www.elsevier.com/locate/jconrel

Journal of Controlled Releas

Inulin is a promising cryo- and lyoprotectant for

PEGylated lipoplexes

W.L.J. Hinrichsa,*, N.N. Sandersb, S.C. De Smedtb, J. Demeesterb, H.W. Frijlinka

aDepartment of Pharmaceutical Technology and Biopharmacy, University of Groningen, Antonius Deusinglaan 1,

9713 AV Groningen, The NetherlandsbLaboratory of General Biochemistry and Physical Pharmacy, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium

Received 2 November 2004; accepted 16 December 2004

Available online 15 January 2005

Abstract

The aim of this study was to investigate whether the oligosaccharides dextran and inulin are able to prevent aggregation of

lipoplexes based on 1,2-dioleoyl-3-trimethylammonium-propane and dioleoylphosphatidyl-ethanolamine with and without

distearoylphosphatidylethanolamine-polyethyleneglycol (PEGylated and nonPEGylated lipoplexes, respectively) during

storage. The lipoplexes, dispersed in the oligosaccharide solution were frozen and subsequently stored at subzero temperature

or freeze dried and subsequently stored at 37 8C. When lipoplexes in frozen dispersions were stored below the glass transition

temperature of the maximally freeze concentrated fraction (Tg’) of the oligosaccharide solutions severe aggregation of the

nonPEGylated lipoplexes was prevented for 3 months by both inulin and dextran. However, while dextran failed to stabilize the

frozen PEGylated lipoplexes (as in most cases full aggregation occurred in short time) inulin successfully protected them

against aggregation. Compared to dextran, inulin was also a superior lyoprotectant of PEGylated lipoplexes: during freeze

drying and subsequent storage at 37 8C of the dried powders for 3 months the PEGylated lipoplexes maintained their original

size when dispersed in inulin matrices while in dextran matrices they fully aggregated in most cases. It is hypothesized that the

aggregation of the PEGylated lipoplexes in dextran solutions is caused by the well known incompatibility between dextrans and

PEG. This is further supported by the observation that inulins and PEG are compatible. It is concluded that oligosaccharides can

prevent severe aggregation of nonPEGylated lipoplexes. The same holds for PEGylated lipoplexes provided that the

oligosaccharide is compatible with PEG. Finally, this work also shows that the higher Tg’ of oligosaccharides makes them more

versatile cryoprotectants than disaccharides like sucrose or trehalose as the frozen dispersions can be stored at higher

temperatures for prolonged periods of time. Furthermore, it is proposed that oligosaccharides are also more versatile

lyoprotectants than the disaccharides because they can be exposed to higher relative humidities without passing the glass

transition temperature.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Freeze drying; Inulin; Lipoplex; Particle size; Stability

0168-3659/$ - s

doi:10.1016/j.jco

* Correspondi

E-mail addr

e 103 (2005) 465–479

ee front matter D 2004 Elsevier B.V. All rights reserved.

nrel.2004.12.011

ng author. Tel.: +31 50 363 3134; fax: +31 50 363 2500.

ess: W.L.J.Hinrichs@farm.rug.nl (W.L.J. Hinrichs).

W.L.J. Hinrichs et al. / Journal of Controlled Release 103 (2005) 465–479466

1. Introduction

The elucidation of the human genome has boosted

the idea of using DNA as a therapeutic agent [1–3]. To

act as a therapeutic agent, DNA has to reach the

nucleus of the target cell. However, when DNA as

such is contacted with cells spontaneous introduction

of DNA in the nucleus hardly occurs. This limited

transfection efficiency can be partially ascribed to the

large hydrodynamic diameter and the negative charge

of DNA [4]. Moreover, considering in-vivo admin-

istration, many substances such as DNases are present

in the body, which metabolize the foreign DNA [4–6].

One way to solve these problems is to complex DNA

with cationic liposomes [7,8]. These lipid based gene

delivery systems, also known as lipoplexes, are

relatively small, neutral, or positively charged, and

protect DNA against degradation.

Lipoplexes are prepared in an aqueous environ-

ment, thus obtaining an aqueous dispersion. A major

drawback of these dispersions is their thermodynamic

driven tendency to lower their interfacial surface area

with the environment and thus to aggregate. Aggre-

gation strongly diminishes transfection [9]. This

physical instability is even more apparent when

during storage or shipping the dispersion is subjected

to freeze thaw cycles. At subzero temperatures, water

crystallizes through which the concentration of

remaining dispersion is strongly increased. By this

so-called freeze concentration the lipoplex particles

are forced towards each other through which aggre-

gation is facilitated.

Therefore, it would be advantageous to find a way

to prevent or at least slow down aggregation during

freezing. It would even be more advantageous to

prevent aggregation during freeze drying because,

once in a stable dry state, increased shelf lives can be

expected even at ambient temperatures. Having the

lipoplexes in the dry state would also offer the

possibility to develop dosage forms which are not

possible with aqueous dispersions e.g. tablets for oral

administration or dry powder formulations for pulmo-

nary delivery [10–12].

It is well known that sugars can stabilize a large

variety of unstable drug substances during freeze

thawing and freeze drying [13–16]. Furthermore, it

has been reported that lipoplexes can be stabilized by

sugars [17,18]. Stabilization has been explained by the

particle isolation hypothesis [19] and the vitrification

theory [20]. The particle isolation hypothesis refers to

the formation of a sugar matrix which acts as a

physical barrier between the lipoplex particles. The

vitrification theory refers to the formation of a glassy

sugar matrix in which diffusion on a realistic time

scale is inhibited. Both the physical barrier and lack of

translational movement prevent aggregation. To

ensure vitrification the sample temperature should be

below its glass transition temperature (Tg). Therefore,

for storage of aqueous dispersions at subzero temper-

atures or for freeze drying, sugars with a high glass

transition temperature of the maximally freeze con-

centrated fraction (Tg’) are preferred. Additionally,

also a high Tg (of the dry sugar) is beneficial for the

subsequent storage of powders obtained after freeze

drying. Storing the sample below the Tg is on the one

hand essential for vitrification. On the other hand, it is

important to maintain the particles isolated as above

the Tg the sugar is in the rubbery phase and

crystallization can occur. This crystallization will

force the particles towards each other. Therefore, it

is favorable when the sugar has a low tendency for

crystallization in case the Tg is (accidentally) passed.

A review of the literature reveals that especially small

sugars like the disaccharides sucrose and trehalose can

be successfully used for stabilization of lipoplexes

[19,21,22]. In contrast, polysaccharides like hydrox-

yethyl starch [19,21] and high molecular weight

dextrans [22] were found to act as poor stabilizers.

