Biodegradable Alginate Polyelectrolyte Capsules As Plausible

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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

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Abstract Polyelectrolyte capsules made of different biodegradable and non-biodegradable polymers can be designed as systems for effective encapsulation and delivery of compounds. The objective of this work was to synthetize biocompatible and biodegradable capsules '= 1 µm) by the Layer-by-Layer (LbL) approach using alginate (ALGI) and Poly-L-arginine (PARG) polyelectrolytes with a pHsensitive outer layer of EUDRAGIT L 100 (EuL) polymer. Those capsules were loaded with curcumin as model therapeutic drug, which possesses antioxidant, anti-inflammatory, and anticancer activity. Encapsulation of drugs inside capsules protects its therapeutic activity and increases its bioavailability. We report the capsule stability, loading efficiency, drug release, as well as capsule degradation studies as a function of pH. Furthermore, in vitro biocompatibility studies of capsules including cell viability and uptake studies were performed using HeLa cells. The here synthesized capsules exhibited good reproducibility, spherical shape and highly monodispersed. The capsules showed good loading efficiency and drug release profile dependent upon pH environment. The in vitro studies indicate that the capsules exhibited acceptable biocompatibility and highly internalized by cells. Our study thus suggests that alginate LbL capsules could be used as efficient drug carrier with effective encapsulation and successful in vitro release of cargo in the cell. Key words: Biocompatible, biodegradable polyelectrolyte capsules, layer-by layer assembly, drug release, nanomaterials. 1. Introduction In recent years, capsules made out of different biodegradable and non-biodegradable polyelectrolytes by the Layer-by-Layer (LbL) technique have gain interest and been reported as an attractive approach for effective encapsulation and delivery of therapeutics.1-4 Furthermore, the first in vivo approaches also have been reported.5-6 The basic characteristics of the capsules are determined by several factors such as the size of the template core (which will influence the final capsule size), the choice of polyelectrolytes used for making capsule shell, the molecules loaded into the cavity of the capsules i.e molecular cargo etc. All these factors will determine and define the performance of the capsules. For potential in vivo applications, the use of biocompatible polyelectrolytes for the capsule assembly is advantageous. In general, as stated above, the size of the capsule is predetermined by the size of the template core.7 A wide range of size distributions from nm to mm, along with a series of different materials for template core formation has been reported.8 Calcium carbonate (CaCO3) particles are well established as template cores. They are suitable candidates (as core templates) for preparing polymer capsules due to their high porosity, biocompatibility, easy to perform synthesis, the ability to embed molecular cargo and the possibility of core removal using nontoxic ethylene diamine


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tetra acetic acid (EDTA) solution, after synthesis. Monodisperse spherical CaCO3 particles of 4-12 um size can be easily synthesized by mixing different equivalent ratios of calcium chloride and sodium carbonate solutions.8 The capsules prepared from these micron size particles have been tested towards in vivo delivery of drugs9 and vaccines.10 However, for drug delivery applications a smaller overall size is preferable. Recently, the synthesis of smaller submicron size (400-500 nm) CaCO3 particles has been reported in presence of comparatively toxic water-ethylene glycol or water-glycerol medium.11-14 The control over CaCO3 particle size can also be achieved by using different biodegradable polymers in the aqueous medium during the synthesis.15 A large variety of different polyelectrolytes has been used for LbL assembly around template cores. Apart from the frequently used synthetic polymers polystyrene sulfonate (PSS) and poly(allyamine hydrochloride) (PAH),16-17 also biological molecules have been used, such as polyarginine (PARG),18 proteins,19 oligonucleotides,20-21 or alginate. Alginate is a naturally occurring anionic polysaccharide extensively found in the cell walls of brown seaweeds.22 It has received attention in microencapsulation and biomedical applications due to its high biocompatibility and unique physicochemical properties.23 Aqueous solutions of alginate can easily form gels in presence of divalent cationic cross-linkers.24-25 Different macromolecules or drugs can be loaded into such ionically cross-linked alginate networks.23, 26-27Furthermore, alginate gels have interesting swelling properties dependent on the pH of the environment, which has been extensively explored to prepare drug delivery systems enabling controlled release.28 However, limited long-term stability is a major challenge of this system owing to the loss of mechanical strength due to ion release from the matrices at physiological conditions. 22 Recently a hollow silver alginate hydrogel capsule has been reported which was synthesized using CaCO3 particles as sacrificial templet. Laser-induced remote controlled in vivo delivery of cargo from this capsule achieved by taking advantage of silver nanoparticle’s response to NIR laser.29 Alginate has a pHdependent ionic nature, thus spherical capsules based on alginate also have been synthesized using LbL assembly, resulting in capsules with improved mechanical strength and encapsulation efficiency.30 In addition, a large variety of different compounds has been used as molecular cargo inside polyelectrolyte capsules. This involve molecular probes for sensing, 31-32 oligonucleotides for gene delivery,33 molecules for molecular signalling,5 or pharmaceutical agents such as doxorubicin for drug delivery.34-36 One interesting pharmaceutical agent is curcumin. Curcumin is a naturally occurring polyphenolic compound. Despite its possible therapeutic effects, the poor solubility of curcumin limits it clinical applicability. For this reason, different approaches have been utilized to enhance the solubility of curcumin, including solid dispersion techniques,37 nanoparticle encapsulation38 and others, which enables the use of curcumin for treatment of several diseases. For instance, Priya et al. encapsulated curcumin into carboxymethyl cellulose and casein Nano gels and studied its potent anticancer effect in a melanoma skin cancer cell model.39 In addition,




