Cyclodextrin-enabled δ-Tocotrienol for Treating Stress and Memory Dysfunction: Molecular Insights, Biotechnological Applications and Biomanufacturing Implications

Abstract

Background

Antioxidants possess substantial health benefits owing to their ability to alleviate cellular oxidative stress, reactive oxygen species generation and inflammation. Vitamins, dietary fatty acids, minerals, micronutrients and plant-derived flavonoids have attracted significant attention for their primary role in maintaining brain health by attenuating oxidative stress and neuroinflammation, the key culprits in neurodegenerative pathologies and age-related cognitive decline.

Purpose

Vitamin E is a fat-soluble antioxidant with isomers categorised as tocopherols and tocotrienols. Delta-tocotrienol (δ-T3) is an isomer with an unsaturated isoprenoid side chain that confers better membrane penetration and enhanced bioactivities, including scavenging of free radicals, anti-inflammatory and cholesterol-lowering effects. However, the neurodifferentiation and neurocognitive potential of δ-T3 and its bioactive conjugate δ-T3–γCD beyond its classical antioxidant properties is not known.

Methods

In the present study, using rodent-derived PC12 neuroblastoma and C6 glioblastoma cell lines, we assessed the differentiation potential of δ-T3 and its unique complex with γCyclodextrin (δ-T3–γCD). Further, an animal model of scopolamine (SC)-induced memory dysfunction was employed to evaluate the effect of δ-T3 and δ-T3–γCD on memory processes and key molecular markers.

Results

Here, we report that brain-derived cells cultured in low, nontoxic doses of δ-T3 exhibit differentiation. In the SC-induced amnesic mouse model, δ-T3 reversed object recognition memory impairment, concomitantly increasing cortical and hippocampal neural progenitor and differentiation markers, mature neuron markers and neurotrophic factors. Of note, δ-T3–γCD (δ-T3 complexed with γCyclodextrin), containing only 20.7% δ-T3, showed a stronger effect.

Conclusion

δ-T3 showed neurodifferentiation and anti-amnesic potential with improved efficacy when complexed with γCyclodextrin (δ-T3–γCD) in both cellular and mouse model systems. Cyclodextrin-enabled δ-T3 might be a useful resource for the treatment of stress- and age-related memory dysfunction.

Introduction

Oxidative stress, an imbalance between the production of reactive oxygen species (ROS) and their detoxification through the body’s innate defence mechanisms, largely contributes to cardiovascular and neurological disorders, diabetes, pulmonary disease, rheumatoid arthritis and cancer.^1–3 The brain is particularly susceptible to oxidative stress owing to high oxygen consumption and abundant lipid content that is readily oxidised. ⁴ Brain disorders, predominantly Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis and stroke, are the deteriorative outcomes of oxidative stress resulting from genetic and epigenetic factors. ROS induce damage to mitochondrial DNA and protein content, compromising adenosine triphosphate (ATP) production and eventually leading to neuronal death. ROS also activates microglia, resident immune brain cells, in response to brain injuries and infections, which release inflammatory cytokines that further augment oxidative stress and neuronal and synaptic atrophy.^5–7 Protein oxidation results in misfolding and aggregation, hallmarks of neurodegeneration, especially dementia. These toxic aggregates further enhance ROS, generating a vicious cycle. ⁸

Accumulating evidence demonstrates a key role of oxidative stress in memory and cognitive deficits associated with ageing and neurodegeneration.^{9, 10} Oxidative stress induces structural and functional abnormalities, including neuronal atrophy, diminished neurotransmitter release and deficits in synaptic membrane depolarisation, leading to cognitive dysfunction.^{11, 12} Neurodifferentiation is crucial for synaptic plasticity, cognitive abilities, learning and memory, and overall brain health. Neurodegenerative diseases (such as Alzheimer’s, Parkinson’s and Huntington’s) are predominantly marked by protein misfolding and aggregation, reactive gliosis, and compromised neurodifferentiation affecting memory and cognition.^{13, 14}

