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. Author manuscript; available in PMC: 2014 Sep 28.
Published in final edited form as: J Control Release. 2013 Jun 2;170(3):325–333. doi: 10.1016/j.jconrel.2013.05.024

PEG Length and Chemical Linkage Controls Polyacridine Peptide DNA Polyplex Pharmacokinetics, Biodistribution, Metabolic Stability and In Vivo Gene Expression

Sanjib Khargharia 1, Koby Kizzire 1, Mark Ericson 1, Nicholas J Baumhover 1, Kevin G Rice 1,*
PMCID: PMC3904502  NIHMSID: NIHMS503000  PMID: 23735574

Abstract

The pharmacokinetics (PK), biodistribution and metabolism of non-viral gene delivery systems administered systemically are directly related to in vivo efficacy. The magnitude of luciferase expression in the liver of mice following a tail vein dose of a polyplex, composed of 1 μg of pGL3 in complex with a polyethylene glycol (PEG) polyacridine peptide, followed by a delayed hydrodynamic (HD) stimulation (1–9 h), depends on the HD stimulation delay time and the structure of the polyacridine peptide. As demonstrated in the present study, the PEG length and the type of chemical linkage joining PEG to the polyacridine peptide dramatically influence the in vivo gene transfer efficiency. To understand how PEG length, linkage and location influence gene transfer efficiency, detailed PK, biodistribution and HD-stimulated gene expression experiments were performed on polyplexes prepared with an optimized polyacridine peptide modified through a single terminal Cys or Pen (penicillamine) with a PEG chain of average length of 2, 5, 10, 20, or 30 kDa. The chemical linkage was examined by attaching PEG5kDa to the polyacridine peptide through a thiol-thiol (SS), thiol-maleimide (SM), thiol-vinylsulfone (SV), thiol-acetamide (SA), penicillamine-thiol-maleimide (PM) or penicillamine-thiol-thiol (PS). The influence of PEG location was analyzed by attaching PEG5kDa to the polyacridine peptide through a C-terminal, N-terminal, or a middle Cys residue. The results established rapid metabolism of polyplexes containing SV and SA chemical linkages leads to a decreased polyplex PK half-life and a complete loss of HD-stimulated gene expression at delay times of 5 hrs. Conversely, polyplexes containing PM, PS, and SM chemical linkages were metabolically stable, allowing robust HD-stimulated expression at delay times up to 5 hrs post polyplex administration. The location of PEG5kDa within the polyacridine peptide exerted only a minor influence on the gene transfer of polyplexes. However, varying the PEG length from 2, 5, 10, 20, or 30 kDa dramatically altered polyplex biodistribution, with a 30 kDa PEG maximally blocking liver uptake to 13% of dose, while maintaining the ability to mediate HD-stimulated gene expression. The combination of results establishes important relationships between PEGylated polyacridine peptide structure, physical properties, in vivo metabolism, PK and biodistribution resulting in an optimal PEG length and linkage that leads to robust HD-stimulated gene expression in mice.

Keywords: Gene Delivery, Hydrodynamic Dosing, Polyethylene glycol

Introduction

The PEGylation of a nonviral gene delivery system is considered essential in order to improve blood compatibility of DNA polyplexes[1, 2]. Without PEGylation, upon i.v. administration the cationic surface of most polyplexes binds albumin and other serum proteins causing a rapid increase in particle size and entrapment in the capillary bed of the lung [3, 4]. In the absence of PEGylation, anionic polyplexes and plasmid DNA are rapidly removed from the circulation by liver Kupffer cells via the scavenger receptor [5, 6].

Several prior studies have examined the influence of PEG length and loading density on nanoparticle biodistribution in vivo[7–11]. In general, increasing the PEG length and loading density decreases serum protein binding and biodistribution to the lung. However, even with a stealth layer of PEG, polyplexes are still primarily recognized by Kupffer cells, accounting for the majority of biodistribution to liver immediately after i.v. administration[12]. Although these relationships are well understood, to date no studies have reported the development of long-circulating DNA nanoparticles that avoid liver uptake and maintain the ability to mediate gene transfer in vivo.

A major difficulty in PEGylating DNA polyplexes is that increasing the PEG loading density can also compromise polyplex stability. This is because PEGylation of PEI[13, 14], polylysine, chitosan[15] or dendrimers simultaneously results in a proportional decrease in the number of primary amines, thereby decreasing the binding affinity of PEGylated polymers for DNA, resulting in their dissociation in the circulation[16]. In an effort to overcome this limitation, PEGylated adamantane was incorporated through binding to cationic cyclodextrin polyplexes[17]. While this approach preserved amines to maintain binding affinity for DNA, it failed to achieve a high PEG loading density.

The cross-linking of residual amines on Lys residues of peptides with glutaraldehyde was previously used to stabilize PEGylated peptide DNA polyplexes from dissociation in the circulation[18]. Likewise, HPMA was used previously to cross-link amines on the surface of polyplexes resulting in laterally stabilized DNA nanoparticles that possessed a long circulatory half-life[19]. However, both of these approaches resulted in polyplexes that failed to mediate appreciable gene expression in vivo, presumably due in part to their inability to release DNA inside the cell [20, 21].

We have previously reported on the unique attributes of PEGylated polyacridine peptides that bind to DNA to form polyplexes that overcome the limitations of other strategies by providing a long circulatory half-life and demonstrated in vivo activity. In prior studies we first optimized the properties of the peptide portion of PEGylated polyacridine peptides to achieve efficient in vivo gene expression following a delayed hydrodynamic (HD)-stimulatory dose (saline only) after i.v. administration of the polyplex[22]. Short PEGylated polyacridine peptides of the general structure (Acr-Lys)n=2,4,6-Cys, where Acr is a Lys residue modified on its ε-amine with acridine, were found to bind pGL3 with high affinity through a combination of polyintercalation and ionic interaction. PEGylation of the C-terminal Cys residue resulted in PEGylated polyacridine peptides that bind to pGL3 to form polyplexes that are stable in the circulation for up to two hrs[22]. Further optimization of the peptide sequence revealed that four Acr residues spaced by four Lys residues, (Acr-Lys4)3-Acr-Lys-Cys-PEG, dramatically increased the stability of a 1 μg i.v. dose of pGL3 polyplex in the circulation for 5 hrs[23].

The present investigation examines the influence of PEG length, linkage and location on DNA polyplex pharmacokinetics (PK), biodistribution and gene expression in vivo. The identified relationships were found using high-affinity DNA binding polyacridine peptides possessing a single Cys or Pen attachment site for PEG[22, 23]. A PEG was linked to either Cys or Pen using six different chemical linkages which included thiol-maleimide (SM), thiol-thiol (SS), thiol-acetamide (SA), thiol-vinyl sulfone (SV), penicillamine-thiol-thiol (PS), and penicillamine-thiol-maleimide (PM). In addition, five different PEG lengths (2, 5, 10, 20, and 30 kDa) and three different attachment sites (N-terminal, C-terminal and middle) were compared for their influence on polyplex mediated in vivo gene delivery.

