Liposomes containing mannose-6-phosphate-cholesteryl conjugates for lysosome-specific delivery

Article abstract of DOI:10.1039/c4ra08681c

Lysosomes are promising targets for cancer and enzyme replacement therapy. In this area of interest we present a novel liposomal nanocarrier containing mannose 6-phosphate-cholesteryl conjugates and show its ability to reach the lysosomes by means of confocal and fluorescence microscopy measurements.

Full text of DOI:10.1039/c4ra08681c

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Liposomes containing mannose-6-phosphate-
cholesteryl conjugates for lysosome-specific
delivery†
Cite this: RSC Adv., 2014, 4, 58204
E. Crucianelli,a P. Bruni,b A. Frontini,c L. Massaccesi,a M. Pisani,b A. Smorlesic
Received 14th August 2014
Accepted 30th October 2014
a
*
and G. Mobbili
DOI: 10.1039/c4ra08681c
www.rsc.org/advances
Lysosomes are promising targets for cancer and enzyme replacement the Golgi and endosomal compartments where acidity induces
therapy. In this area of interest we present a novel liposomal nano- release of enzymes from M6P receptor which is recycled in the
carrier containing mannose 6-phosphate-cholesteryl conjugates and Golgi apparatus. A minor fraction of CI-M6PR is also present on
show its ability to reach the lysosomes by means of confocal and the cell surface where it mediates the endocytosis and the
fluorescence microscopy measurements.
transfer to lysosomes of secreted acid hydrolases, but also
internalizes extracellular ligands,13–16 including M6P-containing
proteins.17–21 CI-M6PR can therefore be considered an extremely
interesting candidate that could be used to specically target
the transport of molecules into the lysosomes.
In the last years our research has been focused on the
development of neutral liposomal delivery systems,22–24 articial
and biologically inert phospholipid vesicles that, by virtue of
their amphiphilic nature, are capable of transporting both
water-soluble and water-insoluble drugs inside the cells. Lipo-
somes can increase the stability of the transported molecules by
retarding their processing,25 delaying drug clearance and, in
general, improving the pharmacokinetic of the administered
drug.
We propose here a new delivery system based on liposomes
containing dioleoylphosphatidilcholine (DOPC) and M6P func-
tionalized cholesterol. By virtue of its affinity for the CI-M6PR
receptor, M6P group enables liposomes to carry bioactive
molecules along the route leading to lysosomes. One of the
principal therapeutic applications for M6P functionalized lipo-
somes could be enzyme replacement therapy (ERT) for LSDs.
ERT is usually performed by administering the enzyme to the
patient through intravenous infusion.26 An interesting
approach27 among the used ERT strategies is the administration
of lysosomal hydrolases obtained through a protein expression
system that provides the enzyme of higher levels of phospho-
rylated oligomannose residues correlating with increased
binding to M6P receptors. However, this therapy presents some
drawbacks such as the enormous cost and the limited life of
enzymes in the circulation. The administration of lysosomal
enzymes through the use of a M6P functionalized liposomes-based
carrier system could improve the transport of the therapeutic
contents into lysosomes through the CI-M6PR pathway while
protecting the enzyme from a too rapid deactivation in vivo.
