This document summarizes research on synthesizing hollow mesoporous silica nanoparticles (HMSN) functionalized with poly(ethylene glycol) (PEG). HMSN were first synthesized then aminated. Monomethoxy PEG was activated and conjugated to the aminated HMSN via reaction. The PEGylated HMSN were characterized using techniques like SEM, TEM, FT-IR, TGA, XRD, and zeta potential analysis. Doxorubicin was loaded into the PEGylated HMSN and showed higher loading capacity and more controlled release compared to unmodified HMSN due to PEG capping of the pores.

1. Surface PEGylation of hollow mesoporous silica
nanoparticles via aminated intermediate
Research title
 Presented by:- Debasish Sahoo
 M. Pharm 1ST Year
 Department of pharmaceutics
 ISF College of Pharmacy, Moga, Punjab
Authors:-Thi Ngoc Tram Nguyen, Diem-Huong Nguyen-Tran, Long Giang Bach, Tran Hoang Du Truong,
Ngoc Thuy Trang Le, Dai Hai Nguyen
 Journal:-Progress in Natural Science:
Materials International
 Publisher- ELSEVIER
https://doi.org/10.1016/j.pnsc.2019.10.002

2. Flow of presentation
 Abstract
 Introduction
 Materials & method
 Results and discussion
 Conclusion
 Reference
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3. 1.Abstract
 A hybrid of organic/inorganic system, hollow mesoporous silica nanoparticles functionalized
with poly(ethylene glycol), was synthesized in the presence of coupling agent and aminated
ends.
 In this study, mPEG conjugated HMSN was successfully synthesized by the reaction between
aminated HMSN and activated mPEG.
 The obtained products were characterized by a set of experiments including SEM, TEM, FT-IR,
TGA, XRD, nitrogen adsorption-desorption and zeta potential.
 DOX was encapsulated and calculated for drug loading efficiency and capacity. In vitro release
behavior of DOX was also examined to evaluate the advantages of PEGylation on both loading
capacity and release pattern of the HMSN system.
 Due to the capping effect of PEG molecules over the pores in the silica structure, the synthesized
system was able to retain higher amount of cargo and establish a controlled manner when
releasing.
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4. 2.Introduction
 ADVANTAGES OF HMSN
o Superior features for delivering active molecules.
o High drug loading capacity compared to the conventional porous nano silica (PNS).
o Controllable particle size and porosity.
o High chemical stability.
o Available surface modification.
 DRAWBACKS OF HMSN
o Burst release of cargo in dispersion due to the uncapped pores, resulting in untargeted release manner
that limit the amount of drug actually reaching tumour site.
o Coating of serum proteins, opsonins and ions in a physiological environment, which influences cellular
uptake and induce agglomeration and hence clearance by the mononuclear phagocytic system (MPS).
MERITS OF PEG
o With a mechanism of the “stealth” effect of PEGylated particles involving rapid movement of the hydrated chains and
reduced opsonization due to the masking of particle's shape and surface.
o PEGylation can significantly increases the circulation time of the particles inside the bloodstream. PEGylation can
improve systemic circulation from a few hours to more than 24 h, in some cases even up to a few days.
o The presence of PEG on the surface of PNS and HMSN can play the role of capping in the loaded drug, resulting in a
much more sustainable release profile in physiological fluids.
o silica-based nanoparticles(PNS and HMSN) that are functionalized with PEG have negligible toxicity risk.
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5.  About Doxorubicin(DOX)
o Doxorubicin (DOX) approved as first line therapy for the treatment of various cancers, including breast,
ovarian, bladder, and lung.
o The antitumor effect of DOX has been associated with gene expression disruption, DNA synthesis and
replication prevention via down-regulation of topoisomerase II, and apoptosis induction via production of
reactive oxygen species.
o The most significant clinical activity of DOX is administration method dependent and cell-cycle specific,
arresting it at G0/S and G2/M through the above proposed mechanisms.
Contd….
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6. 3.Material Used and Method
o Tetraethyl orthosilicate (TEOS, 98%)
o Cetyltrimethylammonium bromide (CTAB, 99%)
o Diethyl ether (≥99.7%)
o 4-nitrophenyl chloroformate (NPC, 97%)
o Monomethoxy polyethylene glycol (mPEG, MW 5 kDa)
o Doxorubicin (DOX, 99%)
o (3-aminopropyl)triethoxysilane (APTES)
o Ammonia solution (NH3 (aq), 28%)
o Ethanol (≥99.9%)
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7. 3.1:-Synthesis of HMSN
First, sSiO2 templates were prepared
The mixture was then freeze-dried and HMSN spheres were obtained. The spheres were further immersed in acetic acid / ethanol
solution (1:1, v/v) for another 24 h then washed with deH2O to remove the CTAB.
After dialysis against deionized water (deH2O) for 4 days, a core shell structure consisting of a layer coating the sSiO2
templates was obtained.
Next, CTAB (0.05 M) was dissolved in deH2O for 15 min, then added the prepared sSiO2 and stirred for 30 min at 50 °C. A
mixture of ethanol/ammonia solution (1.43:0.05, M/M) was then added into the CTAB solution, followed by adding TEOs
solution (0.27 M) and stirring continued for another 6 h at 50 °C.