Based on the above described stabilization mecha-

nisms, this is rather unexpected because polysacchar-

ides usually have excellent physico-chemical

characteristics: besides a high Tg’ and Tg, they also

show a low tendency for crystallization compared

with disaccharides [23]. It has been hypothesized that

polysaccharides are too bulky to effectively incorpo-

rate the drug particles [21], i.e. the large molecules are

not able to accommodate to the surface of the lipoplex

and no tight coating can be obtained. This suggests

that oligosaccharides may be superior stabilizers since

they combine the advantageous properties of both

small saccharides and polysaccharides for optimal

stabilization of lipoplexes i.e. they have a high Tg’

and Tg, and low tendency for crystallization but are

small or flexible enough to effectively incorporate

lipoplexes. Indeed, in a preliminary study we have

found that the transfection efficiency of lipoplexes

W.L.J. Hinrichs et al. / Journal of Controlled Release 103 (2005) 465–479 467

(based on 1,2-dioleoyl-3-trimethylammonium-pro-

pane/dioleoylphosphatidyl-ethanolamine liposomes

and plasmid DNA encoding for alkaline phosphatase)

freeze dried without additives was fully lost while

substantial transfection was observed when they were

freeze dried in the presence of oligosaccharides.

The aim of this study was to evaluate the effects

of oligosaccharides on the particle size and zeta

potential of lipoplexes during subzero temperature

storage and freeze drying followed by storage. It has

been reported that maintenance of particle size (and

zeta potential) is not the only determinant for

preservation of biological activity [20,24]. Treatment

may also induce structural changes in the tertiary

structure of plasmid which may affect the biological

activity but not particle size. However, determination

of particle size can be used as a screening method

because when severe aggregation of the lipoplexes

occurs, the transfection efficiency will always be

strongly diminished.

Four oligosaccharides were evaluated: inulin 1.8

and 4 kDa and dextran 1.5 and 5 kDa. Sucrose and

trehalose were used as positive controls and no sugar

and dextran 40 kDa as negative controls. Stability of

lipoplexes prepared with nonPEGylated and PEGy-

lated liposomes was considered. It can be envisaged

that during freeze thawing and freeze drying the

degree of compatibility of the oligosaccharide and

PEG will affect the stabilization of PEGylated lip-

oplexes by the oligosaccharide [25]. Therefore, in the

present work the compatibility of the oligosaccharides

with PEG was also studied and related to the extent

oligosaccharides prevent PEGylated lipoplexes from

aggregation.

2. Materials and methods

2.1. Materials

The lipids, 1,2-dioleoyl-3-trimethylammonium-

propane (DOTAP), dioleoylphosphatidyl-ethanol-

amine (DOPE), and distearoylphosphatidyletha-

nolamine-polyethyleneglycol (DSPE-PEG) with a

molecular weight of polyethylene glycol 2000 were

obtained from Avanti Polar Lipids (Alabaster, AL,

USA). Dextran 1.5 kDa and PEG 6 kDa were obtained

from Fluka (Zwijndrecht, the Netherlands). Dextran 5

and 40 kDa were purchased from Dextran Products

Limited (Scarborough, Ontario, Canada). Inulin 1.8

and 4 kDa (inulin types HD001111 and TEX!803,

respectively) were gifts of Sensus, Roosendaal, the

Netherlands. Plasmid DNA (pDNA) of 5803 base

pairs encoding the reporter gene secretory alkaline

phosphatase was used. The pDNA was amplified in

Escherichia coli, purified and dissolved in 20 mM

Hepes at pH 7.4 as previously described [9]. All other

chemicals were of analytical grade and purchased

from commercial suppliers.

2.2. Methods

2.2.1. Preparation of liposomes

Unilamellar nonPEGylated liposomes composed of

DOTAP/DOPE (molar ratio 1:1) and PEGylated

liposomes composed of DOTAP/DOPE/DSPE-PEG

(molar ratio 1:1:0.08) were prepared by the thin film-

extrusion method as reported previously [9]. Briefly,

appropriate amounts of lipids were dissolved in

chloroform in a round bottom flask. The solvent

was removed by rotary evaporation at 40 8C followed

by purging the flask with dry nitrogen for 30 min at

room temperature. The lipids were hydrated by adding

20 mM Hepes pH 7.4. Glass beads were added and

swirled around to facilitate detachment of the lipid

layer from the wall of the flask. Subsequently, the

formed vesicle dispersion was stored for 1 day at 4 8Cafter which it was extruded 11 times through two

stacked 100 nm polycarbonate membrane filters

(Whatman, Brentfort, UK) at room temperature.

2.2.2. Preparation of lipoplexes

Lipoplexes were prepared at +/� charge ratio of 4

(i.e. weight ratio DOTAP/pDNA of 8.5). A pDNA

solution of plasmid (in 20 mM Hepes at pH 7.4) was

added to a dispersion of liposomes (compositions see

above) and then vortexed for a few seconds as

described previously [9]. The final pDNA concen-

tration was 126 Ag/ml and the final lipid concentration

was 2.21 mg/ml (DOTAP/DOPE) or 2.46 mg/ml

(DOTAP/DOPE/DSPE-PEG).

2.2.3. Freeze thawing

From the lipoplex dispersions, as described above,

50 Al was added to 1.5 ml eppendorf cups and

subsequently either 50 Al of a sugar solution in water

W.L.J. Hinrichs et al. / Journal of Controlled Release 103 (2005) 465–479468

(12.6 wt.%; thus final weight ratio sugar/plasmid was

1000) or 50 Al of pure water was added and shortly

vortexed. The resulting dispersions were frozen by

placing the samples in a refrigerator of �20 8C(sample temperature was decreased to below �15 8Cwithin 20 min) or �85 8C (sample temperature was

decreased to below �50 8C within 10 min). After 6

days, 1, or 3 months, the samples were rapidly thawed

by adding 0.9 ml Hepes (20 mM at pH 7.4) of room

temperature and then analyzed.

2.2.4. Freeze drying

From the lipoplex dispersions, as described above,

50 Al was added to 4 ml glass vials. Subsequently,

either 50 Al of sugar solution in water (12.6 wt.%; thus

final weight ratio sugar/plasmid was 1000) or 50 Al ofpure water was added and shortly vortexed. The

resulting dispersions were frozen by placing the glass

vials in a refrigerator of �85 8C (sample temperature

was decreased to below �50 8C within 10 min). After

16–24 h, the glass vials were placed in the freeze

dryer (Amsco-Finn Aqua GT4 freeze dryer) and

subsequently lyophilized at shelf temperature of

�35 8C, a pressure of 0.9 mBar for 2 h, and a

condensor temperature of �60 8C. Then, the shelf

temperature was raised to �15 8C while keeping the

pressure the same. After 13 h the pressure was

decreased to 0.15 mBar while the temperature was

gradually raised to 10 8C during 9 h. Subsequently,

the samples were removed from the freeze dryer.

Thus, freeze dried lipoplexes were either immediately

rehydrated with 1 ml Hepes (20 mM at pH 7.4) and

then analyzed or first placed in an oven at 37 8C for 6

days, 1, or 3 months. Furthermore, sugar solutions

(6.3 wt.%) in water and in Hepes (10 mM at pH 7.4)

were freeze dried according to a procedure described

before [23].

