A Facile Strategy to Prepare Hyperbranched Hydroxyl-Rich

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A Facile Strategy to Prepare Hyperbranched HydroxylRich Polycations for Effective Gene Therapy Shun Duan, Bingran Yu, Chunxiao Gao, Wei Yuan, Jie Ma, and Fu-Jian Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11029 • Publication Date (Web): 11 Oct 2016 Downloaded from http://pubs.acs.org on October 17, 2016

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ACS Applied Materials & Interfaces

A Facile Strategy to Prepare Hyperbranched Hydroxyl-Rich Polycations for Effective Gene Therapy Shun Duan,a,b,c Bingran Yu,a,b,c Chunxiao Gao,a,b,c,d Wei Yuan,d,* Jie Ma,d and Fu-Jian Xua,b,c,* a

State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029 China b Key Laboratory of Carbon Fiber and Functional Polymers (Beijing University of Chemical Technology), Ministry of Education, Beijing 100029 China c Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029 China d State Key Laboratory of Molecular Oncology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences, Beijing 100021, China * To whom correspondence should be addressed E-mail addresses: [email protected] (F.J.X); [email protected] (W. Yuan)

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ABSTRACT: For effective gene therapy, non-viral gene carriers with low toxicity and high transfection efficiency are of much importance. In this work, we developed a facile strategy to prepare hyperbranched hydroxyl-rich polycations (denoted by TE) by the one-pot method involving ring-opening reactions between two commonly used reagents, ethylenediamine (ED) with two amino groups and 1,3,5-triglycidyl isocyanurate (TGIC) with three epoxy groups. The hyperbranched TEs with different molecular weights were investigated on their DNA condensation ability, protein absorption property, biocompatibility, transfection efficiency and in vivo cancer therapy and toxicity. TE exhibited low cytotoxicity and protein absorption property due to the plentiful hydroxyl groups. The optimal transfection efficiency of TE was significantly higher than that of the gold standard of polycationic gene carrier, branched polyethylenimine (PEI, 25 kDa). Furthermore, TE was applied for in vivo tumor inhibition by the delivery of antioncogene p53, which showed good anti-tumor efficiency with low adverse effects. The present work provided a new concept on facile preparation of hyperbranched hydroxyl-rich polycationic carriers with good transfection performances. Keywords: Gene therapy, carrier, hyperbranched, ring-opening, hydroxyl-rich.

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1. INTRODUCTION Gene therapy is a promising strategy for cancer treatment.1 To realize efficient gene therapy, the key issue is to safely and effectively deliver exogenous genes into target cells, where delivery carriers play an essential role. Though viral carriers possess high transfection efficiency, they have many drawbacks, such as immunological problems, which limits their further clinical application.2 Thus, non-viral gene carriers had drawn great attention due to their low immunogenicity, good stability, and high feasibility of molecular design.3-5 The majority of non-viral carriers are polycations which could electrostatically interact with negatively-charged nuclear acids to form nanoparticles for gene delivery. In recent years, various polycationic carriers have been developed, including polyethylenimine (PEI),6 poly (L-lysine),7 polypeptide,8 and nanohybrid.9 However, polycationic carriers also have some shortcomings, such as unsatisfactory cytotoxicity and insufficient transfection efficiency. Moreover, the positively-charged

polycations

also

could

non-specifically

interact

with

negatively-charged proteins and tend to aggregation, which would hinder their in vivo application.10 In general, transfection efficiencies of polycations can be improved with the molecular weight increasing, but the associated cytotoxicity also increases.11 To reduce the cytotoxicity of gene carriers, a series of hydroxyl-rich polycaitons were developed

based

on

ethanolamine-functionalized

polyglycidyl

methacrylate

(PGMA).12-15 Hydroxyl units could benefit transfection performances.16 Due to the plentiful hydroxyl groups, PGMA-based polycations also have low protein absorption 3



