Discovery of 2-deoxy glucose surfaced mixed layer dendrimer: a smart neuron targeted systemic drug delivery system for brain diseases

The availability of non-invasive drug delivery systems capable of efficiently transporting bioactive molecules across the blood-brain barrier to specific cells at the injury site in the brain is currently limited. Delivering drugs to neurons presents an even more formidable challenge due to their lower numbers and less phagocytic nature compared to other brain cells. Additionally, the diverse types of neurons, each performing specific functions, necessitate precise targeting of those implicated in the disease. Moreover, the complex synthetic design of drug delivery systems often hinders their clinical translation. The production of nanomaterials at an industrial scale with high reproducibility and purity is particularly challenging. However, overcoming this challenge is possible by designing nanomaterials through a straightforward, facile, and easily reproducible synthetic process. Methods: In this study, we have developed a third-generation 2-deoxy-glucose functionalized mixed layer dendrimer (2DG-D) utilizing biocompatible and cost-effective materials via a highly facile convergent approach, employing copper-catalyzed click chemistry. We further evaluated the systemic neuronal targeting and biodistribution of 2DG-D, and brain delivery of a neuroprotective agent pioglitazone (Pio) in a pediatric traumatic brain injury (TBI) model. Results: The 2DG-D exhibits favorable characteristics including high water solubility, biocompatibility, biological stability, nanoscale size, and a substantial number of end groups suitable for drug conjugation. Upon systemic administration in a pediatric mouse model of traumatic brain injury (TBI), the 2DG-D localizes in neurons at the injured brain site, clears rapidly from off-target locations, effectively delivers Pio, ameliorates neuroinflammation, and improves behavioral outcomes. Conclusions: The promising in vivo results coupled with a convenient synthetic approach for the construction of 2DG-D makes it a potential nanoplatform for addressing brain diseases.

All starting materials and reagents were purchased from Sigma-Aldrich and Merck.Commercially available reagents and solvents for synthesis were analytical grade and used without further purification.Thin-layer chromatography was performed on a film of silica gel that contained a fluorescent indicator F254 supported on an aluminum sheet (Merck).Spectra/Por dialysis membranes were purchased from Repligen.

Instrumentation.
Nuclear Magnetic Resonance (NMR) spectra of samples were recorded on a Bruker spectrometer operating at 500 MHz at 25 °C.The samples were prepared in deuterated chloroform (CDCl3), deuterated DMSO (DMSO-d6) or deuterated water (D2O).
Microwave reactions were performed in a Biotage Initiator+ instrument using a sealed 10 mL/ 20 mL process vials.Reaction times refer to irradiation time at the target temperature, not the total irradiation time.The temperature was measured with an IR sensor.
The purity and drug release studies were analyzed using high-performance liquid chromatography (HPLC).The HPLC was performed using a Waters Acquity Arc system (Milford, MA, USA), equipped with binary pumps, 2998 PDA detector, and a 2475 fluorescence detector.The analyses were performed using Waters Empower software.The samples were run using Waters C18 symmetry 300, 5 μm, 4.6 × 250 mm column using a gradient flow method starting with 90:10 (Solvent A: 0.1% TFA and 5% ACN in water; Solvent B: 0.1% TFA in ACN), gradually increasing to 50:50 (A:B) at 20 minutes, 10:90 (A:B) at 38 minutes finally returning to 90:10 (A:B) at 40 minutes.A flow rate of 1 mL/min was maintained during the run.The dendrimers and drug conjugates were detected at 210 and 254 nm.The 2DG-D-Cy5 was detected at 650nm.
The particle size and zeta potential distribution were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano 90 (Westborough, MA).To measure the size, the 2DG-D was dissolved in deionized water (18.2Ω) to create a solution with a final concentration of 0.5 mg/mL.This solution was then passed through 0.2 μm syringe filters (Pall Corporation, 0.2 μm HT Tuffryn membrane) directly into the cell (UV transparent disposable cuvette, dimensions: 12.5 × 12.5 × 45 mm).For zeta potential measurement, a sample at a concentration of 0.2 mg/mL in 10 mM NaCl was prepared using the same procedure.The measurements were conducted using a Malvern Zetasizer Nanoseries disposable folded capillary cell.