Ethanol (13.5 M), deH2O (6.0 M) and NH3 (0.38 M) were stirred together for 30 min at 50 °C. Then TEOS solution (0.29 M)
was mixed in under stirring at room temperature for 6 h. After membrane dialysis the obtained solution was freeze-dried.
Na2CO3 etching was performed by mixing the dialyzed solution with free CTAB and aqueous Na2CO3 solution (0.2 M) under
stirring at 50 °C for 9 h.
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8. After cooling down the mixture, THF (50 mL) was added and
stirred at room temperature for another 16 h. The mPEG-NPC
was obtained by precipitation using diethyl ether.
The amination of HMSN was carried out by reaction with
APTES. Briefly, a mixture of ethanol and APTES (1:4, v/v)
was added into a dispersion of HMSN in ethanol and stirred
continuously for 24 h at room temperature.
mPEG in solid state (0.01 mol, 50 g) was melted at 65 °C
in vacuum environment for 2 h, then NPC (0.012 mol, 2.42
g) was added into the melted mPEG and stirred for 5 h in a
nitrogen atmosphere.
3.2:-Synthesis of mPEG-NPC and HMSN-NH2
PEGylation of HMSN (HMSN-mPEG)
Then, dialysis and lyophilization were used to collect the
HMSN-NH2.
A solution of mPEG-NPC in deH2O was also prepared. Then,
mPEG-NPC solution was added into the HMSN-NH2 solution
in a dropwise manner under continuous stirring for 2h. The
final product of HMSN-mPEG was collected by dialysis
against distilled water.
First, HMSN-NH2 (50 mg) was dissolved in 10 mL of
deH2O.
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9.  XRD
o XRD measurement was performed under Cu/Ka
radiation, scanning rate set at 4°/min (λ=0.154056 nm,
40 kV, 40 mA) (Bruker D2 Phaser diffractometer,
Germany).
 TGA
o Thermal gravimetric analysis (TGA) was
performed with a TGA Analyzer under a nitrogen
flow. The heating rate was set at 10°C per minute
and temperature ranged from 30 °C to 800 °C.
 FT-IR
o FT-IR was carried out by Bruker Equinox 55 FTIR
spectrometer, using KBr pellet method.
 TEM
o A TEM instrument operating at an accelerating
voltage of 300 kV was employed to image the
particle size and morphology of samples.
o TEM samples were prepared by allowing a
carbon-copper grid containing a drop of solution
in deH2O to dry for 10 min.
3.3:-Characterization
 ZETA POTENTIAL
o Zetasizer nano ZS (SZ-100, Horiba, Kyoto, Japan)
was used to determine the zeta potential of the
product, set at 37 °C and 532 nm wavelength.
 N2 adsorption-desorption isotherms
o A TRISTAR 3000 analyzer was employed to produce
N2 adsorption-desorption isotherms at 77K under
continuous adsorption conditions.
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10. To load DOX into HMSN-mPEG, equilibrium dialysis method was employed. First, 20 mg of HMSN-mPEG was
dissolved in 8 mL of deionized H2O.
Then, DOX solution at 1000 ppm concentration was added into the mixture, which was directly followed by stirring at
room temperature.
After 24 h, the loaded particles were dialyzed against deionized water for 6 h.
The water was changed until no free drug was detected by UV–Vis in the solution outside the dialysis membrane.
Free DOX of two series of known concentrations (5–50 ppm and 0–5 ppm) was dissolved in deH2O. Absorbance of these
samples was measured at 570 nm using a UV–Vis spectrophotometer to establish the standard curve, which is used to
calculate the concentration of unloaded DOX.
DOX loading efficiency (DLE) and loading capacity (DLC) were calculated using the equations below:
3.4:-Preparation of HMSN-mPEG/DOX and drug loading
capacity
20 (mg):-the initial amount of DOX used in the
loading experiment
Wnon-DOX:-total amount of unloaded DOX
detected in the dialysis solution
Wparticles:- the dried weight of the carrier system.
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11. o The release profile of the loaded HMSN-
mPEG/DOX was assessed in PBS buffer (0.01
M) at physiological temperature (37°C) using
dialysis method.
o First, a dialysis bag containing 1 mL of HMSN-
mPEG/ DOX suspended in PBS was immersed in
a vial containing 20 mL of the release medium.
o The vial was then subjected to continuous
shaking at 100 rpm and 37°C in an orbital shaker
bath.
o At pre-specified intervals, 2 mL of the release
medium was substituted by an equal volume of
fresh medium. The collected samples were
analyzed by UV–Vis spectroscopy.
3.5In vitro release study
Fig:-In vitro release profile of HMSN (triangle) and
HMSN-mPEG (round) at pH 5.5. Each marked point
corresponds to 0, 1, 3, 6, 9, 12, 24, 36, and 48 h.
