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8 Cationic Lipospheres as
Delivery Systems for
Nucleic Acid Molecules
Rita Cortesi, Elisabetta Esposito, and
Claudio Nastruzzi

CONTENTS

8.1 Introduction ..................................................................................................143

8.2 Nucleic Acid Stability: General Considerations and Improvement............145
8.3 Preparation of CLS ......................................................................................147
8.3.1 Materials and Preparation Methods.................................................147
8.3.2 Formation of CLS/DNA Complex...................................................148
8.4 Cytotoxicity of Nucleic Acids from CLS....................................................151
8.5 Transfection Efficiency ................................................................................153
8.6 Conclusions ..................................................................................................156
References..............................................................................................................156

8.1 INTRODUCTION
Gene delivery, or the release of exogenous genetic material into cells or tissues at
a pathological state, has recently received much attention as a therapeutic method-
ology for a number of acquired and inherited diseases, including cancer [1–6].
Several diseases can, in fact, be traced back to defective or missing genes. Thus,
bringing an appropriate gene into the appropriate cells could prevent or mitigate
manifestations of the disease. In vivo cell transfection with foreign genes could be
a promising pharmacologic treatment for a variety of diseases, including cancer,
cystic fibrosis [7], and viral infections [6]. A large number of abnormally expressed
genes have, in fact, been cloned and identified, allowing the prevention or the
treatment of many human diseases [8], thus making gene therapy an active field that
has progressed rapidly into clinical trials.
Because polydeoxyribonucleotides do not penetrate easily into the cell by them-
selves, the use of gene delivery vehicles has been proposed to facilitate the transport
of genes to cellular targets. Gene transfer vehicles can be divided into viral and
nonviral systems [9,10]. In the first case, the virus uses normal tactics to enter the
cell and to begin to transcript its genome, but the absence of a key gene prevents

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144 Lipospheres in Drug Targets and Delivery

the packaging of more infectious viral particles. At the same time, the gene of interest
gets expressed by the cell and compensates for the lack of cellular proteins related
to genetic defects. Viral delivery systems, such as retroviruses, adenoviruses, len-
tiviruses, and so forth, provide high transfection efficiency, and despite the deletion
of vital genes, viruses can induce immune responses that are able to abolish trans-
genic expression. Moreover, viral vectors, because of their viral tropism, can infect
only certain cells expressing receptors for proper replication, thus limiting their
activity to specific tissues or compartments. In addition, engineered viruses may
induce an immune response that compromises transfection resulting from subsequent
injections and that lacks target specificity.
Obviating these problems, a large array of nonviral transfection agents have
emerged as potential safe and effective gene vectors for in vitro applications. These
nonviral systems include cationic lipids, peptides, glycopeptides, liposomes,
micelles, glycosylated polymers, dendrimers, and micro- and nanoparticles [10–18].
The efficiency of nonviral transfection systems has, in general, improved several
orders of magnitude in the last decade, even if none of them has yet proven to be
efficient enough in vivo [10–18].
Thus, the key to success for any gene therapy strategy is to design a vector able
to provide safe and efficient gene transcription of the transgene in a variety of cells
and tissues. In this view, the development of protocols aimed at obtaining optimal
and efficient genetic transfer has been studied [19–21] and has led to the production
of many delivery vehicles that are able to bind to DNA. The optimal carrier has to
accumulate at sites of diseases such as infections, inflammations, and tumors and
has to be a small, neutral, and highly serum-stable particle. Moreover, it has to be
not readily recognized by the fixed and free macrophages of the reticuloendothelial
system.
Among nonviral systems, particulate carriers (e.g., polymeric nano- and micro-
particles, fat emulsion, liposomes) possess specific advantages and disadvantages.
For instance, the relatively slow degradation of polymeric particles might possibly
cause systemic toxic effects by impairment of the reticuloendothelial system or by
accumulation at the injection site; cytotoxic effects have been indeed observed
in vitro after phagocytosis of particles by macrophages and human granulocytes [20].
In addition, organic solvent residues deriving from preparation procedures, such as
the solvent evaporation technique often used for liposome and polyester micropar-
ticles, could result in severe acceptability and toxicity problems [22].
However, with respect to other delivery systems, microparticles could maintain
their physicochemical characteristics unaltered for long periods, allowing long-term
storage; they can be administered through different ways (orally, intramuscularly,
or subcutaneously), depending on their composition; and they are suitable for indus-
trial production [23,24].
All together, these findings have encouraged the development of neutral and
cationic lipospheres (CLS) as nonviral DNA-mediated gene transfer techniques
because CLS enable the extemporaneous production of pharmaceutical formulations.
Like emulsions and liposomes, lipospheres (LS) consist of physiologically well-
tolerated ingredients that have often already been approved for pharmaceutical use

