PEI and PEGylated PEI Polyplex-Mediated Gene Delivery to the Live

Bioconjugate Chem. 2008, 19, 693–704

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Extracellular Barriers to in ViWo PEI and PEGylated PEI Polyplex-Mediated Gene Delivery to the Liver Rob S. Burke and Suzie H. Pun* Department of Bioengineering, University of Washington, 1705 NE Pacific Street, Seattle, Washington 98195. Received October 20, 2007; Revised Manuscript Received November 10, 2007

Polyplex-mediated gene therapy is a promising alternative to viral gene therapy. One challenge to these synthetic carriers is reduced transfection efficiencies in ViVo compared to those achieved in Vitro. Many of the intracellular barriers to gene delivery have been elucidated, but similar quantification of extracellular barriers to gene delivery remains a need. In this study, the unpackaging of polyplexes by serum proteins, soluble glycosaminoglycans, and an extracellular matrix extract was demonstrated by a YOYO-1 fluorescence quenching assay. Additionally, exposing polyplexes to serum or proteoglycans before in Vitro transfection caused decreased cellular uptake of DNA. Lastly, PEI polyplexes and PEGylated PEI polyplexes were injected into the portal vein of mice, and the intrahepatic distributions of labeled DNA and polymer were assessed by confocal microscopy. PEI polyplexes delivered DNA to the liver, but extensive vector unpackaging was observed, with PEI primarily colocalized with the extracellular matrix. PEGylated polyplexes mediated less DNA delivery to the liver, possibly due to premature vector unpackaging in the blood. Through this work, both the blood and the extracellular matrix have been determined to be significant extracellular barriers to polyplex-mediated in ViVo gene delivery to the liver.

INTRODUCTION The field of gene therapy first gained momentum with the use of viral gene delivery vectors (1, 2). However, interest in the development of nonviral vectors remains high because of challenges associated with the use of viruses in humans, including safety and large-scale production issues (3, 4). One of the main classes of nonviral gene delivery vectors that has been extensively investigated is complexes of cationic polymer and DNA, termed polyplexes (5). Polycations condense DNA through electrostatic interactions to form particles with sizes on the order of 100 nm. One of the most popular cationic polymers used to form polyplexes is the commercially available polyethylenimine (PEI) (6). The use of PEI to deliver DNA has become widespread after several groups showed that it generates particularly high in Vitro transfection efficiencies compared to those of other nonviral gene delivery vectors by mediating endosomal release (6–11). Branched PEI (bPEI) and linear PEI (lPEI) can both be used effectively for gene delivery. Although lPEI is generally preferred for in ViVo applications because of its advantageous toxicity profile (12), bPEI contains a higher percentage of primary amines and is thus more suitable to modifications. Examples of modifications include incorporation of targeting ligands to increase the specificity of delivery, labeling the polymer with fluorophore to track its location through fluorescence microscopy, or grafting of poly(ethylene glycol) (PEG) to the polymer to increase polyplex stability. Incorporation of PEG in polyplex formulation, a process known as PEGylation, increases the salt stability of the particles and reduces interaction with blood components, resulting in decreased uptake by the reticuloendothelial system and prolonged circulation time of the PEGylated polyplexes (13). However, at the same time, PEGylation has been shown to reduce uptake by cells, which in turn reduces the level of gene expression (14, 15). * To whom correspondence should be addressed. Tel: (206) 6853488. Fax: (206) 616-3928. E-mail: [email protected].

Much of the work investigating the mechanism of nonviral gene delivery has focused on the intracellular barriers that a delivery vector must overcome to deliver its cargo to the nucleus, including binding to the cell surface and internalization (16–19), escape from the endosomal pathway (9, 10, 20, 21), transport in the cytoplasm (22–25), nuclear entry (26–29), and vector unpackaging (30, 31). However, the extracellular barriers to gene delivery are important to consider for in ViVo applications. For organ-specific delivery after systemic administration, the vectors must successfully extravasate from the vasculature and maneuver through the extracellular matrix (ECM) to reach the target cells. Because polyplexes are held together electrostatically, other charged species may disrupt the interaction between DNA and polymer. Indeed, Huth et al. showed that polyplexes released from endosomes could unpackage in the cytoplasm by competition with cytosolic RNA (31). Additionally, Schaffer et al. found that chromosomal DNA inside the cell nucleus is capable of participating in vector unpackaging (30). Taken together, these studies illustrate that polymeric properties such as the molecular weight, which affects how tightly the polymer binds DNA, influence where the polyplexes unpackage after intracellular delivery. However, charged species that are able to compete the polymer off the DNA do not only exist inside cells. Polyplex vector interactions with extracellular and cell surface glycosaminoglycans (GAGs) also result in vector unpackaging and a decrease in gene delivery efficiency (32–35). In Vitro transfection studies with PEI polyplexes have shown high gene delivery efficiencies; in contrast, in ViVo data obtained by various researchers shows that in Vitro transfection is a poor predictor of in ViVo success (12, 36, 37). The rationale for this work is that, although many polymeric vectors have been optimized in Vitro to overcome intracellular barriers, there are many extracellular barriers that must be overcome in order to achieve successful in ViVo gene delivery. An understanding of the limiting barriers to in ViVo gene delivery will facilitate the design of improved vectors able to overcome these barriers, thus increasing the efficiency of gene delivery. In this work, the integrity of PEI and PEGylated PEI polyplexes is examined both

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in Vitro and in ViVo through dual-fluorescence labeling of polymer and DNA. The in ViVo studies focused on the assessment of intrahepatic distributions of polymer and delivered DNA because other studies have shown that polyplexes accumulate primarily in the liver after injection but that transgene expression is relatively low (38–41). Our results reveal that both the serum and the ECM pose barriers to gene delivery. Systemically injected PEI polyplexes are found to be unpackaged in the liver, thus accounting for suboptimal gene delivery efficiencies.