2.2.5. Determination of glass transition temperatures

Glass transition temperatures were determined by

calorimetric measurements using a 2920 differential

scanning calorimeter (DSC, TA instruments, Gent,

Belgium). The Tgs’ of sugar solutions (6.3 wt.%) in

water or in Hepes (10 mM at pH 7.4), and 10–200

mM Hepes buffer (at pH 7.4) were determined via the

following procedure. Solutions (40–50 Al) were

cooled to �60 8C with a cooling rate of 10 8C/min.

Subsequently, the samples were heated to 40 8C with a

rate of 20 8C/min. During these measurements, the

sample cell was purged with helium at a flow rate of

35 ml/min. The midpoint of the transition was taken

as the Tg’. Samples were measured at least in

duplicate.

Also the Tg of sugar glasses (with and without

Hepes) and of pure Hepes was measured. The sugar

glasses were prepared by freeze drying sugar solutions

(6.3 wt.%) in water or in Hepes (10 mM at pH 7.4).

The samples (5–10 mg) were preheated for 30 min at

40 8C (sucrose and trehalose containing samples) or

15 min at 90 8C (inulin or dextran containing samples)

subsequently cooled to 20 8C and then heated to 300

8C at a rate of 20 8C/min. The preheating step was

performed to remove moisture that was absorbed

during sample preparation. The Tg of pure Hepes was

determined as follows: crystalline Hepes (5–10 mg)

was heated to 250 8C (melting temperature of Hepes is

238 8C) and then rapidly cooled to 0 8C. Subse-

quently, the sample was heated at a rate of 20 8C to

100 8C. During the measurements, the sample cell

was purged with nitrogen at a flow rate of 35 ml/min.

The midpoint of the transition was taken as the Tg. All

samples were measured at least in duplicate.

2.2.6. Compatibility between oligosaccharides and

PEG

The compatibility between the oligosaccharides

and PEG was determined by two different procedures.

In the first procedure, which was adopted from Izutsu

et al. [26], the effect of oligosaccharides on the heat of

fusion of PEG hydrate in a frozen solution is

determined. When the compatibility between an

oligosaccharide and PEG increases, they will better

mix at subzero temperatures. Consequently, the

oligosaccharide will increasingly disturb crystalliza-

tion of PEG and thus the heat of fusion of PEG

hydrate will decrease. The heat of fusion was

measured as follows. Aqueous solutions of PEG 6

kDa (2 wt.%) and inulin 1.8 and 4 kDa or dextran 1.5,

5, or 40 kDa of various concentrations were eval-

uated. Trehalose and sucrose were used as controls.

DSC scans were performed in the DSC apparatus

described above. Solutions (40–50 AL) were cooled to

�60 8C with a cooling rate of 10 8C/min. Sub-

sequently, the samples were heated to 40 8C with a

rate of 20 8C/min. The heat of fusion of PEG 6 kDa

hydrate (at �14.5 8C [26]) was related to that of an

W.L.J. Hinrichs et al. / Journal of Controlled Release 103 (2005) 465–479 469

aqueous solution of PEG 6 kDa (2 wt.%) without

additives. During the measurements, the sample cell

was purged with helium at a flow rate of 35 ml/min.

Samples were measured at least in duplicate.

In the second procedure to evaluate compatibility,

freeze dried powders were considered. Similar to what

has been described above, when a solution of PEG

and an oligosaccharide is freeze dried, crystallization

of PEG (anhydrate) is disturbed depending on the

degree of compatibility of both constituents. Again

this can be quantified by measuring heat of fusion of

crystalline PEG. Therefore, aqueous solutions of PEG

6 kDa (2 wt.%) and inulin 1.8 and 4 kDa or dextran

1.5, 5, or 40 kDa (all 5 wt.%) were freeze dried after

which the heat of fusion PEG 6 kDa anhydrate (at 60

8C) was related to that of a freeze dried aqueous

solution of PEG 6 kDa (2 wt.%) without additives.

The calorimetric measurements were conducted on the

same DSC apparatus as described above. The samples

(5–10 mg) were equilibrated for 2 min at 0 8C and

than heated to 100 8C with rate of 20 8C/min. The heat

of fusion of PEG 6 kDa anhydrate was related to that

of freeze dried aqueous solution of PEG 6 kDa (2

wt.%) without additives. During the measurements,

the sample cell was purged with nitrogen at a flow rate

of 35 ml/min. Samples were measured at least in

duplicate.

2.2.7. Particle size measurements

The Z-average particle size was determined by

dynamic laser scattering (DLS) using a Malvern 4700

system equipped with a helium-neon laser (Malvern,

Worcestershire, UK). For the data analysis, the

viscosity and refractive index of water were used. In

control experiments it was found that the presence of

sugars at concentrations used in this study did not

affect the results of the measurements (viscosity and

refractive index of the dispersing medium did not

change significantly). The performance of the instru-

ment was checked with polystyrene standard spheres

with a particle size of 220F6 nm (Duke Scientific,

Palo Alto, CA, USA). As a measure of particle size

distribution, the system reports a polydispersity index

(pd). This index ranges from 0 for a monodisperse

sample up to 1 for an entirely polydisperse sample.

All formulations were measured at least in duplicate

and each measurement consisted of five runs. In this

study a number of different batches of lipoplexes were

used which showed some variation in particle size.

Therefore, the size of the lipoplexes after a particular

treatment was expressed as percentage of the size of

the lipoplex of the same batch before treatment.

Samples were considered as bfully aggregatedQ whenone of the three following observations was made: (1)

large aggregates visible by eye, (2) a pd of 1 in more

than two out of the five runs, and (3) an increase in the

Z-average particle size by at least a factor three in

combination with an average pd higher than 0.9.

2.2.8. Zeta potential measurements

The zeta potential was measured by determining

the electrophoretic mobility using a Malvern zeta-

sizer 2000 unit (Malvern, Worcestershire, UK). The

zeta potential was calculated with the Smoluchowski

equation: f=12.8�le in which f is the zeta potential

and le is the electrophoretic mobility. The perform-

ance of the instrument was verified using a dispersion

of carboxyl modified polystyrene nanospheres with a

zeta potential of �50F5 mV (DTS5050, Malvern,

Malvern, UK). In control experiments it was found

that the presence of sugars at concentrations used in

this study did not affect the results of the measure-

ments (viscosity and refractive index of the dispersing

medium did not change significantly). In this study a

number of different batches of lipoplexes were used

which showed some variation in zeta potential.

Therefore, the zeta potential of lipoplexes after a

particular treatment was expressed as percentage of

the zeta potential of the lipoplex of the same batch

before treatment. All formulations were measured at

least in duplicate and each measurement consisted of

five runs. The zeta potential of samples that were

considered as fully aggregated (see above) was not

measured.

