A bifunctional bortezomib-loaded porous nano-hydroxyapatite/alginate scaffold for simultaneous tumor inhibition and bone regeneration

Abstract

Treatments of osteolytic lesions due to malignant metastasis remain one of the major clinical challenges. The residual tumor cells after surgical resections and an acidic tumor microenvironment are unfavorable for osteogenic induction. Bortezomib (BTZ), a proteasome inhibitor used in chemotherapy, also has an osteogenic potential in concentration- and Ca²⁺-dependent manners. In this study, controlled delivery of BTZ in a novel bifunctional scaffold based on nano-hydroxyapatite (nHA) and sodium alginate (SA) nanocomposite, namely BTZ/nHA@SA, has been explored. By smartly adjusting microenvironments, a sustainable release of Ca²⁺ from nHA could be achieved, which was not only able to cross-link SA but also to regulate the switch between the dual functions of tumor inhibition and bone regeneration of BTZ to promote the osteogenic pathway. The freeze-dried BTZ/nHA@SA scaffold has excellent interconnectivity, is capable to promote the attachment and proliferation of mouse embryonic osteoblast precursor cells, as well as effectively induces breast cancer cell death in vitro. Furthermore, in vivo, studies using a mouse tumor model and a rabbit femoral defect model showed that the BTZ/nHA@SA scaffold could promote tumor ablation, and also enhance bone repair. Therefore, the BTZ/nHA@SA scaffold has unique dual functions of inhibiting tumor recurrence and promoting bone tissue regeneration simultaneously. This smart bi-functional scaffold offers a promising novel approach for oncological treatments by synchronously orchestrating tumor inhibition and tissue regeneration for the repair of neoplastic bone defects.

Keywords: Anti-tumor; BTZ; BTZ/nHA@SA scaffold; Bone regeneration; nHA.

Fig. 1

Schematic illustration of BTZ/nHA@SA scaffold for simultaneous tumor inhibition with bone regeneration

Fig. 2

A Photographs of nHA@SA scaffold; B FESEM images of SA scaffold; C FESEM images of nHA@SA scaffold; D EDS elemental mapping analysis of Ca and P in nHA@SA scaffold; E XRD pattern of nHA and nHA@SA; F Swelling ratio of nHA@SA scaffold at pH 6.5 and 7.4; G Degradation of nHA@SA scaffold at pH 6.5 and 7.4; H Typical compressive stress–strain curves of SA scaffold and nHA@SA scaffold; I Drug release curve of BTZ/nHA@SA scaffold at pH 6.5 and 7.4

Fig. 3

A Viabilities of osteoblast cells (MC3T3) cultured with different materials; B Fluorescence images of osteoblast cells (MC3T3) stained with Calcein AM (green, live cells) and PI (red, dead cells); C SEM images of MC3T3 cells co-cultured with BTZ/nHA@SA scaffold at day 3; D Representative images of ALP staining and ARS staining of MC3T3 cells cultured with nHA@SA and BTZ/nHA@SA extract at day 7 or day 14; E Relative mRNA expression levels of osteogenic genes Alp at day 3, 5 and 7; F Relative mRNA expression levels of osteogenic genes Sp7 at day 3, 5 and 7; G Expression levels of RUNX2 and COL1 in MC3T3 cells after 7 days; H Viabilities of breast cancer cells (4T1) cultured with different materials; I Fluorescence images of breast cancer cells (4T1) stained with Calcein AM (green, live cells) and PI (red, dead cells). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

A Schematic diagram of anti-tumor experiment analysis in mice; B Flow chart of anti-tumor surgery in mice; C Pictures of the isolated tumors collected at day 14 after different treatments; D Tumor growth curves of mice subjected to different treatments; E H&E staining of tumor tissue slices; F H&E staining of major organs (heart, liver, spleen, lung and kidney) slices

Fig. 5

A Schematic diagram of rabbit bone regeneration experiment; B Flow chart of scaffold implantation in rabbit bone defect; C Micro-CT reconstruction images of femoral defect; D Coronal and transverse micro-CT reconstruction images of new bone at defect site; E Volume fraction of new bone in the area of the bone defect (BV/TV); F–I Trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and cortical bone thickness (Ct.Th) of new bone in the defect area by micro-CT quantitative analysis; J H&E staining of new bone tissue slices. *p < 0.05, **p < 0.01, ***p < 0.001