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Cationic Lipospheres as Delivery Systems 145

in humans [25]; in addition, similar to polymeric nanoparticles, their solid matrix

can protect drugs against chemical degradation and allow modulation of drug release
profile [24]. Thus, LS combine the advantages of polymeric nanoparticles, fat emul-
sions, and liposomes, avoiding some of their typical disadvantages, such as cytotoxic
effects after phagocytosis, toxic effects of organic residues after the production of
polymers, and lack of large industrial-scale production.
LS and CLS are solid microparticles with a mean diameter usually between 0.2
and 500 µm, composed of a solid hydrophobic fat matrix in which (in the case of
LS) the bioactive compound or compounds are dissolved or dispersed. Because of
their large range in particle size, LS can be administered by different routes, such
as orally, subcutaneously, intramuscularly, or topically, or they can be used for cell
encapsulation, thus allowing them to be proposed for treatment of a number of
diseases [26–28]. The in vivo distribution of LS demonstrated a high affinity to
vascular wells (including capillaries), to inflamed tissues, and to granulocytes
[29,30].
In addition, LS have several advantages over other delivery systems: good
physical stability, low cost of ingredients, ease of preparation and scale-up, and high
entrapment yields for hydrophobic drugs. Moreover, LS have been successfully used
both for the controlled delivery of various types of drugs and as carriers of vaccines
and adjuvants [29,31,32].
This chapter will discuss the production and characterization of CLS obtained
by different techniques, the formation of the complex between CLS and DNA, and,
finally, the in vitro cytotoxicity on different cell lines and the transfection efficiency
of CLS.

8.2 NUCLEIC ACID STABILITY: GENERAL CONSIDERATIONS

AND IMPROVEMENT
In the past few years, many types of nucleic acid molecules, such as synthetic
oligonucleotides complementary to viral or eukaryotic RNA, have been reported to
inhibit viral replication in cell culture as well as the in vitro and in vivo expression
of genes [33,34], thus indicating the use of synthetic antisense DNA for the modu-
lation of specific gene expression as a novel pharmacological approach for patho-
logical states deriving from an altered expression of a gene or genes. In this respect,
viral infections, including AIDS as well as neoplastic diseases, could represent
possible targets for oligonucleotide therapeutics [35]. Moreover, other DNA mole-
cules — such as triple helix–forming oligonucleotides and double-stranded poly-
merase chain reaction–generated DNA fragments, mimicking genomic regulatory
regions recognized by transcriptional factors — could be efficiently employed molec-
ular tools to study and modulate gene transcriptional activity [36].
Nevertheless, in spite of these interesting pharmacobiological properties, nucleic
acid molecules (i.e., oligonucleotides and polymerase chain reaction–generated DNA
fragments) are, in general, rapidly degraded by cellular and extracellular nucleases.
It is thus necessary that, to carry on their pharmacobiological activities, nucleic acid
molecules should remain stable after in vivo administration, retaining an appreciable

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146 Lipospheres in Drug Targets and Delivery

half-life in the extracellular environment. The stability problems of this class of