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Figure 1. A schematic diagram of the synthesis of biodegradable polyelectrolyte capsules loaded with curcumin. Transmission electron microscope (TEM) images of curcumin-loaded (A) (PARG/ALGI)2 and (B) (PARG/ALGI)2(PARG/EuL) capsules after dissolution of the template cores are shown. Scale bars correspond to 500 nm. Here, PSS, PARG, ALGI and EuL corresponds to sodium poly-(styrene sulfonate), poly-L-arginine hydrochloride, alginic acid sodium salt (ALGI, Sigma) and eudragit L 100 polymers respectively. Briefly, CaCO3 particles of hydrodynamic diameter dh 850 nm, as determined by dynamic light scattering (DLS), were prepared by co-precipitation and were used as core templates (c.f Figure 2F, J). To prepare curcumin-loaded capsules, curcumin was co-precipitated in the CaCO3 particles. The integration of the curcumin in the template core was confirmed by both, fluorescence microscopy and fluorescence measurements (c.f. Figure 2A-D,). Note that the CaCO3 particles were synthesized under the presence of PSS in order to reduce size. Next, the core particles were coated with PARG and ALGI using the well-described LbL method.31 As seen in Fig. 2K-L, a reversal of the surface charge between negative to positive zeta potential values could be observed, which verified the adsorption of polyelectrolytes in each layer. Capsules of two bilayers were prepared and are in the following referred to as (PARG/ALGI)2 capsules. Furthermore, in order to support and preserve the capsules in harsh pH environment (e.g. promote the efficient delivery capability through oral route), the capsules were complemented with an additional pH-sensitive EuL polymer layer and are then referred to as (PARG/ALGI)2(PARG/EuL) capsules (c.f. Figure 2L). Finally, the template cores were removed, and the capsules were washed and stored in sterilized water. Then, the capsule concentration was determined by confocal laser scanning microscopy (CLSM) (as described in the Supporting Information (SI)), and capsules were stored in dark at 4 °C until further used. The resulting capsules (with dissolved template core) exhibited dh of around 885 nm ((PARG/ALGI)2) and 916 nm ((PARG/ALGI)2(PARG/EuL)) respectively. Capsules were uniform, well dispersed, and presented spherical shape as shown by TEM and CLSM images (c.f. Figure 2 and the SI for further synthesis details). A summary of the capsule characterization including the colloidal stability and surface charge of the capsules determined using DLS in Milli-Q water, are presented in the Table 1. The found size variation between DLS measurement and TEM image analysis could be attributed to little deformation or flattening of spherical capsules under dry condition for TEM imaging. When these capsules are dispersed in solution, they are prone to get sediment within very short time due to their size 42-43. Experimentally, in this particular case, this effect could be monitoring following the fluorescence intensity of capsules over time. Indeed, the fluorescence intensity of capsule solution starts to drops down within just 5 min upon capsule dispersion. However, further homogenization with pipetting brings back its initial fluorescence. (c.f Figure S9A, B), thus this results indicates that the



decrease of fluorescence was related to the sedimentation effect of the capsules and not do degradation. Therefore, during each spectroscopic analysis all capsule solutions were mixed properly before measurements.

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I e G I L or R G G G G u C PA AL PAR AL AR E P

Figure 2. Characterization of the biodegradable (PARG/ALGI)2 and (PARG/ALGI)2 PARG/EuL) capsules. (A, B) Confocal microscopy images of curcumin-loaded (A) (PARG/ALGI)2 and (B) (PARG/ALGI)2(PARG/EuL) capsules after removal of the template cores. (C, D) Fluorescence spectra Icur'N( of curcumin-loaded (C) (PARG/ALGI)2 and (D) (PARG/ALGI)2(PARG/EuL) capsules after removal of the template cores at different wavelengths of excitation Nex. (E-I) TEM image of curcumin-loaded (E) (PARG/ALGI)2 and (I) (PARG/ALGI)2(PARG/EuL) capsules after removal of the template cores. (F-H) Number distributions N(dh) of the hydrodynamic diameters dh of (F) PSS-CaCO3 cores, (G) (PARG/ALGI)2, and (H) (PARG/ALGI)2(PARG/EuL) capsules as measured by DLS. Data are presented as number distributions N(dh). (J-L) Zeta potential distributions N( ) of (J) PSS-CaCO3 cores, and capsules coated as function of the additional PARG/ALGI layers for (K) (PARG/ALGI)2 and (L) of (PARG/ALGI)2(PARG/EuL) capsules. DLS and measurements were carried out in Milli-Q water.


