Implications of pharmacokinetic behavior of lipoplex for its inflammatory toxicity

Advanced Drug Delivery Reviews 57 (2005) 689 – 698 www.elsevier.com/locate/addr

Implications of pharmacokinetic behavior of lipoplex for its inflammatory toxicity Jing-Shi Zhang, Feng Liu, Leaf Huang* Center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, 15213, USA Received 6 January 2004; accepted 18 December 2004

Abstract Inflammatory toxicity represents a typical toxicity associated with systemic administration of cationic liposome/DNA complex (lipoplex). Collected information indicates that the lipoplex gene delivery system mediates an uptake of plasmid DNA by the liver, mainly by Kupffer cells, in which a large amount of cytokine is produced. Therefore, many efforts have been made to overcome this problem. Previous reports by our laboratory demonstrated that sequential injection of cationic liposome and DNA could dramatically decrease the toxicity. In comparison with lipoplex injection, this method significantly suppresses the uptake of DNA by the liver. Opsonization effect in the stimulation of Kupffer cell uptake is proposed as an explanation for the differences in the pharmacokinetic properties of plasmid DNA after lipoplex injection and sequential injection. In this review, we cover the current understanding of the mechanisms underlying inflammatory toxicity and the several attempts to overcome this toxicity. The mechanism related to the pharmacokinetic properties of the lipoplex is focused on here for discussion. D 2005 Elsevier B.V. All rights reserved. Keywords: Cationic liposomes; Cytokine induction; Plasmid DNA; Nonviral vectors; Immune response; Liver uptake; Kupffer cells; Opsonization

Contents 1. 2.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . Inflammatory toxicity of lipoplex via systemic administration . . 2.1. General observations on the lipoplex-induced toxicity . . 2.2. Inflammatory toxicity and its relation to gene expression . 2.3. Role of unmethylated CpG motif . . . . . . . . . . . . . 2.4. Strategies to reduce the inflammation induced by lipoplex

* Corresponding author. Tel.: +1 412 648 9667; fax: +1 412 648 1664. E-mail address: [email protected] (L. Huang). 0169-409X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2004.12.004

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3.

Relation between pharmacokinetic properties of lipoplex and its inflammatory toxicity . 3.1. Biodistribution of DNA delivered via lipoplex . . . . . . . . . . . . . . . . . . 3.2. Contribution of cationic liposome vectors to the phagocytosis of lipoplex . . . . 3.3. Relation of liver uptake of lipoplex with cytokine induction . . . . . . . . . . . 4. Pharmacokinetic significance of sequential injection in suppression of immune response 4.1. Reduction in inflammatory toxicity with sequential injection . . . . . . . . . . . 4.2. Change in liver uptake of DNA with sequential injection . . . . . . . . . . . . . 4.3. Possible mechanism related to the reduced immune response . . . . . . . . . . . 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Cationic lipids are the most extensively studied nonviral vectors for gene delivery. This kind of vectors, in virtue of their positive charges, is capable of complexing with negatively charged DNA. The resulting cationic liposome/DNA complex (lipoplex) has shown a great promise in introduction of DNA into cells both in vitro and in vivo [1,2]. Several lipoplex formulations have been evaluated in clinical treatment of cancer [3,4] and cystic fibrosis [5–7]. Specially, systemic administration of lipoplex symbolizes a progress in the development of liposomal vectors, which could efficiently express a therapeutic gene in the lung in animal models, as well as other major organs with moderate levels of gene expression [8–11]. However, the application of lipoplex has been complicated by the occurrence of acute inflammatory toxicity, which was observed in animal studies and human clinical trials at effective dose [12]. Further improvements in the nonviral gene delivery formulations rely on understanding the lipoplex-associated inflammatory toxicity [13]. Currently, many studies have focused on exploring the mechanism of the inflammatory toxicity and looking for means to overcome the toxicity [12]. A valuable progress has been made in understanding the lipoplex-induced the immune response [14]; thereafter, several strategies have been successful in animal models [12]. In this review, we provide an overview of the understanding of systemic lipoplex-induced inflammatory toxicity. Emphasis is placed on the discussion of the correlation between the pharmacokinetic behavior of DNA and the inflammatory toxicity by comparing the differences in the pharmacokinetics of DNA following lipoplex injection and sequential

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injection. Sequential injection is one of the strategies used to ablate diminish the toxicity associated with the application of cationic liposomes for gene transfection in vivo [15]. This review intends to provide a more comprehensive understanding of the relationship between the pharmacokinetic behavior of lipoplex and their inflammatory toxicity. We also want to demonstrate the usefulness of pharmacokinetics in evaluating the benefit of a novel gene delivery system.

