Development of a Nonviral Gene Delivery Vehicle for Systemic

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Bioconjugate Chem. 2002, 13, 630−639

Development of a Nonviral Gene Delivery Vehicle for Systemic Application Suzie Hwang Pun†,‡ and Mark E. Davis*,† Chemical Engineering, California Institute of Technology, Pasadena, California 91125 and Insert Therapeutics Inc., 2585 Nina Street, Pasadena, California 91107. Received November 2, 2001; Revised Manuscript Received January 30, 2002

Polycation vehicles used for in vitro gene delivery require alteration for successful application in vivo. Modification of polycations by direct grafting of additional components, e.g., poly(ethylene glycol) (PEG), either before or after DNA complexation, tend to interfere with polymer/DNA binding interactions; this is a particular problem for short polycations such as linear, β-cyclodextrin-containing polycations (βCDPs). Here, a new method of βCDP polyplex (polycation/DNA composite structures) modification is presented that exploits the ability to form inclusion complexes between cyclodextrins and adamantane. Surface-PEGylated βCDP polyplexes are formed by self-assembly of the polyplexes with adamantane-PEG conjugates. While unmodified polyplexes rapidly aggregate and precipitate in salt solutions, the PEGylated βCDP polyplexes are stable at conditions of physiological salt concentration. Addition of targeting ligands to the adamantane-PEG conjugates allows for receptor-mediated delivery; galactosylated βCDP-based particles reveal selective targeting to hepatocytes via the asialoglycoprotein receptor. Galactosylated particles transfect hepatoma cells with 10-fold higher efficiency than glucosylated particles (control), but show no preferential transfection in a cell line lacking the asialoglycoprotein receptor. Thus, surface modification of βCDP-based polyplexes through the use of cyclodextrin/adamantane host/guest interactions endows the particles with properties appropriate for systemic application.

Cationic polymers are routinely used as transfection reagents for DNA delivery to cultured cells (1-3). These polycations self-assemble with and condense DNA into small particles termed polyplexes (4). When formulated with a net positive charge, polyplexes bind to cell surfaces and are internalized (5). Despite laboratory success with polyplexes, cationic polymers are often highly toxic and therefore currently not used in human trials for gene delivery. In addition, polyplex formulations optimized for in vitro delivery are typically not amenable for in vivo use because successful systemic delivery requires very different particle characteristics. After intravenous injection, cationic polyplexes interact with serum proteins and are quickly eliminated from the bloodstream by phagocytic cells (6, 7). Additionally, these polyplexes rapidly aggregate at physiological ionic strengths (150 mM salt concentration). Finally, gene delivery vehicles should ideally target specific cells in the body while cationic polyplexes tend to be primarily taken up by liver Kupffer cells (8, 9). Therefore, cationic polyplexes require modification before they can be successfully applied for systemic gene delivery. One method of preventing polyplex self-aggregation and undesired interactions with non-self-entities employs steric stabilization of polyplexes with hydrophilic polymers. Poly(ethylene glycol) (PEG) has been conjugated to different nonviral gene carriers, and the resulting PEGylated particles have demonstrated increased salt and serum stability (10-14). PEG can be grafted onto the reactive primary amine functional groups of cationic * To whom correspondence should be addressed. E-mail: [email protected]. † California Institute of Technology. ‡ Insert Therapeutics Inc.

polymers. This method has been used for PEGylating polyethylenimine (PEI) and poly(L)lysine (PLL) in order to impart steric stabilization to the particles thus formed (11, 15). In doing so, the charge density of the polymers is reduced. Therefore, this method is often ineffective for short polymers that have lower DNA binding constants, i.e., the polymer-DNA condensation interactions are disrupted (16, 17). An alternative PEGylation approach requires complexation of the cationic polymer with DNA followed by reaction of surface-available amines with activated PEG (14). While postcomplexation PEG grafting successfully stabilizes PEI-based polyplexes, PEGylation of low molecular weight polymers by this method may result in disruption of polyplex structure. Stabilized particles may be directed to the desired organ or cells of interest by the attachment of a ligand for receptor-mediated targeting. For example, conjugation of galactose or other ligands for the asialoglycoprotein receptor can result in targeting to hepatocytes (6, 1820). A family of β-cyclodextrin-containing polymers (βCDPs) suitable for DNA delivery has been described (21, 22). These polymers contain β-cyclodextrins (cyclic oligomers of glucose) in the polymer backbone and are cationic due to repeating amidine groups. βCDP-based polyplexes show low in vitro (IC50 ) 1.1 mM) and in vivo (LD40 ) 200 mg/kg of polymer alone in mice) toxicity and are therefore promising in vivo agents. These polyplexes are subject to the same aggregation and nonspecific interactions noted above for other polyplexes. The goal of our work is to develop a method of modifying βCDP-based polyplexes for in vivo use that mitigates nondesirable

10.1021/bc0155768 CCC: $22.00 © 2002 American Chemical Society Published on Web 04/20/2002

Nonviral Gene Delivery Vehicle

Bioconjugate Chem., Vol. 13, No. 3, 2002 631

Chart 1

Chart 2

interactions that would be encountered after i.v. administration. Cyclodextrins have the capability of forming inclusion compounds with hydrophobic moieties. With some molecules, this association can be quite strong. For example, adamantane derivatives have β-cyclodextrin association constants (Ka) on the order of 104-105 (23). Complexes between adamantane end-capped poly(ethylene oxide) and cyclodextrin copolymers have also been described (24, 25). Here, we exploit cyclodextrin/guest compound complexation to develop a new method for modifying βCDPbased polyplexes. Adamantane is conjugated to a desired polyplex modifier such as PEG, and the resulting compounds are exposed to βCDP-based polyplexes for selfassembly between adamantanes and cyclodextrins. This method is applied to βCDP-based polyplexes to provide particles that are stable in salt solutions and to formulate galactose-modified particles that demonstrate targeted delivery to hepatoma cells. EXPERIMENTAL PROCEDURES

