Glioblastoma (GBM) is one of the most aggressive and common primary brain tumors in adults with median survival time less than 15 months upon diagnosis. The standard treatment for newly diagnosed GBM is surgical resection to remove tumor, followed by a combination with radiotherapy and chemotherapy [1]. Although there have been standard therapy and numerous ongoing clinical trials for GBM patients, therapeutic benefits were still limited with standard treatment and better therapeutic options are still needed [2].

Temozolomide (TMZ), a DNA alkylating agent, is the standard chemotherapy drug commonly prescribed for newly diagnosed GBM. TMZ can be spontaneously converted to the active compound (5-(3-Methyl-1-triazeno)imidazole-4-carboxamide, MTIC) under physiological conditions and exert antitumor effect by DNA methylation [3]. Oral administration of TMZ can achieve nearly 100 % of bioavailability, and some studies reported that TMZ have good blood–brain barrier (BBB) permeability [4], [5]. However, TMZ is the substrate of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), both of them are expressed in BBB and may efflux transporting TMZ out of the brain, leading to reduced local TMZ concentration and therefore diminish therapeutic effect in GBM patients [6], [7]. To solve this issue, combination of transporter inhibitor may be a good choice to increase TMZ concentration in brain.

Tariquidar (XR9574, TQD) is a potent and selective third-generation P-gp inhibitor that has demonstrated the ability to increase penetration of P-gp substrates through BBB and overcome multiple drug resistance (MDR) in animal studies and clinical trials [8], [9], [10], [11]. While TQD has not yet been approved for market use, since the risks was bigger than benefits in cancer treatment and the excipient of TQD injection also provoked some adverse events during clinical trials investigation [10], [12]. Suitable formulation design for TQD may preserve its pharmacological activity and minimize potential side effects when combined with chemotherapy drugs.

TQD is administered by IV infusion in clinical trials investigation. However, for combined use with TMZ, long acting injectable (LAI) formulation may be suitable for TQD to align with the chemotherapy regimen of TMZ and improve patient compliance. Biodegradable inorganic carriers are common LAI and each of them have distinct advantages for formulation design. An ideal LAI carrier should exhibit characteristics including good biocompatibility and high biodegradability to ensure biosafety, as well as controlled drug release profile to avoid drug related side effects and maintain suitable drug concentration in plasma [13], [14].

Hydroxyapatite (HAP) is a calcium-based particle that has been applied and investigated as a biomaterial and cellular drug delivery system in many studies due to its biocompatibility and biodegradability [15], [16], [17]. HAP is stable in physiological conditions but can undergo degradation into Ca2+ and PO43- ions in acidic environment. Previous study has shown that drug-loaded HAP can be taken up by immune cells such as macrophage, then release the drug from cytoplasm into extracellular matrix and blood circulation through exocytosis [18]. Drug releasing rate is controlled by macrophage endocytosis, exocytosis, and HAP degradation rate, thus exhibiting sustained release characteristic. The surface of HAP can be modified with different materials to increase hydrophilicity or hydrophobicity, making it capable of loading different drugs with various characteristics [19]. Aside from these advantages, HAP can be easily prepared in large quantities with precipitation method, making scaling up possible [16], [20].

In this study, we aimed to use HAP as a carrier for TQD in combination with TMZ therapy for GBM management (Fig. 1). We used stearic acid (SA) to modify the surface of HAP, and TQD were loaded on to SA-modified HAP (SA-HAP-TQD) with hydrophobic interaction. SA-HAP-TQD was characterized for its in vitro release, cellular uptake and in vivo degradation study. Brain penetration enhancement effect of TMZ with the aid of SA-HAP-TQD was assessed using brain microdialysis. The possible side effects of TMZ and combination therapy were evaluated using hematology analyzer. Antitumor effect of this combination therapy was also examined. The SA-HAP-TQD were consistently released TQD to enhance TMZ brain concentration and facilitated antitumor effect of TMZ.