Biocompatibility and Efficacy of Oligomaltose-Grafted Poly(ethylene

Article pubs.acs.org/molecularpharmaceutics

Biocompatibility and Efficacy of Oligomaltose-Grafted Poly(ethylene imine)s (OM-PEIs) for in Vivo Gene Delivery Daniela Gutsch,† Dietmar Appelhans,‡ Sabrina Höbel,† Brigitte Voit,‡,§ and Achim Aigner*,† †

Rudolf-Boehm-Institute for Pharmacology and Toxicology, Clinical Pharmacology, University of Leipzig, Härtelstrasse 16-18, 04107 Leipzig, Germany ‡ Leibniz-Institut für Polymerforschung Dresden e.V., Hohe Strasse 6, 01069 Dresden, Germany § Organische Chemie der Polymere, Technische Universität Dresden, 01062 Dresden, Germany ABSTRACT: Polycationic polymers like poly(ethylene imine)s (PEIs) are extensively explored for the nonviral transfer of DNA or small RNAs (siRNAs). To enhance biocompatibility and alter pharmacokinetic properties, hyperbranched PEI was recently grafted with the nonligand oligosaccharides maltose or maltotriose at various degrees in a systematic study to yield (oligo-)maltose PEIs (OM-PEIs). In this paper, we investigate the in vivo biocompatibility and efficacy of a whole set of (OM-)PEIs and the corresponding (OM-)PEI-based DNA or siRNA complexes upon systemic (intravenous, i.v.) administration in mice. We determine the overall survival and animal welfare, hepatotoxicity, immune stimulation, erythrocyte aggregation, and the efficacy of DNA delivery in vivo. Higher-degree oligomaltose-grafting of PEI substantially decreases weight loss, abolishes lethality upon repeated treatment with the free polymers or with complexes, and abrogates hepatotoxicity, as determined by serum levels of liver enzymes. Immunostimulatory effects (TNF-α, IFN-γ) and erythrocyte aggregation are mainly observed upon treatment with partially maltotriose-grafted PEI or PEI-based complexes and are largely abolished upon higher-degree grafting. In vivo transfection experiments in mice bearing subcutaneous (s.c.) tumor xenografts reveal a strong dependence of reporter gene expression in a given organ on the mode of complex administration (i.v. vs intraperitoneal injection) and the OM-PEI architecture, with high-level maltose-grafted PEI (PEI-(2-Mal)) being most efficient for DNA delivery. We conclude that distinct differences between different patterns of maltose- or maltotriose-grafting are observed with regard to both biocompatibility and in vivo efficacy and identify optimal oligomaltose-PEIs for therapeutic applications. KEYWORDS: poly(ethylene imine) (PEI), maltose grafting, gene delivery, siRNA, biocompatibility somes6−8 as an essential prerequisite for the transfer of the DNA into the nucleus or the siRNA incorporation into the RNAi machinery.9−11 While PEIs are available in linear as well as branched topology and in a wide range of molecular weights, only certain PEIs are qualified for in vitro or in vivo DNA or siRNA delivery (see, e.g., refs 5, 12, and 13). In vivo, the overall positive charge of the PEI complexes may lead to decreased biocompatibility/increased toxicity, complex aggregation, and nonspecific complex interactions with cellular and noncellular components.14−18 To address this issue, modifications to the PEI backbone have been introduced. Most prominent is the grafting of PEI with poly(ethylene glycol) (PEG).19−23 Upon systemic delivery, this has been shown to alter the biodistribution and other in vivo properties dependent on the degree and pattern of PEGylation.17,24 Other approaches for PEI modification include

1. INTRODUCTION The nonviral transfer of DNA or small RNAs (siRNAs) in vivo represents a promising approach in gene therapy, for example in the treatment of cancer. Polymer-based vectors have been explored for the condensation of nucleic acids into nanoscale complexes. These nanoparticles are able to cross biological barriers, protect their payload, and mediate cellular delivery and intracellular release. Additional requirements are favorable pharmacokinetic properties and, most notably, an absence of cytotoxicity and other unwanted side effects like hepatotoxicity, the stimulation of the adaptive immune system or the complement system. Because of their high gene transfer efficacy, poly(ethylene imine)s (PEIs) take a prominent position among polycationic polymers.1,2 Their high cationic charge density at physiological pH, which is due to protonable amino groups in every third position,3,4 allows PEIs to form noncovalent complexes with DNA and other nucleic acids including small interfering RNAs (siRNA) for the induction of RNA interference (RNAi).5 Upon internalization, the so-called “proton sponge effect” facilitates the release of the PEI/nucleic acid polyplexes from endo© 2013 American Chemical Society