Polycation Synthesis. βCDP6. βCDP6 (6 denotes the number of methylene groups separating the charges) was synthesized according to previously described procedures and is shown in Chart 1 (21, 22). The average molecular weight of the polycation corresponds to a “z” of approximately 4 (21, 22). βCDP6-PEG3400. βCDP6 (20.3 mg, 3 µmol) and FMOCPEG3400-NHS (10 equiv, 190 mg, 60 µmol, Shearwater Polymers) were added to a glass vial equipped with a magnetic stirbar and dissolved in 1 mL of 50 mM

NaHCO3, pH 8.5. The solution was stirred in the dark at room temperature for 20 h and then lyophilized. The solid was dissolved in 0.5 mL of 20% piperidine in DMF and stirred for 30 min for FMOC deprotection. The solvent was removed in vacuo and the remaining viscous liquid dissolved in water and the pH brought below 6.0 with 0.1 M HCl. Complete PEG coupling and FMOC deprotection was verified by TNBS amine quantification (26). The polymer was separated from unreacted PEG by anion-exchange chromatography and lyophilized to yield a white fluffy powder (Chart 2). Synthesis of Adamantane Conjugates. Adamantane-PEG5000 (AD-PEG5000). PEG5000-SPA (674 mg, 135 µmol, Shearwater Polymers) was added to a glass vial equipped with a magnetic stirbar. 1-Adamantanemethylamine (5 equiv, 675 µmol, Aldrich) dissolved in 10 mL of dichloromethane was then added and the solution stirred overnight at room temperature. The solvent was removed in vacuo, and water was added to the remaining solution. The solution was centrifuged at 20K rcf for 10 min, whereupon the adamantanemethylamine phase separated as a denser liquid. The aqueous portion was collected and dialyzed for 24 h (Slide-A-Lyzer, MWCO ) 3500) against water. The solution was lyophilized to yield 530 mg of a white, fluffy powder (75% yield, structure of product shown below). The product was analyzed on a Beckman Gold HPLC system equipped with a Richards Scientific ELS detector and a C18 column and found to be pure (retention time of PEG5000-NHS: 10.7 min; retention time of product: 12.0 min; acetonitrile/water gradient). AD-PEG3400 was synthesized using a similar protocol (56% yield; product confirmed by Maldi-TOF analysis).

632 Bioconjugate Chem., Vol. 13, No. 3, 2002

Adamantane-Anionic Peptide-PEG3400-Galactose/ Glucose (AD-pep-PEG-gal/glc). An anionic peptide (sequence: E-A-E-A-E-A-E-A-C) was synthesized by the Biopolymer Synthesis Facility (Beckman Institute, California Institute of Technology) using an automatic synthesizer. Before cleaving the peptide from the resin, adamantanecarboxylic acid (ACA, Aldrich) was conjugated to the N-terminal end of the peptide with DCC coupling chemistry. The resulting peptide (AD-E-A-E-AE-A-E-A-C, MW 1084) was cleaved from the resin and analyzed by Maldi-TOF. Galactose- and glucose-PEG3400-vinyl sulfone (gal/ glc-PEG3400-VS) were prepared with approximately 95% yield by reacting NHS- PEG3400-VS (Shearwater Polymers) with 20 equiv of glucosamine or galactosamine (Sigma) in phosphate-buffered saline, pH 7.2, for 2 h at room temperature. The solution was dialyzed extensively against water and then lyophilized. The thiols of the anionic peptide (2 equiv) were reacted with galactosePEG3400-VS or glucose-PEG3400-VS in 50 mM sodium borate buffer (pH 9.5) containing 10 mM TCEP. The solution was acidified, and the precipitated peptide (insoluble below pH 9.0) was removed by centrifugation. The supernatant was collected, dialyzed extensively, and lyophilized. The desired products were confirmed by Maldi-TOF analysis (schematic shown below).

Polyplex Preparation and Characterization. Plasmids. Plasmid pGL3-CV (Promega, Madison, WI), containing the luciferase gene under the control of the SV40 promoter, was amplified by Esherichia coli and purified using Qiagen’s Endotoxin-free Megaprep kit (Valencia, CA). Physicochemical Characterization. pGL3-CV (2 µg) in 600 µL of dH2O was mixed with an equal volume of βCDP6 (in dH2O) at a charge ratio of 5+/-. Particle size and zeta potential were measured using a ZetaPals dynamic light scattering detector (DLS, Brookhaven Instruments Corporation, Holtsville, NY). Post-DNA-Complexation PEGylation by Grafting. The procedure used was modified from Ogris et al. (14). pGL3CV (5 µg) in 500 µL of dH2O was mixed with an equal

