Bioactive magnetic near Infra-Red fluorescent core-shell iron oxide/human serum albumin nanoparticles for controlled release of growth factors for augmentation of human mesenchymal stem cell growth and differentiation
- Itay Levy†1,
- Ifat Sher†2,
- Enav Corem-Salkmon1,
- Ofra Ziv-Polat1,
- Amilia Meir3,
- Avraham J Treves3,
- Arnon Nagler4,
- Ofra Kalter-Leibovici5,
- Shlomo Margel1 and
- Ygal Rotenstreich2Email author
© Levy et al.; licensee BioMed Central. 2015
Received: 28 September 2014
Accepted: 6 April 2015
Published: 7 May 2015
Iron oxide (IO) nanoparticles (NPs) of sizes less than 50 nm are considered to be non-toxic, biodegradable and superparamagnetic. We have previously described the generation of IO NPs coated with Human Serum Albumin (HSA). HSA coating onto the IO NPs enables conjugation of the IO/HSA NPs to various biomolecules including proteins. Here we describe the preparation and characterization of narrow size distribution core-shell NIR fluorescent IO/HSA magnetic NPs conjugated covalently to Fibroblast Growth Factor 2 (FGF2) for biomedical applications. We examined the biological activity of the conjugated FGF2 on human bone marrow mesenchymal stem cells (hBM-MSCs). These multipotent cells can differentiate into bone, cartilage, hepatic, endothelial and neuronal cells and are being studied in clinical trials for treatment of various diseases. FGF2 enhances the proliferation of hBM-MSCs and promotes their differentiation toward neuronal, adipogenic and osteogenic lineages in vitro.
The NPs were characterized by transmission electron microscopy, dynamic light scattering, ultraviolet–visible spectroscopy and fluorescence spectroscopy. Covalent conjugation of the FGF2 to the IO/HSA NPs significantly stabilized this growth factor against various enzymes and inhibitors existing in serum and in tissue cultures. IO/HSA NPs conjugated to FGF2 were internalized into hBM-MSCs via endocytosis as confirmed by flow cytometry analysis and Prussian Blue staining. Conjugated FGF2 enhanced the proliferation and clonal expansion capacity of hBM-MSCs, as well as their adipogenic and osteogenic differentiation to a higher extent compared with the free growth factor. Free and conjugated FGF2 promoted the expression of neuronal marker Microtubule-Associated Protein 2 (MAP2) to a similar extent, but conjugated FGF2 was more effective than free FGF2 in promoting the expression of astrocyte marker Glial Fibrillary Acidic Protein (GFAP) in these cells.
These results indicate that stabilization of FGF2 by conjugating the IO/HSA NPs can enhance the biological efficacy of FGF2 and its ability to promote hBM-MSC cell proliferation and trilineage differentiation. This new system may benefit future therapeutic use of hBM-MSCs.
Magnetic nanoparticles (NPs) which are known for their very large surface area and magnetic properties (size up to 0.1 μm) have a wide range of potential applications such as drug delivery, MRI, diagnostics, hyperthermia, specific cell labeling and separation, cell tracking and bio-catalysis [1-6]. Iron oxide (IO) NPs of sizes less than approximately 50 nm are superparamagnetic (possess magnetic properties when they are exposed to external magnetic field, and lose their magnetic properties when the magnetic field is removed), allowing therefore the separation of these NPs by using high gradient magnetic columns. IO NPs are also known for their non-toxicity and biodegradability, therefore ideal for biomedical applications [7,8]. Previous studies showed that it is also possible to mark the IO NPs with a fluorescent dye, e.g., near IR (NIR) dye, which further improves the probe capabilities .
NIR light of 700 to 1000 nm, achieves the highest tissue penetration due to minimal absorbency of the surface tissue in this spectral region. In vivo fluorescence imaging has experienced substantial growth with the “opening” of the NIR “window” because of the development of novel NIR fluorescence probes and optical imaging instruments [10-12]. In previous studies we described the generation of IO NPs coated with Human Serum Albumin (HSA) . HSA exhibits an average blood half-life of 19 days and is emerging as a versatile protein carrier for drug targeting and improving the pharmacokinetic profile of peptide or protein-based drugs . These properties combined with lack of toxicity, easy availability, biodegradability and preferential uptake in tumor and inflamed tissues make the core-shell IO/HSA NPs an ideal candidate for drug targeting and delivery. In addition, another important property of the HSA coating onto the IO NPs is that its various functional groups, e.g., carboxylates, amines, hydroxyls and thiols, can easily be used through different activation methods for conjugation of the IO/HSA NPs to various biomolecules such as proteins, amino acids, antibodies, oligonucleotides, etc. [15,16]. Conjugation of proteins to IO/HSA NPs is predicted to reduce their susceptibility to chemical, enzymatic and thermal degradation, thus enhancing the protein biological efficacy [17-20]. Furthermore, it may provide a mean for sustained release of the conjugated proteins.
Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent cells that can differentiate into mesenchymal and non-mesenchymal lineages. They can give rise to osteogenic, chondrogenic, adipogenic, myoegenic, hepatogenic, endothelial and neurogenic cells both in vitro and in vivo [21-26]. BM-MSCs secrete trophic factors that can promote the survival of damaged cells, as well as immunomodulatory cytokines that can suppress T-cell proliferation and function [27-31]. Because of their good proliferation, differentiation and paracrine potential, as well as their relative ease of isolation and low immunogenicity, BM-MSCs have become a main source for tissue engineering of bone, cartilage, muscle, marrow stroma, tendon, fat, and other connective tissues [32-34]. Furthermore, we and others have shown that hBM-MSC transplantation has the potential to ameliorate the symptoms of various neurodegenerative diseases, including retinal degeneration, Alzheimer's disease, Parkinson, familial amyotrophic lateral sclerosis and multiple sclerosis [29,35-37] as well as other disease such as acute liver failure  and pulmonary emphysema . These and other successful animal studies have led to numerous clinical trials using hBM-MSC as a source for cellular therapy for treatment of heart, liver, bone and cartilage repair, foot ulcers, spinal cord injuries, peripheral nerve injuries and acute graft-versus-host disease [40-46]. Since mesenchymal stem cells comprise only 0.001-0.01% of the bone mononuclear cells, extensive in vitro expansion is required to obtain sufficient number of cells for clinical use . Although the cells have high proliferation potential, prolonged culture expansion may reduce the cell differentiation potential. In addition, proliferation and differentiation potential varies between donors . Hence enhancing cell proliferation and differentiation potential could improve their yields for clinical applications.
In addition, following transplantation of hBM-MSc there is a need to repeatedly monitor the cells in vivo in a non-invasive manner. This cannot be achieved using histological and immunohistochemical techniques that require tissue removal. We have previously shown that prelabeling of mesenchymal stem cells with IO NPs enables noninvasive in-vivo tracking following cell transplantation using Magnetic Resonance Imaging (MRI, ).
Several studies have demonstrated that supplementation of basic FGF (also known as FGF2) to BM-MSC culture medium increases cell proliferation rate and cell differentiation [50,51]. However, as the cells are cultivated at 37 degrees, rapid enzymatic degradation and protein denaturation leads to short time life of FGF2 of about 3–10 minutes and reduces its biological activity and functions [52,53]. In a previous study we showed that conjugation of FGF2 to IO/HSA NPs stabilized the factor and significantly improved its ability to promote rat nasal olfactory mucosa cell migration, growth and differentiation . The present article describes a method of preparing FGF2-conjugated IO/HSA NIR fluorescent core-shell NPs that significantly stabilized the FGF2 through its covalent conjugation to the nanoparticle’s surface [55,56]. We also show that FGF2 conjugated to IO/HSA NPs is internalized by hBM-MSCs and promotes the growth and trilineage (neuronal, bone, fat) differentiation capacity of the cells at a higher extent compared with the free FGF2.
Results and discussion
The NIR fluorescent IO/HSA NPs were prepared by nucleation followed by stepwise growth of IO thin films onto the gelatin/IO nuclei as described in the “Methods” section.
Nanoparticles’ characterization: dry and hydrodynamic size and size distribution
The irreversible light-induced destruction of the fluorophore also known as photobleaching is affected by factors such as temperature, exposure time, oxygen, oxidizing or reducing agents and illumination levels . The encapsulation of Cy7 within the NPs significantly reduced the photobleaching as demonstrated in Figure 3. Encapsulation of the dye probably protects the dye against reactive oxygen species, thereby reducing photobleaching [55,57]. Previous work in our lab showed similar results with the dye RITC conjugated to NPs .
