Synthesis of Amphiphilic Poly (β-amino ester) for Efficiently Minicircle

Jun 7, 2016 - Chinese Academy of Sciences, Shenzhen 518055, P. R. China ... Research Center for Biomedical Imaging, Shenzhen Key Laboratory for MRI, ...
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Synthesis of Amphiphilic Poly (#-amino ester) for Efficiently Minicircle DNA Delivery in Vivo Jing Zhao, Ping Huang, Zhiyong Wang, Yan Tan, Xiaohu Hou, Liping Zhang, Cheng-Yi He, and Zhi-Ying Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04412 • Publication Date (Web): 07 Jun 2016 Downloaded from http://pubs.acs.org on June 26, 2016

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Synthesis of Amphiphilic Poly (β-amino ester) for Efficiently Minicircle DNA Delivery in Vivo

Jing Zhao †, Ping Huang †, Zhiyong Wang*†,‡, Yan Tan ‡, Xiaohu Hou †, Liping Zhang†, Cheng-Yi He†, Zhi-Ying Chen *†



Center for Gene and Cell Engineering, Institute of Biomedicine and Biotechnology, Shenzhen

Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, P. R. China



Paul C. Lauterbur Research Center for Biomedical Imaging, Shenzhen Key Laboratory for MRI,

Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, P. R. China.

Corresponding authours:

ZYW, [email protected]; ZYC, [email protected]/(85)755 5839 2257

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ABSTRACT: Minicircle DNA (mcDNA) is a kind of enhanced non-viral DNA vector with excellent profiles in biosafety and transgene expression. Herein, we reported a novel amphiphilic polymer comprising polyethylenimine(PEI) modified Poly (β-amino ester) PEI-PBAE(C16) for efficient mcDNA delivery in vivo. The synthesized polymer could condense mcDNA into nanoscaled structure and exhibited efficient gene transfection ability without detectable cytotoxicity. Importantly, when injected into mouse intraperitoneally, these PEI-PBAE(C16) nanocomplexes were able to result in high level of trangene expression which lasted at least 72 hours. Overall, these results demonstrated the PEI-PBAE(C16) can mediate effective and safe gene delivery in vivo with clinical application potential.

KEYWORDS: Poly(β-amino ester); amphiphilic polymer; non-viral vectors; minicircle DNA; gene delivery; nanoparticles

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1.

INTRODUCTION Gene therapy has shown great potentials for treating some serious diseases.

1

Nowadays, building

efficient and safe gene delivery systems is a major challenge for clinical gene therapy.

2, 3

Although

viral vectors are able to mediate gene transfection with high efficiency, adverse immune responses, risky integration and limited size of the loaded gene hinder their further application. But adverse immune responses, risky integration and limited size of the loaded gene hinder their further application.4,5 Minicircle DNA (mcDNA) is a commonly used non-viral vector which is prepared by elimination of plasmid backbone DNA. 6,7 As compared to regular plasmids, mcDNA possess a smaller size without the detrimental bacterial sequences allowing an increased efficiency in reducing immune responses and prolonging transgene expression in vitro and in vivo.8 However, application of mcDNA still need delivery technologies to avoid degradation by nucleases in the bloodstream and cytoplasm. In recent decades, several types of vehicles, including lipids,9 cationic polymers,10 and dendrimers11,12 have been developed as gene transfection agents. One class of synthetic cationic polymer, poly (β-amino esters) (PBAEs), have attracted significant attentions for gene devliey applications because of their biocompatibility, ease of synthesis and high transfection efficiency

13-15

PBAEs are synthesied by Micheal addition of amine and diacrylate, and have a wide chemical diversity.16,17 In addition, the hydrolysis degradable ester groups offer PBAEs the possibility of low cytotoxicity.18 Comparely, polyethleneimine (PEI) which contains numerous amine groups has been applied as an effective transfection agent for several years. PEI can condense DNA into nanoscale, protect them from nucleases and mediate the intracellular transfection.19 However, PEI is not biodegradable and its

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molecular weight is proportional to cytotoxicity, high molecular weight PEI exhibits high cytotoxicity.20,12 In this work, we prepared a novel cationic polymer combining the superior features of PBAE and low molecular weight PEI cationic polymer. As expected, we found that the PEI-PBAE(C16) delivery system has gained multiple enhancements attributed by each of the component. Compared to our previous work,

22

the new synthesied polymer introduced hexadecylamine chain and exhibited

stronger DNA binding ability. The polymer could condense mcDNA into small nanoparticles effectively and mediate high transfection efficacy in 293T cell with low cytotoxicity. Importantly, the PEI-PBAE(C16)/mcDNA composites were able to efficiently express trangene product when intraperitoneally

delivered

to

mouse

without

detectable

toxicity.