Synthesis of compound 15:
To a stirred solution of compound 14 (52 mg, 1.0 eq, 0.024 mmol) in DMF (4 mL) in a microwave vial was added solution of compound 10 (613 mg, 9.0 eq, 0.191 mmol) dissolved in DMF (4 mL).To this solution, CuSO4.5H2O(6.0 mg, 1.0 eq, 0.024 mmol) and sodium ascorbate (7.0 mg, 1.5 eq, 0.037 mmol) dissolved in minimum amount of deionized water were added.The reaction was performed in microwave condition at 40 o C for 12 h.The completion of reaction was tracked using HPLC.Upon completion, the dialysis was performed using a 3.5 kDa dialysis membrane in DMF for 12h.The final dialysis was performed in DI water.The product was lyophilized to afford compound 15 in 90% yield.

In vitro drug release and stability studies.
Drug release studies were conducted in vitro under both plasma conditions (PBS pH 7.4 and 2% FBS in PBS) and intracellular conditions (citrate buffer pH 5.5 with esterase).2DG-D-Pio was dissolved at a concentration of 1 mg/mL in each buffer and subjected to incubation at 37 °C with continuous shaking to mimic physiological conditions.At designated time intervals, samples were withdrawn, promptly quenched with an equal volume of methanol, and stored at −20 °C until further use.The released drug was subsequently analyzed using HPLC, and the extent of drug release was determined by comparison to the standard curve established for free Pio on HPLC.

2DG-D-Pio formulation stability studies.
2DG-D-Pio was formulated at 50mg/mL in PBS and sterile filtered through 0.4µm filters.The formulations were kept at either room temperature or 4 o C. The aliquots were analyzed via HPLC for the purity at 0h, 1-d, 7-d, and 28-d time points.  .The brain tissues from the injured area (or the matching area from the sham mice) were harvested 24-h post-treatment for gene expression evaluations.A, TNF-α significantly increased in both male and female TBI+saline, TBI+Pio, and TBI+2DG-D-Pio groups, compared with the sham groups.B, IL-1β significantly increased in both male and female TBI+saline groups, compared with the male and female sham and TBI+2DG-D-Pio groups.C, TLR4 significantly increased in

Supplementary Figures.
High resolution mass spectra were performed by Dr. Yue Li in the department of Chemistry and Biochemistry at the University of Maryland-College Park using ESI by direct infusion on a Bruker MALDI-TOF (trans-2-[3-(4-tert-Butylphenyl)-2-methyl-2-propenylidene]malononitrile matrix), and Bruker Q-TOF.

Figure S39 :
Figure S39: Formulation of 2DG-D-Pio in PBS was found to be stable at RT until 28 Days as shown by the HPLC chromatogram @269nm at various time points.

Figure S40 :
Figure S40: Formulation of 2DG-D-Pio in PBS was found to be stable at 4 o C until 28 Days as shown by the HPLC chromatogram @269nm at various time points.

Figure S41 :
Figure S41: 2DG-D-Pio in 2% FBS in PBS was found to be stable at physiological temperature until 7 Days as shown by the HPLC chromatogram @269nm at various time points.

Figure S42 :Figure S43 .
Figure S42: Cytocompatibility of (a) cortical neurons, (b) CATH.a neurons and (c) RAW blue macrophages treated with 2DG-D dendrimer at different concentrations.The 2DG-D dendrimer was found to be nontoxic to all the three cell types as determined by Two-way ANOVA (Turkey's multiple comparisons test), with no significant effect of treatments at all the concentrations.For this, the p-values were calculated between control, DMSO, and 2DG-D treated cells, with **** p < 0.0001 and ns-non significant.

Figure S44 .
Figure S44.Confocal micrographs showing effect of Phloretin inhibitor on the uptake of the 2DG-D-Cy5 dendrimer on (a) cortical and (b) CATH.a mouse nuerons, showng no red fluorescence of Cy5 inside these neuronal cells.The cells stained with blue (DAPI), green (Nestin), yellow (Phalloidin) represent cell nucleus, nestin protein, and actin microflament proteins, respectively.The scale bar shown in the figure is 150 µm (a) and 100 µm (b).Images are representative for three independent experiments.Normalized mean fluorescence intensities (MFIs) of (c) cortical neurons and (d) CATH.a neurons showing uptake of 2DG-D-Cy5 in presence of trafficking inhibitors using ImageJ software.For this, the p-values were calculated between control (no inhibitor) with inhibitor treated cells, with **** p < 0.0001 and ns-non significant.