Cell Biology International ISSN 1065-6995doi: 10.1002/cbin.10051

MINI-REVIEW

PAMAM dendrimer roles in gene delivery methods and stemcell researchNasibeh Daneshvar1, Rasedee Abdullah1,2, Fatemeh T. Shamsabadi1, Chee Wun How1, M. Aizat MH3

and Parvaneh Mehrbod1

1 Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia2 Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia3 Immunology Unit, Pathology Department, Faculty of Medicine and Health Sciences, Universiti PutraMalaysia, 43400 UPM Serdang, Selangor, Malaysia

Abstract

Nanotechnology has provided new technological opportunities, which could help in challenges confronting stem cell research.Polyamidoamine (PAMAM) dendrimers, a new class of macromolecular polymers with high molecular uniformity, narrowmolecular distribution specific size and shape and highly functionalised terminal surface have been extensively explored forbiomedical application. PAMAMdendrimers are also nanospherical, hyperbranched and monodispersive molecules exhibitingexclusive properties which make them potential carriers for drug and gene delivery.

Keywords: gene delivery; PAMAM dendrimer; stem cell

Introduction

Functionally, the heterogeneous character of cells in amulticellular organism is due to the differential expressionof genes, and is supposed to contain the genetic eliminationof the genes that are silenced and those maintained forexpression in specific tissues. Cloning in amphibians andmammals has shown that adult cells are genetically equal toearly embryonic cells (Gurdon, 1962; Campbell and Wilmut,1997) and differential expression of genes is a consequence ofreversible epigenetic modifications that are progressivelyenforced on the genome through development (Gurdon andByrne, 2003). The reversal of the differentiation status of amature cell to one that is unique to the undifferentiatedembryonic status is termed nuclear ‘reprogramming’(Hochedlinger and Jaenisch, 2006).

This review discusses the criteria for reprogramming at thefunctional and molecular stages using different methods, andthe molecular mediators that could facilitate reprogrammingand the perpetuation of pluripotency. Basically, reprogram-ming stays largely conceptual; thus, further efforts should betaken to elucidate reprogramming at the molecular andbiochemical stages.

Techniques for the formation of inducedpluripotent stem cells (iPSCs)

Pluripotent cells of the blastocyst inner cell mass (ICM),embryonic stem (ES) cells and their in vitro derivatives containgenomes in an epigenetic condition on hold for subsequentdifferentiation. In nature their chromatin is hyperdynamicand quite uncondensed (Meshorer et al., 2007). Many genesare expressed at low levels in both the ICM and ES cells. Thechromatin of natural pluripotent cells includes histonemodifications like bivalent domains, which indicate that thegenes are designated for the subsequent developmentallyregulated expression status (Lohmann et al., 2010). Collec-tively, these characteristics of the extremely early embryonicchromatin are necessary for the successful production ofdifferentiated cell lineages. Artificial reprogramming techni-ques like somatic nuclear transfer, ES cell fusion-mediatedreprogramming and induced pluripotency, generate cells thatcontain many characteristics of the natural pluripotent cells,including many of their epigenetic characteristics. Neverthe-less, the way to achieving pluripotent epigenomic status inartificial pluripotent cells differ significantly from that of theirnatural equivalents (Krueger et al., 2010).

*Corresponding author: e-mail: rasedee@vet.upm.edu.myAbbreviations: iPSC, induced pluripotent stem cell; PAMAM, polyamidoamine; ICM, inner cell mass; ESC, embryonic stem cell; pDNA, plasmid DNA; TA,triamcinolone acetonide

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Artificial techniques of reprogramming attempt todevelop autologous cell graft sources and culture methodsfor the treatment of human genetic disorders. However, theeffectiveness of artificially reprogrammed cells is extremelydependent on the accuracy of the reprogramming proce-dures; thus it is very important to determine the epigeneticresemblances between ES cells and iPSCs. Three differenttechniques have been used for the artificial reprogramming ofsomatic cells – nuclear transfer, cell fusion and mediatedinduction of pluripotency in somatic cells by defined factors(Wernig et al., 2007; Jaenisch and Young, 2008; Yamanaka,2008).

