10 1021@acsabm 9b00203

Subscriber access provided by UNIV OF SOUTHERN INDIANA
Article
Biodegradable alginate polyelectrolyte capsules
as plausible biocompatible delivery carriers
Sathi Roy, Nancy Elbaz, Wolfgang J Parak, and Neus Feliu
ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00203 • Publication Date (Web): 11 Jun 2019
Downloaded from pubs.acs.org on July 17, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,

Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.
Page 1 of 27 ACS Applied Bio Materials
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 ACS Paragon Plus Environment
ACS Applied Bio Materials Page 2 of 27
1
2
3 Abstract
4
5
6 Polyelectrolyte capsules made of different biodegradable and non-biodegradable polymers can be
7 designed as systems for effective encapsulation and delivery of compounds. The objective of this
8
9 work was to synthetize biocompatible and biodegradable capsules '= 1 µm) by the Layer-by-Layer
10 (LbL) approach using alginate (ALGI) and Poly-L-arginine (PARG) polyelectrolytes with a pH-
11 sensitive outer layer of EUDRAGIT L 100 (EuL) polymer. Those capsules were loaded with
12
13 curcumin as model therapeutic drug, which possesses antioxidant, anti-inflammatory, and
14 anticancer activity. Encapsulation of drugs inside capsules protects its therapeutic activity and
15 increases its bioavailability. We report the capsule stability, loading efficiency, drug release, as
16
17 well as capsule degradation studies as a function of pH. Furthermore, in vitro biocompatibility
18 studies of capsules including cell viability and uptake studies were performed using HeLa cells.
19 The here synthesized capsules exhibited good reproducibility, spherical shape and highly
20
21
monodispersed. The capsules showed good loading efficiency and drug release profile dependent
22 upon pH environment. The in vitro studies indicate that the capsules exhibited acceptable
23 biocompatibility and highly internalized by cells. Our study thus suggests that alginate LbL
24
25
capsules could be used as efficient drug carrier with effective encapsulation and successful in vitro
26 release of cargo in the cell.
27
28
Key words: Biocompatible, biodegradable polyelectrolyte capsules, layer-by layer assembly, drug
29
30 release, nanomaterials.
31
32
1. Introduction
33
34
35 In recent years, capsules made out of different biodegradable and non-biodegradable
36
polyelectrolytes by the Layer-by-Layer (LbL) technique have gain interest and been reported as an
37
38 attractive approach for effective encapsulation and delivery of therapeutics.1-4 Furthermore, the
39 first in vivo approaches also have been reported.5-6 The basic characteristics of the capsules are
40 determined by several factors such as the size of the template core (which will influence the final
41
42 capsule size), the choice of polyelectrolytes used for making capsule shell, the molecules loaded
43 into the cavity of the capsules i.e molecular cargo etc. All these factors will determine and define
44 the performance of the capsules. For potential in vivo applications, the use of biocompatible
45
46 polyelectrolytes for the capsule assembly is advantageous.
47
48 In general, as stated above, the size of the capsule is predetermined by the size of the template
49
50 core.7 A wide range of size distributions from nm to mm, along with a series of different materials
51 for template core formation has been reported.8 Calcium carbonate (CaCO3) particles are well
52 established as template cores. They are suitable candidates (as core templates) for preparing
53
54 polymer capsules due to their high porosity, biocompatibility, easy to perform synthesis, the ability
55 to embed molecular cargo and the possibility of core removal using nontoxic ethylene diamine
56
57
58
59
1
2
3 tetra acetic acid (EDTA) solution, after synthesis. Monodisperse spherical CaCO3 particles of 4-12
4
5 um size can be easily synthesized by mixing different equivalent ratios of calcium chloride and
6 sodium carbonate solutions.8 The capsules prepared from these micron size particles have been
7 tested towards in vivo delivery of drugs9 and vaccines.10 However, for drug delivery applications a
8
9 smaller overall size is preferable. Recently, the synthesis of smaller submicron size (400-500 nm)
10 CaCO3 particles has been reported in presence of comparatively toxic water-ethylene glycol or
11 water-glycerol medium.11-14 The control over CaCO3 particle size can also be achieved by using
12
13 different biodegradable polymers in the aqueous medium during the synthesis.15
14
15 A large variety of different polyelectrolytes has been used for LbL assembly around template
16
17 cores. Apart from the frequently used synthetic polymers polystyrene sulfonate (PSS) and
18 poly(allyamine hydrochloride) (PAH),16-17 also biological molecules have been used, such as
19 polyarginine (PARG),18 proteins,19 oligonucleotides,20-21 or alginate. Alginate is a naturally
20
21
occurring anionic polysaccharide extensively found in the cell walls of brown seaweeds.22 It has
22 received attention in microencapsulation and biomedical applications due to its high
23 biocompatibility and unique physicochemical properties.23 Aqueous solutions of alginate can
24
25
easily form gels in presence of divalent cationic cross-linkers.24-25 Different macromolecules or
26 drugs can be loaded into such ionically cross-linked alginate networks.23, 26-27Furthermore, alginate
27 gels have interesting swelling properties dependent on the pH of the environment, which has been
28
extensively explored to prepare drug delivery systems enabling controlled release.28 However,
29
30 limited long-term stability is a major challenge of this system owing to the loss of mechanical
31 strength due to ion release from the matrices at physiological conditions. 22 Recently a hollow
32
silver alginate hydrogel capsule has been reported which was synthesized using CaCO3 particles as
33
34 sacrificial templet. Laser-induced remote controlled in vivo delivery of cargo from this capsule
35 achieved by taking advantage of silver nanoparticle’s response to NIR laser.29 Alginate has a pH-
36
dependent ionic nature, thus spherical capsules based on alginate also have been synthesized using
37
38 LbL assembly, resulting in capsules with improved mechanical strength and encapsulation
39 efficiency.30
40
41
42 In addition, a large variety of different compounds has been used as molecular cargo inside
43 polyelectrolyte capsules. This involve molecular probes for sensing, 31-32 oligonucleotides for gene
44 delivery,33 molecules for molecular signalling,5 or pharmaceutical agents such as doxorubicin for
45
46 drug delivery.34-36 One interesting pharmaceutical agent is curcumin. Curcumin is a naturally
47 occurring polyphenolic compound. Despite its possible therapeutic effects, the poor solubility of
48 curcumin limits it clinical applicability. For this reason, different approaches have been utilized to
49
50 enhance the solubility of curcumin, including solid dispersion techniques,37 nanoparticle
51 encapsulation38 and others, which enables the use of curcumin for treatment of several diseases.
52 For instance, Priya et al. encapsulated curcumin into carboxymethyl cellulose and casein Nano gels
53
54 and studied its potent anticancer effect in a melanoma skin cancer cell model.39 In addition,
55
56
57
58
59
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
1
2
3
4
5
6
7
8
9 Figure 1. A schematic diagram of the synthesis of biodegradable polyelectrolyte capsules loaded
10 with curcumin. Transmission electron microscope (TEM) images of curcumin-loaded (A)
11 (PARG/ALGI)2 and (B) (PARG/ALGI)2(PARG/EuL) capsules after dissolution of the template
12
13 cores are shown. Scale bars correspond to 500 nm. Here, PSS, PARG, ALGI and EuL corresponds
14 to sodium poly-(styrene sulfonate), poly-L-arginine hydrochloride, alginic acid sodium salt (ALGI,
15 Sigma) and eudragit L 100 polymers respectively.
16
17
18 Briefly, CaCO3 particles of hydrodynamic diameter dh 850 nm, as determined by dynamic light
19 scattering (DLS), were prepared by co-precipitation and were used as core templates (c.f Figure
20
21 2F, J). To prepare curcumin-loaded capsules, curcumin was co-precipitated in the CaCO3 particles.
22 The integration of the curcumin in the template core was confirmed by both, fluorescence
23 microscopy and fluorescence measurements (c.f. Figure 2A-D,). Note that the CaCO3 particles
24
25 were synthesized under the presence of PSS in order to reduce size. Next, the core particles were
26 coated with PARG and ALGI using the well-described LbL method.31 As seen in Fig. 2K-L, a
27 reversal of the surface charge between negative to positive zeta potential values could be observed,
28
29
which verified the adsorption of polyelectrolytes in each layer. Capsules of two bilayers were
30 prepared and are in the following referred to as (PARG/ALGI)2 capsules. Furthermore, in order to
31 support and preserve the capsules in harsh pH environment (e.g. promote the efficient delivery
32
33
capability through oral route), the capsules were complemented with an additional pH-sensitive
34 EuL polymer layer and are then referred to as (PARG/ALGI)2(PARG/EuL) capsules (c.f. Figure
35 2L). Finally, the template cores were removed, and the capsules were washed and stored in
36
37
sterilized water. Then, the capsule concentration was determined by confocal laser scanning
38 microscopy (CLSM) (as described in the Supporting Information (SI)), and capsules were stored in
39 dark at 4 °C until further used. The resulting capsules (with dissolved template core) exhibited dh
40
of around 885 nm ((PARG/ALGI)2) and 916 nm ((PARG/ALGI)2(PARG/EuL)) respectively.