The results are startling in that linkages anticipated to be most stable (SA, SV) were found to result in short PK half-lives and rapid loss of HD-stimulated gene expression. Likewise, linkages expected to be metabolically less stable (SS, PS) proved to be more stable than SA and SV, resulting in longer PK half-lives and extended HD-stimulated expression profiles. Just as important as PEG linkage, PEG length was found to exert a dramatic effect on PK half-life and the ability to stealth polyplex biodistribution to the liver, resulting in the discovery of DNA polyplexes with a long circulatory half-life that avoid biodistribution to the liver. Together, the results address key parameters regarding the attachment of PEG to DNA polyplexes that improve the performance of i.v. dosed non-viral gene delivery systems in mice.

Materials and Methods

Unsubstituted Wang resin, 9-hydroxybenzotriazole, Fmoc-protected amino acids, O-(7-Azabenzo-triazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), Fmoc-Lysine-OH, and N-Methyl-2-pyrrolidone (NMP) were obtained from AAPPTec (Louisville, KY, USA). FMOC Penicillamine(trityl)-Wang (FMOC-Pen) was from BACHEM, (King of Prussia, PA). N, N-Dimethylformamide (DMF), trifluoroacetic acid (TFA), and acetonitrile were purchased from Fisher Scientific (Pittsburgh, PA, USA). Diisopropylethylamine, piperidine, acetic anhydride, Tris (2-carboxyethyl)-phosphine hydrochloride (TCEP), 9-chloroacridine, thiazole orange, N-hydroxysuccinimide (NHS), iodoacetic acid and dicyclohexylcarbodiimide (DCC) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Agarose was obtained from Gibco-BRL (Carlsbad, CA, USA). mPEG-maleimide (2, 5, 10, 20, 30 kDa), mPEG5kDa-S-2-sulfanylpyridine and mPEG5kDa-amine were purchased from Laysan Bio (Arab, AL, USA), whereas mPEG5kDa-vinylsulfone was from Jenkem Technologies, Beijing, China. D-Luciferin and luciferase from Photinus pyralis were obtained from Roche Applied Science (Indianapolis, IN, USA). pGL3 control vector, a 5.3 kbp luciferase plasmid containing a SV40 promoter and enhancer, was obtained from Promega (Madison, WI, USA). pGL3 was amplified in a DH5α strain of Escherichia coli and purified according to the manufacturer’s instructions.

Synthesis and Characterization of PEGylated Polyacridine Peptides

9-Phenoxyacridine and Fmoc-Lysine(Acridine)-OH were prepared as recently reported [24, 25]. The polyacridine peptides defined in Figure 1 and Table 1S were prepared by solid phase peptide synthesis on a 30 μmol scale using an APEX 396 synthesizer (Advanced ChemTech, Louisville, KY, USA) with standard Fmoc procedures.

Figure 1. Structure of PEGylated Polyacridine Peptides.

Figure 1

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The structure of PEGylated polyacridine peptides used for gene delivery are illustrated. Peptide 1 (Table 1S) was modified with PEG of varying length to generate (Cys-Maleimide) SM12kDa, SM15kDa, SM110kDa, SM120kDa and SM130kDa. Peptide 1 or 2 were reacted with PEG5kDa to generate SS (thiol-thiol), PS (penicillamine-thiol-thiol), PM (penicillamine-thiol-maleimide), SV (thiol-vinyl sulfone) and SA (thiol-acetamide). In addition, Salk was generated by reacting the Cys on peptide 1 with iodoacetic acid. Peptide 3 and 4 (Table 1S) were reacted with maleimide-PEG5kDa to generate geometric isomers of SM15kDa possessing PEG at the N-terminus (SM35kDa) and in the middle (SM45kDa) of the polyacridine peptide.

PEGylation of the Cys residue on (Acr-Lys4)3-Acr-Lys-Cys was achieved by reacting 1 μmol of peptide with 1.1 μmol of PEG5kDa-maleimide in 4 ml of 100 mM HEPES buffer pH 7 for 12 hrs to generate (Acr-Lys4)3-Acr-Lys-Cys-Mal-PEG5kDa (SM15kDa). In addition, S-Mal PEG-peptides were prepared with PEG2kDa (SM12kDa), PEG10kDA (SM110kDa), PEG20kDa (SM120kDa) and PEG30kDa (SM130kDa) (Table 1S).

Similarly, 1 μmol of (Acr-Lys4)3-Acr-Lys-Pen was reacted with 1.1 μmol of PEG5kDa-maleimide to form (Acr-Lys4)3-Acr-Lys-Pen-Mal-PEG5kDa (PM). Reaction of 1 μmol of (Acr-Lys4)3-Acr-Lys-Cys/Pen with 1.1 μmol of PEG5kDa-S-2-sulfanyl pyridine in 4 ml of 100 mM HEPES pH 7 for 12 hrs resulted in the formation of (Acr-Lys4)3-Acr-Lys-Cys-SS-PEG5kDa (SS), and (Acr-Lys4)3-Acr-Lys-Pen-SS-PEG5kDa (PS). The optimized reaction to generate (Acr-Lys4)3-Acr-Lys-Cys-SV-PEG5kDa (SV) was performed by adding 1 μmol of (Acr-Lys4)3-Acr-Lys-Cys and 10 μmol of PEG5kDa-vinyl sulfone in 4 ml of 100 mM HEPES pH 7.5, which was allowed to react for 12 hrs.

The formation of a PEG-peptide with an iodoacetamide linkage was achieved by derivatization of amino-PEG5kDa with NHS-iodoacetic acid. Amino-PEG5kDa (5 μmols) was reacted with 50 μmols of NHS-iodoacetic acid in 5 ml of 0.1 M sodium bicarbonate pH 8.0 for 5 hrs. The reaction product was isolated by Sephadex G10 column chromatography (2.5 × 50 cm) eluted with 0.1 v/v % TFA by collecting the peak eluting at 100 ml, which was concentrated by rotary evaporation, freeze dried, reconstituted in water and quantified by Abs280nm of iodoacetamide (ε = 370 M−1cm−1). Analytical reverse phase high performance liquid chromatographic (RP-HPLC) analysis of the product (10 nmols) eluted at 1 ml min−1 with 0.1 v/v % TFA with a gradient of acetonitrile of 15–65 v/v % over 30 min while monitoring Abs280nm identified iodoacetamide-PEG eluting at 19 min. Reaction of 1 μmol of (Acr-Lys4)3-Acr-Lys-Cys with 5 μmols of iodoacetamide-PEG in a total volume of 4 ml of 0.1 M sodium bicarbonate resulted in the conversion of the peptide into (Acr-Lys4)3-Acr-Lys-Cys-S-Acetamide-PEG (SA). PEGylated peptides were purified by semi-preparative RP-HPLC as eluted at 10 ml min−1 with 0.1 v/v % TFA and a gradient of 20–60 v/v % acetonitrile while monitoring acridine absorbance at Abs409nm. The major peak was collected and pooled from multiple runs, concentrated by rotary evaporation, lyophilized, and stored at −20°C. The counter ion was exchanged by chromatography on a Sephadex G-25 column (2.5 × 50 cm) equilibrated with 0.1 v/v % acetic acid to obtain the peptide in an acetate salt form. The major peak corresponding to the PEG-peptide eluting at 100 ml was pooled, concentrated by rotary evaporation, and freeze-dried. PEG-peptides were reconstituted in water and quantified by Abs409nm to determine isolated yield (Table 1S). PEG-peptides were characterized by matrix assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF MS) by combining 1 nmol with 10 μl of 2 mg ml−1 α-cyano-4-hydroxycinnamic acid (CHCA) in 50 v/v % acetonitrile and 0.1 v/v % TFA. Samples were spotted onto the target and ionized on a Bruker Biflex III Mass Spectrometer (Bruker Daltonics Inc, Billerica, MA, USA) operated in the positive ion mode.