compounds have been approached largely through chemical modification of the
oligonucleotides, and mainly through substitution of the natural phosphodiester
linkage with alternative chemical groups [37]. Although many backbone-modified
oligonucleotides have been synthesized and proposed, methylphosphonates and
phosphorothioates are the most studied and used nuclease-resistant compounds
[37,38].
An alternative approach to chemical modification of oligonucleotides is offered
by attaching a “pendant” group to the 5- or 3-ends of the oligonucleotide [33]. A
large variety of pendant groups has been designed and synthesized, both to study
oligonucleotide cellular uptake and compartmentalization, such as fluorochrome,
and to improve or modify the biological activity of the oligonucleotides. This latest
class comprises intercalating agents playing different roles, such as stabilizing inter-
calating agents, cleaving or photo-induced cleaving reagents, and photo-induced
cross linkers [39], as well as lipophilic groups such as fatty acid or cholesterol
moieties [40].
The potential of the chemically modified nucleic acid molecules has been proven
by in vitro studies; however, the in vivo therapeutic applicability of these molecules
seems to be unsatisfactory because of their possible toxic effects (largely unknown)
and adverse bioavailability. In this view, both antisense and transfection technologies
require reliable and efficient systems for their delivery into target cells. On the basis
of this consideration, the development of an efficient nucleic acid delivery system
represents one of the key steps for these therapeutic agents, which are necessary for
a practical clinical utilization of natural or unnatural oligonucleotides.
In this respect, an interesting approach to reduce degradation and possible
toxicity problems related to nucleic acid use in vivo is offered by their encapsulation
in or association to microcarrier systems, such as neutral or cationic liposome and
polymeric microparticles [41–44].
Microparticles can, in principle, vehiculate nucleic acids in two ways: DNA can
be physically entrapped in the polymeric matrix of the particle, or DNA can be
bound through electrostatic interactions to the positively charged surface of cationic
particles. The surface of these carriers could be positively charged as a result of the
presence of quaternary ammonium cationic lipids in the liposome composition, such
as DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride)
[43,45], DEBDA (diisobutyl-cresoxy-ethoxy-ethyl-dimethyl-benzyl-ammonium
hydroxide) [46], or CTAB (cetyltrimethylammonium bromide) [47]. In this way,
negatively charged nucleic acids are able to complex the surface of preformed
cationic carrier. The association of DNA molecules to preformed cationic micropar-
ticles could provide two important benefits. First, DNA is not exposed to the chem-
ical, thermal, or mechanical stresses often present in the production of microparticles,
and second, the association of DNA to microparticles can be performed extempo-
rarily immediately before their administration [45,48]. In this way, both microspheres
and DNA can be maintained separately in sterile lyophilized forms, thus avoiding
possible long-term instability problems. To confer a positive charge to the micro-
particle surface, different approaches can be proposed, such as a polymeric mixture

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Cationic Lipospheres as Delivery Systems 147

that is constituted of an uncharged copolymer and a cationic one, or a not entirely

polymeric mixture constituted of a neutral copolymer plus a cationic lipid [49,50].
As above reported, among microparticles, CLS have been proposed as a new
type of fat-based encapsulation system developed for drug delivery of bioactive
compounds.

8.3 PREPARATION OF CLS

8.3.1 MATERIALS AND PREPARATION METHODS
CLS can potentially be used for gene delivery and have shown to be less rapidly
cleared from the circulation than negatively charged particles [51]. In addition, the
choice of the lipid matrix also plays an important role on the morphology of the
particles and on the possible formation of aggregates that are important for the
circulations of this carrier [52]. As described in many papers [53–58], LS, and thus
also CLS, can be prepared in a variety of ways by using a wide range of chemical
ingredients [26]. To obtain a formulation suitable for human administration, triglyc-
erides and monoglycerides have been chosen as LS biomaterials because of their
high biocompatibility, high chemicophysical stability, and drug delivery release.
However, all preparation methods make use of surfactants, and the resulting particles
are characterized by an overall positive, neutral, or negative surface charge, which
is determined by the composition, influencing the aggregation tendency in suspension.
The simplest and more used preparation methods are typified by the emulsion–melt
dispersion and the solvent evaporation technique [53].
It has to be underlined that, in comparison to LS containing nucleic acid mol-
ecules inside the particle, the production of CLS may be performed obviously
without considering the stability problems of nucleic acid molecules. In this view,
some preparation procedures are not considered, such as the microemulsion tech-
nique [55] that represents a favorable method when working with substances unstable
because of the high mechanical stress produced by high-pressure homogenization.
The production of CLS by the melt dispersion technique is based on the melting
of the lipid core material together with the lipophilic agent (i.e., phospholipids).
Afterward, a warm aqueous solution is added to the molten material and is mixed
by various methods (i.e., mechanical stirring, shaking, sonication, homogenization).
Then the preparation is rapidly cooled until lipid solidification and the formation of
particle dispersion. This method was used by Olbrich et al. [19] to produce the
cationic solid lipid nanoparticles to use as novel transfection agent.
The solvent evaporation technique is based on the use of organic solvents as
dissolving agents for the lipid matrix (i.e., phospholipids or triglycerides and
monoglycerides) and the subsequent evaporation of the solvent within an aqueous
medium until a CLS dispersion is obtained.
However, Erni et al. [50] have prepared LS with a method established in their
laboratory using a solvent extraction. In particular, the method is based on the disso-
lution of the triglyceride (i.e., tripalmitin) and the cationic lipid in the organic solvent
(i.e., dichloromethane), and on the addition of an aqueous polyvinyl alcohol (PVA)