EXPERIMENTAL PROCEDURES Materials. All materials were purchased from Sigma-Aldrich unless otherwise stated. Synthesis of PEI-Dinitrophenyl (PEI-DNP). bPEI (25,000 g/mol, 12.7 mg) was dissolved in 0.15 M NaHCO3 at pH 8.3, at 10 mg/mL. To this solution was added 5 mg (25 equiv) of 6-(2,4-dinitrophenyl)aminohexanoic acid, succinimidyl ester (DNP-NHS, from Invitrogen) dissolved at 10 mg/mL in DMF, followed by stirring overnight at room temperature. The polymer was then protonated by the addition of HCl and dialyzed in a Slide-A-Lyzer 10K MWCO dialysis cassette (Pierce) against dH2O to remove solvent and excess reactants. The solution was lyophilized to yield a fluffy yellow solid. The polymer was redissolved in dH2O, the PEI concentration was determined by a copper (II) acetate assay as described previously (42), and the DNP concentration was measured by absorbance at 360 nm. Synthesis of PEG-DNP. Thirty-eight milligrams of HCl · H2NPEG-COOH (5000 g/mol, Nektar Therapeutics) and 1.5 equiv of triethylamine (TEA) were dissolved in 1 mL of anhydrous DMF. DNP-NHS (5.4 mg (1.8 equiv)) was dissolved in 250 µL of anhydrous DMF, added to PEG, and stirred for 48 h. Ten milliliters of dH2O was added to the reaction mixture, which was then added to a Slide-A-Lyzer 3.5K MWCO dialysis cassette (Pierce) and dialyzed against dH2O for 2 days to remove unreacted DNP. The reaction solution was recovered from the dialysis cassettes, passed through a glass filter with a piece of grade #50 Whatman filter paper (Fisher), and lyophilized to yield approximately 40 mg of a fluffy yellow solid. 1H NMR (300 MHz Bruker Avance NMR Spectrometer using a BBI inverse probe) was performed on PEG-DNP to determine the labeling ratio of DNP by proton integration of PEG CH2 next to the terminal carboxyl group (2.5 ppm) and of the DNP CH groups in the aromatic ring (7.1, 8.3, and 9.1 ppm). Integration revealed that 84.1% of the PEG was conjugated to DNP (Supporting Information). The remaining amine groups were reacted with Sulfo-NHS acetate (Pierce), irreversibly capping the amines with an acyl group. Amine blocking was confirmed by a 2,4,6trinitrobenzenesulfonic acid (TNBS) assay as described in ref 43. Synthesis of PEI-PEG-DNP. PEG-DNP (10.5 mg) was dissolved in 400 µL of anhydrous DMF. 1,3-Dicyclohexylcarbodiimide (DCC) (2.0 mg (5 equiv)) was dissolved in 50 µL of anhydrous DMF, and 1.12 mg (5 equiv) of N-hydroxysuccinimide (NHS) was dissolved in 50 µL anhydrous DMF. The 50 µL of NHS was added to PEG-DNP, followed immediately by 50 µL of DCC, and the reaction was allowed to stir for 48 h to activate the carboxylic acid group on the PEG with NHS. Then, 6.08 mg of PEI (25,000 g/mol, branched) was dissolved in 608 µL of anhydrous DMF with 150 molar equiv of TEA, and the reaction was stirred for 30 min. The activated PEI solution was added to NHS-PEG-DNP for an 8:1 molar PEG/PEI ratio and allowed to stir for 24 h. Another aliquot of TEA (150 equiv), DCC (5 equiv), and NHS (5 equiv) in anhydrous DMF was added to drive the reaction to completion and stirred for another 24 h. The polymer was protonated by the addition of HCl and dialyzed in a Slide-A-Lyzer 10K MWCO Dialysis Cassette

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against dH2O to remove unreacted PEG-DNP from the PEIPEG-DNP solution. The dialyzed solution was filtered through a 0.22 µm filter and lyophilized to yield a fluffy yellow solid. 1 H NMR (300 MHz Bruker Avance NMR Spectrometer using a BBI inverse probe) was performed on PEI-PEG-DNP to determine the labeling ratio of PEG-DNP by proton integration of the PEI main chain CH2 groups (2.6–3.2 ppm) (44) and of the DNP CH groups as described above. The integration of the DNP, the integration of the PEI, and the 0.841 DNP/PEG relationship were used to determine the labeling ratio as 6.84 mol PEG per mol PEI (Supporting Information). Synthesis of PEI-PEG. bPEI (25,000 g/mol, 6.08 mg) was dissolved in 608 µL of anhydrous DMF with 150 molar equiv of TEA, and the reaction was stirred for 30 min. Then, 10.5 mg of mPEG-SPA (5000 g/mol, Nektar Therapeutics) was dissolved in 400 µL of anhydrous DMF, and the activated PEI solution was added to the mPEG-SPA for an 8:1 molar PEG/ PEI ratio and allowed to stir for 48 h. The polymer was protonated by the addition of HCl and dialyzed in a Slide-ALyzer 10K MWCO Dialysis Cassette (Pierce) against dH2O to remove unreacted PEG from the PEI-PEG solution. The dialyzed solution was filtered through a 0.22 µm filter and lyophilized to yield a fluffy white solid. 1H NMR (300 MHz Bruker Avance NMR Spectrometer using a BBI inverse probe) was performed on PEI-PEG to determine the labeling ratio of PEG by proton integration of the PEI main chain CH2 groups (2.6–3.2 ppm) and of the PEG main chain CH2 groups (3.6 ppm) (44). The integration revealed a labeling ratio of 7.31 mol PEG per mol PEI (Supporting Information). Labeling Plasmid DNA with Quantum Dots. gWiz-Luciferase plasmid (gWiz-Luc, Endotoxin-free) was purchased from Aldevron, and 1 mg of the DNA plasmid was purified by ethanol precipitation and resuspended in 1× TE buffer (10 mM Tris and 1 mM EDTA) at a concentration of 2 mg/mL. The plasmid was labeled with EZ-Link psoralen-PEO3-biotin (Pierce) and then linked to streptavidin-conjugated quantum dots (QD585-SA conjugate from Invitrogen/Molecular Probes) as described by the protocol of Ho et al. (45). Briefly, the DNA was biotinylated by photocross-linking psoralen-PEO3-biotin onto the DNA, and then the QD-585-SA conjugates were attached to the DNA through the biotin. The degree of labeling of the DNA was determined by first creating standard curves of absorbance at 260 nm (A260) versus concentration for both the DNA and the QDs and standard curves of fluorescence (ex ∼350 nm, em ∼585 nm) versus concentration for both the DNA and QDs. The concentration of the QDs could be determined from the fluorescence measurements, the QD concentration could be used to calculate the QDs’ contribution to the A260 measurements, and then the corrected A260 signal could be used to calculate the DNA concentration. It was determined that the DNA plasmid was labeled with one QD per 2 plasmids. Formulation of Polyplexes and Testing Salt Stability. The gWiz-Luc plasmid was diluted in water to a concentration of 0.1 µg/µL and mixed with an equal volume of polymer at the desired polymeric nitrogen to DNA phosphate (N/P) ratio. After mixing, the polyplexes were allowed to incubate for 5 min at room temperature. Ten microliters of each polyplex sample was mixed with either 90 µL of dH2O or PBS, and the 100 µL sample was assayed for particle size by dynamic light scattering (DLS) measured on a Brookhaven Instruments Corp. ZetaPALS instrument. Particle sizing measurements were performed at a wavelength of 659.0 nm with a detection angle of 90° at RT. The samples were then diluted into 1.3 mL dH2O, and the zeta potential was analyzed on the same ZetaPALS instrument. Serum and ECM Components Unpackaging Assay. The gWiz-Luc plasmid was mixed with the bis-intercalating dye YOYO-1 iodide (Invitrogen/Molecular Probes) at a dye/base

