International Journal of Pharmaceutics 465 (2014) 112–119
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The strategy to improve gene transfection efficiency and biocompatibility of hyperbranched PAMAM with the cooperation of PEGylated hyperbranched PAMAM Yangfei Sun a , Yunfeng Jiao b , Yang Wang b , Daru Lu a , Wuli Yang b, * a b
State Key Laboratory of Genetic Engineering and School of Life Sciences, Fudan University, Shanghai 200433, China State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Fudan University, Shanghai 200433, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 September 2013 Received in revised form 4 January 2014 Accepted 8 February 2014 Available online 12 February 2014
As a promising non-viral gene vector, cationic polyamidoamine (PAMAM) dendrimer could form complexes with negative charged DNA to mediate efficient gene delivery in vitro and in vivo. However, complicated synthesis technology and potential cytotoxicity limited their application in clinical translational researches. Hyperbranched polyamidoamine (h-PAMAM), which could be synthesized by a simpler one-pot method, has similar properties with PAMAM, and PEGylation modification of h-PAMAM has been used to reduce cytotoxicity. Here we prepared gene delivery system with h-PAMAM and hPAMAM derivative h-PAMAM-g-PEG, respectively and found that the viability of cells with h-PAMAM-gPEG was quite higher in comparison with cells with unmodified h-PAMAM. However, gene delivery efficiency was lower with h-PAMAM-g-PEG. Then we used mixture composed of h-PAMAM and hPAMAM-g-PEG and such composition was designed to reduce cytotoxicity while maintaining high transfection efficiency. Our results indicated that this mixture system of h-PAMAM and h-PAMAM-g-PEG achieved higher transfection efficiency and lower cytotoxicity compared with h-PAMAM-only system. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Gene delivery Hyperbranched polyamidoamine PEGylation Cytotoxicity Mixture system
1. Introduction Reliable and efficient gene delivery system is essential for successful gene therapy. Currently gene delivery researches mainly focus on two systems: viral vector-based and non-viral gene delivery system. Nowadays many basic researches and clinical trials employ viral vectors for gene delivery in view of their higher gene transfection efficiency. However, viral vectors have several limitations such as lower DNA carrying capability, potential immunogenicity and recombination. The safety of viral vectors was challenged severely with gene therapy death (Marshall, 1999) and leukemia development (Buckley, 2002) in patients (Giacca and Zacchigna, 2012). With such considerations, non-viral gene delivery system becomes more and more attractive and many investigations are underway to design and improve non-viral gene delivery system for future clinical trials (Ibraheem et al., 2014; Luo and Saltzman, 2000).
* Corresponding author at: State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Fudan University, No. 220 Handan Road, Shanghai 200433, China. Tel.: +86 21 6564 2385; fax: +86 21 6564 0293. E-mail address: [email protected] (W. Yang). http://dx.doi.org/10.1016/j.ijpharm.2014.02.018 0378-5173/ã 2014 Elsevier B.V. All rights reserved.
Of non-viral vectors, cationic polymers are particularly remarkable because of their positive surface charges making sufficient interaction with plasmid DNA (pDNA) and efficient gene delivery (De Smedt et al., 2000; Shcharbin et al., 2013), and versatile controlled surface modifications make cationic polymers more biocompatible and suitable for targeted therapy (Shcharbin et al., 2013). One of the cationic polymers, polyamidoamine (PAMAM) dendrimer, predominates the research field of non-viral vectors, which owes to its special characteristics: gene delivery with PAMAM could result in higher gene transfection efficiency and lower cytotoxicity compared with other cationic polymers (Cloninger, 2002; Daneshvar, 2013; Lee et al., 2005). While PAMAM dendrimer is dramatic in non-viral gene delivery research and commercially available, it cannot be denied that their synthesis technology is quite complicated and costly (Tomalia and Frechet, 2002), which limits their application in clinical translational researches. Hyperbranched polyamidoamine (h-PAMAM), which could be prepared by a simple one-pot method, has similar chemical and physical characteristics with PAMAM dendrimer (Cao et al., 2007). It could also mediate efficient gene delivery into skeletal myoblasts (Zhu et al., 2011) and cell lines such as COS7 and 293T cells (Zhu et al., 2012). Although h-PAMAM-mediated gene delivery has lower cytotoxicity in comparison to other cationic
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polymers, further biocompatibility improvement is still required for possible clinical trials (Chauhan et al., 2010; Fischer et al., 2003; Malik et al., 2000; Roberts et al., 1996). As mentioned previously, surface modification of PAMAM, such as acetylation (Kolhatkar et al., 2007), L-arginine (Bai et al., 2013; Son et al., 2013) or PEGylation (Kim et al., 2008; Luo et al., 2002), could reduce cytotoxicity significantly and increase the circulation time in vivo (Jevprasesphant et al., 2003; Shah et al., 2013). Here, in order to develop an efficient and reliable gene delivery system, we synthesized h-PAMAM derivative h-PAMAM-g-PEG with h-PAMAM surface PEGylation (Chen et al., 2012). Plasmid DNA expressing EGFP was transfected into 293T cells with h-PAMAM-gPEG and h-PAMAM, respectively. Results showed that the viability of cells with h-PAMAM-g-PEG-mediated transfection was quite higher in comparison with cells transfected with unmodified h-PAMAM. However, the gene delivery efficiency was lower with h-PAMAM-gPEG, which was consistent with previous studies (Fant et al., 2010). To achieve higher gene transfection efficiency while maintain lower cytotoxicity, we prepared the composites consist of h-PAMAM-gPEG and unmodified h-PAMAM, hoping that such a mixed system would be better than any individual system (Debus et al., 2010). We found that the result was in consistent with our expectations and to our surprise, a mixed system consisting of 70% h-PAMAM and 30% hPAMAM-g-PEG achieved even higher transfection efficiency compared with other h-PAMAM-g-PEG/h-PAMAM ratios. 