Received: Revised: Accepted: Published: 4666

August 13, 2013 October 14, 2013 October 31, 2013 October 31, 2013 dx.doi.org/10.1021/mp400479g | Mol. Pharmaceutics 2013, 10, 4666−4675

Molecular Pharmaceutics

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2.2. (OM-)PEI/DNA and (OM-)PEI/siRNA Complex Formation. (OM-)PEI complexes were prepared at PEI/ nucleic acid mass ratios 30 for PEI and 180 for OM-PEIs, respectively. This has been previously44 determined as optimal, thus taking into account the decrease in PEI mass content upon grafting. While optimal N/P ratios appear to remain rather similar over a wider range of the degree of grafting, as determined by complexation efficacies, the constant mass ratios still translate into some variations in N/P ratios; see ref 44 for details. To this end, dependent on the experiment and as detailed in the respective section, 10, 30, or 100 μg of DNA (plasmid pCpG-LucSH45) or chemically unmodified siRNA (luciferase siLuc3 targeting pGL3 (Promega, Madison, WI); sense: 5′-CUUACGCUGAGUACUUCGAdTdT-3′, antisense: 3′-dTdTGAAUGCGACUCAUGAAGCU-5′; Thermo Scientific, Schwerte, Germany) were dissolved in 75 μL of 150 mM NaCl buffered with 10 mM HEPES, pH 7.4, and in a separate vial, (OM-)PEIs from 300 mg/mL stock solutions were dissolved in the same buffer to the appropriate amounts in 75 μL. After 5 min incubation, the PEI solution was pipetted to the DNA or siRNA solution, and after vortexing, the mixture was incubated for 30 min at room temperature. To firmly exclude any effects on cytokine response, the CpG-free plasmid DNA pCpG-LucSH (purified using a commercially available kit that is also intended to prevent contamination by LPS or bacterial genomic DNA) or the siGL3 (chemically synthesized and the sequence checked for the absence of immunostimulatory motifs) were used throughout the study. 2.3. Treatment of Mice and Sample Preparation. Immunocompetent, heterocygous FoxN1 nu/+ mice were purchased from Harlan Winkelmann (Borchen, Germany) and kept at 23 °C in a 12 h light/dark cycle, with standard rodent chow and water ad libitum. Experiments were performed according to the national regulations and approved by the Regierungspräsidium Giessen, Germany. Mice were treated by i.v. or i.p. injection of various amounts of (OM-)PEI/(nucleic acid) complexes in 150 μL or the corresponding amounts of (OM-)PEI alone, as indicated in the figures. Unless indicated otherwise, mice were treated by repeated injections (treatment start, after 24 h and after 72 h). The mouse body weight was measured after 96 h and compared to the treatment start. For other analyses, mice were sacrificed at the time points indicated in the figures, 1 h after the last application of (OM-)PEI/ (nucleic acid) complexes, by inhalation anesthesia and cortical dislocation. Lungs and livers were removed, washed with icecold saline, and immediately transferred to 3-methyl-propane precooled in liquid nitrogen, prior to storage at −80 °C. Blood was taken from the heart, stored for 10 min at room temperature, and centrifuged. The blood serum (supernatant) was collected and aliquotted and stored frozen, prior to the assessment of liver enzyme activities and markers of immunostimulation. Unless indicated otherwise, results are based on n = 3−4. 2.4. Determination of Liver Enzyme Activity and Immunostimulation. A sample of 50 μL of blood serum was analyzed for the activity of the liver enzymes ASAT and ALAT, using kits according to the manufacturer’s protocols (DiaSys Diagnostic Systems, Holzheim, Germany). Briefly, 50 μL of serum were mixed in 96-well plates with 200 μL of substrate solution. After 5 min, the absorption at 340 nm was measured in a microplate reader (BioTek Instruments, Wisnooski, VA) to determine the product formation and thus the enzyme activity.