Formulation and In Vivo Analysis of PEGylated Polyacridine Peptide Polyplexes

The relative binding affinity of PEGylated polyacridine peptides for pGL3 was determined by a fluorophore exclusion assay[26]. Pharmacokinetic and biodistribution analysis of PEGylated polyacridine polyplexes were performed using radioiodinated pGL3 as previously described [22, 23, 27]. HD-stimulated expression of luciferase with quantitative bioluminescence imaging in liver were performed as previously described [22, 23].

The metabolic stability of PEGylated polyacridine peptides was determined by incubating 10 nmols with 500 μl of either EDTA (2 mg ml−1) whole mouse blood, freshly prepared mouse liver or spleen homogenate prepared in 0.25 M sucrose[28]. After incubation at 37°C for 4 h, the blood, liver or spleen homogenates were precipitated by adding 250 μl of lysate with 125 μl 20 v/v% TFA/ddH2O, followed by incubation at 4°C for 30 min, and then centrifuging at 10,000 g for 30 min. The soluble fraction containing the PEGylated acridine peptide was analyzed by Abs409nm to determine recovery (95%). Recovered PEGylated peptides were analyzed by RP-HPLC eluted at 1 ml min−1 with 0.1% TFA and a gradient of 15–60% acetonitrile over 30 min while detecting at Abs409nm. The retention time of PEG-peptide digests were compared to untreated PEG-peptide standards and a non-PEGylated peptide to determine metabolic status.

Results

The development of nonviral gene delivery systems that function in vivo requires careful optimization using realistic doses in animal models with reliable gene expression readouts. We have chosen to optimize PEGylated polyacridine peptide delivery systems using a standardized tail vein dose of 1 μg of pGL3 in 50 μl in 15–20 g male ICR mice. pGL3 is a standard commercially available 5.3 kbp plasmid expressing luciferase under the control of an SV40 promoter.

It has been previously demonstrated that pGL3 is rapidly metabolized by endogenous DNAse in the blood resulting in no detectable luciferase expression when delivered i.v. to mice[18, 22, 23]. Alternatively, a rapid (5 sec) direct-hydrodynamic dose of 1 μg of pGL3 in approximately 1.8 ml of saline via the tail vein in a 20 g mouse results in the bioluminescence imaging (BLI) detection of 108 photons sec−1cm−2seradian−1 for luciferase in the liver after 24 h [22, 29]. Results were analyzed for statistical significance (p ≤ 0.05) based on Dunnett T3 test using SPSS 19.0 analysis of variance software.

The delivery of 1 μg of pGL3 PEGylated polyacridine polyplex to mice via the tail vein in 50 μl also leads to no detectable expression in the liver. However, following polyplex delivery, the administration of a blank (saline only) HD-stimulatory dose at times ranging from 5 min to 9 hrs post-DNA delivery, results in appreciable luciferase expression in the liver. Both the magnitude of expression and the length of the stimulation time window are influenced by the structure of the PEGylated polyacridine peptide[22, 23]. Increasing the affinity of PEGylated polyacridine peptides for DNA results in polyplexes with greater stability in the circulation and a longer stimulation time window[22, 23]. Using an optimized PEGylated polyacridine peptide polyplex, the magnitude of expression mediated by HD-stimulation is equivalent to that of direct-HD dosing of 1 μg of pGL3[22].

In the present study we examined the influence of PEG linkage by attaching PEG5kDa to (Acr-Lys4)3-Acr-Lys-Cys/Pen (1 or 2, Table 1S), using six unique linkages which included thiol-maleimide (SM), thiol-thiol (SS), thiol-acetamide (SA), thiol-vinyl sulfone (SV), penicillamine-thiol-maleimide (PM), and penicillamine-thiol-thiol (PS) (Fig. 1, Table 1S). PEGylated polyacridine peptides were prepared by first synthesizing peptides 1–4, which were RP-HPLC purified in approximately 20% isolated yield and characterized by LC-ESI-MS prior to derivatization with PEG (Table 1S).

Monofunctionalized PEGs with a terminal maleimide, S-2-sulfanylpyridine, vinyl sulfone, or amine were selected based on their commercial availability. A maleimide-PEG5kDa was reacted with the C-terminal Cys or Pen on 1 or 2 to produce PEG-peptides SM15kDa and PM (Fig. 1A). An S-2-sulfanylpyridine-PEG5kDa was reacted with a C-terminal Cys or Pen to produce PEG-peptides SS and PS (Fig. 1A). A vinyl sulfone PEG5kDa was reacted with the C-terminal of Cys to produce SV. Conversion of an amino-PEG5kDa to an iodoacetamide, followed by reaction with a C-terminal Cys produced SA (Fig. 1A). In addition, a control non-PEGylated polyacridine peptide was prepared by alkylating the Cys on peptide 1 to form Salk (Fig. 1A, Table 1S). Furthermore, the influence of PEG length on polyplex PK and in vivo gene transfer was evaluated by modification of peptide 1 with maleimide modified PEG2, 5, 10, 20, or 30kDa to prepare SM12kDa, SM15kDa, SM110kDa, SM120kDa and SM130kDa (Fig. 1A). In addition, peptides 3 and 4 were designed as geometric isomers of peptide 1 with either an N-terminal or middle Cys residue. Peptides 3 and 4 were reacted with maleimide-PEG5kDa to prepare SM35kDa and SM45kDa, to test the influence of PEG location on the ability of PEGylated polyacridine peptides to mediate gene expression in vivo (Fig. 1B and C and Table 1S).