2. Material and methods 2.1. Materials MeO-PEG-OH (99%, Mw = 2000) was purchased from Fluka. Methyl acrylate (MA) (99%) and diethylene triamine (DETA) (99%) were purchased from Shanghai Chemical Reagent Company. N,N0 Dimethylformamide and diethyl ether were analytical purity, and N,N-dimethylformamide was dried by distillation with CaH2 before used. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and other biological reagents were purchased from Invitrogen Corp. The pEGFP-N2 was prepared with MACHEREY-NAGEL NucleoBond Xtra Midi Plus kit. 2.2. Synthesis of h-PAMAM h-PAMAM was synthesized according to the method previously reported by our lab (Cao et al., 2007). 20 g of diethylene triamine was measured and dissolved to 30 mL of methanol in a 100 mL of three-necked bottle. Then, the mixture of 30 mL of methanol and 20 g of methyl acrylate was added drop-wise into the reaction flask in nitrogen atmosphere for 30 min. The nitrogen flow was removed and the mixture was stirred at room temperature for other 72 h. Then the flask was equipped onto a rotary evaporator to remove the methanol under the vacuum. The process was performed for 1 h at 60 C, for 1 h at 80 C, for 1.5 h at 100 C, for 1.5 h at 120 C, and for 3 h at 140 C. The product was dissolved in 30 mL of deionized water, and the resulted solution was dialyzed against water for 3 days to eliminate any unreacted monomers. Finally, the product was precipitated in acetone and dried under the vacuum. 2.3. Synthesis of h-PAMAM-g-PEG Before the condensation reaction between h-PAMAM and MeOPEG-OH, poly(ethylene glycol) succinimidecarbonate (PEG-SC) was synthesized first as described briefly below. Ten grams of MeOPEG-OH (Mw = 2000) and 7.69 g of N,N0 -disuccinimide carbonate (DSC) were added to 30 mL of N,N0 -dimethylformamide (DMF) to form a white emulsion in a 100 mL flask. Then, 6.07 g of triethylamine was added to the mixture as the catalyst. The reaction
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mixture was incubated at 30 C for 24 h with stirring. The product was precipitated in diethyl ether, and the obtained sample was dissolved in toluene to remove the insoluble substance. The supernatant was precipitated in diethyl ether again, and then the product was dried under the vacuum. h-PAMAM-g-PEG was synthesized through the substitution reaction of succinimide groups of PEG-SC with amino groups of h-PAMAM (Ji et al., 2009). The solution of 0.56 g of PEG-SC in 2 mL of DMF was added drop-wise to the solution of 0.63 g of h-PAMAM in 3.5 mL of DMF. The mixture reaction was stirred at 30 C for 72 h. The product was precipitated in diethyl ether, and was further purified via dialysis in water for 3 days. Finally, the product was retrieved by freeze drying from the aqueous solution and kept in the sealed container. 2.4. Gel retardation assay To investigate the DNA condensation effect of h-PAMAM, hPAMAM-g-PEG and h-PAMAM/h-PAMAM-g-PEG mixture, gel retardation assay was performed. Briefly, we prepared h-PAMAM solution, h-PAMAM-g-PEG solution, h-PAMAM/h-PAMAM-g-PEG mixture with 70% h-PAMAM solution and DNA (pEGFP-N2) solution respectively. Different volumes of solutions were mixed to achieve specified N/P ratios (the ratios between moles of the amine groups of h-PAMAM/h-PAMAM-g-PEG and those of the DNA phosphate groups). Mixture was vortexed slightly for 30 s and incubated at room temperature for 30 min. And then, all these samples were mixed with loading buffer and loaded on 0.8% (w/v) agarose gel for electrophoresis. The gel was ran at 100 V for 40 min and photographed under UV light. 2.5. Cytotoxicity analysis The cytotoxicity of h-PAMAM, h-PAMAM-g-PEG and the mixture of h-PAMAM and h-PAMAM-g-PEG with a molar ratio of 7/3 was determined in 293T cells using MTT assay. Briefly, 293T cells were seeded in 96-well plates (Corning) and incubated at 37 C and 5% CO2 for 24 h. Then, different materials were added into wells at variant concentrations including 0.01, 0.05, 0.2, 0.5, 1 and 2 mg/mL. Twenty-four hours later, 200 mL of MTT solution (0.1 mg/mL) was added into each well, and the well was allowed to react for 4 h at 37 C and 5% CO2. Then the medium of each well was replaced with 200 mL of DMSO and the plate was under constant shaking until formazan dissolved. The absorbance at 570 nm was measured with a microplate reader, while the absorbance at 630 nm was measured as the background. Untreated cells were defined as 100% viable. All toxicity assays were carried out in triplicate. 2.6. In vitro gene transfection with 293T cells In vitro gene transfection efficiency with the polymers was tested in 293T cells. Briefly, 293T cells were seeded in 12-well plate at a density of 1.5 105 cells per well. Gene transfection was performed 24 h later. And h-PAMAM/h-PAMAM-g-PEG–DNA complex was prepared at different N/P ratios as described in gel retardation assay. After incubation for 30 min at room temperature, polymer–DNA complex was transfected into 293T cells at a final concentration of 2 mg DNA/well. Culture medium was changed after incubation for 4 h and cell culture was continued for another 48 h. Then cells were washed with 1 PBS (pH 7.4) and imaging was performed with fluorescent microscope. 2.7. Flow cytometry Gene transfection with h-PAMAM/h-PAMAM-g-PEG was quantified by flow cytometry. 293T cells were transfected with hPAMAM/h-PAMAM-g-PEG–DNA complex as described above. After
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Fig. 1. The structures of (a) h-PAMAM and (b) h-PAMAM-g-PEG polymers.