Taken

together,

our

PEI-PBAE(C16)/mcDNA system is promising in clinical application and deserve further investigation.

2.

EXPERIMENTAL SECTION 2.1 Materials Dimethyl sulfoxide (DMSO, ≥99.5%), 4-amino-1-butanol (98%), 1,4-butanediol diacrylate (90%),

hexadecylamine (98%) and X-tremeGENE HP DNA Transfection Reagent were purchased from Sigma-Aldrich (Shanghai, China). Chloroform, diethyl ether, cyclohexane and ethanol were bought from Lingfeng Chemical Reagent Co. Ltd (Shanghai, China). Branched PEI with molecular weight of 600 Da was bought from Aladdin Reagent (Shanghai, China). Cy 5.5 (mono-reactive NHS ester) was purchaesd from Fanbo Biochemical (Beijing, China). All these reagents were used without further purification. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo, (Kumamoto, Japan). Fetal bovine serum (FBS) and Dulbecco's Modified Eagle Medium (DMEM) were bought from Shanghai

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Biotch (Shanghai, China). 293T cell (human hepatocarcinoma cell) line was ordered from cell bank of Chinese Academy of Science (Shanghai, China). BALB/C mice (female, 6-10 weeks) were obtained from Guangdong Province Laboratory Animal Center, China (Guangzhou, China). 2.2 Preparation of Minicircle DNA mcDNAs encoding either enhanced green fluorescent protein (mcDNA-eGFP) or luciferase (mcDNA-Luc) reporter genes were prepared as described previously.23 2.3 Synthesis of PBAEs The PEI-PBAE(C16) was synthesized by a two-step reaction as illustrated in Figure 1. Firstly, the poly (β-amino ester) terminated with acrylate (denoted as Acrylate-PBAE(C16)) was prepared as described previously.18 Briefly, 0.167g (0.69 mmoL) of hexadecylamine was placed in a 50 mL flask and incubated at 60oC until it melted. Subsequently, 0.125g (1.4mmoL) of 1,4-Butanediol diacrylate and 0.436g (2.2 mmoL) of 4-amino butanol were added into the above reaction and kept stirring for 48 hours at 90 oC in the argon atmosphere. The product Acrylate-PBAE(C16) was washed twice with diethyl ether and cyclohexane mixed solvent. Secondly, dissolve 0.406g acrylate-PBAE(C16) and 0.551g PEI into 5 mL CHCl3 respectively, and then transfer both of the solution into a round bottom flask. The reaction mixture was kept at room temperature overnight under argon protection. After passing through a Sephadex G-10 size exclusion chromatography column, purified PEI-PBAE(C16) polymer was obtained finally. The chemical structures of the products were confirmed by 1H nuclear magnetic resonance (NMR) analysis (AVANCE III 400 spectrometer, Bruker, Switzerland). Cy 5.5 is a traditional dye with excitation wavelength at 673 nm. Cy 5.5-labeled PEI-PBAE(C16) was synthesized by dissolving 200 mg PEI-PBAE(C16) and 1 mg Cy 5.5 dye in anhydrous ethanol and stirring