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148 Lipospheres in Drug Targets and Delivery

solution (0.5% w/w) used as extraction fluid. The solution and the extraction fluid
are pumped into a static microchannel mixer, leading to the production of an O/W
(oil in water) emulsion. The mixing leads to the production of fine lamellae, which
subsequently disintegrate into droplets, allowing the formation of solid lipid micro-
particles dispersed in the extraction aqueous medium.
In a recent set of experiments [56], LS composed of triglycerides and monoglyc-
erides were alternatively produced by melt dispersion technique, by solvent evapo-
ration, or by the w/o/w double-emulsion method. The influence of preparation
parameters, such as type and amount of lipids, presence and concentration of sur-
factants, stirring speed, and type of stirrer was studied. In the case of LS prepared
by melt dispersion, the use of a lipid composition of cetyl alcohol/cholesterol (2:1,
w/w), a 5% (w/w) gelatin solution (50 Bloom grades), and a 1000-rpm stirring speed
resulted in the production of spherical particles with a high percentage of recovery
(82%, w/w), a mean diameter of 80 µm, and a narrow size distribution. In the case
of LS prepared by solvent evaporation, the best results in terms of LS morphology,
recovery, and size distribution were obtained by the use of a lipid composition of
tristearin/monostearate (66:34, w/w), a 1% (w/w) PVA solution, a 750-rpm stirring
speed, and a 55-mm three-blade turbine rotor.
The solvent evaporation method resulted in the production of LS characterized
by a smaller size (20 µm mean diameter) but poor mechanical properties in respect
to particles with the same composition that were obtained by the melt dispersion
technique (170 µm mean diameter). The use of a combination of lipids and a
methacrylic polymer (Eudragit RS100) overcame this problem, resulting in the
production of spherical particles with a narrower size distribution and good mechan-
ical properties [53,56].
Concerning the SLN produced by hot homogenization as described by Olbrich
et al. [19], as lipidic matrix Compritol ATO 888 or paraffin were used, as tenside a
mixture of Tween 80 and Span 85 was used, and as charge carrier either EQ1 [N,N-
di-(β−steaorylethyl)-N,N-dimethylammonium chloride] or cetylpyridinium chloride
were used. The resulting particles were characterized by size between 101 and
105 nm and showed zeta potentials around 40 mV at pH 7.4.
In contrast, solid lipid microparticles consisting of a tripalmitin matrix and
cationic lipids prepared using the micromixer-based solvent extraction process as
described by Erni et al. [50] were of monomodal size, showing a narrow size
distribution in the submicrometer range (Table 8.1).

8.3.2 FORMATION OF CLS/DNA COMPLEX

To our knowledge, only a few papers have been recently published concerning CLS
for gene therapy. In particular, to investigate the ability of CLS to bind nucleic acids,
different nucleic acid molecules were considered as models, namely, two double-
stranded plasmid DNA [19,50] and defibrotide (DFT, Mw 26,200 Da), a single-
stranded polydeoxyribonucleotide (DNA) sodium salt extracted from mammalian
organs [56]. This last molecule was chosen because it is included in a pharmaceutical
product presently on the market as an antithrombotic agent, it is a low–molecular
weight nucleic acid mimicking the behavior of polymerase chain reaction products
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Cationic Lipospheres as Delivery Systems 149