3. Results and discussion

3.1. Characteristics of starting materials

3.1.1. Glass transition temperatures

Aqueous sugar solutions and the corresponding

freeze dried sugars were analyzed by DSC (see Table

1). For sucrose and trehalose, Tgs’ of �30.7 and

�28.9 8C, respectively, were found which agree well

with literature values [27]. Substantially higher values

Table 1

Tgs’ (in 8C) of sugars solutions (6.3 wt.%) in water and in Hepes (10 mM at pH 7.4) and Tgs of the corresponding fried dried powders

Tg’ of the solution

in water

Tg’ of the solution

in Hepes

Tg of the

powder (water)

Tg of the

powder (Hepes)

Sucrose �30.7F0.1 �31.6F0.3 77.2F0.6 77.7F0.8

Trehalose �28.9F0.2 �30.4F0.1 120.9F0.5 119.7F0.1

Dextran 1.5 kDa �21.8F0.0 �23.2F0.2 152.5F0.4 147.0F0.1

Dextran 5 kDa �17.1F0.1 �18.5F 0.0 175.6F3.1 171.9F1.1

Dextran 40 kDa �11.9F0.1 �13.6F0.1 226.6F0.7 214.0F0.1

Inulin 1.8 kDa �21.2F0.2 �22.6F0.1 132.9F0.1 130.7F0.6

Inulin 4 kDa �17.8F0.1 �19.3F0.2 156.9F0.9 151.9F1.2

Pure Hepes b�60 8C 52.6F1.5

Table 2

Particle size and zeta potential of liposomes and lipoplexes

Particle

size

in nm

pd Zeta

potential

in mV

Liposome DOTAP/DOPE 124F5 0.12F0.03 43F9

Liposome DOTAP/DOPE/

DSPE-PEG

124F8 0.11F0.02 24F8

Lipoplex DOTAP/DOPE 227F24 0.16F0.04 47F5

Lipoplex DOTAP/DOPE/

DSPE-PEG

195F28 0.13F0.08 14F4

W.L.J. Hinrichs et al. / Journal of Controlled Release 103 (2005) 465–479470

were found for inulin as well as for dextran.

Furthermore, in accordance with the Fox–Flory theory

[28–31], the Tg’ increased with molecular weight for

both inulin and dextran. As described above, lip-

oplexes were dispersed in sugar solutions (6.3 wt.%)

containing 10 mM Hepes at pH 7.4. Therefore, the

Tgs’ of sugar solutions (6.3 wt.%) in Hepes (10 mM

at pH 7.4) were also measured. As can be seen in

Table 1, the presence of Hepes lowers the Tg’ of the

sugars in all cases. A change in the Tg’ can be

ascribed to the incorporation of buffer species in the

maximally freeze concentrated fraction [32]. A

decrease of the Tg’ indicates that the Tg’ of a pure

buffer solution is lower than that of the pure sugars

solutions. Indeed DSC scans from �60 8C to 40 8C of

Hepes solutions with concentrations up to 200 mM

showed no Tg’ indicating that the Tg’ is lower than

�60 8C.The Tg of the freeze dried sugars showed a similar

trend (see Table 1): the Tg increased in the order

sucrose, trehalose followed by both oligosaccharides

inulin and dextran. Again in accordance with the Fox–

Flory theory [29,31], the Tg of the oligosaccharides

increased with molecular weight. The values found

correspond well with literature values [23,30,33,34].

Also the Tgs of freeze dried sugar solutions (6.3

wt.%) prepared in Hepes (10 mM at pH 7.4) were

determined. They were lower than the corresponding

freeze dried sugar solutions prepared in pure water

(except for sucrose). Furthermore, a Tg of pure Hepes

was found to be 52.6 8C. These results indicate that

Hepes forms homogeneous mixtures with each of the

sugars which is in agreement with the observation that

Hepes is incorporated in the maximally freeze

concentrated fractions.

3.1.2. Size and zeta potential of the liposomes

In this study eight batches of each liposome

composition (DOTAP/DOPE and DOTA/DOPE/

DSPE-PEG) were independently prepared. Table 2

shows that the particle size was highly reproducible

and averaged at about 125 nm. The liposome

dispersions also had a low polydispersity as the pd

was 0.11–0.12 (see Table 2). The zeta potential of the

DOTAP/DOPE liposomes (43 mV) was substantially

higher than that of the DOTA/DOPE/DSPE-PEG

liposomes (24 mV). The decrease of the zeta potential

by the incorporation of DSPE-PEG was expected and

can be ascribed to two different mechanisms. First,

DSPE-PEG contains a negative charge decreasing the

zeta potential. Secondly, the PEG chains increased the

distance between the surface of the liposome and the

plane of shear through which the positive charge is

increasingly neutralized by ions present in the

dispersion medium [35].

3.1.3. Size and zeta potential of the lipoplexes

Lipoplexes were prepared at a +/� charge ratio of

four by complexing the liposomes with pDNA at a

-60 -40 -20 0 20 40

Temperature (°C)

Hea

t fl

ow

(ex

oth

erm

up

)

CrystallizationPEG hydrate Melting

PEG hydrate

Melting H2O

Fig. 1. DSC scan of 2 wt.% solution of PEG 6 kDa in water.

W.L.J. Hinrichs et al. / Journal of Controlled Release 103 (2005) 465–479 471

lipid/pDNA weight ratio of 17.5. In this study fifteen

batches of each type of lipoplexes (i.e. nonPEGylated

and PEGylated lipoplexes) were independently pre-

pared. As can be seen in Table 2, particle size analysis

showed some interbatch variation. Upon complex-

ation the liposomes increased in size. Such an increase

is usually found and indicates that the lipoplex

contains more than one of the original liposomes

[36]. The zeta potential of the nonPEGylated lip-

oplexes did not differ significantly from the zeta

potential of the nonPEGylated liposomes. This indi-

cates, as also observed by others, that the plasmid

DNA became sandwiched between two lipid

(bi)layers [9,37]. On the other hand, the slight

decrease in the zeta potential of the PEGylated

liposomes upon DNA complexation indicates that at

least part of the plasmid DNA becomes bound to the

surface of the lipoplexes. Finally, gel electrophoresis

did not reveal free plasmid DNA in both the

nonPEGylated and PEGylated lipoplexes (data not

shown).

3.2. Compatibility between oligosaccharides and

PEG

Besides the Tgs’ and Tgs of the sugar solutions and

freeze dried sugar solutions, respectively, the stabili-

zation of especially PEGylated lipoplexes may also be

dependent on the compatibility of the sugar and PEG.

The compatibility between the sugars and PEG was

characterized by measuring the effect of sugars on the

heat of fusion of PEG hydrate in frozen solutions and

the heat of fusion of PEG anhydrate in freeze dried

solutions.