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overnight. Obtained polymers were dissolved in DMSO at a concentration of 100mg/mL and stored at -20 oC. 2.4 Characterization of PEI-PBAE(C16)/mcDNA complex The PEI-PBAE(C16)/mcDNA complex was prepared by mixing PEI-PBAE(C16) polymer with mcDNA in aqueous solution at different nitrogen to phosphate (N/P) ratios and incubating for 30 minutes. The complexes were subjected to electrophoresis to examine the association between the mcDMA and polymers. Herein, electrophoresis assay was performed on 1% agarose gel and run in Tris–acetate (TAE) buffer at 90 V for 45 minutes. To characterize the morphology of the corresponding composites, transmission electron microscope was performed on FEI Tecnai G2 F20 S-Twin, 100kV. Dynamic light scattering system (DLS, Malvern Zetasizer Nano ZS, USA) was used to measure particle size and zeta potential. 2.5 In vitro cytotoxicity and gene transfection In vitro cytotoxicity of PEI-PBAE(C16)/mcDNA-eGFP complex, acrylate-PBAE(C16)/mcDNA complex and Roche/mcDNA complex were measured by CCK8 assay, and the transfection efficiencies were determined by fluorescence microscope and flow cytometry. 293T cells were seeded in 96-well plates at an initial density of 1.0 × 104 per well and incubated at 37 oC before treatment. The samples in 100 µL serum-free media were added into each well and incubated for 4 hours. And then, the culture media were replaced with fresh serum-containing media. The cytotoxicities were determined using CCK-8 kits. According to the instruction of the kit, the cells were transfected, washed, and cultured in CCK8 solution at 37 oC for 4 h before the absorbance of each well was measured at 450 nm using a multimode plate Reader (Synergy 4, BioTek). Furthermore, the eGFP positive expressions were

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monitored by fluorescence microscope and the quantitative transfection efficiencies were analyzed using flow cytometry (FC) on a BD FACSCantoTM II. 2.6 In vivo gene expression and biodistribution The in vivo experiments were maintained according to the Guidelines set by the Laboratory Animal Committee of the institute. PEI-PBAE(C16)/mcDNA-luc complexes with different amount of mcDNA (20 ~ 40 µg) in 400 µl 5% glucose solution were prepared and injected into the peritoneal cavity of the BALB/C mice. Meanwhile, 40µg naked mcDNA-Luc served as polymer-free control and Roche/mcDNA-luc complex was applied as positive control. To quantify the luciferase expression level, mice were anesthetized with isoflurane and injected with 200 µl luciferin (1.6 mg/kg body weight). And then, expression level of luciferase in vivo was conveniently monitored by IVIS imaging system (Xenogen IVIS Spectrum, USA). Luciferase fluorescence level in selected areas was denoted as the number of photons/s/cm2/steradian (p/s/cm2/sr). To further determine the tissue types that were transfected, the mice were injected with cy5.5 labeled PEI-PBAE(C16)/mcDNA complex and sacrificed at 24 h post-injections. The cells in the peritoneal fluids were analyzed by flow cytometry. The major organs and tissues (heart, liver, lung, spleen, kidney and fat) were resected and washed before the measurement of luciferase activity.

3.

RESULTS AND DISCUSSION 3.1 Synthesis of PBAE(C16) polymers. As illustrated in Figure 1, the synthesis of PEI-PBAE(C16) polymer was performed by a two-step

reaction. Firstly, acrylate-terminated PBAE(C16) polymer was synthesized via Michael addition

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reaction. Then, this polymer was end-capped with PEI (MW 600). The primary amine groups in PEI could increase the positive charge and improve the hydrophilic property of PBAE. 1H NMR analysis was used to characterize the chemical structures of synthesized polymers. As shown in Figure 2, the structure of PEI-PBAE(C16) was confirmed by the disappearance of peaks at 5.6-6.4 ppm, which represented typical double bonds of acrylate group (Figure 2). The amphiphilic PEI-PBAE(C16) polymer can self-assemble into nano-scaled particles in water and the hydrophilic PEI part form the shell to combine with negative mcDNAs. Usually, when negative charged DNA binds to positively charged polymer via electrostatic interaction. In the electric field, combined DNA may lose their mobility. The Nitrogen/Phosphate ratio (N/P ratio) is an important factor which could affect the combination between polymers and DNA. Agarose gel electrophoresis was performed to evaluate the DNA-binding capability of PEI-PBAE(C16). As shown in Figure 3A, the mcDNA mobility was fully inhibited at N/P ratio 5, suggesting high affinity binding of mcDNA with PEI-PBAE(C16) polymer. Moreover, surface charge and particle size of the vector/DNA complex also affect the cellular uptake and satisfactory transfection.24 Thus, DLS method was used to analyze the particle size and zeta potential of PEI-PBAE(C16)/mcDNA nanocomplexes (Figure 3B). Naked DNA is negatively charged and incompact in aqueous solution. When combine with PEI-PBAE(C16) polymer at N/P ratio 2, the nanocomplexes was nearly electrically neutral, that leaded some mcDNAs to dissociate from nanoparticles and migrate in agarose gel electrophoresis assay (Figure 3A), indicating a poorly compact structure of the complex due to the weak electrostatic attraction. At N/P ratio of 5, stable nanocomplexes with size of 50 nm and high surface charge were obtained. The size and morphology of PEI-PBAE(C16)/DNA nanocomplexes were further conformed by transmission electron microscope

