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Tumor-Targeted Gene Delivery Using Molecularly Engineered Hybrid Polymers Functionalized with a Tumor-Homing Peptide
Kris C. Wood, Samira M. Azarin, Wadih Arap, Renata Pasqualini, Robert Langer, and Paula T. Hammond
Bioconjugate Chem., 2008, 19 (2), 403-405 DOI: 10.1021/bc700408r Publication Date (Web): 12 January 2008 Downloaded from http://pubs.acs.org on November 16, 2008

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Tumor-Targeted Gene Delivery Using Molecularly Engineered Hybrid Polymers Functionalized with a Tumor-Homing Peptide
Kris C. Wood, Samira M. Azarin, Wadih Arap, Renata Pasqualini, Robert Langer,*,, and Paula T. Hammond*,
Department of Chemical Engineering, Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, Department of Genitourinary Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030. Received November 3, 2007; Revised Manuscript Received December 12, 2007

Before gene therapy can be used in clinical settings, safe and efcient DNA delivery systems must be developed to overcome a range of extra- and intracellular transport barriers. As a step toward the development of a modular, multifunctional gene delivery system to overcome these diverse barriers, we have developed a family of linear-dendritic hybrid polymers which contain functionalities for tissue targeting, minimization of nonspecic interactions, endosomal buffering, and DNA binding. Here, we demonstrate the rapid three-step, room-temperature, aqueous synthesis of hybrid polymers, as well as the functionalization of these polymers with a peptide targeting ligand that specically binds to glucose-regulated protein-78 kDa (GRP-78), a clinically relevant tumor antigen identied in human cancer patients. These polymer systems can condense plasmid DNA into small nanoparticle structures (<210 nm) and transfect cells expressing GRP-78 with efciencies that exceed that of branched polyethylenimine (bPEI), one of the best commercially available polymers for in Vitro transfections. The synthetic approach described here may be useful for the rapid synthesis and optimization of polymer gene delivery systems bearing a range of diverse functional domains, and the specic GRP-78-targeted systems developed in this study may potentially have clinical applications in cancer gene therapy.

One approach toward the development of improved nonviral nucleic acid delivery systems involves the rational design of integrated, multifunctional systems containing domains which can be used to impart diverse functions such as DNA binding, endosomal buffering, and tissue or intracellular targeting (1, 2) Unfortunately, many early attempts to build such multifunctional systems have been unsuccessful because the addition of new domains results in the direct modication of existing domains, adversely affecting their properties. For example, the conjugation of poly(ethylene glycol) (PEG) to polyethylenimine (PEI) via primary and secondary amines, while improving the colloidal stability of polymer-DNA complexes, also dramatically decreases transfection efciency by directly altering the polymers DNA binding and endosomal buffering properties (3). In response to these well-established problems, we set out to design a gene delivery system possessing multiple functional domains arranged together in a modular fashion, such that new domains could be added, modied, or removed while only minimally affecting existing properties. In order to make this possible, we required that all domains be physically separated
* To whom correspondence should be addressed. 77 Massachusetts Avenue, Room 66-546, Cambridge, MA 02139. Tel: 617-258-7577; Fax: 617-258-5766; Email: hammond@mit.edu. 77 Massachusetts Avenue, Room E25-342, Cambridge, MA 02139; Tel: 617-253-3123; Fax: 617-258-8827; Email: rlanger@mit.edu. Department of Chemical Engineering, Massachusetts Institute of Technology. Biological Engineering Division, Massachusetts Institute of Technology. University of Texas M.D. Anderson Cancer Center.

