key: cord-0796995-0mdineht
authors: Ede, Benjamin C.; Diamanti, Paraskevi; Williams, David S.; Blair, Allison
title: Non-toxic polymer nanovectors for improved delivery of dexamethasone
date: 2021-08-26
journal: Sci Rep
DOI: 10.1038/s41598-021-96797-4
sha: 5b1813632adb1e915a4f258fd5bcfde1a07777eb
doc_id: 796995
cord_uid: 0mdineht

Dexamethasone (Dex) is a highly insoluble front-line drug used in cancer therapy. Data from clinical trials indicates that the pharmacokinetics of Dex vary considerably between patients and prolonging drug exposure rather than increasing absolute dose may improve efficacy. Non-toxic, fully biodegradable Dex loaded nanovectors (NV) were formulated, via simple direct hydration within 10 min, as a vehicle to extend exposure and distribution in vivo. Dex-NV were just as effective as the free drug against primary human leukemia cells in vitro and in vivo. Importantly, high levels of DMSO solvent were not required in the NV formulations. Broad distribution of NV was seen rapidly following inoculation into mice. NV accumulated in major organs, including bone marrow and brain, known sanctuary sites for ALL. The study describes a non-toxic, more easily scalable system for improving Dex solubility for use in cancer and can be applied to other medical conditions associated with inflammation.

Using an optimised protocol for the direct hydration of NV, it was established that the upper limit of Dex encapsulation was 5 wt% with 100% loading efficiency, confirmed using dynamic light scattering (DLS). Therapeutically relevant concentrations of Dex (0.25-0.5 mg/mL) giving 2.5-5 mg/kg doses in mice (equivalent to 5-10 mg/m 2 in humans) were encapsulated, which contained ≤ 0.5% DMSO to enhance its dissolution during the direct hydration process (Fig. 1A) . The prepared 5 wt% Dex-NV had an average size of 41 ± 1 nm and narrow polydispersity www.nature.com/scientificreports/ index (< 0.14), which did not significantly change after 0.2 µm filtration (Fig. 1B) . Without NV, free Dex solutions contained large, heterogeneous precipitates that were almost entirely removed by filtration (Fig. 1C, Fig. S1 ). The presence of Dex in NV formulations, after filtration, was confirmed by the elevated 242 nm absorbance signal ( Figure S1 ). There was no significant difference in the critical micelle concentration (CMC) between Dex-NV and empty NV, with NV forming stably above 0.025 mg/mL copolymer ( Figure S1 ), well below the concentration that would be reached in vivo in this study. Treating primary T-ALL samples with Dex-NV revealed this system was equipotent to free Dex (dispersed in medium from DMSO stock solution at 0.5 mg/mL). The sensitivity to Dex, varied between the 6 samples tested, with pts. 1, 2, 5 and 6 being very sensitive, < 16% cell survival at the highest dose tested (100 μM) with half maximal inhibitory concentration, (IC 50 ) , < 100 nM. In contrast, pts. 3 and 4 were resistant, both failing to reach an IC 50 value ( Fig. 2A ). Superior leukaemia cell killing was observed in pt. 6 using Dex-NV compared to free drug (P < 0.04). Results in normal bone marrow controls did not differ between the two formulations. Crucially, these data demonstrate that NV formulation did not reduce Dex efficacy and confirmed our previous findings using parthenolide in this system 11 . This represents a major advantage over other carriers, such as Pluronic F-127, where drug performance can be reduced following encapsulation 15 .

For in vivo studies, cells from 3 of these cases were inoculated into NOD.Cg-Prkdc scid Il2rγ tm1Wjl /SzJ (NSG) mice. Once ≥ 0.1% human CD45 + cells were detected in peripheral blood (PB) aspirates, separate cohorts of animals were treated with Dex-NV, free Dex (IP, 2.5 mg/kg,) or placebo daily, for 5 days/week over a 4 week period. Both Dex-NV and free Dex delayed disease progression until day 18 in pt.1 (Fig. 2B ) and until after treatment cessation in pts. 2 and 3 (Fig. 2C,D) . In contrast, disease progression was rapid in all control animals ( Fig. 2B-D) . Dex-NV and free Dex significantly improved the survival of NSG by up to 27 days (P < 0.01, Fig. 2E ). Dex-NV administered IV (5 mg/kg, 3 days/week for 4 weeks), was also equipotent to free Dex ( Figure S2 ) and there were no differences in NV efficacy using IP or IV administration. Importantly empty NV and drug loaded had no adverse effects on healthy animals, regardless of administration route ( Figure S3 ).

