids and examined by TEM (JEOL JEM-2100). For G1-CM/DNA polyplexes, G1-CM was mixed with DNA at various N/P ratios (N represents the number of residual primary amine groups on G1-CM and P represents the number of phosphate groups in the DNA backbone). The G1-CM/DNA complexes were diluted with de-ionized water, and maintained at room temperature for 30 min before measurement. Size and zeta-potential of the polyplexes were characterized at 25 °C using DLS and TEM. DNA binding capability of G1-CM was evaluated by an agarose gel retardation assay. The G1-CM/DNA or G1/DNA polyplexes were run on an agarose gel (1.2% (w/v), 90 V, 50 min). UVIpro Gel documentation system was used to photograph the DNA bands. Preparation of 5-Fu loaded G1-CM. G1-CM and 5-Fu were dissolved in DMSO, and then added dropwise into distilled water. The final concentration of 5-Fu is 1.2 mM. The drug-loaded G1-CM complex was maintained at room temperature for 1 h before further experiments. The drug loading efficacy (LE) and encapsulation efficiency (EE) of 5-Fu by G1-CM were calculated. LE (%)=[weight of drug in the complex solution/weight of polymer and drug in the complex solution]*100% EE (%)= [(Drug added - Unentrapped free drug)/Drug added] *100%


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In vitro release of 5-Fu from G1-CM. In vitro drug release was carried out by an equilibrium dialysis method reported in our previous study

37

. The 5-Fu loaded G1-CM was treated under

various UV irradiation conditions before the release studies (irradiated by a 365 nm UV light for 20 min, or irradiated by a 365 nm UV light for 20 min, followed by irradiation with a 254 nm UV light for 30 min). Cell culture and gene transfection. MDA-MB-231 (a human breast adenocarcinoma cell line, ATCC) and HEK293 (a human embryonic kidney cell line, ATCC) cells were cultured in MEM (for MDA-MB-231 cells) or DMEM (for HEK293 cells) containing 10% heat-inactived fetal bovine serum (FBS, Gemi Inc.), and penicillin sulphate (100 units/mL) and streptomycin (100 µg/mL) at 37 °C and 5% CO2. The cells cultured in 24-well plates overnight (70-80% confluent) were incubated with G1-CM/EGFP plasmid polyplexes for 6 h. After that, 500 µL fresh media containing 10% FBS were added and the transfection experiments were continued for another 42 h. The expression of EGFP plasmids was examined by fluorescent microscopy (Olympus, Japan) and flow cytometry (BD FACSCalibur, San Jose). Three repeats were conducted for each transfection. Cellular uptake. Cellular uptake was detected using a well-established YOYO-1 assay. DNA was labeled with YOYO-1 (Y3601, Invitrogen) for 10 min and then conjugated with the polymers. MDA-MB-231 cells were incubated with the above polyplexes for 2 h and 6 h, respectively, and the cellular uptake of YOYO-1-labeled polyplexes was measured by flow cytometry. Cytotoxicity assay. The cytotoxicity of G1-CM and G1-CM/TRAIL plasmid polyplexes were measured by a well-established MTT assay. MDA-MB-231 cells were incubated with G1CM/TRAIL polyplexes at various N/P ratios (the G1-CM concentration is kept constant at 40 µM) at 37 °C for 48 h. For the co-delivery experiments, the toxicity of G1-CM/5-Fu/TRAIL complexes on MDA-MB-231 cells was determined by the same assay. The concentration of G1CM in the complexes was 40 µM. The N/P ratio of G1-CM/TRAIL complex is 20:1. Free 5-Fu, G1-CM/5-Fu complex and G1-CM/TRAIL polyplex were tested as controls. Cells without any treatment were set as 100% cell viability. Five repeats were conducted for each sample.


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RESULTS AND DISCUSSION G1-CM was synthesized by reacting G1 PAMAM dendrimer with CM at room temperature as shown in Figure 1a. According to the 1H-NMR spectrum in Figure 1b, the average number of coumarin conjugated on each G1 dendrimer was calculated to be 1.2. The conjugation of coumarin molecules to G1 PAMAM dendrimer was also confirmed by UV-vis spectrum (Figure S1 in Supporting Information). The conjugation of more CM on G1 dendrimer yielded insoluble conjugates in aqueous solution and thus only G1-CM was investigated in later studies. We then investigated the assembled structures for G1-CM by DLS and TEM. As shown in Figure 1c, G1CM assembled into nanoparticles in aqueous solution (∼137 nm by TEM and ∼176 nm by DLS). The assembly property of G1-CM facilitates the formation of stable polyplexes when binding with DNA for gene delivery25, 49. As shown in Figure 1d, G1-CM was able to condense plasmid DNA into nanoparticles around 200 nm at N/P ratios above 8:1, while the value for unmodified G1 dendrimer was 32:1. Moreover, compared to G1/DNA complexes, the zeta potentials of G1CM/DNA complexes were relatively higher at the same N/P ratios (Figure 1e). This is probably due to the stronger DNA binding capacity of G1-CM compared to unmodified G1 dendrimer (Figure 1f). G1-CM efficiently retarded the mobility of plasmid DNA at an N/P ratio of 0.6:1, while unmodified G1 dendrimer completely bound the DNA at a ratio of 0.8:1. Though the modification of a coumarin on G1 reduced the surface charge density of dendrimer, the assembly property of G1-CM could compensate the effect of reduced charge density via cooperative binding. Therefore, G1-CM showed improved DNA condensation in comparison with unmodified G1 dendrimer in DLS and agarose gel electrophoresis.


