Cationic star copolymers based on β

Showcasing latest research on gene delivery from Xian Jun Loh’s Laboratory at the Institute of Materials Research and Engineering, A*STAR, Singapore. The graphic shows the process of gene transfection to a cell, drawn by Cally Owh.

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Cationic star copolymers based on β-cyclodextrins for efficient gene delivery to mouse embryonic stem cell colonies In this work, a cationic star copolymer with a β-cyclodextrin core was developed for nonviral gene transfer to mouse embryonic stem cells (mESCs). These materials have very low toxicity and show highly efficient transfection to mESC colonies. See Xian Jun Loh and Yun-Long Wu, Chem. Commun., 2015, 51, 10815.

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Cite this: Chem. Commun., 2015, 51, 10815

Cationic star copolymers based on b-cyclodextrins for efficient gene delivery to mouse embryonic stem cell colonies†

Received 6th May 2015, Accepted 21st May 2015

Xian Jun Loh*abc and Yun-Long Wud

DOI: 10.1039/c5cc03686k www.rsc.org/chemcomm

A cationic star copolymer with a b-cyclodextrin core was developed for nonviral gene transfer to mouse embryonic stem cells (mESCs). The copolymer comprises poly(2-dimethyl aminoethyl methacrylate) as the cationic component and poly(2-hydroxyethyl methacrylate) as the non-toxic stealth component. These materials have very low toxicity and show highly efficient transfection to mESC colonies.

Gene therapy has been mooted as a possible strategy for treating various human disorders because most of these disorders are related to the genetic makeup of the patient. There is immense potential for the treatment of diseases such as HIV, inherent genetic disorders, cancers, and cardiovascular diseases.1–7 Gene delivery can be subdivided into viral or non-viral modes of delivery. Viral gene delivery is currently the method that transfects cells most efficiently. However, safety concerns regarding the use of viruses for gene delivery have remained unsolved. There have been reports of the side effects arising from the use of viral gene vectors in clinical trials.8–10 For this reason, a safer alternative, using synthetic polymeric vectors, is sought. Synthetic cationic polymers such as the homopolymers or derivatives of polyethylenimine (PEI),7 poly(L-lysine),11 and poly((2-dimethyl amino)ethyl methacrylate) (PDMAEMA)12–15 and poly(b-amino esters)16–18 have been widely explored as potential alternatives. The main bottleneck in the use of the synthetic polymeric vectors is the poor transfection efficiency as well as the cytotoxicity of these polymers. Recent improvements have resulted in a polymeric vector having comparable transfection efficacy to a viral vector in the transfection of human embryonic stem cells.19 Very few other papers have a

Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 3 Research Link, Singapore 117602, Singapore. E-mail: [email protected] b Department of Materials Science and Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore c Singapore Eye Research Institute, 11 Third Hospital Avenue, Singapore 168751, Singapore d School of Pharmaceutical Sciences, Xiamen University, Xiamen, Fujian, P. R. China † Electronic supplementary information (ESI) available. See DOI: 10.1039/c5cc03686k

This journal is © The Royal Society of Chemistry 2015

reported transfection of pDNA to embryonic stem cells.20–22 Several papers have reported the synthesis of cyclodextrins and polyrotaxanes conjugated with polyethylenimine.23–25 By using cyclodextrin as a core, polyethyleneimines with a star architecture were obtained. By delocalizing the positive charge, these modified polyethyleneimines were found to have lower cytotoxicity than linear PEI of molecular weight 25 kDA, while at the same time having the transfection efficiency comparable to that of linear PEI. The star structure of polymers has been shown to be useful for increasing the gene transfection efficiency. Twenty-four hours after transfection, polymer fluorescence (indicating successful gene transfection) was almost exclusively found in close proximity but never inside the nucleus. Gene transfection of star-shaped PDMAEMA resulted in possible localization of DNA delivery to specific sub-cellular structures.26 Biodegradability is also important for a biomaterial to be excreted from the body after a desired period of use.27–30 Biodegradable star shaped PDMAEMA structures based on poly(e-caprolactone) and poly(lactic acid) have been also developed for co-drug and gene delivery.31,32 Stem cells as gene delivery targets are an attractive research topic. In the aspects of vascularization and tissue regeneration, cell-based therapies using stem cells are highly promising. Naldini et al. showed that by delivering an inhibiting gene, Tie2-expressing mononuclear (TEM) cells were selectively eliminated and achieved substantial inhibition of angiogenesis and slower tumor growth without systemic toxicity.33 This work demonstrated that TEM cells may represent new targets for drug development and may provide the means for selective gene delivery and targeted inhibition of tumor angiogenesis. Anderson et al. developed genetically modified stem cells which express a high concentration of the vascular endothelial growth factor (VEGF) for promoting angiogenesis.34 The modified stem cells demonstrated markedly enhanced hVEGF production, cell viability, and engraftment into target tissues showing its potential utility for therapeutic applications. There remain a lot of unknown factors in research in the transfection of embryonic stem cells using polymeric gene delivery vectors. The objective of our work is to develop a safe nonviral gene delivery vector for