PEG-peptides SM12kDa, SM15kDa, SM110kDa, SM120kDa, SM130kDa, SM35kDa, SM45kDa, PM, SS, PS, SV, SA and Salk were isolated using preparative RP-HPLC in yields ranging from 26–86 % based on peptides 1–4. MALDI-TOF MS analysis of PEGylated polyacridine peptides resulted in observed masses ranging from 5174-36459 Da. The difference between the calculated and observed mass is related to PEG polydispersity in the commercially available functionalized PEGs. The ability of PEGylated polyacridine peptides to bind pGL3 was evaluated using a thiazole orange dye exclusion assay [26]. The results established that each PEG-peptide completely displaced the fluorophore at a stoichiometry of 0.15 nmol μg−1 of pGL3, suggesting that the identity of the linker joining PEG to the peptide did not influence DNA binding affinity (Fig. 2A). However, the location of PEG exerted a minor influence on the ability of PEG-peptides to displace thiazole orange (Fig. 2A).

Figure 2. Binding of PEGylated Polyacridine Peptides to DNA.

Figure 2

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The displacement of thiazole orange from pGL3 was monitored by loss of fluorescence intensity as a function of peptide to DNA stoichiometry. Panel A illustrates the results of titrating SS, PS, PM, SV, Salk, SM15kDa, SM35kDa and SM45kDa. Panel B illustrates the result of titrating SM12kDa, SM15kDa, SM110kDa and SM120kDa, and SM130kDa Each result represents the mean and standard deviation of three determinations.

Attachment of PEG to Cys located in the middle (SM45kDa) or N-terminus (SM35kDa) of the polyacridine peptide resulted in slightly lower apparent affinities for pGL3 based on the decreased ability of these peptides to displace thiazole orange from DNA at peptide stoichiometries of 0.05–0.1 nmol μg−1 of pGL3 (Fig. 2A). Similarly, a PEG length of 2, 20, or 30 kDa resulted in a similar decrease in affinity at low stoichiometries for SM12kDa, SM120kDa and SM130kDa relative to SM15kDa and SM110kDa (Fig. 2B).

We have previously shown that PEGylated polyacridine peptides form DNAse stable electronegative polyplexes at a stoichiometry of 0.2 nmol μg−1 of pGL3, but that further addition of PEG-peptide results in more fully condensed electropositive DNAse stable polyplexes at 0.8 nmols of peptide μg−1 of pGL3[22]. While both electronegative and electropositive polyplexes are able to mediate gene expression in vivo following hydrodynamic stimulation, electropositive polyplexes are more tightly condensed and protect pGL3 from DNAse in the circulation for a longer time [23].

For this reason, the relative particle size and zeta potential were compared for polyplexes prepared at a standard stoichiometry 0.8 nmols of peptide μg−1 of pGL3 (Fig. 3). This stoichiometry was also used for all PK, biodistribution and in vivo expression experiments. Polyplexes prepared with PM, SM, PS, SS, SV and SA possessed a mean diameter of 170–200 nm (Fig. 3A) whereas polyplexes prepared with Salk were determined to be smaller, with a diameter of 105 nm (Fig. 3B). Alternatively, polyplexes prepared with SM12kDa, SM15kDa, and SM110kDa were approximately 170 nm in diameter, whereas SM120kDa and SM130kD produced polyplexes that were 200 and 250 nm in diameter, respectively (Fig. 3B). The zeta potential of polyplexes prepared with PM, SM, PS, SS, SV and SA ranged from +10–14 mV and was independent of PEG-linkage and PEG-location (Fig. 3C). In contrast, polyplexes prepared with either Salk or SM12kDa both produced zeta potentials of +28–30 mV, establishing that relative to longer PEGs, a 2kDa PEG was unable to mask polyplex charge (Fig. 3D). A significantly lower zeta potential of +10 mV was achieved using 5 and 10 kDa PEG on SM15kDa and SM110kDa (Fig. 3D). Increasing the PEG length further to 20 or 30kDa using SM120kDa and SM130kDa to form polyplexes resulted in progressive decrease in zeta potential to +5 and 0 mV, respectively (Fig. 3D).

Figure 3. Size and Charge of PEGylated Polyacridine Peptide Polyplexes.

Figure 3

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The mean particle size and charge for pGL3 polyplexes (30 μg ml−1) prepared at 0.8 nmol of PEG-peptide per μg of pGL3 determined by QELS are illustrated. Panels A and B establish the mean particle size of 105–250 nm for varying PEG-peptide linkage, location of PEG, and PEG length. Panel C illustrates a mean zeta potential peptide polyplex possessing a 5 kDa PEG with different linkages. Panel D illustrates the zeta potential of peptide polyplexes with a maleimide linkage with different PEG length. Each result represents the mean and standard deviation of three determinations.

The ability of PM, SM, PS, SS, SV, and SA polyplexes to mediate stimulated gene expression as a function of circulation time were compared (Fig. 4). The results revealed that PM, SM and PS each produced maximal gene expression of ≥108 photons sec−1cm−2seradian−1 when applying HD-stimulation at times up to 4–5 h post DNA polyplex delivery (Fig. 4A).

Figure 4. HD-Stimulated Gene Expression.

Figure 4

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The relative gene expression in the liver was determined after tail vein administration of a 1 μg dose of pGL3 peptide polyplex in triplicate mice followed by delayed HD-stimulation (tail vein administration of 1.5–2 ml of saline in 5 sec) at times ranging from 1–9 hrs. The magnitude of luciferase expression was determined by BLI at 24 hrs after HD-stimulation. Panel A illustrates the influence of varying PEG5kDa-peptide linkage (SM, PM, PS, PS, SS, SA, SV) to peptide 1. Panel B illustrates the influence of varying PEG length (2, 5, 10, 20, and 30 kDa) linked to peptide 1 with a maleimide linkage. Panel C illustrates the influence of C (SM1) versus N-terminal (SM3), versus middle (SM4) attachment of PEG5kDa to peptide 1 via a maleimide linkage, relative to no PEG (Salk). Each data point represents the mean and standard deviation resulting from BLI measured for three independently dosed mice. At 1 hr the asterisks indicate a statistical significance of p ≤ 0.05 compared with SM15kDa (+).

The stimulated expression profiles from time zero to 4–5 h were stable suggesting the polyplexes were stable in the circulation, followed by a steady-decay in expression to background when applying HD-stimulation at times ranging from 5–9 hrs. The loss of gene expression at stimulation times greater than 5 hrs is most likely the result of peptide shedding and exposing polyplexes to DNAse in the circulation as described previously[23]. The SS PEG-peptide polyplex demonstrated maximal expression of 108 photons sec−1cm−2ser−1 at stimulation times up to 2 hrs, followed by a decay to zero at stimulation times of 2–5 hrs (Fig 4A). Surprisingly, SA and SV each produced ten-fold lower expression at 1 h relative to PM, SM, PS and SS, suggesting a decreased stability. The expression profile for SA revealed maximal expression of 107 photons sec−1cm−2seradian−1 when applying HD-stimulation at times ranging from 1 to 3 hrs post-polyplex delivery, followed by a decay to background at 3–5 hrs (Fig. 4A). By comparison, SV PEG-peptide polyplexes only achieved 107 photons sec−1cm−2seradian−1 after hydrodynamic stimulation times of 1 hr followed by a decay in expression to background at stimulation times of 2 hrs (Fig. 4A).