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volume of PEI (in dH2O) at 3+/- or 6+/- (stage 1). βCDP6 complexes were prepared in the same manner at a charge ratio of 5+/-. The polyplex diameters were measured by DLS. After complex formation, 10 mg/mL of PEG5000-SPA in DMF (Shearwater Polymers) was added to the solution and mixed at room temperature for 2 h (stage 2). After particle size determination, 500 µL of PBS, pH 7.2, were added to the solution. The solution was incubated for 30 min at room temperature before final particle sizes were measured by DLS (stage 3). A schematic of this procedure is illustrated in Scheme 1. Post-DNA-Complexation PEGylation by Inclusion Compound Formation. pGL3-CV (2 µg) in 600 µL of dH2O was mixed with an equal volume of βCDP6 (in dH2O) at a charge ratio of 5+/-. The desired amount of AD-PEG (10 mg/mL in dH2O) was added and particle size determined by DLS. PBS (600 µL), pH 7.2, was added to the solution and particle size monitored in 1 min intervals for 10 min. For serum stability determination, polyplexes (0.05 mg/mL DNA, CDP at 3+/-) and polyplexes PEGylated by inclusion compound formation (CDP at 3+/-, 100% adamantane to cyclodextrin by mol %) containing 5 µg of plasmid DNA were prepared by adding equal volumes of polymer solutions (50 µL) to DNA solutions. An aliquot of MEM media containing 10% fetal bovine serum (900 µL) was added to the polyplex solutions and allowed to incubate for 5 min before centrifuging at 14000 rpm for 100 min. Media was then removed, and the remaining pellet was washed with water and then resuspended in 1 mL of dH2O. The amount of DNA in the pellet was quantified by UV adsorbance at 260 nm. Formulation of Glycosylated Particles. Cyclodextrinbased polyplexes were prepared as described previously (22) at 5+/-. Adamantane-conjugated modifier was dissolved in dH2O at the desired concentrations and added to the polyplexes at 5 µL of AD-pep-PEG-sugar/µg DNA. For physicochemical characterization, glycosylated particles containing 2 µg of pGL3-CV plasmid were prepared as described and diluted with the addition of 1.2 mL of dH2O. Particle size and zeta potential of the modified polyplexes were determined by DLS. Transmission Electron Microscopy. Polyplexes, PEGylated polyplexes, and glycosylated polyplexes were prepared as described in the previous sections in 10 µL final volume. For salt stability studies, 5 µL of PBS was added to the solutions and used 10 min after salt addition. Grids were prepared and visualized as described previously (21). Cell Culture and Transfection Experiments. Cells. HepG2 (hepatoma) and HeLa (cervical carcinoma) cells were purchased from the ATCC, and HuH-7 cells (hepatoma) were purchased from the Japan Health Sciences Foundation. Cells were cultured according to recommended procedures. Media and supplements were purchased from Gibco BRL (Gaithersburg, MD). Transfections. HepG2, HeLa, and HuH-7 cells were transfected in the presence of serum with modified and unmodified βCDP6-based particles containing the pGL3CV plasmid as described previously for other adherent cell lines (22). Determination of cell viability and total protein concentration was conducted by Lowry protein assay, also described previously (22). For inhibition studies, transfections were conducted following a similar protocol except cells were preincubated for 15 min at 4 °C with 1 mg/mL asialofetuin (Sigma), after which polyplexes were added directly to the AF-containing media. Transfection media was removed after 1 h and replaced with 1 mL complete media/well.



Scheme 1

Table 1. Particle Sizes of PEI and βCDP6 Polyplexes during Post-DNA-Complexation Pegylation by Graftinga,b polyplex

PEG

stage 1 (nm)

stage 2 (nm)

stage 3 (nm)

PEI 3+/PEI 6+/βCDP6 5+/βCDP6 5+/βCDP6 5+/βCDP6 5+/-

10:1 10:1 100% 150% 200% 100% PEG**

58 55 70 70 70 67

65 60 67.4 X* X* 81

115 78 303 N/A N/A 700

a For stage 1, 5 µg of DNA plasmid (in 500 µL of dH O) was 2 mixed with PEI or βCDP6 (in 500 µL of dH2O) at the indicated ratios. PEG5000-SPA (10 mg/mL in DMF) was then added to the polyplexes in stage 2. The PEG5000-SPA was allowed to react with the polymer primary amines. After 2 h, 500 µL of PBS, pH 7.2, was added to solution. The solution was incubated for 30 min before particle size measurement. All particle sizes were determined by dynamic light scattering and reported as the mean of three measurements. b *Poor correlation function; no size measurements possible. **PEG10000 added instead of PEG5000-SPA.

RESULTS

Salt Stabilization by PEGylation. The following PEGylation approaches to the βCDP6 polycation were examined: pre-DNA-complexation PEGylation, postDNA-complexation PEGylation by grafting, and postDNA-complexation PEGylation by inclusion compound formation. The results from each method are provided below. Pre-DNA-Complexation PEGylation. PEG3400-NHS was coupled to the βCDP6 amine end groups to give the polymer CDP6-PEG3400. The PEGylated polymer was mixed with plasmid DNA for particle size measurements. While βCDP6 condenses plasmid DNA to uniform particles with hydrodynamic diameter, ∼130 nm, PEGylated βCDP6 is unable to condense DNA. Post-DNA-Complexation PEGylation by Grafting. Ogris et al. PEGylated transferrin/PEI (polyethylenimine) and PEI after complexation with plasmid DNA by reacting some of the primary amino groups of PEI with PEG5000SPA (14). PEI polyplexes were formulated at 3+/- and 6+/- while the βCDP6 polyplexes were formulated at 5+/- for stage 1. PEG5000-SPA was added to PEI at 10:1 w/w according to the procedure published by Ogris et al. (14). βCDP6 was PEGylated with 100%, 150%, and 200% PEG:amine (mol %). As a control, PEG10000 (terminated with hydroxyl groups) was also added to βCDP6 at 100%. The particle diameters obtained at each stage are presented in Table 1. The PEI polyplexes increased slightly in size upon PEGylation (58 nm to 65 nm for 3+/- and 55 nm to 60 nm for 6+/-). PEGylation protected the PEI polyplexes against salt-induced aggregation. Although unmodified PEI particles increase in diameter to ∼800 nm after salt addition, PEGylated PEI polyplexes increased slightly in size to 78 nm (for 6+/-) and 115 nm (for 3+/-).