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(TEM) image. As shown in Figure 3C and D, nanocomplexes at N/P ratio 20 were monodispersed and spherical in shape with an average diameter of 45.2 nm. 3.2 In vitro transfection and cytotoxicity of PEI-PBAE(C16)/mcDNA nanocomplexes. Biocompatibility is one of the most critical properties for drug and gene delivery system. In this study, the cytotoxicities of PEI-PBAE(C16)/mcDNA nanocomplexes were evaluated by CCK8 assay in 293T cell line in the presence of 10% FBS. Herein, the commercial available Roche DNA transfection reagent, acrylate-PBAE(C16) polymer without PEI modification and naked mcDNA were applied as contrasts. As shown in Figure 4A, even after 48 h co-inhibition within 293T cells, no obvious toxicity was observed in the PEI-PBAE(C16)/mcDNA nanocomplexes groups even at a high N/P ration of 50, and this result may attribute to the relatively low positive charge density and good degradation property of PEI-PBAE(C16) polymer. Meanwhile, commercial Roche transfection reagent showed significant cytotoxicity in 293T cells, and there are only 60% cells survived after 48 h. It has reported that the end-modification with PEI could enhance the cellular uptake and endosomal escape of DNA from lysosome.

20,25

In order to evaluate the transfection ability, the in vitro gene

expression test were studied by expressing mcDNA-eGFP at various N/P ratios in 293T cell line. The transfection efficiencies were detected using flow cytometer and fluorescent microscopy. As shown in Figure 4B and C, there was no eGFP expression in Acrylate-PBAE(C16)/mcDNA and naked mcDNA groups. Roche transfection reagent displayed the highest transfection activity (70.7%). In the PEI-PBAE(C16)/mcDNA nanocomplexes group, the highest gene transfection was observed when the N/P ratio was 20 (53.56%), which may be due to the nanocomplexes’ appropriate surface charge and

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relative good biocompatibility. These results demonstrate that introducing of high positive charge density PEI to PBAE(C16) polymer could improve their gene transfection ability.

3.3 Bioluminescent imaging of gene expression in vivo.

Intraperitoneal delivery of drugs and functional gene is thought to be a promising approach to treat some diseases such as peritoneal dissemination and peritoneal fibrosis.26,27 Herein, PEI-PBAE(C16) polymer was employed to deliver bioluminescent reporter gene into mice abdominal cavity. Bioluminescent imaging (BLI) technology was employed for non-invasive evaluation of luciferase expression in small laboratory animal models.28 Firefly-luciferase expression in vivo can be quantitatively measured by IVIS imaging system.29 As shown in Figure 5A, there was no bioluminescence signal from the naked mcDNA and Roche reagent groups. After treated with PEI-PBAE(C16)/mcDNA nanocomplexes, the bioluminescent signals were clearly observed in the abdominal cavity. With the incensement of DNA mass, the bioluminescent expression was obviously enhanced (Figure 5A and B). Then, it was found that the highest gene expression was obtained when the N/P ratio was 20 (Figure 5C and D), which was consistent with the in vitro transfection in cell line. The maximal bioluminescent signal presented at 8 h post-injection and then gradually decreased in the next 3 days. Importantly, those injections did not cause any mice death even at high PEI-PBAE(C16) amount and high N/P ratio. These results revealed that PEI-PBAE(C16) cationic polymer can encapsulate and deliver mcDNA into mice abdominal cavity with high transfection efficiency in vivo. To further ascertain the transfected tissues, another group of mouse was sacrificed and cut abdomens open at 24 h post-injection. The main mcDNA-luc expression was found in the abdominal cavity as

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shown in Figure 6A. Then, the liver, lung, kidney, heart, spleen and mesenteric adipose tissue were isolated and the bioluminescence intensity readings were performed on each tissue. It was found that the transgene expression signals were mainly detected from mesenteric adipose tissue and the signal intensities were more than 10-fold higher than the expression levels in other organs (Figure 6B and C).