transfection for 48 h, 293T cells were washed with 1 mL PBS and detached by adding 200 mL 0.025% trypsin per well. After incubation for 2 min at 37 C, 800 mL culture medium was added to resuspend 293T cells. Then after centrifuging for 5 min at 500 rpm, cells were washed with PBS and resuspended with 200 mL PBS and finally incubated on ice. GFP expression was analyzed by flow cytometry system from Beckman Coulter. Experiments were performed at least in triplicates. 2.8. Characterization The zeta potential and the hydrodynamic diameter of the particles were measured by a Zetasizer Nano-ZS (Malvern) at pH 7.4 at 25 C. Transmission electron microscopy (TEM) images were obtained on a Hitachi H-600 transmission electron microscope. Flow cytometry was performed with Moflo XDP (Beckman Coulter) and cell imaging experiments were performed with Nikon microscope DS-Ri1. To perform MTT experiments, we used SpectraMax M5 Microplate Reader (Molecular Devices Corporation) to measure related absorbance. 3. Results 3.1. Characterization of polymer–DNA complexes h-PAMAM was synthesized through the Michael addition between MA and DETA. Its structure was shown in Fig 1a and its molecular weight was about 5600 Da measured by GPC (Chen et al., 2012). Then, the PEGylation of h-PAMAM (h-PAMAM-g-PEG) was achieved by the condensation reaction between h-PAMAM and MeO-PEG-OH. The molecular weight of the obtained product was about 11700 Da measured by GPC (Chen et al., 2012), suggesting the number of the grafted PEG was 3 (Fig. 1b). The interactions between the h-PAMAM and plasmid DNA could lead DNA embedded in the polymers. h-PAMAM/pDNA complex was prepared with the N/P ratio of 10/1. First, the structure of hPAMAM/pDNA complexes was investigated by TEM. As shown in Fig. 2a, the complexes formed spherical nanoparticles and appeared between 50 and 150 nm under TEM. The hydrodynamic diameter measured by DLS was 406 nm and the polydispersity index was 0.12. The hydrodynamic diameter measured by DLS was much larger than the size obtained from the TEM image because of a
hydrate layer existing on the surface of the complex in the medium. The zeta-potential of the complexes was +42.1 mV due to the abundant amino groups on the surface. In addition, the influence of the addition of h-PAMAM-g-PEG to the complex structure was also investigated. As shown in Fig. 2b, when the mixture of h-PAMAM and h-PAMAM-g-PEG with a molar ratio of 7/3 was employed as the carrier, the complex still appeared spherical nanoparticles and the similar size with pure h-PAMAM/pDNA complex. Its hydrodynamic diameter measured by DLS was 489 nm and the polydispersity index was 0.21. The zeta-potential showed +32 mV and was lower than the h-PAMAM/pDNA complex. This result might be attributed to the grafted uncharged PEG chains, which was proved by the result that the zeta-potential of the pure h-PAMAM-g-PEG/pDNA complex was just +18 mV. 3.2. DNA condensation and encapsulation by h-PAMAM and hPAMAM-g-PEG High DNA condensation and encapsulation is essential for gene delivery by cationic polymers. Here we used gel retardation assay to analyze h-PAMAM or h-PAMAM-g-PEG-mediated DNA condensation. Results showed that DNA could be condensed efficiently by hPAMAM and h-PAMAM-g-PEG (Fig. 3a and b). Complete DNA condensation could be achieved at N/P 3. We also noticed that DNA condensation mediated by the composites with 70% h-PAMAM was quite similar with either h-PAMAM or h-PAMAM-g-PEG (Fig. 3c). 3.3. Cytotoxicity of the materials An ideal gene delivery system should have maximal delivery efficiency with minimal cellular toxicity. So here we used MTT assay to check the influence of h-PAMAM, h-PAMAM-g-PEG and the mixture of h-PAMAM and h-PAMAM-g-PEG on cell survival. As shown in Fig. 4, at low concentrations such as 0.05 mg/mL, the relative viability of h-PAMAM-treated 293T cells was about 90%, and h-PAMAM showed concentration-dependent toxicity on 293T cells. We also noticed that PEGylation lowered the cytotoxicity of hPAMAM significantly. Even at the concentrations of 2 mg/mL, the relative cell viability was still around 90% compared with 60% in hPAMAM treated cells. The cytotoxicity of mixture system composed of h-PAMAM and h-PAMAM-g-PEG was similar to that of h-PAMAMg-PEG only system when the concentration was lower than 1 mg/
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Fig. 2. TEM images of (a) h-PAMAM/pDNA complex at N/P ratio of 10 and (b) the mixture of h-PAMAM and h-PAMAM-g-PEG with a molar ratio of 7/3 combined with plasmid DNA at N/P ratio of 10.
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Fig. 3. Gel retardation assay of (a) h-PAMAM, (b) h-PAMAM-g-PEG, (c) h-PAMAM and h-PAMAM-g-PEG mixture with a molar ratio of 7/3. M: size marker; 1: naked DNA; 2: N/ P = 1; 3: N/P = 3; 4: N/P = 4; 5: N/P = 6; 6: N/P = 8; 7: N/P = 10; 8: N/P = 15.
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Fig. 4. MTT assay for cellular toxicities of h-PAMAM, h-PAMAM-g-PEG and the mixture of h-PAMAM and h-PAMAM-g-PEG with a molar ratio of 7/3 at different concentrations in 293T cells.
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Fig. 5. The EGFP expression in 293T cells transfected with h-PAMAM/pDNA complexes for 4 h at variant N/P ratios: 3:1, 6:1, 8:1, 10:1 and 15:1.
mL. Previous researches have shown that PEGylation modification of PAMAM could reduce the cellular toxicity of PAMAM (Fant et al., 2010). These results indicated that PEGylation would be a promising modification for the toxicity improvement of hPAMAM-mediated gene delivery. 3.4. In vitro gene delivery efficiency analysis To achieve higher gene delivery efficiency and lower cytotoxicity with h-PAMAM and h-PAMAM-g-PEG, we initially investigated the influence of N/P ratio on gene delivery with h-PAMAM. As gel retardation assay indicated that DNA could be condensed
completely at N/P 3, we prepared complexes with different N/P ratios including 3, 6, 8, 10, 15, 20 and 30. In vitro transfection with pEGFP-N2 in 293T cells (4 h) showed that higher N/P ratio resulted in higher gene delivery efficiency and from N/P 3 to N/P 10, this efficiency increase was dose-dependent (Fig. 5). However, it was improved little when N/P ratio was changed from 10 to 15 (Fig. 5), indicating that gene delivery was saturated when N/P ratio achieved 10 in 293T cells. On the other hand, partial cell float at N/P 20 and massive cell shrinkage at N/P 30 were observed for hPAMAM system. In contrast, almost all cells had normal morphology when the transfection was performed with h-PAMAM-g-PEG even at N/P 30.
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Fig. 6. The transfection efficiency of the h-PAMAM and h-PAMAM-g-PEG mixed system at N/P 10:1 for 4 h with the various molar ratios between h-PAMAM and h-PAMAM-gPEG: 0:10, 3:7, 5:5, 7:3 and 10:0.