the chemical coupling of hydrophobic chains, fatty acid residues, cholesterol, hyaluronic acid, or polyglycerol, but also the grafting of other polymers like chitosan, dextran, or hydroxyethyl starch.25−34 The grafting of PEI with mono- or oligosaccharides has been explored in greater detail. Several studies focused on the chemical coupling of galactose, mannose, or lactose aimed at introducing ligands to the PEI surface for enhanced cell-specific uptake through selective binding.35−42 Recent work has described the grafting of maltose or oligomaltose molecules as nonligand oligosaccharides. This approach allowed the tailor-made alteration of physicochemical properties of PEI-based complexes and the establishment of structure−function relationships with regard to biological activity in vitro, independent of specific recognition patterns. More specifically, hyperbranched PEIs with various oligosaccharide architectures were synthesized and shown to mediate the uptake of free nucleotides, DNA, or siRNA in vitro.43 This larger set of PEIs, systematically modified through grafting with maltose, maltotriose, or maltoheptaose at various degrees (OMPEIs, (oligo-)maltose-modified PEIs), was analyzed with regard to the physiochemical and biochemical properties of the grafted polymers as well as of OM-PEI-based DNA and siRNA complexes.44 While these studies mainly focused on the applicability of these complexes for DNA or siRNA transfection in vitro, the assessment of the siRNA biodistribution profile also indicated their possible usefulness in vivo. Their in vitro cytotoxicity was found to be generally low; however, no in vivo studies on toxicity or unwanted side effects have been performed so far, and biological activities of the OM-PEIbased complexes have not been determined yet. In this paper, we analyze the biocompatibility of a whole set of (OM-)PEIs and the corresponding (OM-)PEI-based DNA or siRNA complexes upon systemic administration in mice, with regard to overall survival and animal welfare, hepatotoxicity, immune stimulation, and erythrocyte aggregation. Furthermore, utilizing a luciferase reporter gene vector, we demonstrate for the first time their efficacy in DNA delivery in vivo. Notably, distinct differences between different patterns of maltotriose- or maltose-grafting are observed with regard to both in vivo efficacy and biocompatibility. Thus, optimal oligomaltose−PEIs for therapeutic in vivo applications are available.

2. EXPERIMENTAL SECTION 2.1. Oligomaltose-Grafted PEIs (OM-PEIs). OM-PEIs were synthesized by oligomaltose-grafting of branched poly(ethylene imine) (Lupasol G100 with Mw 5000 g/mol, obtained from BASF SE, Ludwigshafen, Germany) as described previously.43 Briefly, 0.2 g of PEI was dissolved in a 0.1 M sodium borate solution in distilled water. Upon addition of the corresponding oligosaccharide (Maltose (Mal) or maltotriose (Mal-III) from Fluka, Taufkirchen, Germany) and the borane− pyridine complex, the solution was stirred at 50 °C for 7 days. The crude product was then purified by dialysis against doubledistilled water for 3 days, and the different oligosaccharidemodified PEIs were obtained through freeze-drying with yields between 50 and 98%. For the synthesis of the various OMPEIs, molar ratios between oligosaccharide units and amino groups (primary and secondary) were used as follows: 10/1 for structure A, thus translating into a large excess of oligosaccharide units, or substoichiometric amounts of oligosaccharide, that is, ratios 0.5/1 for structure B and 0.2/1 for structure C. 4667

dx.doi.org/10.1021/mp400479g | Mol. Pharmaceutics 2013, 10, 4666−4675


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Figure 1. Overview of the various OM-PEIs employed in this study, based on different degrees and patterns of (oligo-)maltose substitution of PEI. “Structure A OM-PEIs” synthesized in the presence of an excess of maltose or maltotriose, respectively, are characterized by a dense oligosaccharide shell with most primary and secondary amines being converted into tertiary amino groups. “Structure B OM-PEIs” are characterized by mostly monosubstitution of primary amino groups; “structure C OM-PEIs” are substituted with maltose or maltotriose to an even lesser degree, resulting in ∼50% unreacted primary amino groups. Mal = maltose, Mal-III = maltotriose, NH2 = containing free amino groups. Note that “1” and “2” do not reflect stoichiometric numbers. Lower panel: Structural properties of (OM-)PEIs and zeta potentials of (OM-)PEI/DNA complexes employed in this study according to ref 44.