It is well known that dextran and PEG are

incompatible in an aqueous solution i.e. when a

concentrated aqueous dextran solution is added to a

concentrated aqueous PEG solution, the system forms

two phases, one rich in dextran and the other rich in

PEG [38,39]. At low concentrations, dextran and PEG

can form homogeneous solutions in water. However,

when such a solution is frozen the concentration of

both solutes in the freeze concentrated fraction is

strongly increased (by ice formation) which may

result in phase separation [26,40–42]. The degree of

phase separation can be quantified as follows. When a

solution of PEG in water is rapidly cooled and then

heated in a DSC pan, the following observations can

be made. At around �45 8C an exothermic peak is

observed followed by two endothermic peaks around

�14.5 and 4 8C (see Fig. 1). These thermal events can

be ascribed to the crystallization of PEG-hydrate and

the fusion of PEG-hydrate and the fusion of water,

respectively. When an oligosaccharide, which is

compatible with PEG, is added to the solution and

the same DSC scan is performed, both solutes remain

mixed during freeze concentration. As a result, the

oligosaccharide is able to disturb the formation of

PEG-hydrate upon heating. Consequently, both the

heat of crystallization at �45 8C and the heat of fusion

at �15 8C will decrease. These changes will be more

pronounced with increasing oligosaccharide concen-

tration. In contrast, when the added oligosaccharide is

incompatible with PEG, the freeze concentrated

fraction will separate in two different phases, one

rich in PEG and the other one rich in oligosaccharide.

Being predominantly present in another phase, the

oligosaccharide is unable to disturb the formation of

PEG-hydrate crystals. Consequently, the heat of

crystallization at �45 8C as well as the heat of fusion

at �15 8C will not decrease or decrease to a lesser

extent.

Aqueous solutions of 2 wt.% PEG 6 kDa and

sugars of various concentrations were evaluated.

Sucrose and trehalose were used as controls because

it has been reported that these disaccharides are

compatible with PEG [26]. The results confirm this

compatibility as crystallization of PEG was fully

inhibited at a sucrose or trehalose concentration of

already 2.5 wt.% (see Fig. 2). As expected the results

show that dextran and PEG are highly incompatible

as the heat of fusion of PEG hydrate remained

substantial at dextran concentrations up to 10 wt.%.

In contrast, both inulins strongly disturbed crystal-

020406080

100120140

0 2 4 6 8 10 12

[sugar] in wt-%

Hea

t o

f fu

sio

n o

fP

EG

hyd

rate

(%

)

Fig. 2. Effect of sugars on the crystallization of 2 wt.% PEG 6 kDa

in frozen solutions. Sucrose (o), trehalose (5), inulin 1.8 kDa (4),

inulin 4 kDa (w), dextran 1.5 kDa (.), dextran 5 kDa (n), and

dextran 40 kDa (E).

020406080

100120

inulin

1.8

kDa

inulin

4 kD

a

dextr

an 1

.5 kD

a

dextr

an 5

kDa

dextr

an 4

0 kD

aCry

stal

linit

y P

EG

(%

)

Fig. 3. Effect of sugars on the degree of crystallinity of PEG 6 kDa

after freeze drying of a solution of 5 wt.% sugar and 2 wt.% PEG 6

kDa as calculated from the heat of fusion measured by DSC.

O

CH2OH

HH

OH

OH

H

H

OH

HO

O

OH

OHCH2OH

H

H

HCH2

O

O

OH

OH

CH2OH

H

H

HCH2

O

O

OH

OHCH2OH

H

H

HCH2OH

n

C

C

C

Fig. 4. Structure of inulin with emphasis on its PEG-like backbone

W.L.J. Hinrichs et al. / Journal of Controlled Release 103 (2005) 465–479472

lization of PEG. In fact, the extent of crystallization

inhibition by inulins and disaccharides was compa-

rable (see Fig. 2), indicating that inulin and PEG are

fully compatible.

In the second procedure to evaluate compatibility,

freeze dried samples were considered. When using

most lyophilizators, PEG crystallizes (as an anhydrate)

during freeze drying. This is due to the fact that the

sample temperature is usually higher than the Tg’ of

PEG (�85 to �65 8C, depending on its molecular

weight [43]). Crystallization is manifested by an

endothermic (melting) peak in a DSC scan at 65–70

8C or lower depending on the molecular weight of

PEG. Similar to what has been described above, when a

solution of PEG and an oligosaccharide is freeze dried,

crystallization is disturbed depending on the degree of

compatibility of both constituents. Again this can be

quantified by measuring heat of fusion of crystalline

PEG. The heat of fusion of PEG of freeze dried aqueous

solutions of PEG 6 kDa (2 wt.%) and dextran or inulin

(5 wt.%) was determined and related to the heat of

fusion of a freeze dried aqueous solution of PEG 6 kDa

(2 wt.%) without additives. The incompatibility of

dextran and PEG was evident as the crystallinity of

PEG was 80–100% compared to PEG freeze dried

without additives (see Fig. 3). Furthermore, these

experiments showed that inulin and PEG are compat-

ible as the crystallinity of PEG was below 20%

compared to PEG freeze dried without additives.

As the phase behavior of polymer mixtures is a

delicate issue, we can only hypothesize at this point,

why in contrast to dextran, inulin is compatible with

PEG. One way to approach this question is to

compare their structural characteristics. It is well

known that compatibility increases when oligomers

are more alike [38]. When the structures of inulin and

PEG are considered, two characteristics attract atten-

tion because they point to similarities. First of all, both

oligomers are highly flexible when dissolved in water

[44]. Although many factors play a role, in many

cases polymers are compatible when they have

comparable flexibilities [38]. Secondly, the molecular

structure of inulin closely resembles PEG since it

consists of a PEG backbone with sugar moieties as

side groups (see Fig. 4).

3.3. Stability of lipoplexes

3.3.1. Effects of storage at �20 8C on particle size of

lipoplexes

The effects of various sugars on the aggregation

behavior of nonPEGylated and PEGylated lipoplexes

upon storing the frozen dispersions at �20 8C were

evaluated. This temperature was chosen because

.

W.L.J. Hinrichs et al. / Journal of Controlled Release 103 (2005) 465–479 473

freezers at �20 8C belong to standard laboratory

equipment. The results are summarized in Fig. 5a and

b. Without sugars nonPEGylated lipoplexes severely

aggregated already within 6 days of storage indicating

the necessity of using a cryoprotectant (see Fig. 5a). In

the presence of the sugars investigated in this study,

no matter which one, severe aggregation did not occur

within 6 days of storage as the particle size only

increased to 125–150% of its original value. However,

at prolonged storage (1 or 3 months) sucrose and

trehalose were not able to prevent severe aggregation.

These results can be explained by the Tgs’ of these

sugars (see Table 1), which are much lower than the

storage temperature. During the lowering of the

temperature to �20 8C substantial freeze concentra-

tion will occur. However, at �20 8C the concentrated

solution is not yet in its glassy but in its rubbery state.

