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Interaction between immobilized polyelectrolyte complex nanoparticles and human mesenchymal stromal cells

Background Implant loosening or deficient osseointegration is a major problem in patients with systemic bone diseases (eg, osteoporosis). For this reason, the stimulation of the regional cell population by local and sustained drug delivery at the bone/implant interface to induce the formation of a mechanical stable bone is promising. The purpose of this study was to investigate the interaction of polymer-based nanoparticles with human bone marrow-derived cells, considering nanoparticles’ composition and surface net charge. Materials and methods Polyelectrolyte complex nanoparticles (PECNPs) composed of the polycations poly(ethyleneimine) (PEI), poly(L-lysine) (PLL), or (N,N-diethylamino)ethyldextran (DEAE) in combination with the polyanions dextran sulfate (DS) or cellulose sulfate (CS) were prepared. PECNPs’ physicochemical properties (size, net charge) were characterized by dynamic light scattering and particle charge detector measurements. Biocompatibility was investigated using human mesenchymal stromal cells (hMSCs) cultured on immobilized PECNP films (5–50 nmol·cm−2) by analysis for metabolic activity of hMSCs in dependence of PECNP surface concentration by MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) assay, as well as cell morphology (phase contrast microscopy). Results PECNPs ranging between ~50 nm and 150 nm were prepared. By varying the ratio of polycations and polyanions, PECNPs with a slightly positive (PEC+NP) or negative (PEC−NP) net charge were obtained. The PECNP composition significantly affected cell morphology and metabolic activity, whereas the net charge had a negligible influence. Therefore, we classified PECNPs into “variant systems” featuring a significant dose dependency of metabolic activity (DEAE/CS, PEI/DS) and “invariant systems” lacking such a dependency (DEAE/DS, PEI/CS). Immunofluorescence imaging of fluorescein isothiocyanate isomer I (FITC)-labeled PECNPs suggested internalization into hMSCs remaining stable for 8 days. Conclusion Our study demonstrated that PECNP composition affects hMSC behavior. In particular, the PEI/CS system showed biocompatibility in a wide concentration range, representing a suitable system for local drug delivery from PECNP-functionalized bone substitute materials.


Introduction
The self-repair ability of bone tissue allows for healing of small defects. Large bone defects, namely critical size defects caused by difficult fractures or osteolysis, often show impaired healing. In osteoporotic patients, the diminished bone mass and altered microarchitecture of bone results in increased fragility and susceptibility to fractures. [1][2][3] Fracture fixing in predestined fracture sites of those patients, such as hip and vertebrae, is a challenge for orthopedic surgeons. 4,5 Implant loosening and deficient osseointegration are frequent problems. For this purpose, materials are needed that assist and guide the healing process by mechanical and biochemical control.
In particular, bone-forming osteoblasts, which arise from mesenchymal stromal cells, should be pushed to migrate, proliferate, and differentiate in order to regenerate the defective tissue. 6,7 A strategy for this aim is the modification of common biomaterials: eg, titanium-based implants, [8][9][10] scaffolds, and composite materials made of biodegradable polymers, calcium phosphate-based biomaterials, [11][12][13] bioactive glasses, 14 and glass ceramics, 15 with bioactive molecules and therapeutic drugs that can act locally at the site of the bone defect. For a controlled release of bioactive molecules and therapeutic drugs, polymer-based nanoparticles can be used as a modification of bone substitute and replacement materials. 16,17 In this study, we have selected polyelectrolyte complex (PEC) nanoparticles (NPs) as the polymer-based NPs. PECNPs can be prepared by controlled mixing of oppositely charged polyelectrolytes (PELs), originally established by Kabanov and Zezin 18 and later followed by Philipp et al 19 and others. 20,21 PECNPs have apparent advantages over other polymer-based NP systems concerning easy availability and preparation, usage of biorelated and water-based educts and products, and adhesiveness with bone substitute materials. Up to now, PECNPs have not been assigned to one of the nanomedicine classes denoted or defined in classical review articles like that of, for example, Duncan and Gaspar. 22 However, their potential in respect of sizing (20-500 nm), shaping (spheres, rods), and surface chemistry variation (eg, charge sign) match well with those of nanomedicines, as we pointed out in a recent review. 21 Recently, we reported on successful loading and retarded release of the bisphosphonates pamidronate and zoledronate by the branched/linear PECNP system poly(ethyleneimine) (PEI)/cellulose sulfate (CS) from model substrates (like ATR-crystals of germanium and silicon). 23,24 Based on this work, in this study heteropolymeric PECNPs composed of the polycations PEI, poly(L-lysine) (PLL), or (N,N-diethylamino)ethyldextran (DEAE) and the polyanions dextran sulfate (DS) or CS, respectively, were immobilized and used as a substrate for human mesenchymal stromal cells (hMSCs). The investigation of these PECNPs was based on the anticipation that the combination of a branched PEL with an oppositely charged linear PEL results in PECNPs whose internal structure is more compact than branched/branched or linear/linear combinations. 21 Such compact or dense PEL particles are envisioned to show higher retention and slower release of loaded drugs. The investigated PECNPs are aimed at both release of bone therapeutic drugs and binding on relevant bone substitute materials.
The aim of this study was to identify PECNP systems that are highly biocompatible and accepted in an immobilized manner in a broad concentration range by hMSCs and to study their putative internalization. Thereby, we focused on empty PECNPs and undifferentiated hMSCs. hMSCs were cultured onto PECNP films and analyzed for metabolic activity, morphological changes, and internalization features. To the best of our knowledge, there is no report published yet on the interaction of PECNPs in the used compositions with hMSCs.  25 water was used instead of Tris buffer. The anionic sodium poly(ethylenesulfonate) and cationic poly(diallyldimethylammonium chloride) PEL solution, which were used for particle charge detector (PCD) titration, were purchased from BTG Instruments GmbH (Herrsching, Germany).

