Formulation development of tocopherol polyethylene glycol nanoengineered polyamidoamine dendrimer for neuroprotection and treatment of Alzheimer disease

Abstract Amyloid-beta (Aβ) aggregates deposition at extra neuronal sites induces neurotoxicity and major hallmarks of Alzheimer’s disease (AD). To reduce the Aβ fibril toxicity, multi-functional polyamidoamine (PAMAM) dendrimer was conjugated with tocopheryl polyethylene glycol succinate-1000 (TPGS) which acts as a carrier matrix for the delivery of neuroprotective molecule piperine (PIP). This PIP-TPGS-PAMAM dendrimer was fabricated to mitigate the Aβ1–42 fibril toxicity on SHSY5Y cells. TPGS-PAMAM was fabricated through carbodiimide coupling reaction, and PIP was encapsulated in dendrimer through solvent injection method to prepare PIP-TPGS-PAMAM. Antioxidant assay of PIP-TPGS-PAMAM showed 90.18% inhibition of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radicals compared to free PIP, which was 28.27%. The SHSY5Y cells showed 37.25% for negative control group and 82.55% cell viability for PIP-TPGS-PAMAM treated group against Aβ1–42 toxicity. PIP-TPGS-PAMAM reduced the ROS activity to 15.21% and 48.5% for free PIP treated in cell group. Similarly, extent of Aβ1-42-induced apoptosis also reduced significantly from 38.2% to 12.36% in PIP-TPGS-PAMAM treated group. In addition, PIP-TPGS-PAMAM also disaggregated the Aβ1–42 fibril in SHSY5Y cells. Our findings suggested that PIP-TPGS-PAMAM showed mitigation of Aβ1-42-induced toxicity in neuronal cells, which can offer excellent prospect of neuroprotection and AD therapy.


Introduction
Alzheimer's disease (AD) is associated with degeneration of neuronal cells, which lead to cognitive decline brain. AD majorly affects hippocampus and cerebral cortex of brain due to aggregates of amyloid beta (Ab) protein [1]. Ab monomers oligomerise to form aggregates via self-recognition binding of monomers of protein. In AD, production of the Ab in brain higher compared to the clearance rate results in deposition of aggregates at extra neuronal sites [2]. The extracellular deposition of Ab aggregates triggers formation of reactive oxygen species (ROS), which leads induction of apoptosis in neuronal cells [3]. Current treatment interventions like acetylcholine esterase inhibitor and NMDA (Nmethyl D-aspartate) antagonist gives only symptomatic relief in AD [4]. Therefore, inhibition of Ab aggregate formation and scavenging of ROS can be alternative therapeutic method to reduce neurotoxicity in AD models.
Piper longum active constituent piperine (PIP) is known for its profound effect on the central nervous system (CNS) has been reported to improve cognitive function in AD. PIP is a nitrogenous pungent alkaloid compound that is extracted from the fruit of Piper longum having an aqueous solubility of 40 mg/L. Various reported experimental results have shown that PIP can increase cognitive function, the structural activity relationship (SAR) of PIP due to a polyene double bond system in chemical structure that acts as potent antioxidant. The tertiary nitrogen involved in PIP moiety mimics acetylcholine esterase's inhibitor drug in brain [5,6]. Reportedly, PIP demonstrated protective effect on neuronal SHSY5Y cells against Ab mediated cytotoxicity by scavenging the ROS generation [7]. Although with aforementioned advantages, CNS delivery of PIP is challenging due to its poor water solubility which restricts entry in neuronal cells [8]. Nanomedicines have wide application in encapsulation of hydrophobic drugs and increase their availability to targeted site. Nanocarriers comprising of polymers, lipids and inorganic materials are developed, which demonstrated excellent properties for targeting CNS with neuroprotective agents [9]. Among all nanocarriers, dendrimers exhibited desired properties of drug delivery systems such to increased solubility, improved half-life, greater permeation of drugs, ability to encapsulate hydrophobic and hydrophilic molecules, site-specific targeting and superficial passage across cell membrane by transcytosis [10][11][12][13][14]. Polyamidoamine (PAMAM) dendrimers of G4 generation are highly branched and monodisperse macromolecule consists of free amine groups. These free amines facilitates covalent attachment of ligands, targeting agents and drugs. The hydrophobic core of PAMAM is advantageous for physical encapsulation of lipophilic drugs [15][16][17][18][19][20][21][22][23][24]. PAMAM dendrimer has ability to inhibit the Ab fibril formation by attaching to free end of peptide and terminates fibril growth thermodynamically. These dendrimers exhibit generation dependent disaggregation effect on the Ab 1-42 fibrils; in addition, PAMAM can also reduce the preformed Ab fibrils [25,26]. However, toxicity associated with PAMAM has been the major concern due to positively charged free amine groups during intracellular drug delivery [27]. In another study, grafted PAMAM dendrimer has shown the Ab disaggregation through nucleophile reaction with lysine terminal end of Ab protein [28], whereas hydroxyl groups modified PAMAM dendrimer are non-cytotoxic and disaggregate Ab fibrils of human islets. The multiple hydroxyl group on the surface of dendrimers has shown their ability to interact with Ab with no significant cell death. Grafting the surface of PAMAM dendrimer enhances cellular biocompatibility and showed interesting anti-amyloidogenic properties [29].
To overcome the toxicity associated with free amine without hampering the amyloid plaque disaggregation activity, the PAMAM dendrimer surface was modified with a-D-Tocopheryl Polyethylene Glycol Succinate 1000 (TPGS). TPGS is amphiphilic biocompatible macromolecule polymer that includes polar head and lipophilic tocopherol tail. Tocopherol imparts antioxidant property in TPGS and functions as free radical scavenger of ROS. It enhances the drug permeation through lipophilic cell membranes and improves the availability of drugs at intra-neuronal sites. In addition, tocopherol prevents amyloid-induced neurotoxicity including oxidative stress and lipid peroxidation in SH-SY5Y cells [30].
In this work, PAMAM G4 generation dendrimer is conjugated with TPGS to form TPGS-PAMAM in two steps reactions via esterification and amide coupling. This TPGS-PAMAM was used to encapsulate PIP molecules through solvent injection method to form PIP-TPGS-PAMAM. This nanocarrier was screened for neuroprotective and Ab 1-42 plaque disaggregation activity. The Ab 1-42 disaggregation activity was monitored by various spectral, chemical and microscopy-based techniques. Further neuroprotection activity of PIP-TPGS-PAMAM was evaluated on Ab 1-42 -induced cytotoxicity on SHSY5Y cell line and assessed for cell viability assay, ROS scavenging assay, apoptosis and amyloid aggregated detection using fluorescent microscopy.