Extracellular Barriers to Polyplex-Mediated Gene Delivery

pair (D:BP) ratio of 1:25 and incubated at 50 °C for 2 h to equilibrate the bis-intercalator complexes with DNA (46). Polyplexes were formed at N/P ratios of 0 and 3 by complexing unlabeled or YOYO-labeled DNA with either PEI or PEI-PEG. Type I collagen from rat tail (Upstate/Millipore) was prepared according to the manufacturer’s instructions at pH 7.4. Fetal bovine serum (FBS, from Mediatech) was serially diluted with 1:10 dilutions into either dH2O or OptiMEM I (Invitrogen), while heparan sulfate (HS, from Sigma), collagen, and Matrigel (BD Biosciences) were serially diluted in OptiMEM. Twenty microliters (containing 1 µg DNA) of polyplex was added to each well of a 96-well plate, followed by 80 µL of one dilution of FBS, Matrigel, collagen, or HS. The fluorescence from each well was measured on a Tecan Safire2 plate reader with excitation at 491 nm and emission at 510–520 nm. The fluorescence signal from the N/P ) 3 polyplexes was corrected by subtracting the signal from unlabeled samples and then normalizing to the N/P ) 0 (DNA only) signal. For the ECM components, a concentration for 50% unpackaging was determined by fitting a trendline to the data points by linear regression and using the equation of that line to calculate the concentration at 50% unpackaging. NBD-Oligo Internalization. Mouse fibroblast cells (NIH3T3, passage 91, ATCC # CRL-1658) were resuspended in complete cell culture medium (DMEM + 10% FBS + 1% antibiotic/antimicrobial) to a concentration of 200,000 cells/mL. The cells were plated at 200,000 cells per well in a 12-well plate. Polyplexes at N/P ) 3 with 3 µg of DNA (NBD-labeled oligonucleotide, prepared as described previously (19)) in 60 µL were formulated, and the polyplex samples were brought up to 600 µL with OptiMEM or with OptiMEM containing 0.01 µg/µL HS, 10% FBS, or 30% FBS. The cells were incubated with the polyplex solutions for 2 h, and then the cells were washed with 10 mM dithionite/10 mM Tris in cold PBS for 2 min at RT to quench any extracellular NBD fluorescence. The cells were washed with PBS, permeabilized with Triton X-100, and NBD uptake quantified as described previously (19). In Vitro Transfection. Human cervix epithelial adenocarcinoma cells (HeLa, passage 56, ATCC # CCL-2) were resuspended in complete cell culture medium (DMEM + 10% FBS + 1% antibiotic/antimicrobial) to a concentration of 2.5 × 105 cells/mL, and 5 × 105 cells were added to each well of a 6-well plate, each containing a 22 × 22 mm sterilized glass coverslip. The 6-well plates were placed in a 37 °C incubator with 5% CO2 for 24 h to allow the HeLa cells to attach to the glass coverslips. Polyplexes were formed at an N/P of 3 using 5 µg of DNA-QD and PEI-DNP in 100 µL total volume. Controls containing polymer or DNA only were also prepared. For a polyplex + FBS sample, 300 µL of FBS was added to the 100 µL polyplex (75% v/v FBS), and for a polyplex + HS sample, 100 µL of HS at a concentration of 0.1 µg/µL was added to the 100 µL polyplex (2 µg HS/µg DNA). Each sample was brought up to 1 mL with OptiMEM. The cells were washed twice with PBS, and the transfection mixtures were added. After a 2 h incubation at 37 °C, the cells were fixed with 4% paraformaldehyde (PFA, diluted in PBS) for 7 min, followed by permeabilization with 0.1% Triton X-100 (diluted in PBS) for 7 min and blocking with 10% normal goat serum (Sigma) diluted in PBS + 1% bovine serum albumin (PBS/BSA) for 30 min. The slides were each incubated with 100 µL of a primary rat antiDNP monoclonal antibody (Zymed) diluted 1:100 (starting concentration 1 mg/mL) in PBS/BSA for 1 h and 100 µL of a secondary antibody (Ab) solution for 30 min. The secondary Ab solution contained goat anti-rat IgG Ab that was labeled with Alexa Fluor 633 (Invitrogen/Molecular Probes) and diluted 1:100 (starting concentration is 2 mg/mL), phalloidin labeled with Alexa Fluor 488 (to stain actin, from Invitrogen/Molecular