Furthermore, PEI-PBAE(C16)/mcDNA nanocomplexes labeled with Cy 5.5 were injected slowly to peritoneal cavity and the biodistribution of complex was detected at 24 h post-injection. The mice were sacrificed and scanned by IVIS fluorescence imaging system. It was found that the majority of red fluorescence signal (Ex = 700 nm) were detected mainly in liver, kidney and mesenteric adipose tissue (Figure 7A). Then, we diluted mice ascites with phosphate buffered saline (PBS) and analyzed by flow cytometry. The peritoneal macrophage (P1, 16.26 %) of naked mcDNA treated mice was selected (Figure 7B(1)).

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As showed in Figure 7B(3), the proportion of peritoneal macrophage (P1) in ascites

was obvious growth after treated with PEI-PBAE(C16)/mcDNA nanocomplexes, which can be attributed to the stimulation of the positive charged nanoparticles. The fluorescence intensity value of 102.2-105 was selected as the threshold of Cy 5.5 positive to deduct the background of untreated cells. Among these selected cells, the Cy 5.5 positive ratio was 62.13% in PEI-PBAE(C16)/mcDNA treated mice (Figure 7B(4)) while only 1.80% positive in naked mcDNA treated group (Figure 7B(2)). Herein, we hypothesized that a part of the PEI-PBAE(C16)/mcDNA nanoparticles were taken up by mesenteric endothelial cells and then successfully expressed, while another part of the nanoparticles were taken by peritoneal macrophage, but degraded rapidly by the enzyme system of macrophage without transgene. In the end, the PEI-PBAE(C16) polymers were absorbed through the peritoneal capillaries entering the

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blood circulation and metabolized by liver and kidney in this mouse model, protecting other organs from side effects.

4.

CONCLUSION In summary, we successfully synthesized a novel amphiphilic PEI-modified PBAE polymer for

efficient mcDNAs delivery. This cationic polymer binds with mcDNA and compress mcDNA into stable nanocomplexes with positive zeta potential. Compared to the commercial DNA transfection reagent, the PEI-PBAE(C16) polymer showed lower cytotoxicity in 293T cell in vitro. When injected into mice abdominal cavity, PEI-PBAE(C16) could efficiently delivery mcDNA into abdominal mesenteric adipose tissues and continuously express functional gene for more than 72 hours. In the end, the PEI-PBAE(C16) polymer can be metabolized by liver and kidney, and protect other healthy organs from cumulative toxicity. Overall, the PEI-PBAE(C16) polymer might have a promising potential as the non-viral gene carrier for future clinical use.

AUTHOR INFORMATION

Corresponding Authors: ZYW, [email protected]; ZYC, [email protected]

J Z and P H contributed equally to this work.

ACKNOWLEDGMENT

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The authors gratefully acknowledge the support for this research by Natural Science Foundation of GuangDong Province of China (2014A030310335), the National Natural Science Foundation of China (NSFC) (Grant no. 51203177 and 81471778), the National High Technology Research and Development Program (863 Program) of China (Grant no. 2014AA020708), the China Postdoctocal Science Foundation (2015M582446) and Science and Technology Foundation of Shenzhen, China (Grant No. SFG2012.566, SKC2012.237, JCYJ20150521094519466 and JCYJ20140417113430662)