Antioxidants of natural and synthetic origin that promote differentiation, facilitating tissue growth, maturation and maintenance, are at the forefront of regenerative medicine and the treatment of a wide range of diseases, especially neurodegeneration.^{15, 16} Retinoids (vitamin A derivatives), thyroid hormones, curcumin, epigallocatechin, cyclo-glycylproline, and vitamin D3 analogues are attractive candidates owing to their ability to influence key signalling cascades such as MAPK, Notch, Wnt, BMP, MDM2-p53 and TGF-β, directing stem cell fate and augmenting tissue regeneration.^16–18 Vitamin E, particularly α-tocopherol and delta-tocotrienol (δ-T3), has been extensively studied for its antioxidant properties. A study conducted by Osakada et al. ¹⁹ observed that δ-T3 and other tocotrienol-rich fractions markedly inhibit hydrogen peroxide (H₂O₂)-induced neuronal death in cultures of rat striatal neurons. This effect was not found with α-tocopherol, suggesting that tocotrienols have greater neuroprotective potential. Chronic treatment with tocotrienols in rats that were injected with intracerebroventricular streptozotocin mitigated cognitive deficits alongside oxidative and nitrosative stress. ²⁰ Selvaraju et al. ²¹ reported that α-tocopherol and tocotrienol-rich fractions protected astrocytes from glutamate-mediated cell death.^{22, 23} Furthermore, tocotrienol lowered the cholesterol levels and the production of amyloid-β, which are closely associated with Alzheimer’s disease.

In the present study, using rodent-derived PC12 neuroblastoma and C6 glioblastoma cell lines, we assessed the differentiation potential of δ-T3. Molecular characterisation of control and δ-T3-treated cells revealed that it is a potent neurodifferentiating agent. In an animal model of stress-induced memory dysfunction, δ-T3-treated mice showed enhanced recovery. Of note, δ-T3-γCyclodextrin (γCD) showed a significantly greater effect, suggesting its potential to intervene in memory dysfunction, often associated with stress and age-related brain disorders.

Material and Methods

In Vitro Assays

Cell Culture and Reagents

Rat PC12 (pheochromocytoma/neuroblastoma) and C6 (glioblastoma) were cultured in the Dulbecco’s Modified Eagle’s Medium (DMEM) low-glucose medium (Fujifilm WAKO Pure Chemical Corporation, Osaka, Japan), supplemented with 5% foetal bovine serum (Thermo Fisher Scientific, Japan) and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA, United States). Cells were cultured in an incubator maintained at 37 °C and 5% CO₂ and treated at ~60% confluency with δ-T3 prepared in cell culture-grade dimethylsulfoxide (DMSO). The morphology of control and treated cells was recorded using a phase-contrast microscope (Nikon, Tokyo, Japan).

Western Blotting

Cells were treated with δ-T3 at ~70% confluency in six-well plates and were lysed with RIPA lysis buffer (Thermo Fisher Scientific). The protein lysate was quantified using the BCA assay. Protein samples (20 µM) were resolved by SDS-PAGE, transferred to nitrocellulose membrane (Millipore) by electroblotting using a semidry transfer blotter and probed with antibodies including MAP2 (M3696; Sigma-Aldrich), PSD (MA1-046; Thermo Fisher Scientific), NeuF-H/NF-200 (N4142; Sigma-Aldrich), GFAP (G9269; Sigma-Aldrich), GAP43 (sc-17790; Santa Cruz), NCAM (sc-106; Santa Cruz), Vimentin (sc-6260; Santa Cruz), Survivin (71G4B7; Cell Signalling), Nestin (sc-23927; Santa Cruz) and Mortalin. ²⁴ The immune complexes were detected using horseradish peroxidase-conjugated anti-rabbit/mouse/goat immunoglobulin G and ECL (Amersham Pharmacia Biotech, NJ) and quantified using ImageJ (National Institutes of Health).

Immunostaining

Cells were cultured on coverslips placed in a 12-well culture plate and treated with δ-T3 at about 60% confluency. The control and treated cells were fixed in pre-chilled acetone: methanol (1:1 v/v) and processed for immunostaining using primary antibodies against MAP2, PSD95, NeuF-H, GAP43, GFAP, Vimentin, Nestin, Neuropsin, Survivin and Mortalin, as described. ²⁴ Immunostaining was observed by secondary staining with Alexa-594 or Alexa-488-conjugated antibodies (Molecular Probes) using a Carl Zeiss Axioplan-2 microscope and captured with a Zeiss AxioCam HRc camera.

