Chapter 1 General Introduction
1.6 Lysosome-Targeting Nanocarriers for Lysosomal Storage Disorder Therapy 19
1.6.2 The Trafficking of MSNs in Living Cells
The cell membranes are usually the most important barrier for intracellular drug
delivery. Clear understanding of the pathways for the cellular internalization of MSNs is
a significant for many of its applications in biomedicine and biotechnology. There are
various pathways for internalization of external materials in mammalian cells. In general,
these mechanisms can be divided in two categories: pinocytosis and phagocytosis (Figure
1.12).50 The cells can utilize any of these internalization process depending on the size of the particles. The cellular uptake of small particles (< 200 ─ 300nm) such as MSNs is
usually involved in endocytosis for the majority of cases.51 The mechanisms of
endocytosis that transport the MSNs into the cells include clathrin-dependent,
caveolin-dependent, receptor-mediated, and clathrin- and caveolin-independent processes (Figure
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1.12).50 So far there is no specific endocytic pathways that either MSNs or functionalized
MSNs materials will follow when they are internalized by cells. Hence thorough
understanding and controlling the internalization pathway is of extreme importance in the
development of drug delivery platforms. The endocytic pathways of MSNs with different
functional groups have been studied (Table 1.2).50 In addition to the different
functionalization, the MSNs with various surface charge, shape or size also have effect
on the cellular uptake.50
After the MSNs overcome the cell membrane barrier and enter cells by endocytic
pathways, the series of events can be divided into the following sequence: the MSNs are
first transported to primary endosomes followed by transport to sorting endosomes. And
then, a fraction of MSNs are directed back to the cell exterior through recycling
endosomes, while the remaining fraction is transported to secondary endosomes that fuse
with lysosomes. And then the MSNs escape from the endolysosomes into the cytosolic
compartment (Figure 1.13).50 On the other hand, MSNs with different surface properties
could quickly escape the early-endosomes before they reach the lysosomes. For example,
the effect of the surface charge of MSNs were studied by S.-Y. Lin et al..52 The
FITC-MSNs with the diameter of 150 nm separately modified with different functional groups
(AP-, GP-, GEGP-, and FAP-) have the different surface charge and 50% effective dose
(ED50) as shown in Table 1.3.52 The results exhibit the more negatively charged FITC-
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and AP-MSNs appear to be able to escape from endosomes within 6 hours, while those
with more positive surface charge, such as GP-, GEGP- and FAP-MSNs, remained
trapped within endosomes (Figure 1.14)52. This behavior demonstrate the more negatively
charged materials have the better Proton Sponge effect or buffering capacity which is
important for the endosome escape.53 The proton sponge effect implies that the weak acid
or basic compounds such as polyethyleneimine (PEI) buffers the protons being pumped
into the lysosomal compartment by the v-ATPase (proton pump). This results in
heightened pump activity, leading to the accumulation of a Cl─ and a water molecule for
each proton that is retained; ultimately, this leads to osmotic rupture of the endosome.54
The negatively charged MSNs by surface attachment of phosphonate groups (MSN-PP)
and positively charged MSNs by conjugating quaternary ammonium groups (MSN-TA)
to FITC&RITC@MSN were also researched by C.-Y. Mou et al.40 The results suggest the
presence of positively charged TA in the cytoplasm, but negatively charged
MSN-PP is probably trapped in the endosome/lysosome analyzed by the pH detection (Figure
1.15) and confocal images (Figure 1.16).40 The MSNs coated with polyethyleneimine
(PEI) for the delivery of siRNA and DNA construct is also reported by Andre E. Nel et
al.54 PEI (10k)-coated MSN (100-130 nm in diameter) is a versatile delivery system that
can facilitate cellular uptake to increase drug delivery payload and also be utilized to
improve nucleic acids delivery into cytoplasm with the proton sponge effect. David
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Oupicky et al. reported the polycation- (PDMAEMA or PDEAEMA) and PEG-coated
mesoporous silica nanoparticles were able to successfully deliver plasmid DNA and
siRNA in cell culture.55 Shi Zhang Qiao et al. also reported that the
poly-L-lysine-functionalized large-pore MSNs with cubic mesostructured have a true potential for
delivery of nucleic acids into HeLa cells for gene therapy applications.56 The effect of
different sizes and surface charge of the naked MSNs on the cellular trafficking were also
studied by Ciro Isidoro et al.57 They found the 10 nm naked MSNs can quickly
accumulate in lysosome in 5 minutes with the almost 98% co-localization, while more
90% of MSNs escaped the lysosomes at 30 minutes (Figure 1.17)57. Moreover, the
R6GFITCMSNs with post modification of PEG (110 nm in diameter, surface charge :
-6.76 mV) are site-specifically delivered into lysosomes (Figure 1.19).58 The effect of
spherical mesoporous silica (MS) nano- and microparticles with the treatment of
extraction (E-MS) or calcination (C-MS) on the intracellular localization was also
investigated by Yaping Li et al.59 The results qualitatively indicate the intracellular
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distribution of RhB-labeled MS-1, 2, and 3 particles (190 nm, 420 nm, and 1220 nm) in
lysosomes, but there is a very limited number of the E-MS-3 can enter the MDA-MB-468
cells and locate in lysosomes, compared to the E-MS-1 and E-MS-2 with a much larger
number in lysosomes. However, the behavior of the C-MS is largely different from that
of the E-MS. Only a limited number of C-MS particles can go into cells and accumulate
in lysosomes, even at the particle size of 190 nm (Figure 1.20).59 From the above research,
we found the MSNs reaching the lysosomes involve the various factors such as size, shape,
surface charge, surface functionalization, and even the method of surfactant removing.