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Probes) diluted 1:10 (starting concentration is 1 unit in 5 µL), and DAPI diluted 1:100 (to stain the nucleus, starting concentration is 1 mM). For the secondary Ab solution, all materials were diluted into PBS/BSA. Cells were washed twice with PBS between all steps. The slides were mounted on Gold Seal Ultra Frost glass microscope slides (Becton Dickinson) with ProLong Antifade reagent (Invitrogen/Molecular Probes) and sealed with nail polish. Cells were imaged on a Zeiss LSM 510 confocal system equipped with an inverted Axiovert 200 motorized microscope using a Zeiss plan-apochromat 63×/1.4 oil objective and taking 2 µm slices in the z-plane. In ViWo Liver Delivery. Polyplexes were prepared at N/P ) 3 using 25 µg of DNA-QD per mouse, where both the polymer and DNA were diluted in 5% dextrose to a total volume of 200 µL (400 µL after mixing). The polyplex (PPX) sample was formed using PEI-DNP and the PEGylated polyplex (PEG-PPX) sample was formed using PEI-PEG-DNP. A DNA only sample was made with 25 µg of DNA in 400 µL of 5% dextrose, a PEI only sample was made with an amount of PEI-DNP equivalent to N/P ) 3 in 400 µL of 5% dextrose, and a PEG-PEI only sample was made with an amount of PEI-PEG-DNP equivalent to N/P ) 3 in 400 µL of 5% dextrose. Each sample was assayed for particle size in dH2O and PBS before injection into animals. Female BALB/c mice (8 weeks, from Jackson Laboratories) were anesthetized with isoflurane, and the sample solutions were injected into the portal vein. After 20 min or 1 h, a Saf-T-Intima IV catheter (22 GA, 0.75 in, from BD) was inserted into the inferior vena cava, the portal vein was clipped, and the liver was perfused with 10 mL of PBS at a flow rate of 1 mL/min. During perfusion, the liver was periodically gently massaged to enhance the clearance of blood out of the vasculature in the liver. After perfusion, the liver was excised and fixed in 4% PFA for 3 h. The liver was then cut in half, with one-half remaining in 4% PFA overnight. The other half was placed in 25% sucrose (a cryoprotectant) and stored at 4 °C overnight. Histology, Immunohistochemistry, and Imaging. The livers in PFA were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The livers in sucrose were snap-frozen in Tissue-Tek Optimal Cutting Temperature embedding medium (OCT, from Sakura Finetek) and sectioned into 8 µm thick sections on a Leica CM 1850 cryostat (Meyer Instruments). Slides were rehydrated in PBS for 10 min, nonspecific Ab binding was blocked by incubating slides in 10% normal goat serum in PBS/BSA for 1 h, and then a primary antibody solution was applied to the slides for 1 h. The primary antibody solution consisted of a 1:100 dilution of rat anti-DNP monoclonal Ab and a 1:100 dilution of rabbit anti-mouse collagen IV polyclonal Ab (Chemicon International) in PBS/BSA. The slides were washed twice with PBS, and a secondary antibody solution was applied to the slides for 30 min. The secondary antibody solution consisted of a 1:100 dilution of goat anti-rat IgG conjugated to Alexa Fluor 633, a 1:100 dilution of goat anti-rabbit IgG conjugated to Alexa Fluor 350, and 2 U per section phalloidinAlexa488 (stock concentration is 1 Unit/5 µL) with all reagents diluted in PBS/BSA. Glass microscope coverslips were mounted on the tissue sections with ProLong Antifade reagent, then sealed with nail polish. Tissue sections were imaged on a Zeiss LSM 510 confocal system equipped with an inverted Axiovert 200 motorized microscope using either a Zeiss plan-apochromat 63×/1.4 oil objective or an F Fluar 40×/1.3 oil objective and taking 3 µm slices in the z-plane. Colocalization Analysis and DNA Quantification. The 4-channel LSM image files were imported into Volocity Visualization software from Improvision, and the colocalization function was used to assess the colocalization of two channels. A threshold was set on each channel to exclude the low-signal autofluorescence associated with each channel, and then Vo-


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locity was used to compute the colocalization coefficients. The calculated colocalization coefficients were M1 and M2 as reported by Manders et al., and these coefficients were used because they are proportional to the amount of fluorescence in the colocalizing objects and independent of the signal intensities of each channel (47). Volocity software was also used to quantify the amount of DNA in each tissue section. A threshold was set on the DNA channel, and then Volocity was used to calculate the number of voxels in the DNA channel above the threshold.

RESULTS Polymer and Plasmid Labeling. PEGylated PEI was synthesized by conjugating an activated PEG (PEG-SPA, MW 5000) to PEI (MW 25,000). Graft copolymers with approximately 7 PEG polymers per PEI polymer were synthesized to allow comparisons with previous reports (39–41). For fluorescence microscopy studies, polymers were also labeled with 2,4-dinitrophenyl (DNP) for antibody recognition. PEI was tagged with DNP-NHS at a labeling ratio of 23.2 DNP molecules per PEI polymer. PEI-PEG-DNP was synthesized by grafting DNP-PEG-COOH to PEI by DCC/NHS coupling. Graft copolymers with approximately 7 PEG polymers and 5.75 DNP molecules per PEI polymer were synthesized. Initial experiments were conducted with AlexaFluor568labeled plasmid DNA. However, the fluorescence quenching observed in condensed DNA resulted in fluorescence levels that were only slightly above background levels in tissue sections. Thus, plasmids were labeled with red fluorescent quantum dots (QDs). DNA was labeled with psoralen-biotin and then reacted with streptavidin-coated QDs, resulting in the incorporation of one QD for every two DNA plasmids. The fluorescence from polyplexes formed with QD-labeled DNA (QD-DNA) at N/P ) 3 was approximately 5-fold higher than that from polyplexes formulated with Alexa568-labeled DNA at 6 dye molecules per plasmid (data not shown). Polyplex Characterization. Polyplexes were formulated at N/P ) 3 on the basis of particle size, zeta potential, and gel retardation studies that showed complete DNA condensation at N/P ) 3 with only small amounts of free polymer after complex formation (data not shown). The polyplexes used in this study were characterized by measuring particle size in water and physiological salt concentrations and particle surface charge (Supporting Information). Polyplex diameter was ∼100–150 nm in water. The unPEGylated polyplex rapidly aggregated to sizes greater than 1 µm in phosphate-buffered saline (PBS), whereas the PEGylation of polyplex conferred improved salt stability. All polyplexes had positive surface charges. Effect of ECM and Serum Components on Vector Unpackaging. The effect of serum on polyplex stability was assessed using a YOYO-1-based unpackaging assay (Figure 1). The unpackaging assay is based on the properties of YOYO binding to DNA. First, the fluorescence of YOYO is over 1000fold greater when bound to DNA than when in solution, and second, when YOYO-labeled DNA is condensed with a cationic polymer such as PEI, the dye molecules become closer in proximity and begin to interact electronically, causing a dramatic decrease in fluorescence signal (i.e., YOYO dye self-quenching occurs) (48). Complexation of YOYO-labeled plasmid with PEI or PEI-PEG results in a ∼75–90% decrease in YOYO fluorescence. Polyplexes incubated with increasing concentrations of fetal bovine serum (FBS) show corresponding increases in YOYO-1 fluorescence until, at 80% FBS, nearly all the fluorescence is recovered, indicating complete unpackaging. Interestingly, the YOYO signal of polyplexes in the absence of serum is significantly higher when diluted in OptiMEM versus water (3-fold higher for unPEGylated and 2-fold higher for PEGylated). The YOYO signal of the polyplexes in the absence

Figure 1. Polyplex (N/P ) 3) unpackaging mediated by FBS diluted in water and OptiMEM. The x-axis shows the volume percent of FBS in solution. Eighty microliters of the indicated solution was added to 20 µL of polyplex (N/P ) 3) containing 1 µg of DNA. Table 1a HS Matrigel collagen

PPX (µg)

PEG-PPX (µg)

fold increase

0.27 6.30 301.03

0.01 0.57 28.50

26.3 11.0 10.6

a Various concentrations of extracellular matrix components (HS, Matrigel and Collagen I) were incubated with PEI and PEGylated PEI polyplex (PPX and PEG-PPX, respectively) labeled with YOYO-1. The amount of the ECM component that was required to unpackage polyplexes containing 1 µg of DNA to recover 50% of uncomplexed DNA fluorescence is reported.