Reverse Transcriptase Polymerase Chain Reaction

Control and treated cells were lysed with TRIzol (15596018, Ambion) for 5 min, incubated with chloroform (038-02606, Wako) for 5 min, and centrifuged at 12,000 rpm for 15 min at room temperature. Clear supernatant was transferred into fresh vials, washed with isopropanol (166-04836, Wako) for 10 min at room temperature, and centrifuged at 12,000 rpm for 15 min. The pellet was extracted, resuspended in 70% ice-cold ethanol, centrifuged at 8,000 rpm for 5 min twice, and then air-dried. RNA was resuspended in nuclease-free water. RNA (1 µg; quantity and quality evaluated through an ND-1000 spectrophotometer, Nanodrops, Wilmington, NC, USA) was transcribed to cDNA using the ThermoScript Reverse Transcriptase (QIAGEN) following the manufacturer’s instructions. Mastermix for amplification was prepared by mixing 1 µL cDNA with 0.1 µL exTAQ (RR001, Takara), 2 µL 10× TAQ buffer, 2 µL dNTP, and 1 µL each of forward and reverse primers (Table 1) in 12.9 µl nuclease free water, and amplified with ‘denaturation - 95 °C, 10 min → amplification - 95 °C, 45 s - 60 °C, 1 min - 72 °C, 45 s (32–40 cycles) → annealing - 72 °C, 10 min → 4 °C’ protocol. The amplified products were separated on a 1% agarose gel containing 0.06 µg/ml ethidium bromide (15585-011, Invitrogen) and were acquired using a Lumino Image Analyzer equipped with a CCD camera (LAS3000-mini; Fuji Film, Tokyo, Japan).

Table 1. Primer Sequences Used for Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and Quantitative Polymerase Chain Reaction (qPCR).

	Gene	Direction	Sequence
1	GAP43	Forward	5′-ATGCTGTGCTGTATGAGAAGAACC-3′
		Reverse	5′-GGCAACGTGGAAAGCCGTTTCTTAAAG-3′
2	GFAP	Forward	5′-GGCGCTCAATGCTGGCTTCA-3′
		Reverse	5′-TCTGCCTCCAGCCTCAGGTT-3′
3	PSD-95	Forward	5′-AGCTGGAATGGCCTTAGTCA-3′
		Reverse	5′-CAGGAGTTGGTAGTACCCAAATC-3′
4	NeuF-H	Forward	5′-CCTTGAACGCAAAGTGGAATC-3′
		Reverse	5′-GACATGCTGTTCCTGAATCTGAG-3′
5	MAP2	Forward	5′-CAAGGAACCCAGCAAACC-A-3′
		Reverse	5′-GGCCTCTGTCTTGGGTTTCTC-3′
6	Vimentin	Forward	5′-TCTGTGCGAGAGGTAGCAGA-3′
		Reverse	5′-AAGCACTCCGTGAACTCCTG-3′
7	Nestin	Forward	5′-CGAACTTTATATTTTACCACTTCCTTG-3′
		Reverse	5′-CCGTTCATCTGCCATTCTTC-3′
8	Mortalin	Forward	5′-GAATCAGATCGCTCAGATCC-3′
		Reverse	5′-GCACGACACCAGTAGAACTGG-3′
9	Survivin	Forward	5′-AGAACTGGCCCTTCTTGGAGG-3′
		Reverse	5′-CTTTTTATGTTCCTCTATGGGGTC-3′
10	GAPDH	Forward	5′-TGGAAATCCCATCACCATCT-3′
		Reverse	5′-TTCACACCCATGACGAACAT-3′
11	BDNF	Forward	5′-TGCCAGAGCCCCAGGTGTGA-3′
		Reverse	5′-CTGCCCTGGG CCCATTCACG-3′
12	Nestin	Forward	5′-TCTCCAGAAGAGGAGGACCA-3′
		Reverse	5′-TTCGAGAGATTCGAGGGAGA-3′
13	NeuN	Forward	5′-CGTGTATCAGGATGGATTTTATGG-3′
		Reverse	5′-CCATAACTGTCACTGTAGGCTGCT-3′
14	GAPDH	Forward	5′-TGCACCACCAACTGCTTAGC-3′
		Reverse	5′-GGCATGGACTGTGGTCATGAG-3′

For quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), RNA isolated from the cerebral cortex and hippocampus of mice (n = 9 per group) across different experimental groups was first reverse-transcribed into cDNA using RT. The cDNA was used as a template for subsequent semi-quantitative PCR amplification using gene-specific primers (Table 1). To analyse qRT-PCR data, the 2^−∆∆Ct method was used to calculate relative fold changes in mRNA expression, which were plotted as histograms.

