Liposome encapsulated polyethylenimine/ODN polyplexes for brain targetingYoung Tag Ko1, Raktima Bhattacharya, Ulrich Bickel?Department of Pharmaceutical Sciences, Texas Tech University Health Science Center, Amarillo, TX 79106, United Statesa b s t r a c ta r t i c l ei n f oArticle history:Received 27 May 2008Accepted 8 October 2008Available online 26 October 2008Keywords:In vivo gene deliveryOligodeoxynucleotidesPEGylated liposomesPolyethylenimineBlood-brain barrierDespite high in vitro transfection efficiency, the use of the cationic polymer polyethylenimine (PEI) for systemicapplication is limited due to its rapid blood clearance and accumulation by RES sites upon intravenousadministration of PEI/DNA polyplexes. Therefore, it is important to improve the properties of the PEI/DNAcomplexwith respect to extendingthe systemiccirculationtime and suppressionof RES uptake.Inthisstudy,weapplied PEGylated liposome technology for systemic delivery of PEI polyplex of oligodeoxynucleotides (ODN),basedonencapsulationofthePEI/ODNpolyplexesintoPEGylatedliposomes.ThePEI/ODNpolyplexwaspreparedwith a low-branched PEI with MW 2.7 kDa and 20-mer double stranded ODN and was then entrapped intoPEGylated liposomes with 95% loading efficiency, leading to a virus-like structure with ∼130 nm diameter. ThePEG-stabilized liposome (PSL) entrapping PEI/ODN polyplexes remained stable in the presence of serum. Uponintravenousadministration,theDNAinthePSLwasclearedfromsystemiccirculationatasignificantlyslowerrateas compared to the naked PEI/ODN complex. Furthermore, targeting of the PSL with antibody specific totransferrin receptor redirected biodistribution of the entrapped ODN, leading to significant accumulation in thetargetedorgan, i.e.brain.Encapsulation of the PEI/ODN polyplexeswithina long-circulatingliposome providedapromising ODN delivery system for in vivo application.© 2008 Elsevier B.V. All rights reserved.1. IntroductionAmong polycationic polymers, the polyethyleneimines (PEI) havebeen widely explored for gene delivery due to their high gene transferefficiency [1–4]. This efficiency of PEI depends mainly on theircharacteristic chemical structure. PEI contain one amino group perevery two carbons (ethylene group) and about 20% of the amino groupsare protonated at physiological pH [5] resulting in high positive chargedensity. Due to this high positive charge density, PEI form dense nano-sizedparticulatecomplexeswithnegativelychargedDNAbyelectrostaticinteractions. The PEI/DNA complexes take overall positive charge andinteract with negatively charged components of cell membranes andenter cells by endocytosis. The PEI/DNA complexes enter the cells bynonspecific adsorption-mediated endocytosis while the condensed DNAin the complexes is protected from enzymatic degradation. Uponendocytosis, PEI are subject to further protonation as the endosomalcompartment acidifies. Protonation of PEI by capturing protons, the socalled ‘proton sponge’ mechanism [1,2], leads to osmotic swelling andsubsequentendosomaldisruption.Hence,genedeliveryusingPEIisbasedon (i)condensation of thenegativelychargedDNAintocompactparticlesby electrostatic interactions, thus protecting the DNA from enzymaticdegradation, (ii) endocytosis of the particles intothe cells and (iii) releaseof the DNA from endosomes via the ‘proton sponge’ mechanism.Despite high transfection efficiency in vitro, PEI/DNA complexeshave not shown significant therapeutic efficacy for in vivo applicationdue to rapid plasma clearance and accumulation by RES sites. Thisinstability of PEI/DNA complexes is mainly due to the overall positivecharge of the complexes. While the cationic complexes interact withanionic components of cell membranes and thus trigger cellularuptake by absorptive mediated endocytosis, they also interact withblood components and are subject to clearance by the RES. As a result,PEI/DNA complexes are cleared from the circulation within a fewminutes and accumulate mainly in RES organs such as liver and spleen[6]. The short blood circulation time limits the possibility of effectivedelivery of these complexes to target organs other than RES, e.g.central nervous system which represents a particular difficult organfor targeted delivery due to the presence of the blood-brain barrier(BBB). Therefore, it is desirable to improve the in vivo behavior of thePEI/DNA complexes by reducing the nonspecific charge interactionsand thus prolong circulation time. Several approaches have been triedto increase stability of PEI/DNA complexes in the blood circulation[3,7], including covalent attachment of polyethylene glycol (PEG) toPEI. The PEG-PEI/DNA complexes showed reduced surface charge andnon-specific interaction in blood, resulting in prolonged circulationtime. Although the PEG-PEI was able to change in vivo biodistributionand pharmacokinetics, the stabilizing effect by PEG was modest [6,8].In the present study, we applied PEI to form polyplexes with ODNand combined it with PEG-stabilized liposomes. We hypothesized thatencapsulation of PEI/ODN complexes inside PEG-stabilized liposomesJournal of Controlled Release 133 (2009) 230–237? Corresponding author. Department of Pharmaceutical Sciences, School of Phar-macy, Texas Tech University Health Sciences Center, 1300 Coulter Dr. Amarillo, TX79106, United States. Tel.: +1 806 356 4015; fax: +1 806 356 4034.E-mail address: Ulrich.Bickel@ttuhsc.edu (U. Bickel).1Present address: Center for Pharmaceutical Biotechnology and Nanomedicine,Northeastern University, Boston, MA 02115, United States.GENE DELIVERY0168-3659/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.jconrel.2008.10.013Contents lists available at ScienceDirectJournal of Controlled Releasejournal homepage: www.elsevier.com/locate/jconrel