of serum is also significantly higher for PEGylated polyplexes versus that for unPEGylated polyplexes (25% of the fluorescence of unpackaged DNA vs 10% for water, and 50% vs 30% for OptiMEM). These data indicate that not only does PEGylation slightly loosen the polyplexes but also the presence of counterions from the salt in OptiMEM loosens the polyplexes. The stability of polyplexes was also assessed in the presence of extracellular matrix proteins. Gene delivery vehicles entering the liver will pass out of the blood vessels and into the hepatic sinusoids that flow past rows of hepatocytes, where they will encounter an extracellular matrix (ECM) composed of both collagen and heparan sulfate proteoglycan (HSPG) (49–55). Thus, Matrigel (an ECM extract that contains HSPGs), heparan sulfate (HS, a negatively charged proteoglycan component), and collagen were investigated for their ability to unpackage PEI polyplexes and PEGylated PEI polyplexes (PPX and PEG-PPX, respectively). The amounts of each ECM component required to unpackage polyplexes containing 1 µg of YOYO-1abeled DNA to recover 50% of uncomplexed DNA fluorescence are reported in Table 1. HS most effectively unpackaged the polyplexes, requiring only 0.27 µg of HS and 0.01 µg of HS per µg DNA to unpackage PPX and PEG-PPX, respectively. Comparatively, approximately 25–50 times more Matrigel than HS (by weight) was required to unpackage the polyplexes, with 6.3 µg of Matrigel and 0.57 µg of Matrigel per µg DNA needed to unpackage PPX and PEG-PPX, respectively. Similarly, about 50-fold more collagen than Matrigel (by weight) was required to unpackage the polyplexes, with 301.03 µg of collagen and 28.5 µg of collagen per µg DNA needed to unpackage PPX and PEG-PPX, respectively. A general trend also appears in that approximately 10-fold more ECM component (by weight) is required to unpackage PPX compared to that required to unpackage PEG-PPX. This trend holds true for FBS unpackaging as well, with PEG-PPX unpackaging to 50% of uncomplexed DNA fluorescence around 1% FBS and PPX unpack-



Figure 2. (A) Cellular internalization of PEI and PEGylated PEI polyplexes (PPX and PEG-PPX) incubated in the presence of heparan sulfate or FBS. The amount of internalized DNA for each formulation was normalized to that of cells incubated with PEI polyplex in OptiMEM. The internalization with PEI polyplexes in OptiMEM was statistically significantly different from PEI polyplexes in 30% FBS with p < 0.05 and all other samples (except the 10% FBS) with p < 0.01. (B) Confocal images from DNA-QD and PEI-DNP polyplex transfections. On the left is the PEI-DNP channel (green), in the middle is the DNA-QD channel (red), and on the right is an overlay of PEI-DNP, DNA-QD, and DAPI (blue). Punctate (yellow, thin arrows) and diffuse (white, thick arrows) fluorescence is observed. Some examples of free DNA are also found near the cell nuclei in the areas of diffuse fluorescence. The scale bars are 20 µm.

aging to 50% of uncomplexed DNA fluorescence around 10% FBS (Figure 1). Polyplex Internalization. Polyplex-mediated DNA internalization in cultured mammalian cells was quantified in the presence of both FBS and HS by using an environmentally sensitive fluorophore, NBD, conjugated to DNA (Figure 2A). NBD is converted into a nonfluorescent molecule by dithionite, a membrane-impermeable reducing agent. Measurement of fluorescence from internalized NBD-labeled DNA is a facile method to quantify polyplex uptake (19). Exposure of cells to polyplexes in OptiMEM or OptiMEM + 10% serum resulted in efficient DNA internalization. However, significantly decreased polyplex uptake was observed in the presence of 30% serum (43% reduction in internalized DNA, p < 0.05). Similarly, PPX incubated with HS also led to a statistically significant decrease in the amount of DNA uptake (95% reduction in internalized DNA, p < 0.01). As is consistent with previous observations, PEG-PPX did not mediate much internalization of DNA. HeLa cells were transfected with polyplexes formed with DNA-QD and PEI-DNP to verify dual fluorescence imaging of

the polyplexes and to investigate intracellular distributions of polyplexes after internalization. Cells were stained with DAPI to visualize the nucleus and anti-DNP antibodies to visualize PEI and then imaged by confocal microscopy. As can be seen in Figure 2B, PPX were successfully delivered, with both cellassociated polyplexes and internalized polyplexes present. The cell-associated polyplexes are mainly visible as large aggregates attached (primarily through electrostatic interaction between the positively charged particles and the negatively charged cell surface) to the periphery of the actin network. The internalized polyplexes are present in two forms: first (and primarily) in punctate dots characteristic of vesicular localization (yellow, thin arrows) and second in diffuse fluorescence (white, thick arrows). Furthermore, it is clear that much of the PEI is colocalized with DNA, and nearly all of the DNA is colocalized with PEI. Most of the polyplex unpackaging observed occurs in and around the nucleus (in the areas of diffuse fluorescence), indicating that the majority of polyplexes remain intact until the very end stages of delivery. Low uptake of QD-DNA and significant unpackaging was observed with polyplexes incubated


with FBS or HS, consistent with the NBD internalization results (data not shown). In ViWo Liver Delivery. The intrahepatic distribution of duallabeled PPX and PEG-PPX after portal vein delivery was next assessed. Eight-week old female BALB/c mice were injected with 400 µL of polyplexes in 5% dextrose, and liver tissue was collected either 20 min or 1 h after injection. The liver tissue was first analyzed by hematoxylin and eosin (H and E) staining for gross morphology. Tissues from a few mice had areas of sinusoidal dilation and hepatocyte death, but most of the tissue in each of the samples was normal and healthy. For fluorescent confocal imaging, tissue sections were stained with phalloidin to visualize actin and an antibody against collagen IV to represent ECM. PEI and PEG-PEI were visualized using an antibody against DNP, and the plasmid was fluorescently labeled with QD. Figure 3A shows confocal images of liver sections harvested 20 min after the portal vein injection of PPX. Both PEI and DNA can be detected in the sections. PEI is primarily colocalized with the collagen lining the sinusoidal structures and rarely colocalized with DNA. DNA was observed to be located near the cell periphery, but no cell-internalized DNA was observed. Figure 3B shows confocal images from liver that was removed 1 h postinjection, and again both PEI and DNA can be detected in the tissue. Similar to the 20 min time point, at 1 h the PEI and DNA are not colocalized with each other; interestingly, intracellular delivery of DNA was observed. The distribution of PEG-PPX in liver 20 min after administration is shown in Figure 4. The distributions of the polymer and DNA in the tissue are very similar to that of PPX delivery. Again, both polymer and DNA can be detected in the tissue. Notably, with PEG-PPX, much less DNA is observed in the tissue compared to that in PPX (Figures 3 and 4). The relative amount of DNA in the tissues was determined using Volocity software to calculate the number of voxels containing fluorescence in the DNA channel in at least five images from each sample (Figure 5). The amount of DNA fluorescence in the liver 20 min after PPX administration is ∼4-fold higher than levels from PEG-PPX or naked DNA injection (p < 0.01). Fluorescence from DNA in PPX samples decreases by over 50% between 20 min and 1 h (p < 0.05). Interestingly, PEG-PPX did not mediate a statistically significant increase in DNA in the tissue compared to that in the tissue of mice treated with DNA plasmid only, though both PPX time points showed a statistically significant increase in DNA compared to that of the DNA only sample (p < 0.01) (Figure 5). Unlike PPX, PEGPPX does not appear to lead to any observable intracellular DNA delivery, even 1 h after delivery (Supporting Information). With the delivery of plasmid DNA or polymer alone (Supporting Information), it can be seen that very little DNA is present in the tissue when it is not complexed with polymer, although there is a large amount of polymer present in the tissue even when it is not complexed with DNA. No evidence of either DNA or polymer entering cells when delivered alone was found. Colocalization Analysis. The extent of colocalization between polymer and DNA, collagen, or actin, and DNA and polymer, collagen, or actin was quantified by measuring colocalization coefficients using Volocity image analysis software. The coefficients M1 and M2 (as reported by Manders and Aten) are used to represent the degree to which two images overlap because they (i) do not depend on the ratio of the number of objects in both channels, (ii) do not depend on the signal intensities of the channels, and (iii) are proportional to the amount of fluorescence in the colocalizing objects (47). For both PPX and PEG-PPX formulations at 20 min and 1 h, DNA is most highly colocalized with actin, with colocalization coefficients ranging from 0.5 to 0.7 as shown in Figure 6. For PPX at 20 min, low colocalization of DNA with PEI or collagen