erythrocyte aggregates by two blinded observers and photographed. All experiments were performed in triplicate. 2.6. Determination of Reporter Gene Activity in Animals Treated with (OM-)PEI/DNA Complexes. B16− F10 mouse melanoma cells (ATCC/LGC Standards, Wesel, Germany) were cultivated under standard conditions (37 °C, 5% CO2 in a humid atmosphere) in IMDM medium (PAA, Cölbe, Germany), supplemented with 10% fetal calf serum (PAA). To generate tumor xenografts, 0.75 × 106 cells were injected subcutaneously into both flanks of mice. Upon establishment of tumors ∼2 × 3 × 5 mm in size, mice were treated with (OM-)PEI/DNA complexes containing 10 μg (PEI) or 100 μg (OM-PEIs) of the plasmid pCpG-LucSH, prepared as described above. Complexes were injected intraperitoneally or intravenously as indicated in the figure, and mice were euthanized 24 or 48 h after injection as described above. Organs (lung, liver, spleen, kidney, brain, and tumor) were removed and immediately frozen, ground in liquid nitrogen in a mortar, and then incubated in lysis buffer (Promega Luciferase assaying kit) for 15 min at room temperature. After a 10 min centrifugation at 13 000 rpm, the supernatant was transferred to fresh tubes, and protein concentrations were measured using the BioRad protein detection kit (DC Protein Assay, BioRad, Hercules, CA). Samples were diluted at 10 mg/mL in lysis buffer, prior to

Serum levels of TNF-α and IFN-γ as a means of determining immune-stimulation in treated mice were measured using the ELISA-Kits from PreproTech (Hamburg, Germany) according to the manufacturer’s protocols. Briefly, 96-well round-bottom plates were coated with antibody overnight, then blocked, incubated with blood serum for 2 h, washed several times, and incubated with detection antibody. After washing steps, wells were incubated with avidin-HRP conjugate and washed again prior to the addition of ABTS substrate. Color intensities were monitored after 20 min in a Dynatech MRX microplate reader at 405 nm with a wavelength correction set at 650 nm and compared to a standard curve. 2.5. In Vitro Determination of Erythrocyte Aggregation. Erythrocyte aggregation induced by (OM-)PEI/DNA complexes was analyzed as described previously.17 Briefly, 200 μL of blood was collected from untreated mice and dissolved in Ringer’s solution (containing 0.5% (w/v) sodium citrate, pH 7.5) to prevent coagulation. The blood was washed several times with Ringer’s solution until the supernatant became clear and colorless. Washed erythrocytes were again resuspended 1:50 in Ringer’s solution, and 50 μL of the suspension was mixed with 50 μL of complex solution corresponding to 1 μg of DNA. After 2 h incubation at 37 °C, the mixtures were carefully applied onto coverslips, and at a 63× magnification five different fields of view were microscopically examined for 4668



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Table 1. Influence of Mice Treatment with (OM-)PEIs or (OM-)PEI-Based Complexes on Survivala carrier polymer (PEI/OM-PEI) PEI PEI-(2-Mal) PEI-(2-Mal-III) PEI-(1-Mal) PEI-(1-Mal-III) PEI-(1-Mal-NH2) PEI-(1-Mal-III-NH2)

a

amount of nucleic acid per injection (μg)

amount of (OM-)PEI per injection (mg)

survival rate (PEI)

survival rate (PEI/DNA)

survival rate (PEI/siRNA)

10 30 100 100 100 100 10 30 10 30 100

0.3 0.9 18 18 18 18 1.8 5.4 1.8 5.4 18

0/2* n.d. 2/2 2/2 2/2 2/2 2/2# 1/2 2/2 0/2# 0/2#

2/2 n.d. 2/2 2/2 2/2 2/2 2/2 0/2# 0/2 0/2# n.d.

2/2 0/2 n.d. n.d. 2/2 n.d. 3/3 0/2# 2/2 0/2# n.d.

Three injections, except for § = one single injection; # = hepatotoxicity; n.d. = not done.

Figure 2. Changes in mouse body weight upon repeated treatment with the free (OM-)PEI polymers (A, left), the corresponding (OM-)PEI/DNA complexes (A, right), or (OM-)PEI/siRNA complexes (B).

1-Mal) or maltotriose (PEI-1-Mal-III). Finally, “structure C OM-PEIs” are substituted with maltose or maltotriose to an even lesser degree, resulting in ∼50% unreacted primary amino groups that are indicated in their acronyms, PEI-(1-Mal-NH2) and PEI-(1-Mal-III-NH2), respectively. To analyze the effects of oligomaltose-grafting of PEIs with regard to the biocompatibility of the complexes in vivo, mice were i.v. injected 3 times over 72 h with PEI-based or OM-PEIbased complexes, respectively, corresponding to 10−100 μg of nucleic acid as presented in Table 1. Since mixing ratios are critical for complexation efficacy, complex stability, and biological activity, complexes were prepared according to the N/P ratios determined previously as optimal,44 corresponding to (grafted) polymer/nucleic acid mass ratios 30 for PEI and 180 for OM-PEI complexes, respectively. Mice were monitored over a 96 h period for survival and weight loss. Since noncomplexed PEI or OM-PEI may exert more profound toxic effects, the biocompatibilities of the corresponding amounts of the free (grafted) polymers were determined as well. Overall, whenever differences between PEI/DNA and PEI/siRNA complexes were observed, PEI/DNA complexes proved to be somewhat more toxic than their siRNA counterparts (see Table 1, PEI-(1-Mal-III-NH2) at 10 μg of