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Nanoparticles with Reduced Cytotoxicity for Gene Delivery Applications. Nanosci. Nanotechnol. 2015, 15, 4094-4105. 13. Harris, T. J.; Green, J. J.; Fung, P. W.; Langer, R.; Anderson, D. G.; Bhatia, S. N. Tissue-specific Gene Delivery via Nanoparticle Coating. Biomaterials 2010, 31, 998-1006. 14. Zugates, G. T.; Little, S. R.; Anderson, D. G.; Langer, R. Poly(beta-amino ester)s for DNA delivery. Isr. J. Chem. 2005, 45, 477-485. 15. Chen, Y.; Li, Y.; Gao, J.; Cao, Z.; Jiang, Q.; Liu, J.; Jiang, Z. Enzymatic PEGylated Poly(lactone-co-beta-amino ester) Nanoparticles as Biodegradable. Acs Appl. Mater. Interfaces 2016, 8, 490-501. 16. Segovia, N.; Dosta, P.; Cascante, A.; Ramos, V.; Borros, S. Oligopeptide-terminated Poly(beta-amino ester)s for Highly Efficient Gene Delivery and Intracellular localization. Acta Biomater. 2014, 10, 2147-2158. 17. Memanishvili, T.; Zavradashvili, N.; Kupatadze, N.; Tugushi, D.; Gverdtsiteli, M.; Torchilin, V. P.; Wandrey, C.; Baldi, L.; Manoli, S. S.; Katsarava, R. Arginine-Based Biodegradable Ether-Ester Polymers with Low Cytotoxicity as Potential Gene Carriers. Biomacromolecules 2014, 15, 2839-2848. 18. Lynn, D. M.; Langer, R. Degradable Poly(beta-amino esters): Synthesis, Characterization, and Self-assembly with Plasmid DNA. J. Am. Chem. Soc. 2000, 122, 10761-10768. 19. Godbey, W. T.; Wu, K. K.; Mikos, A. G. Poly(ethylenimine) and Its Role in Gene Delivery. J. Controlled Release 1999, 60, 149-160. 20. Lv, H.; Zhang, S.; Wang, B.; Cui, S.; Yan, J., Toxicity of Cationic Lipids and Cationic Polymers in Gene Delivery. J. Controlled Release 2006, 114, 100-109. 21. Meneksedag-Erol, D.; Bahadur, R. K. C.; Tang, T.; Uludag, H. A Delicate Balance when Substituting a Small Hydrophobe onto Low Molecular Weight Polyethylenimine to Improve its Nucleic Acid Delivery Efficiency. Acs Appl. Mater. Interfaces 2015, 7, 24822-24832. 22. Zhao, J.; Yang, L.; Huang, P.; Wang, Z.; Tan, Y.; Liu, H.; Pan, J.; He, C.-Y.; Chen, Z.-Y., J. Synthesis and Characterization of Low Molecular Weight Plyethyleneimine -terminated Poly(beta-amino ester) for Highly Efficient Gene Delivery of Minicircle DNA. J. Colloid Interf. Sci. 2016, 463, 93-98. 23. Hou, X. H.; Guo, X. Y.; Chen, Y.; He, C.-Y.; Chen, Z.-Y., Mol. Increasing the Minicircle DNA Purity Using an Enhanced Triplex DNA Technology to Eliminate DNA Contaminants. Mol. Ther. --Methods Clin. Dev. 2015, 1, 14062-14062. 24. Shan, Y.; Luo, T.; Peng, C.; Sheng, R.; Cao, A.; Cao, X.; Shen, M.; Guo, R.; Tomas, H.; Shi, X. Gene Delivery using Dendrimer-entrapped Gold Nanoparticles as Nonviral Vectors. Biomaterials 2012, 33, 3025-3035. 25. Tang, S.; Yin, Q.; Zhang, Z.; Gu, W.; Chen, L.; Yu, H.; Huang, Y.; Chen, X.; Xu, M.; Li, Y. Co-delivery of Doxorubicin and RNA using pH-sensitive Poly (beta-amino ester) Nanoparticles For Reversal of Multidrug Resistance of Breast Cancer. Biomaterials 2014, 35, 6047-6059. 26. Hallaj-Nezhadi, S.; Dass, C. R.; Lotfipour, F. Intraperitoneal Delivery of Nanoparticles for Cancer Gene Therapy. Future Oncol. 2013, 9 (1), 59-68. 27. Flessner, M. F. The Transport Barrier in Intraperitoneal Therapy. Am. J. Physiol-Renal 2005, 288, 433-442.