Long term stability of free versus conjugated neurotrophic factors
Conjugated-FGF2 promotes hBM-MSC cell expansion
Uptake of Nanoparticles by hBM-MSCs
Cells incubated with non-conjugated IO/HSA NPs were also positive for Prussian Blue staining, but to a lower extent compared with the FGF2-conjugeted IO/HSA NPs (Figure 7B,E,H). This data strongly suggest that at concentration of 50 ng/ml and lower, the majority of FGF2-conjuagted NPs were most probably internalized by the cells via receptor-mediated endocytosis. Endocytosis of FGF2-conjugated NPs could be mediated by FGF receptor 1 (FGFR1) that is expressed in hBM-MSCs and mediates FGF2 internalization and signaling [62,63]. Our findings are supported by various studies that demonstrated that conjugating a variety of ligands to NP surfaces facilitates receptor-mediated endocytosis of the NPs . TUNEL staining revealed that there were no apoptotic cells in cultures supplemented with 100 ng/ml conjugated or free FGF2, 90 μg/ml non-conjugated NPs or control cells, supporting the biocompatibility of the IO/HSA NPs (data not shown).
Effect of conjugated FGF2 on the clonal expansion capacity of hBM-MSCs
Effect of conjugated FGF2 on neurogenic differentiation
Effect of conjugated FGF2 on adipogenic differentiation
Effect of conjugated FGF2 on Osteogenic differentiation
Taken together we have shown here that IO/HSA NPs are biocompatible and that FGF2-conjugated IO/HSA NPs significantly enhanced hBM-MSC growth and trilineage differentiation compared with the same concentration of free FGF2. Our findings suggest that these FGF-coupled NPs may possibly be used for expanding hBM-MSCs and enhancing their differentiation potential for future therapeutic use. As the cells endocytose the FGF2-IO/HSA NPs, it is very likely that these NPs will facilitate in-vivo detection of transplanted cells using MRI and as an added benefit, the labeled cell may be imaged by NIR fluorescence using optical coherence tomography (OCT) and other in vivo imaging systems equipped with NIR fluorescence. In this work we tested the biological activity of conjugated FGF2 as there is a vast amount of literature supporting the use of this growth factor for enhancing growth and differentiation of hBM-MSCs. The effect of supplementing the growth media of hBM-MSCs with FGF2-conjugated IO/HSA NPs on the cell therapeutic effect in vivo in animal models of neuroretinal degeneration will be investigated in future studies.
In future work we will also test the effect of other conjugated factors such as Bone Morphogenetic Protein 1 (BMP1) for bone defect applications and Heparin binding Epidermal Growth Factor-like Growth Factor (HB-EGF) for neuroretinal degeneration applications.
The following analytical-grade chemicals were purchased from commercial sources and used without further purification: bicarbonate buffer (BB; 0.1M, pH 8.4), ferric chloride hexahydrate, hydrochloric acid (1 M), sodium hydroxide (1 M), sodium nitrate, Triton X-100, gelatin from porcine skin, human serum albumin (HSA), NHS-Cy7, rhodamine isothiocyanate (RITC), divinyl sulfone (DVS), triethylamine (TEA), D-glucose from Sigma (Israel); FGF2 ELISA kit and recombinant human FGF2 from PeproTech (Israel); Midi-MACS magnetic columns from Almog Diagnostic (Israel); phosphate-buffered saline (PBS free of Ca+2 and Mg+2; 0.1 M, pH 7.4) from Biological-Industries (Israel); tissue culture plates (96 wells) and plastic tips from Greiner bio-one (Germany); Water was purified by passing deionized water through an Elgastat Spectrum reverse osmosis system (Elga, High Wycombe, UK). All tissue culture reagents were from Biological Industries (Israel). B27 and DAPI were from Invitrogen. Dexamethasone, insulin, β-glycophostphate, ascorbate phosphate, neuron-specific microtubule-associated protein 2 mouse monoclonal antibody and dyes were from Sigma. Glial Fibrillary Acidic Protein rabbit monoclonal antibody was from Cell Signaling. TUNEL TMR Red was from Roche. Secondary antibodies were from Jackson ImmunoResearch. PCNA (pc1-0) mouse monoclonal IgG2a was from Santa Cruz, USA.
Preparation of the non-fluorescent and fluorescent core IO nanoparticles
Core IO NPs of narrow size distribution were prepared by nucleation in the initial part, followed by stepwise controlled growth of IO thin films onto gelatin/IO nuclei. Briefly, IO NPs of 18 ± 1 nm diameter were prepared by adding FeCl2 solution (10 mmol/5 ml H2O, 1 N HCl 0.5ml) to 80 ml aqueous solution containing 240 mg gelatin (during the whole procedure, the aqueous suspension is agitated at 60°C), followed by NaNO3 solution (7 mmol/5 ml H2O). Next, 1N NaOH aqueous solution was added up to pH 9.5. This procedure was repeated three times with 10 min intervals. The formed magnetic NPs were then washed from excess reagents with water using high gradient magnetic field (HGMF) technique. As soon as the washing step was completed, the column was removed from the magnetic field and the NPs were eluted by adding an aqueous bicarbonate buffer (BB, 0.1M, pH = 8.3) . NIR core IO NPs were prepared similarly, by substituting the gelatin for gelatin covalently conjugated with NHS Cy7 to obtain NIR-IO NPs .