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property and good stability.17 Different PGMA-based polycations with special molecular and topological structures, such as star-shaped, comb-shaped and branched carriers, were studied.18-20 It was reported that hyperbranched polycations have high charge density, resulting in high transfection efficiency.21-23 To combine the advantages

of

hyperbranched

and

hydroxyl-rich

structures,

hydroxyl-rich

supramolecular polycations have been constructed.24-26 However, the preparation processes of hyperbranched hydroxyl-rich polycations are still complicated. Herein, a facile strategy to prepare hyperbranched hydroxyl-rich polycations was developed by the one-pot reaction method. Hyperbranched polycations with plentiful hydroxyl groups were readily synthesized by the reagents with multiple amino groups or epoxy groups via the direct ring-opening reaction. In this work, two commonly used reagents, ethylenediamine (ED) with two amino groups and 1,3,5-triglycidyl isocyanurate (TGIC) with three epoxy groups, were selected as the model species to produce a series of hyperbranched hydroxyl-rich polycations (denoted by TE) (Figure 1). The DNA condensation ability, protein absorption property, biocompatibility and transfection performances of TE were investigated in detail. TE was also utilized for in vivo tumor therapy via the delivery of antioncogene p53.

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Figure 1 Schematic illustration of the preparation process of hyperbranched hydroxyl-rich TE. 2. EXPERIMENTAL SECTION 2.1 Materials. Branched polyethylenimine (PEI, Mw=25 kDa), anhydrous dimethyl sulfoxide

(DMSO),

heparin

sodium,

ethylenediamine

(ED,

>98%)

and

1,3,5-triglycidyl isocyanurate (TGIC) were purchased from Sigma-Aldrich Chemical Co., St. Louis, MO. Cell Counting Kit-8 (CCK-8) was purchased from Beyotime Biotechnology, Shanghai, China. COS7 and SMMC 7721 cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD). 2.2 Synthesis of Hyperbranched Polycation (TE). For the preparation of TE, the molar feed ratio [TGIC]:[ED] was controlled at 2:3. In a 50-mL round flask, 5 mL of DMSO was introduced, and then different amounts of ED and TGIC were added. The reaction mixture was degassed by nitrogen for 5 min and reacted at 50 °C for 2 d under stirring. Later, 200 µL of ED was added into the flask and reacted for another 2 h. Then, the crude products were dissolved in 50 mL of deionized water and dialyzed against deionized water with a dialysis membrane (MWCO=3500 Da) for 48 h. Finally, white powders were obtained after freeze drying. For the preparation of TEs with different molecular weights, three different combinations of TGIC and ED were 5



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applied (148.5 mg of TGIC/50 µL of ED for TE1, 297 mg of TGIC/100 µL of ED for TE2, and 594 mg of TGIC/200 µL of ED for TE3). The final masses of TE1, TE2 and TE3 were 79.5, 178.4 and 412.1 mg (yields: 41%, 46% and 53%), respectively. 2.3 Polymer Characterization. The molecular weights of polycations were characterized using gel permeation chromatography (GPC, YL9100, Korea, with UV/Vis detector). In the GPC measurement, a pH = 3.5 acetic buffer solution was used as the eluent at the flow rate of 0.5 mL/min at 25 °C. Hyperbranched polysaccharide standards were used to obtain a calibration curve. The chemical structures of polycations were determined by proton nuclear magnetic resonance (1H NMR) spectrometer (400 MHz, Bruker, USA), and D2O was used as the solvent. 2.4 Characterization of TE/pDNA Complexes. The TE solution was prepared at the concentration of 5 mg/mL in deionized water, and the concentration of pDNA was 0.1 mg/mL in Tris-EDTA buffer (pH=7.4). The ratio of TE to pDNA was represented as the mass ratio (w/w). To prepare TE/pDNA complexes, TE was mixed with pDNA at different mass ratios, vortexed and incubated for 30 min at room temperature. The abilities of pDNA condensation were evaluated by agarose gel electrophoresis as described in our previous work.27 The particle sizes and zeta-potentials of the complexes were measured by Zetasizer Nano ZS (Malvern Instruments, UK). The morphologies of complexes were observed by atomic force microscope (AFM, Bruker, USA). The stability of TE/pDNA complexes at the mass ratio of 30 was evaluated as described in the literature.28 2.5 Protein Absorption Assay. The protein absorption assay was performed as 6