Succinylation of TPGS to TPGS-COOH
The carboxylic group introduced in TPGS molecule through esterification reaction by adding succinic anhydride (SA), briefly TPGS (500 mg, 0.869 mmol), SA (173 mg, 1.73 mmol) and DMAP (122 mg, 0.869 mmol) were co-dissolved in 10 mL anhydrous DCM. The reaction mixture was stirred for 24 h in an inert atmosphere at room temperature. The product was suspended in cold DCM to filter out the unreacted SA. Then TPGS-COOH precipitated in 10 mL of diethyl ether and vacuum dried [31].

Preparation of PIP encapsulated TPGS-PAMAM dendrimer (PIP-TPGS-PAMAM)
Briefly weighed 5 mg of PIP was co-dissolved with 50 mg of TPGS-PAMAM dendrimer in 2 mL of methanol. This organic phase was added in an aqueous solution dropwise through syringe. The mixture is stirred at 700 rpm on magnetic stirrer overnight at ambient temperature to evaporate the methanol from solution. Excess saturated PIP was removed from solution via filtration through 0.4 mm syringe filter to obtain PIP-TPGS-PAMAM nanosuspension. Similarly, non-conjugated PIP-PAMAM were also prepared for further studies.

H NMR
The NMR spectra of TPGS, TPGS-COOH, PAMAM and TPGS-PAMAM were recorded on NMR spectrometer (JEOL 500 MHz) using solvents such as CDCl 3 , DMSO-d 6 and D 2 O. The value of chemical shift of proton is denoted in ppm (d). The degree of grafting is calculated by following equation, whereas S represents the degree of conjugation in equation.

S ¼
integral areas ofÀCH3 at 0:8 ppm in TPGS integral area of À CH2CO À in PAMAM at 2:3 ppm (1) Matrix-assisted laser desorption ionisation time of flight (MALDI-TOF) The mass of unconjugated PAMAM and TPGS-PAMAM dendrimer samples were determined using MALDI-TOF (AB Sciex 4800 plus spectrophotometer). Beta indole acrylic acid was used as matrix in MeOH/water solvent and 10 wt % of the dendrimer samples were prepared.

Fourier transform infrared red spectroscopy (FTIR)
In order to understand functionalisation between TPGS and PAMAM G4 dendrimer, infra-red spectroscopy was used. The spectra of TPGS, TPGS-COOH, PAMAM and TPGS-PAMAM samples were analysed on FTIR (Bruker, USA) instrument based on attenuated total reflection mode. The pre-dried samples were placed under diamond tip for analysis and recording of spectra.

Quantification of free amine by TNBS assay
To quantify number of TPGS-COOH attached with primary amines, group on PAMAM was analysed through TNBS assay. The extent of conjugation was evaluated by the difference in amount of free amine between TPGS conjugated PAMAM and unconjugated PAMAM. The standard curve is prepared by using aqueous TNBS solution of 25 mg/mL was added to the different concentrations (25-200 mg/mL) of PAMAM sample. Absorbance of the samples was analysed using UV-visible spectrophotometer (Agilent carry system). The well containing TNBS reagent was considered void sample to measure the baseline absorbance. For determination of TPGS-PAMAM concentration, carrier (C) (mg/mL) per well was acquired by interpolation of the calibration equation of PAMAM [32]. The percentage conjugation (X) for TPGS-PAMAM NPs was calculated through following equation: The amount of primary amine groups on PAMAM G4 generation dendrimer is 64, therefore to quantify the free amine groups on each PAMAM carrier is denoted (n) and the M.W of attached substituent TPGS-COOH is 1613.12 Da (S).

Differential scanning calorimetry (DSC)
For thermal analysis of PAMAM, TPGS-COOH and TPGS-PAMAM were done through DSC (Thermo analytical Instrument, USA). Precisely weighed samples were sealed into aluminium T-zero pans. The ramp temperature for samples was set at 5 C/min ranged from 0 C to 180 C. During analysis, the flow of ultrapure nitrogen gas 50 mL/min was maintained in sample holding chamber [33].

UV-visible spectroscopy
The UV absorption spectra of the PIP-TPGS-PAMAM, PIP and TPGS-PAMAM were recorded. Using 100.0 mL samples were diluted in 1.0 mL methanol solutions. The spectra were measured using a UV-visible spectrophotometer (LAB INDIA-3000) using cuvette of quartz having an optical path length of 10.0 mm. Methanol was used to subtract the background noise [34].

