Enantiomorphous Periodic Mesoporous Organosilica-Based

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Enantiomorphous Periodic Mesoporous Organosilica based Nanocomposite Hydrogel Scaffolds for Cell Adhesion and Cell Enrichment Nermin Seda Kehr Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01739 • Publication Date (Web): 26 Jan 2016 Downloaded from http://pubs.acs.org on January 30, 2016

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Enantiomorphous Periodic Mesoporous Organosilica based Nanocomposite Hydrogel Scaffolds for Cell Adhesion and Cell Enrichment

Nermin Seda Kehr* Physikalisches Institut and CeNTech, Westfälische Wilhelms-Universität Münster, Heisenbergstraße 11, D-48149 Münster, Germany

KEYWORDS: nanocomposite hydrogels, cell adhesion, cell enrichment, drug delivery, enantiomerically functionalized nanoparticles

ABSTRACT: The chemical functionalization of nanomaterials with bioactive molecules has been used as an effective tool to mimic extracellular matrix (ECM) and to study the cell-material interaction in tissue engineering applications. In this respect, this study demonstrates the use of enantiomerically functionalized periodic mesoporous organosilicas (PMOs) for the generation of new multifunctional 3D nanocomposite (NC) hydrogels to control the affinity of cells to the hydrogel surfaces and so to control the enrichment of cells and simultaneous drug delivery in 3D network. The functionalization of PMOs with enantiomers of bioactive molecules, preparation of

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their nanocomposite hydrogels, and the stereoselective interaction of them with selected cell types are described. The results show that the affinity of cells to the respective NC hydrogel scaffolds is affected by the nature of the biomolecule and its enantiomers, which is more pronounced in serum containing media. The differentiation of enantiomorphous NC hydrogels by cells is used to enrich one cell type from a mixture of two cells. Finally, PMOs are utilized as nanocontainers to release two different dye molecules as a proof of principle for multi-drug delivery in 3D NC hydrogel scaffolds.

1. INTRODUCTION Nanocomposite (NC) hydrogels1-7 are inorganic-organic functional hybrid materials which can be generated by the incorporation of inorganic nanometer-scaled objects with organic polymers in water. NC hydrogels show enhanced mechanical, thermal, electrical, optical and biological properties due to the presence of functional nanometer-scaled objects.4-13 Owing to the superior properties, NC hydrogels have been used as artificial scaffolds for the development of e.g. tissue engineering applications. Besides the physical properties of NC hydrogels, chemical functionalization of nanometer-scaled objects with bioactive molecules is an effective tool to mimic extracellular matrix (ECM) and to control the cell-material interaction.12-14 For example, Khademhosseini et al. reported the controlled and confined gene therapy using NC methylacrylated gelatin hydrogel in which the vascular endothelial growth factor plasmid DNA coordinated graphene oxide (GO) nanosheets were embedded.14 We described NC alginate hydrogels with biomolecule functionalized nanoparticles (NPs), and the use of the respective NC hydrogels as models for 3D scaffolds for controlled and enhanced cell growth.13


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The enantiomers of bioactive molecules have been utilized by us15-17 and other groups18-27 for the functionalization of material surfaces (e.g. nanoparticles, hydrogels, polymers, gold substrates) to investigate the chiral surface/biosystem interactions. The different cell adhesion behavior15-21,26 and single stranded DNA and protein adsorption23-25,27 behavior on enantiomophous surfaces were studied. These results show significantly that cells or biological systems interact differently on enantiomerically functionalized surfaces and that enantiomers of the same molecule can be used to control the affinity of biosystems to biomaterial surfaces. Although these promising results, there is still less studies on the behavior and the interaction of cells in enantiomorphous 3D (NC) hydrogel scaffolds. Cell enrichment and cell separation28-30 is particularly important in cell biology and in biomedical applications such as diagnostic, sensoring, stem cell research etc. Recently, different cell adhesion behaviours on functional material surfaces was used to separate cell mixtures.16,17,3133

