TOWARDS PERSONALIZED DRUG DELIVERY

Preparation of an Encapsulated Multicompartment System

Maik Hadorn

Department of Informatics, Artificial Intelligence Laboratory, University of Zurich, Zurich, Switzerland

Peter Eggenberger Hotz

The Mærsk Mc-Kinney Møller Institute, University of Southern Denmark, Odense M, Denmark

Keywords: Personalized healthcare, Drug delivery, Encapsulation, Compartmentalization, Vesicle, Liposome.

Abstract: Single liposomes and vesicles are successfully utilized as delivery vehicles of pharmaceuticals. However

limitations of these unilamellar, single compartments led to the development of encapsulated multicom-

partment systems that establishes the prospect of multicomponent or multifunctional drug delivery systems.

So far compartmentalization is restricted to binary systems. To realize a personalized drug delivery, a pro-

grammable linkage of n-entities of different content will be needed. Here we present both a programmable

DNA-mediated linkage of three distinct vesicle populations and a novel encapsulation protocol. We discuss

how the techniques established in this study might be used in personalized healthcare based on custom-

tailored encapsulated multicompartment vesicular drug delivery systems.

1 INTRODUCTION

Biological as well as artificial vesicles feature an

aqueous compartment partitioned off an aqueous

surrounding by a lipid membrane that is nearly im-

permeable for hydrophilic substances. The mem-

brane organizes processes by compartmentalizing

them. The compartmentalization enables segregation

of specific chemical reactions for the purposes of

increased controllability, observability, stability, and

biochemical efficiency by restricted dissemination

and efficient storage of reactants, and/or reaction

products. Thus, wide usage of artificial vesicles is

found in analytics (Hotani, Nomura and Suzuki,

1999; Jesorka and Orwar, 2008; Limozin, Roth and

Sackmann, 2005; Luisi and Walde, 2000) and syn-

thetics, where they are used as bioreactors (Bolinger,

Stamou and Vogel, 2008; Michel et al., 2004;

Noireaux and Libchaber, 2004), and drug delivery

systems (Allen and Cullis, 2004; Bonacucina, Cespi,

Misici-Falzi and Palmieri, 2009; Torchilin, 2005).

Vesicles featuring biocompatibility, biodegradabil-

ity, low toxicity, and structural variability are suc-

cessfully utilized as therapeutic agents for the deliv-

ery of antibacterial, antiviral, and anticancer drugs,

as well as of hormones, enzymes, and nucleotides

(Eckstein, 2007; Lasic, Vallner and Working, 1999;

Weissig, Boddapati, Cheng and D'souza, 2006).

Generally, single unilamellar vesicles apply in

therapeutic systems. However premature content

release in physiological environments limits their

reliability (Bakker-Woudenberg, Schiffelers, Storm,

Becker and Guo, 2005). Extending the circulation

time of vesicles that results in accumulation at tu-

mors or inflammation sites due to the enhanced

permeability and retention (EPR) effect (Allen and

Cullis, 2004) is realized at the molecular level via

monomer design (Torchilin, 2009) or at the

mesoscopic level via encapsulation. The bilayer-

within-a-bilayer structure of encapsulated vesicles

not only prevents a premature degradation and con-

tent release (Boyer and Zasadzinski, 2007) but offers

a division of different membrane functions (biocom-

patibility, cargo release, targeting, and protection)

among several membranes of different compositions

and dimensions. Encapsulated vesicles are fre-

quently used in pharmaceutical and cosmetic appli-

cations (Lasic, 1993). The applicability of single

vesicles is limited further by the need for a simulta-

neous entrapment of a given set of (pharmaceutical)

components in one single compartment, which is

Hadorn M. and Eggenberger Hotz P. (2010).

TOWARDS PERSONALIZED DRUG DELIVERY - Preparation of an Encapsulated Multicompartment System.

In Proceedings of the Third International Conference on Biomedical Electronics and Devices, pages 5-12

DOI: 10.5220/0002691400050012

 SciTePress

Figure 1: Schematic representation of the vesicle formation/encapsulation procedure and micrographs of internally com-

partmentalized vesicles. (A) A water droplet (blue) is added to a phospholipid suspension (light gray, cp. D.1). (B) A water-

in-oil emulsion is produced by mechanical agitation and sonication. (C) The emulsion is placed over an aqueous solution.