Lysosomes are the waste disposal system of the cell. Containing
about 60 different hydrolytic enzymes, they are able to process
all kinds of biomolecules internalized by the cell or obsolete
cellular components.1,2 Lysosomal enzymes are acid hydrolases
that are activated in the acidic environment of these organelles
to ensure the normal function of the cell. Their deciency leads
to accumulation of substrates such as glycosphingolipids,
mucopolysaccharides or glycogen in various tissues and
generates over 50 different human lysosomal storage diseases
(LSDs),3 for example lipid storage disorders, mucopolysacchar-
idoses, glycoprotein storage disorders and mucolipidoses. Once
synthesized, lysosomal hydrolases are provided of a mannose-6-
phosphate (M6P) group through the action of a UDP-N-
acetylglucosamine-1-phosphotransferase
(GlcAc-1-phospho-
transferase).4–8 The presence of the M6P tag makes lysosomal
hydrolases recognizable by M6P receptors9 in the Golgi appa-
ratus so that they can be transported to the lysosomal system via
the secretory pathway through clathrin-coated vesicles. In
mammalian cells two specic receptors of the trans-Golgi
network recognize M6P-functionalized hydrolases: the cation-
independent M6P receptor (CI-M6PR) and the cation-
dependent M6P receptor (CD-M6PR), both type
membrane glycoproteins.10–12 CI-M6PR is especially localized in
I trans-
a
`
Di.S.V.A. Department, Universita Politecnica delle Marche, Via Brecce Bianche, 60131
Ancona, Italy. E-mail: g.mobbili@univpm.it
b
`
SIMAU Department, Universita Politecnica delle Marche, Via Brecce Bianche, 60131
Ancona, Italy
c
`
Department of Experimental and Clinical Medicine, Universita Politecnica delle
Marche, Via Tronto, 60126 Ancona, Italy
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra08681c
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A further interesting application of lysosomotropic lipo- insertion into the lipid bilayer, (ii) a linker and (iii) at the
somes could be the induction of apoptotic pathways in cancer opposite end of the spacer, a hydrophilic M6P group. With the
cells through the delivery of molecules, able to destabilize aim to connect a M6P moiety to a steroid structure we chose a
lysosomes and induce the release of lysosomal enzymes.28,29 Cell sufficiently rigid aryl-incorporated linker both to ensure an
apoptosis can be caused by the activation of mitochondrial efficient exposure of the M6P function and to favour tight
death pathway,30 triggered by cathepsins,31–33 specic enzymes association of the liposome with the receptor by reducing the
that are released from lysosomes following the permeabiliza- entropic cost of the process.36 The apolar structure of the steroid
tion of lysosomal membranes (LMP). Relying on the fact that provides stability to the drug delivery system because it is
the majority of solid tumors, particularly breast cancers,34 over- embedded into the lipid bilayer of liposomes spanning about
express CI-M6PR receptor, M6P functionalized liposomes could half a layer. The tetracyclic apolar ring structure is compact and
be used to transport and release into lysosomes specic mole- very rigid so that it increases the degree of order (cohesion and
cules35 that are able to induce LMP and consequently lead to the packing) in the liposome membrane and generally reduces the
death of the cancer cell.
passive permeability to drugs encapsulated inside the lipidic
In the present study a M6P cholesteryl conjugate (Chol-M6P) vectors. The synthesis of Chol-M6P is drawn in Scheme 1 and
was synthesized (Scheme 1) and employed with DOPC in the described in detail in the ESI.† The 4-nitrophenyl-a-D-
preparation of functionalized vesicles. To demonstrate the mannopyranoside 1 was selectively protected on the primary
potential of our novel vector system, specically designed to hydroxyl with a trityl group and on the secondary hydroxyl
target lysosomes, we loaded the M6P functionalized liposomes functionalities with benzoyl groups in one-pot reaction to give 2.
with the model compound calcein and investigated the intra- Aer purication by chromatography, the nitro group of
cellular trafficking in a 3T3-NIH cell line using confocal and compound 2 was converted by hydrogenation into an amino
uorescence microscopy technique.
group to give 3. Meanwhile, in order to synthesize the steroidal
The design of Chol-M6P is based on three requirements: it portion of the molecule, a cholesteryl chloroformate was added
comprises (i) a lipophilic part acting as an anchor, enabling its to a solution of the triuoro acetate salt of a methyl ester glycine
to give 4. Subsequent hydrolysis of the ester furnished the cor-
responding acid 5. Then compounds 3 and 5 were joined
together via carbodiimide coupling to give 6, which was selec-
tively deprotected at C-6 with catalytic amount of p-toluene
sulfonic acid. The resulting primary alcohol 7 was rstly con-
verted into phosphate 8 and successively debenzoylated with
NaOCH3. Final treatment of compound 9 with Dowex cation
exchange resin (Na+ form) afforded Chol-M6P as disodium salt.
