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Dendritic Poly(ether imine) Based Gene Delivery Vector Ullas Padinjaremattathil Thankappan,† Shampur Narayan Madhusudana,*,† Anita Desai,† Govindasamy Jayamurugan,‡ Yamajala B. R. D. Rajesh,‡ and Narayanaswamy Jayaraman*,‡ † ‡
Department of Neurovirology, National Institute of Mental Health & Neurosciences, Bangalore 560029, India Department of Organic Chemistry, Indian Institute of Science, Bangalore 560012, India
bS Supporting Information ABSTRACT: The nonviral vector based gene delivery approach is attractive due to advantages associated with molecularlevel modifications suitable for optimization of vector properties. In a new class of nonviral gene delivery systems, we herein report the potential of poly(ether imine) (PETIM) dendrimers to mediate an effective gene delivery function. PETIM dendrimer, constituted with tertiary amine branch points, n-propyl ether linkers and primary amines at their peripheries, exhibits significantly reduced toxicities, over a broad concentration range. The dendrimer complexes pDNA effectively, protects DNA from endosomal damages, and delivers to the cell nucleus. Gene transfection studies, utilizing a reporter plasmid pEGFP-C1 and upon complexation with dendrimer, showed a robust expression of the encoded protein. The study shows that PETIM dendrimers are hitherto unknown novel gene delivery vectors, combining features of poly(ethylene imine)-based polymers and dendrimers, yet are relatively nontoxic and structurally precise.
T
he process of gene therapy aims at generation of an optimal therapeutic outcome, by either production or inhibition of specific intracellular proteins, mediated by exogenously delivered DNA.1 Critical to the success of gene therapy is the requirement of a safe and efficient gene delivery system.2 Viral and nonviral vectors are the general approaches to address gene delivery requirements. Whereas viral vectors are prominent, including in clinical gene therapies,3 development of nonviral vectors is being pursued actively in the effort to ameliorate undesirable effects of viral vectors. Nonviral vectors are considered attractive due to advantages associated with molecular-level modifications suitable to optimizing vector properties.4-7 Nonviral vectors based on cationic lipids, chitosan, poly(lactide-co-glycolide), gelatin, poly(ethylene imine), and poly(methyl methacrylate) were studied extensively.8-16 Synthetic dendrimer macromolecules have also been explored in gene delivery, accompanied with many attributes of dendrimers suitable to the functions of a vector.17-20 Highergeneration poly(amido amine) series of dendrimers are by far the most studied dendrimers for gene delivery studies among dendrimers.21-26 Primary requirements of gene carriers are their buffering capacities, DNA condensation abilities, and reduced toxicities. Poly(ethylene imines) and poly(amido amines) fulfill the requirements, although impediments relating to low gene expression levels and toxicities are a concern, for which modifications of generic structures provide a clue.27,28 In an entirely new class of dendrimer r 2010 American Chemical Society
delivery systems, we herein report the potential of poly(ether imine) (PETIM) dendrimers to mediate an effective gene delivery function. PETIM dendrimers are able to condense the DNA effectively, protect it from endosomal damage, and deliver to the cell nucleus for effective gene expression. Experiments29 that uncover the in vitro gene delivery potential are (i) cytotoxicity, (ii) DNA condensation, (iii) nuclease protection, (iv) tracking intracellular localizations, and (v) transfection assays. PETIM dendrimer generation four, presenting 32 amine peripheral functionalities (Figure 1) was chosen for the study. Synthesis of the dendrimer involved two alternate Michael addition reactions and two alternate functional group reductions, performed by a divergent growth methodology, and amine functionalities were obtained upon reduction of nitrile groups present at the peripheries of dendrimer.30,31 Synthesis of dendrimer undertaken for the present study is given in the Supporting Information. There are 32 primary amine and 30 tertiary amine sites. The molecular structure also presented 61 oxygens in the form of ether functionalities. With pKa of ∼9, the amine sites are expected to be in the protonated form at physiological pH. The dendrimer generation in the neutral form is estimated to be ∼3.5 nm in diameter.32 Received: July 8, 2010 Revised: December 8, 2010 Published: December 30, 2010 115
dx.doi.org/10.1021/bc1003108 | Bioconjugate Chem. 2011, 22, 115–119
Bioconjugate Chemistry
COMMUNICATION
Cytotoxicity of PETIM dendrimer was evaluated first in the BHK-21 cell line by an MTT assay. A concentration-dependent
cytotoxicity was observed, with cell viability reducing to ∼50%, when dendrimer concentration was ∼1 mg mL-1 (Figure 2a). Gel retardation assay on agarose gel was performed subsequently on complexes formed with a pDNA, namely, pEGFP-C1 and PETIM dendrimer, prepared at varying charge (N/P) ratios, so as to identify the DNAbinding abilities of dendrimer. A gradual retardation of pDNA mobilities was observed, as evidenced by gradual disappearance of pDNA band, as a function of increasing dendrimer ratio (Figure 2c). At a charge ratio of 3.5:1, a complete and effective complexation of the added pDNA could be observed clearly. Upon observing complexation of dendrimer with DNA, assessing sizes and shapes of the complexes was undertaken by TEM technique, wherein the complex was observed to be in a toroidal shape with