Particle size and zeta potential determination
The hydrodynamic size and zeta potential of PIP-TPGS-PAMAM and PIP-PAMAM were determined using Zetasizer (Malvern Nano-ZS UK). Nanoparticle suspension was diluted 10 times with TDW prior to analysis. The responses were measured in triplicate (n ¼ 3) [35].
High resolution transmission electron microscopy (HR-TEM) The particle size and morphology of PIP-TPGS-PAMAM and PIP-PAMAM were analysed using HRTEM (JEOL, Tokyo, Japan) on 200 kV voltage. The nanosuspension of dendrimers was diluted to 1 mg/mL and were placed on carbon coated grid and dried at room temperature prior to measurements [36].

Estimation of drug content
The amount of PIP encapsulated in dendrimers was analysed through high-performance liquid chromatography (HPLC) (Waters, USA) attached with photodiode array detector and reverse phase C 18 column. Methanol and water were used as mobile phase at 60:40 with flow rate of 1.0 mL/min and column temperature was set at 25 ± 0.5 C. The injection volume adjusted to 20 mL at 343 nm wavelength for 7.4 min retention time. The calibration curve of PIP from 0.5 to 20 mg/mL concentration range showed regression coefficient of 0.992. The percentage encapsulation efficiency (% EE) was analysed through dialysis method. The 2 mL of PIP-TPGS-PAMAM and PIP-PAMAM nanosuspension was placed into a dialysis bag having 12 kDa MW cut off. The nanosuspension was dialysed against TDW (1000 mL) at room temperature for 4 h to remove the free PIP [37]. To extract PIP from PIP-TPGS-PAMAM, nanocarrier was solubilised in methanol and ultrasonicated for the removal of encapsulated PIP and filtered through 0.2 mm pore size syringe filter prior to HPLC analysis. The %EE of PIP in TPGS-PAMAM dispersion was obtained from following equation: Whereas percentage drug loading was calculated after lyophillisation of PIP-TPGS-PAMAM using 1% mannitol. The amount of PIP is estimated after reconstituting the briefly weighed powder in methanol and analysed on HPLC.

Stability studies of PIP-TPGS-PAMAM
The stability of the PIP-TPGS-PAMAM dendrimers was conducted at room temperature in phosphate buffer pH 7.4 for 30 days. The zeta potential and particle size for PIP-TPGS-PAMAM were monitored using DLS [38].

In-vitro studies
In-vitro drug release The PIP release study from PIP-TPGS-PAMAM, PIP-PAMAM and free PIP was investigated through dialysis technique. Briefly, free PIP, PIP-TPGS-PAMAM and PIP-PAMAM containing equal amount 3 mg of pristine PIP was introduced into bag of dialysis membrane having MW cut off 12,000 Da. The release of PIP was conducted in 50 mL PBS of pH 7.4 containing tween-80 1% w/v at 37 ± 0.5 C temperature. The media was continuously agitated at 100 rpm on magnetic stirrer. For analysis, samples were withdrawn at scheduled time period and replenished with the equivalent volume of fresh PBS medium to maintain the sink conditions. The amount of PIP in samples was determined through HPLC. The percentage cumulative amount of drug released (% CDR) was determined through the following equation [39].
% CDR ¼ Amount of PIP Released at Specific Time Total Amount of PIP in Dendrimers Â 100 (5) Antioxidant activity studies DPPH assay was done to investigate the antioxidant property of Free PIP, TPGS-PAMAM and PIP-TPGS-PAMAM. These formulations were diluted with methanol to obtain concentration of 1 mg/mL stock solution. The stock samples were serially diluted to prepare different concentrations range of 10, 50 and 100 mg/mL in PBS. The diluted samples were mixed with an equal volume of DPPH methanolic solution (100 mM) followed by incubation for 30 min in a dark place. The absorbance of samples was recorded at 517 nm using microtiter plate reader and DPPH solution without sample was considered as control. % Antioxidant activity of samples was calculated using the formula: where A DPPH and A sample are the absorbances of DPPH without and with samples, respectively. All the experiments were performed in triplicate [40].

Ab 1-42 monomer preparation
For preparation of Ab 1-42 monomers, lyophilised powder was dissolved in HFIP using microcentrifuge tube. The HFIP was removed using high vacuum apparatus from Ab 1-42 solution. The film of Ab 1-42 peptide forms at inner wall of microcentrifuge tube which was stored at À20 C temperature. DMSO (Dimethylsulphoxide) was used to dissolve the Ab 1-42 film and diluted in PBS to obtain appropriate concentration [41].

Ab fibril disaggregation ThT assay
For disaggregation studies the Ab 1-42 solution (10 mM) was incubated at 100 rpm for 72 h at 37 C to attain the sufficient amount of Ab 1-42 aggregates [29]. To monitor the disaggregation of Ab 1-42 fibrils, thioflavin-T (ThT) (10 mM) was used as a probe. The equivalent concentration of PIP (50 mg/mL) was used in PIP-TPGS-PAMAM, free PIP and void TPGS-PAMAM added into the preformed Ab 1-42 aggregates solutions with ThT for further incubation. The absorption of ThT reagent was measured at 480 nm emission and 450 nm excitation. The measurement was done on ELISA plate reader (synergy) and experiments were done in triplicates.

Ab monomer kinetics ThT assay
In fibrillation, kinetics studies Ab 1-42 (10 mM) monomers were incubated with group of PAMAM, PIP-PAMAM, void PIP-PAMAM, PIP-TPGS-PAMAM, free PIP and TPGS-PAMAM containing (50 mg/mL PIP) for 72 h. For this study at predetermined time interval, fluorescence was analysed on ELISA plate reader. The fluorescence of ThT reagent was set at 480 nm emission, and 450 nm excitation wavelength and the experiments were done in triplicates.
The samples were dilute 10 folds in triple distilled water prior to analysis, and measurements were taken in triplicates [42].