mixing of a 10 μL sample with 2.5 μL of 1 M ATP and 25 μL of substrate solution (Promega). Bioluminescence was immediately monitored for 10 s in a luminometer (Berthold, Bad Wildbad, Germany). To determine luciferase amounts in the tissue samples from the measured RLUs, a serial dilution of recombinant luciferase (Sigma-Aldrich) as a standard was prepared from 10 ng/mL to 1 pg/mL in the respective organ lysates taken from untreated animals.

3. RESULTS 3.1. Survival Rates and Weight Loss of Mice after i.v. Injection of (OM-)PEI/Nucleic Acid Complexes. Derivatives of branched PEI with different degrees and patterns of oligomaltose-grafting were synthesized as described previously44 (Figure 1). Briefly, OM-PEIs formed in the presence of an excess of maltose or maltotriose, respectively, are characterized by a dense oligosaccharide shell with most primary and secondary amines being converted into tertiary amino groups and are termed “structure A OM-PEIs” (PEI-2-Mal and PEI-2-Mal-III). In contrast, “structure B OM-PEIs” are characterized by mostly monosubstitution of primary amino groups with maltose (PEI4669



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Figure 3. Hepatoxicity of various (OM-)PEIs and (OM-)PEI-based complexes. Serum levels of the hepatic marker enzymes alanine− aminotransferase (ALAT; A) and aspartate−aminotransferase (ASAT; B)

These findings regarding lethality were paralleled by data on the mouse body weight after 3 d (Figure 2). Untreated mice gained ∼1 g over this period. Quite substantial ∼2−3 g weight losses were observed upon injection of free PEI (only one injection due to acute lethality) or free structure C OM-PEIs, PEI-(1-Mal-NH2) and PEI-(1-Mal-III-NH2); Figure 2A, left. An even more profound up to ∼4 g reduction of body weight was determined for the corresponding DNA complexes (Figure 2A, right), while no negative effects of (OM-)PEI/siRNA complexes were observed (Figure 2B). In contrast to the partially substituted PEIs, the highly oligomaltose-grafted structure A and structure B OM-PEIs did not exert substantial effects on mouse body weight. This was true for the free polymers as well as for the DNA complexes. In particular, in mice treated with the maltotriose-substituted PEIs, PEI-(1-Mal-III) and PEI-(2-Mal-III), or with the DNA complexes based on structure B OM-PEIs, PEI-(1-Mal) and PEI-(1-Mal-III), the body weight remained indistinguishable from untreated mice (Figure 2A). From these data, we conclude that maltose- or maltotriosegrafting of PEI substantially decreases or abolishes lethality and weight loss upon treatment with the free polymers or with complexes, as compared to the parental PEI. While structure C modifications are insufficient, oligomaltose-grafted PEIs with structure A and especially with structure B show markedly better profiles. 3.2. Hepatotoxicity and Immunostimulation. To further analyze possible underlying effects of the observed differences in biocompatibility, we next assessed the hepatotoxicity of the various free polymers or complexes by measuring serum levels of the hepatic marker enzymes alanine-amino-

nucleic acid, and data not shown). This prompted us to rather focus on OM-PEI/DNA biocompatibility as the more critical issue. In the case of PEI complexes without grafting, no lethality was observed at amounts corresponding to 10 μg of DNA or siRNA (Table 1). Notably, however, the same amount of free PEI showed already acute toxicity with 100% lethality after one injection, indicating the toxic potential of this nongrafted PEI. The dose escalation toward 30 μg of nucleic acid led to lethality even with PEI/siRNA complexes and prompted us, for ethical reasons, to refrain from testing PEI/DNA or free PEI at these dosages. In contrast, DNA complexes based on OM-PEIs with structures A or B (i.e., no free primary amines after grafting) exhibited no lethality even upon escalation of the dosage to as much as 100 μg of DNA. For the reasons stated above, experiments were focused on OM-PEI/DNA rather than OMPEI/siRNA complexes, with one exception, PEI-(1-Mal), for the confirmation of previous findings that siRNA-containing complexes do not show higher toxicity. Notably, the corresponding (large) amounts of free OM-grafted PEIs did not induce any lethality as well, contrasting the results for nongrafted PEI at already smaller amounts. This effect was dependent on the absence of free primary amines, since structure C OM-PEIs, PEI-(1-Mal-NH2) and PEI-(1-Mal-IIINH2), showed reduced toxicity when compared to PEI, but did not allow a dose escalation beyond amounts corresponding to 10 μg of nucleic acid. Of note, the larger maltotriose units in structure C OM-PEIs did not improve biocompatibility compared to maltose grafting but led in some cases to an even increased lethality. 4670