The particle size of CDP-based polyplexes was maintained at 67 nm after PEGylation with 100% PEG5000SPA. However, the monitoring of particle size as a function of time revealed that the particles were disrupted for approximately 30 s after PEG addition. The small particles were again observed after that time. Therefore, the addition of 100% PEG5000-SPA may PEGylate a fraction of the βCDP6. Because the polycation is added in excess with respect to the DNA (at a 5+/-), the particles could then rearrange such that the unmodified polycations form polyplexes with the plasmid DNA while most of the PEGylated polycation remains free in solution. Salt addition to these particles results in particle aggregation (300 nm), although not to the extent of unmodified βCDP6 polyplexes (700 nm). The addition of 150% and 200% PEG5000-SPA to βCDP6-based polyplexes results in particle disruption; particle counts drop drastically and no consistent correlation function was observed. Post-DNA-Complexation PEGylation by Inclusion Compound Formation. The approach to post-complexation PEGylation developed here is to use the ability of cyclodextrins to form inclusion complexes with guest molecules, and the method is schematically illustrated in Figure 1A. Adamantane was conjugated to various PEG molecules. The adamantane-PEG (AD-PEG) molecules were added to solutions of preformed polyplexes at 100% adamantane to cyclodextrin (mol %). A schematic of this self-assembly process for post-DNA-complexation PEGylation by inclusion compound formation is depicted in Figure 1B. PBS was then added to the solutions and the particle size monitored by DLS in 1 min intervals (Figure 2A). The average diameter of βCDP6 polyplexes increases from 58 to 272 nm within 10 min after salt addition. The presence of free PEG5000 in solution does not prevent aggregation (average diameter of 278 nm after salt addition). However, PEGylation with AD-PEG molecules reduces particle aggregation in a length dependent matter. Ten minutes after salt addition, particles PEGylated with AD-PEG3400 aggregate to 204 nm in diameter while particles PEGylated with AD-PEG5000 only increased in diameter to 95 nm 10 min after salt addition. Additionally, the particles PEGylated with ADPEG5000 demonstrate sustained stability in physiological salt concentration (150 mM). The stabilization also occurs in a PEG density-dependent manner (Figure 2B). The average particle diameter measured 10 min after salt addition increases by 4.7-fold for unmodified polyplexes (58 nm to 272 nm) but only 1.2-fold for polyplexes modified with the addition of 150% or 200% PEGadamantane to cyclodextrin. The stabilization in salt by PEGylation of βCDP6 polyplexes via inclusion compound formation was verified by transmission electron microscopy. βCDP6-based polyplexes formulated in water appear as discrete, uniform


Figure 1. (A). Schematic of inclusion compound formation. (B). Schematic for post-DNA-complexation PEGylation by inclusion compound formation by PEG-AD and ligand-PEG-AD. PEG is conjugated to adamantane. Adamantane forms an inclusion complex with β-cyclodextrin on the polyplex surface and brings with it PEG or PEG-L to decorate the polyplex and provide steric stabilization and targeting.

particles with diameters around 100 nm (Figure 3A). PEGylated βCDP6-based polyplexes (100% PEG:CD by mole) formulated in water are also uniform particles with diameters around 100 nm (Figure 3B). An aliquot of PBS (150 mM salt concentration) was then added to the polyplexes to give a final salt concentration of 50 mM. After 10 min, the solutions were loaded onto a grid and visualized by electron microscopy. Unmodified polyplexes in 50 mM salt aggregated; no discrete particles were observed (Figure 3C). Electron micrographs similar to Figure 3C have been observed previously by Goula et al. for PEI-based polyplexes (27). To the contrary, βCDP6based polyplexes modified with AD-PEG5000 were resistant to salt-induced aggregation. Electron microscopy revealed discrete particles (∼110 nm in diameter, Figure 3D). Thus, successful stabilization of βCDP6-based polyplexes by AD-PEG5000 modification is demonstrated by both DLS measurements and electron microscopy. The PEGylated polyplexes are also stable in culture media containing 10% serum. Unmodified and PEGylated polyplexes were exposed to serum-containing media for 10 min and then centrifuged to pellet aggregated complexes. The pellets were resuspended in water and the amount of DNA in the pellet was quantified by UV adsorption. All of the DNA from the unmodified polyplexes were recovered in the pellets, indicating that the polyplexes aggregate rapidly in the presence of physiological salt and serum. In addition, proteins were precipitated with the unmodified polyplexes (on average 6 µg protein/µg DNA). However, only ∼18% of the DNA from PEGylated polyplexes was recovered in the pellets, demonstrating increased stabilization in the presence of serum. Galactose-Mediated Delivery to Hepatocytes. Preparation and Characterization of Galactose and Glucose-Modified Polyplexes. βCDP6-based polyplexes re-