Vesicles are produced in 96-well microtiter plates, providing parallel formation of up to 96 distinct vesicle populations. (D)

Induced by centrifugation, the droplets pass the oil/water interface. Due to the density difference of the inter- and intrave-

sicular fluid and the geometry of the microplate bottom, vesicles pelletize in the centre of the well and become easily acces-

sible for pipetting. (D.1) Amphiphilic phospholipids, solved in mineral oil, stabilize water-oil interfaces by forming

monolayers. Two monolayers form a bilayer when a water droplet passes the interface. (E) A droplet of the aqueous solu-

tion that hosts the vesicles is added to a phospholipid suspension. After mechanical agitation (F) and placing over an aque-

ous solution (G), internally compartmentalized vesicles are produced by centrifugation. Lower color saturation indicates

lower density of the aqueous solution. (H.1, H.2) Differential interference contrast micrographs of internally compartmen-

talized vesicles. Scale bar represents 10μm.

“not an easy matter” (Luisi, de Souza and Stano,

2008, p. 14660). Multicompartment systems can

overcome this limitation by conciliating smaller

subsets of components entrapped in different com-

partments. Thus, encapsulated multicompartment

systems could provide stable vehicles for a multi-

component or multifunctional drug delivery.

Zasadzinski et al. established a protocol to en-

capsulate a multicompartment system of tethered

liposomes (Boyer and Zasadzinski, 2007; Kisak,

Coldren, Evans, Boyer and Zasadzinski, 2004;

Walker, Kennedy and Zasadzinski, 1997). Both

tethering and encapsulation of these vesosomes are

based on the molecular recognition process of the

biotin-streptavidin complex. Like most of the current

tethering strategies (Berti, Baglioni, Bonaccio, Bar-

sacchi-Bo and Luisi, 1998; Chiruvolu et al., 1994;

Constable, Meier, Nardin and Mundwiler, 1999;

BIODEVICES 2010 - International Conference on Biomedical Electronics and Devices

Marchi-Artzner et al., 2001; Menger, Seredyuk and

Yaroslavov, 2002; Paleos, Sideratou and Tsiourvas,

1996; Sideratou et al., 2002; Vermette, Taylor, Dun-

stan and Meagher, 2002; Weikl, Groves and Li-

powsky, 2002), tethering is based on single ligand-

receptor pairs and result in systems binary at most.

DNA-mediated linkage (Beales and Vanderlick,

2007; Chan, van Lengerich and Boxer, 2009) offers

multiple ligand-receptors, as needed in a multicom-

ponent or multifunctional drug delivery system.

However neither a coupling of more than two vesi-

cle populations is realized, nor a procedure to encap-

sulate such a system is established.

In this study, we present both a programmable

DNA-mediated linkage of three distinct vesicle

populations and a novel encapsulation mechanism.

Based on the results of this study, we formulate a

scenario how encapsulated multicompartment sys-

tems might be used to realize custom-tailored ve-

sicular drug delivery systems.

2 MATERIALS AND METHODS

Technical modifications of the vesicle formation

protocol reported by Pautot et al. (Pautot, Frisken

and Weitz, 2003) were: (i) the introduction of 96-

well microtiter plates U96 to increase procedural

manageability in laboratory experimentation and (ii)

a density difference between inter- and intravesicu-

lar solution induced by isomolar solutions of mono-

saccharids (glucose: inter) and disaccharids

(sucrose: intra). For a description of the modified

vesicle protocol see Figure 1A-D. For the membrane

composition of the vesicles used in the encapsulation

and the self-assembly experiments see Table 1. All

phospholipids were solved in mineral oil.

Table 1: Membrane composition of vesicles used in ex-

perimentation.

ncapsulation

100%

PC(16:0/18:1(Δ9-Cis)) =

1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine

Self-Assembly

99%

PC(16:0/18:1(Δ9-Cis)) =

1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine

0.75%

methyl-PEG2000-PE(18:0/18:0) =

1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-

[Methoxy (Polyethylene glycol)-2000]

0.25%

biotin-PEG2000-PE(18:0/18:0) =

1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-

[Biotinyl (Polyethylene Glycol) 2000]

For details of the encapsulation procedure of

untethered vesicles see Figure 1. Encapsulated vesi-

cles exhibited quick random motion within the

boundaries of the surrounding vesicle.