2. Inflammatory toxicity of lipoplex via systemic administration 2.1. General observations on the lipoplex-induced toxicity Little toxicity was observed, in early studies, when lipoplex was administered in moderate doses by either local or systemic injection [12]. However, mortality occurs in animals after systemic administration of several cationic lipid-based gene delivery systems at high dose [9,11,16]. The systemic toxicity of lipoplex has been comprehensively studied, in which various cationic lipids were explored, such as GL-67, GL-62, DMRIE, DOTMA, DOTAP [17–19]. The toxicities observed in these studies include (1) inflammatory toxicity, which we will discuss in detail later; (2) hepatotoxicity, as evidenced by elevated serum levels of the transaminases, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) [17,20]; and (3) hematologic and serologic toxicity, as typified by leukopenia and thrombocytopenia [17]. It has been found that the complexes formed between cationic liposomes and DNA is likely to be responsible for these toxicities. The mechanism underlying lipoplex-

J.-S. Zhang et al. / Advanced Drug Delivery Reviews 57 (2005) 689–698

induced inflammatory toxicity was extensively studied. However, the mechanisms of hepatotoxicity, leukopenia and thrombocytopenia are still not completely revealed. 2.2. Inflammatory toxicity and its relation to gene expression Systemic administration of lipoplex induces a rapid activation of the innate immune system [17]. The inflammatory response was observed in patients as a flu-like symptom: myalgia, headache and high temperature 6 h after nebulization of lipoplex into the lung in a clinical trial of cystic fibrosis [21]. Studies on animals have shown that lipoplex is immunologically active when administered locally [18,22,23] or systemically [17,24–26]. The immune response is characterized as induction of large quantities of proinflammatory cytokines, such as tumor necrosis factor a (TNF-a), interferon-g (IFN-g), interleukin 6 (IL-6) and interleukin 12 (IL-12). TNF-a is the earliest produced cytokine with peak value of 2000–3000 pg/ ml in serum after 2 h of injection; while IL-12 and IFN-g peak at 6 h with a value of 2000 pg/ml and 50000 pg/ml, respectively [15]. Cytokines mediate the early inflammatory reactions by stimulating the cellular reaction of innate immune system, resulting in accumulation of leukocytes and plasma proteins. Therefore, accompanying the cytokine production, observations of complement protein activity and depletion [17], and influx of leukocytes have been reported [18]. Besides the above immune responses, systemic administration of lipoplex simulate rapid and marked activation of several immune cells, including T cells, B cells, NK cells and macrophages, as illustrated by strong up-regulation of CD69 expression on these cells [24]. Several studies have demonstrated that lipoplex is responsible for this series of inflammatory toxicity, because equivalent dose of cationic liposomes or DNA alone not generate comparable effects as their complexes [17,24,25]. The effect was found to be independent of the cationic species and the level transgene expression [17], but it was highly related to the injected dose of the lipoplex [17,24,27]. Several studies have demonstrated that the inflammation induced by systemic administration of lipoplex also leads to transient gene expression with single injection and a refractory period for repeated dosing