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transfection into embryonic stem cells. As a cell model, we have chosen to work with mouse embryonic stem cells, EB3, from the RIKEN Bioresource Centre. In our system, b-cyclodextrin (b-CD) is chosen as the core. b-CD offers the option of forming inclusion complexes of the CD-drug for drug solubilization, stabilization and delivery options to the stem cell. Star structures, either based on CDs or other core molecules, have been reported to delocalise the cationic charges and lower toxicity, providing another benefit and purpose of using this as the core material.35–37 Poly(2-hydroxyethyl methacrylate) (P(HEMA)) is one of the first polymers used as biomaterial in tissue engineering. It has excellent biocompatibility and is in the form of a hydrogel, its physical properties are similar to those of living tissues.38 Its incorporation is likely to improve the biocompatibility of the polymer. Finally, PDMAEMA will be chosen as the cationic block as it is readily polymerised by ATRP, a polymerisation technique that generates polymers with low polydispersity. Well-defined star-shaped CD-(PDMAEMA-co-PHEMA)4, consisting of a b-CD core and four PDMAEMA-co-PHEMA arms, was synthesized via ATRP from the macroinitiator, Br-b-CD, bearing 4 initiator arms (Fig. 1). The molecular weights of the copolymers are summarized in Table 1. Mn of the copolymers were determined to be in the range of 21.9 to 65.7 103 g mol 1. The number of DMAEMA repeat units per arm raised accordingly from 16 to 89, whereas the HEMA repeat units per arm were kept fairly constant at 16–20 units. In addition, narrow molecularweight distribution (polydispersity index (PDI) B1.2) of the copolymers indicated that the ATRP processes of DMAEMA and

Fig. 1 Synthesis of cationic cyclodextrin based copolymers by Atom Transfer Radical Polymerization (ATRP) from 2-bromoisobutyryl-functionalized b-CD macroinitiator (Br-b-CD) (CD = cyclodextrin, DMAEMA = 2-(dimethylamino)ethyl methacrylate, HEMA = 2-hydroxylethyl methacrylate, HMTETA = 1,1,4,7,10,10-hexamethyl triethylenetetramine).

Table 1

HEMA were well-controlled. An effective gene delivery system calls for the condensation of pDNA into nanoparticles which are small enough to be taken up by cells. Agarose gel electrophoresis, dynamic light scattering (DLS) and zeta potential measurements were used to confirm the pDNA binding ability of the star-shaped polycations. AFM was also utilised to observe the morphology of the resultant NPs in the present work. An agarose gel was used to prove the formation of the polymer–pDNA nanocomplexes at different N/P ratios. The gel retardation results of all nanocomplexes with increasing N/P ratios are demonstrated in Fig. S1 (ESI†). The control PEI can bind pDNA completely at an N/P ratio of 2. H1 binds completely to pDNA at an N/P ratio of 5. H2 binds completely to pDNA at an N/P ratio of 6. H3 binds completely to pDNA at an N/P ratio of 7. These results show that the incorporation of the biocompatible PHEMA could improve the biocompatibility of the copolymer but could also retard the complexation ability of the copolymer. The particle size of the nanocomplexes was determined from DLS measurements. Nanoparticles with diameters of less than 200 nm have a longer circulation time and less susceptible to clearance by the reticuloendothelial system (RES).39 Fig. 2a shows that all the copolymers can effectively bind pDNA into nanoparticles, and that the particle size decreased

Fig. 2 (a) Particle size and (b) zeta potential of the complexes between the cationic copolymers and pDNA at different N/P ratios. AFM images of (c) H3–pDNA (at an N/P ratio of 1), and (d) H3–pDNA (at an N/P ratio of 20) complexes.

Molecular characteristics of the cationic copolymers

Chemical compositiona (monomer repeat units per arm)

Weight compositiona (%)

Copolymer

DMAEMA

HEMA

DMAEMA

HEMA

Mn 103 a (g mol 1)

Mn 103 b (g mol 1)

H1 H2 H3

89.4 30.9 16.2

16.2 18.6 20.4

87.0 67.5 49.8

13.0 32.5 50.2

65.7 30.2 21.9

52.3 22.4 15.4

a

Calculated from 1H NMR.

b

Obtained from GPC.