In the compartmentalization experiments, three

distinct vesicle populations were prepared by doping

their surface with binary combinations of six ssDNA

populations (1

population: α, β; 2

: α’, γ; 3

: β’,

γ’; for the sequence of biotinylated ssDNA strands

see Table 2). The DNA strands were biotin-labeled

and anchored to biotinylated vesicular membrane via

streptavidin as a cross-linking agent. For a detailed

protocol of the surface doping and the self-assembly

procedure see Figure 2. The sequences of the ssDNA

were produced by a genetic algorithm and optimized

for minimal DNA-DNA-hybridization among the

three pairs. The specificity was verified on a com-

mercial program.

Light and confocal laser scanning microscopy

was performed using an inverted Leica DMR IRE2

SP2 confocal laser scanning microscope.

Table 2: Sequence of complementary biotinylated DNA

single strands used in the self-assembly experiments.

α biotin-TGTACGTCACAACTA-3’

α’ 3’-ACATGCAGTGTTGAT-biotin

β biotin-AGAAAGAGCCCTCCA-3’

β’ 3’-TCTTTCTCGGGAGGT-biotin

γ biotin-AAAGATTACACACGA-3’

γ’ 3’-TTTCTAATGTGTGCT-biotin

3 RESULTS

The density difference between the inter- and in-

travesicular solution induced vesicle pelletization at

the centre of the well. The size distribution of vesi-

cles produced in the first round of vesicle formation

(Figure 1 A-D) was shifted to the left when com-

pared to vesicles produced in the second round (Fig-

ure 1 E-H). The two vesicle formation protocols

differed only in the presence (Figure 1 B) or absence

(Figure 1 F) of the sonication of the water-in-oil

emulsion. To indicate independence of the tethering

and encapsulation process, vesicles to be encapsu-

lated were not tethered. Tethered assemblies were

encapsulated without any modification of the encap-

sulation procedure (results not shown). As seen in

Figure 1 H.1 and H.2 the ratio of vesicles internally

compartmentalized to vesicles uncompartmentalized

was high. Most of the vesicles produced in the first

round were found to be enclosed – encapsulation

efficiency was high.

TOWARDS PERSONALIZED DRUG DELIVERY - Preparation of an Encapsulated Multicompartment System

Figure 2: Schematic representation of the self-assembly process and micrographs of adhesion plaques. (A) For vesicle

formation see Figure 1.(B) Vesicle populations become distinct by incubating them with single stranded DNA (ssDNA) of

different sequence (α, α’, β, β’, γ, γ’) and streptavidin differing in fluorescence labeling (Alexa Fluor 488 / 532 conjugate

(AF488 / AF532) or unlabeled). Monohomophilic oligonucleotide-doping of streptavidin is provided by separated incuba-

tions. (C) The vesicle populations are merged. (C.1) The lateral distribution of linkers in the lipid membrane is homogene-

ous. Vesicles doped with complementary ssDNA come into contact. (C.2) Hybridization of DNA strands results in double

stranded DNA and induces the assembly process. Due to their lateral mobility, linkers accumulate in the contact zone form-

ing an adhesion plaque – the lateral distribution of linkers in the outer leaflet becomes inhomogeneous (situation shown for

α-AF532, α’-AF488). (D) CLSM (confocal laser scanning microscope) and DIC (differential interference contrast) micro-

graph of a vesicular aggregate that emerged in real-world experimentation. Accumulation and depletion of linkers are

clearly visible in the CLSM micrograph. Scale bar represents 10μm.

When vesicles doped with complementary ssDNA

came into contact, hybridization of single DNA

strands resulted in double stranded DNA. Linkers

accumulated in the contact area of the two vesicles

formed an adhesion plaque (Figure 2 D). Adhesion

plaques were found exclusively, when DNA strands

were complementary and inorganic ions were pre-

sent (data of control experiments not shown). No

transfer of linkers between the membranes of differ-

ent vesicles was observed (data not shown).