from one another, that no domains possess overlapping functions, and that domains be assembled using complementary (orthogonal) chemistries (Figure S1a). Here, we demonstrate the rapid synthesis and in Vitro validation of a system which fullls all of the above criteria. This system, based on linear-dendritic hybrid polymers consisting of linear PEG and dendritic poly(amidoamine) (PAMAM), possesses functional domains for electrostatic DNA condensation, endosomal escape (via the proton sponge mechanism), reduction of nonspecic tissue interactions, and tissue targeting (Figure S1b) (4). In order to functionalize hybrid polymers with sensitive biological functional domains such as peptides, antibodies, and nucleic acids, we have developed a synthetic scheme that uses commercially available starting materials and a rapid (three reaction steps, two days), aqueous chemical conjugation (1, 5). To examine the utility of this approach, we synthesize hybrid polymers modied with a peptide ligand (denoted peptide 1, sequence ) WIFPWIQL) capable of selectively targeting glucose-regulated protein-78 kDa (GRP-78) (6). GRP-78 is a functional tumor antigen identied in human cancer patients, and peptide 1 has been used to target GRP-78 in tumors in Vitro, in ViVo (in mouse models of breast and prostate cancer), and in human patient-derived tumor samples (6, 7). We show here that peptide 1 functionalized hybrid polymer systems can electrostatically condense DNA into small nanoparticle structures with low, positive zeta potentials and the ability to transfect cells expressing GRP-78 more efciently than branched polyethylenimine (bPEI), one of the best commercially available polymers for in Vitro transfections.

10.1021/bc700408r CCC: $40.75 2008 American Chemical Society Published on Web 01/12/2008

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Scheme 1. Synthesis of Peptide-Functionalized Hybrid Polymers

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The aqueous synthetic approach developed in this work is shown in Scheme 1. Critical to this approach is the availability of cystamine-core PAMAM dendrimers (generation 4.0), which can be reduced to yield dendrons with a single, free thiol in the interior (8). Briey, dendrimers were rst reduced by addition to a molar excess of dithiothreitol (DTT) with overnight stirring under N2 gas, followed by dialysis against pure water to remove the reducing agent. Ellmans assay was used to quantify the fraction of dendrimers reduced prior to the PEG conjugation (see Supporting Information). In a separate vessel, peptide 1 was added to a bifunctional maleimide-PEG-NHS ester for 90 min, followed by addition of a molar equivalent of reduced dendrimer at pH 7.4 (phosphate buffered saline) with stirring for 2448 h to yield the nal conjugate. Note that peptide 1 was dissolved in dimethylsulfoxide (DMSO) prior to the PEG conjugation reaction only because it is insoluble in water. Quantitatively, PAMAM reduction proceeded at conversions of 95100% (based on total thiol concentration as measured by Ellmans method). The peptide-PEG conjugation reaction proceeded at 78% conversion (Figure S2), and the nal PEG-PAMAM conjugation step went at 74% conversion (based on the disappearance of thiols; see Table S1). To ensure that the disappearance of free thiols during the PEG-PAMAM conjugation step was a result of a reaction with the PEG maleimide group instead of the reformation of dendritic disuldes, a sample of the nal product was incubated overnight with a 10-fold molar excess of DTT, dialyzed, and analyzed again for thiol content. No additional thiols were observed,

suggesting that all originally reacted thiols had formed stable thioether linkages with the maleimide group. Additionally, each conjugate was also veried using NMR (see Supporting Information). A range of techniques was used to measure the physical properties of polymer-DNA complexes. In Figure 1a, gel electrophoresis demonstrates binding and charge neutralization of plasmid DNA (pCMV-Luciferase) by hybrid polymers at polymer/DNA mass ratios greater than 1:1 (N:P 0.8:1), with complete charge neutralization occurring at mass ratios equal to or greater than 5:1 (N:P 3.9:1). Zeta potential measurements indicate that complexes formed at polymer/DNA ratios of up to 40:1 (N:P 31:1) have low, positive zeta potentials, and dynamic light scattering shows that small particles (<210 nm) are formed with diameters equal to or less than the reported cutoff of 200 nm for efcient cellular uptake in ViVo (Figure 1b) (9). Particle size showed some sensitivity to the polymer/ DNA mass ratio, with small ratios yielding smaller (140150 nm) particles and larger ratios yielding slightly larger particles (180210 nm), likely due to increased steric or charge repulsion in complexes formed at high mass ratios. To test for the ability of these peptide-functionalized hybrid polymers to target and deliver plasmid DNA to cells expressing GRP-78, we transfected DU145 prostate carcinoma cells, which have been shown to bind and internalize peptide 1 in a GRP78-dependent manner (6). As shown in Figure 2, peptidefunctionalized hybrid polymers can transfect DU145 cells at levels nearly 10-fold higher than an optimized formulation of