Circular dichroism spectroscopy (CD)
The CD spectra of final Ab 1-42 aggregates (10 mM) incubated with free PIP, PAMAM, void TPGS-PAMAM, PIP-PAMAM and PIP-TPGS-PAMAM containing 10 mM equivalent of PIP monitored after 72 h. The spectra of samples were measured at scan rate of 1 nm/sec from range 190 to 250 nm recorded on (JASCO J810, Tokyo Japan) spectropolarimeter. Constant nitrogen purging was maintained during analysis of the sample [43].

Atomic force microscopy (AFM)
The PIP-TPGS-PAMAM incubated with Ab 1-42 aggregate for 12, 48 and 72 h samples were applied over freshly cut mica (1 cm 2 ) of sheet and incubated for 3 min at room temperature. The nanocarrier suspension was then washed with water to remove any traces of residues and dried using nitrogen gas. A Nanoscope V Multimode scanning probe was used to capture the images of samples.

Scanning electron microscopy (SEM)
The Ab 1-42 disaggregation was also monitored using SEM (JEOL), PIP-TPGS-PAMAM incubated Ab 1-42 fibrils were dried and sputter coated with gold layer. After placing the sample in vacuum chamber at 3 kV, the images of samples were observed at 12, 48 and 72 h.

In-vitro cell line studies
Cytotoxicity assays SHSY5Y cells were procured from national centre for cell sciences (NCCS Pune), which was grown in DMEM. The cell culture medium includes 10% foetal bovine serum, 1% streptomycin and incubated in 5% CO 2 . The Ab 1-42 fibril-induced cytotoxicity studies was performed on SHSY5Y cells using MTT reagent. The cells were seeded in 96-well plate having cell count of 1 Â 10 3 cells/well which are incubated for 24 h to adhere in wells. Fresh culture media was added in cells prior to treatment with Ab 1-42 precultured fibrils (10 mM). Then cells are treated with PBS (control), Ab 1-42 (10 mM) (negative control), void TPGS-PAMAM þ Ab 1-42 (10 mM), PIP (PIP concentration 10, 50 and 100 mg/mL) þ Ab 1-42 (10 mM), and PIP-TPGS-PAMAM (PIP concentration 10, 50 and 100 mg/mL) þ Ab 1-42 (10 mM). Cells were incubated in CO 2 incubator after treatment for 24 h at 37 C. Cells were further washed with PBS prior to addition of MTT reagent (10 mL) with concentration 4 mg/mL. After 4 h, incubation cells were treated with 100 mL DMSO to dissolute formazan crystals. ELISA microtiter plate reader (synergy model) was used to analyse the absorbance of MTT reagent in cells at 570 nm. Results were shown in % cell viability [8]. Experiments were performed in triplicates for statistical significance analysis of variance was implemented.
Determination of the intracellular ROS levels H 2 DCF-DA reagent was used to analyse the amount of free radical concentration in Ab 1-42 treated cells [44]. The SHSY5Y cells were grown in six-well plate for 24 h with coverslip prior to treatment with Ab 1-42 ( . Then cells were incubated in serum free medium for 24 h at 37 C. Then cells were fixed in 4.0% paraformaldehyde for 10 min at room temperature. The cells was incubated for 30 min with a 0.05% solution of ThT and DAPI (1 mg mL À1 ) and washed to remove any excess stain. The images of cells were taken on fluorescence microscopy (Olympus, Japan) at 40Â magnification [44].

Apoptosis studies
For apoptosis studies, SHSY5Y cells were grown in six-well plate and treated with control (PBS),  [45,46].

Statistical analysis
The data were shown in standard deviations (SD±), and data were statistically analysed using variance analysis (ANOVA) as appropriate, (ÃÃ) p < .01 and (Ã) p < .05, respectively, indicating significant differences between the treated and control sample.

Results and discussion
This PAMAM dendrimer was grafted with TPGS to reduce the toxicity and increase biocompatibility without hampering in-vitro Ab 1-42 disaggregation activity. The hypothesis for the fabrication of TPGS-PAMAM was used to encapsulate and deliver PIP in neuronal cells with reduced Ab 1-42 -induced toxicity.

Synthesis and conjugation of TPGS-PAMAM
The functionalisation of TPGS with PAMAM is two-step process, the first step involves conjugation of succinic ester to TPGS for introduction of carboxylic group shown in a schematic representation ( Figure 1). In second step, free amines at periphery of PAMAM G4 enable the formation of amide bond with TPGS-COOH to form TPGS-PAMAM. The conjugation of TPGS with succinic anhydride was done using DMAP which is weak base that act as catalyst for the ring-opening and esterification. TPGS was reacted with excess amount of SA to avoid crosslinking with PAMAM. The proton NMR analysis of TPGS-COOH showed the succinoyl methylene peaks at 2.5-2.6 ppm and a-tocopherol signal at 0.8 ppm represented in Figure 2(a,b). The succinoylation is confirmed by integration of the proton peaks at 3.5 ppm polyethylene glycol peak and supporting table ST [30]. The internal amides of PAMAM dendrimer were observed at 5.2 ppm shown in Figure 2  of PAMAM was observed at 7.82-8.06 ppm in Figure 2(d). The amount of TPGS group grafted in TPGS-PAMAM was calculated from the integral ratio of the signal at 2.3 ppm exhibit the -CH 2 adjacent to the C¼O group of PAMAM and -CH 3 proton in the TPGS at 0.8 ppm. The reaction of TPGS-COOH with PAMAM was done at two different molar ratios such as 25:1 which showed 12 TPGS molecules attached to dendrimer. At reactant molar ratio of 50:1, integral ratio calculation exhibited 20 TPGS molecule is conjugated to per PAMAM dendrimer. The TPGS-COOH reaction efficiency with PAMAM G4 dendrimer was found to be 40%.