For example, we described self-assembled monolayers of enantiomerically functionalized

nanoparticles for the separation of health cells from canceregeneous cells.16,17 In another contribution, Okano et al. developed thermoresponsive surfaces for label-free cell separation.32,33 With this respect, the design of functional (bio)materials which are able to simultaneously, effect the cell behavior, separate or enrich cells from each other will be useful for the production of advanced biomedical devices such as biosensors, biomembranes, devices for diagnostic etc. In this content, here I reported the enantiomerically pure surface-functionalized periodic mesoporous organosilicas (PMOs) with chiral (bio)molecules and loaded (in the pores) with dyes and their use for the generation of multifunctional nanocomposite (NC) hydrogel scaffolds. This new advanced biomaterial (NC hydrogel) provided 3D environment to cells like in native tissue and used additively the stereochemistry of the PMO surfaces to control the affinity of cells to the


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hydrogel network and so e.g. to enrich cells from cell mixtures. Additionally, the advantage of pores of the PMO was used for simultaneous release of two different dyes to adhered cells inside of the 3D hydrogel network as a model for multi-drug delivery systems. Selective cell experiments showed, to the best of my knowledge, for the first time the effect of the stereochemistry of the particle surface on the interaction of cells within 3D NC hydrogel scaffolds. The results demonstrated that cells differentiate the type of enantiomers on the PMO surfaces and show different affinity to the respective enatiomerically functionalized 3D NC hydrogel scaffolds. This property was utilized to separate partially one cell type from a mixture of two cells to show the proof of concept of cell enrichment by using enantiomorphous PMO surfaces. Additionally, two different fluorescence dye loaded PMOs were used as nanocontainers to stain adhered cells in the NC hydrogel scaffold by simultaneous release of fluorescence dye molecules as proof-of-principle for multi-drug delivery in 3D networks. The enantiomers of aminoacid D(L)-penicillamine (PEN) and carbonhydrate mannose derivative D(L)-MAN) were used as chiral end groups due to their importance for biological systems.34 Alginate was used as 3D hydrogel owing to its high biocompatibility.35 PMO36-39 was used as porous nano-meter-scale object and as a nanocontainer for the preparation of NC hydrogels.

2. EXPERIMENTAL SECTION Materials: D- and L-penicillamine [D(L)-PEN], D- and L-mannose pentaacetate, (purum, ≥97.0%),

calcium

D-gluconate

monohydrate,

alginic

acid

sodium

salt,

hexadecyltrimethylammonium bromide (CTAB, 98%), 1,2-bis(trimethoxysilyl)ethane (BTME, 96%), 3-aminopropyltrimethoxysilane (APTES, 99%), paraformaldehyde (PFA), N-hydroxysuccinimide (NHS), and trypsin, were purchased from Sigma-Aldrich. Ethanol (absolute for


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analysis), ammonia solution (32%, pure) and hydrochloric acid (32%, for analysis), were purchased from Merck. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) was obtained from ABCR. Hoechst 33342 and FM™ 4-64FX were purchased by Invitrogen. Trypan blue solution was purchased by Life Technologies GmbH. The cell medium [supplemented with 1% (v/v) Penicillin/Streptomycin, 2% (v/v) L-Glutamate, and 10% (v/v) fetal bovine serum (FBS)], phosphate buffered saline (PBS), ethylenediaminetetraacetic acid (EDTA) 1 % in PBS, without Ca2+ / Mg2, Penicillin/Streptomycin, L-Glutamate and FBS were obtained from Biochrom, Germany. Functionalization of PMO-NH2: A solution of 10 mM D(L)-PEN or D(L)-MAN, 30 mM EDC, and 60 mM NHS in 1 mL DMSO was added dropwise to a suspension of the PMO-NH2 (20 mg) in 1 mL DMSO. The reaction mixture was stirred for 16 h at room temperature. Subsequently the suspension was centrifuged 10 min at 6500 rpm and the isolated solid was washed with water x 2 and ethanol x 2. Finally the solid was dried at room temperature. General procedure for the preparation of Alg and PMO-Alg hydrogels: Alg hydrogel: Stock solutions of alginate (1.25 g) in 100 mL double distilled water and calcium D-gluconate monohydrate (1.00 g) in 100 mL double distilled water were prepared. 3 mL of alginate stock solution was calcium cross-linked by adding a 3 mL stock solution of calcium D-gluconate monohydrate while homogenizing the solution to obtain homogeneous calcium ion distribution and crosslinking. PMO-Alg hydrogels: PMO-NH2 or PMO-D(L)-PEN, or PMO-D(L)-MAN (6 mg) was sonicated with calcium D-gluconate monohydrate (3 mL of the stock solution) for 5 min and then added into the alginate stock solution (3 mL). General procedure for the preparation of Alg and PMO-Alg hydrogels scaffolds: The crosslinked PMO-Alg hydrogels or Alg hydrogel (0.250 mL) was transferred into a Teflon beaker (0.5