41
42 Capsules were uniform, well dispersed, and presented spherical shape as shown by TEM and
43 CLSM images (c.f. Figure 2 and the SI for further synthesis details). A summary of the capsule
44
characterization including the colloidal stability and surface charge of the capsules determined
45
46 using DLS in Milli-Q water, are presented in the Table 1. The found size variation between DLS
47 measurement and TEM image analysis could be attributed to little deformation or flattening of
48
spherical capsules under dry condition for TEM imaging. When these capsules are dispersed in
49
50 solution, they are prone to get sediment within very short time due to their size 42-43.
51 Experimentally, in this particular case, this effect could be monitoring following the fluorescence
52 intensity of capsules over time. Indeed, the fluorescence intensity of capsule solution starts to
53
54 drops down within just 5 min upon capsule dispersion. However, further homogenization with
55 pipetting brings back its initial fluorescence. (c.f Figure S9A, B), thus this results indicates that the
56
57
58
59
1
2
3 decrease of fluorescence was related to the sedimentation effect of the capsules and not do
4
5 degradation. Therefore, during each spectroscopic analysis all capsule solutions were mixed
6 properly before measurements.
7
8
9
10
11
12
13
14 (A) (B) (C) (D)
15 2x107 2x107
ex 420 nm ex 420 nm
16 ex 488 nm
ex 488 nm
Icur [a.u.]
17
Icur [a.u.]
1x107 1x107
18
19 5x106 5x106
20
0
21 5 +m 5 +m 0
600 700 600 700
22 (E) (G)
[nm]
(H)
[nm]
(F)
23 40 40
40
24
25
N(dh)
N(dh)
N(dh)
26 20 20 20
27
28
0 0 0
29 500 nm
5x103 1x104 5x103 1x104 5x103 1x104
30 (I) (J) dh [nm] (K) dh [nm] (L) dh [nm]
31
3x105 20 20
32
33
2x105 0 0
34
N( )
[mV]
[mV]
35 1x105 -20
-20
36
37 0 e G I I L
or R G G G G u
500 nm
-100 0 100
re
RG
RG
C PA AL PAR AL AR E
I
I
G
38
Co
AL
AL
PA
PA
[mV] P
39
40
41 Figure 2. Characterization of the biodegradable (PARG/ALGI)2 and (PARG/ALGI)2 PARG/EuL)
42 capsules. (A, B) Confocal microscopy images of curcumin-loaded (A) (PARG/ALGI)2 and (B)
43
(PARG/ALGI)2(PARG/EuL) capsules after removal of the template cores. (C, D) Fluorescence
44
45 spectra Icur'N( of curcumin-loaded (C) (PARG/ALGI)2 and (D) (PARG/ALGI)2(PARG/EuL)
46 capsules after removal of the template cores at different wavelengths of excitation Nex. (E-I) TEM
47
image of curcumin-loaded (E) (PARG/ALGI)2 and (I) (PARG/ALGI)2(PARG/EuL) capsules after
48
49 removal of the template cores. (F-H) Number distributions N(dh) of the hydrodynamic diameters dh
50 of (F) PSS-CaCO3 cores, (G) (PARG/ALGI)2, and (H) (PARG/ALGI)2(PARG/EuL) capsules as
51 measured by DLS. Data are presented as number distributions N(dh). (J-L) Zeta potential
52
53 distributions N( ) of (J) PSS-CaCO3 cores, and capsules coated as function of the additional
54 PARG/ALGI layers for (K) (PARG/ALGI)2 and (L) of (PARG/ALGI)2(PARG/EuL) capsules.
55
56
DLS and measurements were carried out in Milli-Q water.
57
58
59
1
2
3
4
5 Capsule Type dTEM [µm] dh [nm] 1 [mV] PDI
6 PSS-CaCO3 -- 856 ± 29 -20 ± 5 0.15 ± 0.05
7
(PARG/ ALGI)2 1.07 ± 0.32 885 ± 34 -37.3 ± 3.7 0.23 ± 0.15
8
9 (PARG/ ALGI)2 (PARG/ EuL) 1.17 ± 0.52 916 ± 46 -39.2 ± 4 0.27 ± 0.035
10
11
12
Table 1. Colloidal properties of the capsules: The capsule diameter (dTEM) obtained from TEM
13 images, average hydrodynamic diameter dh, zeta potential , and PDI of the cores and capsules
14 with dissolved cores. The results of the dh and the are presented (as mean ± standard deviation
15
16 (SD)) from three independent batches of capsules. DLS and measurements were carried out in
17 Milli-Q water.
18
19
20 2.2 Drug loading and encapsulation efficiency of capsules
21
22
The encapsulation efficiency ( EE) and loading capacity ( LC) of curcumin loaded capsules were
23
24 evaluated by both, indirect and direct methods, which are described in the methods section. Note
25 that the spectral and photo physical properties of curcumin are highly sensitive to pH and solvent
26 conditions.44 For direct quantification of encapsulated curcumin the capsule walls were dissolved
27
28 by pronase before spectroscopy. Interference of the optical properties of curcumin with those of
29 pronase was controlled by UV-Vis absorption spectroscopy 'N( and fluorescence spectroscopy
30 ?'N( after 24 h incubation of curcumin in pronase solution at 37°, as shown in Figure S3. The
31
32 absorption A420 'N = 420 nm) was used to calculate EE and LC, see the methods section. The
33 results are presented in Table 2, indicating similar EE and LC of curcumin in both types capsules.
34
35
Spectroscopic measurements of the fluorescent signal of released curcumin after pronase mediated
36 degradation (i.e. the "direct" method) indicated EE values of approx. 70 % for both capsules. The
37
LC values found for (PARG/ALGI)2 and (PARG/AlGI)2(PARG/EuL) capsules were
38
39 approximately 15.57 and 11.24 pg curcumin/capsule as measured by the indirect method and 11.24
40 and 11.94 pg curcumin/capsule as measured by the direct method, respectively. Since, both types
41 of capsules were prepared from one initial batch of CaCO3 cores these similar results of curcumin
42
43 loading were expected. The slightly different fluorescence and consequent variance of LC
44 observed maybe attributed to the additional washing steps involved in the synthesis of the
45 (PARG/ALGI)2(PARG/EuL) capsules, which may result in the loss of encapsulated curcumin or
46
47 due to the counting errors of the capsules. However, it is important to mention that the LC results
48 of both types of capsules showed that the direct and indirect methods agree remarkably well. These
49 results are summarized in Table 2. The full set of data using UV/vis absorption spectroscopy and
50
51 fluorescence spectra are presented in the SI.
52
53
54
55
56
57
58
59
1
2
3 Capsules EE[%] LC [pg/capsule] LC [pg/capsule]
4
5 (direct method) (indirect method) direct method)
6 (PARG/ALGI)2 75.75 ± 2.25 15.57 ± 1.05 13.54 ± 1.86
7
8 (PARG/ALGI)2(PARG/EuL) 71.00 ± 2.80 11.24 ± 2.15 11.94 ± 1.37
9
10
11 Table 2. Encapsulation efficiency ( EE) and loading capacity ( LC) of (PARG/ALGI)2 and
12 (PARG/ALGI)2(PARG/EuL) capsules with curcumin (number of capsule batches n=2).
13
14
15
16 2.3 In vitro release study from different capsules
17
18
19 (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules were loaded with curcumin as model
20 drug to investigate the effect that the surface modifications (EuL layer) and the number of bilayers
21
22 of the capsules may have on the drug release kinetics. Thus, the in vitro release profile of curcumin
23 ( CR) loaded (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules was investigated by two
24 different approaches: the dialysis and the ultrafiltration method (see the methods section).45-46 For
25
26 those investigations 1 mL (nc = 4×107 capsules/mL) of (PARG/AlGI)2 and (nc = 4.6 ×107
27 capsules/mL) of (PARG/ALGI)2(PARG/EuL) capsule solution were used. In both types of the
28 capsules, the curcumin concentration used for the release study was 40 g/mL. The release profile
29
30 was evaluated at different environmental conditions: in acidic (pH 3 and pH 5) as well as in neutral
31 conditions (pH 7). Free curcumin was used as standard to correlate absorptions and intensity
32 values to curcumin concentration (c.f. Figure S4 - S6). Spectroscopic measurements of the
33
34 fluorescence signal of released curcumin as obtained after capsules were incubated at different
35 conditions over time are shown in Figure 3 and in the SI.47 The results show that (PARG/ALGI)2
36 and (PARG/ALGI)2(PARG/EuL) capsules had an opposite pH-dependent drug release profile. (c.f.
37
38 Figure 3A-B, C-D). (PARG/ALGI)2 capsules exhibited higher release of encapsulated curcumin
39 following incubation at lower pH, while (PARG/ALGI)2(PARG/EuL) capsules released more
40 curcumin after incubation at higher pH. Amount of released curcumin increased over the time.
