E.K. Efthimiadou, et al.
Journal of Drug Delivery Science and Technology 55 (2020) 101481
solvent was removed in vacuum. The resulted product (Fig. S1B), was
were mixed in dry DMF and left under stirring at 0 °C for 30min.
Afterwards, the mixture of activated fluorescein was added in the
mixture NCs, under nitrogen, and the final mixture was left for over-
night reaction. In order to purify the final product, the FITC functio-
nalized NCs mixture was centrifuged and washed with DMF three times.
The functionalization was further confirmed by confocal microscopy
after FITC conjugation (Fig. S1B).
purified by recrystallization from a 1:2 ethyl acetate/hexane mixture.
1
Yield: 2.62 g, 98%). H NMR (CDCl
3
): 6.7 (sb, 2H, –NH), 5.72 (d, 2H,
), 2.85 (q, 4H,
): 168.9 (C]O), 139.3
C]C), 119.1 (C]C), 39.6 (-NHCH), 37(-SCH), 19.9 (-CH ).
=
–
CH, cis), 5.35 (d, 2H, =CH, trans), 3.61 (q, 4H, -NHCH
2
SHCH ), 1.96 (s, 6H, -CH ). C NMR (CDCl
2 3 3
1
3
(
3
2
2
.5. Synthesis of fitc/folic acid functionalized NCs (tNCs)
2
.5.5. Synthesis of yolk-shell PMMA@P(MMA-co-HPMA-co-DS-co-DVB)
Fe @APTES@FA/FITC (tNCs)
In a mixture of DMF (1 ml) and Et
(MMA-co-HPMA-co-DS-co-DVB)Fe @APTES@FITC NCs were dis-
.5.1. Synthesis of PMMA seed-core particles
In a typical emulsifier free radical polymerization, 1 ml of monomer
3 4
O
3
N (10 μl), 4 mg of PMMA@P
MMA was added to 12 ml of distilled water in a 25 ml spherical flask.
The flask was then placed under nitrogen over a magnetic stirrer and
the temperature was increased to 80 °C. When temperature reached
3 4
O
persed. In another mixture of DMSO (0.5 ml) and DIC (50 μl) 3.4 mg of
folic acid was dissolved under nitrogen and the solution was left for
30 min under stirring, in order for the reaction to be completed. The
suspended NCs were then added dropwise in the mixture, which was
left overnight under stirring. The reaction mixture was centrifuged,
aiming at the isolation of the solid material. The unconjugated folic acid
remained in the supernatant and its percentage was determined by
UV–Vis via the standard curve method. From the difference between
the final and initial concentration of folic acid in the supernatant, it was
possible to calculate the amount that actually conjugated onto the NCs
(0.39 μg/mg of the polymer).
8
0 °C and after 30 min of nitrogen supply, KPS is added (2% of the
monomers). After 20 h of the polymerization, the resulted polymer was
purified by centrifugation (3 times at 10000 rpm for 10 min).
2
.5.2. Synthesis of PMMA@P(MMA-co-HPMA-co-DS-co-DVB) core-sell
NCs
The PMMA@P(MMA-co-HPMA-co-DS-co-DVB) spheres were pre-
pared by emulsion copolymerization of methyl methacrylate (MMA), N-
Hydroxy propyl methylacrylamide (HPMA), and N,N'-(disulfanediylbis
(
(
ethane-2,1-diyl))bis-(2-methyl acryl amide) (DS). Divinyl benzene
DVB) served as a crosslinker. According to our procedure, 0.3 g of
2.6. Loading and release study
seeds were dispersed in a solution of water/ethanol (25:2) and in the
dispersant 60 mg of HPMA, 640 μl of MMA and 300 μl of crosslinker
DVB 45 μl and 60 mg of DS were added; the mixture was left under
stirring for 2 h. Following, the spherical vial was covered by a septum
and the solution was stirred for additional 30 min under nitrogen.