The unexpected rapid loss of HD-stimulated gene expression for SV and SA PEG-peptide polyplexes was in part explained by the rapid loss of pGL3 polyplexes from the blood. PK analysis revealed a much shorter α half-life for SA and SV polyplexes relative to PM, PS and SS (Fig. 5A, Table 2S). The short α half-life is similar to that determined for naked pGL3 which is rapidly acted upon by DNAse in the circulation (Fig. 5A)[22, 23]. In contrast, the longer α half-life of PM, PS and SS polyplexes correlated with their greater stability in the circulation and their extended stimulated expression (Fig. 4A, 5A). Gel electrophoretic and autoradiographic analysis of blood time points confirmed that SV and SA polyplexes were also rapidly degraded (Fig. 5). An attempt to determine if polyplex metabolism occurred in the blood, liver or spleen resulted in no detectable metabolism of PEGylated peptides SV and SA when analyzing blood or tissue homogenate digests of polyplexes in vitro by RP-HPLC. The only metabolically sensitive PEG-peptides were SS and PS which underwent partial reduction of the disulfide in liver and spleen homogenate, presumably due to reduction by glutathione.

Figure 5. Pharmacokinetic Analysis of PEGylated Polyacridine Peptide Polyplexes.

Figure 5

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The concentration of 125I-pGL3 polyplexes and 125I-pGL3 in the circulation versus time is compared as a function of differing PEG-peptide. Panel A illustrates the influence of PEG-peptide linkage by comparing the PK profile for PM, PS, SS, SV, and SA polyplexes, relative to pGL3. Extraction of 125I-pGL3 from PK time points, followed by agarose gel electrophoresis and autoradiography demonstrates that PM polyplexes are stable in the blood over 2 hrs. SV and SA polyplexes are only stable for 20–30 minutes, and pGL3 is digested within 1 min. Panel B illustrates the relative PK profile for PEG-peptides of differing length. Panel C illustrates the influence of C-terminal (SM15kDa), N-terminal (SM35KDa) and middle (SM45kDa) PEG attachment in comparison to Salk polyplexes that lack PEG. The corresponding calculated PK parameters are included in Table 2S. Each curve represents the mean and standard deviation from dosing triplicate mice serially sampled at the times indicated.

Comparison of SM110kDa, SM120kDa and SM130kDa polyplexes established that each mediated a 5-fold lower HD-stimulated gene expression relative to SM15kDa polyplexes at a delay time of 1hr (Fig. 4B). Delay times of up to 4 hrs post-polyplex delivery resulted in a stable level of expression for SM15kDa polyplexes, which then declined to background over 5 h. Alternatively, SM12kDa polyplexes mediated nearly 500-fold lower gene expression compared to SM15kDa polyplexes. The HD-stimulated expression profile was stable for 3 hrs and then rapidly declined to background (Fig. 4B).

The differences in the magnitude of HD-stimulated gene expression when varying PEG length were partially explained by analysis of the polyplex PK and biodistribution. SM12kDa polyplexes possessed the shortest α half-life, whereas SM15kDa, SM110kDa, SM120kDa and SM130kDa polyplexes possessed a coincident PK profile (Fig 5B, Table 2S).

The short α half-life of SM12kDa polyplexes is likely the result of the higher (+30 mV) surface charge revealed by zeta potential analysis (Fig. 3D) which correlates with greater liver uptake (70% of dose) compared to 50% of dose for SM15kDa (Fig. 6B′).

Figure 6. Biodistribution Analysis of PEGylated Polyacridine Peptide Polyplexes.

Figure 6

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The biodistribution of polyplexes was determined by dosing triplicate mice. Panel A illustrates the percent of dose recovered in the major tissues at a biodistribution time of 5 min. The asterisks indicates statistical significance of p ≤ 0.05 compared with SM15kDa. Panels B, C, and D illustrate the percent of dose in the liver, lung, and spleen at biodistribution times ranging from 5 min to 6 hrs. Inset B′ illustrates a subset of the results from Panel B. Each bar represents the mean and standard deviation of three determinations.

Presumably, this is due to Kupffer cell recognition of the electropositive polyplex. Interestingly, as the PEG length was increased from 10 to 30 kDa, the percent of dose captured by the liver decreased, resulting in only 13% of dose associated with liver following administration of SM130kDa polyplexes (Fig. 6B′). The biodistribution of SM120kDa polyplexes resulted in an increase in spleen uptake, which reached a maximum of 18% of dose at a biodistribution time of 2 hr (Fig. 6D, Table 3S).

The fundamental importance of PEG to protect polyplexes from protein binding, Kupffer cell recognition in the liver, uptake in the spleen, or entrapment in the lung in vivo can be ascertained by analysis of the PK, biodistribution and stimulated expression for Salk polyplexes. The complete loss of stimulated gene expression (Fig. 4C) is directly correlated with rapid and nearly complete loss of Salk polyplexes from the blood as revealed by PK analysis (Fig. 5C). The biodistribution properties of Salk polyplexes are distinctive in their immediate uptake by the liver and lung (Fig. 6B and C), with a high percentage of the dose (30%) in lung that remains constant over time (Fig. 6C). This biodistribution profile is characteristic of a stable cationic non-PEGylated polyplex that ionically attracts albumin, resulting in larger particles that become entrapped in the capillary beds of the lung[30–32].

Attaching PEG via a maleimide linkage to either the C-terminal (SM15kDa) or N -terminal (SM35kDa) Cys residue resulted in only small differences in DNA binding affinity (Fig. 2A), and no significant change in particle size, zeta potential (Fig. 3A and C), PKs (Fig. 5C) or HD-stimulated gene expression profiles (Fig. 4C). While attachment of PEG to a Cys placed in the center of the polyacridine peptide (SM45kDa) resulted in a slight decrease in the apparent DNA binding affinity (Fig. 2A), this did not did influence the particle size, zeta potential (Fig. 3A and C) or PKs (Fig. 5C). SM45kDa polyplexes did mediate 5-fold less expression versus SM15kDa and SM35kDa polyplexes at HD-stimulation times of 4, 5, and 7 hrs (Fig. 4C). Thereby, it can be concluded that PEG attachment at the C or N-terminus has a negligible effect on polyplex performance in HD-stimulated gene expression whereas placement of the PEG in the middle of polyacridine peptide results in decreased gene transfer efficiency.