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[25,28]. Among the inflammatory cytokines, IFN-g and TNF-a play a critical role in directly inactivating transgene expression, due to their function of inhibiting transcription and destabilizing mRNA [29,30]. It was further confirmed that addition of IFN-g and TNF-a added to cultured mouse lung endothelial cells could inhibit lipofection [19,25]. If mice were pretreated with cytokine-neutralizing antibodies, the levels of gene expression as well as the duration of gene expression were considerably increased following i.v. administration of a cationic lipid-based gene vector [25]. It was also found that dexamethasone, an immunosuppressant, was effective in improving gene expression, which is due to its activity reducing the cytokine induction [19]. In addition, it was observed by Li et al. that cytokines, at a high levels, could lead to apoptosis of lung endothelial cells in vivo [25]. This observation is consistent with those reports that proinflammatory cytokines can trigger damage and apoptosis of vascular endothelial cells [31–33]. Apoptosis caused by the lipoplex-induced cytokines may be partially contributed to the reduction of the gene transfection in vivo. However, Sakurai et al. found that there was no significant improvement in gene transfection despite of almost completely suppressing TNF-a production in gadolinium chloride (GdCl3)-pretreated mice [34]. GdCl3 was able to block phagocytosis function of Kupffer cells and eliminate these cells by causing their apoptosis [35]. They claimed that IFN-g, which was not completely eliminated by GdCl3, might play a critical role in the reduced gene expression of lipoplex. 2.3. Role of unmethylated CpG motif The plasmid DNA carrying therapeutic gene is proven to associate with the inflammatory toxicity of lipoplex [18,24]. The immune response is due to the fact that bacterial DNA is recognized as foreign in mammals, because its structure differs from mammalian DNA. In bacterial DNA, the frequency of CpG dinucleotides is much higher than those in mammalian cells and the sequence is usually unmethylated, whereas the cytosine in most of the CpG sequences in mammalian DNA is methylated to become 5Vmethylcytosine [36]. The unmethylated CpG sequence primarily induces a strong immunostimulatory effect in different cell types of the innate immunity, such as

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macrophages, dendritic cells and NK cells, to induce the production of inflammatory cytokines [37–39]. The important role of unmethylated CpG motifs in lipoplexinduced inflammatory toxicity is implied from the following studies: (1) methylation of plasmid DNA using DNA methylase resulted in reduced cytokine production [18]; (2) synthetic unmethylated CpGcontaining ODN could induce in cytokine production, and the methylated or CpG-free ODN was inert [19]. TLR9 is the receptor recognizing immunostimulatory CpG motifs [40]. The immunostimulatory effect of lipoplex was largely absent in TLR / mice [41]. Therefore, it is clear that unmethylated CpG motifs of plasmid DNA contribute significantly to the inflammatory toxicity of lipoplex. While methylating these CpG sequences results in greatly reduced cytokine levels, this modification does not eliminate their ability to generate the other systemic toxicities. Examples of non-CpG DNA sequences that induce distinct toxicity profiles when administered systemically in the form of cationic lipid/DNA complex are also identified [42,43]. 2.4. Strategies to reduce the inflammation induced by lipoplex In recent years, many strategies have been developed for overcoming the inflammatory toxicity of lipoplex. These strategies can be summarized as three different categories: (1) eliminating immunostimulatory CpG motifs in the plasmid DNA; (2) decreasing the interactions of lipoplex with immune cells; (3) suppressing the immune response to lipoplex by using immunosuppressant agents [14]. Since CpG motif of plasmid DNA is the major source of immune stimulation, direct modification or reduction of the number of unmethylated CpG motifs should be an effective approach. In vitro methylation by methylase was effective in reducing cytokine induction, but the gene expression was also severely inhibited due to non-specific methylation by the enzyme [18]. As an alternative approach, construction of a plasmid DNA expression vector containing 50% fewer CpG motifs than the conventional plasmid could decrease the cytokine induction by 40 to 70% while the levels of reporter gene expression were not affected [27]. Using synthetic genes is another approach to eliminate the immunostimulatory CpG motifs. PCRamplified fragments, without bacterial sequence,

showed threefold reduction in cytokine induction with similar transfection efficiency as the plasmid DNA [44]. To selectively deliver DNA to target cells and decrease the uptake by immune cells is an ultimate goal of developing nonviral gene delivery system. Li et al. have tried to actively target pulmonary endothelium by conjugating polyethyleneimine with an antibody against the platelet endothelial cell adhesion molecule (PECAM) which is over-expressed in the pulmonary endothelial cells [45]. This targeted gene delivery system led to 20-fold improvement in lung transfection and reduced TNF-a induction [45]. As a simple and effective approach, sequential injection of cationic liposomes and plasmid DNA not only resulted in higher gene expression in the lung, but also minimized cytokine induction [15]. We will detail the features of this approach below. Besides the above two approaches, using an immunosuppressant such as dexamethasone, or inhibitors of endocytosis such as chloroquine and quinacrine, also proved to be efficient in suppressing the cytokine response after lipoplex injection and improving the gene transfection efficiency [19,27]. However, the safety of using these drugs is the critical concern, particularly for the long-term treatment of chronic diseases. Cytokine production could also be controlled by co-delivery of an NF-nB decoy-related oligonucleotide, which potentially blocked NF-nB activity as a regulator of inflammatory and immune response [46].