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cm in diameter and 1 cm length), frozen at -20 oC for 16 h and then lyophilized with freeze dryer for 16 h. Cell Experiments in Alg, PMO-NH2-Alg, PMO-D(L)-PEN-Alg, or PMO-D(L)-MAN-Alg: The cells were carefully thawed and resuspended in their specific medium (dulbecco's modified eagle's medium) for cell experiments in serum free medium. The cell medium was supplemented with fetal bovine serum (FBS) for cell experiments in serum containing medium. Cells were seeded into the hydrogel scaffolds (approximately 50.000 cells in approx. 20 µl media for each scaffold). The cells/media mixture was soaked immediately by hydrogel scaffolds. The scaffolds were covered with cell culture media (1 ml) and incubated for 1d and 4d at 37 °C and 5% CO2 in an incubator. After the incubation periods scaffolds were transferred to another cell culture plate and washed twice with PBS++ (supplemented with 0.5 mM MgCl2, 0.9 mM CaCl2) to remove non-adhered cells. Subsequently, scaffolds were dissolved in EDTA (0.04 % w/v in PBS, without Ca2+/Mg2+) with gentle mixing and cells were counted immediately with Neubauer chamber (trypan blue solution was used to detect death cells). Cell enrichment experiment in PMO-D-PEN-Alg and PMO-L-PEN-Alg: The HeLa and 3T3 cells were carefully thawed and resuspended in their specific medium (dulbecco's modified eagle's medium). Hoechst 33342 dye (0.1 mM) was added to HeLa cells suspension in cell culture media and incubated for 30 min at 37 °C and 5% CO2 to stain HeLa cell nucleus. After the incubation HeLa cells were washed with cell culture media x 3. HeLa (approximately 50.000 cells) and 3T3 (approximately 50.000 cells) cells were mixed in 1 mL cell culture media and seeded into PMOD-PEN-Alg and PMO-L-PEN-Alg. The scaffolds were covered with cell culture media and incubated for 4d at 37 °C and 5% CO2 in an incubator. After the incubation periods scaffolds were transferred to another cell culture plate and washed twice with PBS++ (supplemented with 0.5 mM


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MgCl2, 0.9 mM CaCl2) to remove non-adhered cells. Subsequently, scaffolds were dissolved in EDTA (0.04 % w/v in PBS, without Ca2+/Mg2+) with gentle mixing and cells were counted with Neubauer chamber. HeLa and 3T3 cells were distinguished from each other using fluorescence microscopy. General procedure for the co-staining of cells: The cells were carefully thawed and resuspended in their specific medium. Cells were seeded into the PMO-NH2-Alg hydrogel scaffold and incubated for 1d at 37 °C and 5% CO2. After that PMO-NH2-Alg hydrogels were washed twice with PBS++ (supplemented with 0.5 mM MgCl2, 0.9 mM CaCl2) and fixed with 4% PFA solution. After fixation PMO-NH2-Alg hydrogels were washed twice with PBS and kept in 0.1% Triton X100 in PBS for 10 minutes and afterwards in 3% bovine serum albumin (BSA), in PBS for 20 min. The cells in the PMO-NH2-Alg hydrogels were stained with Phalloidin Alexa Fluor® 488 (for factin staining) for 40 min, in the dark at room temperature, and washed twice with PBS. Finally, cell nucleus in the PMO-NH2-Alg hydrogel was stained with 4',6-diamidino-2-phenylindole carboxamidine (DAPI) and washed twice with PBS. Hoechst 33342 or FM™ 4-64FX loading of PMO-NH2: PMO-NH2 (10 mg) was suspended in 1 mL water and mixed with Hoechst 33342 (0.1 mg) or FM™ 4-64FX (0.1 mg). This reaction mixture was stirred for 1 day at room temperature. The final product HoechstPMO-NH2 or FMPMONH2 was obtained by centrifugation, washed with water x 2 and dried at room temperature. Hoechst

PMO-NH2-FMPMO-NH2-Alg hydrogels:

Hoechst

PMO-NH2-Alg (1 mg) and

FM

PMO-NH2-

Alg (1 mg) was mixed and sonicated with calcium D-gluconate monohydrate (1 mL of the stock solution) for 5 min and then added into the alginate stock solution (1 mL).


