Biodegradable Nanovectors As a Direct Platform for the Controlled Delivery of Chemotherapeutic Drugs Against Childhood ALL
Authors: Ede Benjamin C , Diamanti Paraskevi , Williams David C , Blair Allison 
Published: November 2020

Drugs used to treat childhood acute lymphoblastic leukemia (ALL), both clinically and experimentally, are often highly insoluble making drug delivery inefficient due to precipitation and poor distribution. Toxicity is also a major issue owing to the high levels of harmful solvents used to solubilize drugs for administration. Therefore, identifying strategies to enhance the stability and solubility of poorly soluble drugs is an ongoing challenge. Data from clinical trials indicates that the pharmacokinetics of dexamethasone (Dex), commonly used in induction regimens for ALL, vary considerably between patients (pts) and prolonging drug exposure rather than increasing absolute dose may improve efficacy. Copolymeric nanovectors (NV) are an excellent candidate for the delivery of insoluble drugs. However, despite reports of multiple systems, the fabrication processes often rely on complex methodologies with purification steps to remove harmful chemicals used to load drugs. To bypass these fabrication issues, we have shown that fully biodegradable copolymer NV comprised of poly(ethylene glycol)-block-poly (trimethylene carbonate) (PEG-PTMC) can sequester highly insoluble drugs (with almost total efficiency) through a direct hydration process in 5 minutes, eliminating the need for purification and use of harmful solvents. The aim of this study was to assess the effects of this systemin vitroandin vivousing Dex. NV were formulated with Dex or 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR, a near infrared insoluble dye) to model NV distributionin vivo. Both Dex and DiR solubilization was initiated using residual (&lt;0.025%) amounts of dimethyl sulfoxide (DMSO) prior to addition to PEG-PTMC, oligo ethylene glycol and stirring into phosphate buffered saline. The average hydrodynamic radii of NV were ca. 40nm when loaded with Dex or DiR, as measured by dynamic light scattering. All formulations had low polydispersity index values (&lt;0.18), indicating highly uniform monodispersed populations of NV. DiR-NV were optimised to 0.125wt% to give the best linearity in fluorescence signal (r2 = 0.97) over a wide range of copolymer concentrations (0.1-20 mg/ml).In vitro, Dex-NV were equipotent with free Dex (dispersed in medium with DMSO) against primary T-ALL cells from all pts tested. The highest dose (100 µM) reduced cell viability to 45±38% in Dex-NV and 46±40% in free Dex treated cells. Crucially, the NV formulation did not reduce Dex efficacy and confirmed our previous findings using parthenolide in this system. As a control, empty (unloaded) NV were not toxicin vitro. To assess the effects of NVin vivo, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were injected with primary ALL cells from 3 pts and engraftment was measured in peripheral blood (PB) aspirates using flow cytometry. Once engrafted (&gt;0.1% ALL cells) animals were treated daily for 5 days, over 4 weeks with 2.5 mg/kg Dex via intraperitoneal injection (IP). Both Dex-NV and free Dex delayed disease progression until day 18 in pt 1, and until after treatment cessation in pts 2 and 3 with disease levels of 0.24±0.1% and 0.55±0.1% near the end of treatment, respectively. In contrast, leukemia engraftment increased immediately following engraftment in placebo controls. Dex-NV and free Dex significantly improved the survival of NSG by up to 27 days (P&lt;0.01) with no significant differences between treatments. Ten minutes after intravenous injection (IV) with DiR-NV, ventral imaging showed that fluorescence was detected throughout NSG with accumulations in the bone marrow, lungs, liver, spleen and the head area, indicative of uniquely broad distribution properties. Peak fluorescence was also detected in PB aspirated from the opposite tail vein to that injected, from the first time point (452±225 RFU, 30 minutes). The half-life of DiR-NV in PB was 2.25 hours for IV treated mice. As expected, in IP treated mice fluorescence levels took longer to peak (2 hours, 138±83 RFU) and had a half-life of 3.2 hours from this point. In conclusion, these results demonstrate the usefulness of PEG-PTMC NV for the delivery and consequent retention of insoluble chemotherapeutic drugs; without hindering their performancein vitroorin vivo. The generated NV are biocompatible, do not require purification and can be fabricated within 5 minutes. These data support the testing of NV against more pt samplesin vivowith Dex and other insoluble drugs.

Disclosures
No relevant conflicts of interest to declare.