This was consistent with the cytotoxicity results, which showed that h-PAMAM had higher cellular toxicity while h-PAMAM-g-PEG was well tolerated by 293T cells. But previous studies indicated that gene delivery efficiency mediated by PAMAM-g-PEG was quite lower (Fant et al., 2010). So we prepared mixtures composed of hPAMAM and h-PAMAM-g-PEG with different ratios and investigated their effect on gene delivery. This mixed system resulted in much better transfection efficiency compared with h-PAMAM-g-PEGonly system. As the peak of h-PAMAM-mediated gene delivery efficiency arose at N/P 10, and previous studies have showed that
DNA could be condensed sufficiently at N/P 3 with PEI while gene delivery efficiency was highest only when N/P 10, and they proposed that 7 portions of PEI chains were in free, which were essential for gene delivery in cell lines (Yue et al., 2011), we also tested the transfection efficiency of mixed system at N/P 10, while changing the ratio of h-PAMAM and h-PAMAM-g-PEG from 1/0 to 0/ 1 (Fig. 6). We found that mixed system containing 70% h-PAMAM at N/P 10 achieved even higher pDNA delivery efficiency compared to 100% h-PAMAM. As fluorescent microscopy could not provide quantitative evaluation of GFP transfection efficiency, we used
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Fig. 7. Percentage of GFP-positive cells analyzed by flow cytometry. (a) Negative control cells; cells transfected with h-PAMAM and h-PAMAM-g-PEG mixture at N/P 10 with different molar ratios: (b) 5:5, (c) 7:3, (d) 10:0.
sensitive flow cytometry to evaluate the percentage of GFP-positive cells transfected with nanoparticles at different ratios. In consistent with fluorescent imaging results, flow cytometry showed that the percentage of GFP-positive cells was 67.4% in cells transfected with h-PAMAM/h-PAMAM-g-PEG mixed system containing 70% hPAMAM, which was the highest compared with those transfected with the system containing 50% h-PAMAM (35.5%) or 100% hPAMAM (46.5%) (Fig. 7). 4. Discussion Many genetic disorders are caused by loss-of-function mutations in specific genes and the delivery of normal genes is viewed as a promising therapy method for such diseases. An efficient pDNA delivery system with low cellular toxicity is necessary to achieve preconceived gene therapy effects for disorders mentioned above. In current study, we chose h-PAMAM to investigate the gene delivery strategies with higher transfection efficiency, which has similar characteristics with PAMAM dendrimer but only need much simpler and low cost preparation method.
On the basis of above results, we demonstrated that PEGylation of h-PAMAM could reduce the cellular toxicity quite significantly, and both h-PAMAM and h-PAMAM-g-PEG possessed the high DNA condensation and encapsulation capacity. Though h-PAMAM-g-PEG-mediated gene transfection had lower cytotoxicity compared with h-PAMAM, its gene delivery efficiency reduced simultaneously. This coincided well with previous studies (Fant et al., 2010). Initially we tried to improve transfection efficiency by increasing the molar ratio of nitrogen from h-PAMAM-g-PEG to phosphate from DNA (N/P) but little improvement could be achieved. Several factors may contribute to this reduction: firstly, PEGylated h-PAMAM has lower capacity to condense pDNA and keep complexes stable for periods of time (Fant et al., 2010); secondly and mainly, as PAMAM endocytosis is dependent on surface charge, PEGylation modification reduces the surface charges and may affect h-PAMAM-g-PEG mediated cell transfection, just as showed above that the zeta-potential of the hPAMAM-g-PEG/pDNA complex was much lower than h-PAMAM /pDNA complex because of the existence of grafted uncharged PEG chains.
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In view of their respective features, we considered that the hPAMAM/h-PAMAM-g-PEG mixture system could deliver pDNAs safely and effectively in 293T cells. In fact, we indeed found that the addition of h-PAMAM to the h-PAMAM-g-PEG system improved gene delivery efficiency quite significantly and a mixture system containing 70% h-PAMAM achieved even higher gene transfection efficiency compared to h-PAMAM-only system. The results were in consistent with previous researches showing that blends of PEI and PEI-PEG resulted in higher mRNA transfection efficiency and lower cytotoxicity than PEI or PEI-PEG alone (Debus et al., 2010). Previous studies explored the possible mechanisms of PEI-mediated gene transfection, suggesting that 3 portions of PEI would condense DNA completely while extra 7 portions of free PEI were essential for efficient gene transfection (Yue et al., 2011). So it is quite possible that in our system DNA may be encapsulated by h-PAMAM/hPAMAM-g-PEG while free h-PAMAM mediated sufficient gene delivery in 293T cells. All these data indicated that blends composed of unmodified polymers and surface-modified polymers were critical for more efficient and safe gene delivery. 5. Conclusions In this work, we present that a mixture system composed of hPAMAM and h-PAMAM-g-PEG possesses the advantages of both hPAMAM and h-PAMAM-g-PEG, resulting in higher gene delivery efficiency and lower cellular toxicity in 293T cells. As an ideal gene delivery tool, our mixture system has potential applications in clinical translational researches. Further researches to analyze the usefulness of this mixture gene delivery system in vivo and related mechanism would provide deep insight for system improvement. This work represents a promising candidate for further analysis and researches. Acknowledgments We are grateful for the support of the National Science Foundation of China (Grant No. 20874015, 51273047), the Shanghai Rising-Star Program (10QH1400200), and the “Shu Guang’’ project (12SG07) supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation. References Buckley, R.H., 2002. Gene therapy for SCID-a complication after remarkable progress. Lancet 360, 1185–1186. Bai, C.Z., Choi, S., Nam, K., An, S., Park, J.S., 2013. Arginine modified PAMAM dendrimer for interferon beta gene delivery to malignant glioma. International Journal of Pharmaceutics 445, 79–87. Cao, L., Yang, W.L., Wang, C.C., Fu, S.K., 2007. Synthesis and striking fluorescence properties of hyperbranched poly(amido amine). Journal of Macromolecular Science Part A 44, 417–424. Chauhan, A.S., Jain, N.K., Diwan, P.V., 2010. Pre-clinical and behavioural toxicity profile of PAMAM dendrimers in mice. Proceedings of the Royal Society A: Mathematical, Physical and Engineering Sciences 466, 1535–1550. Chen, J., Guo, J., Chang, B.S., Yang, W.L., 2012. Blue-emitting PEGylated hyperbranched PAMAM: transformation of cross-linked micelles to hollow spheres controlled by the PEG grafting density. Colloid and Polymer Science 290, 517–524. Cloninger, M.J., 2002. Biological applications of dendrimers. Current Opinion in Chemical Biology 6, 742–748.