TABLE 8.1
Composition, Particle Size, Zeta Potential, and Loading Efficiency of Nucleic
Acid of the Cationic Lipospheres Considered in the Chapter
Zeta
Cationic Lipospheres Composition Method of Particle Potential
(% of component) Preparation Size (nm) (mV) Reference
Solid paraffin (4%), Tween 80/Span 65 Melt dispersion 103 ± 2.3 +41.2 ± 0.9 [19]
(7:3) (4%), cetyltrimethylammonium
bromide (0.5%)
Compritol (4%), Tween 80/Span 65 (7:3) Melt dispersion 105 ± 1.7 +40.2 ± 0.5 [19]
(4%), cetyltrimethylammonium
bromide (0.5%)
Compritol (4%), Tween 80/Span 65 (7:3) Melt dispersion 101 ± 2.3 +42.3 ± 1.0 [19]
(4%), N,N-di-(β-steaorylethyl)-N,N-
dimethylammonium chloride (1%)
Stearylamine (1.8%), cetyltrimethyl- Melt dispersion 189.5 +17.9 [54]
ammonium bromide (1.2%), Trimyristin (unwashed) (bimodal)
(1.8%), Polysorbate 80 (19.0%)
Stearylamine (1.8%), cetyltrimethyl- Melt dispersion 211.7 10.1 [54]
ammonium bromide (1.2%), Trimyristin (dialyzed)
(1.8%), Polysorbate 80 (19.0%)
Tripalmitin (95%), cetyltrimethyl- Solvent 1890 +21.4 ± 7.3 [50]
ammonium bromide (5%), PVA (0.5%) extraction
Tripalmitin (95%), DDAB12 (5%) Solvent 5150 +24.9 ± 7.9 [50]
PVA (0.5%) extraction
Tripalmitin (95%) Solvent 2370 +30.9 ± 8.7 [50]
DDAB18 (5%) extraction
PVA (0.5%)
Tripalmitin (95%) Solvent 6830 8.4 ± 2.3 [50]
PVA (0.5%) extraction
Tristearin (66%) Melt dispersion 1260 ± 150 n.d.a [56]
Glyceryl monostearate (33%)
cetyltrimethyl-ammonium bromide (5%)
PVA (1%)
Tristearin (66%) Melt dispersion 1650 ± 90 n.d. a [56]
Glyceryl monostearate (33%)
DDAB12 (5%)
PVA (1%)
Tristearin (66%), Glyceryl monostearate Melt dispersion 1420 ± 210 n.d. a [56]
(33%), DDAB18 (5%), PVA (1%)
a n.d.: not determined

and synthetic oligonucleotides, and its complex with cationic liposomes is a patented
pharmaceutical formulation.
The formation of the CLS/nucleic acid complex was performed by mixing an
aqueous suspension of CLS with a solution containing the nucleic acids [19,50,56].
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150 Lipospheres in Drug Targets and Delivery

