Cellular Uptake of Modified Mesoporous Bioactive Glass Nanoparticles for Effective Intracellular Delivery of Therapeutic Agents

Abstract

Introduction: There has recently been a surge of interest in mesoporous bioactive glass nanoparticles (MBGNs) as multi-functional nanocarriers for application in bone-reconstructive and -regenerative surgery. Their excellent control over their structural and physicochemical properties renders these nanoparticles suitable for the intracellular delivery of therapeutic agents to combat degenerative bone diseases, such as bone infection, or bone cancer. Generally, the therapeutic efficacy of nanocarriers strongly depends on the efficacy of their cellular uptake, which is determined by numerous factors including cellular features and the physicochemical characteristics of nanocarriers, particularly surface charge. In this study, we have systematically investigated the effect of the surface charge of MBGNs doped with copper as a model therapeutic agent on cellular uptake by both macrophages and pre-osteoblast cells involved in bone healing and bone infections to guide the future design of MBGN-based nanocarriers.

Methods: Cu-MBGNs with negative, neutral, and positive surface charges were synthesized and their cellular uptake efficiency was assessed. Additionally, the intracellular fate of internalized nanoparticles along with their ability to deliver therapeutic cargo was studied in detail.

Results: The results showed that both cell types internalized Cu-MBGNs regardless of their surface charge, indicating that cellular uptake of nanoparticles is a complex process influenced by multiple factors. This similarity in cellular uptake was attributed to the formation of a protein corona surrounding the nanoparticles when exposed to protein-rich biological media, which masks the original nanoparticle surface. Once internalized, the nanoparticles were found to mainly colocalize with lysosomes, exposing them to a more compartmentalized and acidic environment. Furthermore, we verified that Cu-MBGNs released their ionic components (Si, Ca, and Cu ions) in both acidic and neutral environments, leading to the delivery of these therapeutic cargos intracellularly.

Conclusion: The effective internalization of Cu-MBGNs and their ability to deliver cargos intracellularly highlight their potential as intracellular delivery nanocarriers for bone-regenerative and -healing applications.

Keywords: bioactive glass; copper; intracellular delivery; nanocarrier; surface charge.

Graphical abstract

Figure 1

Synthesis and characterization of Cu-MBGNs. (A) SEM and (B) TEM of Cu-MBGNs. (C) Size distribution of Cu-MBGNs obtained from SEM pictures. (D) Nitrogen adsorption-desorption isotherms and pore size distribution (insert) of Cu-MBGNs. (E) FTIR spectra and (F) XRD patterns of synthesized nanoparticles.

Figure 2

(A) Schematic illustration indicating preparation of differently charged Cu-MBGNs through surface modification with APTES and labeling with FITC. (B) FTIR spectra of Cu-MBGNs before and after amine modification. (C) Variation of the surface charge of aminated Cu-MBGNs depending on the applied FITC: aminated Cu-MBGNs ratio. The scale bar in panel A represents 20 µm.

Figure 3

Interaction between differently charged Cu-MBGNs with MC3T3-E1 and RAW 264.7 cells. Cytocompatibility of differently charged Cu-MBGN to (A) MC3T3-E1 and (B) RAW 264.7 cells as a function of nanoparticle concentration. Dashed red lines in panel A and B indicate the control group (cells without particles). (C) Intracellular uptake of differently charged Cu-MBGNs by MC3T3-E1 and RAW 264.7 cells. Cu-MBGNs were labeled using FITC (green), while actin filaments of the cytoskeleton and nuclei were stained with rhodamine-phalloidin (red) and DAPI (blue), respectively. Intracellular uptake efficiency of differently charged Cu-MBGNs by (D) MC3T3-E1 and (E) RAW 264.7 cells. Scale bars represent 10 µm.

Figure 4

Zeta potential measurement of the nanoparticles after 4 h of incubation in media containing FBS.

Figure 5

(A) Colocalization of Cu-MBGNs. MC3T3-E1 and RAW 264.7 cells were treated with differently charged Cu-MBGNs for 24 h and then stained with LysoTracker^® Red. Scale bars represent 10 µm. (B) In vitro ion release profiles of Cu-MBGNs in PBS and ALF for up to 10 days.