41
42 Notably, the tendency of the results were confirmed by the two different approaches used (the
43 dialysis and the ultrafiltration method), which corroborates reproducibility of the found pH-
44 dependent drug release kinetics. Moreover, the dialysis method showed that after 48 h incubation,
45
46 the (PARG/ALGI)2 and PARG/ALGI)2(PARG/EuL) capsules exhibited a maximum CR of 50% at
47 pH = 3 environment and CR of 52% at pH = 7 environment, respectively (c.f. Figure 3A, 3B).
48
Besides, the cumulative curcumin release CR from capsules observed at 48h by the ultrafiltration
49
50 method was 70% in pH = 3 environment and 71% in pH = 7 environment for the (PARG/ALGI)2
51 and PARG/ALGI)2(PARG/EuL) capsules respectively (c.f. Figure. 3D, 3E). The two used
52
53
approaches provided similar trends in maximum CR release from (PARG/ALGI)2 and
54 PARG/ALGI)2(PARG/EuL) capsules pH 3 and 7, respectively but differed in the absolute CR
55 value. The variation can be attributed to the nature of the different methods, some loss of
56
57
58
59
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
1
2
3
4
5 2.4 Biodegradation study of capsules
6
7 The biodegradation of the capsules under different environment conditions over time was
8
9 investigated by means of changes in hydrodynamic diameter dh, zeta potential , and visual
10 inspection by confocal laser microscopy (CLSM) images (c.f Figure 4). Briefly, 10 µL of
11 (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules incubated with 100 µL 2 mg/mL
12
13 pronase in phosphate buffered saline (PBS, pH 7) and in PBS buffer at pH = 7 and pH = 3 for 2, 4,
14 6, 24, 48, and 72 h. The biodegradation was monitored in terms of dh(t), (t), and changes in
15
CLSM images of capsules over time.
16
17 The degradation kinetics of (PARG/ALGI)2 capsules was found to be pH depend. The average size
18 (dh) of capsules incubated at pH 3 initially decreased but slightly increased over time as shown by
19 the dh measurements. Furthermore, (PARG/ALGI)2 capsules looses curcumin fluorescence at pH
20
21 3, whereas it was slightly preserved at pH 7 as revealed by the CLSM images, suggesting the loss
22 of curcumin at pH 3 (c.f. Figure 4). In contrast, (PARG/ALGI)2(PARG/EuL) capsules looses
23 curcumin fluorescence at pH 7 after 48h incubation. Those results are in accordance with the pH-
24
25 dependent drug release profile (c.f. Figure 3). The dh of (PARG/ALGI)2(PARG/EuL) capsules
26 increases over time for incubation in both pH 3 and 7 buffers, which may be due to aggregation of
27 capsules. Also for both capsules a decrease in (-) ve surface charge was observed at pH 3. A
28
29 possible explanation can be address to the protonation of carboxylate group present in both ALGI
30 and EuL polymers at low pH.
31 As control, both capsules were incubated with pronase, a decreasing trend in capsule size (as
32
33 observed by the dh measurements and CLSM images) and lowering of outer charge of capsules
34 over time, indicates degradation of capsules even at short periods of time. These data suggest that
35 (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) are biodegradable capsules, but exhibit different
36
37 degradation kinetics which was found to be pH dependent. The different release profiles of
38 capsules could be attributed to the effect of the additional PARG/EuL bilayer. In an earlier study,
39 Skirtach et al. group investigated enzymatic degradation of poly(L-arginine) (PARG)/ poly(L-
40
41
glutamic acid) (pGlu) based capsule using pronase and found capsules degrades already after 60
42 min of incubation with pronase.52 Whereas, in our case after 2h incubation capsule structure starts
43 to deform and after 4h almost completely loss its fluorescence indicating degradation of the
44
45
capsule shell. The relatively slower capsule degradation kinetics observed in our study can be
46 because of the presence of an inner PSS layer, which forms during small CaCO3 core synthesis.
47 The ionic strength of the dispersion solution highly affects capsules stability. The DLS
48
49
measurement of capsule size revealed both the capsule size decreases with increasing ionic
50 strength of solution. At higher ionic concentration, polyelectrolytes are prone to coiled due to
51 screening effects of counter ions present in solution. This causes poor interaction between
52
polymers, which can affect the overall capsule structure. Although, both the capsules were quite
53
54 stable in cell culture media, as confirmed by DLS size measurement of capsules in cell media
55 without FBS after 24h incubation. (c.f. Figure S8)
56
57
58
59
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
1
2
3 agglomeration. Data correspond to (mean ± SD) of 3 measurements. (E) Confocal images of
4
5 curcumin-loaded (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules at each time point of
6 the degradation study. For imaging 10 µL of incubated capsule solution was used and around 150
7 capsule images were captured. The scale bars correspond to 5 Km.
8
9
10 2.5 Biocompatibility studies of capsules
11
12
13 In order to investigate the biocompatibility of (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL)
14 capsules, cell viability studies of HeLa cells exposed to those capsules were carried out by using
15 the traditional resazurin assay as previously reported 53-54. HeLa cells were derived from a cervical
16
17 carcinoma, and those cells have been frequently employed in toxicology studies,55-57 and thus were
18 used as model system in this work. As seen in Figure 5, the cell viability (V) results indicated that
19 both capsules types are not acutely toxic at the time and doses used. Only a slight decrease of cell
20
21
viability was observed at higher exposure concentrations of capsules/cell added.
22
23
(A) (B)
24 120 120
25
26 100
100
27
28
V [%]
V [%]
29 80 80
30
31 60 60
32 (PARG/ ALGI)2 24 h (PARG/ ALGI)2 (PARG/ EuL) 24 h
(PARG/ ALGI)2 (PARG/ EuL) 48 h
33 (PARG/ ALGI)2 48 h
40
40
34 10 100 1000 10 100 1000
35 Ncapsules / cell Ncapsules / cell
36
37
38 Figure 5. Biocompatibility study of curcumin-loaded biodegradable capsules. HeLa cells were
39 incubated with various numbers capsules per cell (Ncapsules/cell) in serum-supplemented media for
40 t = 24 h and 48 h. Cell viability V was assessed using the resazurin assay, for (A) (PARG/ALGI)2
41
42 and (B) (PARG/ALGI)2(PARG/EuL) capsules. Results are presented as percent of cell viability V
43 [%] (mean ± SD) from three independent experiments.
44
45
46 2.6 Uptake studies of the capsules
47
48 Taking advantage of the intrinsic fluorescence of curcumin, uptake studies of curcumin-loaded
49
50 biodegradable capsules were conducted in HeLa cells by flow cytometry and CLSM. Flow
51 cytometry is a commonly used strategy to monitor and quantify internalization of particles by
52 cells.58-60 Briefly, different concentrations of (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL)
53
54 capsules (exposure concentrations Ncapsules/cell) were exposed to HeLa cells for t = 24 h and 48 h in
55 serum-supplemented medium. After exposure, the curcumin fluorescence Icur in the cells was
56
57
58
59
1
2
3 monitored. Internalized of capsule into cell happens into major extend, via endocytosis process.
4
5 Capsule uptake may trigger the initial formation of membrane protrusions in their vicinity
6 followed by restructuring of these protrusions into smooth thin layer extending over the surface of
7 the capsule.61 The internalization studies by flow cytometry indicated that capsules uptake
8
9 occurred in a dose dependent manner (c.f. Figure 6). Notably, time (24 versus 48 h) did not
10 influence the uptake results, which might be due to cell proliferation. Moreover, the internalization
11 of (PARG/ALGI)2(PARG/EuL) capsules reached a plateau as seen in Figure 6B, which was not
12
13 observed for (PARG/ALGI)2 capsules. Internalization of (PARG/ALGI)2(PARG/EuL) capsules
14 was higher at lower exposure dose (25 caps/ cell, Icur = 1075 and 996 a.u. at 24 h and 48 h
15 respectively) and gradually, at higher dose a saturation in capsule uptake was observed. Whereas,
16
17 internalization of (PARG/ALGI)2 capsules was comparatively less at lower dose (25 caps/ cell, Icur
18 = 818 and 842 a.u. at 24 h and 48 h respectively) and also capsule uptake did not achieved
19 saturation limit within the range of exposed dose. In addition, the uptake was also monitored by
20
21
CLSM (c.f. Figure 7). For that, cells were exposed to capsules at the concentration of 50
22 capsules/cell for 24 h. Co-localization studies were conducted and reveled that the majority of the
23 capsules were found in the lysosomal compartments as shown by the Manders’ coefficient m1.58
24
25
The coefficient mx indicates the degree of overlap between pixels from two different fluorescence
26 channels (i.e. yellow = curcumin inside capsule and red = Lysotracker® red stained lysosomes)
27 ranging from 0 to 1. Here, 0 correlates to no overlap and 1 correlates to complete overlap. The m1
28
and m2 corresponds to the amount of capsule, which co-localized with lysosomes, and the amount
29
30 of lysosomes that co-localized with capsules respectively. The results indicate that the number of
31 (PARG/ALGI)2 capsules internalized is slightly higher than the (PARG/ALGI)2(PARG/EuL)
32
capsules. (c.f Figure 7B) Those results agree and complement the flow cytometry studies.