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quire formulation at a net positive charge for complete condensation of DNA (21). However, these positively charged polyplexes indiscriminately transfect cultured cells. To differentiate between charge-mediated transfection and receptor-mediated transfection, the polyplexes should ideally possess neutral or negative zeta potentials and contain a targeting ligand directed at the receptor of interest. Adamantane-anionic peptide-PEG3400galactose (AD-pep-PEG-gal) was prepared for βCDP6 polyplex modification. The adamantane serves as the guest for forming inclusion complexes with the βCDP6based polyplexes, the anionic peptide increases the binding affinity of the AD-pep-PEG-gal compound for the cationic polyplex while also decreasing the zeta potential of the polyplex to eliminate charge-mediated entry, and the PEG provides steric stabilization and serves as a spacer between the polyplex and the galactose molecules that act as the targeting ligands to hepatocytes. A glucose analogue (AD-pep-PEG-glc) was prepared as a control. The zeta potentials of βCDP6 polyplexes (prepared at 5+/-) modified with various amounts of AD-pep-PEGgal/glc were determined and are shown in Figure 4A. Unmodified polyplexes are positively charged, with zeta potential around +10 mV. The addition of the glycosylated adamantane compounds to the complexes decreases the zeta potential of the particles. The alteration of charge plateaus at around 50% AD-pep-PEG-gal/glc (% PEG to CD by mole) with zeta potential approximately -7 mV. Little differences between the zeta potentials of the galactose and glucose-modified polyplexes are observed. The size of the two types of particles is also very similar. Electron micrographs of the galactosylated particles (50% galactose:CD by mole) show particles with average diameters around 100 nm (Figure 4B), while measurements by DLS reveal particles with diameters around 120 nm (data not shown). Although these particles are negatively charged, the particles look similar to the unmodified polyplexes (Figure 3A), demonstrating that modification by inclusion compound formation does not significantly affect polyplex morphology. Galactosylated and glucosylated βCDP6 particles containing the luciferase gene were prepared at 0, 20, 40, 60, 80, and 100% PEG and exposed to HepG2 (hepatoma) cells (Figure 5A). Unmodified polyplexes yielded the highest transfection (2 × 108 RLU/mg protein). At 20% PEG, the galactosylated and glucosylated particles have reduced but positive zeta potentials and gave similar transfection efficiencies. At 40% PEG, the particles are anionic (∼-5 mV) and the transgene activity from glucosylated polyplexes is reduced by 2 orders of magnitude from that of the polyplex alone, while activity from galactosylated polyplexes is reduced by 1 order of magnitude. As the PEG concentration increases to 100%, the luciferase activity of glucosylated polyplexes continues to decrease; however, activity of galactosylated polyplexes remains 1 order of magnitude higher than the glucosylated polyplexes. Another hepatoma cell line, HuH-7, was transfected with the modified polyplexes and the same trend is observed. The glycosylated polyplexes transfect HuH-7 cells with the same efficiency when formulated at net positive charges. However, when formulated at net negative charges, galactosylated polyplexes consistently transfect with higher efficiencies, with up to 15-fold higher luciferase activity (at 50% PEG). HeLa cells that do not express the asialogylcoprotein receptor were transfected with the modified polyplexes. No preferential transfection of galactosylated polyplexes to HeLa cells was observed (Figure 5B).



Figure 2. Salt stabilization of polyplexes by PEGylation. Plasmid DNA was mixed with βCDP6 at 5+/-, and then PEG compounds were added to the complexes for a total volume of 1.2 mL. PBS (600 µL) was added to the solutions, and the particle sizes were monitored by dynamic light scattering. (A) The effect of PEG length on salt stabilization. Polyplexes were mixed with various PEG compounds. Particle sizes were monitored by dynamic light scattering for 10 min after salt addition. ((: polyplex, ): polyplex + PEG5000, 2: polyplex + AD-PEG3400, 9: polyplex + AD-PEG5000). (B). The effect of AD-PEG5000 concentration of salt stabilization. Various amounts of AD-PEG5000 were added to polyplexes before exposure to 50 mM PBS. Particle sizes were measured 10 min after salt addition.

Figure 4. (A). Zeta potential of glycosylated polyplexes. Polyplexes were prepared at 5+/- in water and modified with various amounts of AD-pep-PEG-gal or AD-pep-PEG-glc (100% PEG ) 1 mol PEG/1 mol CD). Polyplexes were diluted in water to 2 µg DNA/1.2 mL dH2O and zeta potential determined by DLS measurements. (Solid line, galactosylated polyplexes; dashed line, glucosylated polyplexes.) (B). Transmission electron micrograph of galactosylated particles modified with 50% AD-pep-PEG-gal (bar is 100 nm). Figure 3. Transmission electron micrographs of (A) unmodified βCDP6 polyplexes, (B) βCDP6 polyplexes PEGylated by ADPEG5000 modification, (C) unmodified βCDP6 polyplexes in 50 mM salt, and (D) PEGylated βCDP6 polyplexes in 50 mM salt. (Bar is 100 nm for A, B, D and 1000 nm for C).

To verify cell entry via the asialoglycoprotein receptor, transfections were performed in the presence of an excess of asialofetuin, a high affinity ligand for the asialoglycoprotein receptor (Figure 6). Asialofetuin did not affect polyplex or glucosylated polyplex transfection but decreased transfection with galactosylated polyplexes by 4-fold. The toxicities of the βCDP polyplexes and modified particles were measured by determining total cellular protein 48 h after transfection. βCDP-based polyplexes have been shown to be well-tolerated to high doses both in vitro and in vivo (21, 22). The adamantane-based compounds do not impart significant added toxicity to the βCDP particles (93% cell survival with galactosylated polyplexes vs 96% survival with unmodified polyplexes). DISCUSSION