Direct reprogramming by defined transcriptionfactors

Mammalian development is characterised by limitations inthe continuous developmental capability during thetransport of totipotent zygote through the pluripotentICM, which ultimately emerge as a mass of differentiatedcell types that usually cannot revert to the less specialisedstate. ES cells are obtained from the ICM of the blastocyst-phase embryos (Dey and Evans, 2011), which can be grownin vitro while conserving pluripotency (Rossant, 2008).The characteristics of the ESCs have made them aninteresting tool for initial developmental research and as asource of supply of stem cells for use in regenerativemedicine (Nori et al., 2011; Okamoto and Takahashi,2011; Ruan et al., 2011). Although, differentiation isnormally irreversible under physiological conditions,several techniques have been used to reprogramme somaticcells. Reprogramming here refers to the reversion of somaticcells into a less differentiated or stem cell-like state.Reprogramming methods, for example somatic nucleartransfer, have been successful for many species (Campbellet al., 1996, 2007; Iii et al., 2002). Nevertheless, thereare technological and ethical issues in applying thismethod to human medicine (Takahashi et al., 2007; Hannaet al., 2009). In an innovative study, mouse somatic cellswere transformed to ESC-like cells with extended develop-mental capability using only four transcription factors(Takahashi and Yamanaka, 2006; Brambrink et al., 2008).Direct transduction of Klf4, Oct4, Sox2 and c-Myc willproduce the stem-like state in mouse somatic cells. Theresultant iPSCs are similar to ES cells in their pluripotentstate (Stadtfeld et al., 2008). The pluripotency of mouseiPSC has been confirmed by assays to include germlinetransmission, in vitro differentiation into cell types of thethree germ layers, contribution to chimeras, teratomaformation and tetraploid complementation (Nakagawaet al., 2008). Subsequent studies also showed that humaniPSCs can be generated by similar transduction approaches.Human fibroblasts were reprogrammed using a different

set of transcription factors, including OCT4, SOX2,NANOG and LIN28 (OSNL), showing that KLF4 andc-MYC could be replaced by NANOG and LIN28 (Yuet al., 2007).

The development of human iPSCs has created oppor-tunities for use in autologous regenerative medicine bywhich patient-specific pluripotent cells can be obtainedfrom adult somatic cells. Consequently, there is a needto improve the efficiency and reliability of the iPSCsand develop a sound gene (RNA or DNA) deliverymethod for application in the medical setting (Draperet al., 2004).

Viral vectors seem to be the main gene carriers fordelivery because of their high gene transfer abilities. Thesevectors have been used in many gene delivery researchesand clinical tests (Warnock et al., 2011). It is possiblethat the viral gene carriers will insert in the initial codingregion of the genome of the host. Furthermore, virusesare intrinsically immunogenic; as a result the repeatedunits in the virus facilitate the induction of immunereactions in the host (Marshall, 2002). In contrast, non-viral vectors have an advantage in being non-pathogenic,non-immunogenic and more manageable. For thesereasons, development of the non-viral vectors for thetransfection of somatic cells to produce iPSCs aredesirable (Jia et al., 2010). The non-viral procedure iscomposed of physical and chemical delivery techniques.The chemical delivery techniques include lipofection,polyfection and lipopolyfection. Currently several den-drimers and polymer micelles have been used for theinsertion of the genes, as complexes called ‘polyplexes’(Thomas and Klibanov, 2003; Eliyahu et al., 2005; Huanget al., 2012).

Polyamidoamine dendrimer (PAMAM)

PAMAM dendrimers were first introduced in the mid-1980s (Tomalia et al., 1985). The dendrimers are of lowmolecular weights and remain particularly small, usuallybetween 1.5 (generation 1, G1) and 14 nm in diameter(generation 8, G8) (Tomalia et al., 2006), are biocompati-ble, water-soluble, star-like patterned and non-immuno-genic (Jain et al., 2010). They also have terminal changeableamine functional groups, which serve as sensors for thebinding of different target or guest molecules. Unlikeclassical polymers, dendrimers have a high level ofmolecular regularity, narrow molecular weight range andare stable in size and shape. Dendrimers can developcomplexes with nucleic acid-like plasmid DNA (pDNA)through electrostatic interactions and bind to glycosami-noglycans (chondroitin sulphate, hyaluronic acid andheparan sulphate) on the surface of cell (Fant et al.,2008).