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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
The strategy to improve gene transfection efficiency and biocompatibility of hyperbranched PAMAM with the cooperation of PEGylated hyperbranched PAMAM Yangfei Sun a , Yunfeng Jiao b , Yang Wang b , Daru Lu a , Wuli Yang b, * a b
State Key Laboratory of Genetic Engineering and School of Life Sciences, Fudan University, Shanghai 200433, China State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Fudan University, Shanghai 200433, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 September 2013 Received in revised form 4 January 2014 Accepted 8 February 2014 Available online 12 February 2014
As a promising non-viral gene vector, cationic polyamidoamine (PAMAM) dendrimer could form complexes with negative charged DNA to mediate efficient gene delivery in vitro and in vivo. However, complicated synthesis technology and potential cytotoxicity limited their application in clinical translational researches. Hyperbranched polyamidoamine (h-PAMAM), which could be synthesized by a simpler one-pot method, has similar properties with PAMAM, and PEGylation modification of h-PAMAM has been used to reduce cytotoxicity. Here we prepared gene delivery system with h-PAMAM and hPAMAM derivative h-PAMAM-g-PEG, respectively and found that the viability of cells with h-PAMAM-gPEG was quite higher in comparison with cells with unmodified h-PAMAM. However, gene delivery efficiency was lower with h-PAMAM-g-PEG. Then we used mixture composed of h-PAMAM and hPAMAM-g-PEG and such composition was designed to reduce cytotoxicity while maintaining high transfection efficiency. Our results indicated that this mixture system of h-PAMAM and h-PAMAM-g-PEG achieved higher transfection efficiency and lower cytotoxicity compared with h-PAMAM-only system. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Gene delivery Hyperbranched polyamidoamine PEGylation Cytotoxicity Mixture system
1. Introduction Reliable and efficient gene delivery system is essential for successful gene therapy. Currently gene delivery researches mainly focus on two systems: viral vector-based and non-viral gene delivery system. Nowadays many basic researches and clinical trials employ viral vectors for gene delivery in view of their higher gene transfection efficiency. However, viral vectors have several limitations such as lower DNA carrying capability, potential immunogenicity and recombination. The safety of viral vectors was challenged severely with gene therapy death (Marshall, 1999) and leukemia development (Buckley, 2002) in patients (Giacca and Zacchigna, 2012). With such considerations, non-viral gene delivery system becomes more and more attractive and many investigations are underway to design and improve non-viral gene delivery system for future clinical trials (Ibraheem et al., 2014; Luo and Saltzman, 2000).
* Corresponding author at: State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Fudan University, No. 220 Handan Road, Shanghai 200433, China. Tel.: +86 21 6564 2385; fax: +86 21 6564 0293. E-mail address: [email protected] (W. Yang). http://dx.doi.org/10.1016/j.ijpharm.2014.02.018 0378-5173/ã 2014 Elsevier B.V. All rights reserved.
Of non-viral vectors, cationic polymers are particularly remarkable because of their positive surface charges making sufficient interaction with plasmid DNA (pDNA) and efficient gene delivery (De Smedt et al., 2000; Shcharbin et al., 2013), and versatile controlled surface modifications make cationic polymers more biocompatible and suitable for targeted therapy (Shcharbin et al., 2013). One of the cationic polymers, polyamidoamine (PAMAM) dendrimer, predominates the research field of non-viral vectors, which owes to its special characteristics: gene delivery with PAMAM could result in higher gene transfection efficiency and lower cytotoxicity compared with other cationic polymers (Cloninger, 2002; Daneshvar, 2013; Lee et al., 2005). While PAMAM dendrimer is dramatic in non-viral gene delivery research and commercially available, it cannot be denied that their synthesis technology is quite complicated and costly (Tomalia and Frechet, 2002), which limits their application in clinical translational researches. Hyperbranched polyamidoamine (h-PAMAM), which could be prepared by a simple one-pot method, has similar chemical and physical characteristics with PAMAM dendrimer (Cao et al., 2007). It could also mediate efficient gene delivery into skeletal myoblasts (Zhu et al., 2011) and cell lines such as COS7 and 293T cells (Zhu et al., 2012). Although h-PAMAM-mediated gene delivery has lower cytotoxicity in comparison to other cationic
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polymers, further biocompatibility improvement is still required for possible clinical trials (Chauhan et al., 2010; Fischer et al., 2003; Malik et al., 2000; Roberts et al., 1996). As mentioned previously, surface modification of PAMAM, such as acetylation (Kolhatkar et al., 2007), L-arginine (Bai et al., 2013; Son et al., 2013) or PEGylation (Kim et al., 2008; Luo et al., 2002), could reduce cytotoxicity significantly and increase the circulation time in vivo (Jevprasesphant et al., 2003; Shah et al., 2013). Here, in order to develop an efficient and reliable gene delivery system, we synthesized h-PAMAM derivative h-PAMAM-g-PEG with h-PAMAM surface PEGylation (Chen et al., 2012). Plasmid DNA expressing EGFP was transfected into 293T cells with h-PAMAM-gPEG and h-PAMAM, respectively. Results showed that the viability of cells with h-PAMAM-g-PEG-mediated transfection was quite higher in comparison with cells transfected with unmodified h-PAMAM. However, the gene delivery efficiency was lower with h-PAMAM-gPEG, which was consistent with previous studies (Fant et al., 2010). To achieve higher gene transfection efficiency while maintain lower cytotoxicity, we prepared the composites consist of h-PAMAM-gPEG and unmodified h-PAMAM, hoping that such a mixed system would be better than any individual system (Debus et al., 2010). We found that the result was in consistent with our expectations and to our surprise, a mixed system consisting of 70% h-PAMAM and 30% hPAMAM-g-PEG achieved even higher transfection efficiency compared with other h-PAMAM-g-PEG/h-PAMAM ratios. 