This work describes the development of a new method for modifying the surface properties of βCDP-based

polyplexes that will now allow for their study as systemic, in vivo, gene delivery agents. Two features necessary for a successful systemic carrier are stability at physiological conditions and selective delivery to sites of interest (28). These issues are addressed here by PEGylation of the βCDP-based polyplexes and by the attachment of a galactose ligand for delivery to hepatocytes. PEGylation has been shown to stabilize colloidal particles (28). Three different approaches to βCDP-based polyplex PEGylation were assessed: pre-DNA-complexation PEGylation, post-DNA-complexation PEGylation by grafting, and post-DNA-complexation PEGylation by inclusion compound formation. PEG was covalently conjugated to the amine termini of βCDP6. While βCDP6 self-assembles with and condenses DNA to small particles (21, 22), the presence of PEG3400 at the polymer termini inhibits DNA condensation. Choi et al. report successful albeit reduced DNA binding with PEGylated PLL (11). However, they used PLL with MW 25000, that contains on average 170 amines/polymer strand. The long polycation provides for strong DNA binding. The βCDP6 possesses approximately 10 charges/polymer strand, and PEGylation doubles the molecular weight. Therefore, it


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Figure 6. HepG2 cells were transfected with polyplexes (unmodified or modified with 50% AD-pep-PEG-galactose/ glucose) prepared at 5+/-. Luciferase protein activity was determined 48 h after transfection and reported as RLU/mg total protein (total protein determined by Lowry assay). Data are presented as mean ( SD of three samples. Competitive inhibition of galactose-mediated delivery. HepG2 cells were transfected in the presence (hatched bars) and absence (black bars) of asialofetuin (amounts larger than 1 mg/mL significantly altered cell proliferation behavior and therefore were not used).

Figure 5. A. Galactose- and glucose-mediated transfection of HepG2 hepatoma cells. Cells were transfected with 1 µg of luciferase gene-containing plasmid complexed with βCDP6 at 5+/- and modified with various amounts of AD-pep-PEGgalactose or AD-pep-PEG-glucose (100% PEG ) 1 mol PEG/1 mol CD). Luciferase protein activity is reported as the mean +/- SD of three samples in RLU/mg of total protein (protein concentration determined by Lowry protein assay). Black bars, galactosylated polyplexes; hatched bars, glucosylated polyplexes. DNA alone ) 1,000 RLU/mg protein. B. Galactose- and glucosemediated transfection of HeLa cells that do not express the asialoglycoprotein receptor. Cells were transfected with 1 µg of luciferase gene-containing plasmid complexed with βCDP6 at 5+/- and modified with various amounts of AD-pep-PEGgalactose (Gal-Polyplex) or AD-pep-PEG-glucose (Glc-Polyplex, 100% PEG ) 1 mol PEG/1 mol CD). Luciferase protein activity is reported as the mean ( SD of three samples in RLU/ mg of total protein (protein concentration determined by Lowry protein assay).

is not surprising that PEGylation of the βCDPs eliminates its ability to condense to plasmid DNA. Post-complexation PEGylation as described by Ogris et al. (14) and Oupicky et al. (29) first requires the formation of the polyplex followed by PEGylation of the particles. This method involves the conjugation of surfaceavailable, primary amines and thus reduces polyplex instability that can result from the interference in the DNA condensation process when using PEG-grafted polycations. This approach is effective for polymers such as PEI and HPMA but results in particle disruption for βCDP6-based polyplexes (Table 1). Thus, post-DNAcomplexation PEGylation by reaction with accessible polymer functional groups is likely to be effective for high molecular weight polymers with high charge densities. However, reaction with low molecular weight βCDP6 destabilizes the polyplex structure. The approach to surface modification developed here involves the use of PEG conjugated to moieties that are in turn exploited as anchors to the polyplex surface by inclusion compound formation in cyclodextrins (Figure 1). We hypothesized that use of the cyclodextrin cups for

the site of modification would cause minimum disturbance to preformed polyplexes. This is because the βCDPs bind to DNA via electrostatic interactions of the cationic amidine groups on the βCDP and the anionic phosphate groups on the DNA. The cyclodextrin cups should therefore be available on the polyplex surface for complexation. Adamantane-PEG conjugates were added to βCDP6based polyplexes, and the resulting particles remained small and disperse with only slightly increased diameters (Figures 2 and 3A). PEGylation introduced salt stability to the polyplexes in a PEG length and PEG densitydependent manner (Figure 2). The salt stability of PEGylated βCDP6-based polyplexes was confirmed by electron microscopy. While unmodified polyplexes form large aggregates in 50 mM salt, PEGylated βCDP6-polyplexes remain discrete and condensed (Figure 3A-D). In addition, PEGylated βCDP6based polyplexes have increased stability in the presence of serum. Other investigators have reported interference of polymer/DNA condensation by PEGylation. For example, PEGylation of PEI (MW 2000) results in thick thread and donut morphologies rather than densely compacted structures (16) and PEGylation of PLL (MW 4000) altered polyplex structure from toroids to rods (12). Here, it is shown that PEGylation by inclusion compound formation stabilizes βCDP6-based polyplexes without disrupting the polyplex structure. The PEGylated particles maintained small sizes in the presence of 150 mM salt for hours. The adamantane is integral to forming protected polyplexes; the addition of free PEG5000 has no effect on salt stabilization. The addition of adamantane (in DMSO) eliminates the salt stability of the modified polyplexes by displacing the ADPEG from the polyplexes. Thus, it is clear that the adamantane in the AD-PEG5000 forms an inclusion complex with the β-cyclodextrins on the surface of the polyplexes. PEGylation of polyplexes alleviates salt-induced aggregation by providing a steric layer around the particles. The steric layer prevents close contact between particles by keeping the van der Waals attraction forces, that are dependent on the interparticle distance, lower than the


