Ascorbic acid tethered polymeric nanoparticles enable efficient brain delivery of galantamine: An in vitro-in vivo study

The aim of this work was to enhance the transportation of the galantamine to the brain via ascorbic acid grafted PLGA-b-PEG nanoparticles (NPs) using SVCT2 transporters of choroid plexus. PLGA-b-PEG copolymer was synthesized and characterized by 1H NMR, gel permeation chromatography, and differential scanning calorimetry. PLGA-b-PEG-NH2 and PLGA-b-mPEG NPs were prepared by nanoprecipitation method. PLGA-b-PEG NPs with desirable size, polydispersity, and drug loading were used for the conjugation with ascorbic acid (PLGA-b-PEG-Asc) to facilitate SVCT2 mediated transportation of the same into the brain. The surface functionalization of NPs with ascorbic acid significantly increased cellular uptake of NPs in SVCT2 expressing NIH/3T3 cells as compared to plain PLGA and PLGA-b-mPEG NPs. In vivo pharmacodynamic efficacy was evaluated using Morris Water Maze Test, Radial Arm Maze Test and AChE activity in scopolamine induced amnetic rats. In vivo pharmacodynamic studies demonstrated significantly higher therapeutic and sustained action by drug loaded PLGA-b-PEG-Asc NPs than free drugs and drug loaded plain PLGA as well as PLGA-b-mPEG NPs. Additionally, PLGA-b-PEG-Asc NPs resulted in significantly higher biodistribution of the drug to the brain than other formulations. Hence, the results suggested that targeting of bioactives to the brain by ascorbic acid grafted PLGA-b-PEG NPs is a promising approach.

NH2 was terminally functionalized by the free NH2 group. Similarly, PLGA-b-mPEG copolymer was also synthesized using mPEG-NH2 (methoxy-PEG-NH2; Mol. wt. 2000 Da). 1 H NMR spectroscopy of the PLGA-b-PEG-NH2 was carried out at 300 MHz in CDCl3. Gel Permeation Chromatography (GPC) was carried out by a system having a refractive index detector, and the analysis was carried out at room temperature (RT, 27±2⁰C) using two serially aligned TSKGEL columns. Dimethylformamide (DMF) was used as an isocratic mobile phase with a flow rate of 1 ml/min.

Preparation and optimization of PLGA-b-PEG NPs
Nanoprecipitation method was utilized for the preparation of amino group functionalized PLGA-b-PEG-NH2 NPs 3,4 . Before preparation of drug loaded NPs, optimization of empty PLGA-b-PEG-NH2 NPs was carried out by varying different parameters which control the size of NPs e.g. different solvent (acetone and acetonitrile), the various ratio of solvent to water (keeping polymer concentration constant) and different polymer concentration. Acetone and/or acetonitrile solvents were selected due to their miscibility with water and low boiling point so that they can be removed easily during formulation development to avoid toxicity. Solvent to water ratio was varied from 0.1 to 1 i.e. from 1:1 to 1:10 (keeping 10 mg/mL polymer concentration constant). NPs optimization was also carried out by varying polymer concentrations (from 5-20 mg/mL) in the organic phase.
Briefly, the polymer was dissolved in organic solvent (either acetone or acetonitrile).
The NPs were prepared by the addition of polymer solution drop wise to de-ionized water (a non-solvent) followed by sonication for 60 sec. The NPs suspension was stirred for 6 hrs at RT to facilitate complete evaporation of the organic solvent (acetone). In the case of acetonitrile, the NPs suspension was stirred for 2 hrs, and the organic solvent was evaporated in a rotary evaporator at reduced pressure. The NPs were purified by centrifugation (15 min at 18,000 rpm). The PLGA-b-PEG-NH2 NPs were washed with water thrice and lyophilized.
The effects of the different parameters were observed on overall particle size, Pdi and zeta potential of the NPs and were characterized using dynamic light scattering (ZetaSizer Nano ZS90, Malvern Instrument, USA).

Preparation and optimization of GLM loaded PLGA-b-PEG NPs
The pre-optimized parameters of acetone and acetonitrile formulations from above studies were selected to synthesize GLM loaded PLGA-b-PEG-NH2 NPs. The NPs formulations were further optimized on the basis of the drug to polymer ratio for optimum drug loading. Briefly, GLM was dissolved in organic solvent, either acetone or acetonitrile.
The polymer was likewise dissolved in the same solvent and mixed with the drug. The effect of the different drug to polymer ratio (from 1:1 to 1:10) was optimized (keeping polymer concentration constant and varying drug concentration). The NPs were prepared by the addition of drug-polymer solution drop wise to de-ionized water (a non-solvent) followed by sonication for 60 sec. The resulting NPs suspension was processed as discussed above. The drug loaded NPs were characterized for size, Pdi and zeta potential by the same procedure as  Fig. S4 online).
Firstly, a reactive imidazole carbamate intermediate was synthesized via reacting hydroxyl group of Asc with carbonyldiimidazole (CDI) using the reported method 5,6 with slight modification. For the above reaction, Asc was taken in DMSO, and the solution was stirred at room temperature for 2 hrs under N2 atmosphere to generate a reactive imidazole carbamate.
The product was precipitated with chilled diethyl ether, filtered and vacuum dried. The addition of a highly nucleophilic group (e.g. NH2) to the reactive imidazole carbamate removes imidazole to forms a stable carbamate. The PLGA-b-PEG NPs possess highly nucleophilic terminal NH2 groups of PEG. The NPs were suspended in 10M borate buffer (pH 11), and reactive imidazole carbamate of Asc was added, and the mixture was stirred at RT for 2 hrs to form carbamate bond between hydroxyl group of Asc and amino group of PEG. The NPs were collected via centrifugation and washed with de-ionized water thrice to remove un-reacted Asc followed by lyophilization. PLGA-b-PEG-Asc NPs were characterized for particle size, Pdi and zeta potential using DLS technique. Surface morphology was evaluated using SEM and TEM. Asc conjugation on PLGA-b-PEG NPs was further confirmed by FTIR spectroscopy and thermogravimetric analysis (TGA).