Preparation of PecNPs
Polycations and polyanions were dissolved in sterile water to achieve concentrations of 2 mM related to their monomer units. As not all monomer units are necessarily charged, the true concentration of charged units (n − , n + ) can be determined by colloid titration (see next section). Related to these charged units, PECNPs composed of n − /n + =0.9 (positively charged PECNPs, PEC + NP) and n − /n + =1.1 (negatively charged PECNPs, PEC − NP) were prepared by mixing respective volumes of 2 mM polycation and 2 mM polyanion solutions. To prepare PECNPs, the defined pH value settings of the respective PEL solution were used and no additional salt (ionic strength) was added. For PEC + NP dispersions,

characterization of Pel solutions and PecNPs
The actual amount of charges in PEL solutions was determined by colloid titration using the PCD (BTG Instruments GmbH, Herrsching, Germany). The PCD consists of the components BTG Mütek TM PCD-T3 Titrator and PCD-04. 1 mM sodium poly(ethylenesulfonate) solution was used to titrate the polycation; 1 mM poly(diallyldimethylammonium chloride) solution was used as a standard to titrate the polyanion. Based on the titrated volume (V TIT ) of the respective 1 mM standard solutions, the factor F of the given PEL solution with c PEL =1 mM related to monomer units and with V PEL =1 mL was calculated by F=V TIT /V PEL (mL/mL). PECNP diameters were determined by dynamic light scattering (DLS) at the Zetasizer 3000 of Malvern Instruments Ltd (Malvern, UK) equipped with an He-Ne laser with 5 mW at 632.8 nm at 25°C and a detection angle of 90°. The measurements were performed applying an automatic duration time and the automatic analysis mode. The samples were held in 10 mm cuvettes. The hydrodynamic diameter D H was estimated using the Stokes-Einstein equation. Intensity-weighted DLS data were considered, as they were applied earlier therein. 26 Errors of D H are related to the standard deviation of at least three different sample measurements. The Zetasizer 3000 Software (PCS v.1.61 Rev 1) of Malvern Instruments Ltd was used for recording and calculating DLS parameters.
Immobilization of Pel and PecNPs onto tissue culture polystyrene PEL as well as PECNPs were immobilized onto TCPS plates by adding aliquots of an aqueous PEL solution or PECNP dispersion (each 2 mM) in 100 µL of sterile water (Milli-Q PF Plus water) (Millipore, Monsheim, France) to finally get 5-50 nmol⋅cm −2 PELs or PECNPs, respectively. Both, PEL solutions and PECNP dispersions were air-dried overnight under laminar flow and sterilized with ultraviolet light. Immobilized FITC-labeled PECNPs were not sterilized. The spatial distribution of PECNPs after immobilization was visualized by scanning force microscopy (SFM). spatial distribution of PecNPs SFM images were recorded from PECNP films on TCPS using Nanostation II of Bruker Nano GmbH (Karlsruhe, Germany) with silicon probe tips from Nanosensors ( Darmstadt, Germany) with apex radii of around 10 nm. The bottoms of the 24-well TCPS plates were carefully sawn out and placed on the SFM sample table. PECNP samples were measured in "noncontact mode" at 25°C in topography and error and phase modes, and scanning parameters were optimized by minimizing the signal in the error mode. Topographical images and surface profiles were generated from SFM raw data by either SISCANPro software (Bruker Nano GmbH) or SPIP software (Image Metrology A/S, Hørsholm, Denmark).

