Hydroxyapatite (HA) has been widely used as a bone substitute and more recently as a carrier for local delivery of bone targeted drugs. Majority of the approved HA based biomaterials and drug carriers comprise of micrometer sized particulate HA (mHA) or granules and can therefore only be used for extracellular drug release. This shortcoming could be overcome with the use of cell penetrating HA nanoparticles (nHA) but a major concern with the clinical use of nHA is the lack of data on its in vivo biodistribution after implantation. In this study, we aimed to study the in vivo biodistribution of locally implanted nHA in a clinically relevant tibial void in rats and compare it with mHA or a combination of mHA and nHA. To enable in vivo tracking, HA particles were first labelled with 14C-zoledronic acid (14C-ZA), known to have a high binding affinity to HA. The labelled particles were then implanted in the animals and the radioactivity in the proximal tibia and vital organs was detected at various time points (Day 1, 7 and 28) post-implantation using scintillation counting. The local distribution of the particles in the bone was studied with micro-CT. We found that majority (>99.9%) of the implanted HA particles, irrespective of the size, stayed locally at the implantation site even after 28 days and the findings were confirmed using micro-CT. Less than 0.1% radioactivity was observed in the kidney and the spleen at later time points of day 7 and 28. No pathological changes in any of the vital organs could be observed histologically. This is the first longitudinal in vivo HA biodistribution study showing that the local implantation of nHA particles in bone is safe and that nHA could potentially be used for localized drug delivery.

Osteosarcoma is a malignant cancer of the bone mainly affecting adolescents. Despite progress, the clinical management of osteosarcoma is still challenging. With the current chemotherapy protocol being used for more than 30 years, the number of poor responders is increasing. Although new treatments have been explored since then, no improved tumor eradication effect have been found. In the present thesis, we have developed a new treatment method for osteosarcoma, using hydroxyapatite (HA) based materials as a platform for local delivery of cytostatics. Doxorubicin (DOX), a cornerstone osteosarcoma drug, was chosen as a drug candidate, due to its binding capacity to HA. Different types of HA-based biomaterials were tested for local or targeted delivery of DOX. The efficacy of the developed system was evaluated in-vitro, in osteosarcoma cells as well as in-vivo, in mice bearing an aggressive osteosarcoma.
In Study 1, a clinically approved calcium sulphate (CaS)/HA biomaterial achieved a sustained and controlled release of DOX up to 28 days, both in-vitro and in-vivo. Compared to no treatment or the clinical standard with systemic DOX administration, the local delivery of DOX using a CaS/HA biomaterial significantly hindered tumor progression by inhibiting angiogenesis and cell proliferation.
In Study 2, we investigated the physicochemical interactions between DOX and different sizes of HA particles, both in-vitro and in-vivo. When delivered by HA nanoparticles, DOX is routed to the mitochondria causing insufficient ATP synthesis, less cell migration and cell apoptosis. This leads to stronger in-vivo tumor eradication compared to systemic administration of DOX. Furthermore, nHA mediated delivery of DOX may prevent further metastases in- vivo, which was indirectly verified by PET/CT data.
In Study 3, HA particles (nHA, mHA or n/mHA) were labelled with carbon 14 (14C) to detect particle migration in- vivo. During the observational time of 28 days, the majority (>99.9%) of implanted HA particles, irrespective of the size, stayed in the implantation site (proximal tibia), without migrating to other vital organs. No pathological changes were detected in the vital organs.
In summary, we describe a new and efficient method to supplement osteosarcoma treatment, with a possible rapid translational potential, using clinically approved constituents. By using a hydroxyapatite-based biomaterial, DOX could be routed to the tumor site, more efficiently and with less side effects compared to systemic administration. The chemical interaction between DOX and HA lead to a sustained and controlled DOX release which further improved its tumor eradication effect. When using HA nanoparticles, DOX could be directed to the mitochondria causing tumor cell starvation, reduced migration and apoptosis, jointly leading to improved tumor eradication. The local administration of HA particles, irrespective of size, was confirmed as safe without damage to vital organs. In the future, chemotherapeutics with multi-release profile potentially could be applied by using a combination of nHA and mHA.

Efficient systemic pharmacological treatment of solid tumors is hampered by inadequate tumor concentration of cytostatics necessitating development of smart local drug delivery systems. To overcome this, we demonstrate that Doxorubicin (DOX), a cornerstone drug used for osteosarcoma treatment, shows reversible accretion to hydroxyapatite (HA) of both nano (nHA) and micro (mHA) size. nHA particles functionalized with DOX get engulfed in the lysosome of osteosarcoma cells where the acidic microenvironment causes a disruption of the binding between DOX and HA. The released DOX then accumulates in the mitochondria causing cell starvation, reduced migration and apoptosis. The HA + DOX delivery system was also tested in-vivo on osteosarcoma bearing mice. Locally delivered DOX via the HA particles had a stronger tumor eradication effect compared to the controls as seen by PET-CT and immunohistochemical staining of proliferation and apoptosis markers. These results indicate that in addition to systemic chemotherapy, an adjuvant nHA could be used as a carrier for intracellular delivery of DOX for prevention of tumor recurrence after surgical resection in an osteosarcoma. Furthermore, we demonstrate that nHA particles are pivotal in this approach but a combination of nHA with mHA could increase the safety associated with particulate nano materials while maintaining similar therapeutic potential.