The structure of Chol-M6P was conrmed by NMR Spectroscopy
and Mass Spectrometry (ESI†).
Liposomes were prepared by the “thin lm hydration”
method as reported in ESI.† Appropriate amounts of chloro-
form solutions of DOPC and cholesterol-M6P were mixed to
obtain liposomes with different molar ratio (mol/mol) of DOPC/
Chol-M6P (95 : 5, 90 : 10, 85 : 15, 80 : 20).
In order to verify the insertion of Chol-M6P inside the lipo-
some bilayers we carried out z-potential measurements of all
liposomal formulations by means of electrophoretic light scat-
tering; in fact, the z-potential is correlated to the vesicle surface
charge. The size of vesicles was also determined by dynamic
light scattering. As reported in Table 1, the liposome mean
diameter was in the range between 86 and 106 nm, with a low
polydispersity index (PdI) between 0.18 and 0.25, which proved
Table 1 Size and z-potential of DOPC/Chol-M6P liposomes
DOPC/Chol-M6P
mol/mol
Size Æ SD (nm)
z-potential Æ SD (mV)
Scheme 1 Reagents and conditions: (a) TrCl, DMAP, dry Py, 70 ꢀC then
BzCl, rt, on, 86%; (b) Pd/C 10%, H2, dry CH3OH, reflux, 73%; (c) NaOH 1
M, THF, 0 ꢀC, 2 h then HCl 1 M, 95%; (d) DCC, DMAP, DCM, rt, 63%; (e)
p-TsOH, CH3OH, CHCl3, 67%; (f) POCl3, TEA, dry DCM, 0 ꢀC, rt, 72%;
(g) NaOCH3, dry CH3OH then Dowex Na cation exchange resin.
100 : 0
95 : 5
90 : 10
85 : 15
80 : 20
103.5 Æ 1.5
86.5 Æ 0.6
106.0 Æ 1.4
101.1 Æ 1.4
99.8 Æ 0.8
À13.2 Æ 1.6
À40.5 Æ 5.0
À57.1 Æ 7.2
À65.7 Æ 7.0
À72.1 Æ 8.8
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that all dispersions used had very narrow vesicle size distribu-
tion. By increasing the concentration of the Chol-M6P in the
DOPC liposomes, the negative z-potential values decreased,
thus demonstrating the presence of negative M6P group on the
liposome surface (Table 1).37
The capability of M6P functionalized liposomes to be inter-
nalized by M6P-receptor expressing cells has been assessed
through intracellular tracing experiments in NIH-3T3 cells.38
For this purpose we decided to test the DOPC liposomes con-
Fig. 2 Co-localization study performed by confocal microscopy. (A)
Calcein visualized in green; (B) lysosomes labeled in red using Lyso-
taining the highest percentage of Chol-M6P (as evidenced by tracker; (C) by merging the previous two panels, calcein clearly
resulted localized in lysosome structures (yellow).
their z-potential) in order to increase the cellular uptake.
We performed preliminary experiments by epi-uorescence
microscopy using DOPC/Chol-M6P liposomes encapsulating
(positive) appeared to be in equal proportion with negative ones
but sometimes they appeared clustered in specic chambers
and other time they showed a different pattern i.e. positive cells
scattered among negative ones. Overall, we have estimated an
average of 40–50% of cells that showed calcein internalization
aer treatment with functionalized liposomes.