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is observed (colocalization coefficients of ∼0.2 and ∼0.15, respectively). For PPX at 1 h and both PEG-PPX at 20 min and 1 h, the colocalization coefficients of DNA with polymer and collagen are two to three times greater than the respective coefficients from PPX at 20 min. As can be seen in Figure 7, PEI is highly colocalized with collagen IV and actin (with colocalization coefficients ranging from 0.4 to 0.8), while it is only partially colocalized with the DNA (colocalization coefficients of 0.1 or less). Comparing Figures 6 and 7, it can be noted that even when DNA is highly colocalized with PEI, PEI is still not highly colocalized with the DNA, indicating that there is an abundance of free polymer in the tissue not associated with DNA. It should also be noted that the colocalization coefficients for each sample sum to greater than unity because there is colocalization among all the channels; therefore, some colocalized voxels can be counted twice.

DISCUSSION Electrostatic condensation of polyanionic DNA and polycationic PEI into polyplexes occurs by a polyelectrolyte addition reaction driven by an increase in system entropy due to the displacement of counterions and the final formation of the polyelectrolyte complex, as described by Kabanov and coworkers (56). However, the addition of free polyanions can then cause dissociation of the initial complexes as a result of a polyelectrolyte exchange reaction, which is driven by a decrease in free energy due to the redistribution of interpolymeric salt bonds between the complexed polyanion and the free polyanion (56). Indeed, unpackaging of polyplexes delivered to cultured mammalian cells has been proposed to occur by competitive binding interactions with RNA in the cytosol or DNA in the nucleus (30, 31, 57). Polyplexes administered in ViVo by systemic injection may be subject to premature unpackaging through interactions with serum proteins or ECM components, which have been shown in Vitro to bind to and/or unpackage polyplexes (32, 33, 58–61). Rice and co-workers demonstrated that plasmid is rapidly released from cationic peptide vectors in blood unless the vectors are stabilized by cross-linking (62). Recently, two reports investigated the biodistribution of PEI and PEGylated PEI polyplexes by radioactive labeling of both DNA and polymer, finding that polyplexes formed with PEGylated PEI or low molecular weight PEI separated in blood in a dose-dependent manner compared to polyplexes formed with high molecular weight PEI, which remained intact (40, 41). The studies also showed that PEGylated polyplexes were more likely to unpackage when the PEG grafting on PEI was high or when injected in small quantities but that PEI polyplexes were likely to remain intact even when injected in small quantities (40, 41). Additionally, both studies found that large amounts of both DNA and polymer primarily distributed to the liver, with the spleen and lung also having measurable amounts of DNA and polymer (40, 41). Although PEI and plasmid were shown to exhibit similar organ distributions and clearance kinetics and therefore assumed to remain packaged, the intrahepatic distributions of the polyplexes has, to date, not been investigated. In this work, the distribution of PEI polyplexes and PEGylated PEI polyplexes in the liver after intravenous delivery was investigated with duallabeled polymer and plasmid with analysis by fluorescence confocal microscopy. For systemic administration, the polyplexes will first encounter serum proteins in the blood. The polyplexes will then enter the liver via the portal vein and move into a parallel system of vascular channels called the hepatic sinusoids, which emanate radially from the portal triad (portal vein, hepatic artery, and bile duct) and conform to the surfaces of the hepatic epithelial cell laminae that compose the liver (51). The sinusoids



Figure 3. (A) 40× confocal images from a polyplex-injected mouse liver section from a liver that was removed after 20 min. On the top left is the PEI-DNP (green) channel; at the top right is the DNA-QD (red) channel; at the bottom left is the anti-Collagen IV Ab (blue) channel, DNA-QD, and PEI-DNP; and on the bottom right is anti-Collagen, DNA-QD, PEI-DNP, and Phalloidin (anti-actin, colored pink). The scale bars are 20 µm. (B) 63× confocal images from a polyplex-injected mouse liver section from a liver that was removed after 1 h. At the top left is the PEI-DNP (green) channel; at the top right is the DNA-QD (red) channel; on the bottom left is the anti-Collagen IV Ab (blue) channel, DNA-QD, and PEI-DNP; and on the bottom right is anti-Collagen, DNA-QD, PEI-DNP, and Phalloidin (anti-actin, colored pink). The scale bars are 10 µm; the arrows show the internalized DNA-QD.

intercommunicate through fenestrations to form an interconnected system of thin-walled vessels coupled to a large surface area of liver parenchyma to facilitate material transfer from the

vessels to the cells (51). The hepatic sinusoids are lined with endothelial cells, and hepatic stellate cells lie in the space between the sinusoidal endothelial cells and the parenchymal


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Figure 4. 40× confocal images from PEGylated polyplex-injected mouse liver section from a liver that was removed after 20 min. At the top left is the PEI-DNP (green) channel; on the top right is the DNA-QD (red) channel; on the bottom left is the anti-Collagen IV Ab (blue) channel, DNA-QD, and PEI-DNP; and on the bottom right is anti-Collagen, DNA-QD, PEI-DNP, and Phalloidin (anti-actin, colored pink). The scale bars are 20 µm.