Finally, 30 mg of KPS (2% w/w of monomers) was used as the initiator
in order for the polymerization to start. The reaction proceeded for
In order to investigate the tNCs loading capacity (LC%) and their
encapsulation efficiency (EE%) we used the anthracycline drug DOX as
a model drug. The procedure is as follows; equal amounts of tNCs
(5 mg) and DOX (5 mg) were suspended in phosphate buffer saline
(PBS, 5 ml) at pH = 7.4. The mixture was left under gentle stirring for
three days in the dark, at room temperature. The mixture was then
isolated and the supernatant was removed. In a second step of pur-
ification, the nanocontainers were resuspended in PBS (5 ml) and then
the mixture was centrifuged again. The second supernatant was re-
moved also. This purification method was repeated twice. The drug
release was assumed to start as soon as the containers were suspended.
The concentration of loaded NCs and released DOX from tNCs was
quantified by using UV–vis spectroscopy, while the loading of DOX was
further confirmed by confocal microscopy (Fig. S2A).
12 h at 70 °C. The resulting product was washed by three cycles of
centrifugation with deionized water and then the solid was dried at the
vacuum. Hollow microspheres are prepared during the multi shell
production due to Oswald mechanism. The cavity is created due to the
migration of the seed polymer, towards the polymer surface as the
polymerization proceeds, as supported in our previous work [47,48].
2
.5.3. Synthesis of PMMA@P(MMA-co-HPMA-co-DS-co-DVB)@Fe
core-shell NCs
The isolated hollow NCs (100 mg) were dispersed in a solution
consisting of ethylenoglycol (EG) and water (EG/H O = 65/35 ml).
The mixture remained for 1 h under nitrogen, while stirring, and then
00 mg of HETM and 80 mg of FeCl ·4H O were added. In continuation,
after 20 min of stirring 80 mg of KNO was introduced in the reaction
3 4
O
2.7. Characterization
2
2.7.1. Morphological and structural characterization
1
3
2
The following SEM and TEM images depict the resulting functio-
nalized tNCs. The size of the tNCs is approximately 308 ± 16.21 nm
and the sample is monodispersed. The NCs core surface is smooth, while
after the shell growth the surface increases in size and becomes
rougher. The observed outer layer cavity of the tNCs is due to shell
collapse stemming from an internal cavity that was created during shell
formation [11,12,16,46,49]. The cavity inside the spheres is verified
through TEM images (Figs. 1 and 2). The black dots on the surface of
the nanocontainers (Fig. 1D) are the embedded iron oxide nanoparticles
with a mean size of 22.43 ± 3.93 nm.
3
solution and the mixture was heated up at 80 °C for 4 h. The reaction
was left to cool down at room temperature and the resulted magneti-
cally functionalized hollow NCs were purified by water via cen-
trifugation [12,49]. After the incorporation of magnetic nanoparticles
the structure of the microspheres has no considerable changes, except
the fact that more raspberry-like structures are observed. Full char-
acterization of the incorporated mNPs is included in the Supplementary
file.
According to DLS study the NCs behave in the desired way; when pH
is increased the NCs’ size augments significantly which indicates pH
sensitivity of the structure. This phenomenon can be justified by the
protonation/deprotonation equilibrium (pKa of Hydroxyl HPMA group
is almost 6). In lower pH values, the hydroxylic acid groups are pro-
tonated (Fig. S3) whereas at higher pH values they are deprotonated
causing the swelling of the NCs due to repulsive interactions.
According to these findings, the NCs can function as an ideal pH
responsive drug delivery system, because the NCs’ protonation in this
environment can lead to the release of the encapsulated drug (Fig. S2B).
As has already mentioned different interactions have been performed
like hydrogen bonding and electrostatic interactions. Based on that at
2
.5.4. Synthesis of yolk-shell PMMA@P(MMA-co-HPMA-co-DS-co-DVB)
Fe @APTES@FITC core-shell NCs
To start with, 10 mg of the iron functionalized NCs were dispersed
in a mixture solution of water/ethanol (10:50). Following, 100 μl
APTES was added dropwise, then a catalytic amount of NH was also
3 4
O
3
added and the mixture was left under stirring for 4 h, at room tem-
perature. The silica coated magnetic NCs were then collected by cen-
trifugation, washed with EtOH twice and centrifuged again. The iso-
lated material was dried under vacuum and then 4 mg were re-
dispersed in dry DMF with catalytic amount of triethylamine (Et
another vial, 2 mg of fluorescein isothiocianate (FITC) and 50 μl of DIC
3
N). In
4
Efthimiadou