33
34
35 (A) (B)
36 1750
1750
37
38 1400 1400
39
40
Icur [a.u.]
1050
Icur [a.u.]
1050
41
42 700 700
43
350 350
44 (PARG/ ALGI)2 24 h (PARG/ ALGI)2 (PARG/ EuL) 24 h
45 (PARG/ ALGI)2 48 h
0
(PARG/ ALGI)2 (PARG/ EuL) 48 h
0
46 0 25 50 75 100 0 25 50 75 100
47 Ncapsules / cell Ncapsules / cell
48
49
50 Figure 6. Uptake study of curcumin-loaded biodegradable capsules. HeLa cells were exposed to
51 different concentrations of capsules (Ncapsules/cell) for t = 24 h and 48 h in serum-supplemented
52 medium. (A) (PARG/ALGI)2 capsules. (B) (PARG/ALGI)2(PARG/EuL) capsules. Cells were then
53
54 analyzed by flow cytometry and the mean curcumin fluorescence Icur in cell was determined. Data
55
56
57
58
59
1
2
3 were derived from the curves shown in Figure S10 and represent (mean ± SD) from three
4
5 independent experiments.
6
7 (A) Curcumin Lysotracker Hoechst Overlay (B)
8
9 1.0
10
Ctrl
11
12
m1
13 0.5
14
Ctrl(PARG/ALGI)2
15
16
17 0.0
(PARG/ALGI)2 (PARG/ALGI)2
18
(PARG/EuL)
19 (C)
(PARG/ALGI)2
20 1.0
21
22
23
24
m2
0.5
25
(PARG/ALGI)2
(PARG/EuL)
26
27
28
0.0
29 (PARG/ALGI)2 (PARG/ALGI)2
30 (PARG/EuL)
31
32
33
34 Figure 7. (A) CLSM images of cells exposed to (PARG/ ALGI)2 and (PARG/ ALGI)2(PARG/
35 EuL) capsules for 24 h along with controls at 50 capsules/cell added. LysoTracker-red-DND-99
36
and Hoechst were used for lysosome and nucleus staining respectively. Ctrl and Ctrl(PARG/ ALGI)2
37
38 are cells without capsules added and cell with capsules added but without membrane staining,
39 respectively. (B, C) Quantification of Manders’ coefficients m1 corresponding to the fraction of
40 capsules in co-localization with lysosomes and m2 corresponding to the fraction of lysosomes in
41
42 co-localization with capsules. Around 40 cells were analyzed from two independent experiments.
43 Scale bars corresponds to 20 µm.
44
45
46 3. Conclusions
47
48 The present work provides an assessment of the in vitro biocompatibility, stability, and
49
50 biodegradability of (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules. Capsules were
51 shown to be biodegradable. Hereby (PARG/ALGI)2 capsules degraded faster at acidic pH = 3, in
52 contrast to (PARG/ALGI)2(PARG/EuL) capsules, which exhibit better stability at lower pH.
53
54 However, both capsules were degraded in presence of pronase. In addition, the drug release studies
55 indicated reverse drug release kinetics. The maximum release profile found for (PARG/ALGI)2
56
57
58
59
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
1
2
3 eudragit L 100 (EuL, Evonik), ethylenediaminetetraacetic acid disodium salt (EDTA, Sigma),
4
5 phosphate-buffered saline (PBS, Biochrom AG), curcumin (cur, Sigma), polyvinyl pyrrolidone
6 (PVP, Mw = 55 kDa, Sigma), polyethylene glycol (Mw = 6 KDa), and sodium dodecyl sulfate
7 (Sigma) were used as received.
8
9
10 5.2 Synthesis of biodegradable (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules
11
12
13 Synthesis of PSS calcium carbonate cores
14 Calcium carbonate cores (PSS-CaCO3) 2 were prepared by mixing 0.615 mL of 0.33 M aqueous
15 solution of calcium chloride (CaCl2) and 0.75 mL of 25 mg/mL polystyrene sulfonate (PSS)
16
17 solution under magnetic stirring (1000 rpm, stirrer "Magnetrührer IKA-RO 5 power") at room
18 temperature (RT) for 5 min. Next, 0.615 mL of 0.33 M aqueous solution of sodium carbonate
19 (Na2CO3) was added to the mixture under vigorous stirring (1000 rpm, "Magnetrührer IKA-RO 5
20
21
power") and stirred for 60 s at RT. Then the solution was transferred into an Eppendorf tube of 2
22 mL. PSS-CaCO3 particles of 800 – 1000 nm size were formed. The solution was centrifuged at
23 1400 rcf for 2 min to separate the precipitate. The precipitated PSS-CaCO3 particles were then
24
25
washed three times with 1 mL Milli-Q water to remove unreacted salts and were then re-suspended
26 in 1 mL of Milli-Q water. The PSS-CaCO3 cores were used to synthesize the capsules by the layer
27 by layer (LbL) approach 62 as described in the following.
28
29
30 Synthesis of (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules
31
32
Biodegradable polyelectrolyte capsules were prepared by LbL assembly of oppositely charged
33
34 polyelectrolytes around small PSS-CaCO3 cores. Two different types of capsules were prepared,
35 i.e. (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL). A general sketch of the synthesis route is
36
depicted in Figure 1.
37
38
39 Synthesis of LBL poly-L-arginine/alginate (PARG/ALGI)2 capsules
40
41
42 Capsules were prepared using a modified synthesis from literature.31, 63 Briefly, 0.5 mL of poly-L-
43 arginine solution (8 mg/mL) dissolved in 0.05 M NaCl (pH 6) was mixed with the as-synthesized
44 PSS-CaCO3 cores in a 2 mL Eppendorf tube and sonicated for 5 min, followed by shaking for 20
45
46 min at RT. Then, the excess polyelectrolyte solution was removed by centrifugation at 1400 rcf for
47 2 min at RT. Capsules were washed three times with 1 mL Milli-Q water and by centrifugation,
48 similar to the washing procedure as described before for the PSS-CaCO3 particles. Afterwards, 0.5
49
50 mL of alginate solution (8 mg/mL) dissolved in 0.05 M NaCl (pH 6) was added 64 and a similar
51 washing process was followed as described before. These synthesis steps of adding alternately
52 charged polyelectrolyte layers were repeated until forming 2-bilayered (PARG/ALGI)2 capsules.
53
54 Next, hollow capsules were obtained by dissolving the CaCO3 cores in 0.2 M aqueous EDTA
55 buffer (pH 7.4) overnight at 4 °C. Finally, the sample was centrifuged at 175 rcf for 40 min and
56
57
58
59
1
2
3 washed twice with 1 mL of Milli-Q water as described before. The capsules were then re-dispersed
4
5 in 1 mL of Milli-Q water and kept at 4 °C until further use.
6
7 Synthesis of LBL Eudragit-100 poly-L-arginine-alginate (PARG/ALGI)2(PARG/EuL)
8
9 capsules
10
11 (PARG/ALGI)2 PARG capsules were prepared as described above, with an additional layer of
12
13 PARG added. Then 0.5 mL of Eudragit-100 (8 mg/mL) dissolved in 0.05 M NaCl (pH 7) was
14 added to the (PARG/ALGI)2PARG capsules in an Eppendorf tube, followed by 5 min sonication
15 and 20 min shaking at RT. The excess of polyelectrolyte was separated by performing three
16
17 washing steps as described before. Finally, the CaCO3 core was removed by incubation with
18 EDTA buffer (pH 7.4) overnight at 4 °C. The sample was finally centrifuged at 175 rcf for 40 min
19 and washed twice with 1 mL of Milli-Q water. Then the capsules were re-dispersed in 1 mL of
20
21
Milli-Q water and kept at 4 °C until further use.
22
23
24
25
Solid dispersion of curcumin
26
27 The goal of the here described investigation was to enhance the aqueous solubility of curcumin and
28
thus to enhance its bioavailability. Solid dispersion formulations of curcumin were prepared using
29
30 different surfactants such as polyethylene glycol (PEG), sodium dodecyl sulfate (SDS), and
31 polyvinyl pyrrolidone (PVP). Solid dispersion was conducted using two different methods, the
32
melting method and the solvent evaporation method.37 Briefly, the melting method includes
33
34 melting 50 mg of PEG followed by mixing with 50 mg of curcumin at 60 °C and then drying in
35 air. The solvent evaporation method was carried out by dissolving 50 mg of SDS or PVP in 20 mL
36
ethanol in a beaker. 50 mg of curcumin was added and the solution was stirred overnight.