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described earlier.16,17,29 Briefly, 1 mL of TE solution (1 mg/mL) was mixed with 0.2 mL of bovine serum albumin solution (BSA, 5 mg/mL), vortexed thoroughly, and incubated at 37 °C for 0.5, 1, 5, 10, 30 and 60 min, taking branched PEI (25 kDa) solution (1 mg/mL) as the control group. At each time point, the mixture was centrifuged at 4 °C with full speed. The supernatant was collected and the concentration of BSA was determined using bicinchoninic acid assay (Biorad Lab, Hercules, CA). The protein absorption abilities were calculated by the equation:

‫=݌‬

݉଴ − ݉୲ × 100% ݉଴

Where mt was the mass of BSA in the supernatant after the incubation for different times and m0 was the initial mass of BSA in the solution. 2.6 Cytotoxicity Assay. The cytotoxicity assay of TE was performed in COS7 and SMMC 7721 cell lines using the CCK-8 method. COS7 and SMMC 7721 cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Hyclone, USA) with 10% fetal bovine serum (FBS, Sigma, USA), 100 IU/mL of penicillin (Sigma, USA) and 100 mg/mL streptomycin (Sigma, USA) in an CO2 incubator (Sanyo, Japan) at 37 °C and saturated humidity. The cells were seeded into 96-well plates at the density of 104 cells/well with 100 µL of culture medium. After 24 h, the media were replaced by 100 µL of culture media containing 10 µL of TE/pDNA complexes and cultured for 4 h, taking branched PEI (25kDa) as the control. The media were then replaced by fresh media and incubated for another 20 h. As described in our earlier work, the cell viability was evaluated by CCK-8 method.30 Briefly, the absorbance ([A]) of each well was measured by a microplate reader (Bio-rad 680, USA) at the wavelength of 7



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450 nm. The relative cell viability was calculated by [A]sample/[A]control×100%. 2.7 In Vitro Transfection Assay. The in vitro transfection efficiency of TE was first determined by using pRL-CMV plasmid as a reporter gene in COS7 and SMMC 7721 cell lines. Briefly, the cells were seeded into 24-well plates at the density of 5×104 cells/well in 500 µL of culture medium containing 10% of FBS. After the incubation for 24 h, 20 µL of TE/pDNA complexes (containing 1 µg of pDNA) at various mass ratios, were added into each well. The cells were incubated for 4 h, and then the culture medium containing 10% of FBS was replaced with fresh one and the cells were cultured for another 20 h. To measure the gene expression of luciferase, a commercial kit (Promega Co., CergyPontoise, France) was used. The gene expression level was measured by a luminometer (Berthold Lumat LB 9507, Berthold Technologies GmbH. KG, Bad Wildbad, Germany). The amount of protein was quantified via bicinchoninic acid assay. The efficiency of gene transfection was expressed as relative light units (RLUs) per milligram of protein. Furthermore, to visualize the gene transfection performance of TE, the enhanced green fluorescent protein (EGFP) was taken as another reporter gene in SMMC 7721 cells. The processes were the same as described above. The gene expression of EGFP athe optimal mass ratios was observed by a confocal laser scanning microscope (CLSM, SP8, Leica, Germany) and the percentage of EGFP-positive cells was measured by flow cytometry (Beckman Coulter, Pasadena, CA). 2.8 In Vivo Antitumor Assay. Four-week-old female nude mice with the weight of ~20 g were used in the animal experiment. All the animal experimental procedures 8


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followed the guideline of Cancer Hospital, Chinese Academy of Medical Science, Beijing, China. 2×106 SMMC 7721 cells were suspended in 100 µL of PBS and injected subcutaneously into the middle of the back of nude mice. When the diameters of tumor blocks reached about 5 mm, the mice were randomly divided into three groups and each group contained six mice. TE3/p53 complexes (mass ratio=30) with 30 µg of pDNA in the total volume of 100 µL were administered intratumorally for six times at the preset time points. And 100 µL of saline and 30 µg of p53 plasmid in 100 µL of saline were also injected as the controls. The treatment and tumor size/body weight measurements were performed at preset time points. The detailed timetable of in vivo anti-tumor experiment could be seen in Figure 4a. The tumor volume was calculated by the formula: tumor volume = (tumor length × tumor width)2/2. After the treatment for 20 d, the mice were sacrificed, and the tumor were imaged and weighed before fixation. The tumor inhibition ratios were calculated: (W-Wsaline)/Wsaline × 100%, where W is the tumor weight in the experiment groups and Wsaline meant the tumor weight in the control group treated with saline. For each group, 55 µL of blood was collected after treatment for 20 d, and then analyzed by a hematouogy analyzer (MEK-7222K, Nihon Kohden, Japan) to evaluate the in vivo toxicity. The in vivo toxicity of TE3 was evaluated by the tests of liver and kidney functions, including the activities or concentrations of glutamic-pyruvic transaminase (GPT), glutamic oxalacetic transaminase (GOT), creatinine (CRE) and blood urea nitrogen (BUN) in serum. 2.9 Histological and Immunohistochemical Assay. The tumors, livers and 9