2. Material and methods 2.1. Materials MeO-PEG-OH (99%, Mw = 2000) was purchased from Fluka. Methyl acrylate (MA) (99%) and diethylene triamine (DETA) (99%) were purchased from Shanghai Chemical Reagent Company. N,N0 Dimethylformamide and diethyl ether were analytical purity, and N,N-dimethylformamide was dried by distillation with CaH2 before used. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and other biological reagents were purchased from Invitrogen Corp. The pEGFP-N2 was prepared with MACHEREY-NAGEL NucleoBond Xtra Midi Plus kit. 2.2. Synthesis of h-PAMAM h-PAMAM was synthesized according to the method previously reported by our lab (Cao et al., 2007). 20 g of diethylene triamine was measured and dissolved to 30 mL of methanol in a 100 mL of three-necked bottle. Then, the mixture of 30 mL of methanol and 20 g of methyl acrylate was added drop-wise into the reaction flask in nitrogen atmosphere for 30 min. The nitrogen flow was removed and the mixture was stirred at room temperature for other 72 h. Then the flask was equipped onto a rotary evaporator to remove the methanol under the vacuum. The process was performed for 1 h at 60 C, for 1 h at 80 C, for 1.5 h at 100 C, for 1.5 h at 120 C, and for 3 h at 140 C. The product was dissolved in 30 mL of deionized water, and the resulted solution was dialyzed against water for 3 days to eliminate any unreacted monomers. Finally, the product was precipitated in acetone and dried under the vacuum. 2.3. Synthesis of h-PAMAM-g-PEG Before the condensation reaction between h-PAMAM and MeOPEG-OH, poly(ethylene glycol) succinimidecarbonate (PEG-SC) was synthesized first as described briefly below. Ten grams of MeOPEG-OH (Mw = 2000) and 7.69 g of N,N0 -disuccinimide carbonate (DSC) were added to 30 mL of N,N0 -dimethylformamide (DMF) to form a white emulsion in a 100 mL flask. Then, 6.07 g of triethylamine was added to the mixture as the catalyst. The reaction
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mixture was incubated at 30 C for 24 h with stirring. The product was precipitated in diethyl ether, and the obtained sample was dissolved in toluene to remove the insoluble substance. The supernatant was precipitated in diethyl ether again, and then the product was dried under the vacuum. h-PAMAM-g-PEG was synthesized through the substitution reaction of succinimide groups of PEG-SC with amino groups of h-PAMAM (Ji et al., 2009). The solution of 0.56 g of PEG-SC in 2 mL of DMF was added drop-wise to the solution of 0.63 g of h-PAMAM in 3.5 mL of DMF. The mixture reaction was stirred at 30 C for 72 h. The product was precipitated in diethyl ether, and was further purified via dialysis in water for 3 days. Finally, the product was retrieved by freeze drying from the aqueous solution and kept in the sealed container. 2.4. Gel retardation assay To investigate the DNA condensation effect of h-PAMAM, hPAMAM-g-PEG and h-PAMAM/h-PAMAM-g-PEG mixture, gel retardation assay was performed. Briefly, we prepared h-PAMAM solution, h-PAMAM-g-PEG solution, h-PAMAM/h-PAMAM-g-PEG mixture with 70% h-PAMAM solution and DNA (pEGFP-N2) solution respectively. Different volumes of solutions were mixed to achieve specified N/P ratios (the ratios between moles of the amine groups of h-PAMAM/h-PAMAM-g-PEG and those of the DNA phosphate groups). Mixture was vortexed slightly for 30 s and incubated at room temperature for 30 min. And then, all these samples were mixed with loading buffer and loaded on 0.8% (w/v) agarose gel for electrophoresis. The gel was ran at 100 V for 40 min and photographed under UV light. 2.5. Cytotoxicity analysis The cytotoxicity of h-PAMAM, h-PAMAM-g-PEG and the mixture of h-PAMAM and h-PAMAM-g-PEG with a molar ratio of 7/3 was determined in 293T cells using MTT assay. Briefly, 293T cells were seeded in 96-well plates (Corning) and incubated at 37 C and 5% CO2 for 24 h. Then, different materials were added into wells at variant concentrations including 0.01, 0.05, 0.2, 0.5, 1 and 2 mg/mL. Twenty-four hours later, 200 mL of MTT solution (0.1 mg/mL) was added into each well, and the well was allowed to react for 4 h at 37 C and 5% CO2. Then the medium of each well was replaced with 200 mL of DMSO and the plate was under constant shaking until formazan dissolved. The absorbance at 570 nm was measured with a microplate reader, while the absorbance at 630 nm was measured as the background. Untreated cells were defined as 100% viable. All toxicity assays were carried out in triplicate. 2.6. In vitro gene transfection with 293T cells In vitro gene transfection efficiency with the polymers was tested in 293T cells. Briefly, 293T cells were seeded in 12-well plate at a density of 1.5 105 cells per well. Gene transfection was performed 24 h later. And h-PAMAM/h-PAMAM-g-PEG–DNA complex was prepared at different N/P ratios as described in gel retardation assay. After incubation for 30 min at room temperature, polymer–DNA complex was transfected into 293T cells at a final concentration of 2 mg DNA/well. Culture medium was changed after incubation for 4 h and cell culture was continued for another 48 h. Then cells were washed with 1 PBS (pH 7.4) and imaging was performed with fluorescent microscope. 2.7. Flow cytometry Gene transfection with h-PAMAM/h-PAMAM-g-PEG was quantified by flow cytometry. 293T cells were transfected with hPAMAM/h-PAMAM-g-PEG–DNA complex as described above. After
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Fig. 1. The structures of (a) h-PAMAM and (b) h-PAMAM-g-PEG polymers.