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with the increase of the N/P ratio. At an N/P ratio of 2.0, large particles were found due to insufficient complexation by the cationic polymers. The diameter of all NPs was found to be stabilized in the range of 100–150 nm at higher N/P ratios. Zeta potential was used to determine the amount of surface charges on the polymer–pDNA nanoparticles. The surface charge on the nanoparticle is a significant factor affecting the internalisation of the nanocomplexes. The surface charges of nanoparticles were positive above the N/P ratio of 2 in Fig. 2b. Electrostatic interaction between nanoparticles with positively charged and negatively charged cell membranes promotes endocytosis. The morphology of H3–pDNA nanocomplexes at N/P ratios of 1 and 20 was visualized in the AFM image in Fig. 2c and d, respectively. The AFM image showed that the copolymers condensed pDNA into uniform nanoparticles with a diameter of 100–200 nm, which was consistent with the result of DLS measurement. As a quick assessment of the gene transfection ability, luciferase was first used as a gene reporter for assessing the in vitro gene transfection efficiency of star-shaped polycations in HEK293 cells.40 Fig. S2 (ESI†) shows the profile of luciferase expression mediated by the copolymers at different N/P ratios in comparison with that of PEI (25 kDa) in complete serum media. It was found that the gene transfection efficiency was greatly reliant on the N/P ratio of the nanocomplexes as well as the PDMAEMA content of the starshaped polycations. With the increase in the N/P ratio, the transfection efficiency of copolymer–pDNA nanocomplexes showed an increase initially and then subsequently decreased. At low N/P ratios, polycations are not expected to effectively bind DNA and it was difficult for the large aggregates to enter the cells. At high N/P ratios, the presence of the free polycations resulted in increased toxicity of the formulation and leads to a decrease in transfection efficiency. However, overall, the polymeric vectors synthesised in this work appeared to be promising for gene transfection in typical cell lines. The gene transfection efficiency is in fact slightly better than PEI under complete serum conditions. The relatively high N/P ratio required for good transfection indicates that the copolymers may be less cytotoxic than PEI. The good results led us to move towards the testing of the materials using feeder free mouse embryonic stem cells (EB3). The cytotoxicity of gene carriers is critical to the transfection efficiency of the vector for DNA delivery. Factors that may affect the cytotoxicity of gene vectors include molecular weight, polycation structure and charge density. In this work, cytotoxicity of the polymer–pDNA nanocomplexes at different N/P ratios was estimated via MTT assay in feeder free mouse embryonic stem cells (Fig. S3, ESI†). The cell toxicity was slightly lesser for H3 as compared with Lipofectamine 2000. However, at higher concentrations, the toxicity of the polymer increases. At these higher concentrations, the presence of free polymers with positive charge in the transfection formulation leads to damage to cells, resulting in increased toxicity. Luciferase was again used as a gene reporter for assessing the in vitro gene transfection efficiency of star-shaped polycations in EB3 cells. Fig. 3 shows the profile of luciferase expression mediated by the copolymers at different N/P ratios in comparison with that of Lipofectamine 2000 in complete serum media.


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Fig. 3 In vitro gene transfection of the cationic copolymer–pDNA complexes in comparison to Lipofectamine 2000 at various N/P ratios in mouse embryonic stem cell cultures in the absence of serum.

The copolymer H3 performed excellently, transfecting the stem cells with an efficiency close to that of Lipofectamine 2000 which is the standard material used for gene transfection. In order to visually ascertain the effectiveness of our gene delivery systems, we adopted EGFP (enhanced green fluorescent protein) as another kind of gene reporter for gene transfection assay. In this work, EGFP expression was examined by delivering the plasmid pEGFP-N1 encoding GFP in EB3 cell lines. As shown in Fig. 4a–d, the expression of EGFP appeared to be very effective using the polymers synthesized. In fact when compared with Lipofectamine, it appears that H3 transfected cells much more effectively at N/P ratios of 15, 20 and 25. A higher percentage of cells expressing EGFP was observed when transfected with H3

Fig. 4 Fluorescence microscopy images of the transfected mouse embryonic stem cells. The transfection was carried out with (a, c) H3–pDNA (at an N/P ratio of 20) and (b, d) Lipofectamine 2000–pDNA complexes. The top panel shows the cell images under normal field and the bottom panel shows the cell images under fluorescence excitation. (e) Gene transfection efficiency based on the percentage of cells expressing GFP.

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than with Lipofectamine. It appears that in this work, we have uncovered the correct balance between condensation ability of the polymer as well as the optimum cyto-compatibility of this material. The end result is that this leads to excellent transfection ability of the copolymers that rivals that of the commercially available Lipofectamine. In summary, we demonstrated that a star copolymer with a b-cyclodextrin core and poly(2-dimethyl aminoethyl methacrylate) as the cationic component and poly(2-hydroxyethyl methacrylate) as the non-toxic stealth component could be used for nonviral gene transfer to mouse embryonic stem cells (mESCs). The polymers showed excellent DNA condensation capacity. In particular, polymer H3 (with DMAEMA/ HEMA weight content of B50 : 50) showed comparable or better transfection capabilities than the commercially available Lipofectamine. These customisable star-shaped polymers are promising materials for gene therapy to stem cells. Further work will focus on utilising the cyclodextrin cores for the encapsulation of drug molecules for further enhancement of the gene transfection efficiency and a deeper structure property study of this interesting polymer for embryonic stem cell gene delivery.

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