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kidneys of the mice in all groups were harvested after the treatment for 20 d, fixed in 4% paraformaldehyde solution, embedded in paraffin and dissected into slices. The histological slices were stained by hematoxylin and eosin (HE) as described in our previous work,31 and then observed by a light microscope (Olympus CX-41, Japan). For immunohistochemical analysis, P53 protein was detected by anti-P53 primary antibody (sc-6243, Santa Cruz, CA, USA), followed by the incubation with two-step IHC detection kit (ZSGB-Bio, China) and colored by DAB (ZSGB-Bio, China). 2.10 Statistical Analysis. The experiment data were represented as mean ± standard deviation for n=3. Statistics analysis was made based on t-test when two groups were compared. One-way analysis of variance (ANOVA) was performed when more than two groups were compared. 3. RESULT AND DISCUSSION 3.1 Preparation and Characterization of TE. As shown in Figure 1 and Figure S1 (Supporting Information), the hyperbranched hydroxyl-rich polymer, TE, was synthesized by the ring-opening reaction between TGIC with three epoxy groups and ED with two amino groups via a facile one-pot method. ED was used for end-capping reaction. The hyperbranched structure could be formed when the feed molar ratio of TGIC and ED was precisely controlled at 2:3 to keep the amino and epoxy groups at the equivalent molar ratio.32 As shown in Figure S1, plentiful hydroxyl groups were formed when the epoxy rings were opened by the amino groups. The typical 1H NMR spectra of TE were shown and analyzed in Supporting Information (Figure S2). The molecular weights and polydispersity indexes (PDI) of TEs were measured by GPC. 10


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As shown in Table S1 (Supporting Information), the molecular weights of TE1, TE2 and TE3 were 6.5, 10.2 and 14.2 kDa, respectively, which were much lower than that of the gold-standard branched PEI (25 kDa). The above results demonstrated that the hyperbranched hydroxyl-rich TE was successfully synthesized by one-pot ring-opening reaction. The molecular weights could be controlled by the reagent concentrations. 3.2 Biophysical Characterization of TE/pDNA Complexes. An effective gene carrier should possess the ability to condense DNA into nanoparticles with appropriate particle sizes for readily cellular uptake.33 The DNA condensation ability of TE was first assessed by electrophoretic mobility of TE/pDNA complexes in agarose gel at various mass ratios. As shown in Figure 2a, the migration of free pDNA from TE1/pDNA complexes under electronic field was inhibited at the mass ratio of 2.5. For TE2 and TE3 with higher molecular weights, the mass ratios of complete DNA condensation were 1.5 and 1, respectively, indicating the DNA condensation abilities of TEs were enhanced with the increasing molecular weights. To evaluate the stability of TE/pDNA complexes, the pDNA release induced by polyanion exchange was studied by heparin assay. As shown in Figure S3 (Supporting Information), when treated with the proper concentration of heparin sodium, the loaded pDNA could be released from TE/pDNA complexes. This result indicated that pDNA could be gradually released from TE/pDNA complexes to cytoplasm for transfection in the presence of polyanions, such as negatively-charged proteins.