37
38 Afterwards, the co-precipitates were air-dried for 24 h. Solubility measurements were performed
39 according to the method reported by Higuchi and Connors.65 The solubility of curcumin was
40 measured spectrophotometrically at 430 nm using an UV–Vis absorption spectrophotometer and at
41
42 540 nm with a fluorimeter. For the further experiments, the PVP curcumin solid dispersion was
43 used, because it showed a higher aqueous solubility as compared to PEG- and SDS-curcumin solid
44 dispersion. (cf. Table S2)
45
46
47 5.3 Characterization of biodegradable capsules
48
49
50 Structural characterization
51
52 The fluorescence of the capsules was measured with a Horiba FluoroLog fluorimeter upon
53
54 excitation at ?ex = 420 nm and 488 nm (cf. Figure 2(C), (D)). The geometry of the different
55 capsules was analyzed with transmission electron microscopy (TEM, cf. Figure 2(E), (I)) and
56
57
58
59
1
2
3 CLSM (cf. Figure 2(A), (B)). TEM images of the curcumin-loaded capsules were taken by using a
4
5 field emission gun JEM 2100F UHR (JEOL, Japan) TEM equipped with a high angle annular dark
6 field (HAADF) detector. The TEM was operated at an accelerating voltage of 200 kV along the
7 full study. Fluorescence images of curcumin-loaded capsules were taken with a LSM 510 META
8
9 confocal microscope from Zeiss, equipped with lasers allowing excitation at 405, 488, 543, and
10 633 nm. Capsules were excited with the 488 nm laser and a band pass (BP) filter of 500 - 580 nm
11 was used to collect the emission at 540 ± 20 nm. The hydrodynamic diameters dh of the spherical
12
13 capsules were measured by dynamic light scattering (DLS) and the zeta-potential (1) with laser
14 Doppler anemometry, as carried out in a Malvern ZetasizerNano particle analyzer ZEN 3600
15 instrument (cf. Figure 2(F)-(H) and (J)-(L)). The results are summarized in Table 1, (cf. Figure S2)
16
17
18 Determination of capsule concentrations
19
20
21
Capsule concentrations were determined as average number ncapsules of capsule/mL from three
22 independent dilution series by counting. The dilutions were prepared from the stocks solution. For
23 the counting, 10 K0 of curcumin-loaded capsule solution was evaluated by using a Hemocytometer
24
25
(counting chamber (Neubauer-improved 0.1 mm), Marienfeld) under the confocal microscope,
26 using 488 nm laser excitation and a BP filter 500-580 nm, and the capsules were counted. Results
27 are shown in Figure S1. See the SI for detail information.
28
29
30 5.4 Drug loading and encapsulation efficiency of capsules
31
32
Curcumin was loaded into the capsules by the co-precipitation method as previously described. 34
33
34 Briefly, 0.5 mL (Ccur = 1 mg/mL) of aqueous curcumin solution was mixed under stirring for 5 min
35 (1000 rpm, stirrer "Magnetrührer IKA-RO 5 power") with 0.615 mL 0.33 M CaCl2 and 0.75 mL
36
25 mg/mL PSS solution in a glass vial. After that, 0.615 mL 0.33 M sodium carbonate (Na2CO3)
37
38 was added to the mixture under vigorous stirring (1000 rpm, Magnetrührer IKA-RO 5 power) for
39 60 s at RT. The particle precipitate was washed 2 times with Milli-Q water. Next, (PARG/ALGI)2
40 and (PARG/ALGI)2(PARG/EuL) capsules were prepared as described above. The encapsulation
41
42 efficiency ( EE) and loading capacity ( LC) of the capsule were calculated by both, indirect and
43 direct methods, respectively.66 See the SI for further information and Table 2 for the encapsulation
44
efficiency and loading capacity.
45
46
47 EE [%] = [(Ccur(bound)/Ccur(added)] 100%
48
49
50 LC = Ccur(bound)/ncapsules
51
52
53
54
55
56
57
58
59
1
2
3 5.5. In vitro release study of different capsules
4
5
6 A release study of curcumin from (PARG/ALGI)2 and (PARG/AlGI)2(PARG/EuL) capsules was
7 performed by two different methods, either using dialysis tubes (Spectra/Pro, Folat-A-Lyzer G2,
8
9 MWCO 100 kDa) or centrifuge filters (Amicon centrifuge filter, Ultracel- 100 kDa) for separating
10 released curcumin from the capsules.
11
12
13 Dialysis tubes containing 1 mL solution of curcumin-loaded capsule solution (Ccur(added) = 40
14 g/mL) were immersed in 6 mL buffer solutions of different pH (pH 3, 5, and 7). Dialysis was
15 performed at 37 ºC under mild stirring conditions at 40 rcf. At different time intervals (1, 2, 4, 6,
16
17 24, and 48 h), 1 mL released media was withdrawn for analysis and it was replaced with the same
18 volume of buffer to maintain conditions. The amount of released curcumin (Ccur(released)) in the
19 buffer solution was quantified by using a calibration curve of absorbance and fluorescence spectra
20
21 of curcumin as prepared at 0 h, 24 h, and 48 h in different pH buffers (cf. Figures S4-5).
22
23 The release study with centrifuge filters was performed by incubating capsules in 0.5 mL of
24
25 buffers of different pH (pH 3, 5, and 7) at 37 ºC for 24 h and 48 h. After 24 h incubation the
26 supernatant of the capsule solution (Ccur = 80 ug/mL) was removed by centrifugation at 175 rcf for
27 40 min as flow through. The retained capsules were re-dispered in different pH buffers and were
28
29
incubated with fresh pH buffers for another 24 h at 37 ºC. The supernatant was again collected by
30 centrifugation with a centrifuge filter (1400 rcf, 15 min) as flow through. The amount of released
31 curcumin Ccur(released) found in the flow-throughs with the different pH buffers was quantified
32
33 using the above described calibration curves. The amount of cumulative release CR of curcumin
34 was calculated using the following equation:
35
36
37 CR [%] = (Ccur(released)/ Ccur(added)) 100%
38
39 Results are shown in Figure 3, Figure S5-6 and Table 2. In figure S7, the hydrodynamic diameter
40
41 and confocal microscope images of the biodegradable ((PARG/ALGI)2 and
42 (PARG/ALGI)2(PARG/EuL)) capsules after the release study carried out by dialysis are shown.
43
44
45 5.6. Degradation study of capsules
46
47 An enzymatic degradation study of curcumin-loaded (PARG/ALGI)2 and
48
49 (PARG/ALGI)2(PARG/EuL) capsules was carried out using pronase as example of a proteolytic
50 enzyme. 15 K0 of (PARG/ALGI)2 capsules (ncapsules = 2.4×108 capsules/mL) and
51 (PARG/ALGI)2(PARG/EuL) capsules (ncapsules = 2×108 capsules/mL) were incubated with 75 uL
52
53
of 2 mg/mL pronase (pH = 7) at 37 U$ for 2, 4, 6, 24, 48, and 72 h. A series of similar sets at
54 different time points was prepared for each capsule at pH = 7 in PBS. Variation in hydrodynamic
55 diameter and zeta potential of capsules at each time point were measured with a Malvern
56
57
58
59
1
2
3 ZetasizerNano particle analyzer ZEN 3600 instrument. CLSM images of capsules at each time
4
5 point were also taken. Data are presented in Figure 4.
6
7
8
9 5.7. Biocompatibility studies of capsules
10
11 Cell culture
12
13
14 Human cervical adenocarcinoma cells (HeLa) were obtained from American Type Culture
15 Collection (ATCC) (Manassas, USA). Cells were cultured in Dulbecco's Modified Eagle's medium
16
17 Minimum (DMEM Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Biochrom,
18 Germany), and 1% penicillin/streptomycin (P/S, Fisher Scientific, Germany). The cells were kept
19 at 37 °C in a humidified atmosphere of 5% CO2 in air. When cells reached 90% of confluence,
20
21
cells were washed once with PBS and detached with 0.05 % trypsin ethylenediaminetetraacetic
22 acid (EDTA) solution (Fisher Scientific, Germany). Cells were then seeded in flasks for cell
23 passaging or seeded in plates for performing the in vitro experiments.
24
25
26 Cell viability studies
27
28
Cell viability studies of HeLa cells exposed to two different types of curcumin-loaded
29
30 biodegradable capsules were investigated by the resazurin assay as previously reported.53, 59 For
31 that, 7,500 HeLa cells were seeded into 96 well plates (area per well: 3.4 mm2, medium per well:
32
33
100 L) and were kept overnight at 37 °C, 5% CO2. The next day, the HeLa cells were exposed to
34 (PARG/ALGI)2 and to (PARG/ALGI)2(PARG/EuL) capsules at different concentrations
35 Ncapsules/cell (4 - 2000 capsules/cell) in serum-supplemented media for 24 h and 48 h. In each
36
37
capsules encapsulated curcumin concentration was 14 and 12 pg/ caps in (PARG/ALGI)2 and
38 (PARG/ALGI)2(PARG/EuL) capsules respectively. Therefore, maximum exposed curcumin to a
39 single cell was around 0.028 µg (2000 capsules/ cell). After incubation, HeLa cells were washed
40
41
three times with PBS (100 L). Then, 100 L of 10% of resazurin solution (Sigma-Aldrich) in
42 complete cell media was added to the cells and cells were incubated for 4 h at 37 °C, 5% CO2.