thermal energy of the particles. Therefore, there should be a critical polymer length greater than the range of van der Waals attraction between particles that results in polymer stabilization. For many colloidal systems, the necessary distance is ∼5 nm (30). The lengths of PEG3400 and PEG5000 are estimated to be 3.5 and 4.3 nm, respectively (30). PEG5000 is long enough to stabilize βCDP6 polyplexes in salt while PEG3400 does not provide enough steric stabilization under the experimental conditions. Interestingly, Gref et al., working with PEG-coated poly(lactic acid) (PLA) nanoparticles of 160-270 in diameter, also found 5000 to be the minimum PEG molecular weight necessary for significant reductions in the plasma protein binding to the nanoparticles (31). The association between the AD-PEG molecules and the βCDP6 polyplexes was empirically found to be very strong. PEGylated particles with DNA concentrations as dilute as 1 µg/mL remained stable under physiological salt conditions. The high association may be a result of decreased diffusional mobility of the adamantane away from the cyclodextrin because it is tethered to the PEG molecule and the high local concentration of cyclodextrin molecules on the polyplex surface, i.e., when an adamantane molecule exits one cyclodextrin cup, it is likely to go back in the same or a nearby cyclodextrin rather than going into solution (distances between cyclodextrins is very small relative to what they would be for individual cyclodextrins in solution). Based on the success of PEGylating βCDP-based polyplexes by inclusion compound formation, an anionic, galactosylated adamantane compound was evaluated as a modifier to target βCDP-based polyplexes to hepatocytes. These adamantane modifiers contain four elements: (i) the adamantane, to attach the compound to the polyplex surface, (ii) an anionic peptide to change the polyplex surface charge and thereby reduce nonspecific transfection due to charge, (iii) PEG to serve as a tether for the ligand and as a stabilizing agent, and (iv) galactose as a targeting ligand to the asialoglycoprotein receptor (ASGP-R) or glucose as a low affinity ligand as a control. As demonstrated by the data shown in Figure 4A, the addition of these compounds to polyplexes causes the zeta potential to decrease. The drop in surface charge confirms modification of polyplexes. Additionally, the zeta potentials and particle sizes of the galactose and glucosemodified polyplexes are very similar (approximately -7 mV, 120 nm). Electron microscopy reveals that the galactosylated particles, although having different surface charge, retain approximately the same morphology and size as the unmodified polyplexes (Figures 3 and 4B). Therefore, differences in transfection ability between these particles are due to the ligand rather than physicochemical differences in the particles. The glycosylated particles were exposed to the HepG2 cell line for uptake and transgene expression experiments. Uptake experiments were conducted at a variety of conditions (different degrees of polyplex modification, polyplex charge, and polyplex exposure time to cells) using fluorescently labeled plasmid DNA for analysis by flow cytometry. As expected, uptake efficiency was dependent on several parameters. However, uptake of galactosylated polyplexes into the hepatoma cells was consistently higher than uptake of glucosylated polyplexes. When formulated at positive zeta potentials (0, 10, or 20% PEG), no difference in transfection efficiency is observed between glucosylated and galactosylated polyplexes. This is not surprising because when the polyplexes are positively charged, they are efficiently and indiscriminantly internalized by nonspecific interactions


with proteoglycans (5). When the particles are formulated at net negative charges (PEG-pep g 40%), selective transfection by the galactosylated formulation is observed. In fact, the luciferase activity from galactosemediated transfection is consistently an order of magnitude higher than from glucosylated polyplexes. The glucosylated polyplexes also demonstrate a ADpep-PEG-glc-dependent response to surface modification. Addition of the anionic adamantane derivative reduces the surface charge, resulting in a precipitous drop in transfection ability beyond 20% PEGylation. In contrast, the effect of PEGylation on transfection observed with galactosylated polyplexes is not so pronounced most likely because the cells continue to internalize these particles by receptor-mediated endocytosis via the ASGPR. The gene expression from galactosylated polyplexes is approximately 1 order of magnitude lower than that of unmodified polyplexes. Other investigators report increased transfection to hepatocytes upon galactosylation of polyplexes (18, 32, 33). However, in those systems, charged-mediated delivery is not eliminated upon galactosylation, and uptake should occur by both charge and receptor-mediated delivery. Here, charge-mediated delivery of galactosylated βCDP particles is minimized by formulating anionic βCDP6 particles with the galactose ligand. Galactosylated βCDP particles are mainly endocytosed by the ASGP-R-mediated pathway. The reduction in expression from galactosylating the βCDP particles is therefore not surprising as uptake studies with unmodified and galactosylated polyplexes demonstrated that receptor-mediated endocytosis provides for less cellular uptake than nonspecific, charge-mediated uptake (data not shown). Reduction of nonspecific, chargemediated uptake and gene expression is likely to be essential for effective, systemic, in vivo application. Delivery via the ASGP-R is confirmed by two methods. First, transfection in the presence of asialofetuin, a high affinity ligand to the receptor, specifically decreases transfection of galactosylated polyplexes without affecting transfections with unmodified and glucosylated polyplexes (Figure 6). Second, luciferase activity is not observed for transfections of HeLa cells (do not express the ASGP-R) with galactosylated polyplexes (Figure 5B). Luciferase activity is observed only with unmodified polyplexes. The targeted delivery system demonstrates no toxicity over the unmodified polyplex. The low toxicity of these particles make them attractive for in vivo delivery applications. For future work, receptor-ligand interactions can be further optimized by using different conjugation chemistries between the galactose ligand and the PEG. In this study, galactosamine was conjugated to the adamantane modifier via the C2 position. David et al. report that conjugation via the galactose C1 position results in higher uptake into hepatoma cells than conjugation via the C2 position (34). In addition, asialofetuin, a natural ligand for the ASGP-R binds to the receptor via a triantennary galactose residue. In fact, the affinity constants for the ASGP-R has been established with tri- . bi- . monoantennary galactose with affinity constants of about 10-9, 10-6, and 10-3 M, respectively (35). Therefore, incorporation of a C1-conjugated galactose residue or a multiantennary galactose ligand into the adamantane-PEG modifier could further improve the targeting ability of the galactosylated polyplexes. In conclusion, a new method of modifying the surface of βCDP-based polyplexes through the use of cyclodextrin/ adamantane host/guest interactions is presented. The inclusion complex formation is not restricted to adaman-