3. Relation between pharmacokinetic properties of lipoplex and its inflammatory toxicity 3.1. Biodistribution of DNA delivered via lipoplex While it is clear that CpG motifs in the plasmid DNA contribute to the inflammatory toxicity of lipoplex, it is also important to understand the biodistribution of plasmid DNA after lipoplex injection. The methods for monitoring plasmid DNA include Southern blotting, PCR analysis and isotope tracking of 32P- or 125I-labeled plasmid DNA [9,47– 49]. Although the organ distribution of DNA varies with different cationic lipids, a general distribution profile can be illustrated in Table 1. After intravenous injection, lipoplex is rapidly cleared from the blood-

J.-S. Zhang et al. / Advanced Drug Delivery Reviews 57 (2005) 689–698 Table 1 Distribution of plasmid DNA in the lung and liver after intravenous injection of naked DNA and lipoplex formulations into mice Formulation

Naked DNA Lipoplex DOTMA DDAB DOTIM DOTAP

Percent of injected DNA (post-injection time) Lung

Liver

b10%

60% (1.5 min) 60%–50% (15–30 min) 35%–30% (5–30 min) 50%–36% (2–4 h) 31%–26% (10–60 min)

40%–b5% (1–15 min) 40%–30% (1–30 min) 42%–4.4% (5 min–2 h) 62%–33% (2–10 min)

Reference

[48] [48] [48] [49] [72]

stream. For example, with DOTIM–cholesterol–DNA complex, less than 10% of the injected dose was detectable in the blood at 1 min post-injection [48]. Southern blot analysis indicated that blood retained DNA was rapidly degraded with a half-life of less than 5 min [47]. Most lipoplex was immediately distributed into the lung after injection due to the first-pass capillary retention. From then on, lipoplex was removed away from the lung until less than 30% of DNA resided in the lung, and redistributed to the liver for over 50% of the injected dose for some lipids. The pharmacokinetic analysis revealed that the apparent clearance of lipoplex through the hepatic uptake was near to the hepatic plasma flow rate, indicating that lipoplex was taken up once they passed through the liver [48]. Kupffer cells were identified as the major site of uptake in the liver by using fluorescently labeled lipid/DNA complex [50]. Sakurai et al. also found the hepatic uptake of lipoplex was reduced twofold in GdCl3-pretreated mice, in which most Kupffer cells were killed by GdCl3 [34]. Comparing the distribution in the liver, lipoplex accumulated to a less extent in the spleen [48,49]. 3.2. Contribution of cationic liposome vectors to the phagocytosis of lipoplex As mentioned above, systemic injection of naked DNA does not result in cytokine induction even though it contains immunostimulatory CpG motifs. Moreover, several studies have demonstrated that liver also is the