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12.  SEM AND TEM
o SEM and TEM imaging revealed the size
the morphology of the obtained particles.
o Both the HMSN and HMSN-mPEG were
shown to be spherical in shape and no
major change in the sizes was seen.
o In average, the diameter of the naked
HMSN was 134.8 ± 0.3 nm, while that of
HMSN-mPEG was found out to be 149.3
± 0.9 nm.
o However, the shell of the PEG-coated
particles was relatively thicker than the
uncoated ones, which is reasonable since
PEG has been known to thicken the size of
particles.
4.Results and discussion
Fig:-SEM (a, c), TEM images (b, d) and size distribution
histograms for HMSN and HMSN-mPEG (b’, d’), respectively.
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13. o Using X-ray diffraction can reveal information about the
crystalline structure of materials. The XRD patterns
shown in figure confirmed the amorphous nature of silica
particles, even after PEGylation since low molecular
weight PEG was used as the coating, while crystalline
phase of PEG only existed, and its molecular weight
reached 800 or above.
o In Fig. c and d, the specific characteristic peak of APTES
which appeared at values of about 2θ=14° could be
clearly observed in the diffractograms of HMSN-NH2
and HMSN-mPEG,
o Silica peak with much lower intensity that is overlapped
with another characteristic peak of APTES was observed
as well.
o After PEGylation, the XRD spectrum of HMSN-mPEG
showed no significant difference as compared to HMSN-
NH2, indicating that conjugated mPEG has no impact on
the structure of silica. The samples were also prepared
with high purity, as indicated by the lack of other peaks.
 XRD
Fig:-Large-angle powder XRD patterns of HMSN
(a), APTES (b),
HMSN-NH2(c),
HMSN-mPEG (d).
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14. Fig:-FTIR spectra of HMSN-mPEG (a) mPEG
(b), and HMSN (c).
o FT-IR spectra of (a) HMSN-mPEG, (b) mPEG and (c)
HMSN.
o The successful conjugated of mPEG onto HMSN was
indicated by the presence of characteristics peaks of both
mPEG and HMSN in the final product.
o The strong peaks representing the stretching vibrations of Si–
O–Si (1088 cm−1), asymmetric bending and stretching of Si–
OH (958 cm−1 and 800 cm−1) were observed in the spectrum
of HMSN, though in the mPEG-HMSN spectrum, the peaks
were overlapped with the signals from the mPEG coat.
o Besides, a peak attributed to the OH stretching of water
present in HMSN at 1636 cm−1 and a broad band around 3400
cm−1 recognized as the OH stretching of the silanol group
were observed in both spectra. In terms of mPEG, its presence
in the synthesized particles was clearly shown by the C–H
stretching at 2884 cm−1 and bending at 1468 cm−1.
o The introduction of PEG onto the particles surface is therefore
verified by these observations.
 FT-IR spectra 13

15. Fig:-Zeta potentials of HMSN and HMSN-mPEG,
respectively
o The change in surface charge of HMSN after mPEG
conjugation is shown in Fig. mPEG molecules with
ethylene oxide groups could contribute more positive
charge to the particles.
o This is the case of the zeta potential of HMSN and HMSN-
mPEG, in which the former having a negatively charged
surface of −26.8 mV due to the Si–OH, and the latter
having positive charge of 6.8 mV.
 ZETA POTENTIAL 14

16. Fig.-Thermogravimetric analysis for HMSN
(solid line), mPEG (dotted line) and HMSN-
mPEG (dashed line).
o Fig. presents the TGA curves of mPEG, HMSN and
HMSN-mPEG.
o The initial loss at temperature below 100 °C in both
HMSN and HMSN-mPEG samples could be attributed
to the evaporation of water residues from the
hydrolysis reaction during preparation.
o In the range of 100–800 °C, the total weight loss of
HMSN-mPEG was 21.2%, which was much higher
than that of HMSN (9.1%).
o This difference in weight loss could be accounted for
the amount of conjugated mPEG (about 12.1%).
o The functionalized sample also offered better stability
to the polymer than pure mPEG, which is shown to
have a complete degradation around 600°C. Together
with FT-IR and Zeta potential, the data from TGA
traces confirmed the coating of mPEG onto HMSN
surface.
 Thermogravimetric analysis
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17. Fig.-Nitrogen adsorption–desorption isotherms
of HMSN-mPEG.
o These are typical for mesoporous materials,
confirming that even after mPEG coating, the silica
nanoparticles still retained their porous nature.
o The specific surface area was also found out to be
527.6m2/g, lower than that of naked HMSN
(983.7m2/g), which could be accounted for by the fact
that mPEG conjugation may have shielded over the
pores.
 Nitrogen adsorption–desorption isotherms
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18. o The conjugation of HMSN with mPEG has been an
improvement to the system, resulting in various
development, including drug loading enhancement and
more controlled release.
o These findings prove the potential as a nanocarriers of
mPEGylated HMSN. Furthermore, this study also
approves the benefits of surface functionalization in
delivery system development.
 CONCLUSION
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19. o Nguyen TN, Nguyen-Tran DH, Bach LG, Du Truong TH, Le NT, Nguyen DH. Surface
PEGylation of hollow mesoporous silica nanoparticles via aminated intermediate. Progress
in Natural Science: Materials International. 2019 Dec 10.
 Reference 18