In a comparable study, Krishnan et al. used Dex-loaded mPEG-poly(caprolactone) (PEG-PCL) particles (fabricated by nanoprecipitation) to treat mice engrafted with an adult ALL cell line (RS4;11) and demonstrated improved survival by 3 days compared to free Dex 12 . However, PEG-PCL particles, unlike mPEG-PTMC used here, necessitate greater efforts in particle fabrication and purification, with greatly reduced encapsulation efficiency. In contrast, Dex-NV, used in this report, were prepared in 10 min via simple direct hydration (with very low levels of organic solvent) and have now been shown to effectively treat primary T-ALL in vivo, without loss of efficacy.

As stated above, extending the circulation time and distribution of Dex may be key to improving outcomes. To study distribution in vivo, fluorescent NV were prepared loaded with the lipophilic dye 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) that only fluoresces in hydrophobic microenvironments, www.nature.com/scientificreports/ such as lipid bilayers and the NV core. Following the seminal work of Meng et al., the loading content of DiR within NV was optimised to ensure linear concentration dependence and avoid any subsequent inaccuracies due to fluorophore de-quenching 16 . DiR-NV were prepared with a range of wt% of fluorophore (0.05-1) and tested over a wide range of copolymer concentrations (0.1-20 mg/mL), with 0.125 wt% giving the best linearity (r 2 = 0.97, Fig. 3A ). Without being stabilised by NV, DiR aggregated and was removed by 0.2 µm filtration, as indicated by the loss of solution colour (Fig. 3B ) and DLS data ( Figure S4 ). Filtration of DiR-NV did not change fluorescence intensity, whereas free DiR in solution (aggregates) showed no fluorescent signal (Fig. 3C ). Furthermore, DiR-NV were identical to their Dex counterpart in terms of size (Fig. 1B) . Cells treated with DiR-NV showed a steady increase in fluorescence for up to 8 h, suggesting controlled cellular endocytosis and uptake ( Figure S5 ), in agreement with our previous study 11 . Ten minutes after IV injection, DiR-NV fluorescence was detected throughout NSG mice using ventral imaging (Fig. 4A) . Fluorescence was also detected in PB aspirated from the opposite tail vein to that injected, from the first time point (452 ± 225 RFU, 30 min), proving evidence of NV circulation (Fig. 4B) . The half-life of DiR-NV in PB was 2.25 h for IV treated mice. As would be expected, peak fluorescence took longer in IP treated mice (2 h, 138 ± 83 RFU) and had a half-life of 3.2 h from peak fluorescence. Fluorescence remained undetectable in free DiR treated mice over the 48-h period. Dorsal imaging also showed accumulation of DiR-NV in the head area, indicative of uniquely broad distribution properties (Fig. 4A) . In contrast, free DiR, used as a low solubility control, accumulated in the liver over the 45 min of imaging and showed very low levels in the PB (Fig. 4A,B) . After 24 h, NV accumulated in the major organs including lungs, liver and spleen, regardless of administration route (Fig. 4C) . Interestingly, significant accumulations in the bone marrow (BM) and brain were also observed, which are known sanctuary sites for childhood ALL. Mononuclear cells from femoral BM, harvested from mice 24 h after treatment with DiR-NV, had increased median fluorescence intensity (676) compared to cells from animals treated with free DiR (366) or empty NV (284), confirming the presence of NV in BM cavities ( Figure  S6 ). Due to the small size of NV used in this study (40 nm diameter) they are well-suited to permeate diverse biological layers that can impede larger particles. Indeed, pegylated nanoparticles < 100 nm are known to display www.nature.com/scientificreports/ reduced hepatic filtration, longer circulation time and a high rate of extravasation into permeable tissues 17 . Importantly, we have recently shown that NV encapsulation results in more controlled release of Dex compared to the free drug, confirming the advantages of this system 18 .