Focusing more in detail on the confocal imaging of tracing
experiment performed on NIH-3T3 cells (Fig. 1C inset) we
observed that calcein and liposomes co-localize in among 80%
of the vesicles (yellow). About 10% of the elements are instead
represented only by calcein loaded vesicles (white arrow) and,
the remaining 10% of the elements, are vesicles loaded with
lipid alone (white arrowhead). We could then try to propose a
model for the internalization pathway of our carrier liposomes
by hypothesizing that, following the internalization of calcein-
containing M6P-liposomes into the lysosomes (yellow vesi-
cles), carrier lipids begin to be processed and degraded
releasing the transported molecule in the lumen of the organ-
elles (green vesicles) as suggested also by the fact that calcein
itself is not internalized into cells.
the reporter molecule calcein. In order to exclude non-specic
endocytosis we used free calcein and calcein-loaded DOPC/
cholesterol liposomes (80 : 20 mol/mol) as controls. We veri-
ed that, in the absence of M6P moiety, vesicles are not able to
carry the uorescent molecule into cells. Neither calcein alone
is able to cross cell membrane and enter the cells (Fig. S1 in
ESI†).
With the aim of better understanding the intracellular traf-
cking of DOPC/Chol-M6P liposomes we carried out confocal
microscopy experiments. Calcein loaded Rhodamine-labeled
DOPC/Chol-M6P liposomes were prepared and used in
delivery experiments as described in ESI section.† Confocal
microscopy analysis of treated cells revealed a clear localization
of calcein (Fig. 1A) and liposomes (Fig. 1B) inside the cells at
peri-nuclear position into small vesicles resembling lysosomes
(Fig. 1C).
In order to establish the nature of the vesicles where calcein
accumulated, we performed further assays on NIH-3T3 cells and
used the lysosome-specic red uorescent marker Lysotracker
to label treated cells. Confocal microscopy analysis showed the
presence of calcein (Fig. 2A) in the same vesicles that were
labeled by Lysotracker (Fig. 2B) proving that calcein-loaded
liposomes reached lysosomes according to our hypothesis. In
the merged image, the yellow uorescence showed the co-
localization of calcein and lysosomal compartments (Fig. 2C).
Based on several trials, that we performed in the same
experimental conditions, we have estimated a good efficiency
for our system. For each performed assay, calcein loaded cells
were counted in culture chambers. Calcein containing cells
At this stage of our work, liposomes containing Chol-M6P
appears to be promising vector in selective targeting of lyso-
somes. Experiments in vitro and in vivo aimed at demonstrating
the potential application of the system as pharmaceutical
carrier in ERT and in anticancer therapy are being planned.
Acknowledgements
This work was supported by Genzyme Corporation.
Notes and references
1 R. Castino, M. Demoz and C. Isidoro, J. Mol. Recognit., 2003,
16, 337.
2 T. Braulke and S. Bonifacino, Biochim. Biophys. Acta, 2009,
1793, 605.
¨
3 T. Lubke, P. Lobel and D. E. Sleat, Biochim. Biophys. Acta,
2009, 1793, 625.
4 M. L. Reitman and S. Kornfeld, J. Biol. Chem., 1981, 256,
11977.
5 A. Waheed, A. Hasilik and K. von Figura, J. Biol. Chem., 1981,
256, 5717.
6 A. Varki and S. Kornfeld, J. Biol. Chem., 1980, 255, 10847.
Fig. 1 Confocal microscopy analysis of NIH-3T3 treated cells. (A)
Calcein visualized in green; (B) liposome visualized in red; (C) co-
localization of calcein and liposome within individual cells (yellow).
The complex are mainly found at peri-nuclear position and this is
particularly evident in the inset of panel C, where nuclei were coun-
terstained in blue using TO-PRO-3.
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7 M. L. Reitman and S. Kornfeld, J. Biol. Chem., 1981, 256, 24 G. Angelini, M. Pisani, G. Mobbili, M. Marini and
4275. C. Gasbarri, Biochim. Biophys. Acta, 2013, 1828(11), 2506.
8 A. Waheed, A. Hasilik and K. von Figura, J. Biol. Chem., 1982, 25 T. M. Allen, M. S. Newman, M. C. Woodle, E. Mayhew and
257, 12322. P. S. Uster, Int. J. Cancer, 1995, 62, 199.
9 A. Kaplan, D. T. Achord and W. S. Sly, Proc. Natl. Acad. Sci. U. 26 G. A. Grabowski and R. J. Hopkin, Lancet, 2003, 372, 1263.