Figure 5. Quantification of DNA in liver tissue sections. Volocity image analysis software was used to quantify the total number of voxels with DNA fluorescence from tissue sections of each sample type. The amount of DNA fluorescence in the liver from PPX administration is ∼4-fold higher than levels from PEG-PPX or naked DNA injection (p < 0.01). Fluorescence from DNA in PPX samples decreases by over 50% between 20 min and 1 h (p < 0.05). PEG-PPX did not mediate a statistically significant increase in DNA in the tissue compared to DNA only. Both PPX time points showed a statistically significant increase in DNA compared to the DNA only sample (p < 0.01).

cells, where it has been shown that these stellate cells are mainly responsible for synthesizing the ECM surrounding the sinusoids (51, 55). The ECM associated with the hepatic sinusoids is composed of collagen I, collagen IV, and heparan sulfate proteoglycan (HSPG) (49–55). Thus, both the serum and the ECM in the hepatic sinusoids present potential threats for premature polyplex unpackaging. We first determined the ability of serum to unpackage the polyplexes in Vitro. The assay that was employed uses the quenching of YOYO-labeled DNA fluorescence to report

Figure 6. Colocalization coefficients (calculated from images of tissue sections taken 20 min or 1 h after injection) for DNA colocalized with polymer, collagen, or actin, as determined by Volocity. A coefficient value of 1 corresponds to perfect colocalization, while a value of 0 corresponds to no colocalization. For all groups, n > 3.

the compaction of DNA in polyplexes. Since YOYO intercalates into DNA before the polyplexes are formed and could potentially affect polyplex structure, it was first confirmed through a secondary approach, ethidium bromide (EtBr) exclusion, that YOYO-1 labeling does not affect polyplex packaging (Supporting Information). Figure 1 clearly shows that increasing the concentration of serum (the composition of FBS is variable, but the main protein components of FBS are albumins, globulins, and fibrinogens) in either water or OptiMEM causes the unpackaging of both PPX and PEG-PPX. The unpackaging of PEG-PPX occurs more readily at lower concentrations of FBS compared to that of PPX, indicating that PPX are more tightly complexed. Next, Table 1 shows the ability of ECM components to unpackage the polyplexes: collagen, Matrigel (the main


Figure 7. Colocalization coefficients (calculated from images of tissue sections taken 20 min or 1 h after injection) for polymer colocalized with collagen, actin, or DNA, as determined by Volocity. A coefficient value of 1 corresponds to perfect colocalization, while a value of 0 corresponds to no colocalization. For all groups, n > 3.

components of Matrigel are laminin, collagen IV, entactin, and heparan sulfate proteoglycans), and HS all effectively unpackage the polyplexes. Again, the unpackaging of PEG-PPX occurs more readily at lower concentrations of ECM components compared to that of PPX. For comparison with Table 1, the weight ratios for 50% unpackaging by FBS are approximately 500 µg FBS protein and 50 µg FBS protein per µg DNA for PPX and PEG-PPX, respectively; thus, the unpackaging mediated by FBS is comparable to that of collagen. In water, collagen I has an isoelectric point around neutral pH, but in the presence of salt, the isoelectric point can change; thus, since the collagen used in these assays was diluted in PBS, the ionic interactions could have shifted the isoelectric point, causing the collagen to be slightly charged (63). HS was expected to most effectively unpackage the polyplexes because of its high negative charge density. Matrigel was also expected to mediate the unpackaging of polyplexes because of the fact that it contains HSPG, but its intermediate ability to unpackage is consistent with the fact that its main component is collagen and that HSPG is only a minor component. Neither collagen nor Matrigel was allowed to gel, eliminating the possibility that the unpackaging was caused by the gelation process. Though it was determined that a large excess of FBS protein compared to DNA was required for unpackaging, the ratios necessary are within the physiological range normally found in serum. Thus, soluble GAGs, the GAGs of proteoglycans in the ECM, and serum proteins can all potentially facilitate polyplex unpackaging in ViVo. A series of in Vitro transfections was performed with polyplexes formed from the DNP-labeled polymer and QDlabeled DNA, and the cells were imaged to assess uptake and intracellular distributions. The dual-labeled polyplexes were tested by the same EtBr unpackaging assay as described above, and the labeled PPX were found to be slightly more resistant to unpackaging than unlabeled PPX, while the labeled PEGPPX were found to unpackage at levels similar to that of unlabeled PEG-PPX (Supporting Information). The dual-labeled polyplexes also exhibited slightly higher zeta potential than the unmodified polyplex, which may be responsible for the higher resistance to unpackaging seen with the labeled PPX (Supporting Information). As can be seen from Figure 2, PPX are efficiently internalized, and most of the internalized of the polymer and DNA are colocalized, while a small fraction has separated intracellularly after escape from endosomes (as evidenced by diffuse fluorescence rather than punctate). In contrast, minimal amounts of internalized DNA is observed with cells treated with plasmid only or with PPX exposed to HS or FBS before delivery, whereas levels of polymer association with cells is similar for all polyplex formulations (data not shown). These