transfection for 48 h, 293T cells were washed with 1 mL PBS and detached by adding 200 mL 0.025% trypsin per well. After incubation for 2 min at 37 C, 800 mL culture medium was added to resuspend 293T cells. Then after centrifuging for 5 min at 500 rpm, cells were washed with PBS and resuspended with 200 mL PBS and finally incubated on ice. GFP expression was analyzed by flow cytometry system from Beckman Coulter. Experiments were performed at least in triplicates. 2.8. Characterization The zeta potential and the hydrodynamic diameter of the particles were measured by a Zetasizer Nano-ZS (Malvern) at pH 7.4 at 25 C. Transmission electron microscopy (TEM) images were obtained on a Hitachi H-600 transmission electron microscope. Flow cytometry was performed with Moflo XDP (Beckman Coulter) and cell imaging experiments were performed with Nikon microscope DS-Ri1. To perform MTT experiments, we used SpectraMax M5 Microplate Reader (Molecular Devices Corporation) to measure related absorbance. 3. Results 3.1. Characterization of polymer–DNA complexes h-PAMAM was synthesized through the Michael addition between MA and DETA. Its structure was shown in Fig 1a and its molecular weight was about 5600 Da measured by GPC (Chen et al., 2012). Then, the PEGylation of h-PAMAM (h-PAMAM-g-PEG) was achieved by the condensation reaction between h-PAMAM and MeO-PEG-OH. The molecular weight of the obtained product was about 11700 Da measured by GPC (Chen et al., 2012), suggesting the number of the grafted PEG was 3 (Fig. 1b). The interactions between the h-PAMAM and plasmid DNA could lead DNA embedded in the polymers. h-PAMAM/pDNA complex was prepared with the N/P ratio of 10/1. First, the structure of hPAMAM/pDNA complexes was investigated by TEM. As shown in Fig. 2a, the complexes formed spherical nanoparticles and appeared between 50 and 150 nm under TEM. The hydrodynamic diameter measured by DLS was 406 nm and the polydispersity index was 0.12. The hydrodynamic diameter measured by DLS was much larger than the size obtained from the TEM image because of a
hydrate layer existing on the surface of the complex in the medium. The zeta-potential of the complexes was +42.1 mV due to the abundant amino groups on the surface. In addition, the influence of the addition of h-PAMAM-g-PEG to the complex structure was also investigated. As shown in Fig. 2b, when the mixture of h-PAMAM and h-PAMAM-g-PEG with a molar ratio of 7/3 was employed as the carrier, the complex still appeared spherical nanoparticles and the similar size with pure h-PAMAM/pDNA complex. Its hydrodynamic diameter measured by DLS was 489 nm and the polydispersity index was 0.21. The zeta-potential showed +32 mV and was lower than the h-PAMAM/pDNA complex. This result might be attributed to the grafted uncharged PEG chains, which was proved by the result that the zeta-potential of the pure h-PAMAM-g-PEG/pDNA complex was just +18 mV. 3.2. DNA condensation and encapsulation by h-PAMAM and hPAMAM-g-PEG High DNA condensation and encapsulation is essential for gene delivery by cationic polymers. Here we used gel retardation assay to analyze h-PAMAM or h-PAMAM-g-PEG-mediated DNA condensation. Results showed that DNA could be condensed efficiently by hPAMAM and h-PAMAM-g-PEG (Fig. 3a and b). Complete DNA condensation could be achieved at N/P 3. We also noticed that DNA condensation mediated by the composites with 70% h-PAMAM was quite similar with either h-PAMAM or h-PAMAM-g-PEG (Fig. 3c). 3.3. Cytotoxicity of the materials An ideal gene delivery system should have maximal delivery efficiency with minimal cellular toxicity. So here we used MTT assay to check the influence of h-PAMAM, h-PAMAM-g-PEG and the mixture of h-PAMAM and h-PAMAM-g-PEG on cell survival. As shown in Fig. 4, at low concentrations such as 0.05 mg/mL, the relative viability of h-PAMAM-treated 293T cells was about 90%, and h-PAMAM showed concentration-dependent toxicity on 293T cells. We also noticed that PEGylation lowered the cytotoxicity of hPAMAM significantly. Even at the concentrations of 2 mg/mL, the relative cell viability was still around 90% compared with 60% in hPAMAM treated cells. The cytotoxicity of mixture system composed of h-PAMAM and h-PAMAM-g-PEG was similar to that of h-PAMAMg-PEG only system when the concentration was lower than 1 mg/
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Fig. 2. TEM images of (a) h-PAMAM/pDNA complex at N/P ratio of 10 and (b) the mixture of h-PAMAM and h-PAMAM-g-PEG with a molar ratio of 7/3 combined with plasmid DNA at N/P ratio of 10.
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Fig. 3. Gel retardation assay of (a) h-PAMAM, (b) h-PAMAM-g-PEG, (c) h-PAMAM and h-PAMAM-g-PEG mixture with a molar ratio of 7/3. M: size marker; 1: naked DNA; 2: N/ P = 1; 3: N/P = 3; 4: N/P = 4; 5: N/P = 6; 6: N/P = 8; 7: N/P = 10; 8: N/P = 15.
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Fig. 4. MTT assay for cellular toxicities of h-PAMAM, h-PAMAM-g-PEG and the mixture of h-PAMAM and h-PAMAM-g-PEG with a molar ratio of 7/3 at different concentrations in 293T cells.
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Fig. 5. The EGFP expression in 293T cells transfected with h-PAMAM/pDNA complexes for 4 h at variant N/P ratios: 3:1, 6:1, 8:1, 10:1 and 15:1.
mL. Previous researches have shown that PEGylation modification of PAMAM could reduce the cellular toxicity of PAMAM (Fant et al., 2010). These results indicated that PEGylation would be a promising modification for the toxicity improvement of hPAMAM-mediated gene delivery. 3.4. In vitro gene delivery efficiency analysis To achieve higher gene delivery efficiency and lower cytotoxicity with h-PAMAM and h-PAMAM-g-PEG, we initially investigated the influence of N/P ratio on gene delivery with h-PAMAM. As gel retardation assay indicated that DNA could be condensed
completely at N/P 3, we prepared complexes with different N/P ratios including 3, 6, 8, 10, 15, 20 and 30. In vitro transfection with pEGFP-N2 in 293T cells (4 h) showed that higher N/P ratio resulted in higher gene delivery efficiency and from N/P 3 to N/P 10, this efficiency increase was dose-dependent (Fig. 5). However, it was improved little when N/P ratio was changed from 10 to 15 (Fig. 5), indicating that gene delivery was saturated when N/P ratio achieved 10 in 293T cells. On the other hand, partial cell float at N/P 20 and massive cell shrinkage at N/P 30 were observed for hPAMAM system. In contrast, almost all cells had normal morphology when the transfection was performed with h-PAMAM-g-PEG even at N/P 30.
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Fig. 6. The transfection efficiency of the h-PAMAM and h-PAMAM-g-PEG mixed system at N/P 10:1 for 4 h with the various molar ratios between h-PAMAM and h-PAMAM-gPEG: 0:10, 3:7, 5:5, 7:3 and 10:0.