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Figure 2 (a) Electrophoretic retardation assay. (b) particles sizes and zeta potentials of TE/pDNA complexes at various mass ratios. (c) AFM images of TE/pDNA complexes at the mass ratio of 30. For efficient cellular uptake, proper sizes and surface charges of the polycation/pDNA complexes are essential. The particle sizes and zeta potentials of TE/pDNA complexes at various mass ratios were measured by dynamic light scattering. As shown in Figure 2b, the particle sizes of TE1/pDNA, TE2/pDNA and TE3/pDNA complexes at the mass ratio of 5 were about 170, 160 and 150 nm, respectively, and then decreased with the increasing mass ratios. At the mass ratio of 30, TE3 with highest molecular weight could condense pDNA to form nanoparticles with the diameter of ~120 nm, which was beneficial for cellular uptake.34 The morphologies of TE/pDNA complexes were imaged by AFM. As shown in Figure 2c, 12


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at the mass ratio of 30, TE could condense pDNA into nanoparticles. The particle size of TE3/pDNA was smallest among all the TE/pDNA complexes, also showing the strongest DNA condensation ability of TE3. On the other hand, the zeta potentials of TE/pDNA complexes increased with increasing molecular weights of TEs at the same mass ratios. For the same TE, the zeta potential increased with increasing mass ratios, which presented the typical characteristics of polycation/pDNA complexes. The potential of TE3/pDNA complexes at the mass ratio of 30 was larger than 40 mV, which was higher than PEI/pDNA complexes at its optimal transfection condition (~35 mV),12 probably because the positive charge density of TE was higher than that of PEI (25 kDa), the gold standard of non-viral gene carrier. The complexes with high zeta potential would have high affinity with negatively-charged cell membranes. The above results indicated that TE could condense pDNA tightly, due to the hyperbranched molecular structure with high positive charge density.35 3.3 Cytotoxicity Assay. For safe gene delivery, gene carriers should have low cytotoxicity. To evaluate the biocompatibility of TE, COS7 and SMMC 7721 were used for the cytotoxicity assay. As shown in Figure 3a, the cytotoxicity of TE/pDNA complexes increased with the molecular weight and mass ratios. Remarkably, the cytotoxicity of TE was significantly lower than that of the gold-standard branched PEI (25 kDa) in both cell lines. Even at the high mass ratio of 30, the COS7 and SMMC 7721 cell lines still showed more than 60% relative cell viabilities, while PEI demonstrated less than 20% cell viabilities.36 In addition, at the mass ratio of 30, TE also showed comparable or lower cytotoxicity in comparison with branched PEI (25 13



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kDa) at its optimal transfection condition (N/P=10). The molecular structure of TE contained plentiful hydroxyl groups generated from the ring-opening of the epoxy groups, which could shield parts of excessive detrimental positive charges and reduce the cytotoxicity.12,16 Moreover, the low cytotoxicity of TE with high zeta potential was also due to the much lower molecular weights (Table S1, Supporting Information) than branched PEI (25 kDa).

Figure 3 (a) Relative cell viabilities of COS7 and SMMC 7721 cells treated with different TE/pDNA and PEI(25 kDa)/pDNA complexes. (b) Protein absorption properties of TE and branched PEI (25 kDa). (c) In vitro luciferase gene transfection 14


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efficiency of TE/pDNA complexes at various mass ratios in comparison with branched PEI (25 kDa) at its optimal transfection condition. (d) Typical fluorescent images of EGFP expression in SMMC 7721 cells treated by TE/pDNA complexes at the mass ratio of 30 and PEI/pDNA complexes at its optimal transfection condition (scale bar = 200 µm). 3.4 In Vitro Gene Transfection Assay. To evaluate the transfection efficiency of TE in the presence of serum, the in vitro transfection assay was performed in complete culture medium. With the presence of serum, the positively-charged polycations might electrostatically absorb the negatively-charged proteins, and then induce the loss of positive surface charges, which would result in the decrease of transfection efficiency.10

To

investigate

the

protein

absorption

property,

a

typical

negatively-charged protein, BSA, was used. TE and PEI solutions with the same concentration were added into BSA solution and incubated at 37 °C. The quantity of absorbed protein was determined at different time points. As shown in Figure 3b, PEI showed the rapid and strong behavior of protein absorption. After incubation for only 30 s, more than 90% of BSA has been absorbed by PEI and obvious precipitation was formed because of its high positive charge. On the contrary, the amounts of protein absorbed by TE were significantly lower than those absorbed by PEI, which was due to the abundant hydrophilic hydroxyl groups in the molecular structure of TE.37 The plentiful nonionic hydroxyl groups provided antifouling properties to TE, leading to low protein absorption abilities. The above result suggested that the transfection efficiency of TE might be less influenced by the negatively-charged proteins in serum. Therefore, TE could be applied in protein-containing environment, such as complete culture medium. Because of the low protein absorption ability of TE, the transfection efficiencies of 15