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Capsule Type PSS-CaCO3 (PARG/ ALGI)2 (PARG/ ALGI)2 (PARG/ EuL)

dTEM [µm] -1.07 ± 0.32 1.17 ± 0.52

dh [nm] 856 ± 29 885 ± 34 916 ± 46

1 [mV] -20 ± 5 -37.3 ± 3.7 -39.2 ± 4

PDI 0.15 ± 0.05 0.23 ± 0.15 0.27 ± 0.035

Table 1. Colloidal properties of the capsules: The capsule diameter (dTEM) obtained from TEM images, average hydrodynamic diameter dh, zeta potential , and PDI of the cores and capsules with dissolved cores. The results of the dh and the are presented (as mean ± standard deviation (SD)) from three independent batches of capsules. DLS and measurements were carried out in Milli-Q water. 2.2 Drug loading and encapsulation efficiency of capsules The encapsulation efficiency ( EE) and loading capacity ( LC) of curcumin loaded capsules were evaluated by both, indirect and direct methods, which are described in the methods section. Note that the spectral and photo physical properties of curcumin are highly sensitive to pH and solvent conditions.44 For direct quantification of encapsulated curcumin the capsule walls were dissolved by pronase before spectroscopy. Interference of the optical properties of curcumin with those of pronase was controlled by UV-Vis absorption spectroscopy 'N( and fluorescence spectroscopy ?'N( after 24 h incubation of curcumin in pronase solution at 37°, as shown in Figure S3. The absorption A420 'N = 420 nm) was used to calculate EE and LC, see the methods section. The results are presented in Table 2, indicating similar EE and LC of curcumin in both types capsules. Spectroscopic measurements of the fluorescent signal of released curcumin after pronase mediated degradation (i.e. the "direct" method) indicated EE values of approx. 70 % for both capsules. The LC values found for (PARG/ALGI)2 and (PARG/AlGI)2(PARG/EuL) capsules were approximately 15.57 and 11.24 pg curcumin/capsule as measured by the indirect method and 11.24 and 11.94 pg curcumin/capsule as measured by the direct method, respectively. Since, both types of capsules were prepared from one initial batch of CaCO3 cores these similar results of curcumin loading were expected. The slightly different fluorescence and consequent variance of LC observed maybe attributed to the additional washing steps involved in the synthesis of the (PARG/ALGI)2(PARG/EuL) capsules, which may result in the loss of encapsulated curcumin or due to the counting errors of the capsules. However, it is important to mention that the LC results of both types of capsules showed that the direct and indirect methods agree remarkably well. These results are summarized in Table 2. The full set of data using UV/vis absorption spectroscopy and fluorescence spectra are presented in the SI.



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Capsules

[%] (direct method)

[pg/capsule] (indirect method)

[pg/capsule] direct method)

(PARG/ALGI)2

75.75 ± 2.25

15.57 ± 1.05

13.54 ± 1.86

(PARG/ALGI)2(PARG/EuL) 71.00 ± 2.80

11.24 ± 2.15

11.94 ± 1.37

EE

LC

LC

Table 2. Encapsulation efficiency ( EE) and loading capacity ( LC) of (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules with curcumin (number of capsule batches n=2).

2.3 In vitro release study from different capsules (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules were loaded with curcumin as model drug to investigate the effect that the surface modifications (EuL layer) and the number of bilayers of the capsules may have on the drug release kinetics. Thus, the in vitro release profile of curcumin ( CR) loaded (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules was investigated by two different approaches: the dialysis and the ultrafiltration method (see the methods section).45-46 For those investigations 1 mL (nc = 4×107 capsules/mL) of (PARG/AlGI)2 and (nc = 4.6 ×107 capsules/mL) of (PARG/ALGI)2(PARG/EuL) capsule solution were used. In both types of the capsules, the curcumin concentration used for the release study was 40 g/mL. The release profile was evaluated at different environmental conditions: in acidic (pH 3 and pH 5) as well as in neutral conditions (pH 7). Free curcumin was used as standard to correlate absorptions and intensity values to curcumin concentration (c.f. Figure S4 - S6). Spectroscopic measurements of the fluorescence signal of released curcumin as obtained after capsules were incubated at different conditions over time are shown in Figure 3 and in the SI.47 The results show that (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules had an opposite pH-dependent drug release profile. (c.f. Figure 3A-B, C-D). (PARG/ALGI)2 capsules exhibited higher release of encapsulated curcumin following incubation at lower pH, while (PARG/ALGI)2(PARG/EuL) capsules released more curcumin after incubation at higher pH. Amount of released curcumin increased over the time. Notably, the tendency of the results were confirmed by the two different approaches used (the dialysis and the ultrafiltration method), which corroborates reproducibility of the found pHdependent drug release kinetics. Moreover, the dialysis method showed that after 48 h incubation, the (PARG/ALGI)2 and PARG/ALGI)2(PARG/EuL) capsules exhibited a maximum CR of 50% at pH = 3 environment and CR of 52% at pH = 7 environment, respectively (c.f. Figure 3A, 3B). Besides, the cumulative curcumin release CR from capsules observed at 48h by the ultrafiltration method was 70% in pH = 3 environment and 71% in pH = 7 environment for the (PARG/ALGI)2 and PARG/ALGI)2(PARG/EuL) capsules respectively (c.f. Figure. 3D, 3E). The two used approaches provided similar trends in maximum CR release from (PARG/ALGI)2 and PARG/ALGI)2(PARG/EuL) capsules pH 3 and 7, respectively but differed in the absolute CR value. The variation can be attributed to the nature of the different methods, some loss of