FTIR and DSC
Spectra of TPGS-COOH in comparison to TPGS showed a peak at C ¼ O at 1750 cm À1 and sharp peak of carboxylic acid -OH was observed at 3200 cm À1 shown in Figure 3(a) [32]. However, peak observed at 1700 cm À1 in TPGS is an ester carbonyl stretch, which also observed in TPGS-COOH. Whereas the spectra of TPGS-PAMAM showed disappearance of C ¼ O carboxylic at 1750 cm À1 and appearance of C ¼ O amide stretch at 1618 cm À1 showed successful conjugation of TPGS-PAMAM. The intensity of peak at 3400 cm À1 is an indicator for enhancement of secondary -NHamides in the PAMAM.
The formation of covalent bond between polymers moieties affects the melting point of compound, which can analysed using DSC [33]. The DSC thermogram of PAMAM displayed the sharp endothermic melting point peak (Tm) at 144.55 C and glass transition temperature peak (Tg) at 57.23 C. However, the TPGS-COOH showed Tm at 25.34 C and Tg at 102.45 C exhibited Figure 3

MALDI-TOF
MALDI-TOF reflectron mode exhibits higher resolution which is used to analyse the purified product samples of G4 generations dendrimers and high MW polymers up to the mass of 100,000 Da [44]. The MW of marketed TPGS is 1513.55 Da approximately, after reaction with succinic anhydride the weight increased to 1613.25 Da for TPGS-COOH which is predicted by CHEM BIODRAW Software. PAMAM dendrimer exhibited 14214.18 m/z molecular weight through MALDI-TOF analysis. Due to MW >10,000 Da the mass of TPGS-PAMAM and PAMAM were determined using on linear delayed extraction mode displayed in Supporting Figure S1. Due to high molecular weight, the mass spectra of PAMAM and TPGS-PAMAM m/z peaks cover a broad region ± 10,000 units in mass spectra. At reaction molar ratio of 25:1 (TPGS-COOH: PAMAM), the mass of TPGS-PAMAM was found to be 35183.25 m/z indicated 12 TPGS-COOH groups are attached per PAMAM molecule. Further reaction at different molar ratios between TPGS-COOH and PAMAM was estimated through TNBS assay.

TNBS assay and dendrimer conjugate zeta potential
In this assay, primary amine displaces the sulphonic acid group on TNBS reagent and shows absorbance at 410 nm [47]. Reaction at molar ratio 25:1 and 50:1 of TPGS-COOH to PAMAM in second step showed attachment of 12.19 to 20.12 TPGS molecule per PAMAM dendrimer shown in Table 1. The attachment of TPGS-COOH reduces the absorbance of TNBS reagent due to reduction in number of free amine sites at PAMAM dendrimer surface. This reduction in number of free amines at PAMAM due to grafting, which resulted in shift of zeta potential from 11.33 mV to À6.05 mV. The TPGS groups at periphery of TPGS-PAMAM shield the positive charge of free amine and leads to reduction in zeta potential from positive to negative. The molar ratio 50:1 TPGS-PAMAM was selected for further studies due to higher TPGS surface attachment, and this shielding of PAMAM free amine can reduce the fabricated nanocarrier cytotoxicity.

HR-TEM analysis
The morphology of PIP-TPGS-PAMAM and PIP-PAMAM was characterised using HR-TEM. Granular spherical-shaped particle of PIP-PAMAM was observed having particle size of 15.42 nm in Figure  4(a). But the morphology of PIP-TPGS-PAMAM observed to be spherical and rigid with an average particle diameter less than 50 nm displayed in Figure 4(b). meanwhile, the size of grafted dendrimer was also investigated by DLS which indicated that hydrodynamic diameter detected averagely 43.0 nm showed in Figure 4(c) which was approximately similar comparison to TEM micrographs. Due to TPGS, attachment on peripheral amines of PAMAM increased the MW and size of PIP-TPGS-PAMAM in comparison to PIP-PAMAM. However, HRTEM imaging revealed PIP-TPGS-PAMAM is monodisperse in nature.

Physical characterisation of conjugated denrimers
The encapsulation of PIP in TPGS-PAMAM was done through solvent injection method to form PIP-TPGS-PAMAM. Methanol evaporation from the organic phase leads to the formation of PIP encapsulated PIP-TPGS-PAMAM due to nanoprecipitation. Aqueous solubility of free PIP is reported to be poor which is not favourable for achieving drug concentration efficacy at target site [6]. The internal structure of the dendrimer is usually non-polar due to the combination of hydrophobic and hydrogen bond formation enables incorporation of lipophilic drugs in PAMAM matrix. PIP-PAMAM showed only 73.23% EE; however, TPGS conjugation with PAMAM increases the % EE to 80.35% for PIP which is 10% higher than PIP-PAMAM shown in Table 1. This increment in entrapment is due to tocopherol-mediated hydrophobicity in TPGS-PAMAM compared to non-conjugated PAMAM [48,49]. DLS and zeta potential results revealed the particle size and surface charge characteristics of dendrimers. DLS showed 15.32 nm hydrodynamic radius of PIP-PAMAM, and zeta potential was found to be 11.5 mV data provided in Table 1. After TPGS grafting with PAMAM dendrimer, particle size of PIP-TPGS-PAMAM increased to 43.23 nm with reduction in zeta potential to À6.2 mV shown in Supporting Figure S2a. The macromolecular nature of TPGS provides steric stability of nanocarriers and polyethylene glycol group of TPGS forms hydrogen bonding with water molecules in aqueous medium [27].
For PIP-PAMAM, the % DL was found to 6.8%, whereas the TPGS grafting with PAMAM increased the PIP loading to 10.3% shown in Table 1. Increment of the hydrophobic moiety such as tocopherol group in the nanocarrier enhanced the encapsulation of PIP. The entrapment of PIP in TPGS-PAMAM matrix was analysed by UV-visible spectrophotometer. The comparative absorption spectra of TPGS-PAMAM, PIP-TPGS-PAMAM and PIP were measured for their lambda max peaks shown in Figure 4(d). It was observed that TPGS-PAMAM and PIP showed intense absorption peak at 281 nm and 345 nm. In the spectra of PIP-TPGS-PAMAM dendrimer, the intensity is reduced peaks at 345 nm compared to free PIP which showed the encapsulation PIP having equivalent concentration of (5 mg/mL). The PIP containing PIP-TPGS-PAMAM was observed at different concentration (1, 5, and 10 mg/ml) in methanol shown in Supporting Figure S2b.