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Figure 4. Immunostimulatory effects of various (OM-)PEIs and (OM-)PEI-based complexes. Serum levels of TNF-α (A) and IFN-γ (B).

transferase (ALAT) and aspartate−aminotransferase (ASAT). Notably, upon i.v. treatment of mice with OM-PEIs previously identified as rather toxic (structure C), or with the corresponding complexes, visible liver damage was observed (“#” in Table 1). In line with this, profound ∼2−4-fold (ALAT) or 4−7-fold (ASAT) increases were observed upon treatment with free PEI or structure C OM-PEIs, PEI-(1-MalNH2) and PEI-(1-Mal-III-NH2); Figure 3A, B, left panels. The same was true for the corresponding (OM-)PEI/DNA complexes (Figure 3A, B, center panels). In most cases, enzyme activities even reached the levels of the positive control, that is, treatment with lipopolysaccharide, while again overall smaller effects were observed with siRNA complexes (Figure 3A, B, right panels). More importantly, however, the higherdegree maltose- and maltotriose-grafting of PEI (structure A and B OM-PEIs) abrogated hepatotoxicity, as determined by the absence of increased ALAT levels upon treatment of mice. Likewise, ASAT levels in structure B OM-PEI- or OM-PEI/ DNA-treated mice were only slightly elevated over untreated mice, and not at all in the case of structure A OM-PEIs or OMPEI/DNA complexes. Immune stimulation has been shown previously to be a crucial parameter in the assessment of biocompatibility and

unwanted side effects. To determine long-term as well as shortterm immune stimulation, mice were i.v. injected three times with the free polymer or the corresponding complex and sacrificed 1 h after the last injection. A profound >2-fold increase in TNF-α levels was observed upon treatment with free PEI-(1-Mal-III-NH2) or with PEI-(1-Mal-III-NH2)/DNA complexes, which was in the range of the LPS positive control. A slight increase was also determined for PEI/DNA complexes, but not for free PEI. All other free OM-PEIs or OM-PEI/DNA complexes as well as all (OM-)PEI/siRNA complexes did not lead to results above TNF-α levels in untreated animals (Figure 4A), thus also excluding LPS or bacterial genomic DNA contaminations in the (OM-)PEI/DNA complexes that may exert a general immunostimulatory effect. Likewise, a ∼2-fold increase in IFN-γ levels was detected upon treatment of mice with PEI-(1-Mal-III-NH2) or the corresponding DNA complexes. While this remained below the positive control, similar levels were also reached with nongrafted PEI/DNA complexes and, notably, with structure A OM-PEIs (PEI-(2-Mal) and PEI-(2-Mal-III)) and their corresponding DNA complexes. In contrast, no IFN-γ stimulation was observed in mice treated with PEI-(1-Mal)/ DNA or PEI-(1-Mal-III)/DNA complexes, the corresponding 4671



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Figure 5. Induction of erythrocyte aggregation by various (OM-)PEI/DNA complexes. Quantitation of numbers of aggregates in an in vitro assay (A) and pictures of representative samples (B; some aggregates indicated by white circles).

Figure 6. In vivo efficacy of various (OM-)PEI/DNA complexes as indicated by luciferase transgene expression. Luciferase activity was determined 24 h (black bars) and 48 h (gray bars) after intravenous (A) or intraperitoneal (B) complex injection.

free structure B OM-PEIs, free PEI, or PEI/siRNA complexes (Figure 4B). Taken together, this identifies the structure B OM-PEIs PEI-(1-Mal) and PEI-(1-Mal-III) and their corresponding complexes as particularly nonimmunostimulatory. 3.3. Erythrocyte Aggregation. The capability of (OM-)PEI/DNA complexes to induce hemagglutination was studied in an in vitro erythrocyte aggregation assay. The

treatment of erythrocytes with PEI-based complexes prepared at the optimal mass ratio 30 did not lead to significant aggregation, as determined by the number of aggregates in the microscopic picture (Figure 5A and B, upper left). In contrast, complexes based on the structure C OM-PEIs, PEI-(1-Mal-III-NH2) or PEI-(1-Mal-III-NH2), and prepared at the optimal mass ratio 180, led to marked aggregate formation. 4672