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Figure 1. Synthesis and characterization of G1-CM (a). The synthesized G1-CM was characterized by 1H NMR (b). Self-assembled nanostructures of G1-CM characterized by DLS and TEM (c). The G1-CM/DNA complexes were characterized by DLS (d, e). Agarose gel retardant assay of G1-CM (f). *p < 0.05, **p < 0.01 and **p < 0.001 analyzed by student’s t-test. The amount of plasmid DNA in each sample was 0.5 µg.


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Figure 2. EGFP expressions in HEK293 (a, c) and MDA-MB-231 (b, d) cells by G1-CM and unmodified G1 dendrimer. Positive EGFP cells (%) and mean fluorescence intensity of the transfected cells were determined by flow cytometry. Lipo 2000 was tested as a positive control. Cellular uptake of G1/DNA and G1-CM/DNA polyplexes by MDA-MB-231 cells for 2 h and 6 h (e). The N/P ratio for each sample is 20:1.

n.s.

p > 0.05, **p < 0.01 and

***

p < 0.001 analyzed by

student’s t-test. The dose of DNA in each well is 0.5 µg. We further tested the transfection efficacy of G1-CM on HEK293 and MDA-MB-231 cells using EGFP plasmid as the reporter gene. As shown in Figure 2a-d and Figure S2-S3, G1 dendrimer showed significantly improved gene transfection efficacy after coumarin modification. The mean fluorescence intensity of transfected cells was improved by 181-fold and 119-fold for HEK293 cells and MDA-MB-231 cells, respectively after coumarin modification. The efficacy of G1-CM was comparable to that of commercial transfection reagents Lipo 2000 in both HEK293 and MDA-MB-231 cells. To investigate the possible transfection mechanism of G1-CM, we measured the cellular uptake behaviors of the polyplexes. Compared to unmodified G1


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dendrimer, G1-CM showed significantly increased cellular uptake at an equal N/P ratio of 20:1 (Figure 2e). The superior cellular uptake of G1-CM/DNA polyplexes is probably due to the hydrophobic coumarin modified on G1, which is beneficial for improved interactions of the polyplexes with cell membranes. The cytotoxicity of G1-CM was further investigated. As shown in Fig. S4, G1-CM showed minimal toxicity on MDA-MB-231 cells. Even at a relatively high concentration of 80 µM, the material was well tolerated by the treated cells (>90% viability). This is attribute to the low molecular weight of G1 PAMAM dendrimer and the non-toxic coumarin conjugated on dendrimer. These results suggested that G1-CM allows high efficacy while less toxicity to the transfected cells. It is known that photo-dimerization of coumarin groups occurs upon UV light irradiation above 300 nm, and this property can be used to generate cross-linked nanostructures for drug encapsulation

47, 50, 51

. By further exposure of the cross-linked nanoparticle to a shorter

wavelength UV light, photocleavage of coumarin dimers will result in the decreased crosslinking degree and increased drug release from the assembled structures (Figure 3a). We then tested the light-responsive character of G1-CM in the delivery of 5-Fu. As shown in Figure 3b, the olefinic proton peak at 6.22 ppm (H3) disappeared upon irradiation at 365 nm, suggesting the complete dimerization of coumarin on G1-CM. Further irradiation of G1-CM with a 254 nm UV light, the coumarin dimers degraded into monomers, and the peak for H3 appeared in the 1H NMR spectrum. The light-responsive behavior of G1-CM was also confirmed by UV-Vis spectra. As shown in Figure 3c, an absorption peak at 320 nm was observed for G1-CM, which corresponds to the coumarin moieties in the polymer. Upon irradiation at 365 nm for 20 min, the peak at 320 nm significantly decreased, indicating the dimerization of coumarin on G1-CM. Further exposing the material to 254 nm for 30 min resulted in increased peak intensity at 320 nm. It is worth noting that the dimerized coumarin in the structures cannot be fully cleaved into monomers upon UV irradiation at 254 nm. This is because the photo-dimerization of coumarin also occurs at 254 nm, and an equilibrium between photo-dimerization and photo-cleavage will be reached, thus the coumarin moieties could be only recovered by 50% 42, 52. We then investigated the light-responsive behaviors G1-CM in drug delivery. 5-Fu was used as the model anticancer drug (Table S1). As shown in Figure 3d and S5, the size of assembled G1CM nanoparticles was slightly increased after 5-Fu loading. Further exposing the G1-CM/5-Fu