In particular, Erni et al. [50] used a gentle shaking in phosphate-buffered salt with
pH 7.4 or Dulbecco’s Modificatin of Eagle’s Medium (DMEM) for 2 h at 41˚C,
whereas Olbrich et al. [19] prepared the CLS/DNA complexes by mixing 20 mg/mL
of plasmid in 200 mL of 25 mM Hepes (pH 7.4).
In the first case [50], the loading efficiency was in the range of 65 to 95% for
CLS and was up to a maximum of 56% for neutral particles, showing that adsorption
was enhanced by the cationic surface of the particles. In addition, it was proven that
loading efficiency was further dependent on the composition of the medium used
for dispersing the particles. With respect to the second case [19], the binding of the
CLS to polyanionic DNA was studied by analysis of the electrophoretic mobility of
the DNA within an agarose gel, the so-called electrophoretic mobility shift assay.
Addition of the tenside mixtures or the cationic modifier molecules alone did not
result in a change of DNA migration during electrophoresis of the cationically
modified CLS; however, one mixture (SII-13) resulted in a shift in DNA mobility,
whereas the other CLS formulations (SII-4, SII-5, SII-9, SII-10), as well as the
surfactant mixture (SII-17), were not able to immobilize detectable amounts of DNA
at w/w ratios up to 10,000. At ratios above 50, 100% of the DNA was shifted to a
higher apparent molecular weight or was even completely immobilized within the
wells. Twenty to 50 weight equivalents were sufficient to bind most of the DNA,
and five or fewer weight equivalents of SII-13 were practically inactive.
Concerning DFT association to CLS (third case) [56], Figure 8.1 shows the
ability of the different types of CLS to ionically bind the nucleic acid when used at
a different positive to negative molar charge ratio (+/–) comprised between 1:1 to
16:1. In particular, CLS containing DDAB18 (dioctadecyl-dimethylammonium bro-
mide) prepared at 500 rpm in the presence of PVA (white diamond) or gelatin 50
Bloom (black diamond), both at 1% w/v, were considered. As reported in Figure
8.1A, the association of DFT to CLS of both types showed a similar trend, reaching
at the highest molar charge ratio an association around 90%, namely, 87.3% and
90.4% for CLS obtained with PVA and gelatin 50 Bloom, respectively. The associ-
ation ability of CLS containing DDAB12 (didodecyl-dimethylammonium bromide)
and CTAB were evaluated only on lipoparticles obtained in the presence of PVA.
As demonstrated by the data of Figure 8.1B for DDAB12 and CTAB, the association
capacity of CLS was only scarcely affected by their size; in fact, smaller particles
(mean size 0.66 µm) displayed an association profile almost identical to that of
larger particles (mean size 0.87 µm).
Cortesi et al. [56] have conducted a study to evaluate the strength of the inter-
action occurring between DFT and CLS and to evaluate whether different cationic
detergents could cause a variation in binding strength. Briefly, CLS containing
increased concentrations of cationic lipid were incubated with DFT for 10 min, and
then samples were electrophoresed to determine the electrophoretic migration of
DNA complexed to liposome. The results reported in Figure 8.2 indicate that DFT
migration is only slightly retarded by low amounts of CLS, whereas higher CLS
concentration (especially in the case of DDAB18 ) causes the formation of
high–molecular weight complexes with DFT molecules that precipitated within the
well. These nonmigrating complexes were attributed to interparticle bridges formed
by DNA molecules [59].
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Cationic Lipospheres as Delivery Systems 151

100

DFT associated to microspheres (%)

A.

80

60

40

20

0
1:1 1:2 1:4 1:8 1:16
Molar charge ratio (+/–)

100
DFT associated to microspheres (%)

B.

80

60

40

20

0
1:1 1:2 1:4 1:8 1:16
Molar charge ratio (+/–)

FIGURE 8.1 Percentages of DFT association to CLS containing as cationic lipid. (A) DDAB18
prepared in the presence of PVA (open diamonds) or gelatin (closed diamonds); (B) DDAB12
(open circles) or CTAB (open squares).

8.4 CYTOTOXICITY OF NUCLEIC ACIDS FROM CLS

To evaluate the cytotoxic activity of CLS, different types of experiments were
performed. For instance, Erni et al. [50] tested the cytotoxicity of the different
cationic lipids used for the preparation of CLS in two different cell lines (293
embryonic kidney cells and RAW macrophages) for compromised membranes of
dead cells indicating necrosis [60]. All cationic lipids displayed comparable con-
centration-dependent cytotoxicity profiles, all being nontoxic at concentrations up
to 2 mg/well. Slightly increased cytotoxicities were observed with the short alkyl
chain–length DDAB12. Interestingly, a lower necrosis was observed in the RAW
macrophage cell line as compared to the 293 cell line. Significant cytoxicity was
found in primary macrophages after adding CLS or cationic lipid at amounts that
did not cause detectable cytotoxicity in 293 cells or RAW macrophages. In addition,
the cytotoxicity of the various CLS formulations turned out to be comparable to the

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152 Lipospheres in Drug Targets and Delivery

1:2
1:1

1:8
1:4
C
A.

B.

C.