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28. Xia, J.; Martinez, A.; Daniell, H.; Ebert, S. N. Evaluation of Biolistic Gene Transfer Methods in vivo using Non-invasive Bioluminescent Imaging Techniques. Bmc Biotechnol. 2011, 11, DOI: 10.1186. 29. Kumar, J.; Kale, V.; Limaye, L. Umbilical Cord Blood-derived CD11c(+) Dendritic Cells Could Serve as an Alternative Allogeneic Source of Dendritic Cells for Cancer Immunotherapy. Stem Cell Res. Ther. 2015, 6, 1-15. 30. Dimitrijevic, M.; Pilipovic, I.; Stanojevic, S.; Mitic, K.; Radojevic, K.; Pesic, V.; Leposavic, G. Chronic Propranolol Treatment Affects Expression of Adrenoceptors on Peritoneal Macrophages and Their Ability to Produce Hydrogen Peroxide and Nitric Oxide. J. Neuroimmunol. 2009, 211, 56-65.

Figures:

Figure 1: Scheme illustration of the preparation of PEI-PBAE(C16) polymer.

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Figure 2. (A) The 1H NMR spectra and (B) structural formulas of PBAEs. (1) : acrylate-PBAE(C16) and (2): PEI-PBAE(C16).

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Figure 3. (A) Agarose gel electrophoretic of naked mcDNA and PEI-PBAE(C16)/mcDNA nanocomplexes (N/P ratio of 2, 5, 10, 20, 50, 100); (B) Average Size and zeta potential profiles PEI-PBAE(C16)/mcDNA complexes; (C) TEM image and (D) particle size distribution profile of PEI-PBAE(C16)/mcDNA (N/P ratio 20).

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Figure 4. (A) Cell viability profiles, (B) cell transfection efficiency and (C) Fluorescent microscope images of eGFP expressions in 293 T cell line treated with PEI-PBAE(C16)/mcDNA nanocomplexes over 48h. Naked mcDNA, acrylate-PBAE(C16)/mcDNA composites and Roche/mcDNA composites were used as contrast. Ⅰ: 293T cells without any treatment as blank control; Ⅱ:293T cells incubated with naked mcDNA (0.5 µg per well); Ⅲ: 293T cells incubated with acrylate-PBAE(C16)/mcDNA composites (N/P of 20); Ⅳ: 293T cells incubated with Roche/mcDNA composites (1µg sample mixed with 1µg mcDNA).

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Figure 5. In vivo luciferase transgene expressions in mice after intraperitoneal injection of naked DNA, Roche/mcDNA composites and PEI-PBAE(C16)/mcDNA nanocomplexes respectively. (A) Whole mouse bioluminescence imaging at 24 h post-injection and (B) luciferase expression analysis of mice injected with different dose of mcDNA-Luc which combined with PEI-PBAE(C16) (N/P ratio 20). (C) Whole mouse bioluminescence imaging after 24 h injections and (D) luciferase expression analysis of mice injected with totally 20 µg of mcDNA-Luc which combined with PEI-PBAE(C16) (N/P ratio of 5, 10, 15, 20, 30). Here, Roche/mcDNA complex and the naked DNA were employed as contrast ones.

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Figure 6. (A) Whole mouse bioluminescence imaging after intraperitoneal injection of PEI-PBAE(C16)/mcDNA-luc nanocomplexes (20 µg mcDNA-Luc, N/P ratio 20) and 20 µg naked mcDNA as contrast group at 24 h post-injection. (B) Luciferase expression analysis and (C) bioluminescence images of heart, liver, spleen, lung, kidney and abdominal adipose tissues of mouse after intraperitoneal injection of PEI-PBAE(C16)/mcDNA nanocomplexes (20 µg mcDNA-Luc, N/P ratio 20) at 24 h post injection.

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Figure 7. (A) Florescence imaging of heart, liver, spleen, lung, kidney and abdominal adipose tissues after treatment of PEI-PBAE(C16) nanocomplexes at N/P 20 with 20µg mcDNA and 20 µg naked mcDNA in 5% glucose solution as negative control. (B) Flow cytometry measurement of peritoneal macrophage harvested from the mice ascitic fluid. (1) and (2) were ascitic fluid from the untreated mice as contrast, (3) and (4) were ascitic fluid from the mice treated with PEI-PBAE(C16)/mcDNA nanocomplexes at 24 h post-injection.

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