Discussion

Optimization of the PK and biodistribution properties of non-viral gene delivery systems administered systemically is key to their successful improvement in mediating gene transfer in vivo. Until now, there have been few, if any, delivery platforms that allowed systematic manipulation of PK and biodistribution properties to improve the level of gene expression in vivo. The PEGylated polyacridine peptide polyplex delivery platform with delayed HD-stimulation is unique in its ability to match the efficiency of direct-HD dosing of plasmid DNA [29, 33]. The application of HD-stimulation to complete the delivery of DNA to the liver at extended times is a highly stringent experimental tool to establish the viability of a gene delivery system during circulation. Consequently, it has been established that many commonly used gene delivery systems such as PEI, Lipofectamine, chitosan and PEGylated polylysine peptides fail to produce appreciable gene expression following HD-stimulation, even at very short circulation times [23]. While HD-stimulation has utility in facilitating the optimization of the stability and circulatory properties of gene formulations in mice, it is essential to develop more medically acceptable methods that replace HD-stimulation to complete gene delivery in order to find utility in humans.

In a prior study we systematically improved the polyacridine peptide design by increasing the spacing of four Acr residues with four Lys residues to arrive at (Acr-Lys4)3-Acr-Lys-Cys-PEG5kDa (SM15kDa)[23]. pGL3 polyplexes prepared with SM15kDa mediated maximal expression when applying HD-stimulation at up to 4 hrs post-polyplex administration[23]. In the present study we have examined the influence of PEG length, linkage and location on polyacridine peptide mediated gene delivery. Notably, polyplexes prepared with Salk, which lacks PEG, were completely inactive in mediating in vivo gene expression (Fig. 4C). The rapid removal of Salk polyplexes from the blood is likely the result of serum protein binding to these polyplexes with a +30 mV surface charge, resulting in very rapid and complete loss from the blood, and combined accumulation of 83% of the dose in the liver and lung. It is clear that even though a large percentage (50%) of the dose is associated with liver for up to 60 min following systemic delivery, HD-stimulation fails to mediate any luciferase expression, suggesting that entrapment of cationic polyplexes by liver Kupffer cells inhibits HD-stimulated gene expression.

The PEG length is known to influence the PK of liposomal formulations[34]; however this relationship is less well defined for DNA polyplexes. The results establish that SM12kDa mediates a significantly shortened PK half-life, increased liver uptake (70%) and 100-fold loss in HD-stimulated gene expression relative to SM15kDa. The reason why SM110, 20 and 30 kDa mediate 5-fold lower gene expression relative to SM15kDa at delay times of 1 hr is not clear, but is most likely due to less efficient delivery to hepatocytes and release of pGL3 in the nucleus, and not related to biodistribution or premature shedding of PEG-peptide in the circulation. The finding that a 30 kDa PEG is able to stealth DNA polyplexes to nearly completely avoid liver uptake (13 % of dose) is of fundamental importance for the development of targeted delivery systems[35]. A clear systematic progression of decreasing liver uptake as PEG length increases suggests that stable polyplexes with a stealth layer of 30 kDa PEG can avoid Kupffer cell recognition in the liver (Fig. 6B′).

The influence of PEG location on a peptide in relationship to polyplex PK and in vivo gene expression has not been previously reported. Locating PEG5kDa on the C-terminal, N-terminal residue on a polyacridine peptide results in no detectable change in PK or level of HD-stimulated gene expression (Fig. 4C, 5C). Conversely locating the PEG in the middle of the peptide does not influence the level of gene expression at HD-stimulation times of 1 hr but does decrease expression five-fold at HD-stimulation times of 4–7 hours. The reason for the observed decrease in expression is likely due to a weaker binding affinity of the peptide for DNA, caused by a steric interruption of the binding sequence of four acridines spaced by four Lys residues with PEG.

With the exception of linkers designed to undergo reductive or pH sensitive cleavage [36, 37], few studies have considered the ramification of linker metabolic stability when attaching PEG to peptides or polymers used for in vivo gene delivery. It is stunning to realize that such subtle changes can have a dramatic influence on the efficiency of in vivo gene transfer. We presumed that the PEG linker is sterically inaccessible to metabolic enzymes and would have little influence over gene transfer efficiency. It is most likely that the mechanism involves dissociation of the PEG-peptide, followed by metabolism to release PEG from the peptide. Deactivation could result from DNAse digestion of partially uncondensed polyplex. For SV and SA the metabolic deactivation is surprisingly fast, especially considering that a thiol-vinyl sulfone and thiol-acetamide linkage are significantly more chemically stable to reduction and elimination compared to SS, PS, PM and SM. However, the PK and HD-stimulation results establish SV and SA polyplexes have both a short half-life in the circulation and are much less active in mediating gene expression. We can deduce that SV and SA PEG-peptide metabolism is not occurring in the blood, since the de-shielded polyplex products would most likely accumulate in the lung, as was observed for Salk. We hypothesize that SV and SA polyplexes are metabolized in liver Kupffer cells, where both removal of PEG and digestion of pGL3 by DNAse could occur. This hypothesis is supported by biodistribution results that establish that SV and SA polyplexes highly accumulate in the liver (66%) and are eliminated at a similar fast rate compared to other polyplexes.

The comparative metabolic stability of SS polyplexes established that disulfide bond reduction occurs more slowly than metabolism of SV and SA. However, it also leads to a shortened PK half-life and HD-stimulated expression profile. The magnitude of gene expression mediated by SS polyplexes at 1 and 2 hr HD-stimulation time was nearly coincident with that determined for SM, indicating PEG is likely not removed by intracellular reduction under the rapid delivery mediated by HD-stimulation. However, the loss of hydrodynamic stimulated expression for SS polyplexes at circulation times of longer than 2 hrs may be overcome by incorporation of a more reductively stable PS bond [38]. The results clearly establish that both SM and PM are the most metabolically stable chemical linkages resulting in long PK half-lives and an extended HD-stimulation profile.

It is likely that the underlying mechanism of HD-stimulated gene expression is the same as direct-HD dosing [39–42], both of which transfect liver hepatocytes following a rapid delivery of a large volume of saline. Likewise, it is also likely that direct-HD and HD-stimulation both by-pass all of the intracellular barriers to gene delivery. The primary difference is that HD-stimulation utilizes PEGylated polyplexes that are stable in the circulation for hrs, whereas direct-HD dosing uses naked plasmid DNA which has a metabolic half-life of minutes in the circulation.

In conclusion, an optimal PEG length of 5 kDa linked to a C or N terminal Cys through a maleimide linkage (SM15kDa), results in PK and biodistribution properties that produce maximal HD-stimulation when applied at up to 4–5 h following post-DNA delivery. However, even though increasing the PEG length to 30 kDa blocks most of the liver uptake, the majority of the stimulated gene expression in the liver is retained. The reported relationships between PK, biodistribution, metabolism and gene expression should be generally applicable for optimizing other PEGylated DNA and RNAi delivery systems.