FIGURE 8.2 Effect of CLS complexation on the electrophoretic migration of DFT. (A) DDAB18,
(B) DDAB12, (C) CTAB. The following CLS/DFT charge molar ratios, namely, 1:1, 1:2, 1:4,
and 1:8 mol/mol, were used. C: control untreated DFT.

respective soluble cationic lipid, with negligible effects at concentrations less than
2 mg/well (referring to the amount of cationic lipid incorporated in the formulation).
Neutral lipid particles prepared under identical conditions without the addition of
cationic lipid did not show any detectable cytotoxicity when added to the cells in
comparable amounts.
Olbrich et al. [19] tested in vitro the cytotoxicity of the transfection agents,
considering the viability of Cos-1 cell monolayers. Both the initial perturbation of
cell integrity during the 4-h incubation and the influence on the cellular activity after
48 hours were assessed by measuring the release of lactate dehydrogenase and the
mitochondrial conversion of the tetrazolium salt WST-1. Unmodified paraffin parti-
cles (SII-4) did not show cytotoxicity in the LDH (lactate dehydrogenase) release
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Cationic Lipospheres as Delivery Systems 153

assay. The same behavior was observed for paraffin particles modified with EQ1
(SII-5), whereas those containing cetylpyridinium resulted in 50% LDH release at
120 µg/mL. Similar observations were made for modified Compritol-based particles
with no detectable LDH release after incubation with up to 3 mg/mL SII-13 (EQ1
modified) but a LD50 of 150 µg/mL for SII-10, which contains the modifier cetylpy-
ridinium chloride. Interestingly, the mixture of Tween/Span and EQ1 (SII-17) was
more toxic when it was not bound to a suitable particulate matrix, as in batch SII-
13. Assessment of the cell viability after 48 h, using the WST-1 test, was generally
more sensitive than the LDH release assay. However, it produced the same overall
correlations: cetylpyridinium-containing SLN were toxic, and EQ1-modified parti-
cles were less problematic. Moreover, modification of paraffin-based SLN with EQ1
reduced their negative effect on cell viability, resulting in IC50 values of 700 mg/mL
instead of 100 mg/mL. The pure tenside mixture (SII-17) showed similar IC50 values
of 150 mg/mL.
To determine the cytotoxic activity of cationic microparticles, Cortesi et al. [56]
performed an in vitro study treating human leukemic K562 and murine macrophagic
J774 cell lines with different amounts of CLS. After 6 d in cell culture, cells were
electronically counted. Figure 8.3 reports the cytotoxic activity of CLS containing,
alternatively, DDAB18, DDAB12, and CTAB (with an amount between 0 and 300
µg/mL corresponding to a cationic lipid concentration between 0 and 250 µM). The
obtained data demonstrated that cationic lipoparticles are only slightly cytotoxic,
especially when compared with other cationic formulations used for gene therapy,
such as liposomes [49], indicating that DDAB18-based lipoparticles could be safely
used in ex vivo experiments. To assess whether cationic lipoparticles can be effi-
ciently internalized by in vitro cultured cells, an experiment was also conducted with
J774 murine macrophages. Cells were cultured in the presence of CLS and, after
5 min, were fixed with glutaraldehyde and observed by scanning electron microscopy
analysis. As clearly evident in Figure 8.4, CLS are efficiently internalized (probably
by phagocytosis) in J774 cells, indicating that they could be used as a delivery
system for at least ex vivo experiments.
Erni et al. [50] have demonstrated that neutral lipid particles consisting of a
tripalmitin matrix can be efficiently phagocytosed by primary macrophages in vitro.
In particular, complete intracellular degradation was observed within 24 h, making
neutral lipid particles a suitable carrier for the immediate delivery of therapeutics
to antigen-presenting cells. However, CLS also adsorbed plasmid DNA and triggered
the cellular internalization of the macromolecules by phagocytic macrophages. Sur-
prisingly, the CLS also triggered the internalization of these molecules by nonpha-
gocytic 293 cells. This was probably a result of the detachment of nanocomplexes
formed of cationic lipid and DNA from the surface of DNA-loaded CLS and their
subsequent uptake into the cells.