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2.4 Biodegradation study of capsules The biodegradation of the capsules under different environment conditions over time was investigated by means of changes in hydrodynamic diameter dh, zeta potential , and visual inspection by confocal laser microscopy (CLSM) images (c.f Figure 4). Briefly, 10 µL of (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules incubated with 100 µL 2 mg/mL pronase in phosphate buffered saline (PBS, pH 7) and in PBS buffer at pH = 7 and pH = 3 for 2, 4, 6, 24, 48, and 72 h. The biodegradation was monitored in terms of dh(t), (t), and changes in CLSM images of capsules over time. The degradation kinetics of (PARG/ALGI)2 capsules was found to be pH depend. The average size (dh) of capsules incubated at pH 3 initially decreased but slightly increased over time as shown by the dh measurements. Furthermore, (PARG/ALGI)2 capsules looses curcumin fluorescence at pH 3, whereas it was slightly preserved at pH 7 as revealed by the CLSM images, suggesting the loss of curcumin at pH 3 (c.f. Figure 4). In contrast, (PARG/ALGI)2(PARG/EuL) capsules looses curcumin fluorescence at pH 7 after 48h incubation. Those results are in accordance with the pHdependent drug release profile (c.f. Figure 3). The dh of (PARG/ALGI)2(PARG/EuL) capsules increases over time for incubation in both pH 3 and 7 buffers, which may be due to aggregation of capsules. Also for both capsules a decrease in (-) ve surface charge was observed at pH 3. A possible explanation can be address to the protonation of carboxylate group present in both ALGI and EuL polymers at low pH. As control, both capsules were incubated with pronase, a decreasing trend in capsule size (as observed by the dh measurements and CLSM images) and lowering of outer charge of capsules over time, indicates degradation of capsules even at short periods of time. These data suggest that (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) are biodegradable capsules, but exhibit different degradation kinetics which was found to be pH dependent. The different release profiles of capsules could be attributed to the effect of the additional PARG/EuL bilayer. In an earlier study, Skirtach et al. group investigated enzymatic degradation of poly(L-arginine) (PARG)/ poly(Lglutamic acid) (pGlu) based capsule using pronase and found capsules degrades already after 60 min of incubation with pronase.52 Whereas, in our case after 2h incubation capsule structure starts to deform and after 4h almost completely loss its fluorescence indicating degradation of the capsule shell. The relatively slower capsule degradation kinetics observed in our study can be because of the presence of an inner PSS layer, which forms during small CaCO3 core synthesis. The ionic strength of the dispersion solution highly affects capsules stability. The DLS measurement of capsule size revealed both the capsule size decreases with increasing ionic strength of solution. At higher ionic concentration, polyelectrolytes are prone to coiled due to screening effects of counter ions present in solution. This causes poor interaction between polymers, which can affect the overall capsule structure. Although, both the capsules were quite stable in cell culture media, as confirmed by DLS size measurement of capsules in cell media without FBS after 24h incubation. (c.f. Figure S8)


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agglomeration. Data correspond to (mean ± SD) of 3 measurements. (E) Confocal images of curcumin-loaded (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules at each time point of the degradation study. For imaging 10 µL of incubated capsule solution was used and around 150 capsule images were captured. The scale bars correspond to 5 Km. 2.5 Biocompatibility studies of capsules In order to investigate the biocompatibility of (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules, cell viability studies of HeLa cells exposed to those capsules were carried out by using the traditional resazurin assay as previously reported 53-54. HeLa cells were derived from a cervical carcinoma, and those cells have been frequently employed in toxicology studies,55-57 and thus were used as model system in this work. As seen in Figure 5, the cell viability (V) results indicated that both capsules types are not acutely toxic at the time and doses used. Only a slight decrease of cell viability was observed at higher exposure concentrations of capsules/cell added. (A)

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Figure 5. Biocompatibility study of curcumin-loaded biodegradable capsules. HeLa cells were incubated with various numbers capsules per cell (Ncapsules/cell) in serum-supplemented media for t = 24 h and 48 h. Cell viability V was assessed using the resazurin assay, for (A) (PARG/ALGI)2 and (B) (PARG/ALGI)2(PARG/EuL) capsules. Results are presented as percent of cell viability V [%] (mean ± SD) from three independent experiments. 2.6 Uptake studies of the capsules Taking advantage of the intrinsic fluorescence of curcumin, uptake studies of curcumin-loaded biodegradable capsules were conducted in HeLa cells by flow cytometry and CLSM. Flow cytometry is a commonly used strategy to monitor and quantify internalization of particles by cells.58-60 Briefly, different concentrations of (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules (exposure concentrations Ncapsules/cell) were exposed to HeLa cells for t = 24 h and 48 h in serum-supplemented medium. After exposure, the curcumin fluorescence Icur in the cells was