Supplementary Material

01

Acknowledgments

The authors gratefully acknowledge support from NIH Grants GM097093, (KK) T32 GM067795, (ME) T32 GM008365, (NB) T32HL080070.

Footnotes

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References

  • 1.Ogris M, Brunner S, Schuller S, Kircheis R, Wagner E. PEGylated DNA/transferrin- PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Therapy. 1999;6(4):595–605. doi: 10.1038/sj.gt.3300900. [DOI] [PubMed] [Google Scholar]
  • 2.Read ML, Logan A, Seymour LW. In: Advances in Genetics. Hall JC, Dunlap JC, Friedmann T, van Heyningen V, editors. Vol. 53. Academic Press; 2005. pp. 19–46. [Google Scholar]
  • 3.Dash PR, Read ML, Barrett LB, Wolfert MA, Seymour LW. Factors affecting blood clearance and in vivo distribution of polyelectrolyte complexes for gene delivery. Gene Therapy. 1999;6:643–650. doi: 10.1038/sj.gt.3300843. [DOI] [PubMed] [Google Scholar]
  • 4.Oupicky D, Konak C, Dash PR, Seymour LW, Ulbrich K. Effect of albumin and polyanion on the structure of DNA complexes with polycation containing hydrophilic nonionic block. Bioconjug Chem. 1999;10(5):764–772. doi: 10.1021/bc990007+. [DOI] [PubMed] [Google Scholar]
  • 5.Woodle MC, Lasic DD. Sterically stabilized liposomes. Biochim Biophys Acta. 1992;1113(2):171–199. doi: 10.1016/0304-4157(92)90038-c. [DOI] [PubMed] [Google Scholar]
  • 6.Kawabata K, Takakura Y, Hashida M. The Fate of Plasmid DNA After Intravenous Injection in Mice: Involvement of Scavenger Receptors in Its Hepatic Uptake. Pharm Res. 1995;12(6):825–830. doi: 10.1023/a:1016248701505. [DOI] [PubMed] [Google Scholar]
  • 7.Mosqueira VC, Legrand P, Morgat JL, Vert M, Mysiakine E, Gref R, Devissaguet JP, Barratt G. Biodistribution of long-circulating PEG-grafted nanocapsules in mice: effects of PEG chain length and density. Pharm Res. 2001;18(10):1411–1419. doi: 10.1023/a:1012248721523. [DOI] [PubMed] [Google Scholar]
  • 8.Daou TJ, Li L, Reiss P, Josserand V, Texier I. Effect of poly(ethylene glycol) length on the in vivo behavior of coated quantum dots. Langmuir. 2009;25(5):3040–3044. doi: 10.1021/la8035083. [DOI] [PubMed] [Google Scholar]
  • 9.Faure AC, Dufort S, Josserand V, Perriat P, Coll JL, Roux S, Tillement O. Control of the in vivo biodistribution of hybrid nanoparticles with different poly(ethylene glycol) coatings. Small (Weinheim an der Bergstrasse, Germany) 2009;5(22):2565–2575. doi: 10.1002/smll.200900563. [DOI] [PubMed] [Google Scholar]
  • 10.Choi HS, Ipe BI, Misra P, Lee JH, Bawendi MG, Frangioni JV. Tissue- and organ- selective biodistribution of NIR fluorescent quantum dots. Nano Lett. 2009;9(6):2354– 2359. doi: 10.1021/nl900872r. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Maldiney T, Richard C, Seguin J, Wattier N, Bessodes M, Scherman D. Effect of core diameter, surface coating, and PEG chain length on the biodistribution of persistent luminescence nanoparticles in mice. ACS Nano. 2011;5(2):854–862. doi: 10.1021/nn101937h. [DOI] [PubMed] [Google Scholar]
  • 12.Merdan T, Kunath K, Petersen H, Bakowsky U, Voigt KH, Kopecek J, Kissel T. PEGylation of poly(ethylene imine) affects stability of complexes with plasmid DNA under in vivo conditions in a dose-dependent manner after intravenous injection into mice. Bioconjug Chem. 2005;16(4):785–792. doi: 10.1021/bc049743q. [DOI] [PubMed] [Google Scholar]
  • 13.Mao S, Neu M, Germershaus O, Merkel O, Sitterberg J, Bakowsky U, Kissel T. Influence of polyethylene glycol chain length on the physicochemical and biological properties of poly(ethylene imine)-graft-poly(ethylene glycol) block copolymer/SiRNA polyplexes. Bioconjug Chem. 2006;17(5):1209–1218. doi: 10.1021/bc060129j. [DOI] [PubMed] [Google Scholar]
  • 14.Burke RS, Pun SH. Extracellular barriers to in Vivo PEI and PEGylated PEI polyplex- mediated gene delivery to the liver. Bioconjug Chem. 2008;19(3):693–704. doi: 10.1021/bc700388u. [DOI] [PubMed] [Google Scholar]
  • 15.Jaing X, Dai H, Leong K, Goh S, HQM, YYY Chitosan-g-PEG/DNA Complexes Deliver Gene to the Rat Liver Via Intrabiliary and Intraportal Infusions. J Gene Medicine. 2006;8:477–487. doi: 10.1002/jgm.868. [DOI] [PubMed] [Google Scholar]
  • 16.Fitzsimmons RE, Uludag H. Specific effects of PEGylation on gene delivery efficacy of polyethylenimine: Interplay between PEG substitution and N/P ratio. Acta Biomater. 2012;8(11):3941–3955. doi: 10.1016/j.actbio.2012.07.015. [DOI] [PubMed] [Google Scholar]
  • 17.Pun SH, Davis ME. Development of a nonviral gene delivery vehicle for systemic application. Bioconjugate Chemistry. 2002;13(3):630–639. doi: 10.1021/bc0155768. [DOI] [PubMed] [Google Scholar]
  • 18.Collard WT, Yang Y, Kwok KY, Park Y, Rice KG. Biodistribution, metabolism, and in vivo gene expression of low molecular weight glycopeptide polyethylene glycol peptide DNA co-condensates. J Pharm Sci. 2000;89(4):499–512. doi: 10.1002/(SICI)1520-6017(200004)89:4<499::AID-JPS7>3.0.CO;2-V. [DOI] [PubMed] [Google Scholar]
  • 19.Oupicky D, Ogris M, Howard KA, Dash PR, Ulbrich L, Seymour LW. Importance of Lateral an Steric Stabilization of Polyelectrolyte Gene Delivery Vectors for Extended Systemic Circulation. Molecular Therapy. 2002;5:463–472. doi: 10.1006/mthe.2002.0568. [DOI] [PubMed] [Google Scholar]
  • 20.Yang Y, Park Y, Man S, Liu Y, Rice KG. Cross-linked low molecular weight glycopeptide-mediated gene delivery: relationship between DNA metabolic stability and the level of transient gene expression in vivo. J Pharm Sci. 2001;90(12):2010–2022. doi: 10.1002/jps.1152. [DOI] [PubMed] [Google Scholar]