S. A., 1977, 74, 2026.
27 K. Lee, X. Jin, K. Zhang, L. Copertino, L. Andrews, J. Baker-
Malcolm, L. Geagan, H. Qiu, K. Seiger, D. Barngrover,
J. M. McPherson and T. Edmunds, Glycobiology, 2003,
13(4), 305.
10 S. Kornfeld, Annu. Rev. Biochem., 1992, 61, 307.
11 R. Pohlmann, M. W. Boeker and K. von Figura, J. Biol. Chem.,
1995, 270, 27311.
¨¨
¨
12 D. E. Sleat and P. Lobel, J. Biol. Chem., 1997, 272, 731.
13 X. Ni, M. Canuel and C. R. Morales, Histol. Histopathol.,
2006, 21, 899.
28 T. Kirkegaard and M. Jaattela, Biochim. Biophys. Acta, 2009,
1793, 746.
¨¨
¨
29 G. Kroemer and M. Jaattela, Nat. Rev. Cancer, 2005, 5, 886.
14 A. Nykjaer, E. I. Christensen, H. Vorum, H. Hager, 30 P. Boya, K. Andreau, D. Poncet, N. Zamzami, J.-L. Perfettini,
¨¨
¨
C. M. Petersen, H. Roigaard, H. Y. Min, F. Vilhardt,
L. B. Moller, S. Kornfeld and J. Gliemann, J. Cell Biol.,
1998, 141, 815.
D. Metivier, D. M. Ojcius, M. Jaattelaand and G. Kroemer, J.
Exp. Med., 2003, 197(10), 1323.
31 K. Roberg, K. Kagedal and K. Ollinger, Am. J. Pathol., 2002,
161, 89.
32 B. Turk, I. Dolenc, V. Turk and J. G. Bieth, Biochemistry, 1993,
32, 375.
15 Y. Oka, L. M. Rozek and M. P. Czech, J. Biol. Chem., 1985,
260, 9435.
16 J. X. Kang, Y. Li and A. Leaf, Proc. Natl. Acad. Sci. U. S. A.,
1997, 94, 13671.
ˇ
33 B. Turk, V. Stoka, J. Rozman-Pungercar, T. Cirman, G. Droga-
ˇ ˇ
17 S. Kornfeld and I. Mellman, Annu. Rev. Cell Biol., 1989, 5,
483.
Mazovec, K. Oresic and V. Turk, Biol. Chem., 2002, 383, 1035.
¨¨
¨
34 G. Kroemer and M. Jaattela, Nat. Rev. Cancer, 2005, 97(5),
18 M. E. Wowk and J. A. Trapani, Microbes Infect., 2004, 6, 752.
886.
19 C. R. Brunetti, K. S. Dingwell, C. Wale, F. L. Graham and 35 A. Koshkaryev, A. Piroyan and V. P. Torchilin, Cancer Biol.
D. C. Johnson, J. Virol., 1998, 72, 3330. Ther., 2012, 13(1), 50.
20 F. Blanchard, S. Raher, L. Duplomb, P. Vusio, V. Pitard, 36 M. Mammen, E. I. Shakhnovich, J. M. Deutch and
J. L. Taupin, J. F. Moreau, B. Hoack, S. Minvielle,
Y. Jacques and A. J. Godard, Biol. Chem., 1998, 273, 20886.
21 D. Metcalf, Stem Cells, 2003, 21, 5.
G. M. Whitesides, J. Org. Chem., 1998, 63, 3821.
37 C. Sou and E. Tsuchida, Biochim. Biophys. Acta, 2008, 1778,
1035.
22 P. Bruni, O. Francescangeli, M. Marini, G. Mobbili, M. Pisani 38 K. von Figura, A. Waheed and A. Hasilik, J. Biosci., 1983, 5,
and A. Smorlesi, Mini-Rev. Org. Chem., 2011, 8, 38.
23 M. Pisani, G. Mobbili, I. F. Placentino, A. Smorlesi and
P. Bruni, J. Phys. Chem. B, 2011, 115(34), 10198.
19.
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