Stability studies and in-vitro drug release
The 30 days storage stability study of PIP-TPGS-PAMAM dendrimer was monitored via measuring particle size and zeta potential using DLS at room temperature. The particle size of nanocarriers were increased from 40 ± 2 nm to 48 ± 3 nm in one month of storage at room temperature indicated no drastic stability issue. However, zeta potential increased from À6.2 mV to À4.5 mV without change in visible stability of PIP-TPGS-PAMAM presented in Figure 5(a,b). PIP-TPGS-PAMAM showed À6.2 mV zeta potential, but TPGS is a macromolecular neutral amphiphilic structure which that sterically stabilises PAMAM dendrimers and prolongs the stability of nanocarriers [27].
The % CDR of PIP from the PIP-PAMAM and PIP-TPGS-PAMAM dendrimer was observed at cerebrospinal and physiological pH 7.4. The release pattern of PIP was biphasic which includes an early phase with burst release and a late phase of sustained release in case of PIP-TPGS-PAMAM. The 30.98% of PIP was released from the TPGS-PAMAM matrix in the first 8 h. When the second phase-initiated PIP-TPGS-PAMAM dendrimer released 69.85% PIP in 96 h. Due to lipophilic nature free PIP showed release of 27.28% of PIP in dissolution media compared to PIP-TPGS-PAMAM. Whereas PIP-PAMAM exhibited 80.19% PIP release from dendrimer matrix to media in 96 h shown in Figure  5(c). This indicates the aqueous solubility of PIP increased after encapsulation in dendrimers compared to free drug. The rigidity of PIP-TPGS-PAMAM increased due to hydrophobic tocopherol component in aqueous environment that restricted the release of PIP from core branches of dendrimer. TPGS-COOH is conjugated with PAMAM through amide bond which is not prone to hydrolysis and maintains sustain release of drug [10,50]. The HPLC chromatogram showed only retention peak of PIP at 7.3 min, no other degradation peak was observed during release studies shown in Supporting Figure S3. This indicates PAMAM matrix, protects PIP from degradation and provides stability to nanocarrier.

DPPH assay
DPPH assay offers the quantitative estimation of antioxidant potential for radical scavenging compounds [40]. ROS have been described to induce cell death through apoptosis in neuronal cells which can be inhibited via broad range of intra and extracellular antioxidants [51]. PIP and TPGS are known for their antioxidant activity, which are incorporated in the fabricated nanocarrier and are further analysed for free radical scavenging activity. The antioxidant activity of free PIP was observed 28.25 ± 3.4%, whereas due to tocopheryl group the activity increased for TPGS-PAMAM to 54.15 ± 4.3% at 100 mg/mL concentration shown in Figure 5(d).
The PIP-TPGS-PAMAM showed the enhanced antioxidant activity !80% in comparison to free PIP. PIP-TPGS-PAMAM enhanced the direct antioxidant activity against free radicals and the potential Table 1. (a) TNBS assay-based degree of conjugation and zeta potential of dendrimers; (b) physical characteristics of PAMAM dendrimer conjugates; (c) cell viability studies of dendrimer conjugates against amyloid toxicity.   efficacy of PIP to protect cells from oxidative damage. Overall, PIP-TPGS-PAMAM was found to enhance the free radical scavenger activity compared to free PIP.
ThT assay for Ab fibril disaggregation Ab 1 À 42 aggregation is a protein conformational disorder which transforms the Ab 1-42 peptide from monomeric a-helical to b-pleated sheets structure [28,52]. This Ab aggregation was monitored using ThT reagent which binds with Ab fibrils to exhibit hypsochromic shift [41]. For ThT assay of Ab 1-42 , aggregates were prepared and diluted in PBS. Then Ab 1-42 (10 mM) fibrils was coincubated with ThT dye and divided into treatment groups such as free PIP, void TPGS-PAMAM and PIP-TPGS-PAMAM. The fluorescence intensity of only Ab 1-42 group showed 3323 ± 277 au after 72 h incubation. Those Ab 1-42 groups which were treated with PIP-TPGS-PAMAM dendrimer showed remarkable reduction in fluorescence to 764 ± 115 au compared to other group in 72 h as shown in Supporting Figure S4(a). The TPGS-PAMAM also reduced the fluorescence, but the PIP-TPGS-PAMAM showed distinct Ab 1-42 disaggregation. The groups treated with free PIP showed weak reduction of the ThT fluorescence, whereas TPGS-PAMAM reduced the ThT fluorescence intensity to 1435 ± 427 au. The disaggregation of pre-cultured fibrils was observed highest in groups, which are incubated with PIP-TPGS-PAMAM. Encapsulation of PIP in TPGS-PAMAM increased the anti-amyloidgenic activity than other treatment groups. PIP and PAMAM both are Ab fibril inhibitor and combining them in a single nanocarriers PIP-TPGS-PAMAM synergised the activity. PEG is known to have more H-bond sites, which may allow it to engage with Ab fibrils more effectively [42].