In conclusion, this study provides proof of principle that mPEG-PTMC NV can be loaded with Dex at high concentrations, using the direct hydration process, and are effective against ALL in vivo. This NV system is easily prepared, stable for several months and does not require extensive purification, as compared to traditional formulation methods. Importantly, it has been demonstrated that NV circulate to sanctuary sites, including the BM and CNS where increased drug exposure is likely to enhance overall drug efficacy. In addition, NV can cross the blood brain barrier, so this system has the potential to treat the emerging neurological issues in SARS-CoV-2 pts. Future work will aim to enhance NV targeting to sanctuary sites to improve efficacy and minimise off-target toxicity, and to explore the role of morphology upon the circulation/distribution of NV in vivo.

Poly ethylene glycol block poly trimethylene carbonate (PEG-PTMC) copolymers were synthesized as previously reported 11 . Briefly, monomethoxy PEG-OH (Mw = 1 kDa, Đ < 1.1, Rapp Polymers, Tübingen, Germany) was combined with a stoichiometric amount of trimethylene carbonate (3.8 mmol, Thermo Fisher Scientific, Loughborough, UK) to synthesize copolymers comprising 20 wt% PEG. Dry toluene (≈ 50 mL) was then added and evaporated to ensure dryness. To initiate polymerization, dry dichloromethane (DCM, 10 mL) and methanesulfonic acid (0.1 mmol = 6 µL) were added under argon and stirred for 24 h at 30 °C. The reaction mixture was then mixed with DCM, washed with saturated NaHCO 3 , followed by brine and dried over Na 2 SO 4 , concentrated and precipitated into ice cold diethyl ether (≈ 100 mL, all Sigma-Aldrich, Gillingham, UK). The solid was re-dissolved in dioxane and lyophilised.

Drug encapsulation in nanovectors via direct hydration. PEG-PTMC copolymer was dissolved in oligo(ethylene glycol) (OEG, Sigma-Aldrich) at 20 wt% at 45 °C and mixed to ensure homogeneity. To form micelles, 10 µL of PEG-PTMC solution was added to a 1.5 mL Eppendorf and vortexed for 1 min with buffer www.nature.com/scientificreports/ to the desired concentration of copolymer. Dex or 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR, Perkin Elmer, Llantrisant, UK) encapsulation was achieved by dissolving dry drug to 100 mg/mL in dimethyl sulfoxide (DMSO, Origen Biomedical, Austin, USA). A small volume of drug solution was mixed with the copolymer OEG solution to achieve the desired wt% loading of drug, without the need for purification.

Nanovector stability. The stability of NV was assessed using DLS. NV were analysed using a Malvern Instruments Zetasizer Nano (ZSP). Zetasizer software (Malvern Panalytical, Malvern, UK) was used for data analysis. CMC were determined by adding 10 µM of 3,3′-dioctadecyloxacarbocyanine Perchlorate (DiO), which fluoresces in hydrophobic conditions, to different concentrations of NV copolymer (0.00125-0.5 mg/mL). DiO fluorescence was detected using a SynergyNeo2 plate reader (BioTek, Swindon, UK).

Dex-NV and free Dex preparations (0.125 mg/mL) were analysed in a UV-STAR MICROPLATE (Greiner Bio One, Stonehouse, UK). Absorption spectroscopy was performed from 230-500 nm using a SynergyNeo2 plate reader (BioTek, Swindon, UK).

Human samples. Bone marrow samples from 5 children and a young adult diagnosed with T-ALL at disease presentation were used in this study (Supplemental Table 1 

Animal experiments were conducted in the University of Bristol Animal Services Unit, using procedures licensed and approved by the United Kingdom Home Office. All experiments were performed in accordance with Home Office regulations and reported in accordance with ARRIVE guidelines. Human T-ALL cells were injected via the lateral tail vein of 5-8 week old NSG mice and allowed to engraft. Human cell levels were measured by flow cytometric analysis of PB aspirates weekly, using anti-CD45 (BD Biosciences, Bisley UK). Once human CD45 + cells reached ≥ 0.1%, Dex-NV, free Dex or placebo was administered to separate groups of mice (n = 3/group) 5 days/week (2.5 mg/kg, IP) or 3 days/week (5 mg/kg, IV), for 4 weeks. Engraftment levels were monitored weekly and mice were maintained for up to 9 weeks or until they became symptomatic of the disease. NSG were killed, gross anatomy was inspected and femoral BM was assessed by flow cytometry for human cell engraftment using antibodies against human CD45 and CD7 (BD Biosciences). Major organs were collected for fluorescence imaging.