43 Then, the fluorescence intensity of the wells was measured with a microplate-reader equipped
44
45
fluorimeter (Fluorolog-3, from Horiba JobinYvon, Germany). For that, the samples were excited at
46 a wavelength of 560 nm and the emission was recorded in the range of 570 - 620 nm. The
47 background-subtracted mean fluorescence intensity of each sample was normalized to the
48
49
fluorescence of a reference sample to which no capsules had been added (control sample). The
50 control sample was ascribed 100% cell viability V, defined as their normalized fluorescence in
51 respect to the control. The viability data are provided as mean value ± standard deviation (SD)
52
from three independent experiments using HeLa cells at different passage numbers. Data are
53
54 shown in Figure 5.
55
56
57
58
59
1
2
3 5.8 Uptake studies
4
5
6 Uptake studies by flow cytometry
7
8
9 Intracellular uptake of capsules exposed to HeLa cells was determined by flow cytometry
10 according to previous protocols.59 A flow cytometer (BD LSR Fortessa Biosciences, German) was
11 used for the analysis. For that 40,000 Hela cells were seeded per well in 24 well plates (1.9 cm2
12
13 surface area per well; 1 mL of medium added per well) and incubated overnight. The next day,
14 HeLa cells were exposed to the curcumin-loaded (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL)
15 capsules at desired concentrations Ncapsules/cell (3 - 100 capsules/cell) in complete cell medium for
16
17 24 and 48 h. In terms of curcumin concentration it was (42 – 1400) and (36 – 1200) pg/ cell for
18 (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules respectively. After the exposure time,
19 the supernatants of the samples were removed and the HeLa cells were washed three times with
20
21 PBS (500 L). Then, HeLa cells were trypsinated, followed by neutralization of trypsin with
22 complete culture medium. Next, the cells were collected into flow cytometry tubes and centrifuged
23 at 300 rcf for 5 minutes. Finally, the cells were then suspended into 0.3 mL PBS solution. Cells
24
25 were mixed very well with a pipette in order to avoid clamping of cells prior to analysis. The
26 threshold of the forward-scattering signal in the flow cytometer was adjusted to eliminate signals
27 from debris and smaller particles. The cells with internalized capsule were excited by an argon 488
28
29
nm laser and the emission at 540 nm was collected using a 530/30 filter. For each experiment, a
30 minimum of 10,000 cells was sampled with the flow cytometer. A cell sample without capsule was
31 used as control. Results represent the mean value ± standard deviation (SD) from three
32
33
independent experiments using HeLa cells at different passage numbers. Results are shown in
34 Figure 6 and Figure S10.
35
36
37
38
39 Uptake studies of the capsule by confocal microscopy
40
41
42 Internalization of capsules was evaluated by confocal microscopy (CLSM). For that, 12,000 HeLa
43 cells per well were seeded in ibidis 8-well plates (area per well: 1 cm2, medium per well: 300 K0(
44
Cells were incubated overnight. The next day, cells were exposed to curcumin-loaded
45
46 (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules at concentration of Ncapsules/cell = 50
47 capsules/cell in complete cell medium for 24 h. In terms of curcumin concentration it was 700 and
48
600 pg/ cell for (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules respectively. After the
49
50 exposure time, cells were stained using two different approaches. First, the cell membrane was
51 stained with Wheat Germ Agglutinin, Tetramethylrhodamine conjugate from ThermoFisher
52 (WGA-TMRA). For that cell with internalized capsule were washed 3 times with 200 K0 of Hank's
53
54 Balanced Salt Solution (HBSS) and were then incubated with 70 K0 of 25 K I,0 WGA-TMRA
55 solution in HBSS buffer at G4U$ for 10 min. After the incubation cells were washed 3 times with
56
57
58
59
1
2
3 200 K0 HBSS. Next, nucleus staining was performed with Hoechst 33342 solution by incubating
4
5 with 70 K0 of 5 mg/mL Hoechst solution in PBS at RT for 15 min. Finally, cells were washed one
6 time with 200 K0 PBS fresh complete medium was added (See S11, 12). Second, lysosomes were
7 stained. For that, cells were incubated with 75 nM LysoTracker red- DND 99 solution in complete
8
9 media at G4U$ for 120 min. Then, nucleus staining was performed with Hoechst dye as described
10 above.
11
12
13 Fluorescence micrographs of HeLa cells with the internalized capsules were recorded with a
14 confocal microscope (Zeiss LSM 510 Meta Confocal Microscope). Images were taken with a Plan-
15 Apochromat 63×/1.40 Oil DIC M27 objective, and the pin hole was set to 0.86–1.32 airy units. A
16
17 laser diode emitting at 405 nm and a bandpass BP 420-480 emission filter were used to visualize
18 the nucleus labelling with Hoechst. An argon laser with a line at 488 nm and a bandpass emission
19 filter BP 505– 570 were used to visualize curcumin. A helium–neon laser for excitation at 543 nm
20
21
and a BP 560–615 emission filter were used to visualize the WGA-TAMRA. A helium–neon laser
22 for excitation at 543 nm together with a BP 560–615 emission filter were used for recording
23 fluorescence of LysoTracker. All images were further processed with the image J software to
24
25
reduce background noise and to enhance contrast for clear visualization of cellular images. All
26 images were processed with the same settings. Images are shown in Figures 7 and S11, 12.
27
28
Supporting Information
29
30
31 The Supporting information is available free of charge on the ACS Publications website. Details
32
on curcumin solubility, supplemental characterizations of capsule, supplemental cellular uptake
33
34 images and experimental data are provided.
35
36
37
38 6. References
39
40 1. Zhu, D.; Roy, S.; Liu, Z.; Weller, H.; Parak, W.; Feliu, N., Remotely controlled opening of delivery
41
vehicles and release of cargo by external triggers. Adv. Drug Del. Rev. 2019, 138, 117-132.
42
43
2. Antipov, A. A.; Shchukin, D.; Fedutik, Y.; Petrov, A. I.; Sukhorukov, G. B.; Möhwald, H., Carbonate
44 microparticles for hollow polyelectrolyte capsules fabrication. COLLOIDS AND SURFACES A-
45 PHYSICOCHEMICAL AND ENGINEERING ASPECTS 2003, 224 (1-3), 175-183.
46 3. De_Geest, B. G.; De Koker, S.; Sukhorukov, G. B.; Kreft, O.; Parak, W. J.; Skirtach, A. G.; Demeester,
47 J.; De Smedt, S. C.; Hennink, W. E., Polyelectrolyte microcapsules for biomedical applications. Soft Matter
48 2009, 5 (2), 282-291.
49 4. Lee, J. S.; Cho, J.; Lee, C.; Kim, I.; Park, J.; Kim, Y. M.; Shin, H.; Lee, J.; Caruso, F., Layer-by-layer
50 assembled charge-trap memory devices with adjustable electronic properties. Nature Nanotechnology
51
2007, 2 (12), 790-795.
52
53 5. Ambrosone, A.; Marchesano, V.; Carregal-Romero, S.; Intartaglia, D.; J.Parak, W.; Tortiglione, C.,
54 Control of 4$"BC # " $ $ signaling pathway in vivo via light responsive capsules. ACS Nano 2016, 10,
55 4828–4834.
56
57
58
59
1
2
3 6. Peng, L. H.; Zhang, Y. H.; Han, L. J.; Zhang, C. Z.; Wu, J. H.; Wang, X. R.; Gao, J. Q.; Mao, Z. W., Cell
4
Membrane Capsules for Encapsulation of Chemotherapeutic and Cancer Cell Targeting in Vivo. ACS Appl.
5
6
Mater. Inter. 2015, 7 (33), 18628-18637.
7 7. del Mercato, L. L.; Muñoz_Javier, A.; del Pino, P.; Rivera_Gil, P.; Parak, W. J. In Multifunctional
8 polyelectrolyte capsules as carrier systems for biosensing/drug delivery applications in living cells, NaNax 3
9 - Nanoscience with Nanocrystals, Lecce, Italy, 21.-23.5.2008; Manna, L.; Krahne, R.; Parak, W., Eds. Lecce,
10 Italy, 2008.
11 8. Parakhonskiy, B. V.; Yashchenok, A. M.; Konrad, M.; Skirtach, A. G., Colloidal micro- and nano-
12 particles as templates for polyelectrolyte multilayer capsules. Adv. Colloid Interface Sci. 2014, DOI:
13 10.1016/j.cis.2014.01.022, 253-264.
14
9. Navolokin, A. N.; German, V. S.; Bucharskaya, B. A.; Godage, S. O.; Zuev, V. V.; Maslyakova, N. G.;
15
16 Pyataev, A. N.; Zamyshliaev, S. P.; Zharkov, N. M.; Terentyuk, S. G.; Gorin, A. D.; Sukhorukov, B. G.,
17 Systemic Administration of Polyelectrolyte Microcapsules: Where Do They Accumulate and When? In Vivo
18 and Ex Vivo Study. Nanomaterials 2018, 8 (10), 812.