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have been made to further increase their biocompatibility and alter pharmacokinetics. We have performed oligomaltose grafting on hyperbranched PEI scaffold to generate a set of cationic polymers that systematically alters the physicochemical properties of the complexes. Upon grafting, major changes are mainly observed in the zeta potential, the complex stability, and the pH buffering capacity, which will most likely also govern the endosomal escape and thus contribute to transfection efficacies.44 Notably, the maltose or maltotriose shell will not act as a ligand, as known from other saccharides (e.g., galactose), and avoids introducing a non-natural copolymer like polyethyleneglycol. While the shielding of the positive surface charge of the PEI core is generally expected to lead to decreased cytotoxicity in cell culture, which was also true for our OM-PEI derivatives,44 in vitro data may not readily reflect the in vivo situation. This is for example obvious when comparing the (poor) DNA transfection efficacy of PEI-(2Mal) in vitro44 with its most prominent effect in tumor xenografts upon i.p. injection in vivo. Thus, animal studies are required to fully assess the potential of a given carrier for its therapeutic use. Indeed, the dose escalation studies in mice shown here demonstrate improved biocompatibilities over unmodified PEI but also reveal marked differences between the OM-PEIs with regard to lethality, weight loss, immunostimulation, and hepatotoxicity. While the partial retention of free primary amino groups may explain their poorer biocompatibility compared to other OM-PEIs and brings them closer to the unmodified PEI, the systematic variation of degree and pattern of maltose- or maltotriose-grafting also demonstrates that very extensive substitution is suboptimal as well. Our results also reveal that several parameters need to be assessed for the evaluation of in vivo toxicities, as shown before for PEGPEI.17,47 Since PEI-based complexes at optimal N/P ratios are accompanied by free polymer which may be more toxic,48 and it must be taken into consideration that complexes may disintegrate upon injection leading to the further liberation of free polymer, the biocompatibility of the noncomplexed OMPEIs must be determined as well. Notably, we demonstrate similar toxicity profiles of the polymer and its corresponding DNA complex, while siRNA complexes are generally somewhat less toxic. In vitro, PEI-based siRNA complexes have been found to be generally less stable than DNA complexes probably due to the lack of binding cooperativity provided by the longer DNA molecule. Since the siRNA molecule is more rigid, differences in complex structures have also been hypothesized.19,49,50 This may well translate into different pharmacokinetics and biological properties in the in vivo situation. Indeed, when comparing siRNA biodistribution profiles and DNA transgene expression after (OM-)PEI-mediated delivery, major differences are observed. More specifically, previous data after i.v. injection demonstrated, compared to PEI, poor OMPEI-mediated siRNA delivery to the liver, but very similar siRNA levels in the lung, independent of the degree and pattern of (oligo-)maltose grafting.44 In contrast, this study identifies i.v. injected PEI-(2-Mal) with the highest sugar density on the PEI core and the lowest cationic surface charge as optimal candidate for DNA delivery into lung and liver, while other OM-PEIs fail to show bioactivity. Beyond physicochemical differences between PEI-based DNA and siRNA complexes, it should also be noted, however, that the DNA transgene expression requires additional steps. These include not only the complex delivery to the organ, but also the cellular internalization, DNA release, and nuclear uptake. On the other hand, in