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main organ to clear the naked DNA [48,49,51]. Naked DNA was predominantly taken up by the liver nonparenchymal cells and the GdCl3-induced Kupffer cell blockade did not affect the hepatic uptake [51]. It was suggested that liver endothelial cells mainly contribute the uptake of DNA in this organ [51]. Comparing the primary macrophage uptake pattern of lipoplex with that of naked DNA, it is clear that cationic liposomes assist the introduction of DNA into the immune cells, mainly the Kupffer cells, thereby stimulate the inflammatory response. Based on the observation with cryo-electron microscopy, the structure of lipoplex has been proposed as cationic liposomes are exposed on surface of the complex with DNA condensed in the interior of invaginated liposomes between two lipid bilayers [52]. After intravenous injection, lipoplex rapidly formed aggregates with blood proteins, which was observed using fluorescence-labeled lipid [50]. The aggregation also was observed with in vitro studies [53–59]. The interaction of lipoplex with serum proteins, probably by coating lipid on the surface of lipoplex, resulted in a change of zeta potential on the particle surface from positive to negative values [60]. A recent study indicates that the proteins bound to lipoplex are mostly acidic proteins with a pI value ranging from 4 to 6 [61]. Bringing of plasma or serum proteins may opsonize the lipoplex for the uptake by reticuloendothelial system (RES) [59]. However, there is not any report illustrating the action of serum proteins that specifically facilitate the lipoplex uptake in vivo. It has been well documented that serum contains opsonins for cationic liposomes, through which the phagocytic uptake of blood-born liposome–serum complex can be facilitated [62–66]. The opsonin proteins included complement C3, IgG, lipoproteins and fibronectin [67–70]. Lipoplex with DNA condensed in the interior of the particle could similar physicochemical properties with cationic liposomes hence binding some of above proteins. More studies are needed to address the issue. 3.3. Relation of liver uptake of lipoplex with cytokine induction Macrophages play a central role in innate immunity and are major cell source of proinflammatory cytokines in the early immune response [71]. As

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mentioned above, Kupffer cells, the macrophages lining the vascular sinusoid of the liver, are principally responsible for the lipoplex uptake. In a recent study, Sakurai et al. examined the relationship of organ uptake and induction of proinflammatory cytokine by depleting liver Kupffer cells and splenic macrophages with GdCl3 [34]. Their results indicated that the systemic levels of TNF-a and IL-12 in Gd-Cl3pretreated mice were reduced by over 20 times as compared with untreated mice, while GdCl3 caused twofold reduction of lipoplex uptake by the liver but no effect on uptake by the spleen. The study also showed that the spleen contributed as much as the liver in cytokine production while examining the levels of cytokine in the different organs following lipoplex injection. The discrepancy between the spleen uptake and cytokine induction obviously needs further study [34]. Nevertheless, the study by Sakurai at al. suggests that avoiding the uptake of lipoplex by Kupffer cells will be an efficient method to reduce the cytokine induction and consequently the inflammatory toxicity.

4. Pharmacokinetic significance of sequential injection in suppression of immune response 4.1. Reduction in inflammatory toxicity with sequential injection The normal practice of gene transfer by cationic liposome in vivo involves mixing plasmid DNA and cationic liposome to form a complex, i.e., lipoplex, and then inject the complex into the test animal. However, it was reported by Song et al. that equal or higher level of gene expression in the lung could be obtained if one injected cationic liposome first, a few minutes later, injected DNA [72]. Later, it was found by our laboratory that this sequential injection method, with an optimized interval time of 2 min, was also effective in inhibiting the cytokine induction [15]. All cytokine levels, such as TNF-a, IL-12 and IFN-g, were significantly reduced as compared with lipoplex injection. The cytokine levels in mice after sequential injection and lipoplex injection were compared using the area under cytokine level curve (AUC, timeconcentration) from 0 to 4 h, which is more objectively evaluated the exposure of cytokines

to cells in vivo. When comparing the area under the curve (AUC) from 0 to 4 h after injection, sequential injection led to an overall 75–80% lower TNF-a level, 50% for IL-12 level and 60% for IFN-g level. In addition, other types of toxicities associated with lipoplex injection, such as lymphopenia, thrombocytopenia, complement depletion and hepatotoxicity, were also significantly suppressed with sequential injection. Moreover, it has been examined that sequential injection can only benefit cationic liposome vectors, such as DOTAP/chol and DOTMA/chol, but not cationic polymers. In addition, sequential injection not only resulted in higher transfection in the lung following a single dose, but also had a much shorter refractory period with the second dose. Therefore, among all strategies used to overcome the lipoplexinduced cytokine induction, sequential injection is regarded as the best choice with respect to the high transfection efficiency and low inflammatory toxicity. 4.2. Change in liver uptake of DNA with sequential injection To explore the mechanisms involved in the sequential injection, we first compared tissue distribution of the plasmid DNA following sequential injection and lipoplex injection. At 10 min following both injection protocols, 5% of injected DNA was recovered in the liver in case of sequential injection, comparing to about 35% after lipoplex injection, whereas DNA levels in the blood were comparable (Fig. 1). The lung uptake of DNA was significantly higher in the sequential injection than the lipoplex injection (70% vs. 30%). Such distribution pattern persisted for the next hour (data not shown). Significantly lower DNA levels in the spleen were observed by sequential injection than lipoplex injection, even though spleen appeared to contribute less to DNA uptake than the liver. An in vitro experiment, in which macrophage cells were treated by sequentially adding liposome and DNA or directly adding lipoplex to the medium containing 10% of FBS, showed that sequentially adding liposome and DNA on the cells could dramatically suppress the DNA uptake, compared to directly adding lipoplex [unpublished data]. Taken together, the results indicate that sequential injection of liposomes and DNA undoubtedly decreased the introduction of DNA into the RES,

