Abstracts / Journal of Controlled Release 148 (2010) e7e20

[5] Y. Liu, S. Meng, L. Mu, G. Jin, W. Zhong, J. Kong, Novel renewable immunosensors
based on temperature-sensitive PNIPAAm bioconjugates, Biosensors and bioelectronics 24 (4) (2008) 710715.

doi:10.1016/j.jconrel.2010.07.010

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PAGE analysis reveals an enzyme encapsulation efficiency of 10%

and shows a clear incorporation of the porin Tsx. The in vitro
enzymatic activity of the TP-NRs was confirmed spectrophotometrically by measuring a decrease in absorption at 290 nm due to
thymidine consumption.

Polymeric nanoreactors for enzyme replacement therapy

of MNGIE
Caroline De Vocht1,3,, An Ranquin1,3, Jo Van Ginderachter2,3,
Tamara Vanhaecke1, Vera Rogiers4, Patrick Van Gelder1,3,
Wim Verses1,3, Jan Steyaert1,3
1
Structural Biology Brussels, Vrije Universiteit Brussel (VUB),
1050 Brussels (Elsene), Belgium
2
Laboratory of Cellular and Molecular Immunology, Vrije Universiteit
Brussel (VUB), 1050 Brussels (Elsene), Belgium
3
Department of Molecular and Cellular Interactions, VIB, 1050 Brussels
(Elsene), Belgium
4
Department of Toxicology, Vrije Universiteit Brussel (VUB),
1090 Brussels (Jette), Belgium
Corresponding author.
E-mail: Caroline.De.Vocht@vub.ac.be.
Abstract summary
We constructed new polymeric nanoparticles that encapsulate the
enzyme thymidine phosphorylase and are permeabilised for substrates
and products by reconstitution of Tsx porins in their wall. These
nanoreactors are enzymatically active and stable in serum. Moreover,
they do not provoke cytotoxicity when incubated with hepatocytes, nor
do they induce a macrophage-mediated inflammatory response ex vivo
and in vivo. In conclusion, the nanoreactors show potential as drug
delivery vehicles in enzyme replacement therapy of MNGIE [1].
Introduction
Many known genetic deficiencies in human are caused by a
mutation in a gene encoding a crucial enzyme, leading to the
accumulation of its substrates. As a result, toxic amounts of the
substrates appear in the bloodstream, disturbing the normal metabolic degradation pathways. Mitochondrial Neurogastrointestinal
Encephalomyopathy (MNGIE) is such a unique autosomal recessive
disorder caused by loss-of-function mutations in the gene encoding
thymidine phosphorylase, leading to systemic accumulations of the
substrates thymidine and deoxyuridine [2]. A general well-tolerated
strategy to treat such an enzyme deficiency is enzyme replacement
therapy. Nevertheless, the fast elimination of the native enzyme from
the circulation is a major drawback.
In this study, we developed polymeric nanometer-sized nanoreactors for enzyme replacement therapy of MNGIE (Fig. 1). The therapeutic
enzyme, thymidine phosphorylase (TP), is encapsulated in
polymeric particles constructed of the amphiphilic triblock copolymer
PMOXA-PDMS-PMOXA (poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline)). The nanoparticles
are permeabilised for substrates and products by integrating bacterial
channel proteins in their polymeric wall, a strategy introduced by
Meier et al. [3]. The nucleoside specific porin Tsx was selected as
channel-forming protein for the nanoreactors, since it contains specific
nucleoside binding sites. In this way, the Tsx porin allows the efficient
transport of the substrates and products through the capsule wall. This
results in reactors where the enzymatic reaction is restricted to the
compartmentalised inner volume of the polymeric nanocontainer.
Result and discussion
We constructed thymidine phosphorylase encapsulating nanoreactors (TP-NRs) according to Ranquin et al. [4]. Monodisperse
nanoreactors with a mean radius around 100 nm were obtained. SDS-

Fig. 1. Schematic representation of a thymidine phosphorylase encapsulating

nanoreactor.