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TEs in complete culture medium with serum were assayed using luciferase and EGFP as reporter genes in COS7 and SMMC 7721 cell lines (Figure 3c). The transfection efficiencies of luciferase gene increased with the molecular weights of TEs at the same mass ratio. As mentioned above, higher molecular weight TE3 possessed higher pDNA condensability (Figure 2). For TE with the same molecular weight, the transfection efficiencies increased with the mass ratios before the optimal mass ratio of 30, and then slightly reduced beyond the optimal mass ratio. At higher mass ratios, the slight reduction of transfection efficiencies was due to the moderate cytotoxicity of excessive polycations. At the optimal mass ratio of 30, the transfection efficiencies of TEs were significantly higher than the gold standard of polycationic gene carrier, branched PEI (25 kDa) at its optimal transfection condition. As shown in Table S1 (Supporting Information), the molecular weights of TEs were much lower than that of PEI (25 kDa). The abundant hydroxyl groups in the molecular structures of TE could shield excessive positive charges, which decreased the cytotoxicity and protein absorption and improved the gene transfection performance. In addition, the hyperbranched structure of TE also had high charge density, benefiting gene transfection. The positively-charged TE/pDNA complexes were internalized by the cells via endocytosis and then transported by lysosome. In the lysosome, the amino groups of TE molecules could conjugate the protons, and then induced the influx of Cl- and H2O. Subsequently, the large amount of Cl- and H2O caused the swelling and finally induced the rupture of lysosome, making the TE/pDNA complexes release from lysosome to cytoplasm.38 Moreover, the flanking nonionic hydroxyl groups also 16


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could benefit the endosomal escape.12 Therefore, the TE/pDNA complexes could efficiently escape from lysosome to transfect exogenous genes into the target cells. To visualize the gene transfection performance, EGFP was taken as another reporter gene. As shown in Figure 3d and Figure S4 (Supporting Information), the in SMMC 7721 cells treated by TE3 exhibited the highest EGFP expression, which was consistent with the results of luciferase transfection (Figure 3b). Due to its best gene transfection performance, TE3 was selected for further in vivo study. 3.5 In Vivo Antitumor Activity and Toxicity Assay. Based on the above promising in vitro results of gene transfection, the in vivo gene therapy performance of TE was further investigated. It is well known that p53 is a commonly used antioncogene which could be utilized for gene therapy.39 Therefore, p53 was used in this work for the inhibition of the tumor growth. The tumor-bearing nude mice were randomly divided into three groups and treated with saline, p53 plasmid and TE3/p53 under the preset timetable for six times during the long-term treatment (Figure 4a). To analyze the therapeutic effects of different groups, the tumor volumes were measured at different time points. As shown in Figure 4b, the tumors treated with the control saline grew rapidly, which exhibited the typical growth behavior of tumor. When the p53 plasmid was used alone, the growth behavior of tumors had no significant difference from the saline group. Pure p53 plasmid could not effectively enter cells without the help of gene carriers to realize the antitumor function. Notably, the tumor growth of the TE3/p53 group started to decrease after treatment for 10 d, and such inhibition effect continued during the rest of treatment period, leading to continuous decrease of 17



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tumor volume. After the 20 days’ treatment, the mice were sacrificed and the tumors were weighed (Figure 4c) and imaged (Figure 4d). Compared with the tumors of the saline and p53 groups, the weights of the tumors treated with TE3/p53 were significantly lighter, and even several treated tumors totally disappeared, indicating that TE3 exhibited the superior in vivo gene transfection performances (Figure 4e).