Because the lipoplexes are closer in each other’s

vicinity (the volume of the freeze concentrated

fraction is smaller) and because diffusion is possible,

aggregation is facilitated. In addition, above the Tg’

sugars can crystallize through which the lipoplexes

0

100

200

300

a

b

no e

xcipi

ent

sucr

ose

treha

lose

inulin

1.8

kDa

inulin

4 kD

a

dextr

an 1

.5 kD

a

dextr

an 5

kDa

dextr

an 4

0 kD

apar

ticl

e si

ze (

%)

0

100

200

300

no e

xcipi

ent

sucr

ose

treha

lose

inulin

1.8

kDa

inulin

4 kD

a

dextr

an 1

.5 kD

a

dextr

an 5

kDa

dextr

an 4

0 kD

apar

ticl

e si

ze (

%)

Fig. 5. Particle size, as percentage of the value before storage, of

nonPEGylated (a) and PEGylated (b) lipoplexes after storage at

�20 8C of the frozen dispersions for 6 days (n), 1 month ( ), and 3

months ( ). Severely aggregated lipoplexes are indicated by bars

that pass the upper axis.

are forced together which also promotes aggregation.

According to Allison et al. [21] vitrification is not a

prerequisite for maintenance of particle size during

freeze thawing. However, they stored the frozen

dispersions only for one night at subzero temper-

atures. Therefore, these published results are not in

contradiction with our results since we also observed

no substantial aggregation of the lipoplexes dispersed

in sucrose or trehalose within 6 days but after

prolonged storage times.

When dispersed in a solution of dextran 1.5 kDa,

the nonPEGylated lipoplexes also substantially

increased in size after 1 and 3 months of storage.

Again this can be ascribed to the state of the freeze

concentrated sugar solution: the Tg’ of this solution is

a few degrees below storage temperature, however, a

glass transition concerns a gradual change in molec-

ular mobility over a certain temperature range rather

than an abrupt change at a fixed temperature. This

implies that at �20 8C, the mobility of the lipoplexes

may still be significant though reduced. In addition

dextran 1.5 kDa, being an oligosaccharide, will not

crystallize as easily as small sugars like sucrose and

trehalose. Considering these facts, aggregation is

indeed expected to occur but not at such a high rate

as observed with sucrose and trehalose.

Dispersed in an inulin 1.8 kDa solution the particle

size of nonPEGylated lipoplexes remained the same

after 3 months of storage. Similar to the dextran 1.5

kDa solution, the Tg’ of the inulin 1.8 kDa solution is

a few degrees below storage temperature. Possibly,

the glass transition of the inulin 1.8 kDa solution

covers a broader temperature range and translational

movements are already very restricted at �20 8C. Theparticle size of nonPEGylated lipoplexes frozen in the

presence of dextran 5 kDa, dextran 40 kDa, or inulin 4

kDa did not change after 3 months of storage at �20

8C. These solutions have Tgs’ above �20 8Cemphasizing the importance of storing the dispersions

in the glassy state. Remarkably, dextran 40 kDa,

which was due to its bulky nature considered as a

negative control [22], also exhibited sufficient cry-

oprotective properties to prevent aggregation.

Due to their steric repulsion effect PEGylated

particulate systems are generally more stable than

their nonPEGylated counterparts [18]. However, as

also found by Armstrong et al. [45], PEGylated

lipoplexes still require cryoprotectants as a PEGylated

0

50

100

150

200

no e

xcipi

ent

sucr

ose

treha

lose

inulin

1.8

kDa

inulin

4 kD

a

dextr

an 1

.5 kD

a

dextr

an 5

kDa

dextr

an 4

0 kD

a

par

ticl

e si

ze (

%)

0

50

100

150

200

no e

xcipi

ent

sucr

ose

treha

lose

inulin

1.8

kDa

inulin

4 kD

a

dextr

an 1

.5 kD

a

dextr

an 5

kDa

dextr

an 4

0 kD

a

par

ticl

e si

ze (

%)

(a)

(b)

Fig. 6. Particle size, as percentage of the value before storage, of

nonPEGylated (a) and PEGylated (b) lipoplexes after storage a

�85 8C of the frozen dispersions for 6 days (n), 1 month ( ), and 3

months ( ). Severely aggregated lipoplexes are indicated by bars

that pass the upper axis.

W.L.J. Hinrichs et al. / Journal of Controlled Release 103 (2005) 465–479474

lipoplex dispersion without excipients fully aggre-

gated within 6 days when stored at �20 8C (see Fig.

5b). Similar to the results on nonPEGylated lip-

oplexes, sucrose, trehalose, and both inulins were able

to prevent severe aggregation of PEGylated lipoplexes

stored at �20 8C for 6 days as the particle size

increased with only 25–50%. With dextran 1.5 kDa,

however, substantial aggregation occurred while with

both dextrans of higher molecular weight full aggre-

gation was observed within 6 days at �20 8C.Upon prolonged storage at �20 8C, PEGylated

lipoplexes dispersed in sucrose and trehalose strongly

increased in size but in a lower rate than non-

PEGylated lipoplexes under similar conditions. Again

the aggregation is due to the fact that Tgs’ of the

solutions are lower than the storage temperature while

the lower rate of aggregate formation can be ascribed

to steric stabilization caused by PEGylation. Dis-

persed in both inulin solutions, the size of PEGylated

lipoplexes was more or less maintained although some

aggregation was noticed with the inulin 4 kDa

containing samples after 3 months of storage at �20

8C. Given the relatively large SD, the anomalous

behavior of the inulin 4 kDa containing samples after

storage for 3 months is probably due to experimental

errors. Dispersed in the three dextran solutions,

PEGylated lipoplexes fully aggregated within 1 month

of storage at �20 8C.Clearly, in contrast to nonPEGylated lipoplexes,

Tg’ of the dispersion medium is not the only factor

that determines aggregation behavior of PEGylated

lipoplexes during subzero temperature storage. Inter-

estingly, these results can be related to the (in)com-

patibility of the oligosaccharides and PEG i.e. it seems

that oligosaccharides can prevent severe aggregation

of PEGylated lipoplexes on the condition that the

oligosaccharide is compatible with PEG. The follow-

ing mechanism can be proposed. When a dispersion

of PEGylated lipoplexes in a dextran solution is

frozen, the concentration of both lipoplex and dextran

is strongly increased by ice formation. As a result, the

dextran molecules tend to penetrate the PEG shell

surrounding the particles. However, as they are

incompatible, dextran will diffuse away from the

PEG shell thereby forcing the lipoplex particles

together leading to aggregation. Such a process can

be compared with the phase separation during freeze

concentration of dilute aqueous solutions of PEG and

dextran. It has been reported that the degree of

incompatibility increases with molecular weight

[39]. This is indeed reflected in the fact that the

cryoprotective activity of dextran 5 and 40 kDa is

worse than that of dextran 1.5 kDa. This hypothesis is

confirmed by the fact that inulins are compatible with

PEG and indeed these oligosaccharides behaved as

excellent cryoprotectants for PEGylated lipoplexes.