Isolation and cultivation of hMscs
hMSCs were isolated from bone marrow aspirates obtained from healthy donors (average age 32±10 years) at the Bone Marrow Transplantation Center of the University Hospital Dresden (Dresden, Germany) and were characterized as described previously. 27 The donors gave informed consent (Ethic Vote No. EK71022010). The cells were not pooled and used for the experiments in passage 4-6. α-MEM with nucleosides supplemented with 10% heat-inactivated fetal calf serum, 20 U penicillin/mL, 20 µg streptomycin/mL, and 2 mM L-alanyl-L-glutamine was used as expansion medium. For the experiments, hMSCs were subcultured at a density of 5,500 cells⋅cm −2 onto 24-well TCPS plates. After 4 days, a medium change was performed.

analysis of metabolic activity of hMscs
Cells cultured on immobilized PEL, PECNP, and TCPS as a reference were analyzed at day 1 and day 8 after plating for metabolic activity using CellTiter 96 ® AQueous One Solution cell proliferation assay (MTS test; Promega Corporation, Fitchburg, WI, USA) according to the manufacturer's instructions, with a Benchmark Plus Microplate Reader (Bio-Rad Laboratories, Hercules, CA, USA).

statistical analysis
All data were derived from different hMSC preparations and treated as independent biological replicates. Analysis of statistical significance was performed with GraphPad Prism 5 software (Statcon, Witzenhausen, Germany) by analysis of variance (ANOVA) with Bonferroni's post-test.
Two-way ANOVA was performed to compare polycations and polyanions and to analyze the impact of net charge and chemistry of the PECNPs on metabolic activity. The influence of the PEL or PECNP amount compared with TCPS was analyzed by one-way ANOVA. Two-tailed Student's t-test was used to analyze the difference of metabolic activity of hMSCs from day 1 to day 8 and differences amongst the PECNP systems radii dependent on the composition or PECNP net charge. The results are presented as mean ± standard error of the mean (SEM).

Results and discussion characterization of PecNPs
Twelve PECNP systems differing in PEL composition and net charge were prepared and characterized. Both synthetic and biorelated PELs were selected. Their combinations used for the preparation of PECNPs are listed in Table 1. The linear polycation PLL and the branched polycations PEI and DEAE were combined with the linear polyanion CS and the branched polyanion DS. By varying the molar mixing ratio n − /n + of polycationic and polyanionic components, the resulting PECNP systems acquired either a slightly positive (n − /n + =0.9, PEC + NP) or negative (n − /n + =1.1, PEC − NP) net charge. Hydrodynamic radii (R H ) and polydispersity indices of the various PECNP systems were determined by DLS and the net charge sign (±) by the PCD, respectively. R H values in the range between 46 nm and 146 nm were obtained for all compositions of PECNPs. In detail, PEI-containing PECNPs (PEI/DS, PEI/CS) were the smallest (R H =46-59 nm), followed by the PLLcontaining PECNPs (R H =70-106 nm) and DEAE-containing PECNPs (R H =103-146 nm).

