In vivo pharmacodynamic studies

The pharmacodynamic potential of developed targeted formulations was compared with free drugs and plain NPs formulations using scopolamine induced amnesia in albino rats (Sprague Dawley strain, 7–8 weeks old weighing 200 ± 20 g). All the animal experiments were performed according to the protocol approved by the Institutional Animal Ethical Committee of Dr. Hari Singh Gour Central University, Sagar (MP), India (registration no. 379/01/ab/CPCSEA; 13/828). The pharmacodynamic studies were performed using Morris Water Maze Test and Radial Arm Maze Test.

To assess the potential of targeted NPs on reference memory capacities, Morris water maze test was carried out as reported in the literature with slight modification^27–30. The Maze test consisted of training and testing session. Before the test, rats were housed in controlled condition (12 hrs light/12 hrs dark schedule; 25 ± 1 °C). Food and water were provided ad libitum. The water maze apparatus consisted of a rounded pool of 120 cm in diameter and 45 cm in height^{30, 31} with a featureless inner surface (Supplementary Fig. ^S7A). The pool was divided into 4 equal quadrants containing a transparent column platform of 10 cm diameter in one quadrant. Each quadrant was provided with a clue (hint or sign). The pool was filled to a height of 25 cm with water (25 ± 1 °C), and the height of the platform (23 cm) was adjusted such that it was submerged 2 cm below the water surface (hidden platform). The platform was situated in the pool away from the pool wall. The non-toxic white paint was added to the water to make it opaque²⁸. Location of the pool and platform was fixed during the entire experiment period.

Flow chart of the experimental protocol for water maze test is given in Supplementary Fig. ^S7B. Animals were divided into 6 groups each containing 6 animals (n = 6). The first experimental day was dedicated to swimming training in the absence of the platform. During the subsequent days, the rats were given four training-trials sessions per day to locate the platform (with the platform in place). The inter training trial interval of 5 min was kept. During this learning phase, rats were placed randomly in the pool at selected locations and allowed to search the platform. The time taken from starting point to the escape from water onto the hidden platform (escape latency; learning and reference memory test {1^st test}) was measured in each training session. Each trial was ended if the animal arrived at the platform or after 120 s, whichever came first. Animal failing to arrive at the platform in 120 s were guided onto it. The rat was allowed to remain on the platform for 30 s after each hidden-platform trial. The location of the platform was fixed during the entire test period.A probe trial session was carried out on each day after 2 hr of last escape latency test, wherein platform was detached from the pool and rat were allowed to explore for the platform for 120 s. The time spent in the target quadrant was considered as a measure of spatial memory retention (2^nd test).

Animals were divided into 6 groups each containing 6 animals (n = 6). All the treatment were started from the 2^nd day of training as the 1^st day was dedicated to swimming training. Amnesia was induced in all groups except group 1 by intraperitoneal (i.p.) injection of scopolamine hydrochloride (0.4 mg/kg of body weight) in 0.9% normal saline 30 min prior to the training session (once a day)^{21, 31}. Group 1 was injected with normal saline and treated as normal control. Group 2 was injected with scopolamine only (disease control) and didn’t receive any formulation. All other groups were administered with different formulations intravenously via tail vein according to grouping on 2^nd, 3^rd, 4^th and 5^th day of treatment. The escape latency and probe test were done from day 2 to day 8. However, no treatment except scopolamine was given on the 6^th, 7^th and 8^th day. Testing on the 6^th, 7^th and 8^th day was carried out to evaluate any sustained effect exerted by the formulation.

The details of different formulations are as follows: group 3 = GLM solution (GLM), group 4 = GLM loaded plain PLGA NPs (GLM-PLGA), group 5 = GLM loaded PLGA-b-mPEG NPs (GLM-PLGA-b-mPEG), group 6 = GLM loaded PLGA-b-PEG-Asc NPs (GLM-PLGA-b-PEG-Asc) (1.5 mg/kg equivalent of GLM). NPs formulations were suspended in saline and injected intravenously via tail vein 1 hr before testing (30 min before scopolamine treatment)^{21, 31}.

The radial arm maze was designed for evaluating spatial memory retention in the rats. The maze had eight arms radiating from a central platform. A small food cup was placed at the end of each arm (Supplementary Fig. ^S7C). The design of the maze ensures that, after eating the food of one arm, the rat has to return to the central platform before going to the next arm. Each arm was 50 cm in length and 10 cm wide with a 30 cm central platform³². Various extra-maze cues were placed around the maze, and the position of maze and cues were remained same until experiments were complete.

Before training, the rats were kept on a restricted diet to reduce their body weights up to 85–90% of normal weight over a 2 week period to motivate the rats to seek food in the maze^{24, 33}. While the water was freely available to them. The maze test consisted of a 7 day training session and a 7 day testing session (Supplementary Fig. ^S7C). On day 1 of the training session, rats were given 15 min to adapt to the maze arms without any food in the food cups. From days 2 to 7 of the training session, rats received 10 min trials twice a day, separated by an interval of 5 min. During the training, food palette was placed in all cups of eight arms. The rat was placed on the central platform of the maze and was allowed to explore food freely in all arms until 10 min.

During the testing session, food pallets were placed in four cups of four selected arms and other four arms were kept empty³³. The location of food and extra-maze cues were not changed during the experiments. At the beginning of each testing trial, the rat was placed on the central platform and was allowed to explore for the food. The trial was continued until rat consumed all four food pellets or until 10 min had elapsed. Performance parameters that were recorded during testing trials included number of working memory errors (re-entry to earlier visited arm where food was already consumed), the number of reference memory errors (entry to an empty arm) and the number of correct choices (entry to a correct arm to consume food). Time taken to consume all four food pallets (Mean trial time) was also recorded.

Animals were divided into 6 groups each containing 6 animals, and similar treatment regimen was followed as carried out in water maze test. The details of different formulations are also same as mentioned in water maze test. The performance parameters were recorded from day 1 to day 7 of the testing phase.

AChE activity was assessed by the earlier reported method^{24, 30}. Three animals from each group of the radial maze test were sacrificed on 4^th Day and remaining three animals were sacrificed immediately after the last radial maze test (7^th Day) by decapitation. The whole brain was removed and homogenized in sodium phosphate buffer (75 mM, pH 7.4, 4 °C) separately. For the assay of AChE activity, 0.1–0.2 ml homogenate was incubated (37 °C, 20 min) with a 4 ml reaction mixture containing acetylthiocholine iodide (0.3 mM) and 1 ml sodium phosphate buffer (0.1 mM, pH7.4). The reaction was ended by addition of 3% sodium lauryl sulfate (1 mL). Subsequently, 1 ml of 0.2% 5,5′-dithiobis(2-nitrobenzoic acid) was added to give the yellow anion of the 5-thio-2-nitrobenzoic acid. The colour intensity was observed spectrophotometrically at 440 nm, and AChE activity was measured as optical density (OD) value/mg protein for AChE.