Article Cite This: Biomacromolecules 2018, 19, 2194−2201
pubs.acs.org/Biomac
A Self-Assembled Coumarin-Anchored Dendrimer for Efficient Gene Delivery and Light-Responsive Drug Delivery Hui Wang,† Wujun Miao,‡ Fei Wang,§ and Yiyun Cheng*,† †
Shanghai Key Laboratory of Regulatory Biology, East China Normal University, Shanghai, 200241, P. R. China Changzheng Hospital, Department of Orthopedic Oncology, Shanghai, P. R. China § 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 Downloaded via UNIV OF WINNIPEG on June 24, 2018 at 11:11:11 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
‡
S Supporting Information *
ABSTRACT: The assembly of low molecular weight polymers into highly efficient and nontoxic 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 codelivery 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 codelivery of therapeutic genes and anticancer drugs.
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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 nontoxic 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 nontoxic, 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 the 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 © 2018 American Chemical Society
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 cross-linked into nanoclusters with stimuliresponsive 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.30−32 The hydrophobic substitute in the amphiphilic material controls the diameter, surface charge density, Special Issue: Biomacromolecules Asian Special Issue Received: February 11, 2018 Revised: April 18, 2018 Published: April 23, 2018 2194
DOI: 10.1021/acs.biomac.8b00246 Biomacromolecules 2018, 19, 2194−2201
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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 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 codelivery 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 a 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 anticoagulant, antibacterial, anti-inflammatory, and anticancer activities and fluorescent properties.38−43 Coumarin undergoes [2 + 2] cyclodimerization upon irradiation with ultraviolet (UV) light with a 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 crosslinked micelles, nanogels, and self-healing 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 nontoxic 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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EXPERIMENTAL SECTION
Materials. Ethylenediamine-cored and amine-terminated G1 PAMAM dendrimer (molecular weight: 1430 Da) was purchased from Dendritech, Inc. (Midland, MI). 7-Carboxymethoxy-4-methylcoumarin (CM), dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), and 5-fluorouracil (5-Fu) were purchased from Sigma-Aldrich (St Louis, MO). 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium 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. Synthesis and Characterization of G1-CM. The coumarinanchored G1 PAMAM dendrimer was synthesized by reacting the 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 of N,N-dimethylformamide (DMF) at room temperature. Then, the G1 dendrimer (0.070 mmol) and triethylamine (0.157 mmol) dissolved in 3 mL of 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 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 G1CM/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 were added onto carbon grids and examined by TEM (JEOL JEM2100). 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 deionized water and maintained at room temperature for 30 min before measurement. The size and ζ-potential of the polyplexes were characterized at 25 °C using DLS and TEM. The 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). A 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
Scheme 1. Self-Assembled Coumarin-Anchored Low Generation Dendrimer for Efficient Gene Delivery and Light-Responsive Drug Delivery
/weight of polymer and drug in the complex solution] *100% EE (%) = [(Drug added − Unentrapped free drug)/Drug added] *100% 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 sulfate (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. Afterward, 500 μL of fresh media containing 10% FBS were added 2195
DOI: 10.1021/acs.biomac.8b00246 Biomacromolecules 2018, 19, 2194−2201
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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 G1CM (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.
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 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.
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DOI: 10.1021/acs.biomac.8b00246 Biomacromolecules 2018, 19, 2194−2201
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Figure 3. Light-responsive behaviors of G1-CM (a). 1H NMR (b) and UV−vis (c) spectra of G1-CM 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 for 20 min, followed by irradiation at 254 nm for 30 min. The concentrations of 5-Fu and G1-CM in (f) are 120 and 40 μM, respectively. 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 wellestablished YOYO-1 assay. DNA was labeled with YOYO-1 (Y3601, Invitrogen) for 10 min and then conjugated with the polymers. MDAMB-231 cells were incubated with the above polyplexes for 2 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 G1-CM/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 codelivery experiments, the toxicity of G1-CM/5-Fu/TRAIL complexes on MDA-MB-231 cells was determined by the same assay. The concentration of G1-CM 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.
in Figure 1c, G1-CM 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 delivery.25,49 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 the unmodified G1 dendrimer was 32:1. Moreover, compared to G1/DNA complexes, the ζ-potentials of G1-CM/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 the unmodified G1 dendrimer (Figure 1f). G1-CM efficiently retarded the mobility of plasmid DNA at an N/P ratio of 0.6:1, while the 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 the dendrimer, the assembly property of G1-CM could compensate the effect of the reduced charge density via cooperative binding. Therefore, G1-CM showed improved DNA condensation in comparison with the unmodified G1 dendrimer in DLS and agarose gel electrophoresis. We further tested the transfection efficacy of G1-CM on HEK293 and MDA-MB-231 cells using the EGFP plasmid as the reporter gene. As shown in Figure 2a−d and Figures S2− S3, the G1 dendrimer showed significantly improved gene transfection efficacy after coumarin modification. The mean fluorescence intensity of transfected cells was improved by 181fold and 119-fold for HEK293 cells and MDA-MB-231 cells, respectively after coumarin modification. The efficacy of G1CM 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.