19 10. De Geest, B. G.; Willart, M. A.; Hammad, H.; Lambrecht, B. N.; Pollard, C.; Bogaert, P.; De Filette,
20 M.; Saelens, X.; Vervaet, C.; Remon, J. P.; Grooten, J.; De Koker, S., Polymeric Multilayer Capsule-Mediated
21 Vaccination Induces Protective Immunity Against Cancer and Viral Infection. ACS Nano 2012, 6 (3), 2136-
22 2149.
23
11. Parakhonskiy, B. V.; Foss, C.; Carletti, E.; Fedel, M.; Haase, A.; Motta, A.; Migliaresi, C.; Antolini, R.,
24
25
Tailored intracellular delivery via a crystal phase transition in 400 nm vaterite particles. Biomater. Sci.
26 2013, 1 (12), 1273-1281.
27 12. Parakhonskiy, B. V.; Haase, A.; Antolini, R., Sub-Micrometer Vaterite Containers: Synthesis,
28 Substance Loading, and Release. Angew. Chem., Int. Ed. 2012, 51 (5), 1195-1197.
29 13. Trushina, D. B.; Bukreeva, T. V.; Antipina, M. N., Size-Controlled Synthesis of Vaterite Calcium
30 Carbonate by the Mixing Method: Aiming for Nanosized Particles. Crystal Growth & Design 2016, 16 (3),
31 1311-1319.
32 14. Trushina, D. B.; Bukreeva, T. V.; Borodina, T. N.; Belova, D. D.; Belyakov, S.; Antipina, M. N., Heat-
33
driven size reduction of biodegradable polyelectrolyte multilayer hollow capsules assembled on CaCO3
34
35 template. Colloids Surf. B. Biointerfaces 2018, 170, 312-321.
36 15. Hanafy, N. A. N.; De Giorgi, M. L.; Nobile, C.; Rinaldi, R.; Leporatti, S., Control of colloidal CaCO3
37 suspension by using biodegradable polymers during fabrication. Beni-Suef University Journal of Basic and
38 Applied Sciences 2015, 4 (1), 60-70.
39 16. Yap, H. P.; Quinn, J. F.; Johnston, A. P. R.; Caruso, F., Compositional engineering of polyelectrolyte
40 blend capsules. Macromolecules 2007, 40 (21), 7581-7589.
41 17. Skirtach, A. G.; Antipov, A. A.; Shchukin, D. G.; Sukhorukov, G. B., Remote activation of capsules
42
containing Ag nanoparticles and IR dye by laser light. Langmuir 2004, 20 (17), 6988-6992.
43
44 18. Rivera_Gil, P.; Koker, S. D.; Geest, B. G. D.; Parak, W. J., Intracellular processing of proteins
45 mediated by biodegradable polyelectrolyte capsules. Nano Lett. 2009, 9 (12), 4398-4402.
46 19. Kolbe, A.; del Mercato, L. L.; Abassi, A. Z.; Rivera_Gil, P.; Gorzini, S. J.; Huibers, W. H. C.; Poolman,
47 B.; Parak, W. J.; Herrmann, A., De Novo Design of Supercharged, Unfolded Protein Polymers, and Their
48 Assembly into Supramolecular Aggregates. Macromol. Rapid Commun. 2011, 32 (2), 186-190.
49 20. Liao, W.-C.; Riutin, M.; Parak, W. J.; Willner, I., Programmed pH Responsive Microcapsules for the
50 Controlled Release of CdSe/ZnS QDs. ACS Nano 2016, 10, 8683-8689.
51
21. Liao, W.; Lu, C.; Hartmann, R.; Wang, F.; Sohn, Y. S.; Parak, W. J.; Willner, I., Adenosine
52
53
triphosphate-triggered release of macromolecular and nanoparticle loads from aptamer/DNA-cross-linked
54 microcapsules. ACS Nano 2015, 9, 9078–9086.
55 22. Lee, K. Y.; Mooney, D. J., Alginate: properties and biomedical applications. Prog. Polym. Sci. 2012,
56 37 (1), 106-126.
57
58
59
1
2
3 23. Orive, G.; Tam, S. K.; Pedraz, J. L.; Hallé, J.-P., Biocompatibility of alginate–poly-l-lysine
4
microcapsules for cell therapy. Biomaterials 2006, 27 (20), 3691-3700.
5
6
24. Goh, C. H.; Heng, P. W. S.; Chan, L. W., Alginates as a useful natural polymer for
7 microencapsulation and therapeutic applications. Carbohydr. Polym. 2012, 88 (1), 1-12.
8 25. Sergeeva, A. S.; Gorin, D. A.; Volodkin, D. V., In-Situ Assembly of Ca–Alginate Gels with Controlled
9 Pore Loading/Release Capability. Langmuir 2015, 31 (39), 10813-10821.
10 26. Lengert, E.; Yashchenok, A. M.; Atkin, V.; Lapanje, A.; Gorin, D. A.; Sukhorukov, G. B.;
11 Parakhonskiy, B. V., Hollow silver alginate microspheres for drug delivery and surface enhanced Raman
12 scattering detection. RSC Advances 2016, 6 (24), 20447-20452.
13 27. Chiang, C.-Y.; Chu, C.-C., Synthesis of photoresponsive hybrid alginate hydrogel with photo-
14
controlled release behavior. Carbohydr. Polym. 2015, 119, 18-25.
15
16 28. Cong, Z.; Shi, Y.; Wang, Y.; Wang, Y.; Niu, J. e.; Chen, N.; Xue, H., A novel controlled drug delivery
17 system based on alginate hydrogel/chitosan micelle composites. Int. J. Biol. Macromol. 2018, 107, 855-
18 864.
19 29. Lengert, E.; Parakhonskiy, B.; Khalenkow, D.; N O A.; Vangheel, M.; Moreno, J. M. M.;
20 Braeckman, B. P.; Skirtach, A. G., Laser-induced remote release in vivo in C. elegans from novel silver
21 nanoparticles-alginate hydrogel shells. Nanoscale 2018, 10, 17249-17256.
22 30. Ribeiro, C.; Borges, J.; Costa, A.; Gaspar, V.; Bermudez, V.; Mano, J., Preparation of Well-Dispersed
23
Chitosan/Alginate Hollow Multilayered Microcapsules for Enhanced Cellular Internalization. Molecules
24
25
2018, 23 (3), 625.
26 31. del Mercato, L. L.; Abbasi, A. Z.; Parak, W. J., Synthesis and characterization of ratiometric ion-
27 sensitive polyelectrolyte capsules. Small 2011, 7, 351-363.
28 32. Rivera Gil, P.; Nazarenus, M.; Ashraf, S.; Parak, W. J., pH sensitive capsules as intracellular optical
29 reporters for monitoring lysosomal pH changes upon stimulation. Small 2012, 8 (6), 943-948.
30 33. Ganas, C.; Weiß, A.; Nazarenus, M.; Rösler, S.; Kissel, T.; Rivera_Gil, P.; Parak, W. J., Biodegradable
31 capsules as non-viral vectors for in vitro delivery of PEI/siRNA polyplexes for efficient gene silencing. J.
32 Control. Release 2014, 196, 132-138.
33
34. Ott, A.; Yu, X.; Hartmann, R.; Rejman, J.; Schütz, A.; Ochs, M.; Parak, W. J.; Carregal Romero, S.,
34
35 Light-addressable and degradable silica capsules for delivery of molecular cargo to the cytosol of cells.
36 Chem. Mater. 2015, 27, = =R =
37 35. Hu, S. H.; Tsai, C. H.; Liao, C. F.; Liu, D. M.; Chen, S. Y., Controlled rupture of magnetic
38 polyelectrolyte microcapsules for drug delivery. Langmuir 2008, 24 (20), 11811-11818.
39 36. Shen, Y. Q.; Jin, E. L.; Zhang, B.; Murphy, C. J.; Sui, M. H.; Zhao, J.; Wang, J. Q.; Tang, J. B.; Fan, M.
40 H.; Van Kirk, E.; Murdoch, W. J., Prodrugs Forming High Drug Loading Multifunctional Nanocapsules for
41 Intracellular Cancer Drug Delivery. J. Am. Chem. Soc. 2010, 132 (12), 4259-4265.
42
37. Winter, S.; Tortik, N.; Kubin, A.; Krammer, B.; Plaetzer, K., Back to the roots: photodynamic
43
44 inactivation of bacteria based on water-soluble curcumin bound to polyvinylpyrrolidone as a
45 photosensitizer. Photochemical & Photobiological Sciences 2013, 12 (10), 1795-1802.
46 38. Yang, D. H.; Kim, H. J.; Park, K.; Kim, J. K.; Chun, H. J., Preparation of poly-l-lysine-based
47 nanoparticles with pH-sensitive release of curcumin for targeted imaging and therapy of liver cancer in
48 vitro and in vivo. Drug Deliv. 2018, 25 (1), 950-960.
49 39. Kottegoda, N.; Sandaruwan, C.; Priyadarshana, G.; Siriwardhana, A.; Rathnayake, U. A.;
50 Arachchige, D. M. B.; Kumarasinghe, A. R.; Dahanayake, D.; Karunaratne, V.; Amaratunga, G. A. J., Urea-
51
Hydroxyapatite Nanohybrids for Slow Release of Nitrogen. ACS Nano 2017, 11, 1214-1221.