HSA coating onto the fluorescent IO core nanoparticles
HSA coating was performed by shaking the aqueous suspension of the fluorescent IO NPs with 10% HSA (MW ~66,000) at 75°C for 12h. The HSA coated NPs were then washed from excess reagents by magnetic columns with PBS (pH = 7.4).
Activation of the fluorescent IO/HSA nanoparticles
Activation of the fluorescent IO/HSA NPs was performed by functionalization of these NPs with excess DVS. One double bond created a covalent bond with the amino groups of the HSA coating onto the fluorescent IO NPs. The residual activated double bond was then used for covalent binding of ligands containing primary amino groups. Briefly, 20 μl of DVS were added to 1 ml of the fluorescent IO/HSA NPs (5 mg/ml) dispersed in the BB continuous phase. The dispersion was then shaken for 12h at 60°C and the remaining free DVS was then washed from the obtained DVS-conjugated NPs using magnetic columns with BB.
Conjugation of FGF2 to the activated fluorescent IO/HSA nanoparticles
Bioactive ligands such as amino acids, proteins, antibodies and more can be easily conjugated to the DVS activated NPs. Briefly, 200 μl of dissolved FGF2 (0.1 mg/ml) were mixed with 200 μl of the DVS activated fluorescent IO/HSA NPs (5 mg/ml) dispersed in BB (0.1M, pH = 8.3). Next, the dispersion was shaken at room temperature for 60 min in order to allow the nucleophilic attack of primary amino groups (from the bioactive ligand) on the DVS-IO/HSA NPs. Blocking of residual activated DVS groups was then performed with glycine, by adding glycine (1% w/v) and mixing the dispersion for additional 30 min at room temp. Excess of unbound ligands were then removed by magnetic columns and the FGF2 conjugated IO/HSA NPs were then eluted with PBS (pH = 7.4).
Transmission Electron Microscopy (TEM)
The TEM image provides direct information on the dry particle shape and size, in which approximately 200 NPs were measured to determine its average size. The core IO NPs and the core-shell IO/HSA NPs were diluted with H2O to a concentration of 1 mg/ml, dripped on a TEM grid and then dried.
Dynamic Light Scattering (DLS)
Dynamic light scattering measures Brownian motion and relates the intensity fluctuations in the scattered light to the size and size distribution of the particles in its hydrated shape. The fluorescent core IO NPs and core-shell IO/HSA NPs were diluted with H2O inside a cuvette and the average diameter was then measured, while each measurement was repeated 5 times.
Spectrofluorometer uses the fluorescent properties of a molecule to provide information about their concentration and fluorescence intensity, both excitation and emission, in different wavelength. Cy7 and Cy7-IO NPs were diluted with PBS to a concentration of 250 ng/ml of the dye, followed by emission, excitation and stability measurements.
Enzyme-Linked ImmunoSorbent Assay (ELISA)
Enzyme-linked immunosorbent assay (ELISA) is commonly used to determine if a particular protein is present in a sample and its concentration . In the present work the concentration of the free and conjugated FGF2 was determined by FGF2 ELISA kit (PeproTech, Israel) based on a calibration curve of known concentrations of free FGF2, according to the literature and following manufacturer’s instructions . Samples of the Cy7-IO/HSA-DVS-FGF NPs were diluted with ELISA assay diluent to 3 different NPs' concentrations, each concentration was tested in triplicates and the mean value was calculated. The concentration of the bound FGF2 was determined from a calibration curve of free FGF2 and found to be 1.1 μg/mg core-shell NPs.
Comparative stability studies of free versus conjugated FGF2
For stability measurements, free or conjugated-FGF2 were incubated (10 ng/ml) in various concentrations of fetal calf serum in the medium (0–100% of non-heated serum diluted in medium) at 37°C for 1 and 7 days. The concentration of the residual free and conjugated-FGF2 was then determined by FGF2 ELISA kit.