Fluorescence intensity of DiR in vivo. NSG were inoculated IV with unencapsulated DiR (free DiR), empty NV, Free DiR or DiR-NV (n = 3/group). At specified time points, PB was collected into 10 µL heparin solution (360 units/mL, StemCell Technologies, Cambridge, UK) from the opposite tail vein to that used for inoculation. Fluorescence intensity of DiR was measured using a SynergyNeo2 plate reader with excitation and emission wavelengths of 710 and 760 nm, respectively. Separate cohorts of mice, treated as above, were terminated after 24 h and the florescence intensity of femoral BM mononuclear cells was measured by flow cytometry.

Imaging analyses. Animals were anaesthetised using 2.5% isoflurane gas for up to 45 min and imaged using an IVIS Spectrum (Perkin Elmer) at excitation and emission wavelengths of 710 and 760 nm, respectively. Harvested organs were washed in PBS and imaged immediately.

Statistical analyses. One-way analysis of variance followed by Tukey post hoc testing was used to compare fluorescence intensity and polydispersity index values for NV and free drug solutions. Survival data from in vivo experiments were analysed using Kaplan Meier survival analysis using log-rank test of survival distribution.

Half-life of DiR loaded NV in NSG was calculated using one phase exponential decay analysis.

Acute lymphoblastic leukemia in children

Infection-related mortality in children with acute lymphoblastic leukemia: An analysis of infectious deaths on UKALL2003

Dexamethasone in Hospitalized Patients with Covid-19

Effect of alternate-week versus continuous dexamethasone scheduling on the risk of osteonecrosis in paediatric patients with acute lymphoblastic leukaemia: Results from the CCG-1961 randomised cohort trial

Impact of dose and duration of therapy on dexamethasone pharmacokinetics in childhood acute lymphoblastic leukaemia-a report from the UKALL 2011 trial

Asparaginase may influence dexamethasone pharmacokinetics in acute lymphoblastic leukemia

Polymorphisms in the ABCB1 gene and effect on outcome and toxicity in childhood acute lymphoblastic leukemia

ETV6-RUNX1-positive childhood acute lymphoblastic leukemia: Improved outcome with contemporary therapy

Controlled drug delivery: Historical perspective for the next generation

Dexamethasone-loaded, PEGylated, vertically aligned, multiwalled carbon nanotubes for potential ischemic stroke intervention

Biodegradable, drug-loaded nanovectors via direct hydration as a new platform for cancer therapeutics

Dexamethasone-loaded block copolymer nanoparticles induce leukemia cell death and enhance therapeutic efficacy: A novel application in pediatric nanomedicine

Dexamethasone loaded PLGA nanoparticles for potential local treatment of oral precancerous lesions

Dexamethasone-diclofenac loaded polylactide nanoparticles: Preparation, release and anti-inflammatory activity

Functionalized triblock copolymer vectors for the treatment of acute lymphoblastic leukemia

Quantitative assessment of nanoparticle biodistribution by fluorescence imaging. Revisited

Factors affecting the clearance and biodistribution of polymeric nanoparticles

Exploring the impact of morphology on the properties of biodegradable nanoparticles and their diffusion in complex biological medium

The authors wish to thank the patients and their families who gave permission for their cells to be used for research. The samples were provided by the Blood Cancer UK Childhood Leukaemia Cell Bank. This work was supported by the Elizabeth Blackwell Institute for Health Research, University of Bristol and the Medical Research Council.

B.C.E. designed and performed experiments and wrote the report. P.D. designed experiments and commented on the report. D.S.W. designed and performed experiments and wrote the report. A.B. conceived study and wrote the report.

The authors declare no competing interests.

The online version contains supplementary material available at https:// doi. org/ 10. 1038/ s41598-021-96797-4.Correspondence and requests for materials should be addressed to A.B.Reprints and permissions information is available at www.nature.com/reprints.Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.