  • 21.Zhou QH, Miller DL, Carlisle RC, Seymour LW, Oupicky D. Ultrasound-enhanced transfection activity of HPMA-stabilized DNA polyplexes with prolonged plasma circulation. J Control Release. 2005;106(3):416–427. doi: 10.1016/j.jconrel.2005.05.002. [DOI] [PubMed] [Google Scholar]
  • 22.Fernandez CA, Baumhover NJ, Duskey JT, Khargharia S, Kizzire K, Ericson MD, Rice KG. Metabolically Stabilized Long-Circulating PEGylated Polyacridine Peptide Polyplexes Mediate Hydrodynamically Stimulated Gene Expression in Liver. Gene Therapy. 2011;18:23–37. doi: 10.1038/gt.2010.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Kizzire K, Khargharia S, Rice KG. High-affinity PEGylated polyacridine peptide polyplexes mediate potent in vivo gene expression. Gene Therapy. 2013;20:407–416. doi: 10.1038/gt.2012.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Baumhover NJ, Anderson K, Fernandez CA, Rice KG. Synthesis and In Vitro Testing of New Potent Polyacridine-Melittin Gene Delivery Peptides. Bioconjug Chem. 2010;21:74–86. doi: 10.1021/bc9003124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Anderson K, Fernandez CA, Rice KG. N-Glycan Targeted Gene Delivery to the Dendritic Cell SIGN Receptor. Bioconjug Chem. 2010;21:1479–1485. doi: 10.1021/bc1000824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Wadhwa MS, Collard WT, Adami RC, McKenzie DL, Rice KG. Peptide-mediated gene delivery: influence of peptide structure on gene expression. Bioconjug Chem. 1997;8(1):81–88. doi: 10.1021/bc960079q. [DOI] [PubMed] [Google Scholar]
  • 27.Terebesi J, Kwok KY, Rice KG. Iodinated plasmid DNA as a tool for studying gene delivery. Anal Biochem. 1998;263:120–123. doi: 10.1006/abio.1998.2834. [DOI] [PubMed] [Google Scholar]
  • 28.Ehrenreich JH, Bergeron JJ, Siekevitz P, Palade GE. Golgi fractions prepared from rat liver homogenates. I. Isolation procedure and morphological characterization. J Cell Biology. 1973;59(1):45–72. doi: 10.1083/jcb.59.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Therapy. 1999;6(7):1258–1266. doi: 10.1038/sj.gt.3300947. [DOI] [PubMed] [Google Scholar]
  • 30.Mahato RI, Anwer K, Tagliaferri F, Meaney C, Leonard P, Wadhwa MS, Logan M, French M, Rolland A. Biodistribution and gene expression of lipid/plasmid complexes after systemic administration. Human Gene Therapy. 1998;9(14):2083–2099. doi: 10.1089/hum.1998.9.14-2083. [DOI] [PubMed] [Google Scholar]
  • 31.Scheule RK, St George JA, Bagley RG, Marshall J, Kaplan JM, Akita GY, Wang KX, Lee ER, Harris DJ, Jiang C, Yew NS, Smith AE, Cheng SH. Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung. Human Gene Therapy. 1997;8(6):689–707. doi: 10.1089/hum.1997.8.6-689. [DOI] [PubMed] [Google Scholar]
  • 32.Li S, Rizzo MA, Bhattacharya S, Huang L. Characterization of cationic lipid- protamine-DNA (LPD) complexes for intravenous gene delivery. Gene Therapy. 1998;5(7):930–937. doi: 10.1038/sj.gt.3300683. [DOI] [PubMed] [Google Scholar]
  • 33.Zhang G, Budker V, Wolff JA. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Human Gene Therapy. 1999;10(10):1735–1737. doi: 10.1089/10430349950017734. [DOI] [PubMed] [Google Scholar]
  • 34.Torchilin VP. Liposomes as Targetable Drug Carriers. CRC Crit Rev Therap Drug Carrier Sys. 1985;2(1):65–115. [PubMed] [Google Scholar]
  • 35.Kursa M, Walker GF, Roessler V, Ogris M, Roedl W, Kircheis R, Wagner E. Novel shielded transferrin-polyethylene glycol-polyethylenimine/DNA complexes for systemic tumor-targeted gene transfer. Bioconjug Chem. 2003;14(1):222–231. doi: 10.1021/bc0256087. [DOI] [PubMed] [Google Scholar]
  • 36.Walker GF, Fella C, Pelisek J, Fahrmeir J, Boeckle S, Ogris M, Wagner E. Toward synthetic viruses: endosomal pH-triggered deshielding of targeted polyplexes greatly enhances gene transfer in vitro and in vivo. Molecular Therapy. 2005;11(3):418–425. doi: 10.1016/j.ymthe.2004.11.006. [DOI] [PubMed] [Google Scholar]
  • 37.Fella C, Walker GF, Ogris M, Wagner E. Amine-reactive pyridylhydrazone-based PEG reagents for pH-reversible PEI polyplex shielding. Eur J Pharm Sci. 2008;34(4–5):309–320. doi: 10.1016/j.ejps.2008.05.004. [DOI] [PubMed] [Google Scholar]
  • 38.Drummer OH, Routley L, Christophidis N. Reversibility of disulfide formation. Comparison of chemical and enzyme-mediated reduction of penicillamine and captopril disulfides. Biochemical Pharmacology. 1987;36(8):1197–1201. doi: 10.1016/0006-2952(87)90070-0. [DOI] [PubMed] [Google Scholar]
  • 39.Zhang G, Gao X, Song YK, Vollmer R, SDB, Gaskowski JZ, Dean DA, Liu D. Hydroporation as the mechanism of hydrodynamic delivery. Gene Therapy. 2004;11:675–682. doi: 10.1038/sj.gt.3302210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Lecocq M, Andrianaivo F, Warnier MT, Wattiaux-De Coninck S, Wattiaux R, Jadot M. Uptake by mouse liver and intracellular fate of plasmid DNA after a rapid tail vein injection of a small or a large volume. J Gene Medicine. 2003;5(2):142–156. doi: 10.1002/jgm.328. [DOI] [PubMed] [Google Scholar]
  • 41.Crespo A, Peydro A, Dasi F, Benet M, Calvete JJ, Revert F, Alino SF. Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles. Gene Therapy. 2005;12(11):927–935. doi: 10.1038/sj.gt.3302469. [DOI] [PubMed] [Google Scholar]
  • 42.Budker VG, Subbotin VM, Budker T, Sebestyen MG, Zhang G, Wolff JA. Mechanism of plasmid delivery by hydrodynamic tail vein injection. II. Morphological studies. J Gene Medicine. 2006;8(7):874–888. doi: 10.1002/jgm.920. [DOI] [PubMed] [Google Scholar]

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