Figure 4 (a) Detailed timetable of treatment and body weight/tumor volume measurement. (b) Time-dependent growth curves of tumors treated with saline, p53 and TE3/p53. (c) Tumor weights, (d) photographs, and (e) tumor inhibition efficiencies after the treatment for 20 d. (f) The p53 immunohistochemical and (g) HE staining images of the dissected tumors. Moreover, the in vivo expression of p53 was visualized by immunohistochemical 18


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staining (Figure 4f). For the saline and p53 plasmid groups, there was no obvious p53 protein expression in the tumor tissues. However, in the TE3/p53 group, the p53 protein was extensively expressed, which demonstrated the good transfection efficiency of TE3 in vivo. Furthermore, to assess the efficiency of gene therapy, HE staining was also performed to observe the morphologies of the tumor cells (Figure 4g). Most tumor cells in the saline and p53 groups presented the normal morphology. On the contrary, the tumor cells in the TE3/p53 group showed obvious apoptosis morphologies such as nucleus shrinkage, demonstrating that the p53 gene had been delivered by TE3 into tumor cells to induce the cell apoptosis and then inhibit the tumor growth. In addition, when applied in vivo, the gene delivery carriers should have good biocompatibility with few adverse effects. To evaluate the in vivo toxicity of TE, toxicological analysis, including body weights, liver/kidney functions and histological examination of liver and kidney, were also studied (Figure 5a). The body weights of mice treated with TE3/p53 exhibited no significant difference from the saline and p53 groups, which indicated that TE3 had few systematic adverse effects. Moreover, to further investigate the toxicological effects of TE3, blood biochemical analysis was performed at the end of treatment. The liver and kidney functions of mice between the p53 and TE3/p53 groups showed no significant difference (Figure 5b). Compared with the saline group, the higher GOT and GPT activities of p53 group may be induced by the tris(hydroxymethyl) aminomethane in the TE buffer for plasmid solution. In addition, to evaluate the influences of TE3 on the histopathology of the 19



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liver and kidney, the tissue slices were observed after HE staining (Figure 5c). No pathological change was observed in the livers and kidney of mice treated with TE3/p53. The above results of the body weight, blood biochemistry and histological analysis demonstrated the good in vivo biocompatibility of TE3.

Figure 5 (a) Time-dependent body weight curves of the mice treated with saline, p53 and TE3/p53. (b) Liver and kidney function assay and (c) their HE staining images after 20-day treatment with saline, p53 and TE3/p53 (scale bar = 100 µm). 4. CONCLUSIONS In summary, a facile strategy was developed to prepare hyperbranched hydroxyl-rich TEs by the one-pot method involving ring-opening reactions between two commonly used reagents, ED with two amino groups and TGIC with three epoxy groups. TE possessed good DNA condensation ability and low cytotoxicity. 20


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Compared with the gold standard branched PEI (25 kDa), the optimal transfection efficiency of TE was significantly higher in the presence of serum. Furthermore, TE could effectively deliver the antioncogene p53 into tumor cells in vivo for cancer therapy, where the tumor growth of mice models was significantly inhibited with few adverse effects. This work provided a promising concept of facile design and preparation of hyperbranched gene carriers with high efficiency and low toxicity.

ASSOCIATED CONTENT Supporting Information Detailed synthesis route of TE, 1H NMR spectra of TE, results of GPC assay, heparin assay, and merged images of EGFP expression could be found in Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors Email address: [email protected] (F.J.X); [email protected] (W. Yuan) The authors declare no competing financial interests. ACKNOWLEDGEMENTS This work was supported by National Natural Science Foundation of China (grant numbers 51403010, 51325304, 51473014 and 51521062), National High-tech R&D Program (863 Program, 2014AA020519), Innovation and Promotion Project of Beijing University of Chemical Technology, and Collaborative Innovation Center for Cardiovascular Disorders, Beijing Anzhen Hospital Affiliated to the Capital Medical University. REFERENCES (1) Sidaway, P. Lung Cancer: Gene Therapy Can Be Safely Delivered in Mice. Nat. Rev. Clin. Oncol. 2015, 33, 143-150. (2) Kotterman, M. A.; Schaffer, D. V. Engineering Adeno-Associated Viruses For Clinical Gene Therapy. Nat. Rev. Genet. 2014, 15, 445-451. (3) Xu, F. J.; Yang, W. T. Polymer Vectors via Controlled/Living Radical Polymerization for Gene 21



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Table of Contents (TOC)

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A Facile Strategy to Prepare Hyperbranched Hydroxyl-Rich

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