Thus, designing a lysosome-targeting MSNs by adjusting these elements is very
ineffective. In this study, we would modify the MSNs with a lysosome targeting peptide.
If the peptide could successfully deliver MSNs into the lysosomes in living cells by a
biological mechanism, that would be a more convenient and effective manner for
targeting lysosomes of nanoparticles.
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Figure 1.12. Pathways of mesoporous silica nanoparticles for entry into cells. Large
particles are internalized by phagocytosis, whereas fluid uptake occurs by
micropinocytosis. In the case of MSN materials, most internalization is via endocytic
pathways. These pathways differ with regard to the nature of the surface functionalization
and structural properties of MSNs.50
Table 1.2. Endocytic pathways for the internalization of MSNs.50
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Figure 1.13. Intracellular trafficking of mesoporous silica nanoparticles. (a) MSNs are
wrapped through specific (ligand-receptor) and nonspecific (hydrophobic, Coulombic)
binding interaction. (b) Once the MSNs are internalized, depending on the endocytic
pathway, it can be delivered to intermediate compartments (e.g., caveosomes). (c) Later
these compartments are transported to early endosomes and then to sorting endosomes.
From sorting endosomes, a fraction of the MSNs are sorted back to the cell exterior
through recycling endosomes (not shown in the scheme). (d) The remaining fraction is
transported to secondary endosomes, (e) which then fuse with lysosomes. (f) The MSNs
escape the endolysosomes and enter the cytosolic compartment.50
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Table 1.3. Zeta-potentials and ED50 for cellular uptake of the MSNs.52
Figure 1.14. Confocal fluorescence images of HeLa cells stained with FM 4-64 and 40
µg/mL suspensions of (a) FITC-MSN and (b) FAP-MSN after 6 hours of introduction.
The fluorescent images (left) show the MSNs (green) and FM 4-64-labeled endosomes
(red) are shown on the right.52
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Figure 1.15. Ratiometric imaging of pH in various intracellular compartments using
confocal microscopy. HeLa cells were incubated at 37 ℃ with MSN-PP and MSN-TA
for 4 hours, respectively. The images (overlaid on bright field) of pH sensors in HeLa
cells showing (a) MSN-PP, and (b) MSN-TA.40
Figure 1.16. Confocal microscopy analysis of (a) MSN-PP and (b) MSN-TA in HeLa
cells. The living unfixed cells were co-treated with endosome-specific marker FM 4-64
(5 µg/mL) and analyzed by confocal microscopy for an endosomal co-localization image.
The fluorescent images show the MSNs (green, FITC and red, RITC) and FM
4-64-labeled endosomes (blue).40
(a) (b)
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Figure 1.17. Confocal images of 10 nm mesoporous silica nanoparticles with lysosomes.
Cells adherent on coverslips were preincubated for 10 minutes with Lysotracker Green or
Red, then washed and incubated with nanoparticles, and imaged at 1, 5, and 30 minutes.57
Figure 1.18. Comparison of uptake and intracellular localization of 50 nm mesoporous
silica nanoparticles functionalized or not with either COOH or NH2 groups in SKOV3
and NIH-OVCAR cells after incubation times of one and 24 hours with 20 µg of
nanoparticles.57
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Figure 1.19. Intracellular distributions of R6G-FITC-MSNs as compared to LysoTracker
Blue DND-22. Bar, 10 µm.58
Figure 1.20. The intracellular localization of spherical MS nano- and microparticles with
different sizes within lysosomes of MDA-MB-468 cells. (a) 1 (190 nm), (b)
E-MS-2 (4E-MS-20 nm), (c) E-MS-3 (1E-MS-2E-MS-20 nm), (d) C-MS-1 (190 nm), (e) C-MS-E-MS-2 (4E-MS-20 nm), and (f)
C-MS-3 (1220 nm).59
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