monitored. Internalized of capsule into cell happens into major extend, via endocytosis process. Capsule uptake may trigger the initial formation of membrane protrusions in their vicinity followed by restructuring of these protrusions into smooth thin layer extending over the surface of the capsule.61 The internalization studies by flow cytometry indicated that capsules uptake occurred in a dose dependent manner (c.f. Figure 6). Notably, time (24 versus 48 h) did not influence the uptake results, which might be due to cell proliferation. Moreover, the internalization of (PARG/ALGI)2(PARG/EuL) capsules reached a plateau as seen in Figure 6B, which was not observed for (PARG/ALGI)2 capsules. Internalization of (PARG/ALGI)2(PARG/EuL) capsules was higher at lower exposure dose (25 caps/ cell, Icur = 1075 and 996 a.u. at 24 h and 48 h respectively) and gradually, at higher dose a saturation in capsule uptake was observed. Whereas, internalization of (PARG/ALGI)2 capsules was comparatively less at lower dose (25 caps/ cell, Icur = 818 and 842 a.u. at 24 h and 48 h respectively) and also capsule uptake did not achieved saturation limit within the range of exposed dose. In addition, the uptake was also monitored by CLSM (c.f. Figure 7). For that, cells were exposed to capsules at the concentration of 50 capsules/cell for 24 h. Co-localization studies were conducted and reveled that the majority of the capsules were found in the lysosomal compartments as shown by the Manders’ coefficient m1.58 The coefficient mx indicates the degree of overlap between pixels from two different fluorescence channels (i.e. yellow = curcumin inside capsule and red = Lysotracker® red stained lysosomes) ranging from 0 to 1. Here, 0 correlates to no overlap and 1 correlates to complete overlap. The m1 and m2 corresponds to the amount of capsule, which co-localized with lysosomes, and the amount of lysosomes that co-localized with capsules respectively. The results indicate that the number of (PARG/ALGI)2 capsules internalized is slightly higher than the (PARG/ALGI)2(PARG/EuL) capsules. (c.f Figure 7B) Those results agree and complement the flow cytometry studies. (A)

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Figure 6. Uptake study of curcumin-loaded biodegradable capsules. HeLa cells were exposed to different concentrations of capsules (Ncapsules/cell) for t = 24 h and 48 h in serum-supplemented medium. (A) (PARG/ALGI)2 capsules. (B) (PARG/ALGI)2(PARG/EuL) capsules. Cells were then analyzed by flow cytometry and the mean curcumin fluorescence Icur in cell was determined. Data


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were derived from the curves shown in Figure S10 and represent (mean ± SD) from three independent experiments. (A)

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Figure 7. (A) CLSM images of cells exposed to (PARG/ ALGI)2 and (PARG/ ALGI)2(PARG/ EuL) capsules for 24 h along with controls at 50 capsules/cell added. LysoTracker-red-DND-99 and Hoechst were used for lysosome and nucleus staining respectively. Ctrl and Ctrl(PARG/ ALGI)2 are cells without capsules added and cell with capsules added but without membrane staining, respectively. (B, C) Quantification of Manders’ coefficients m1 corresponding to the fraction of capsules in co-localization with lysosomes and m2 corresponding to the fraction of lysosomes in co-localization with capsules. Around 40 cells were analyzed from two independent experiments. Scale bars corresponds to 20 µm. 3. Conclusions The present work provides an assessment of the in vitro biocompatibility, stability, and biodegradability of (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules. Capsules were shown to be biodegradable. Hereby (PARG/ALGI)2 capsules degraded faster at acidic pH = 3, in contrast to (PARG/ALGI)2(PARG/EuL) capsules, which exhibit better stability at lower pH. However, both capsules were degraded in presence of pronase. In addition, the drug release studies indicated reverse drug release kinetics. The maximum release profile found for (PARG/ALGI)2




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eudragit L 100 (EuL, Evonik), ethylenediaminetetraacetic acid disodium salt (EDTA, Sigma), phosphate-buffered saline (PBS, Biochrom AG), curcumin (cur, Sigma), polyvinyl pyrrolidone (PVP, Mw = 55 kDa, Sigma), polyethylene glycol (Mw = 6 KDa), and sodium dodecyl sulfate (Sigma) were used as received. 5.2 Synthesis of biodegradable (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules Synthesis of PSS calcium carbonate cores Calcium carbonate cores (PSS-CaCO3) 2 were prepared by mixing 0.615 mL of 0.33 M aqueous solution of calcium chloride (CaCl2) and 0.75 mL of 25 mg/mL polystyrene sulfonate (PSS) solution under magnetic stirring (1000 rpm, stirrer "Magnetrührer IKA-RO 5 power") at room temperature (RT) for 5 min. Next, 0.615 mL of 0.33 M aqueous solution of sodium carbonate (Na2CO3) was added to the mixture under vigorous stirring (1000 rpm, "Magnetrührer IKA-RO 5 power") and stirred for 60 s at RT. Then the solution was transferred into an Eppendorf tube of 2 mL. PSS-CaCO3 particles of 800 – 1000 nm size were formed. The solution was centrifuged at 1400 rcf for 2 min to separate the precipitate. The precipitated PSS-CaCO3 particles were then washed three times with 1 mL Milli-Q water to remove unreacted salts and were then re-suspended in 1 mL of Milli-Q water. The PSS-CaCO3 cores were used to synthesize the capsules by the layer by layer (LbL) approach 62 as described in the following. Synthesis of (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules Biodegradable polyelectrolyte capsules were prepared by LbL assembly of oppositely charged polyelectrolytes around small PSS-CaCO3 cores. Two different types of capsules were prepared, i.e. (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL). A general sketch of the synthesis route is depicted in Figure 1. Synthesis of LBL poly-L-arginine/alginate (PARG/ALGI)2 capsules Capsules were prepared using a modified synthesis from literature.31, 63 Briefly, 0.5 mL of poly-Larginine solution (8 mg/mL) dissolved in 0.05 M NaCl (pH 6) was mixed with the as-synthesized PSS-CaCO3 cores in a 2 mL Eppendorf tube and sonicated for 5 min, followed by shaking for 20 min at RT. Then, the excess polyelectrolyte solution was removed by centrifugation at 1400 rcf for 2 min at RT. Capsules were washed three times with 1 mL Milli-Q water and by centrifugation, similar to the washing procedure as described before for the PSS-CaCO3 particles. Afterwards, 0.5 mL of alginate solution (8 mg/mL) dissolved in 0.05 M NaCl (pH 6) was added 64 and a similar washing process was followed as described before. These synthesis steps of adding alternately charged polyelectrolyte layers were repeated until forming 2-bilayered (PARG/ALGI)2 capsules. Next, hollow capsules were obtained by dissolving the CaCO3 cores in 0.2 M aqueous EDTA buffer (pH 7.4) overnight at 4 °C. Finally, the sample was centrifuged at 175 rcf for 40 min and