BIODEVICES 2010 - International Conference on Biomedical Electronics and Devices

4 DISCUSSION

Multicomponent or multifunctional custom-tailored

vesicular drug delivery systems have to fulfil several

requirements: (i) the actual drug containing system

should be encapsulated to prevent premature

degradation and content release, (ii) the drug

containing system should consist of more than two

distinct compartments, and (iii) the proper

composition of the drug containing system should be

controlled.

4.1 Encapsulation

The in vitro vesicle formation procedure (Noireaux

and Libchaber, 2004; Pautot et al., 2003; Träuble

and Grell, 1971) enables independent tailoring of

chemical material properties of the inter- and in-

travesicular fluid as well as of the inner and outer

membrane leaflet composition. To our knowledge,

the entrapment efficiency of this vesicle formation

procedure is not analyzed so far. However one may

speculate that its entrapment efficiency is better than

for vesicle formation procedures currently used (for

an overview of the current vesicle formation proce-

dures see Jesorka and Orwar (2008)). The potential

of an asymmetric leaflet composition was exempli-

fied by the production of phospholipid and polymer

hybrids combining biocompatibility and mechanical

endurance in single vesicles (Pautot et al., 2003). We

increased procedural manageability of the formation

procedure by introducing microtiter plates and vesi-

cle pelletization (due to density differences in the

inter- and intravesicular fluid). By introducing soni-

cation of the water-in-oil emulsion, we could shift

the size distribution of the vesicles formed. By re-

feeding the vesicle containing solution, we estab-

lished a novel method to produce multivesicular

assemblies. The protocol provides encapsulation of

either tethered or untethered vesicular assemblies.

The interdependence of tethering and encapsulation,

faced in vesosome formation, is therefore resolved.

4.2 Compartmentalization

Single stranded DNA provides programmability,

specificity, and high degrees of complexity (Licata

and Tkachenko, 2006). Streptavidin offers the

strongest noncovalent biological interaction known

(Green, 1990), an extensive range of possible vesicle

modifications, component modularity, and availabil-

ity off the shelf. Phospholipid-grafted biotinylated

PEG tethers feature lateral mobility (Singer and

Nicolson, 1972), high detachment resistance (Bur-

ridge, Figa and Wong, 2004), and no intermembrane

transfer of linkers. The combination of phosphol-

ipid-grafted biotinylated PEG tethers and strepta-

vidin allows fast production of vesicles differently

doped and avoids problems encountered in other

approaches using cholesterol-tagged DNA to spe-

cifically link different vesicle populations by the

hybridization of membrane-anchored DNA (Beales

and Vanderlick, 2007; Benkoski and Hook, 2005;

Chan et al., 2009): (i) Because the processes of vesi-

cle formation and vesicle modification are not sepa-

rated (the cholesterol-tagged ssDNA has to be pre-

sent during vesicle formation), the formation proce-

dure has to be adjusted anew for each change in the

vesicle modification. The procedural manageability

in laboratory experimentation is therefore reduced.

(ii) As discussed by Beales and Vanderlick (2007)

the cholesterol anchors of the cholesterol-tagged

ssDNA spontaneously leave the lipid bilayer and

incorporate randomly into (other) lipid bilayers.

Thus, the specificity of the linking system is lost

over time.

We presented a DNA-mediated tethering of three

distinct vesicle populations. Linkage of more than

two distinct vesicle populations is realized for the

first time. Thus, restriction to binarism faced in

current donor-acceptor mechanisms is resolved. The

DNA-mediated linkage mechanism offers program-

mability of composition of multicompartment sys-

tems. Thus, custom-tailored vesicular drug delivery

systems seem feasible.

4.3 Composition Control

By loading the vesicular membranes of tethered

assemblies by ligand groups not used in the aggrega-

tion process, a column chromatographic purification

procedure of aggregates may be realized. The ligand

groups would be used to purify aggregates from

single vesicles (for details see Figure 3). Figure 3

depicts the minimal situation of tethered assemblies

of two vesicle populations and two columns in se-

ries. If the tethered assemblies consist of three dif-

ferent vesicle populations bearing three different

ligand groups not used in the aggregation process,

purification of aggregates of proper composition

both from single vesicles and incomplete aggregates

might become possible.

