DOI: 10.1002/chem.201202358 Dendrimer Functionalization with a Membrane-Interacting Domain of Herpes Simplex Virus Type 1: Towards Intracellular Delivery Tom P. Carberry, [a] Rossella Tarallo, [b] Annarita Falanga, [b] Emiliana Finamore, [c] Massimiliano Galdiero, [c] Marcus Weck,* [a] and Stefania Galdiero* [b] Introduction Crossing of the cellular membranes remains a major obsta- cle for the delivery of therapeutics. [1] The aqueous extra- and intracellular matrices are separated by a hydrophobic lipid bilayer, which acts as a barrier separating the cellular components from the extracellular milieu. Although small molecules and ions can diffuse across this barrier, large con- jugates with molecular weight over 500 amu are generally excluded from simple diffusion into the cell. [2] Larger mole- cules can be taken up endocytically, which allows them to exist in the cell. However, the molecule is still separated from the cytoplasm by an endosomal lipid bilayer. [3] In addi- tion to size, membrane permeability is dependent on the hy- drophilicity or lipophilicity of the molecule in question. [2] Even the delivery of a potent drug can be foiled due to its insolubility in either the aqueous cell environment or the lipid membrane. These problems can be alleviated by com- plexation to a macromolecular conjugate which assists the cargo)s solubility and permeability into desired areas. [4] Indeed, there are several recent reports of successful intra- cellular delivery of macromolecular conjugates, which re- lease their cargo through biological triggers (e.g., pH changes or enzymatic cleavage). [5] However, as many macromolecular drug conjugates are internalized via active translocation mechanisms, for exam- ple, endocytosis, they can exhibit poor activity due to the cargo being unable to escape the endosome before lysoso- mal degradation occurs. [1, 3, 4] Certain peptide sequences have been shown to efficiently cross the cellular membrane. [6] These cell-penetrating peptides (CPPs) are useful for the transport of larger macromolecules across the membrane. [7] Many CPPs act by an endocytic mechanism, trapping the conjugate in an endosome. [8] Our approach is based on a membrane-interacting peptide that operates through a different internalization mechanism. In particular, we are interested in viral peptides, such as those derived from the herpes simplex virus type 1 (HSV-1). These peptides are capable of internalizing cargo by induc- ing membrane perturbation. [9] HSV-1 uses four glycopro- teins, glycoproteins B (gB), D (gD), H (gH), and L (gL), to mediate the viral–cell fusion and subsequent infection. [10] We have previously reported that HSV-1 gH contains sever- al domains that are mostly hydrophobic, suggesting that these regions are those that interact with cellular mem- branes. [11] One of these domains, His625–Phe644 (gH625), showed great effectiveness in inducing lipid mixing of large unilamellar vesicles (LUVs), showing its membrane-inter- acting potential. These results suggest that this sequence could be important in the interaction of viruses with cells. [11–12] Figure 1 shows the sequence of this peptide. When folded into an a helix, nonpolar residues tend to concentrate on one side of the helix giving the peptide an amphiphilic Abstract: A polyACHTUNGTRENNUNG(amide)-based den- drimer was synthesized and functional- ized with the membrane-interacting peptide gHACHTUNGTRENNUNG(625–644) (gH625) derived from the herpes simplex virus type 1 (HSV-1) envelope glycoprotein H, which has previously been shown to assist in delivering large cargoes across the cellular membrane. We demon- strate that the attachment of the gH625 peptide sequence to the termini of a dendrimer allows the conjugate to pen- etrate into the cellular matrix, whereas the unfunctionalized dendrimer is ex- cluded from translocation. The pep- tide-functionalized dendrimer is rapidly taken into the cells mainly through a non-active translocation mechanism. Our results suggest that the presented peptidodendrimeric scaffold may be a promising material for efficient drug delivery. Keywords: click chemistry · deliv- ery · dendrimers · membrane trans- location · peptides [a] T. P. Carberry, Prof. Dr. M. Weck Molecular Design Institute and Department of Chemistry New York University,New York, NY 10003 (USA) Fax: (+ 1) 212-995-4895 E-mail: marcus.weck@nyu.edu [b] R. Tarallo, Dr. A. Falanga, Prof.Dr. S. Galdiero Dipartimeno di Scienze Biologiche Università di Napoli “Federico II” Via Mezzocannone 16, Napoli 80134 (Italy) Fax: (+ 39) 081-253-6642 E-mail : sgaldier@unina.it [c] Dr. E. Finamore, Prof. Dr. M. Galdiero Dipartimento di Medicina Sperimentale Seconda Università degli Studi di Napoli Via de Creccio 7, Napoli 80138 (Italy) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201202358. Chem. Eur. J. 2012, 00,0–0 # 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim These are not the final page numbers! ÞÞ &1& FULL PAPER