ThT assay for Ab monomer fibrillation
The prepared monomers of Ab 1-42 were assessed for the fibrillation kinetic assay as shown in Figure 6

Ab disaggregation monitoring using DLS
To validate Ab 1-42 fibril disaggregation of ThT assay DLS was used to determine the particle size of aggragates. Prior DLS analysis these fibril were incubated dendrimer conjugate for 72 h. DLS is normally used to evaluate the hydrodynamic diameter of particles in solution and the % intensity of scattered light is affected by larger size particle in aqueous dispersion. The Ab 1-42 fibril has heterogeneous rod like structure from 100 to 10,000 nm, therefore DLS is used to obtain the qualitative estimation of aggregate particle size [52]. Particle distribution of Ab 1-42 exhibited four main peaks of outsized particles with the diameters of $100 nm, $800 nm, $>5 mm and 10 mm with PDI 0.65 shown in Supporting Figure S5b. Prior measurement of particle size of PIP-TPGS-PAMAM showed 43 ± 2.5 nm diameter which was incubated with Ab 1-42 aggregates of 10 mM for 72 h. Free PIP showed the small extent reduction in particle size of Ab aggregates intensity and PDI reduced from 0.655 to 0.532. Those Ab aggregates group which are incubated with void TPGS-PAMAM showed the reduction of particle size to 700 nm and decreased PDI from 0.6 to 0.3 for aggregates. However, group treated with PIP-TPGS-PAMAM showed of Ab 1-42 aggregates were reduced to smaller fragments and a monodisperse peak observed 63 nm. These smaller fragments of Ab 1-42 have particle size distribution $ PDI of 0.212 showed highest disaggregation of Ab due to PIP-TPGS-PAMAM. Reportedly, surface modified PAMAM dendrimers are inhibitors of Ab 1-42 aggregates via electrostatic interaction blocks the growth of the terminal points and increases the fibril breakage rate [25]. Therefore, the PIP-TPGS-PAMAM NPs showed higher Ab fibrillation inhibitory effect compared to the other groups. However, encapsulation of PIP in TPGS-PAMAM increased the payload to Ab 1-42 fibrils, therefore PIP-TPGS-PAMAM showed highest fibril disaggregation property. DLS analysis revealed large Ab 1-42 aggregates reduced to smaller amorphous species (<100 nm) by PIP-TPGS-PAMAM which is crucial to mitigate neurotoxicity in cells.

CD conformational analysis
Furthermore, the effect of fabricated dendrimer on Ab 1-42 fibril disaggregation was investigated using CD to analyse conversion of secondary b pleated to a-helical conformation [43]. The CD spectrum measurement of PIP-TPGS-PAMAM scanned from 190 to 250 nm and showed straight line used as control which is shown in Figure 6(b). When Ab 1-42 (10 mM) aggregates showed the intense deep negative peak of b-pleated sheets of amyloid fibrils at 214 nm. Further shift of wavelength to 220 nm was observed for PIP-PAMAM and PAMAM indicator of fibril conformational reversal activity. Free PIP showed slight reduction, whereas TPGS-PAMAM reduced the b sheet intensity from À8.6 to À6.1 D a (d meg). Whereas fibrils incubated with PIP-TPGS-PAMAM (10 mM) exhibited negative peak at 220 nm and reduced the fibril from b sheet to a-helical structure. This reduction in peaks intensity of Ab 1-42 aggregates due to PIP-TPGS-PAMAM showed that fabricated nanocarriers are able to change the conformation from secondary b-pleated to a-helical structure; PIP-TPGS-PAMAM enhanced the conformational changes to compare to PIP-TPGS-PAMAM. The macromolecular nature of TPGS-PAMAM and PEG electrostatic charge reversed the fibril conformation [28]. ThT assay, CD and DLS characterisation revealed PIP-TPGS-PAMAM showed highest Ab 1-42 fibril disaggregation activity. Furthermore, PIP-TPGS-PAMAM incubated Ab 1-42 fibrils were studied through microscopy.

Ab fibril disaggregation monitoring AFM and SEM
The nanocarrier-induced disaggregation of Ab aggregates was monitored using AFM at predetermined intervals [53]. The PIP-TPGS-PAMAM incubated aggregates morphology at 12 h showed no significant change in fibril structure. Increment in incubation period leads to fragmentation of Ab 1-42 fibrous aggregate was observed in 48 h. After 48 h, incubation period aggregates fragmented to smaller particle size. PIP-TPGS-PAMAM disaggregated the fibrils converted to amorphous monomeric state in 72 h. The SEM was also used to monitor the disaggregation efficiency of PIP-TPGS-PAMAM on Ab aggregates. The SEM images exhibited distinct Ab 1-42 fibrils at 12 h with PIP-TPGS-PAMAM dendrimers shown in Figure 7(a). The fibrils disaggregation was clearly observed after 48 h showed fibres were fragmented to small length amorphous aggregates. SEM and AFM images confirmed the disaggregation Ab 1-42 fibrils due to PIP-TPGS-PAMAM. We discovered that PIP-TPGS-PAMAM reduced most of the long fibrils to shorter fragments, which reformed into amorphous structures, using both AFM and SEM images.