3.3.2. Effects of storage at �85 8C on particle size of

lipoplexes

The effects of various sugars on the aggregation

behavior of nonPEGylated and PEGylated lipoplexes

were also evaluated upon storing the dispersions at

�85 8C. The results are summarized in Fig. 6a and b.

It appeared that also upon storage at �85 8C,nonPEGylated lipoplexes dispersions fully aggregated

within 6 days when dispersed in pure buffer solution

(see Fig. 6a). In contrast, all sugars under inves-

tigation were able to prevent severe aggregation of

nonPEGylated lipoplexes during storage up to 3

months at �85 8C. Thus, in contrast to storage at

t

050

100150200250300350

no e

xcipi

ent

sucr

ose

treha

lose

inulin

1.8

kDa

inulin

4 kD

a

dextr

an 1

.5 kD

a

dextr

an 5

kDa

par

ticl

e si

ze (

%)

050

100150200250300350

no e

xcipi

ent

sucr

ose

treha

lose

inulin

1.8

kDa

inulin

4 kD

a

dextr

an 1

.5 kD

a

dextr

an 5

kDa

dextr

an 4

0 kD

a

par

ticl

e si

ze (

%)

(a)

(b)de

xtran

40

kDa

Fig. 7. Particle size, as percentage of the value before freeze drying

of nonPEGylated (a) and PEGylated (b) lipoplexes immediately

after freeze drying (n) and after freeze drying and subsequen

storage at 37 8C for 6 days ( ), 1 month ( ), and 3 months ( )

Severely aggregated lipoplexes are indicated by bars that pass the

upper axis.

W.L.J. Hinrichs et al. / Journal of Controlled Release 103 (2005) 465–479 475

�20 8C, also sucrose, trehalose, and dextran 1.5 kDa

behaved as excellent cryoprotectants under these

conditions. Clearly, this is due to the storage temper-

ature of �85 8C which is well below the Tgs’ of the

sugar solutions. Also noteworthy is the fact that

storage at �20 8C induced a particle size increase of

25–50% in the best cases while upon storage at �85

8C the particle size did not increase at all (except for

dispersions in dextran 5 kDa solutions where for

unknown reasons an increase of about 50% was

measured). The high stability of the dispersions

cannot only be explained by the fact that the storage

temperature was far below the Tgs’ of the sugar

solutions but also by the fact that the cooling rate

preceding storage was much higher (see Material and

methods). When slowly cooled to �20 8C, there is

apparently sufficient time available for moderate

diffusion and consequently limited aggregation during

freeze concentration, which is clearly not the case

when rapidly cooled to �85 8C.PEGylated lipoplexes dispersed in pure buffer

stored at �85 8C fully aggregated within 6 days,

similar to what has been found upon storage at �20

8C (see Fig. 6b). As expected, based on the results

found for nonPEGylated lipoplexes, the particle size

of PEGylated lipoplexes dispersed in sucrose, treha-

lose, and both inulins was fully maintained upon

storage at �85 8C up to 3 months. The same result

was also found for PEGylated lipoplexes dispersed in

dextran 1.5 kDa. However, rapid aggregation occurred

with dextrans of higher molecular weights. The results

are in line with those found at a storage temperature of

�20 8C (see Fig. 5b). As hypothesized above, the

failure of dextran as a cryoprotectant of PEGylated

lipoplexes is due to the incompatibility of dextran

with PEG. This incompatibility increases with the

molecular weight of dextran. Indeed, the stabilizing

capacity of dextran 1.5 kDa at �20 8C was not as poor

as that of the other, larger dextrans. Moreover,

stabilization by dextran 1.5 kDa at �85 8C can even

be considered as excellent. Apparently, a not too

strong incompatibility with PEG in combination with

a fast cooling rate and a low storage temperature

prevents extensive diffusion of dextran 1.5 kDa away

from the PEG shell around the particles. As a result

aggregation is prevented. Contrary to 1.5 kDa dextran,

the incompatibility of higher molecular weight dex-

trans with PEG is apparent such that the driving force

for diffusion of dextran from the PEG shell is very

large and can therefore proceed very fast. As a result

with these large dextrans aggregation is also induced

when the solutions are rapidly cooled to �85 8C.

3.3.3. Effects of freeze drying and subsequent storage

at 37 8C on particle size of lipoplexes

The effect of the sugars on the aggregation

behavior of lipoplexes during freeze drying and

subsequent storage at 37 8C was also evaluated. After

freeze drying in the presence of each of the sugars the

samples appeared as porous cakes and remained so

upon storage at 37 8C. As no collapse was observed, itcan be concluded that the samples were kept well

below their Tg’ and Tg during freeze drying and

storage, respectively. The results of particle size

measurements are summarized in Fig. 7a and b.

NonPEGylated lipoplexes without protectant showed

full aggregation immediately after freeze drying (see

Fig. 7a). Sucrose, trehalose, and both inulins were

,

t

.

0

50

100

150

sucr

ose

treha

lose

inulin

1.8

kDa

inulin

4 kD

a

dextr

an 1

.5 kD

a

dextr

an 5

kDa

dextr

an 4

0 kD

a

zeta

po

ten

tial

(%

)

0

50

100

150

sucr

ose

treha

lose

inulin

1.8

kDa

inulin

4 kD

a

dextr

an 1

.5 kD

a

dextr

an 5

kDa

dextr

an 4

0 kD

a

zeta

po

ten

tial

(%

)

(a)

(b)

Fig. 8. Zeta potential, as percentage of the value before freeze

drying, of nonPEGylated (a) and PEGylated (b) lipoplexes

immediately after freeze drying (n) and after freeze drying and

subsequent storage at 37 8C for 6 days ( ), 1 month ( ), and 3

months ( ). The zeta potentials of severely aggregated lipoplexes

(see Fig. 7) were not measured.

W.L.J. Hinrichs et al. / Journal of Controlled Release 103 (2005) 465–479476

able to prevent severe aggregation as the size of the

nonPEGylated lipoplexes only increased up to 125%

of their original size. It can be concluded that the

small but significant increase in particle size occurred

during dehydration and not during freezing because

during storage at �85 8C no such increase was

measured (see above). Furthermore, it was found that

these sugars also acted as excellent stabilizers in the

dry state as no further particle size increase was

observed upon storage at 37 8C up to 3 months. Also

the three dextrans were able to prevent aggregation of

the nonPEGylated lipoplexes during freeze drying.

However, upon storage at 37 8C, the results were

somewhat scattered as in some cases (dextran 1.5

kDa) aggregation occurred while in other cases

particle size was more or less maintained. Similar to

the results of the subzero temperature storage experi-

ments (see Figs. 5a and 6a), dextran 40 kDa appeared

to be a remarkable efficient stabilizer during freeze

drying and storage of nonPEGylated lipoplexes,

despite its bulky nature.

The PEGylated lipoplexes behaved similarly except

that dextran 1.5 kDa induced a twofold increase in

particle size during freeze drying which was main-

tained upon storage. However, similarly as observed

for PEGylated lipoplexes (see Figs. 5b and 6b), in

dextran 5 and 40 kDa the PEGylated lipoplexes fully

aggregated immediately after freeze drying.