The stability of the permeabilised TP-NRs was investigated by

incubating them for several days in 50% naive mouse serum at 37 C. At
different time points both the size distribution and the enzyme activity
was measured. TP containing liposomes were used as control samples.
In mouse serum, the size and dispersity of the TP-NRs stays relatively
constant over time. We can conclude that the nanoreactors are still
intact after 4 days of incubation at 37 C. In contrast to the liposomes,
which show a clear leakage of protein, no leakage is observed for the
TP-NRs. Thus, the enzyme is contained inside the polymeric particles.
The enzyme activity of the TP-NRs decreases slightly over time. After
three days half of the initial activity remained.
Secondly, possible toxic effects of the TP-NRs on hepatocytes were
investigated. Hepatocytes were isolated from rats and culture
medium supplemented with different concentrations of TP-NRs was
added. Cytotoxicity was tested by cellular morphology and membrane leakage of lactate dehydrogenase (LDH assay) as a function of
time. The results show no significant difference in LDH leakage
between the various conditions up to 48 h of incubation. Only after
prolonged exposure and at higher doses of TP-NRs elevated levels of
LDH leakage were measured (Fig. 2). This low cytotoxic effect was
confirmed by visual inspection of the morphology of the hepatocytes.
Finally, the impact of TP-NRs on the inflammatory status of
macrophages was tested ex vivo and in vivo. For ex vivo studies the
effect of nanoreactors on peritoneal macrophages isolated from mice
was analysed. The results show that none of the nanoparticles tested
induce inflammatory cytokine production by nave macrophages.
Interestingly, the free enzyme significantly provoked the secretion of
all inflammatory mediators tested. This means that the encapsulation
of the enzyme in polymer particles indeed protects the protein from
the immune system. However, on IFN- primed macrophages,
complete TP-NRs dose-dependently elevate the amounts of cytokines
produced. Since no significant responses were found with preparations
without porins, we can conclude that the induction of inflammatory
mediators is porin-dependent. To study the inflammatory potency of
the nanoparticles in vivo, mice received a peritoneal injection of TPNRs. Intraperitoneal injection of PBS and a sub-lethal dose of LPS were
used as negative and positive control respectively. Only low amounts of
cytokines could be found in macrophage-supernatant and in serum of
mice treated with TP-NRs, while high concentrations were detected in
mice injected with LPS. Hence, in a physiologically relevant in vivo
setting, the nanoparticles do not provoke acute inflammatory
responses and appear to be immunologically inert.
Conclusion
We successfully constructed active thymidine phosphorylase
encapsulating nanoreactors and evaluated their potential as new
enzyme delivery vehicles. Although a slight decrease in enzymatic
activity was observed over time, the TP-NRs are stable and not leaky

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Abstracts / Journal of Controlled Release 148 (2010) e7e20

at 37 C in serum. At concentrations up to 100 g polymer/ml the TPNRs do not affect the viability of primary hepatocytes. Additionally,
no effect of TP-NRs was observed on naive macrophages, while only a
minor porin-dependent effect at high concentrations was detected on
IFN-primed macrophages in vitro. No effect on inflammation was
seen in vivo. Altogether, these results open realistic hopes and good
prospects for a better and safer enzyme replacement therapy of
genetic deficiencies.

References
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W. Versees, P. van Gelder, J. Steyaert, Assessment of stability, toxicity and
immunogenicity of new polymeric nanoreactors for use in enzyme replacement
therapy of MNGIE J Control Release 137 (2009) 246254.
[2] I. Nishino, A. Spinazzola, M. Hirano, Thymidine phosphorylase gene mutations in
MNGIE, a human mitochondrial disorder, Science 283 (1999) 689692.
[3] W. Meier, C. Nardin, M. Winterhalter, 45994602, Reconstitution of channel
proteins in (polymerized) ABA triblock copolymer membranes, Angew Chem Int Ed
Engl. 39 (2000) 45994602.
[4] A. Ranquin, W. Versees, W. Meier, J. Steyaert, P. van Gelder, Therapeutic
nanoreactors: combining chemistry and biology in a novel triblock copolymer drug
delivery system, Nano Lett 5 (2005) 22202224.

doi:10.1016/j.jconrel.2010.07.011

Fig. 2. LDH leakage of hepatocytes incubated with different concentrations of TP-NRs

as a function of time. Medium and PBS serve as negative controls, a combination of
SuperFasLigand with cycloheximide is used as the positive control. Statistical analyses
were performed using a one-way ANOVA test, followed by a Bonferroni post hoc test.
(* p < 0.05 and *** p < 0.001 compared to medium and PBS control values).