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Biocompatibility of polyelectrolyte complex nanoparticles toward hMscs The particle size of the PECNPs was partially influenced by the net charge. Although DS-containing PECNPs were generally slightly smaller compared with CS-containing PECNP systems, the situation concerning the net charge was more complex. Although positively charged DEAE/DS (R H ≈135 nm; P=0.09) and PEI/DS (R H ≈60 nm; P0.05) were larger than the corresponding negatively charged ones, positively charged PLL/DS (R H ≈70 nm) were significantly smaller than negatively charged PLL/DS (R H ≈80 nm; P0.05). All CS-containing PECNPs, like DEAE/CS (R H ≈ 145 nm), PEI/CS (R H ≈60 nm), and PLL/CS (R H ≈100 nm), showed almost no differences in particle size for PEC + NP compared with PEC − NP. The polydispersity indices were ranging between 0.14 and 0.73, indicating rather broad size distributions for all PECNP systems (Table 1).
For the in vitro experiments, PECNP dispersions were immobilized on 24-well TCPS plates and thereupon incubated in buffered α-MEM cell culture medium. Due to their remarkable adhesive strength, 23,24 stable PECNP films could be generated by simple solution casting. Figure 1 shows  ( Figures 1B-D) at lower spatial resolution (32 µm ×32 µm), whereupon homogenously distributed PECNPs were seen at this scale. Importantly, with increasing initial PECNP concentration (5, 20, and 50 nmol⋅cm −2 ), the density of immobilized PECNPs also increased distinctly and visibly. Finally, a higher spatial resolution SFM image (2 µm ×2 µm) of PEI/DS + at 50 nmol⋅cm −2 bound at TCPS reveals PECNPs between 50 nm and 200 nm due to a partial clustering, which is still consistent with DLS data.

Metabolic activity of hMscs on immobilized Pel and PecNPs
To our knowledge, to date no information with regard to these PEL/PEL combinations interacting with hMSCs has been published. Solely, Tiyaboonchai et al 28 report about drug delivery from PEI/DS, and only Hartig et al 29 reported on the interaction of PECNPs in general with other cell types, in particular human microvascular endothelial cells. 30 In this study, hMSCs were cultured on immobilized PECNP films in various surface concentrations and analyzed at day 1 and day 8 for relative metabolic activity by

3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt (MTS) assay.
To compare the effects of PEL to uncomplexed PEL, immobilized PELs and PECNPs, respectively, in the concentration of 5-50 nmol⋅cm −2 , were used as a substrate for hMSCs. Figure 2A shows that the metabolic activity of hMSCs cultured on TCPS increased from day 1 to day 8 by about 2.8-fold. The data presented in Figures 2B-E were normalized to the metabolic activity on TCPS at day 1 ( Figures 2B and 2D) and day 8 ( Figures 2C and 2E), respectively. The value of 100% metabolic activity is related to untreated TCPS (Figure 2, dotted lines). In all bar plots of Figures 2B-E, the surface concentration was used as a variable. Figures 2B and 2C show the relative metabolic activity of hMSCs cultured on the pure PELs used in this study after 1 day ( Figure 2B) and 8 days ( Figure 2C). For all polycations (PEI, DEAE, PLL), a significant decrease of metabolic activity of hMSCs from 0% to 90% of TCPS in dependence of the surface concentration was seen at day 1 ( Figure 2B) and from 0% to 95% compared with TCPS at day 8 ( Figure 2C). At the highest surface concentration of 50 nmol⋅cm −2 , only hMSCs on PEI and DEAE showed a residual metabolic activity of 40% and 15%, respectively, at day 1. At day 8, only on PEI 25% of hMSCs remained metabolically active on this surface concentration. On the other hand, at the lowest PEL concentration of 5 nmol⋅cm −2 , tolerable values of at least 80% at day 1 and of at least 60% at day 8 were obtained for all polycations. Surprisingly, at this surface concentration, hMSCs on PEI, which is claimed to have a toxic effect on various cell lines and animals, showed almost 95% of viable cells at day 8. In that framework, Moreau et al 31