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Hoechst FM

PMO-NH2-FMPMO-NH2-Alg hydrogel scaffolds: The cross-linked

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Hoechst

PMO-NH2-

PMO-NH2-Alg (0.25 mL) was transferred into a Teflon beaker (0.5 cm in diameter and 1 cm

length), frozen at -20 oC for 16 h and then lyophilized with freeze dryer for 16 h. Cell Experiments in HoechstPMO-NH2-FMPMO-NH2-Alg hydrogel scaffold: The HeLa cells were carefully thawed and resuspended in their specific medium. Cells were seeded into the HoechstPMONH2-FMPMO-NH2-Alg hydrogel scaffold and incubated for 1d at 37 °C and 5% CO2. After that Hoechst

PMO-NH2-FMPMO-NH2-Alg hydrogel scaffolds was washed twice with PBS++

(supplemented with 0.5 mM MgCl2, 0.9 mM CaCl2) and fixed with 4% PFA solution.

3. RESULTS AND DISCUSSION 3.1. Preparation of NC hydrogels with enantiomerically functionalized PMOs. D(L)-MAN was synthesized according to procedure described in the literature.40 Amino functionalized PMOs (PMO-NH2) were synthesized by co-condensation of 3-aminopropyltriethoxysilane (APTES) and 1,2-bis(trimethoxysilyl)ethane (BTME) in the presence of cetyltrimethylammonium bromide (CTAB) and functionalized with D(L)-PEN or D(L)-MAN to obtain PMO-D(L)-PEN or PMOD(L)-MAN (for details see the SI, and Figure S1-2). The obtained functionalized PMOs were characterized by scanning electron microscopy (SEM), dynamic light scattering (DLS), N2-sorption, attenuated total reflection infrared (ATR-IR) spectroscopy, and zeta-potential measurements (for details see the SI). The SEM image (Figure S3) of PMO-NH2 showed that the particles were uniform and spherical shaped with average size of approximately 255 nm (DLS, Figure S4). Porosity and specific surface area of the PMO-NH2 particles were described by a N2-sorption experiment and by BET theory (for details see the SI Figure S5 and Table S1, data are in agreement with previously reported results39).The IR bands of


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the CO) and CN) vibrations of the amide bonds of the PMO-D(L)-PEN or PMO-D(L)-MAN which are characteristic for amide I and amide II absorptions were observed at about 1640 cm-1 and 1530-1540 cm-1 (Figure S6-9). In contrast PMO-NH2 shows no corresponding IR bands (Figure S10). In addition, OH and CH stretching vibrations occur in the 3100-3500 cm-1 and 28502980 cm-1 region respectively. The significant change of the zeta potential from 6.7 mV (PMONH2) to 2.9(2.8) mV [PMO- D(L)-PEN] and -25.5(-24.5) mV [PMO-D(L)-MAN] indicates the successful functionalization of PMO-NH2 with D(L)-PEN or D(L)-MAN as well. Subsequently, the respective NC hydrogels with enantiomerically functionalized PMOs and alginate were prepared. A stock solution of alginate in water was cross-linked by adding a sonicated suspension of calcium D-gluconate monohydrate and the respective PMO particles with well mixing. The obtained NC hydrogel (Figure S11) was frozen and then lyophilized to give the respective PMO-alginate scaffolds [PMO-NH2-Alg, PMO-D(L)-PEN-Alg, PMO-D(L)-MAN-Alg] (Figure S12). The SEM images show the morphology of the alginate (Alg), PMO-NH2-Alg, PMO-D(L)-PENAlg, and PMO- D(L)-MAN-Alg, fabricated by freeze drying (Figure 1). A longitudinal cross section of the scaffolds depicts the regions with large pore structures; the focus images show the comparable embedment of the respective PMOs into the scaffold wall.


