Dendrimer Brain Uptake and Targeted Therapy for Brain Injury in a Large Animal Model of Hypothermic Circulatory Arrest

Treatment of brain injury following circulatory arrest is a challenging health issue with no viable therapeutic options. Based on studies in a clinically relevant large animal (canine) model of hypothermic circulatory arrest (HCA)-induced brain injury, neuroinflammation and excitotoxicity have been identified as key players in mediating the brain injury after HCA. Therapy with large doses of valproic acid (VPA) showed some neuroprotection but was associated with adverse side effects. For the first time in a large animal model, we explored whether systemically administered polyamidoamine (PAMAM) dendrimers could be effective in reaching target cells in the brain and deliver therapeutics. We showed that, upon systemic administration, hydroxyl-terminated PAMAM dendrimers are taken up in the brain of injured animals and selectively localize in the injured neurons and microglia in the brain. The biodistribution in other major organs was similar to that seen in small animal models. We studied systemic dendrimer–drug combination therapy with two clinically approved drugs, N-acetyl cysteine (NAC) (attenuating neuroinflammation) and valproic acid (attenuating excitotoxicity), building on positive outcomes in a rabbit model of perinatal brain injury. We prepared and characterized dendrimer-NAC (D-NAC) and dendrimer-VPA (D-VPA) conjugates in multigram quantities. A glutathione-sensitive linker to enable for fast intracellular release. In preliminary efficacy studies, combination therapy with D-NAC and D-VPA showed promise in this large animal model, producing 24 h neurological deficit score improvements comparable to high dose combination therapy with VPA and NAC, or free VPA, but at one-tenth the dose, while significantly reducing the adverse side effects. Since adverse side effects of drugs are exaggerated in HCA, the reduced side effects with dendrimer conjugates and suggestions of neuroprotection offer promise for these nanoscale drug delivery systems.

Water used in all experiments was purified by a Branstead Nanopure Diamond Lab water purification system with resistivity 18.2 MΩ cm -1 . performed. Elution was simultaneously monitored by PDA detector (collecting UV-Vis spectra from 190-800 nm that allows for obtaining chromatograms at the desired wavelength in this range) as well as fluorescence detector, having 2 channels set for detection of FITC with excitation at 495 and emission at 520 nm as well as Cy5 with excitation at 645 nm and emission at 662 nm. To obtain calibration curves and determine the limits of detection (LOD)

Characterization of D-FITC and D-Cy5 conjugates
for D-FITC and D-Cy5, different amounts of the conjugates (ranging from 1 ng to 100 µg) were injected on the column and used as external standards for their quantification in dog organs, blood, cerebrospinal fluid, serum and urine.
Phosphate buffer (0.1 M, pH 7.4) with 0.025% sodium azide was used as mobile phase (flow rate 0.6 mL/min). Different amount of D-FITC and D-Cy5 were injected on the column ranging from 1 ng to 100 µg and calibration curves were established for the lower limits of detection (LOD).

Quantification of D-FITC and D-Cy5 in the biological specimens
Biodistribution studies D-Cy5 and D-FITC conjugates were done based on fluorescence spectroscopy, HPLC and SEC. To obtain calibration curves and determine the limits of detection (LOD) for both conjugates, their emission spectra with excitation at 495 nm for D-FITC, and 645 nm for D-Cy5 were recorded at concentrations ranging from 1 ng/ mL to 100 µg/mL using appropriate sets of excitation and emission slit widths. High sensitivity for detection of both conjugates (in a mixture) reaching concentration of 0.1 ng/mL was observed. To test the efficiency of the extraction procedure, known amounts of D-FITC and D-Cy5 were injected into tissue samples (around 150 mg of brain, kidney, liver, heart, lungs, and pancreas) which were homogenized in PBS (c = 0.1 M), using 2 mL low DNA binding eppendorf tubes and TissueLyser LT, Giagen homogenizer. Obtained suspensions were extensively vortexed and sonicated. Then samples were centrifuged at 21130 g (15000) rpm for 10 min at 4 °C using an Eppendorf centrifuge 5424 R. Resulting supernatants were filtered using 0.2 µM filters and analyzed by FLS, HPLC and SEC. Around 95% recovery of both conjugates for all tested organ samples was observed. Recovery rates were tested

Preparation of dendrimer-Cy5 conjugate (D-Cy5)
The preparation of dendrimer-Cy5 conjugate is a three steps process and we have established the preparation of the bifunctional dendrimer previously which is the main intermediate [ref 54]. In the final step, Cy5-mono-NHS ester, a near IR dye was reacted with the bifunctional dendrimer in sodium bicarbonate buffer to get Cy5-labelled dendrimer (D-Cy5). The final conjugate was purified by dialysis followed by GPC fractionation. The resulting D-Cy5 conjugate was characterized by proton NMR, HPLC/GPC, MALDI TOF mass, and fluorescence spectroscopy. In NMR spectrum appearance of methylene protons peaks of 7 Cy5 (1.2-2.1 ppm) and aromatic protons (6.3-8.4) along with dendrimer and linker protons confirm the formation of the conjugate. The HPLC trace showed a peak at 16.23 min for D-Cy5 (monitored at 645 nm), different from that of the starting dendrimer and free Cy5, suggesting a pure and stable conjugate. The fluorescence spectrum showed peaks at 645 nm (for excitation) and 665 nm (for emission) which follow a similar pattern to Cy5 also suggesting the conjugation of Cy5 to the dendrimer and did not alter the characteristic nature of the dye. The resulting amide bond between dendrimer and Cy5 dye is stable and did not release any Cy5 in PBS over a week. Only one mole equivalent of Cy5 was used in the reaction and the percentage payload of the Cy5 to the dendrimer is ~ 4-5%.