washed twice with 1 mL of Milli-Q water as described before. The capsules were then re-dispersed in 1 mL of Milli-Q water and kept at 4 °C until further use. Synthesis of LBL Eudragit-100 poly-L-arginine-alginate (PARG/ALGI)2(PARG/EuL) capsules (PARG/ALGI)2 PARG capsules were prepared as described above, with an additional layer of PARG added. Then 0.5 mL of Eudragit-100 (8 mg/mL) dissolved in 0.05 M NaCl (pH 7) was added to the (PARG/ALGI)2PARG capsules in an Eppendorf tube, followed by 5 min sonication and 20 min shaking at RT. The excess of polyelectrolyte was separated by performing three washing steps as described before. Finally, the CaCO3 core was removed by incubation with EDTA buffer (pH 7.4) overnight at 4 °C. The sample was finally centrifuged at 175 rcf for 40 min and washed twice with 1 mL of Milli-Q water. Then the capsules were re-dispersed in 1 mL of Milli-Q water and kept at 4 °C until further use.

Solid dispersion of curcumin The goal of the here described investigation was to enhance the aqueous solubility of curcumin and thus to enhance its bioavailability. Solid dispersion formulations of curcumin were prepared using different surfactants such as polyethylene glycol (PEG), sodium dodecyl sulfate (SDS), and polyvinyl pyrrolidone (PVP). Solid dispersion was conducted using two different methods, the melting method and the solvent evaporation method.37 Briefly, the melting method includes melting 50 mg of PEG followed by mixing with 50 mg of curcumin at 60 °C and then drying in air. The solvent evaporation method was carried out by dissolving 50 mg of SDS or PVP in 20 mL ethanol in a beaker. 50 mg of curcumin was added and the solution was stirred overnight. Afterwards, the co-precipitates were air-dried for 24 h. Solubility measurements were performed according to the method reported by Higuchi and Connors.65 The solubility of curcumin was measured spectrophotometrically at 430 nm using an UV–Vis absorption spectrophotometer and at 540 nm with a fluorimeter. For the further experiments, the PVP curcumin solid dispersion was used, because it showed a higher aqueous solubility as compared to PEG- and SDS-curcumin solid dispersion. (cf. Table S2) 5.3 Characterization of biodegradable capsules Structural characterization The fluorescence of the capsules was measured with a Horiba FluoroLog fluorimeter upon excitation at ?ex = 420 nm and 488 nm (cf. Figure 2(C), (D)). The geometry of the different capsules was analyzed with transmission electron microscopy (TEM, cf. Figure 2(E), (I)) and


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CLSM (cf. Figure 2(A), (B)). TEM images of the curcumin-loaded capsules were taken by using a field emission gun JEM 2100F UHR (JEOL, Japan) TEM equipped with a high angle annular dark field (HAADF) detector. The TEM was operated at an accelerating voltage of 200 kV along the full study. Fluorescence images of curcumin-loaded capsules were taken with a LSM 510 META confocal microscope from Zeiss, equipped with lasers allowing excitation at 405, 488, 543, and 633 nm. Capsules were excited with the 488 nm laser and a band pass (BP) filter of 500 - 580 nm was used to collect the emission at 540 ± 20 nm. The hydrodynamic diameters dh of the spherical capsules were measured by dynamic light scattering (DLS) and the zeta-potential (1) with laser Doppler anemometry, as carried out in a Malvern ZetasizerNano particle analyzer ZEN 3600 instrument (cf. Figure 2(F)-(H) and (J)-(L)). The results are summarized in Table 1, (cf. Figure S2) Determination of capsule concentrations Capsule concentrations were determined as average number ncapsules of capsule/mL from three independent dilution series by counting. The dilutions were prepared from the stocks solution. For the counting, 10 K0 of curcumin-loaded capsule solution was evaluated by using a Hemocytometer (counting chamber (Neubauer-improved 0.1 mm), Marienfeld) under the confocal microscope, using 488 nm laser excitation and a BP filter 500-580 nm, and the capsules were counted. Results are shown in Figure S1. See the SI for detail information. 5.4 Drug loading and encapsulation efficiency of capsules Curcumin was loaded into the capsules by the co-precipitation method as previously described. 34 Briefly, 0.5 mL (Ccur = 1 mg/mL) of aqueous curcumin solution was mixed under stirring for 5 min (1000 rpm, stirrer "Magnetrührer IKA-RO 5 power") with 0.615 mL 0.33 M CaCl2 and 0.75 mL 25 mg/mL PSS solution in a glass vial. After that, 0.615 mL 0.33 M sodium carbonate (Na2CO3) was added to the mixture under vigorous stirring (1000 rpm, Magnetrührer IKA-RO 5 power) for 60 s at RT. The particle precipitate was washed 2 times with Milli-Q water. Next, (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules were prepared as described above. The encapsulation efficiency ( EE) and loading capacity ( LC) of the capsule were calculated by both, indirect and direct methods, respectively.66 See the SI for further information and Table 2 for the encapsulation efficiency and loading capacity. EE