results demonstrate that (i) in the absence of serum proteins or ECM components, PPX are efficiently internalized in a packaged state and that (ii) exposure of PPX to serum or HS results in inefficient DNA delivery, likely due to premature vector unpackaging. The intrahepatic distributions of dual-labeled polyplexes were next assessed. Initial studies used AlexaFluor-labeled plasmid, but the results reported here focus on QD-labeled plasmid because of higher fluorescence yield. However, hepatic distributions were similar for both labeling approaches. The major findings from these studies are that PEI and PEGylated PEI are not colocalized with plasmid DNA in the liver by 20 min postinjection (Figure 3A and 4). The unpackaging of PEG-PPX in the serum has been reported previously, although PEI (25 kD) has been shown to distribute in a manner similar to that of plasmid DNA in the organs (40, 41). On the basis of previous biodistribution studies and these new findings, we propose that PEI polyplexes remain packaged during circulation in serum but unpackage after entering the liver because of competitive binding with ECM components, specifically HS proteoglycans. This conclusion is supported by in Vitro unpackaging studies that show that HS efficiently unpackages PPX and PEG-PPX and by the observation that PEI is primarily localized around the hepatic sinusoids through collagen IV colocalization (Figure 3 and 4). Published images of HS proteoglycan distribution in the liver show fluorescence lining the hepatic sinusoids that bears a striking resemblance to the images in this work showing PEI lining the sinusoids (50–53). Collagen IV has been shown to be present in the space of Disse that lines the hepatic sinusoids, and HSPG has been shown to be present in the same areas. However, antibodies to HS also stain intracellular HS. Because colocalization of PEI with ECM components was quantified in this study by image analysis, collagen IV, which stains in a manner similar to that of extracellular HS was used as an ECM marker instead. In contrast to PEI, DNA is visualized in intercellular spaces and is associated with the outside of the cells (Figure 3A), which is consistent with other reports (64). Previous results combined with the larger amount of DNA observed in the tissue in this study (compared to that in the DNA only sample; see Figure 5) and the fact that DNA disappears from serum very quickly when no PEI protects it (as it has a very short half-life due to nuclease degradation (65)) suggest that the polyplexes remain intact in blood. Thus, we propose that bPEI facilitates liver transfection in ViVo primarily by protecting DNA against nuclease degradation and elimination from circulation, mediating higher levels of delivery to the liver compared with unpackaged DNA. However, because of premature unpackaging of polyplex in the liver, a significant contribution of PEI toward internalization and intracellular trafficking of plasmid DNA is unlikely. The unpackaged DNA was observed to be internalized with kinetics similar to that reported by Wolff and co-workers, who hypothesize that free plasmid DNA can be internalized in hepatocytes by a receptor-mediated process (66). It is worth noting that the colocalization coefficients for DNA with polymer and collagen are higher for PPX at 1 h and PEGPPX at 20 min and 1 h compared to those for the polyplex at 20 min because of an artifact created by a smaller amount of DNA present in the former samples (Figures 5 and 6). Liver sections acquired 20 min after PPX treatment have considerable amounts of free DNA in the tissue, but the other three samples only have small amounts of DNA remaining because of the fact that most DNA has been unpackaged from the polymeric carrier and degraded. The small amount of DNA that remains does so because it is protected by the polymer from nuclease degradation. Analysis of polymer colocalization in Figure 7 reveals that the polymer has poor colocalization with the DNA in any


sample, thus supporting the hypothesis that the high DNApolymer colocalization at 1 h and in PEGylated formulations is simply an artifact derived from the presence of a smaller amount of DNA that has been protected by (and so is physically closer to) the PEI. PEGylation of polyplexes was initially thought to reduce interaction with blood components, thereby increasing the stability of polyplexes in the blood. Subsequent work revealed that the PEGylated polyplexes were actually less stable in blood (40, 41, 59, 67). It is likely that a considerable amount of PEG is trapped within the core of the polyplexes after they form, leading to thermodynamic destabilization (41, 59). In line with these findings, the in Vitro unpackaging assay that compared PPX with PEG-PPX showed that PEG-PPX unpackaged more easily. Additionally, it can be seen in Figure 5 that PEG-PPX carry much less DNA into the tissue, most likely due to early unpackaging of polyplexes in blood and DNA degradation by serum nucleases. It should be noted that in the images for PEG-PPX and PEI-PEG only, the amount of polymer seems to be lower than that seen with PPX. This observation may simply be attributed to the fact that the PEI-PEG contains less DNP (approximately 6 DNP per mol of polymer) than PEI (approximately 23 DNP per mol of polymer). However, the decreased amount of PEG-PEI may also be due to an actual change in biodistribution, similar to the report by Fischer et al. indicating that PEI preferentially accumulated in the liver and spleen, while PEG-PEI preferentially accumulated in the spleen and lung (40). Also of interest is the notion that since preconjugating PEG to PEI causes PEG chains to be trapped in the core of the polyplex during formation, which destabilizes the polyplexes and leads to premature unpackaging, it would be beneficial to add PEG onto the polyplexes after they have been formed. However, this method was investigated, and it was found that coating polyplexes with a hydrophilic polymer similar to PEG generated complexes that were also less stable and unpackaged in the blood (60). In line with this finding, we have constructed similar postformation PEGylated polyplexes and have shown that they unpackage readily and do not aid in delivering more DNA to the tissue (data not shown). The hepatic DNA and PEGPEI distributions observed in these studies were similar to those shown for polyplexes formed from PEG-PEI and DNA condensation. In conclusion, we have demonstrated that both serum proteins and ECM act as extracellular barriers to polyplex-mediated gene delivery because of their ability to unpackage polyplexes. At the same time, polyplexes must unpackage once inside the cell so that transcription of the genes may occur. Thus, a doubleedged sword is presented: the polyplexes must be stable enough to resist unpackaging in blood and ECM, but not too stable that they resist unpackaging once reaching their intracellular destination. Polyplexes will no doubt need to change their form in order to reach their full potential and overcome this serious flaw. One can imagine that if PEI and DNA remained associated extracellularly, PEI would be able to mediate much higher gene expression by increasing the amount of DNA taken up by the cells and aiding in DNA escape from endosomes as it does in Vitro. A potential solution is crosslinking polyplexes with a biodegradable linker that will maintain stability in the blood and degrade in the intracellular environment, as proposed by Neu et al. and Murthy et al. (68, 69) or conjugating the nucleic acid cargo directly to the vector, as demonstrated by Rozema et al. (70).

ACKNOWLEDGMENT Confocal imaging was performed at the NanoTech User Facility (NTUF) at the University of Washington, a member of

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National Nanotechnology Infrastructure Network supported by NSF, with special thanks to Dr. Dong Qin at NTUF for her training and support on the Zeiss confocal microscope. We also thank Dr. Matthew Yeh, from the Department of Pathology at the University of Washington, for his help with analyzing the liver tissue sections. Paraffin embedding, sectioning, and H & E staining were performed by the Pathology Core Immunocytochemistry Lab at UW. Cryosections were prepared using the cryostat at the UW Engineered Biomaterials (UWEB) histology facility. Jamie Bergen kindly donated the NBD-oligo used in the transfection studies. This work was funded by a National Science Foundation Fellowship awarded to R.S.B., an NSF CAREER Award (CBET-0448547) to S.H.P. and a Young Investigator award to S.H.P. by the National Hemophilia Foundation. Supporting Information Available: Table S1 shows particle size and zeta potential characterization of the polyplexes. Figure S1 shows the NMR spectrum of PEG-DNP. Figure S2 shows the NMR spectrum of PEI-PEG-DNP. Figure S3 shows the NMR spectrum of unlabeled PEI-PEG. Figure S4 shows polyplex unpackaging as monitored by an EtBr exclusion assay. Figure S5 contains a 63× confocal image of PEG-PPX injected mouse liver removed after 1 h. Figure S6 contains a 40× confocal image from a PEI-only injected mouse liver removed after 20 min. Figure S7 contains a 40× confocal image from PEI-PEG-only injected mouse liver removed after 20 min. Figure S8 contains a 40× confocal image from DNA-only injected mouse liver removed after 20 min. This material is available free of charge via the Internet at http://pubs.acs.org.

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