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complex to a 365 nm UV light caused a slight decrease in the particle size. The release of 5-Fu from G1-CM/5-Fu complex was investigated. As shown in Fig. 3e, 5-Fu were quickly released from the G1-CM/5-Fu complex in PBS buffer, about 58% of 5-Fu were released within 5 h. If the complex was first irradiated at 365 nm for 20 min before in vitro release, the release rate for 5-Fu was significantly decreased. This is due to the photo-dimerization of the coumarin moieties in the assembled G1-CM at 365 nm, which yields cross-linked and stable nanoparticles. Further irradiation of the complex solution at 254 nm for 30 min sped up the release of 5-Fu, which is attributed to the partially photo-cleavage of coumarin cross-linkage in the assembled G1-CM. The release rate of 5-Fu was not completely recovered as compared to that of non-irradiated G1CM/5-Fu complex, and this phenomenon can be explained by the equilibrium of photodimerization and photo-cleavage at 254 nm.

Figure 3. Light-responsive behaviors of G1-CM (a). 1H NMR (b) and UV-Vis (c) spectra of G1CM upon various UV light irradiation conditions. (d) DLS of the assembled G1-CM/5-Fu complex under various conditions. (e) In vitro release of 5-Fu from G1-CM under various conditions. (f) Cytotoxicity of G1, G1-CM, 5-Fu and G1-CM/5-Fu complex under various light exposure conditions on MDA-MB-231 cells. UV365 means the material was irradiated by a 365 nm UV light for 20 min. UV365UV254 means the material was irradiated by a 365 nm UV light


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for 20 min, followed by irradiation at 254 nm for 30 min. The concentrations of 5-Fu and G1CM in (f) are 120 and 40 µM, respectively. The anticancer activity of G1-CM/5-Fu complex was further evaluated on MDA-MB-231 cells. As shown in Fig. 3f, G1 and G1-CM showed minimal toxicity on the cells, while free 5-Fu efficiently killed the cancer cells. The G1-CM/5-Fu complex showed much reduced toxicity in comparison with free 5-Fu, which is due to the sustained release of anticancer drugs from the complex. Interestingly, irradiation of the G1-CM/5-Fu complex at 365 nm further reduced the toxicity of 5-Fu due to the crosslinking of coumarin in assembled G1-CM and thus decreased release of 5-Fu molecules. Further irradiation of the complex at 254 nm recovered most of the toxicity of G1-CM/5-Fu. These results were in accordance with the in vitro release results in Figure 3e. We then evaluated the gene transfection efficacy of G1-CM/5-Fu complex to investigate the possibility of G1-CM in the co-delivery of 5-Fu and a therapeutic gene. After loading with 5-Fu, the complex showed similar transfection efficacy with G1-CM in the delivery of EGFP plasmid into MDA-MB-231 cells (Figure 4a-4c). These results motivated us to develop G1-CM as a material for the co-delivery of therapeutic genes and anticancer drugs for combination therapy. A plasmid DNA encoding tumor necrosis factor-related apoptosis inducing ligand (TRAIL) was used as the model therapeutic gene. TRAIL has been found to induce apoptosis in tumor cells but less effect on normal cells

53-56

. As shown in Figure 4d, incubation of MDA-MB-231 cells with

G1-CM/TRAIL plasmid complex efficiently suppressed the growth of tumor cells at N/P ratios above 20:1. Though TRAIL-mediated gene therapy showed high selectivity towards cancer cells and normal cells, the expression of TRAIL in the cancer cells only killed 40% cancer cells probably due to limited gene transfection efficacy and TRAIL resistance in the cancer cells

54

.

Therefore, TRAIL plasmid was usually co-administrated with other therapeutics for combination therapy

37, 58-60

. The light-responsive behaviors of G1-CM/5-Fu/TRAIL complex were further

investigated on MDA-MB-231 cells. As shown in Fig. 4e, G1-CM, 5-Fu and TRAIL formed complexes killed more than 70% cells, which is much more efficient than the G1-CM/5-Fu and G1-CM/TRAIL binary complexes. In addition, the G1-CM/5-Fu/TRAIL complex also exhibited light-responsive toxicity on the treated cells. These results together suggested that the coumarin-


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anchored low generation dendrimer can be used for efficient gene delivery and light-responsive drug delivery.