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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 the G1 PAMAM dendrimer was also confirmed by a UV−vis spectrum (Figure S1 in Supporting Information). The conjugation of more CM on the G1 dendrimer yielded insoluble conjugates in aqueous solution, and thus only G1CM was investigated in later studies. We then investigated the assembled structures for G1-CM by DLS and TEM. As shown 2197
DOI: 10.1021/acs.biomac.8b00246 Biomacromolecules 2018, 19, 2194−2201
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Figure 4. Co-delivery of TRAIL plasmid and 5-Fu by G1-CM. Transfection efficacy of G1-CM/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, G1-CM/TRAIL, and G1-CM/5-Fu/TRAIL complexes under various UV irradiation conditions. 5-Fu and G1-CM are fixed at a molar ratio of 2:1. The 5-Fu concentration is 80 μM, and the N/P ratio of the G1-CM/TRAIL complex is 20:1.
noting that the dimerized coumarin in the structures cannot be fully cleaved into monomers upon UV irradiation at 254 nm. This is because the photodimerization of coumarin also occurs at 254 nm, and an equilibrium between photodimerization and photocleavage 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 Figures 3d and S5, the size of assembled G1-CM nanoparticles was slightly increased after 5Fu loading. Further exposing the G1-CM/5-Fu complex to a 365 nm UV light caused a slight decrease in the particle size. The release of 5-Fu from the G1-CM/5-Fu complex was investigated. As shown in Figure 3e, 5-Fu were quickly released from the G1-CM/5-Fu complex in PBS buffer, about 58% of 5Fu was released within 5 h. If the complex was first irradiated at 365 nm for 20 min before in vitro release, the release rate for 5Fu was significantly decreased. This is due to the photodimerization of the coumarin moieties in the assembled G1CM 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 partial photocleavage of the coumarin crosslinkage in the assembled G1-CM. The release rate of 5-Fu was not completely recovered as compared to that of the nonirradiated G1-CM/5-Fu complex, and this phenomenon can be explained by the equilibrium of photodimerization and photocleavage at 254 nm. The anticancer activity of the G1-CM/5-Fu complex was further evaluated on MDA-MB-231 cells. As shown in Figure 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
Compared to the unmodified G1 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 G1CM was further investigated. As shown in Figure 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 attributed to the low molecular weight of the G1 PAMAM dendrimer and the nontoxic coumarin conjugated on the dendrimer. These results suggested that G1-CM allows high efficacy while less toxicity to the transfected cells. It is known that photodimerization 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 crosslinked nanoparticle to a shorter wavelength UV light, photocleavage of coumarin dimers will result in the decreased cross-linking degree and increased drug release from the assembled structures (Figure 3a). We then tested the lightresponsive 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. With 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 G1CM 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 2198
DOI: 10.1021/acs.biomac.8b00246 Biomacromolecules 2018, 19, 2194−2201
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Biomacromolecules the cross-linking 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 the G1CM/5-Fu complex to investigate the possibility of G1-CM in the codelivery 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 the EGFP plasmid into MDAMB-231 cells (Figure 4a−4c). These results motivated us to develop G1-CM as a material for the codelivery 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.53−57 As shown in Figure 4d, incubation of MDA-MB-231 cells with the 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 toward cancer cells and normal cells, the expression of TRAIL in the cancer cells only killed 40% of cancer cells probably due to limited gene transfection efficacy and TRAIL resistance in the cancer cells.54 Therefore, the TRAIL plasmid was usually coadministrated with other therapeutics for combination therapy.37,58−60 The lightresponsive behaviors of the G1-CM/5-Fu/TRAIL complex were further investigated on MDA-MB-231 cells. As shown in Figure 4e, G1-CM, 5-Fu, and TRAIL formed complexes killed more than 70% of 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-anchored low generation dendrimer can be used for efficient gene delivery and lightresponsive drug delivery.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Yiyun Cheng: 0000-0002-1101-5692 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are thankful for financial support from the National Natural Science Foundation of China (21725402, 81601587, 21474030, and 21604052) and the Shanghai Municipal Science and Technology Commission (17XD1401600) for this work.
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REFERENCES
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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 nanoparticles showed light-responsive drug release and anticancer activity. In addition, G1-CM allowed the codelivery of 5-Fu and the TRAIL plasmid for combination cancer therapy. This study provided a facile strategy to develop amphiphilic polymers for efficient gene delivery and light-responsive 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.
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of G1-CM, TEM images of G1-CM/5-Fu under various light exposure conditions (PDF)
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.8b00246. 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 2199
DOI: 10.1021/acs.biomac.8b00246 Biomacromolecules 2018, 19, 2194−2201
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DOI: 10.1021/acs.biomac.8b00246 Biomacromolecules 2018, 19, 2194−2201