Both inulins and in some cases dextrans seem thus

as effective as sucrose and trehalose to prevent

lipoplex aggregation during freeze drying and storage.

However, despite the observed equivalence in stabi-

lizing capacities, oligosaccharides are recommended

over disaccharides as lyoprotectants for lipoplex

dispersions especially when exposed to humidified

air. The freeze thaw experiments clearly indicate that

storage below the glass transition temperature (in this

case the Tg’) is essential for maintenance of particle

size. Extrapolating these results to freeze dried

samples it can be hypothesized that upon storage

above the Tg aggregation will also occur. In the

present study, the samples were stored well below the

Tg. However, when exposed to humidified air sugar

glasses will absorb water. The Tg will strongly

decrease upon water absorption and may drop to

below storage temperature. As a result aggregation is

likely to occur. In a previous study, the physical

stability of amorphous disaccharides and inulins was

compared upon exposure to various relative humid-

ities [23,46,47]. It was found that the hygroscopicity

of all these sugars was more or less the same. Because

the Tgs of the dry inulins were much higher than those

of the disaccharides, inulins could be exposed to much

higher relative humidities before the Tg was passed at

a fixed temperature. Similar results can be envisaged

with other oligosaccharides, e.g. dextrans.

3.3.4. Effects on zeta potential of lipoplexes

The zeta potential of all samples stored at �20 or

�85 8C for 3 months, which were not fully aggregated

were somewhat scattered and were 75–125% of the

original values (data not shown). However, because

there was no clear trend and given the accuracy of the

apparatus, changes induced by storage at subzero

temperatures were not considered as being significant.

The zeta potential of all samples, which were not

fully aggregated, was also not significantly affected by

freeze drying (see Fig. 8a and b). However, upon

storage the zeta potential gradually decreased with all

W.L.J. Hinrichs et al. / Journal of Controlled Release 103 (2005) 465–479 477

the tested lyoprotectants, except trehalose. Because (in

most cases) the particle size remained the same, it can

be suggested that a chemical change occurred at the

surface of the particles. The lipoplexes used in this

study contain the lipid DOPE. At pH 7.4, DOPE is a

Zwitter ion containing a negatively charged phosphate

group and a positively charged primary amine group. It

is well known that amine groups of proteins can react

with reducing groups of sugars to form a Schiff’s base.

Therefore, it can be expected that the amine group of

DOPE will also react with reducing groups of sugars.

This reaction would neutralize the positive charge of

the amine group of DOPE thereby reducing the

positive zeta potential of the particles. Dextrans consist

of a linear a-D-(1Y6) linked glucose oligomer bearing

a-D-(1Y3) linked glucose side chains and contain

therefore reducing end groups. Thus, dextrans allow

the formation of a Schiff’s base. This reaction may be

avoided by chemical modification of the reducing end

groups. Inulins consist of a linear h-D-(2Y1) linked

fructose oligomer ending with a a-D-(1Y2) glucopyr-

anose ring and should therefore contain no reducing

groups. However, commercially available inulins often

also contain reducing sugars like monosaccharides and

inulin species of which the glucose end group is

cleaved [23]. From Fig. 8a and b it can be observed

that the decrease in zeta potential proceeded more

rapidly with the inulin 1.8 kDa containing samples

than with the inulin 4 kDa containing samples. This

can be expected since inulin 1.8 kDa contains more

reducing groups than inulin 4 kDa (data not shown).

These contaminations may be avoided by improved

production and purification methods. Also sucrose is a

non-reducing sugar. However, prolonged storage at

elevated temperatures may induce hydrolysis of the

linkage between the glucose and fructose unit yielding

reducing groups [48]. Indeed, a decrease in zeta

potential is only observed after storage for more than

1 month. Trehalose is also a non-reducing sugar which

is composed of two glucose units. However, it is well

known that trehalose strongly resists hydrolysis [48].

As a consequence, no decrease in zeta potential occurs.

4. Conclusions

The results of this study clearly illustrate that the

oligosaccharides inulin 1.8 and 4 kDa and dextran 1.5,

5, and 40 kDa exhibit better stabilizing properties than

the disaccharides sucrose and trehalose to prevent

aggregation of nonPEGylated lipoplexes during sub-

zero temperature storage of frozen dispersions. It

appeared that the relative high Tgs’ of the oligosac-

charides is the reason for their improved stabilizing

capacity. As a result, during storage at �20 8C particle

size was more or less maintained with the oligosac-

charides (Tg’ around or above �20 8C) while rapid

and severe aggregation occurred in disaccharides

containing solutions (Tg’ below �20 8C). During

freeze drying and storage of the dried powders at 37

8C, both oligosaccharides and disaccharides were able

to preserve particle size of nonPEGylated lipoplexes.

Clearly, particle size was optimally maintained

because in all cases the sample temperature was kept

well below the Tg’ and Tg during freeze drying and

storage, respectively. It is suggested that for storage

purposes, especially upon exposure to humidified air,

oligosaccharides are preferred over disaccharides

because of their higher Tg.

Similar results were found for PEGylated lip-

oplexes with two major differences. First, when stored

above the Tg’, aggregation of the PEGylated lip-

oplexes proceeded at a lower rate than nonPEGylated

lipoplexes, illustrating the steric repulsion effect of

PEG. Second, while inulins showed an adequate

stabilization, dextrans failed to prevent aggregation

of the PEGylated lipoplexes in most cases. This is

most likely caused by the fact that inulins and PEG

are compatible while dextrans and PEG are not.

The zeta potential of all samples remained the same

upon subzero temperature storage. However, the zeta

potential substantially decreased upon storage in the

dry state at 37 8C in all cases except for the trehalose

containing samples where no significant change was

measured. The decrease in zeta potential was most

likely due to the formation of a Schiff’s base.

Concerning the oligosaccharides, this process may

be avoided by improved production and purification

methods of the inulins and by chemical modification

of the end groups of dextran.

In conclusion, oligosaccharides can prevent lip-

oplexes aggregation during subzero temperature stor-

age of frozen dispersions and freeze drying followed

by storage of the dried powder. However, when

PEGylated lipoplexes are considered, the oligosac-

charide should be compatible with PEG. Furthermore,

W.L.J. Hinrichs et al. / Journal of Controlled Release 103 (2005) 465–479478

the oligosaccharide should contain no reducing

groups to prevent change of the zeta potential.

Maintenance of particle size and zeta potential is not

a guarantee but certainly a prerequisite for mainte-

nance of biological activity. Therefore, future experi-

ments should reveal in detail the effects of

oligosaccharides on the transfection efficiency.

Acknowledgements

The authors wish to thank Ms. Hanne Claerhout,

Ms. Lies Peters, and Ms. Eveline Pringels of the

Department of Pharmaceutics of Ghent University for

their technical assistance.

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