8.5 TRANSFECTION EFFICIENCY

Erni et al. and Olbrich et al. [19,50] have data about transfection performed with
CLS as carriers. For instance, Erni et al. tested the transfection efficiency in primary
macrophages and in RAW macrophages. No transfection was observed during 72 h
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154 Lipospheres in Drug Targets and Delivery

A.
100

C ell growth (% of control)

80

60

40

20

0
1 10 100
C ationic lipid (µm)

100
B.
C ell growth (% of control)

80

60

40

20

0
1 1 0 100
C ationic lipid (µm)

FIGURE 8.3 Cytotoxic activity of DDAB18 (circle), DDAB12 (square), and cetyltrimethyl-
ammonium bromide (diamond) cationic lipoparticles on the cultured human K562 (A) and
murine macrophagic J774 (B) cell lines. Data represent the percentage of cell number per
milliliter compared with untreated control K562 cells.

in either type of cells. However, the authors demonstrated that transfection efficiency
of the DNA-loaded CLS was most pronounced in nonphagocytic cells and was not
detected in the macrophage cell line or in primary macrophages. Further studies
have revealed that cytotoxic effects of CLS were more pronounced in the phagocytic
cells, because of the very rapid uptake and degradation of the CLS in these cells.
In particular, free cationic lipid equivalent to the amount of cationic lipid contained
in the tested CLS was mixed with DNA and administered to the various cell cultures
as controls. Detectable transfection in 293 cells was exclusively found with DDAC18.
Its level was about threefold higher than with CLS containing DDAC18 and was in
the range of the commonly used transfectant DOTAP. CTAB and DDAC12 failed to
induce cell transfection. Thus, transfection mediated by CTAB or DDAC12 was only
feasible when enhanced with CLS. However, when CLS containing DDAC18 were
separated by filtration, and the supernatant subsequently mixed with DNA and further
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Cationic Lipospheres as Delivery Systems 155

A.

B.

FIGURE 8.4 Scanning electron micrograph of J774 cells alternatively untreated (A) and
treated (B) with cationic lipospheres containing DDAB18. Black arrows indicate internalized
submicron particles. Bar corresponds to 10 and 30 µm in panels A and B, respectively.

used for transfection, no measurable transfection occurred. This may indicate the
absence of significant amounts of free DDAC18 in the dispersion medium that are
available for complex formation with DNA that induces cell transfection.
As previously stated, Olbrich et al. [19] tested both cytotoxicity and transfection
efficiency on Cos-1 cells in vitro. In particular, the ability of CLS to transfect the
pCMVb reporter gene plasmid at a fixed concentration into Cos-1 cells was tested
in the absence or in the presence of 100 mM of the endosomolytic agent chloroquine.
The obtained results demonstrated that CLS were able to promote transfection in a
wide window of CLS/DNA ratios, whereas incubation with uncomplexed DNA did
not result in detectable levels of β-galactosidase expression in this experimental
setup. In the absence of chloroquine, the strongest reporter gene expression was
observed when the DNA was complexed with 60 weight equivalents of CLS,
although, when the overall transfection efficiency of CLS is compared to established
transfection agents such as poly-L-lysine or polyethylenimine, it has to be ranked
only moderate. Nevertheless, the controls, SII-10 (a CLS not able to immobilize
plasmid DNA in the agarose gel) and SII-17 (the pure cationic modifier unable to
stably bind DNA), were not able to retransfect Cos-1 cells. In all cases, the β-galac-
tosidase activity was within the background range.
Another important aspect of transfection agents, especially for nonviral systems,
was the efficiency/toxicity ratio. The CLS described by Olbrich et al. [19] showed
only moderate transfection efficiencies compared with the established polymers

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156 Lipospheres in Drug Targets and Delivery

poly-L-lysine and polyethylenimine. However, this transfection activity was accom-

panied only by a low degree of cytotoxicity. A number of possibilities exist for opti-
mizing the activity of the system, including choice of matrix lipid, size, and modifier.

8.6 CONCLUSIONS
During recent years, solid lipid nanoparticles have attracted increasing attention.
However, only a few studies that have been aimed to obtain innovative nonviral
transfection systems for gene therapy have been performed on CLS. In the last
decade, the efficiency of nonviral transfection systems has improved several orders
of magnitude. Although as yet none has proven to be effective enough in vivo, new
developments are still ongoing. Among nonviral transfection systems, colloidal carriers
such as CLS represent an alternative drug delivery system to emulsions, liposomes,
and polymeric particles. From the analysis of the results reported in this chapter, it
emerges that CLS may provide a new, efficient means for the immediate intracellular
delivery of therapeutic macromolecules. Nevertheless, caution is warranted for catio-
nic carriers, which may accentuate cytotoxic effects in the phagocytic cells.

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