tane and β-cyclodextrin, as a multitude of guest/host materials are known. Also, the method should be applicable to other cyclodextrin-containing polymers that should be capable of forming of forming polyplexes, e.g., polyamidoamine dendrimers (36), polyallylamines (37), PEI (38), and chitosan (39). Here, we have demonstrated stabilization and galactose-mediated targeting of surfacemodified particles without any increase in cell-associated toxicity. This method of adapting polyplexes for in vivo use imposes no interference with polyplex formation, occurs by self-assembly, and is modularly adaptable for various applications including the addition of agents that effect intracellular trafficking, codelivery of small molecules, different targeting agents, or combinations thereof (40). This system is currently being studied in animal models. ACKNOWLEDGMENT

S.J.H. thanks the Whitaker Foundation for financial support for the portion of research conducted at the California Institute of Technology. S.J.H. is also grateful for helpful discussions with Dr. Nathalie Bellocq (Insert Therapeutics) and Prof. John Brady (Caltech). M.E.D. is a consultant to and has financial interest in Insert Therapeutics. LITERATURE CITED (1) Behr, J.-P. (1993) Synthetic Gene-Transfer Vectors. Acc. Chem. Res. 26, 274-278. (2) Bielinska, A., Kukowska-Latallo, J., and Baker, J. (1997) The interaction of plasmid DNA with polyamidoamine dendrimers: mechanism of complex formation of analysis of alterations induced in nuclease sensitivity and transcriptional activity of the complexed DNA. Biochim. Biophys. Acta 1353, 180-190. (3) Boussif, O., Lezoualcl′h, F., Zanta, M., Mergny, M., Scherman, D., Demeneix, B., and Behr, J.-P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. Proc. Natl. Acad. Sci. U.S.A. 92, 7297-7301. (4) Felgner, P., Barenholz, Y., Behr, J.-P., Cheng, S., Cullis, P., Huang, L., Jessee, J., Seymour, L., Szoka, J., FC, Thierry, A., Wagner, E., and Wu, G. (1997) Nomenclature for Synthetic Gene Delivery Systems. Hum. Gene Ther. 8, 511-512. (5) Mislick, K., and Baldeschwieler, J. (1996) Evidence for the role of proteoglycans in cation-mediated gene transfer. Proc. Natl. Acad. Sci. U.S.A. 93, 12349-12354. (6) Nishikawa, M., Takemura, S., Takahara, Y., and Hashida, M. (1998) Targeted delivery of plasmid DNA to hepatocytes in vivo: Optimization of the pharmacokinetics of plasmid DNA galactosylated poly(L-lysine) complexes by controlling their physicochemical properties. J. Pharm. Exp. Ther. 287, 408-415. (7) Dash, P., Read, M., Barrett, L., Wolfert, M., and Seymour, L. (1999) Factors affecting blood clearance and in vivo distribution of polyelectrolyte complexes for gene delivery. Gene Ther. 6, 643-650. (8) Ward, C. M., Read, M. L., and Seymour, L. W. (2001) Systemic circulation of poly(L-lysine)/DNA vectors is influenced by polycation molecular weight and type of DNA: differential circulation in mice and rats and the implications for human gene therapy. Blood 97, 2221-2229. (9) Collard, W., Yang, Y., Kwok, K., Park, Y., and Rice, K. (2000) Biodistribution, Metabolism, and In Vivo Gene Expression of Low Molecular Weight Glycopeptide Polyethylene Glycol Peptide DNA Co-Condensates. J. Pharm. Sci. 89 (4), 499512. (10) Woodle, M. (1998) Controlling liposome blood clearance by surface-grafted polymers. Biochim. Biophys. Acta 1113, 171199.

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Bioconjugate Chem., Vol. 13, No. 3, 2002 639 the asialoglycoprotein receptor system: A model of receptormediated endocytosis. Int. Rev. Cytol. 97, 47-95. (36) Arima, H., Kihara, F., F., H., and Uekama, K. (2001) Enhancement of Gene Expression by Polyamidoamine Dendrimer Conjugates with a-, b-, and g-cyclodextrins. Bioconjugate Chem. 12, 476-484. (37) Hollas, M., Chung, M. A., and Adams, J. (1998) Complexation of pyrene by poly(allylamine) with pendent b-cyclodextrin side groups. J. Phys. Chem. B 102, 2947-2953. (38) Suh, J., Lee, S. H., and Zoh, K. D. (1992) A novel host containing both binding site and nucleophile prepared by attachment of β-cyclodextrin to poly(ethylenimine). J. Am. Chem. Soc. 114, 7916-7917. (39) Tanida, F., Tojima, T., Han, S. M., Nishi, N., Tokura, S., Sakairi, N., Seino, H., and Hamada, K. (1998) Novel synthesis of a water soluble cyclodextrin-polymer having a chitosan skeleton. Polymer 39, 5261-5267. (40) Hwang, S. (2001) In Chemical Engineering, p 167, California Institute of Technology, Pasadena.

BC0155768

Development of a Nonviral Gene Delivery Vehicle for Systemic

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