Production of BM-MSCs
Fresh human Bone Marrows Mesenchymal Stromal Cells (hBM-MSCs) were collected from 6 healthy donors in the operating room at The Sheba Medical Center, Tel-Hashomer, under sterile conditions. The research was approved by the institutional review board at the Sheba Medical Center. Bone marrow mononuclear cells were separated by Ficoll gradient (1.077g/dl) according to the manufacturer instructions and were seeded in tissue culture flasks with culture media containing low-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 15% FCS, 100U/ml penicillin, 100 ug/ml streptomycin and 2mM L-Glutamine. Tissue culture media was changed after 48 h and then twice a week until 70-80% confluence was reached. Trypan blue staining was performed in every subculturing.
Expansion of hBM-MSCs
Cell expansion in the presence of different supplements was tested in cells derived from 3 BM donors. Cells were grown for 3 passages between passages 2–5. Cells, at concentration of 5 × 103 cell/well, were seeded in 6 well plates in the presence of 0.1 ng/ml free FGF2 or conjugated FGF2 or 90 ng/ml Cy7-IO/HSA NPs in duplicates. Cells were subcultured every 7 ± 2 days and reseeded at 5 × 103 cells per well. Since all plates for each donor were subcultured at the same time for each passage, control and non-conjugated NPs cultures were less confluent than their FGF-treated counterparts during subculturing. To evaluate cell morphology and size, cells were seeded on cover slips and stained with Nuclear Fast Red or analyzed by flow cytometry.
Flow cytometry analysis
Cell surface antigen phenotyping was performed by flow cytometry (FACSCalibur, Becton-Dickinson) using antibodies directed against CD14, CD34, CD45, CD73, CD90, CD105 and HLA-DR to confirm mesenchymal cell phenotype [29,35-37].
The uptake of Cy7 within cells was evaluated by FACSAria III (BD) cell sorting. In order to maximize cell viability and minimize mechanical perturbations, we set the flow rate to 1.1 (minimum). For Cy7 analysis 633nm excitation laser was used with a filter. Data were processed by FlowJo v7.6.4.
Cellular uptake of NPs
Cells were seeded on coverslips precoated with MSC- attachment solution following manufacturer instructions (Biological Industries, Israel). After 24h, NPs were added to cell growth media for 48hr followed by fixation with 4% paraformaldehyde (PFA). Cells were stained with Prussian Blue iron stain and Nuclear Fast Red and visualized by light microscopy (Olympus BX51).
Colony formation assay
For Colony Forming Unit-Fibroblasts (CFU-F) assay, cells were seeded in 6 well plates (250 cell/well) in growth medium. Medium was changed every 3 days. Colonies were formed, analyzed and counted within 7 days after seeding. Cells were washed to remove non adherent colonies. Colonies were fixed in methanol, stained with Giemsa stain, and manually counted. All counting were done in a masked fashion.
Adipogenic differentiation assay
For induction of adipogenic differentiation, cells were cultured for two weeks in growth medium supplemented with 0.6M dexamethasone and 10 mg/l Insulin. Oil-Red-O staining was performed to identify the adipogenic cells followed by hematoxylin counter staining.
Osteogenic differentiation assay
For induction of osteogenic differentiation, cells were cultured for two weeks in growth medium supplemented with 0.1M dexamethasone, 10mM β-glycophostphate and 50 ng/ml ascorbate phosphate. Alizarin red staining was performed to identify the osteogenic cells.
Neurogenic differentiation assay
To induce neurogenic differentiation, cells were grown in DMEM supplemented with 0.5% B27, 1% fetal bovine serum, 5% horse serum, 0.5mM retinoic acid, 20 ng/ml epidermal growth factor and 50 ng/ml nerve growth factor. After two weeks, cells were fixed in 4% PFA, and immunostained with neuron-specific Microtubule-Associated Protein 2 (MAP2) mouse monoclonal antibody or Glial Fibrillary Acidic Protein (GFAP) rabbit monoclonal antibody.
A general linear model was used using the donor, replicate and treatment type as independent parameters and differentiation score or colony number as the dependent variables. In the analysis of adipose differentiation the score was normalized after logarithmic transformation. Equality of variance was tested and maintained in all analyzes. We used the Bonferroni correction for post hoc analyses.
This study was supported by a grant from the Claire and Amedee Maratier Institute for the Study of Blindness and Visual Disorders, Sackler Faculty of Medicine, Tel-Aviv University, and a grant from the Israeli Ministry of Trade and Industry KAMIN–Yeda Program (to YR). IS was partially supported by the Israeli Ministry of Absorption and Immigration. The supporting organizations had no role in the design or conduct of this research.
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