Doxorubicin (DOX) is a cornerstone drug in the treatment of osteosarcoma. However, achieving sufficient concentration in the tumor tissue after systemic administration with few side effects has been a challenge. Even with the most advanced nanotechnology approaches, less than 5% of the total administered drug gets delivered to the target site. Alternatives to increase the local concentration of DOX within the tumor using improved drug delivery methods are needed. In this study, we evaluate a clinically approved calcium sulfate/hydroxyapatite (CaS/HA) carrier, both in-vitro and in-vivo, for local, sustained and controlled delivery of DOX to improve osteosarcoma treatment. In-vitro drug release studies indicated that nearly 28% and 36% of the loaded drug was released over a period of 4-weeks at physiological pH (7.4) and acidic pH (5), respectively. About 63% of the drug had been released after 4-weeks in-vivo. The efficacy of the released drug from the CaS/HA material was verified on two human osteosarcoma cell lines MG-63 and 143B. It was demonstrated that the released drug fractions functioned the same way as the free drug without impacting its efficacy. Finally, the carrier system with DOX was assessed using two clinically relevant human osteosarcoma xenograft models. Compared to no treatment or the clinical standard of care with systemic DOX administration, the delivery of DOX using a CaS/HA biomaterial could significantly hinder tumor progression by inhibiting angiogenesis and cell proliferation. Our results indicate that a clinically approved CaS/HA biomaterial containing cytostatics could potentially be used for the local treatment of osteosarcoma.

Doxorubicin (Dox), an anthracyclin agent, is widely used as a chemotherapy agent for different kinds of malignancies. However, even when entrapped in liposomes, Dox has a terminal half-life in plasma of 69.3 h compared to 17.3 h with free doxorubicin (Rahman et al1 ). And only a small fraction (<5%) of the total administered liposomal formulation is actually delivered to the target site2 . Alternatives to improve efficient and contained delivery of Dox locally within the tissue are needed. Zoledronic acid (ZA) is a new-generation bisphosphonate, and widely used as an adjuvant treatment for bone metastasis, to reduce tumor-related pain and skeletal-related events3 . It could also increase bone formation and enhance repair of large bone defects4 . Furthermore, locally delivered ZA can stay at the target site for up to 6 months due to its affinity to hydroxyapatite (HA) (manuscript in progress). Local dual delivery of Dox and ZA acts synergistically to achieve strong and long-term anti-tumor effect, as well as bone regeneration at the eradicated tumor site. Tumor recurrences and simultaneously an enhanced repair of the bone defect can be achieved. The aim of the present study was to characterize the release kinetics of Dox and ZA from an injectable FDA and CE approved calcium sulfate/hydroxyapatite (CaS/HA) carrier and evaluate the effect on human osteosarcoma cells MG-63. 

  1. Yang L. et al., Longitudinal in vivo biodistribution of nano and micro sized hydroxyapatite particles implanted in a bone defect. Frontiers in Bioengineering and Biotechnology 2022.
  2. Yang Liu., Hydroxyapatite – A trojan horse in the delivery of apatite-binding cytostatics in bone cancer. Doctoral Thesis 2022.
  3. Yang al., Bone mineral: A trojan horse for bone cancers efficient mitochondria targeted delivery and tumor eradication with nano hydroxyapatite containing doxorubicin. Materials Today Bio
  4. Yang L. et al., Sustained and controlled delivery of doxorubicin from an in-situ setting hydroxyapatite carrier for local treatment of osteosarcoma. Acta Biomaterialia 2021.
  5. Yang L. et al., Nano-/micro- sized hydroxyapatite moiety improves doxorubicin delivery and tumor eradication., 2021
  6. Yang L. et al., Dual delivery of doxorubicin and zoledronic acid from an injectable calcium sulphate/hydroxyapatite carrier.ORS 2020 Annual Meeting. Phoenix, Arizona. February 8 – 11, 2020. Abstract number 551.
  7. Yang L. et al., Sequential and sustained release of doxorubicin and paclitaxel from a calcium sulfate/hydroxyapatite carrier.27th Annual meeting of European Orthopedic Research Society (EORS 2019). Maastricht, Netherlands