52
53
40. Krzysztof, S.; Danuta, J.; Marek, P.; Jakub, S.; Monika, L.; Magdalena, R.; S % *S 4 L.; Piotr, W.,
54 Encapsulation of curcumin in polyelectrolyte nanocapsules and their neuroprotective activity.
55 Nanotechnology 2016, 27 (35), 355101.
56
57
58
59
1
2
3 41. De Koker, S.; De Cock, L. J.; Rivera_Gil, P.; Parak, W. J.; Velty, R. A.; Vervaet, C.; Remon, J. P.;
4
Grooten, J.; De Geest, B. G., Polymeric multilayer capsules delivering biotherapeutics. Adv. Drug Del. Rev.
5
6
2011, 63 (9), 748-761.
7 42. Feliu, N.; Sun, X.; Puebla, R. A. A.; Parak, W. J., Quantitative "# R Interaction: Some Basic
8 Physicochemical Pitfalls. Langmuir 2017, 33, EE =REE E
9 43. Sun, X.; Gamal, M.; Nold, P.; Said, A.; Chakraborty, I.; Pelaz, B.; Schmied, F.; Pückler, K. v.; Figiel, J.;
10 Zhao, Y.; Brendel, C.; Hassan, M.; Parak, W. J.; Feliu, N., Tracking stem cells and macrophages with gold
11 and iron oxide nanoparticles – The choice of the best suited particles. Applied Materials Today 2019, 15,
12 267-279.
13 44. Bhopate, D. P.; Mahajan, P. G.; Garadkar, K. M.; Kolekar, G. B.; Patil, S. R., A highly selective and
14
sensitive single click novel fluorescent off–on sensor for copper and sulfide ions detection directly in
15
16 aqueous solution using curcumin nanoparticles. New J. Chem. 2015, 39 (9), 7086-7096.
17 45. Zambito, Y.; Pedreschi, E.; Di Colo, G., Is dialysis a reliable method for studying drug release from
18 nanoparticulate systems?—A case study. Int. J. Pharm. 2012, 434 (1), 28-34.
19 46. Huang, J.; Shu, Q.; Wang, L. Y.; Wu, H.; Wang, A. Y.; Mao, H., Layer-by-layer assembled milk
20 protein coated magnetic nanoparticle enabled oral drug delivery with high stability in stomach and
21 enzyme-responsive release in small intestine. Biomaterials 2015, 39, 105-113.
22 47. Wang, N.; Guan, Y. P.; Yang, L. R.; Jia, L. W.; Wei, X. T.; Liu, H. Z.; Guo, C., Magnetic nanoparticles
23
(MNPs) covalently coated by PEO-PPO-PEO block copolymer for drug delivery. J. Colloid Interface Sci. 2013,
24
25
395, 50-57.
26 48. Hao, S.; Wang, B.; Wang, Y.; Zhu, L.; Wang, B.; Guo, T., Preparation of Eudragit L 100-55 enteric
27 nanoparticles by a novel emulsion diffusion method. Colloids Surf. B. Biointerfaces 2013, 108, 127-133.
28 49. Liu, K.; Li, H.; Williams, G. R.; Wu, J.; Zhu, L.-M., pH-responsive liposomes self-assembled from
29 electrosprayed microparticles, and their drug release properties. Colloids Surf. Physicochem. Eng. Aspects
30 2018, 537, 20-27.
31 50. Joshi, M., Role of eudragit in targeted drug delivery. 2013; Vol. 5, p 58-62.
32 51. Thakral, S.; Thakral, N. K.; Majumdar, D. K., Eudragit: a technology evaluation. Expert Opin Drug
33
Deliv 2013, 10 (1), 131-49.
34
35 52. Marchenko, I.; Yashchenok, A.; Borodina, T.; Bukreeva, T.; Konrad, M.; Möhwald, H.; Skirtach, A.,
36 Controlled enzyme-catalyzed degradation of polymeric capsules templated on CaCO3: Influence of the
37 number of LbL layers, conditions of degradation, and disassembly of multicompartments. J. Control.
38 Release 2012, 162 (3), 599-605.
39 53. Nold, P.; Hartmann, R.; Feliu, N.; Kantner, K.; Gamal, M.; Pelaz, B.; Hühn, J.; Sun, X.; Jungebluth, P.;
40 Pino, P. d.; Hackstein, H.; Macchiarini, P.; Parak, W. J.; Brendel, C., Optimizing conditions for labeling of
41 mesenchymal stromal cells (MSCs) with gold nanoparticles: a prerequisite for in vivo tracking of MSCs.
42
Journal of Bionanotechnology 2017, 15, 24.
43
44 54. Feliu, N.; Kohonen, P.; Palmberg, L.; Nyström, A. M.; Fadeel, B. In Systems biology approaches
45 reveal low-dose effects of dendrimers - abstract, 248th American Chemical Society National Meeing &
46 Exposition, San Francisco, CA, USA, 10.-14.8.2014; San Francisco, CA, USA, 2014.
47 55. Al-Rawi, M.; Diabate, S.; Weiss, C., Uptake and intracellular localization of submicron and nano-
48 sized SiO2 particles in HeLa cells. Arch. Toxicol. 2011, 85 (7), 813-826.
49 56. Mukherjee, S. G.; O'Claonadh, N.; Casey, A.; Chambers, G., Comparative in vitro cytotoxicity study
50 of silver nanoparticle on two mammalian cell lines. Toxicol. In Vitro 2012, 26 (2), 238-51.
51
57. Chakraborty, I.; Feliu, N.; Roy, S.; Dawson, K.; Parak, W. J., Protein-Mediated Shape-Control of
52
53
Silver Nanoparticles. Bioconjugate Chem. 2018, 29, 1261–1265.
54 58. Zhu, D.; Yan, H.; Zhou, Z.; Tang, J.; Liu, X.; Hartmann, R.; Parak, W. J.; Feliu, N.; Shen, Y., Detailed
55 investigation on how the protein corona modulates the physicochemical properties and gene delivery of
56 polyethylenimine (PEI) polyplexes. Biomaterials Science 2018, 6, 1800-1817.
57
58
59
1
2
3 59. Ma, X.; Hartmann, R.; Aberasturi, D. J. d.; Yang, F.; Soenen, S. J. H.; Manshian, B. B.; Franz, J.;
4
Valdeperez, D.; Pelaz, B.; Feliu, N.; Hampp, N.; Riethmüller, C.; Vieker, H.; Frese, N.; Gölzhäuser, A.;
5
6
Simonich, M.; Tanguay, R. L.; Liang, X.-J.; Parak, W. J., Colloidal Gold Nanoparticles Induce Changes in
7 Cellular and Subcellular Morphology. ACS Nano 2017, 11, -8?-R-8 ?
8 60. Summers, H. D.; Brown, M. R.; Holton, M. D.; Tonkin, J. A.; Hondow, N.; Brown, A. P.; Brydson, R.;
9 Rees, P., Quantification of Nanoparticle Dose and Vesicular Inheritance in Proliferating Cells. Acs Nano
10 2013, 7 (7), 6129-6137.
11 61. Chen, Y.; Sukhorukov, G. B.; Novak, P., Visualising nanoscale restructuring of a cellular membrane
12 triggered by polyelectrolyte microcapsules. Nanoscale 2018, 10 (35), 16902-16910.
13 62. Decher, G., Fuzzy nanoassemblies: Toward Layered Polymeric Multicomposites. Science 1997, 277,
14
1232-1237.
15
16 63. Muñoz_Javier, A.; Kreft, O.; Semmling, M.; Kempter, S.; Skirtach, A. G.; Bruns, O.; Pino, P. d.;
17 Bedard, M. F.; Rädler, J.; Käs, J.; Plank, C.; Sukhorukov, G.; Parak, W. J., Uptake of colloidal polyelectrolyte
18 coated particles and polyelectrolyte multilayer capsules by living cells. Adv. Mater. 2008, 20 (22), 4281-
19 4287.
20 64. Schuler, C.; Caruso, F., Decomposable hollow biopolymer-based capsules. Biomacromolecules
21 2001, 2 (3), 921-6.
22 65. Dittert, L. W.; Higuchi, T.; Reese, D. R., Phase solubility technique in studying the formation of
23
complex salts of triamterene. J. Pharm. Sci. 1964, 53 (11), 1325-1328.
24
25
66. Skirtach, A.; Yashchenok, A.; Mohwald, H., Encapsulation, release and applications of LbL
26 polyelectrolyte multilayer capsules. Chem. Commun. 2011, 47 (48), 12736-12746.
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59

10 1021@acsabm 9b00203

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

10 1021@acsabm 9b00203

Uploaded by

Copyright:

Available Formats

Subscriber access provided by UNIV OF SOUTHERN INDIANA

is published by the American Chemical Society. 1155 Sixteenth Street N.W.,

You might also like