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Figure 1. SEM image of Alg (A), PMO-NH2-Alg (B), PMO-L-PEN-Alg (C), PMO-D-PEN-Alg (D), PMO-L-MAN-Alg (E), PMO-D-PEN-Alg (F). Upper right images are the focus image of the polymeric wall. 3.2. Cell experiments in NC hydrogels with enantiomerically functionalized PMOs. After the preparation of the respective NC hydrogels cell adhesion experiments were carried out (Figure 2). Two different cells (HeLa cells; derived from human cervical cancer) and 3T3 cells; mouse embryonic fibroblast cells) were used as selective examples. HeLa and 3T3 cells were seeded separately into Alg, PMO-NH2, PMO-D(L)-PEN-Alg, and PMO-D(L)-MAN-Alg and incubated for 1d and 4d at 37 °C. After the incubation periods scaffolds were dissolved and cells were counted (Figure 2 and Table S2).


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Figure 2. Quantitative number of HeLa (A-B) and 3T3 (C-D) cells after 1d and 4d incubation time at 37°C (see also Table S1). 1: Alg, 2: PMO-NH2-Alg, 3: PMO-L-PEN-Alg, 4: PMO-D-PEN-Alg, 5: PMO-L-MAN-Alg, 6: PMO-D-MAN-Alg [N = 3; * (A, C) and *** (B, D) data show significant differences (t-test: * p≤ 0.05, *** p≤ 0.001)]. The results show that in the first 1d incubation period both HeLa and 3T3 cells have higher affinity to PMO-L-PEN-Alg and PMO-D-MAN-Alg than to PMO-D-PEN-Alg, PMO-L-MANAlg or Alg itself (Figure 2 and Table S2). After an extension of the incubation time for 1d to 4d (37°C) the amount of HeLa cells was increased in the respective hydrogel scaffolds (except PMOL-MAN-Alg), while the number of 3T3 cells decreased. But now HeLa cells show higher affinity to PMO-D-PEN-Alg than 3T3 cells. After 4d incubation twice more HeLa cells was extracted from PMO-D-PEN-Alg than from PMO-L-PEN-Alg. On the other hand twice more 3T3 cells were found in PMO-L-PEN-Alg than in PMO-D-PEN-Alg. As it was already found in 1d incubation


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time, both cell types show higher affinity to PMO-D-MAN-Alg than to PMO-L-MAN-Alg. Moreover, in most cases both cell types have better affection to the NC hydrogels than to Alg itself and higher affinity to the non-functionalized PMO-NH2-Alg than to PMO-D(L)-PEN-Alg and PMO-D(L)-MAN-Alg respectively, show that cells have high affinity not only to biomolecule functionalized PMO embedded hydrogel scaffolds, but also non- functionalized PMO-NH2-Alg. A remarkable difference between the number of HeLa and 3T3 cells was observed: the quantitative number of HeLa cells in hydrogel scaffolds was higher in comparison with the number of 3T3 cells even though approximately same amount of cells was seeded into the respective hydrogel scaffolds. This difference became more significant after 4d incubation time. For example, ca. 3 and 2 times more HeLa cells was found in Alg, PMO-NH2-Alg, and PMO-L-PEN-Alg, PMO-DMAN-Alg, respectively, than 3T3 cells. It should be pointed to the high differentiation between HeLa and 3T3 cells in PMO-D-PEN-Alg after 4d incubation time. The number of HeLa cells was ca. 8 times higher than 3T3 cells (Figure 2 and Table S2). Furthermore, a decrease of the cell viability of both cells with increasing incubation time was observed. The highest cell viability for both cells was found in Alg than in the NC hydrogels showing the PMO dependent cytotoxicity of the NC hydrogel. Similar effect was described previously by us.12 It is known that the behavior of cells is effected by cell-ECM interactions41 which occur mainly in nanometer-scale (e.g. focal adhesions take place in a range of 5-200 nm42) and cells bind to ECM via short amino acid sequences. Thus the used nano-meter scaled PMO particles functionalized with (chiral)bioactive molecules in the 3D hydrogel scaffold provide simultaneously chemical and configurational information and nano(micro)meter-scale roughness (physical property) like in native ECM. Therefore our results demonstrate the complex situation of competing, additive or/and synergetic effects between the 3D material and cells due to the


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functionalization of the particles, the stereochemistry of surfaces, the nano-structural and 3D properties of the hydrogel scaffolds, and external factors as the incubation time.