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PEL concentrations. The overall toxic effect of uncomplexed polycations is claimed to be associated with the high charge density, 36 which is in line with reports claiming that the protonation degree influences cytotoxicity. 36,37 Further studies reported on nonphysiological effects of polycations and related them to their interaction to lipid bilayers, complexation of acidic plasma proteins, and of DNA up to agglutination and lysis of red blood cells. 29,31,38 For the polyanion (CS, DS)-treated TCPS surfaces, the situation was even worse. Although at day 1, constant low to moderate metabolic activity around 20%-30% of TCPS was obtained for all surface concentrations (5-50 nmol⋅cm −2 ), at day 8 metabolic activity decreased to 1%-30%. These results are in line with those of Kamide et al 39 reporting thrombogenicity and acute toxicity of uncomplexed CS in rats. Additionally, Hint and Richter 40 studied the treatment of rabbits with low molecular DS, which caused weight loss, cachexia, and osteoporosis.
Hence, we conclude that for uncomplexed PEI, PLL, and DEAE, only the lowest surface concentration of 5 nmol⋅cm −2 of the pure polycation-modified TCPS was tolerable at day 1. However, after 8 days of culture, only PEI modification of TCPS was well tolerated at this low concentration. CS or DS-modified TCPS even at the lowest concentration of 5 nmol⋅cm −2 was no longer tolerated by hMSCs.

Interaction between PecNPs and hMscs
These results for TCPS modified by the uncomplexed PEL formed the basis for those obtained for TCPS modified by PECNPs, which are shown in Figure 2D (day 1) and Figure 2E (day 8). Generally, for the PECNP-modified TCPS, two types of systems could be identified. Although some of the measured PECNP systems caused a decrease of metabolic activity (DEAE/CS, PEI/DS) at day 1 and day 8, the other PECNP systems did not induce significant alterations of metabolic activity (DEAE/DS, PEI/CS) in dependence of surface concentration, respectively. The former systems were denoted as "variant systems", the latter as "invariant systems" being candidates for rather uncritical interaction between PECNPs and hMSCs. At day 8, this trend was even intensified. Interestingly, the PEL components, like PEI, DEAE, CS, and DS, used in the rather harmless "invariant systems" were found to show only low hMSC metabolic activity dependent on the surface concentration in their uncomplexed pure state. This finding is of great importance to the PECNP concept, as PECNPs, where PELs are complexed one with another, are much more compatible compared with the uncomplexed PEL used in this study. Obviously, complexation of PEL had positive effects on the biocompatibility, presumably because fewer uncompensated charges are present in the neighborhood of hMSCs.
Finally, beside the composition, also the influence of the net charge of PECNPs was studied. These systems are indicated either by the minus and plus sign in Figures 2D/E or denoted as polycation/polyanion + / − , respectively. Obviously, the net charge itself had no effect on the response of hMSCs to PECNP systems of the same chemical composition. With the exception of PLL/DS + (day 1 and 8) and PLL/CS -(day 8), all variant systems remained variant and all invariant systems remained invariant in dependence of the surface concentration regardless of their net charge. This finding is very interesting, as it seems to be in contrast to those on the uncomplexed PEL. According to Figures 2B and 2C, TCPS modified by uncomplexed polyanions showed significantly less metabolic active cells compared with those on uncomplexed polycations. As an explanation, the charge argument raised here might be used, according to which the presence of too much charge reduces metabolic activity.
Complexation, regardless of whether the residual excess charges had a negative or a positive sign, significantly reduces the amount of excess charges. Thereby, it is of minor importance if this low excess charge results in PEC + NP or PEC − NP.