By a downstream fluorescence activated cell

sorting (FACS; for a review of techniques used in

cell separation see Pappas and Wang (2007)) inter

nally compartmentalized vesicles might be purified

from vesicles not equipped properly (Figure 3G).

Based on the fluorescence signal, the chroma-

tographic separation procedure (Figure 3 B-D) might

TOWARDS PERSONALIZED DRUG DELIVERY - Preparation of an Encapsulated Multicompartment System

Figure 3: Schematic representation of the processes to form and purify internally compartmentalized vesicles. (A) For de-

tails concerning the self-assembly process resulting in vesicular aggregates see Figure 2. Vesicle populations become dis-

tinct by incubating them with single stranded DNA (ssDNA) of different sequence (α, α’, δ, ε) and streptavidin differing in

fluorescence labeling (Alexa Fluor 488 (green) / 532 (red) conjugate (AF488 / AF532) or unlabeled (black)) resulting in a

surface doping of (α-AF532, δ-unlabeled; α’-AF488, ε-unlabeled). The DNA strands differ in their melting temperature (T





> T





= T





). Due to the hybridization of DNA strands linkers α-AF532 and α’-AF488 accumulate in the contact zone form-

ing an adhesion plaque. Lateral distribution of linkers δ-unlabeled and ε-unlabeled is not affected by the assembly process.

(B) To dispose vesicles not assembled the mixture is fed to a column whose stationary phase is doped with single stranded

DNA (δ’, ε’). Hybridization of DNA strands results in a retention of aggregates and single vesicles doped with ssDNA (δ).

(C.1) Single vesicles not doped with ssDNA (δ) are eluted at low temperature (T < T





, T





, T





). (C.2) An increase in

temperature (T





, T





< T < T





) results in an elution of aggregates and single vesicles doped with ssDNA (δ). (D.1, D.2)

The procedure of C.1 and C.2 is repeated for a column doped with ε’. (E) Single vesicles can be fed back in the self-

assembly procedure (see Figure 2.C). (F) The purified aggregates are encapsulated in vesicles (for details see Figure 2 E-H).

(G.1, G.2) The fluorescence signal of the internal compartments is exploited to purify internally compartmentalized vesicles

from vesicles not equipped properly.

BIODEVICES 2010 - International Conference on Biomedical Electronics and Devices

be replaced by a final FACS for proper composition

of the tethered assemblies. By introducing an inter-

mediate separation process, a feedback of single

vesicles and incomplete assemblies into the self-

assembly process may be realized before they be-

come encapsulated (Figure 3 E). This may increase

encapsulation efficiency and therefore may econo-

mize the production of custom-tailored vesicular

drug delivery systems.

Encapsulation provides an extended circulation

time resulting in accumulation at tumors or inflam-

mation sites due the EPR effect, without the need of

specific targeting. On the other hand, multiple com-

partments offer segregation of multicomponent

pharmaceuticals that might be released only when

and where they are needed. Permeability control

might be realized either by exploitation of stimuli

inherent to target site (pH, redox potential, tempera-

ture) or externally induced (temperature, magnetic

field, ultrasound). For a recent review on stimuli-

sensitive pharmaceutical nanocarriers see Torchilin

(2009).

5 CONCLUSIONS

Encapsulated multicompartment systems may pro-

vide stable vehicles for a multicomponent or multi-

functional personalized drug delivery. In this work,

we established a novel encapsulation technique and

provide evidence for the first stable DNA-mediated

linkage of more than two vesicle populations. We

discussed how these techniques may personalize the

individual healthcare by providing custom-tailored

vesicular drug delivery systems.

ACKNOWLEDGEMENTS

Maik Hadorn was supported by the Swiss National

Foundation Project 200020-118127 Embryogenic

Evolution: From Simulations to Robotic Applica-

tions. Peter Eggenberger Hotz was partly supported

by the European Union integrated project PACE

(EU-IST-FP6-FET-002035). We thank Eva Bönzli

for careful reading of the manuscript.

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