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Percentage of dose (%)

90

Distribution of DNA at 10 min

80

Lipoplex injection

70

Sequential injection

*

60 50 40 30 20 10 0

* *

Blood

Liver

Spleen

Lung

Fig. 1. Biodistribution of plasmid DNA at 10 min after lipoplex injection or sequential injection. The samples contained 900 nmol of cationic liposomes (DOTAP/Cholesterol, 1:1) and 25 Ag plasmid DNA with trace amount of 125I-labeled DNA. *pb0.05 vs. the group with lipoplex injection. Values are meanFSD (n=4–5).

thus reducing the induction of proinflammatory cytokines. 4.3. Possible mechanism related to the reduced immune response The reduced liver uptake of DNA following sequential injection may explain the effect of sequential injection on decreasing the cytokine

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induction. However, it raises a question of why separate injection of cationic liposomes and DNA can lead to a lower liver uptake and higher lung uptake of DNA than lipoplex injection. As discussed above, the introduction of CpG motifs of plasmid in lipoplex into the Kupffer cells via the opsonization of lipoplexes by some opsonin proteins in the serum is most likely to contribute to the mechanism of inflammatory response associated with lipoplex administration. Therefore, serum proteins seem to play an important role in mediating the liver uptake of the DNA particle in the presence of cationic liposomes. We have investigated the involvement of serum proteins in sequential injection and lipoplex injection. We found that liposome–serum complex, which formed with the initial interaction of liposomes and serum, were able to further complex with DNA, and the resulting ternary complex led to similar low inflammatory response to the sequential injection [73]. Based on the observations from the pharmacokinetics and biophysical study of the formation of the particles in vitro, we propose a model to emphasize the protein binding to the DNA delivery system and its relationship to the uptake by RES, including liver and spleen, in cases of sequential and lipoplex injections (Fig. 2). In case of lipoplex, DNA is condensed by cationic lip-

Fig. 2. Schematic illustration of interactions of serum proteins with liposomes and DNA, as well as the resulting DNA disposition involved in lipoplex injection and sequential injection.

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osomes to form lipoplex, in which most of DNA is in the interior of the lipoplex. When the lipoplex is injected into the circulation, the lipoplex will be recognized and labeled by the blood opsonins, and the labeled complex will be guided to RES. However, after sequential injection, complex of cationic liposomes/serum proteins/DNA is formed in such way that DNA will be exposed on the surface of the complex. Further binding of DNA to the liposome–serum complex might mask the action of opsonization. The resulting ternary complex could also readily release DNA during the first-pass through the lung, and bring less DNA into the liver. As a result, DNA mainly distributes in the lung with the sequential injection, comparing to the major deposition of DNA in the liver with lipoplex injection. This hypothesis obviously requires further experimental support.

[4]

[5]

[6]

[7]

[8] [9]

[10]

[11]

5. Conclusions Pharmacokinetic studies can provide us with useful information about the mechanisms underlying inflammatory toxicity associated with the systemic administration of lipoplex. We assume that opsonization of lipoplex by certain serum proteins may assist the immunostimulatory plasmid DNA in being selectively guided to Kupffer cells; therein resulting in high level of cytokine induction. As a successful example of minimizing the inflammatory toxicity, sequential injection of liposomes and DNA leads to a significant reduction of liver uptake of plasmid DNA and much reduced inflammatory toxicity. When the sequential injection protocol can be applied clinically, we will have to await further trials.

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