In vitro drug release studies
Intracellular pH of the brain is near to 7.2, and the pH of the blood is approximately

Cell Culture
The object of the present work was to develop a formulation which can increase drug delivery to the brain by crossing choroid plexus. The idea was to use SVCT2 transporter expressed on choroid plexus to transport the drug to the brain cell. Therefore, the cell line was selected on the basis of expression of plasma membrane-associated SVCT2 transporter for Asc. NIH/3T3 cell line reported to express plasma membrane-associated SVCT2 transporter for Asc 9,10 . Therefore, NIH/3T3 cell line was selected for cellular uptake studies to assess the targeting potential of the developed nanoparticulate formulations.

Cell uptake using fluorescence microscopy
This assay was carried out to evidence and compare the uptake of non-targeted and targeted NPs formulation by SVCT2 expressing NIH/3T3 cells. The cells were supplemented with 5 mL of culture medium (DMEM; Invitrogen) and maintained in a CO2 incubator at 370.5C and 5% CO2. Rhodamine B (Sigma, USA) was used as fluorescence dye to evaluate cellular uptake. Rhodamine loaded plain PLGA, PLGA-b-mPEG, and PLGA-b-PEG-Asc NPs were prepared according to the optimized method for drug loaded NPs.
However, rhodamine was used instead of GLM. The NIH/3T3 cells were seeded at a density of 2 x 10 4 cells/plate in fibronectin coated tissue culture petri dishes containing 1 mL DMEM, incubated and checked under the microscope for 40-50% confluency. The petri dishes were alienated into four groups, each group containing three petri dishes. The cells were incubated for 48 hrs in 5% CO2 at 37°C with changing of the medium following every 12 th hrs. The medium was replaced then with 2 mL antibiotic-free and serum-free medium. Subsequently, free rhodamine, rhodamine-PLGA NPs, rhodamine-PLGA-b-mPEG NPs and rhodamine-PLGA-b-PEG-Asc NPs were placed separately in a group of plates. The NPs were incubated with the cells for 0.5 and 1 hr at 37ºC. After incubation, the cells were washed with Hank's Balanced Salt Solution thrice to remove extracellular NPs 11 . The fluorescence attributed to uptake of fluorescently labeled formulation was then qualitatively observed under fluorescent microscope (Olympus, Osaka, Japan).

Drug uptake using HPLC
Drug uptake of free GLM and drug loaded formulations (GLM loaded PLGA, PLGA-b-mPEG and PLGA-b-PEG-Asc) was estimated using NIH/3T3 cells via HPLC. The cells were seeded into fibronectin coated tissue culture petri dishes at a density of 5 x 10 3 cells/plate containing DMEM medium and incubated at 37°C and 5% CO2 for 48 hr to acquire more than 75% confluency. Free drug and NPs formulations containing 50 µg/mL of GLM were suspended in the medium and added (100 µL) separately into the wells containing NIH/3T3 cells. After the different incubation period, the culture medium was removed. Cells were detached by cell scrapper by adding 2 mL media and collected from the medium in the form of the pellet by centrifugation at 4000 rpm for 15 min. The cells were washed as mentioned above. To the pellets, 500 µL of 0.5% Triton X 100 was added to rupture the cells 12 .
Internalized drug into the cells was extracted by addition of dichloromethane followed by vortexing for 15 min, and the mixture was incubated at 25°C for 6 hr. The supernatant was filtered and the drug concentration was detected using HPLC 13,14 .

Biodistribution studies
The in vivo biodistribution studies of GLM from various formulations (free drugs and drug loaded NPs formulations) were carried out via HPLC method. Biodistribution studies were performed for quantitative measurement of GLM in various organs. Animals were divided into five groups, every group containing 9 rats. Group 1 was treated as control. The details of samples given via tail vein to each group were as follows: Group 1= PBS (pH 7.4); Group 2= GLM solution; Group 3= GLM-PLGA NPs; Group 4= GLM-PLGA-b-mPEG; Group 5= GLM-PLGA-b-PEG-Asc (1.5 mg/kg equivalent of GLM). Three animals of each group were sacrificed after 1, 6 and 12 hrs post administration. The tissues from brain, lung, liver, kidney, and spleen were carefully removed immediately after sacrificing and weighed.
Subsequently, 5 mL of trichloroacetic acid (10% v/v in water) was added to one gram each of various tissues and vortexed for 1 min. A small quantity of ethanol was added, homogenized in a tissue homogenizer and centrifuged at 5000 rpm for 20 min. The supernatant was collected and analyzed for GLM content by HPLC. Nanoprecipitation