2.3. Cell enrichment in NC hydrogels with enantiomerically functionalized PMOs. Although, HeLa and 3T3 cells are from different organism, the pronounced different affinity of them to PMO-D-PEN-Alg was utilized to enrich one cell type (HeLa) from a mixture of two cells (HeLa and 3T3) to show the prove of concept of cell enrichment. PMO-D-PEN-Alg and for comparison PMO-L-PEN-Alg were prepared. The nucleus of HeLa cells were stained with Hoechst dye molecules for an easy detection by fluorescence microscopy. A heterogeneous cell mixture of HeLa and 3T3 cells (ca. 1:1) was prepared and visualized by microscopy. Both cells can be detected under white light but only HeLa cells were visible by fluorescence microscopy (Figure S13). The heterogeneous cell mixture of HeLa and 3T3 cells was seeded into PMO-DPEN-Alg and PMO-L-PEN-Alg and incubated for 4d. After the incubation period, the adhered HeLa and 3T3 cells in PMO-D-PEN-Alg and PMO-L-PEN-Alg were counted using fluorescence microscopy (Figure 3). Approximately 7 times more HeLa cells was counted than 3T3 cells in PMO-D-PEN-Alg (Table S3). This means close to 90% HeLa cell enrichment from a 1:1 heterogeneous mixture of HeLa and 3T3 cells was achieved by using PMO-D-PEN-Alg. On the other hand the use of L-PEN functionalized NC hydrogel resulted in 65% HeLa cell enrichment. This result shows the proof of principle that cell mixture can be manipulated and so different cell enrichment can be achieved using different enantiomerically functionalized PMO in NC hydrogels. Therefore, in the future the stereochemistry could be a powerful tool to create materials for the effective cell-cell separation or specific cell isolation from blood samples or tissues.


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Figure 3. Fluorescence microscopy (A, C) and white light (B, D) images of HeLa and 3T3 cells mixtures in PMO-D-PEN-Alg (A-B) and PMO-L-PEN-Alg (C-D) scaffolds. E. Quantitative number of extracted cells from PMO-D-PEN-Alg and PMO-L-PEN-Alg scaffolds of a mixture of HeLa and 3T3 cells after 4d incubation time (37°C). 2.4. Effect of protein adsorption on the cell affinity to the enantiomophous NC hydrogels. Cell experiments were performed in serum free [(-)serum] cell culture media to examine the effect of protein adsorption on the cell affinity to the enantiomophous NC hydrogels. HeLa and 3T3 cells


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were separately seeded in Alg, PMO-NH2-Alg, PMO-D(L)-PEN-Alg, and PMO-D(L)-MAN-Alg in (-)serum for 1d and 4d incubation time at 37 °C. In general, the number of adhered cells and the cell viability were lower in (-)serum than in serum containing media [(+)serum] (no viable cells were obtained after 4d incubation time, Table S4). Similar to the results of the (+)serum cell experiment (Table S2) more cells were extracted from the NC hydrogel scaffolds than from Alg. In contrast to the (+)serum cell experiments (Table S2) the affinity of both cell types towards PMO-D(L)-PEN-Alg, and PMO-D(L)-MAN-Alg in (-)serum (Table S4) was less pronounced, but still the trend of better cell adhesion of PMO-L-PEN-Alg and PMO-D-MAN-Alg can be seen compared to PMO-D-PEN-Alg, PMO-L-MAN-Alg. Therefore, these results are in agreement with the results previously described for the cell/chiral surface interaction of (-)serum/(+)serum conditions.15,16,24,25 The different adhesion behaviors of HeLa and 3T3 cells on enantiomerically functionalized surfaces which were observed for longer incubation time and pronounced under (+)serum conditions could be explained by protein-surface interactions. Surfaces with enantiomeric endgroup functionalizations show different affinities toward proteins.43 The electrostatic interactions between proteins (from the media) and chiral surface functionalization could generate stereochemical effects (e.g. diastereotopic effect) on the surfaces resulted in the adsorption of proteins with different orientations on enantiomorphous surfaces. These protein-coated surfaces can vary with time due to the dynamic replacement of adsorb proteins by other ones from the media and so the surface structure is dynamic and based on the concentration and association rate constants of proteins.44 The dependence of cell adhesion on specific cell-receptor/surface-protein interactions could explain the observed different adhesion behaviors of HeLa and 3T3 cells on the respective surfaces.