[%] = [(Ccur(bound)/Ccur(added)] 100%

LC

= Ccur(bound)/ncapsules



5.5. In vitro release study of different capsules A release study of curcumin from (PARG/ALGI)2 and (PARG/AlGI)2(PARG/EuL) capsules was performed by two different methods, either using dialysis tubes (Spectra/Pro, Folat-A-Lyzer G2, MWCO 100 kDa) or centrifuge filters (Amicon centrifuge filter, Ultracel- 100 kDa) for separating released curcumin from the capsules. Dialysis tubes containing 1 mL solution of curcumin-loaded capsule solution (Ccur(added) = 40 g/mL) were immersed in 6 mL buffer solutions of different pH (pH 3, 5, and 7). Dialysis was performed at 37 ºC under mild stirring conditions at 40 rcf. At different time intervals (1, 2, 4, 6, 24, and 48 h), 1 mL released media was withdrawn for analysis and it was replaced with the same volume of buffer to maintain conditions. The amount of released curcumin (Ccur(released)) in the buffer solution was quantified by using a calibration curve of absorbance and fluorescence spectra of curcumin as prepared at 0 h, 24 h, and 48 h in different pH buffers (cf. Figures S4-5). The release study with centrifuge filters was performed by incubating capsules in 0.5 mL of buffers of different pH (pH 3, 5, and 7) at 37 ºC for 24 h and 48 h. After 24 h incubation the supernatant of the capsule solution (Ccur = 80 ug/mL) was removed by centrifugation at 175 rcf for 40 min as flow through. The retained capsules were re-dispered in different pH buffers and were incubated with fresh pH buffers for another 24 h at 37 ºC. The supernatant was again collected by centrifugation with a centrifuge filter (1400 rcf, 15 min) as flow through. The amount of released curcumin Ccur(released) found in the flow-throughs with the different pH buffers was quantified using the above described calibration curves. The amount of cumulative release CR of curcumin was calculated using the following equation: CR

[%] = (Ccur(released)/ Ccur(added)) 100%

Results are shown in Figure 3, Figure S5-6 and Table 2. In figure S7, the hydrodynamic diameter and confocal microscope images of the biodegradable ((PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL)) capsules after the release study carried out by dialysis are shown. 5.6. Degradation study of capsules An enzymatic degradation study of curcumin-loaded (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules was carried out using pronase as example of a proteolytic enzyme. 15 K0 of (PARG/ALGI)2 capsules (ncapsules = 2.4×108 capsules/mL) and (PARG/ALGI)2(PARG/EuL) capsules (ncapsules = 2×108 capsules/mL) were incubated with 75 uL 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 different time points was prepared for each capsule at pH = 7 in PBS. Variation in hydrodynamic diameter and zeta potential of capsules at each time point were measured with a Malvern


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ZetasizerNano particle analyzer ZEN 3600 instrument. CLSM images of capsules at each time point were also taken. Data are presented in Figure 4.

5.7. Biocompatibility studies of capsules Cell culture Human cervical adenocarcinoma cells (HeLa) were obtained from American Type Culture Collection (ATCC) (Manassas, USA). Cells were cultured in Dulbecco's Modified Eagle's medium Minimum (DMEM Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Biochrom, Germany), and 1% penicillin/streptomycin (P/S, Fisher Scientific, Germany). The cells were kept at 37 °C in a humidified atmosphere of 5% CO2 in air. When cells reached 90% of confluence, cells were washed once with PBS and detached with 0.05 % trypsin ethylenediaminetetraacetic acid (EDTA) solution (Fisher Scientific, Germany). Cells were then seeded in flasks for cell passaging or seeded in plates for performing the in vitro experiments. Cell viability studies Cell viability studies of HeLa cells exposed to two different types of curcumin-loaded biodegradable capsules were investigated by the resazurin assay as previously reported.53, 59 For that, 7,500 HeLa cells were seeded into 96 well plates (area per well: 3.4 mm2, medium per well: 100 L) and were kept overnight at 37 °C, 5% CO2. The next day, the HeLa cells were exposed to (PARG/ALGI)2 and to (PARG/ALGI)2(PARG/EuL) capsules at different concentrations Ncapsules/cell (4 - 2000 capsules/cell) in serum-supplemented media for 24 h and 48 h. In each capsules encapsulated curcumin concentration was 14 and 12 pg/ caps in (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules respectively. Therefore, maximum exposed curcumin to a single cell was around 0.028 µg (2000 capsules/ cell). After incubation, HeLa cells were washed three times with PBS (100 L). Then, 100 L of 10% of resazurin solution (Sigma-Aldrich) in complete cell media was added to the cells and cells were incubated for 4 h at 37 °C, 5% CO2. Then, the fluorescence intensity of the wells was measured with a microplate-reader equipped fluorimeter (Fluorolog-3, from Horiba JobinYvon, Germany). For that, the samples were excited at a wavelength of 560 nm and the emission was recorded in the range of 570 - 620 nm. The background-subtracted mean fluorescence intensity of each sample was normalized to the fluorescence of a reference sample to which no capsules had been added (control sample). The control sample was ascribed 100% cell viability V, defined as their normalized fluorescence in respect to the control. The viability data are provided as mean value ± standard deviation (SD) from three independent experiments using HeLa cells at different passage numbers. Data are shown in Figure 5.