Cell line studies
MTT assay MTT was used to analyse the neuroprotective activity of PIP-TPGS-PAMAM against Ab 1-42 fibril-induced cytotoxicity in SHSY5Y cell [54]. Due to the neuroprotective property of PIP, the treatment was done at equivalent concentration (10, 50 and 100 mg/mL) for free PIP and PIP-TPGS-PAMAM shown in Table 1. The group of cells which are treated with only Ab 1-42 (10 mM) aggregates reduced the cell viability to 37.23% in comparison to control shown in Figure 7(b). Void TPGS-PAMAM showed viability of cells to 49.37% compared to control. The tocopherol moiety in nanocarrier inhibits Ab 1-42 -induced free radicals and increases cell viability [17]. Although cells treated with PIP-TPGS-PAMAM at 10 mg/ mL dose of equivalent PIP concentration, increment in cell viability to 69.23% was observed. At 100 mg/mL PIP concentration, SHSYSY cells viability was observed to be 67.83% and 82.55% for free PIP and PIP-TPGS-PAMAM compared to control, respectively, shown in Table 1. PAMAM G4 are Ab 1-42 fibril growth inhibitors, but due to positive surface charge it induces cytotoxicity. Therefore, PAMAM surface is grafted with TPGS to reduce the cytotoxicity and deliver PIP inside the cell. Cell groups which are treated with PIP-TPGS-PAMAM at (100 mg/mL) concentration increased the cell viability to 82.55 ± 2.8% compared to only Ab 1-42 treated group. At 10 mg/ mL containing PIP in PIP-TPGS-PAMAM showed significant increment in viability, therefore in further cell line studies only 50 mg/ mL PIP dose used. PIP-TPGS-PAMAM, in general, transforms fibrils into spherical aggregates that have been found to be less toxic [55]. PIP-TPGS-PAMAM showed the significant reduction in the Ab 1-42 -induced toxicity in the SHSY5Y cells and showed significant increment in cell viability compared to other groups.
Estimation of intracellular ROS H 2 DCF-DA can pass through the lipophilic cellular membrane via passive gradient and cleaved by intracellular ROS to form 2 0 ,7 0dichlorofluorescein and emits fluorescence at 535 nm [56,57]. The Ab 1-42 aggregates induce ROS from mitochondria of cells that leads to neuronal cell death [54]. The H 2 DCF-DA used as probe to analyse the effect of free radical scavenging using flow cytometer Detection of Ab 1-42 fibrils inside SHSY5Y cells by a fluorescence microscope To monitor Ab fibril in the SHSY5Y cells, ThT fluorescence observed in presence of free PIP, TPGS-PAMAM and PIP-TPGS-PAMAM. As earlier described, ThT dye binds with Ab fibrils to emit fluorescence which estimates the amount of aggregation inside the cells. In Figure 9, fibrils were much higher in Ab 1-42 alone treated cells, whereas the group cells were co-incubated with PIP or void TPGS-PAMAM reduced the fluorescence intensity of ThT reagent but not completely. However, PIP-TPGS-PAMAM incubated group showed remarkable reduction in the fluorescence of ThT. ROS inhibition study have pointed out that both free PIP and void TPGS-PAMAM have antioxidant activity but less effective against Ab 1-42 fibril reduction. In case of PIP-TPGS-PAMAM, Ab 1-42 fibril disaggregation was enhanced due to entrapment of PIP in dendrimer matrix; however, PIP has inhibitory effect on self-aggregation of Ab which was earlier observed in ThT assay. Furthermore, TPGS enhanced the cellular uptake of nanocarriers to disaggregate the fibril inside the neurons

Apoptosis studies
The ROS species induces apoptosis in neuronal cells to further evaluate the anti-apoptotic effect of PIP-TPGS-PAMAM. Further for quantitative estimation cells were analysed by Annexin-FITC apoptosis reagent through flow cytometer, FITC bind with membrane phospholipid of apoptotic cell and propdium iodide attaches to nucleic acid. Dot plots of flow cytometer showed PIP-TPGS-PAMAM treated cells reduced to early apoptosis 4.8%, whereas Ab 1-42 treated group exhibited 54.8% early and 1.3% late apoptosis ( Figure 10). In comparison to free PIP and void TPGS-PAMAM, the PIP-TPGS-PAMAM reduced the Ab 1-42 -induced apoptosis sharply. However, void TPGS-PAMAM not showed any sharp decline in apoptotic rate and exhibited 32.83% early apoptosis. This reduction programmed cell death of neurons is via dual mechanism (1) Ab fibril disaggregation and (2) free ROS reduced by formulation. Apoptosis studies displayed the PIP-TPGS-PAMAM was able to inhibit the Ab 1-42 -induced apoptosis and useful to deliver neuroprotective agent.

Conclusion
In summary, The PIP-TPGS-PAMAM dendrimer was fabricated through multistep approach for application for neuroprotection against Ab fibrils. TPGS was covalently attached with PAMAM followed by physical encapsulation of PIP that led to the formation of PIP-TPGS-PAMAM dendrimers. The fabrication of the nanocarrier was confirmed by spectroscopic techniques and chemical assay. The particles size of PIP-PAMAM 15.25 nm and 43.25 nm PIP-TPGS-PAMAM respectively. Time-dependent stability showed no significant increase in particle size and PDI for PIP-TPGS-PAMAM. The in-vitro release of PIP from PIP-TPGS-PAMAM showed sustained release behaviour in 72 h at pH 7.4. PIP-TPGS-PAMAM showed significant disaggregation of Ab fibrils by restraining the protein in its monomeric and nontoxic amorphous state. In PIP-TPGS-PAMAM nanocarrier TPGS and PIP combined antioxidant effect increased the neuroprotective activity via inhibition of radical scavenging and apoptosis activity. Our results showed that PIP-TPGS-PAMAM is possible candidate for the neuroprotection and disaggregation of amyloid fibrils. Ab incubated with PIP-TPGS-PAMAM during Ab fibrillisation is less toxic and can be served as nano chaperones that can prevent and re-direct Ab fibrillisation and potential use in AD.