Morphology of hMscs on immobilized PecNPs
Phase contrast microscopy images were taken to evaluate PECNP-induced morphological changes of hMSCs after 1 day and 8 days, respectively. Figure 3 shows representative images of hMSCs cultured on 20 nmol⋅cm −2 PECNPs. At day 1, after plating, hMSCs appeared as small, thin cells with typical fibroblast-like morphology; partially, hMSCs grew from cell aggregates and developed few cell-cell contacts. With the exception of PEI/DS − , where the majority of the cells stayed round, on all other PECNP films a minor amount of spindleshaped hMSCs spread and flattened to become cuboidal. Within 8 days of culture, hMSCs on TCPS and PECNP films developed filopodial extensions and formed almost confluent cell layers with fibroblast-like cuboidal hMSCs ( Figure 3B). Differences in morphology were obvious when DEAE was present or when DS changed to CS ( Figure 3B). An influence of the net charge was seen for PEI/DS ± and PLL/DS ± ( Figure 3B). These morphological alterations in the presence of PECNPs confirmed MTS data. hMSCs with diminished metabolic activity in the presence of PECNPs also revealed less cell attachment and spreading (images for other PECNP concentrations are not shown). By trend, fewer hMSCs were seen on DEAE-containing PECNPs and the variant PEC systems PEI/DS ± and PLL/DS ± in comparison with TCPS. Fluorescence imaging of hMscs on immobilized FITc-PecNPs hMSCs that were cultured for 8 days on 20 nmol⋅cm -2 FITClabelled PECNPs were analyzed for F-actin and cellular localization of the PECNPs by immunofluorescence imaging (Figure 4). The analyses were performed with PEC + NPs, which were expected to feature a more efficient internalization due to their affinity for the negatively charged cell membrane. 41 Every PECNP system displayed a well-distributed particle film on TCPS, visible as a homogeneous green background staining. An accumulation of FITC-labeled PECNPs beneath hMSCs became apparent by bright dots (see DEAE/DS and PLL/CS, Figure 4C) or areas (DEAE/CS and PEI/CS, Figure 4C). It has to be noted that the focal plane selected for these accumulated dots was situated neither at the TCPS surface below nor on top or outside of the cell body. Hence, these visible PECNPs represent a minor but considerable portion of the initial total amount of immobilized PECNPs that are located within hMSCs.
However, the question arises as to how PECNPs bound at the surface of TCPS can be internalized in cultured hMSCs. Although, generally, cellular internalization has been extensively described in the bulk phase for several nanoparticle types, depending on size, shape, surface charge, chemistry, and functionalization of the nanoparticles and also on the cell type, 42-45 cellular internalization of surface-bound nanoparticles has not been studied to that extent. Actually, we speculate that PECNPs were enclosed in the bound state by the hMSC membrane, detached, and internalized. Thus, due to the small size of PECNPs and the nonphagocytic character of hMSCs, an endocytotic mechanism is assumed.
Furthermore, hMSCs on DEAE/CS + and partially DEAE/DS + showed a deformation of the F-actin cytoskeleton, whereas hMSCs on other PECNP systems displayed a rather similar appearance of the cytoskeleton as seen for the TCPS control ( Figure 4B), where they formed dense cell layers with tight cell-cell contacts ( Figure 4A). Aggregation of PECNPs within the cytoskeleton, particularly at DEAE-and PEIcontaining systems, occurred near the nucleus ( Figure 4C).
Conclusively, the immunofluorescence microscopy data of Figure 4 showed that positively net charged PECNP systems were taken up by hMSCs independently of their effects on metabolic activity of hMSCs. Further microscopic and spectroscopic techniques sensitive to the distance of zones or sections in respect of the bare TCPS surface will be included in future work. The PEL composition of PECNPs significantly affected metabolic activity, whereas the net charge of PECNPs did not. PECNPs could be classified into "variant systems" featuring significant surface concentration dependence on metabolic activity and "invariant systems" lacking such dependence. In particular, the PEI/CS system could be identified to show biocompatibility in a wide surface concentration range, enabling drug delivery from PECNP-functionalized biomaterials. In contrast, pure uncomplexed PEL did not show such biocompatibility at surface concentrations larger than 5 nmol⋅cm −2 . Hence, the complexation of PEL may result in a masking of their toxic properties, which supposedly is associated with charge compensation and the overall reduction of charges.

Conclusion
Analogous to MTS data, phase contrast microscopy revealed that the composition of PECNPs considerably affected hMSC attachment and morphology. Similar to MTS data, cell morphology on PLL/DS but also PEI/DS was influenced by PECNP net charge. Using TCPS-bound FITC-PECNPs as a fluorescent probe, it was shown that PECNPs were internalized by hMSCs and remained stable for at least 8 days. However, the uptake mechanism is still unresolved.
In future work, PECNPs will be used for the modification of bone substitute materials and the delivery of bone therapeutic drugs at the implant/bone interface in order to prevent the first-pass effect in the liver, keeping an effective drug dose, prolonging the pharmacological effects, and reducing putative side effects.