When the degree of maltose- or maltotriose-grafting was further increased toward abolishing free amino groups (OM-PEI structures A and B), erythrocyte aggregation was reduced again, even despite the high mass ratio 180. The structure B OM-PEIs showed best results, and in particular in the case of PEI-(1-Mal)/DNA complexes no aggregation was observed (Figure 5A,B). 3.4. In Vivo Gene Delivery Studies in Tumor-Bearing Mice. Beyond the assessment of biocompatibility, the biological activity of a given nonviral vector is the most critical parameter for its in vivo use. To analyze the efficacy of gene delivery of the OM-PEIs with the most favorable toxicological profiles, structure A and B OM-PEIs, in comparison to unmodified PEI, were further investigated in mice bearing s.c. B16F10 melanoma xenografts. We utilized the CpG-free plasmid pCpG-LucSH and determined the luciferase expression by measuring the luminescence of the organ lysates. Structure C OM-PEIs were omitted because of their suboptimal toxicological properties in the previous experiments (see above). The higher biocompatibility of OM-PEI/DNA complexes of structure A and B allowed us to inject them at larger amounts as compared to PEI/DNA complexes. Since previous studies on the biodistribution of unmodified PEI/siRNA complexes had indicated that the efficacy of siRNA delivery may depend on the mode of administration,46 intravenous and intraperitoneal injections were compared. Furthermore, time-dependent expression kinetics had been described before for other systems, prompting us to analyze expression levels after 24 h as well as after 48 h. Luciferase expression levels in various organs were found to be dependent on the grafting of the PEI, the mode of administration, and on the time point. Upon i.v. injection, gene delivery was observed in liver, lung, and to a lesser extent, in kidney (Figure 6A). Besides PEI, bioactivity was observed for PEI-(2-Mal), but not for its maltotriose-grafted structure analogue PEI-(2-MalIII) or for the lesser grafted structure B for OM-PEIs. In most cases, luciferase expression was more profound after 24 h and decreased subsequently. In line with previous PEI/siRNA studies,46 no expression was observed in s.c. tumor xenografts, which was also found to be true for OM-PEI complexes. To the contrary, upon i.p. injection the most profound transgene expression was observed in tumors (Figure 6B). Besides PEI and PEI-(2-Mal), the structure B OM-PEI derivative PEI-(1Mal) mediated DNA delivery to the xenografts as well, while grafting with maltotriose abolished the biological activity independent of the degree of grafting (structures A or B). Except for PEI, expression levels after 48 h were higher than after 24 h, indicating slower delivery kinetics as compared to i.v. injection. This was also true in the kidney, where all carriers mediated gene delivery, as demonstrated by luciferase activity after 48 h. In liver and lung, all (OM-)PEIs were comparable with little or no expression, respectively, being observed. From these data, we establish the strong dependence of gene expression in a given organ on the mode of complex administration, and we identify the maltose-grafted PEIs PEI(2-Mal) for i.v. injection and PEI-(1-Mal) for i.p. injection as most efficient for DNA delivery.

4. DISCUSSION In the field of gene therapy or gene knockdown, the nonviral delivery of nucleic acids in vivo still represents a formidable task. Based on the favorable properties of PEIs, numerous efforts 4673



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agreement with previous studies on siRNA therapy,46 DNA expression in s.c. tumor xenografts is observed only upon i.p. injection. We show here that this is true for substituted and for nonsubstituted PEIs and is thus independent of the zeta potential. Maltose rather than maltotriose-grafting results in more efficient nanoparticles where PEI-(2-Mal) is again an optimal candidate. Marked differences between the nanoparticle pharmacokinetics after i.v. vs i.p. injection have been observed previously for PEI46 and could be explained by the rapid clearance of i.v. injected complexes from the bloodstream17 vs a depot effect in the peritoneum, leading to a slower release profile. Additionally, it is tempting to speculate that i.p. injected complexes may undergo surface modifications upon injection which require further analysis. This may also explain the marked differences observed between maltose and maltotriose-grafted PEI, despite the similarities in complex sizes and zeta potentials in vitro. Again, this also emphasizes the need to analyze any nanoparticulate delivery system in its in vivo situation. Indeed, a recent study on liposomes that compared in vitro with in vivo data showed that only certain cells (primary hepatocytes) may mimic the in vivo situation.51 The improved biocompatibility of the highly grafted PEIs makes these polymers attractive candidates for therapeutic studies, and among those PEI-(2-Mal), with the lowest cationic surface charge, shows overall the highest efficacy of DNA delivery. At the amounts used here for the assessment of biological activity, the toxicity is acceptable, and thus its use is clearly feasible. The introduction of target cell-specific ligands may further enhance their uptake into target organs, and in this case the absence of “nonspecific” gene delivery largely seen for the maltotriose-grafted PEIs may even provide an advantage.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +49-(0) 341-9724661. Fax: +49-(0)341-9724669. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Andrea Wüstenhagen for expert technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (AI 24/9-1), the Deutsche Krebshilfe, and the VfK Krebsforschung (stipend to D.G.). The authors also acknowledge financial support by the Saxon Ministry for Science and Art and the German Ministry for Education and Science and the SFB-TRR 79 "Werkstoffe für die Geweberegeneration im systemisch erkrankten Knochen". The work was done in part under the COST Action TD0802 “Dendrimers in biomedical applications”.

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ABBREVIATIONS ALAT, alanine−aminotransferase; ASAT, aspartate−aminotransferase; OM, oligomaltose; PEI, poly(ethylene imine) REFERENCES

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Biocompatibility and Efficacy of Oligomaltose-Grafted Poly(ethylene

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