In vitro and in vivo accumulation of magnetic nanoporous silica nanoparticles on implant materials with different magnetic properties

Abstract

Background: In orthopedic surgery, implant-associated infections are still a major problem. For the improvement of the selective therapy in the infection area, magnetic nanoparticles as drug carriers are promising when used in combination with magnetizable implants and an externally applied magnetic field. These implants principally increase the strength of the magnetic field resulting in an enhanced accumulation of the drug loaded particles in the target area and therewith a reduction of the needed amount and the risk of undesirable side effects. In the present study magnetic nanoporous silica core-shell nanoparticles, modified with fluorophores (fluorescein isothiocyanate/FITC or rhodamine B isothiocyanate/RITC) and poly(ethylene glycol) (PEG), were used in combination with metallic plates of different magnetic properties and with a magnetic field. In vitro and in vivo experiments were performed to investigate particle accumulation and retention and their biocompatibility.

Results: Spherical magnetic silica core-shell nanoparticles with reproducible superparamagnetic behavior and high porosity were synthesized. Based on in vitro proliferation and viability tests the modification with organic fluorophores and PEG led to highly biocompatible fluorescent particles, and good dispersibility. In a circular tube system martensitic steel 1.4112 showed superior accumulation and retention of the magnetic particles in comparison to ferritic steel 1.4521 and a Ti90Al6V4 control. In vivo tests in a mouse model where the nanoparticles were injected subcutaneously showed the good biocompatibility of the magnetic silica nanoparticles and their accumulation on the surface of a metallic plate, which had been implanted before, and in the surrounding tissue.

Conclusion: With their superparamagnetic properties and their high porosity, multifunctional magnetic nanoporous silica nanoparticles are ideal candidates as drug carriers. In combination with their good biocompatibility in vitro, they have ideal properties for an implant directed magnetic drug targeting. Missing adverse clinical and histological effects proved the good biocompatibility in vivo. Accumulation and retention of the nanoparticles could be influenced by the magnetic properties of the implanted plates; a remanent martensitic steel plate significantly improved both values in vitro. Therefore, the use of magnetizable implant materials in combination with the magnetic nanoparticles has promising potential for the selective treatment of implant-associated infections.

Keywords: Biocompatibility; Core–shell nanoparticles; Drug targeting; Ferritic steel; Martensitic steel; Mouse model; Nanoporous silica; PEGylation; Superparamagnetic Fe3O4.

Fig. 1

In vitro setup: (1) electromagnet, (2) FITC-linked MNPSNPs, (3) plate, (4) Heidelberger extension, (5) peristaltic pump

Fig. 2

Top: Scheme of synthesis route for MNPSNPs, bottom: scheme for modification of MNPSNPs with PEG and FITC (left) or RITC (right)

Fig. 3

TEM images of (a) oleic acid-capped Fe₃O₄ NPs, (b) unmodified MNPSNPs, (c) MNPSNP@FITC-PEG, (d) MNPSNP@RITC-PEG

Fig. 4

Magnetization curves at 300 K of (a) oleic acid-capped Fe₃O₄ NPs and (b) unmodified MNPSNPs. The inset in bottom right is the low-field magnetization curve

Fig. 5

FT-IR spectra of unmodified MNPSNPs (black), MNPSNP@FITC-PEG (green) and MNPSNP@RITC-PEG (orange)

Fig. 6

Nitrogen adsorption (dots) and desorption isotherm (circles) of unmodified MNPSNPs (black), MNPSNP@FITC-PEG (green) and MNPSNP@RITC-PEG (orange)

Fig. 7

Thermogravimetric curves of unmodified MNPSNPs (black), MNPSNP@FITC-PEG (green) and MNPSNP@RITC-PEG (orange)

Fig. 8

Cell proliferation and cell viability after 24 and 48 h of NIH-3T3 cells. Control group (0 µg MNPSNPs) compared to concentrations of 5, 10, 25, 50 and 100 µg MNPSNPs/mL cell culture medium. Negative controls include 5% DMSO. Mean ± SD, *p < 0.05, ***p < 0.001, n = 6

Fig. 9

Cell proliferation and cell viability after 24 and 48 h of HepG2 cells. Control group (0 µg MNPSNPs) compared to concentrations of 5, 10, 25, 50 and 100 µg MNPSNPs/mL cell culture medium. Negative controls include 5% DMSO. Mean ± SD, *p < 0.05, ***p < 0.001, n = 6

Fig. 10

Percentage in vitro accumulation of MNPSNPs at different implant materials (M martensitic steel, F ferritic steel, T titanium alloy) and in the magnetic field without plate (N no plate in tube systems), *p < 0.05, **p < 0.01

Fig. 11

Percentage MNPSNP mass at the different plates after in vitro accumulation of the MNPSNP suspension in the magnetic field and subsequent 3 min additional circulation time, *p < 0.05

Fig. 12

a–c H.E. staining of the skin-muscle-layer with former plate location (p) showing macrophage infiltration, fibrotic tissue and MNPSNPs associated to cells (black arrows); d Fluorescent MNPSNPs (orange spots, white arrows) in the fibrotic tissue. All scale bars: 50 µm

Fig. 13

a H.E. staining of the Ln. iliacus with histiocytic inclusions (black arrows); b Fluorescent MNPSNPs (orange spots) in a corresponding area associated to cells (white arrows). Scale bars: 50 µm

Fig. 14

Summed score for evaluation of droplets containing MNPSNPs on explanted ferritic steel and titanium alloy plates 1 week after in vivo injection subcutaneously near the implant