Figure 1. Physical properties of hybrid polymer-DNA complexes. (a) 1% agarose gel electrophoresis demonstrates DNA binding at indicated mass ratios. (b) Size (nm, bars) and zeta potential (mV, diamonds) of polymer/DNA complexes measured by dynamic light scattering (DLS). Error bars indicate 1 standard deviation in measured values.

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MIT Center for Cancer Research for a Ludwig Research Fellowship. We thank Drs. Gregory Zugates and Amin Hajitou for helpful discussions and Drs. Donald Tomalia and Ryan Hayes (Dendritic Nanotechnologies, Inc.) for assistance in obtaining cystamine-core PAMAM dendrimers. Supporting Information Available: Schematic of hybrid polymers, conjugation of peptide 1 to Maleimide-PEG-NHS as monitored by the uoraldehyde assay, table of reaction conversions, detailed experimental methods, and NMR characterization. This information is available free of charge via the Internet at http://pubs.acs.org/BC.

LITERATURE CITED
Figure 2. Transfection of DU145 prostate carcinoma cells expressing GRP-78 using peptide-modied hybrid polymers. Polymers with and without the peptide ligand, and peptide-modied polymers transfected in the presence of polyclonal anti-GRP-78 antiserum to block receptor binding, are shown (6). * indicates p < 0.1. Results normalized to an optimized formulation of PEI (2:1 PEI/DNA ratio (m:m)). All results are given as average +/- standard error.

branched PEI (25 kDa). Moreover, this process is receptormediated, as evidenced by the fact that cells transfected using polymers lacking a peptide ligand, as well as cells transfected with peptide-functionalized polymers in the presence of polyclonal anti-GRP-78 antiserum (to block receptorligand binding), yielded signicantly lower levels of transfection (Figure 2). (For more information on serum stability and cytotoxicity of hybrid polymer systems, see ref (4).) In conclusion, we have developed a rapid and simple aqueous synthesis to construct multidomain, biomolecule-functionalized polymers for nonviral gene delivery applications. Unlike previous efforts to functionalize spherical dendrimers with PEG, targeting groups, or other moieties through DNA binding peripheral amines, this approach allows for the synthesis of asymmetric polymers with well-controlled architectures whose DNA binding and endosomal buffering properties are not directly altered in the functionalization process (1012). As a demonstration of this design and its utility, we synthesized hybrid polymers functionalized with short peptides which efciently target and transfect cells expressing GRP-78, a clinically relevant tumor antigen. Preliminary in ViVo bioluminescence imaging experiments performed using mice bearing EF43-FGF4 tumors demonstrate that a single, intravenous injection of these GRP-78-targeted systems can also lead to transient, tumor-specic transgene expression (data not shown). In the future, the peptide-functionalized hybrid polymers developed in this study may potentially nd use in clinical applications involving the targeted delivery of suicide transgenes to solid tumors (13). More broadly, the general synthetic approach described here could be a useful tool for the rapid synthesis and optimization of next-generation gene delivery systems bearing biological functional domains such as nuclear localization sequences, adjuvants, and protein transduction domains (2).

ACKNOWLEDGMENT
This work was supported by the National Institutes of Health (R01-EB00244 and R21-EB004122) and the Division of Materials Research of the National Science Foundation (DMR 9903380). K.C.W. thanks the National Cancer Institute and the

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