5.8 Uptake studies Uptake studies by flow cytometry Intracellular uptake of capsules exposed to HeLa cells was determined by flow cytometry according to previous protocols.59 A flow cytometer (BD LSR Fortessa Biosciences, German) was used for the analysis. For that 40,000 Hela cells were seeded per well in 24 well plates (1.9 cm2 surface area per well; 1 mL of medium added per well) and incubated overnight. The next day, HeLa cells were exposed to the curcumin-loaded (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules at desired concentrations Ncapsules/cell (3 - 100 capsules/cell) in complete cell medium for 24 and 48 h. In terms of curcumin concentration it was (42 – 1400) and (36 – 1200) pg/ cell for (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules respectively. After the exposure time, the supernatants of the samples were removed and the HeLa cells were washed three times with PBS (500 L). Then, HeLa cells were trypsinated, followed by neutralization of trypsin with complete culture medium. Next, the cells were collected into flow cytometry tubes and centrifuged at 300 rcf for 5 minutes. Finally, the cells were then suspended into 0.3 mL PBS solution. Cells were mixed very well with a pipette in order to avoid clamping of cells prior to analysis. The threshold of the forward-scattering signal in the flow cytometer was adjusted to eliminate signals from debris and smaller particles. The cells with internalized capsule were excited by an argon 488 nm laser and the emission at 540 nm was collected using a 530/30 filter. For each experiment, a minimum of 10,000 cells was sampled with the flow cytometer. A cell sample without capsule was used as control. Results represent the mean value ± standard deviation (SD) from three independent experiments using HeLa cells at different passage numbers. Results are shown in Figure 6 and Figure S10.

Uptake studies of the capsule by confocal microscopy Internalization of capsules was evaluated by confocal microscopy (CLSM). For that, 12,000 HeLa cells per well were seeded in ibidis 8-well plates (area per well: 1 cm2, medium per well: 300 K0( Cells were incubated overnight. The next day, cells were exposed to curcumin-loaded (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules at concentration of Ncapsules/cell = 50 capsules/cell in complete cell medium for 24 h. In terms of curcumin concentration it was 700 and 600 pg/ cell for (PARG/ALGI)2 and (PARG/ALGI)2(PARG/EuL) capsules respectively. After the exposure time, cells were stained using two different approaches. First, the cell membrane was stained with Wheat Germ Agglutinin, Tetramethylrhodamine conjugate from ThermoFisher (WGA-TMRA). For that cell with internalized capsule were washed 3 times with 200 K0 of Hank's Balanced Salt Solution (HBSS) and were then incubated with 70 K0 of 25 K I,0 WGA-TMRA solution in HBSS buffer at G4U$ for 10 min. After the incubation cells were washed 3 times with


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200 K0 HBSS. Next, nucleus staining was performed with Hoechst 33342 solution by incubating with 70 K0 of 5 mg/mL Hoechst solution in PBS at RT for 15 min. Finally, cells were washed one time with 200 K0 PBS fresh complete medium was added (See S11, 12). Second, lysosomes were stained. For that, cells were incubated with 75 nM LysoTracker red- DND 99 solution in complete media at G4U$ for 120 min. Then, nucleus staining was performed with Hoechst dye as described above. Fluorescence micrographs of HeLa cells with the internalized capsules were recorded with a confocal microscope (Zeiss LSM 510 Meta Confocal Microscope). Images were taken with a PlanApochromat 63×/1.40 Oil DIC M27 objective, and the pin hole was set to 0.86–1.32 airy units. A laser diode emitting at 405 nm and a bandpass BP 420-480 emission filter were used to visualize the nucleus labelling with Hoechst. An argon laser with a line at 488 nm and a bandpass emission filter BP 505– 570 were used to visualize curcumin. A helium–neon laser for excitation at 543 nm and a BP 560–615 emission filter were used to visualize the WGA-TAMRA. A helium–neon laser for excitation at 543 nm together with a BP 560–615 emission filter were used for recording fluorescence of LysoTracker. All images were further processed with the image J software to reduce background noise and to enhance contrast for clear visualization of cellular images. All images were processed with the same settings. Images are shown in Figures 7 and S11, 12. Supporting Information The Supporting information is available free of charge on the ACS Publications website. Details on curcumin solubility, supplemental characterizations of capsule, supplemental cellular uptake images and experimental data are provided.

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