This was consistent with the cytotoxicity results, which showed that h-PAMAM had higher cellular toxicity while h-PAMAM-g-PEG was well tolerated by 293T cells. But previous studies indicated that gene delivery efficiency mediated by PAMAM-g-PEG was quite lower (Fant et al., 2010). So we prepared mixtures composed of hPAMAM and h-PAMAM-g-PEG with different ratios and investigated their effect on gene delivery. This mixed system resulted in much better transfection efficiency compared with h-PAMAM-g-PEGonly system. As the peak of h-PAMAM-mediated gene delivery efficiency arose at N/P 10, and previous studies have showed that
DNA could be condensed sufficiently at N/P 3 with PEI while gene delivery efficiency was highest only when N/P 10, and they proposed that 7 portions of PEI chains were in free, which were essential for gene delivery in cell lines (Yue et al., 2011), we also tested the transfection efficiency of mixed system at N/P 10, while changing the ratio of h-PAMAM and h-PAMAM-g-PEG from 1/0 to 0/ 1 (Fig. 6). We found that mixed system containing 70% h-PAMAM at N/P 10 achieved even higher pDNA delivery efficiency compared to 100% h-PAMAM. As fluorescent microscopy could not provide quantitative evaluation of GFP transfection efficiency, we used
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Fig. 7. Percentage of GFP-positive cells analyzed by flow cytometry. (a) Negative control cells; cells transfected with h-PAMAM and h-PAMAM-g-PEG mixture at N/P 10 with different molar ratios: (b) 5:5, (c) 7:3, (d) 10:0.
sensitive flow cytometry to evaluate the percentage of GFP-positive cells transfected with nanoparticles at different ratios. In consistent with fluorescent imaging results, flow cytometry showed that the percentage of GFP-positive cells was 67.4% in cells transfected with h-PAMAM/h-PAMAM-g-PEG mixed system containing 70% hPAMAM, which was the highest compared with those transfected with the system containing 50% h-PAMAM (35.5%) or 100% hPAMAM (46.5%) (Fig. 7). 4. Discussion Many genetic disorders are caused by loss-of-function mutations in specific genes and the delivery of normal genes is viewed as a promising therapy method for such diseases. An efficient pDNA delivery system with low cellular toxicity is necessary to achieve preconceived gene therapy effects for disorders mentioned above. In current study, we chose h-PAMAM to investigate the gene delivery strategies with higher transfection efficiency, which has similar characteristics with PAMAM dendrimer but only need much simpler and low cost preparation method.
On the basis of above results, we demonstrated that PEGylation of h-PAMAM could reduce the cellular toxicity quite significantly, and both h-PAMAM and h-PAMAM-g-PEG possessed the high DNA condensation and encapsulation capacity. Though h-PAMAM-g-PEG-mediated gene transfection had lower cytotoxicity compared with h-PAMAM, its gene delivery efficiency reduced simultaneously. This coincided well with previous studies (Fant et al., 2010). Initially we tried to improve transfection efficiency by increasing the molar ratio of nitrogen from h-PAMAM-g-PEG to phosphate from DNA (N/P) but little improvement could be achieved. Several factors may contribute to this reduction: firstly, PEGylated h-PAMAM has lower capacity to condense pDNA and keep complexes stable for periods of time (Fant et al., 2010); secondly and mainly, as PAMAM endocytosis is dependent on surface charge, PEGylation modification reduces the surface charges and may affect h-PAMAM-g-PEG mediated cell transfection, just as showed above that the zeta-potential of the hPAMAM-g-PEG/pDNA complex was much lower than h-PAMAM /pDNA complex because of the existence of grafted uncharged PEG chains.
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In view of their respective features, we considered that the hPAMAM/h-PAMAM-g-PEG mixture system could deliver pDNAs safely and effectively in 293T cells. In fact, we indeed found that the addition of h-PAMAM to the h-PAMAM-g-PEG system improved gene delivery efficiency quite significantly and a mixture system containing 70% h-PAMAM achieved even higher gene transfection efficiency compared to h-PAMAM-only system. The results were in consistent with previous researches showing that blends of PEI and PEI-PEG resulted in higher mRNA transfection efficiency and lower cytotoxicity than PEI or PEI-PEG alone (Debus et al., 2010). Previous studies explored the possible mechanisms of PEI-mediated gene transfection, suggesting that 3 portions of PEI would condense DNA completely while extra 7 portions of free PEI were essential for efficient gene transfection (Yue et al., 2011). So it is quite possible that in our system DNA may be encapsulated by h-PAMAM/hPAMAM-g-PEG while free h-PAMAM mediated sufficient gene delivery in 293T cells. All these data indicated that blends composed of unmodified polymers and surface-modified polymers were critical for more efficient and safe gene delivery. 5. Conclusions In this work, we present that a mixture system composed of hPAMAM and h-PAMAM-g-PEG possesses the advantages of both hPAMAM and h-PAMAM-g-PEG, resulting in higher gene delivery efficiency and lower cellular toxicity in 293T cells. As an ideal gene delivery tool, our mixture system has potential applications in clinical translational researches. Further researches to analyze the usefulness of this mixture gene delivery system in vivo and related mechanism would provide deep insight for system improvement. This work represents a promising candidate for further analysis and researches. Acknowledgments We are grateful for the support of the National Science Foundation of China (Grant No. 20874015, 51273047), the Shanghai Rising-Star Program (10QH1400200), and the “Shu Guang’’ project (12SG07) supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation. References Buckley, R.H., 2002. Gene therapy for SCID-a complication after remarkable progress. Lancet 360, 1185–1186. Bai, C.Z., Choi, S., Nam, K., An, S., Park, J.S., 2013. Arginine modified PAMAM dendrimer for interferon beta gene delivery to malignant glioma. International Journal of Pharmaceutics 445, 79–87. Cao, L., Yang, W.L., Wang, C.C., Fu, S.K., 2007. Synthesis and striking fluorescence properties of hyperbranched poly(amido amine). Journal of Macromolecular Science Part A 44, 417–424. Chauhan, A.S., Jain, N.K., Diwan, P.V., 2010. Pre-clinical and behavioural toxicity profile of PAMAM dendrimers in mice. Proceedings of the Royal Society A: Mathematical, Physical and Engineering Sciences 466, 1535–1550. Chen, J., Guo, J., Chang, B.S., Yang, W.L., 2012. Blue-emitting PEGylated hyperbranched PAMAM: transformation of cross-linked micelles to hollow spheres controlled by the PEG grafting density. Colloid and Polymer Science 290, 517–524. Cloninger, M.J., 2002. Biological applications of dendrimers. Current Opinion in Chemical Biology 6, 742–748.
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