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The cell morphology inside of the 3D NC hydrogel scaffold was determined by fluorescence microscopy. PMO-NH2-Alg was used as a representative example. 3T3 and HeLa cells, respectively, were seeded in the hydrogel scaffold. After 1d incubation time cell membrane and cell nucleus of 3T3 and HeLa cells were co-stained with Phalloidine Alexa Fluor® 488 and 4’,6diamidino-2-phenylindole-6-carboxamidine (DAPI), respectively. The fluorescence microscopy images showed that both cells have spherical shape morphology inside of the 3D hydrogel scaffold (Figure 4A-B) (3T3 and HeLa cells morphology on 2D cell culture plate was shown in Figure S14 for comparison).45-48

Figure 4. 3T3 (A) and HeLa (B) cells in PMO-NH2-Alg scaffold (blue: DAPI stained cell nucleus, green: Phalloidine Alexa Fluor® 488 stained cell membrane). C. Fluorescence microscopy image of stained HeLa cells in hydrogel network (blue: Hoechst dye stained cell nucleus, red: FM™ 4-64FX dye stained cell membrane). 2.5. Dye molecules delivery by nanocontainers to cells in 3D NC hydrogel scaffold. Subsequently, PMO particles were used as nanocontainers to release dye molecules as a proof of principle for multi-drug delivery in 3D NC hydrogel scaffolds. Hoechst 33342 and FM™ 4-64FX loaded PMO-NH2 particles (HoechstPMO-NH2,

FM

PMO-NH2) were prepared (Figure S15). Then

alginate in water was cross-linked by calcium source and the both dye molecules loaded PMO


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particles to obtain the respective NC alginate hydrogel scaffold. HeLa cells were seeded into this scaffold. After 1d incubation time (37 °C) cells in the NC hydrogel were fixed with 4% paraformaldehyde (PFA) solution. The fluorescence microscopy image clearly showed blue and red stained HeLa cells nuclei and membrane, respectively, in the 3D NC hydrogel network caused by Hoechst 33342 and FM™ 4-64FX dye molecule release from the pores of the HoechstPMO-NH2 and FMPMO-NH2 nanocontainers (Figure 4C). The % viability of Hela cells in the respective NC hydrogel was 79 %.

4. CONCLUSION In conclusion, the preparation of enantiomorphous PMOs, their use for the generation of multifunctional NC hydrogel scaffolds and the effect of the stereochemistry of the particle surface on the interaction of cells within 3D NC hydrogel scaffolds were presented. This new advanced NC hydrogel used chirality of nanoparticles as an additional effective tool to control the affinity of cells to the hydrogel network. The results showed that the number of cells in 3D alginate hydrogel networks was affected by the nature of the biomolecule and its enantiomers. Cells recognized and differentiated differently between enantiomers of aminoacid or carbohydrate functionalized 3D NC hydrogel scaffolds. The different differentiation of enantiomorphous NC hydrogels by cells was used to enrich one cell type from a heterogeneous mixture of two cells. Finally, as proof-of-principle different dye molecules loaded PMOs were used both together as nanocontainers to stain nuclei and membranes of cells in NC hydrogel network by the release of dye molecules from the pores of the loaded PMOs. In the long term these results are envisaged for the construction of new biomaterials and their application in the field of bio- and nano-


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technologies e.g. tissue engineering, drug delivery in 3D network, cell-cell separation or specific cell isolation.

ASSOCIATED CONTENT The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. General experimental procedures, SEM, DLS, and ATR-IR of the respective PMOs

AUTHOR INFORMATION Corresponding Author *E-mail. [email protected]

ACKNOWLEDGMENT I thank DFG for funding and Prof. Hans Joachim Galla for providing cells and Hendrik Hermes and Tobias Kemnitzer for N2-soption experiment.

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SYNOPSIS TOC: Multifunctional nanocomposite (NC) hydrogels with enantiomerically functionalized PMO are generated. Cells recognize and differentiate differently in enantiomers of aminoacid or carbohydrate functionalized PMO based 3D NC hydrogel scaffolds resulting in the enrichment of one cell type from a heterogeneous mixture of two cells. PMO are used as nanocontainers to release two different dye molecules as a proof of principle for multi-drug delivery in 3D NC hydrogel scaffolds.


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Enantiomorphous Periodic Mesoporous Organosilica-Based

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