Figure 4. Co-delivery of TRAIL plasmid and 5-Fu by G1-CM. Transfection efficacy of G1CM/EGFP (a) and G1-CM/5-Fu/EGFP (b) complexes in MDA-MB-231 cells. (c) Positive EGFP cells (%) in the transfected MDA-MB-231 cells measured by flow cytometry. (d) Cytotoxicity of G1-CM/TRAIL complexes on MDA-MB-231 cells. The concentration of G1-CM is 40 µM in each sample. (e) Viability of MDA-MB-231 cells treated with G1-CM, G1-CM/5-Fu, G1CM/TRAIL and G1-CM/5-Fu/TRAIL complexes under various UV irradiation conditions. 5-Fu and G1-CM is fixed at a molar ratio of 2:1. The 5-Fu concentration is 80 µM, and the N/P ratio of G1-CM/TRAIL complex is 20:1.

CONCLUSIONS In summary, the coumarin-anchored dendrimer G1-CM was able to assemble into nanostructures via hydrophobic interactions, and exhibited much improved DNA binding, cellular uptake and gene transfection efficacy. The assembled G1-CM was loaded with 5-Fu and the drug-loaded


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nanoparticles showed light-responsive drug release and anticancer activity. In addition, G1-CM allowed the co-delivery of 5-Fu and TRAIL plasmid for combination cancer therapy. This study provided a facile strategy to develop amphiphilic polymers for efficient gene delivery and lightresponsive drug delivery. Considering that the UV light used in this study has possible phototoxicity and poor ability to penetrate deeply in the tissues, we may use upconversion nanoparticles to solve this problem. We are now developing light-responsive nanoparticles based on G1-CM for in vivo cancer therapy.

ASSOCIATED CONTENT Supporting Information. Gene transfection efficacy of G1-CM at various N/P ratios on HEK293 and MDA-MB-231 cells, viability of MDA-MB-231 cells treated with different concentrations of G1-CM, TEM images of G1-CM/5-Fu under various light exposure conditions. This material is available free of charge via the internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT


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We thank financial supports from the National Natural Science Foundation of China (21725402, 81601587, 21474030 and 21604052) and the Shanghai Municipal Science and Technology Commission (17XD1401600) on this work. REFERENCES (1) Dowdy, S. F., Overcoming cellular barriers for RNA therapeutics. Nat. Biotechnol. 2017, 35, 222-229. (2) Wu, S. Y.; Lopezberestein, G.; Calin, G. A.; Sood, A. K., Targeting the undruggable: Advances and obstacles in current RNAi therapy. Sci. Transl. Med. 2014, 6, 240ps7. (3) Wei, H.; Volpatti, L. R.; Sellers, D. L.; Maris, D. O.; Andrews, I. W.; Hemphill, A. S.; Chan, L. W.; Chu, D. S.; Horner, P. J.; Pun, S. H., Dual responsive, stabilized nanoparticles for efficient in vivo plasmid delivery. Angew. Chem. Int. Ed. 2013, 52, 5377-5381. (4) Lächelt, U.; Wagner, E., Nucleic Acid Therapeutics Using Polyplexes: A Journey of 50 Years (and Beyond). Chem. Rev. 2015, 115, 11043-11078. (5) Chen, W.; Meng, F.; Cheng, R.; Deng, C.; Feijen, J.; Zhong, Z., Advanced drug and gene delivery systems based on functional biodegradable polycarbonates and copolymers. J. Controlled Release 2014, 190, 398-414. (6) Cheng, R.; Feng, F.; Meng, F.; Deng, C.; Feijen, J.; Zhong, Z., Glutathione-responsive nano-vehicles as a promising platform for targeted intracellular drug and gene delivery. J. Controlled Release 2011, 152, 2-12. (7) He, H.; Bai, Y.; Wang, J.; Deng, Q.; Zhu, L.; Meng, F.; Zhong, Z.; Yin, L., Reversibly crosslinked polyplexes enable cancer-targeted gene delivery via self-promoted DNA release and self-diminished toxicity. Biomacromolecules 2015, 16, 1390-1400. (8) 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. (9) Chang, H.; Zhang, J.; Wang, H.; Lv, J.; Cheng, Y., A Combination of Guanidyl and Phenyl Groups on a Dendrimer Enables Efficient siRNA and DNA Delivery. Biomacromolecules 2017, 18, 2371-2378. (10) Yang, J.; Qiang, Z.; Hong, C.; Cheng, Y., Surface-Engineered Dendrimers in Gene Delivery.


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Table of contents


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A self-assembled coumarin-anchored dendrimer for efficient gene

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