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The dendrimers are more efficient and reliable thancationic liposomes or cationic polymers for gene transfer(Dutta et al., 2010). High transfection efficiency of dendri-mers is also due to the low pKa of the amines (3.9 and 6.9),which decreases pH changes in the endosome part (Klajnertand Bryszewska, 2001), suggesting that it functions as aproton sponge, which causes osmotic swelling leading to lysisof endosomes/lysosomes. This effect is responsible for theimproved transfection capability of the dendrimers. It isclear that the use of dendrimers as non-viral vectors reliesconsiderably on their generation (G). Dendrimers with highgenerations have greater gene transfer activity than those withlow generations (Braun et al., 2005). However, maximaltransfection efficiency can be best achieved with G6 dendri-mer. Higher generation dendrimers (>G7) have lower trans-fection efficiencies, which could be due to their inflexiblestructures and cytotoxicity (Haensler and Szoka, 1993;Duncan and Izzo, 2005). Improved PAMAM dendrimersnow form a novel class of transfection agents (Tanget al., 1996), and possess improved transfection activitiesbecause of their enhanced flexibility that allows the complexto be compact. A dendrimer returns to its original size uponrelease from the complex. Although PAMAM dendrimerswere initially thought not to produce adverse effects on cells(Lee et al., 2005), a recent study suggests that they haveapoptotic activity and anti-inflammatory effects, but themechanism of action is unclear (Chauhan et al., 2009).

PAMAM dendrimers conjugated to proteins recognisereceptors on the mesenchymal stem cell (MSC) membrane,shown by the generation 5–7 PAMAM dendrimers, whichwere able to deliver the hBMP-2 gene into MSCs andpromote osteogenesis in vitro, even though their transfectionefficiencies were low (Santos et al., 2010a). Their transfectionefficiencies could be improved if native dendrimers anddegraded PAMAM dendrimers were used. The dendrimer–pDNA complexes were transported to the nucleus, integratedinto the host DNA and replicated. Cell colonies wereproduced, their appearance being an indication of successfultransfection (Santos et al., 2010b).

A synthetic corticosteroid (triamcinolone acetonide, TA)was used to conjugate PAMAMdendrimers to form new non-

viral gene vectors (PAMAM–TA), termed polyplexes. Theformation of polyplexes optimises the relationship betweenvector structure and transfection efficiencies. This studyshowed that generation 4 PAMAMdendrimer–TA complexescontain a reasonable number of tertiary amines believed tocontribute to the endosome-buffering effect, which isresponsible for increased transfection efficiencies of thevectors. TA residues in the complexes play a role in thenuclear localisation signal of PAMAMdendrimer–pDNA andincrease transgenic expression (Ma et al., 2009).

One study has shown that small changes in molecule sizecan greatly influence the pharmacokinetics of a compound inthe body. For example, PAMAM dendrimer-based MRIcontrast agents, which are molecules of <3 nm diameter,readily pass through the vascular wall allowing for rapidperfusion throughout the body, whereas molecules of7–12 nm diameter remain in circulation (Kobayashi andBrechbiel, 2005). Based on this study, transvascular deliveryof nanoparticles across the blood–brain tumour barrier intomalignant glioma cells was seen as possible. The pore sizes ofthe blood–brain tumour barrier are limited to 11.7–11.9 nm,which defines the effective size of nanoparticles that can crossthis barrier. Since PAMAM dendrimer diameter sizes usuallyvary between 1.5 nm (G1) and 14 nm (G8), they couldeffectively be used for drug delivery into RG-2 malignantglioma cells (Sarin et al., 2008).

In a stem cell study, the enhancement of transfectionefficiency was investigated using Generation 5 PAMAM-modified magnetic nanoparticles as a delivery system.Fluorescent magnetic nanoparticles were used to label theiPSCs. Transcription factor genes Oct4, LIN28, Nanog andSox2 and plasmids PSPAX2 and PMD2.G were used toproduce the iPSCs from human fibroblasts (Ruan et al.,2011). It was found that a sufficient amount of iPSCs can beproduced using the PAMAM-modified magnetic nano-particles. The labelling of the iPSCs with fluorescentmagnetic nanoparticles allows cells to be observed andtracked, making it an important technique in stem cellresearch.

Conclusion

PAMAM dendrimers are valuable and potential vectorcandidates as a safe gene delivery plasmid, with significantpotential application in development of stem cells research.They have low toxicity, high transfection efficacy andstability, thus making them suitable for gene therapyapplications. PAMAM dendrimer–pDNAs complexes alsoallow for the delivery of desired genes directly to the nucleuswithout loss in the cytoplasm, thereby inducing the expres-sion of genes not normally expressed. There is a need toexplore the pharmacological and physiological characteristicsof dendrimers and determine other functional moieties that

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could be used to conjugate dendrimers for gene deliveryapplications.

Conflict of interest

The authors declare no conflict of interest.

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Received 12 November 2012; accepted 10 January 2013.Final version published online 18 March 2013.

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