In Vivo Assays

Animals and Treatments

Male Balb/C strain mice (10 ± 2 weeks old) from the inbred colony were used for the study. Animal handling and experiments were conducted in accordance with the guidelines of the Institutional Animal Ethics Committee of the CSIR-Institute of Genomics and Integrative Biology (IGIB), New Delhi, India. Approval for the experimental protocols was also obtained from the central animal ethics committees of IGIB and AIST. Balb/c male mice were used for the 7-day experiment and were randomly assigned to 5 groups (n = 9 per group). Scopolamine (SC) hydrobromide (Sigma-Aldrich, USA) was dissolved in 0.9% saline (vehicle) and administered i.p. to mice (3 mg/kg BW). An equal volume of saline was injected into control animals. c-T3 (300 mg/kg BW) and δ-T3–γCD (300 mg/kg BW) compounds were i.p. injected 1 h post-SC hydrobromide treatment. DMSO solution (0.5%) was used as the vehicle for the compounds, which were i.p. administered to the control counterparts. Drugs were administered for 7 days; mice were sacrificed, and brain regions (cerebral cortex and hippocampus) were dissected out for the gene expression studies. A direct dose–response comparison between the pure compound and its cyclodextrin-encapsulated counterpart was not straightforward. The preparation of cyclodextrin inclusion complexes inherently introduces batch-to-batch variability in encapsulation efficiency and compound availability, making it difficult to ensure exact equivalence between the free and complexed formulations. Therefore, we used the most consistent complex containing 20.7% δ-T3 and adopted mathematical normalisation (linear scaling) as a comparative approach to estimate the theoretical biological response under conditions of equivalent δ-T3 delivery. Specifically, because the free δ-T3 preparation contained 100% δ-T3, whereas the δ-T3–γCD formulation contained only 20.7% δ-T3 incorporated within the cyclodextrin complex, direct comparison of the observed bioactivities would not accurately reflect the contribution of δ-T3 itself. To address this limitation, the bioactivity of the δ-T3–γCD complex was linearly normalised to the amount of δ-T3 present, thereby simulating the theoretical response expected if the γCD complex delivered an equivalent amount (100%) of δ-T3. This normalisation enabled a more meaningful comparison of the relative biological effects of the free and cyclodextrin-complexed forms.

Novel Object Recognition Test

Control and treated Balb/c male mice were analysed daily for behavioural properties and anomalies throughout the 7 days of the experiment. Novel object recognition (NOR) test was used to assess the memory consolidation ability of the mice post-treatment with different combinations of drugs (Supplementary Figure 1A). Briefly, control and drug-treated animals (n = 9 mice per group) were habituated in the open field for two consecutive days; on the 5th and 6th days of drug treatment for 5 min each. On the 7th day of drug administration, mice were allowed to interact with two similar objects for 5 min, after which they were returned to their home cage. After 24 h, long-term memory consolidation was assessed by replacing one familiar object with a novel one, and mice were allowed to interact with the objects for 5 min. The time (%) spent with objects was calculated as TNov/(TNov + TFam) × 100 for the novel object and TFam/(TNov + TFam) × 100 for the familiar object. Discrimination Index (DI) for the novel objects was calculated as TNovTFam/(TNov + TFam), where TNov is time spent with the novel object, and TFam is time spent with the familiar object. The data were analysed using ANY-maze software (Ver. 5.1, Stoelting Co., USA).

Figure 1. Delta-Tocotrienol (δ-T3) is Non-toxic and Induces Differentiation in C6 and PC12 Cells. (A) Cell Viability Assay Showing That δ-T3 is Non-toxic to C6 (Glial) and PC12 (Neuronal) Cells Across a Range of Concentrations (0.50–16 µM). Cell Viability Remained Above 100% in Both Cell Types, With No Significant Toxicity Observed (ns = Not Significant; * = p < .05 vs Dimethylsulfoxide [DMSO] Control). (B) Phase-contrast Images Showing Morphological Differentiation in C6 and PC12 Cells After Treatment With 5 µM δ-T3 for 2–4 Weeks. Yellow Arrows Indicate Neurite-like Extensions. Quantification of Differentiated Cells is Shown as Mean ± SEM (n > 3). δ-T3 Significantly Increased the Percentage of Differentiated Cells in Both C6 (100.68 ± 2.68%) and PC12 (100.64 ± 5.43%) Compared to DMSO Controls (*** = p < .001; ns = Not Significant).

Statistical Analyses

For statistical analyses, Microsoft Excel and GraphPad Prism were utilised. All the in vitro experiments were performed more than three times, and the in vivo experiments were performed with n = 9 animals per group. The obtained data were expressed as mean ± SEM of three individual experimental sets. Statistical analyses were executed using Student’s t-test or nonparametric Mann–Whitney U test, whichever was applicable. The data were expressed as mean ± SD. Statistical significance was defined as a p value < .05. The p values were represented as: * p < .05, ** p < .01, *** p < .001.

Results

Delta-Tocotrienol Triggered Differentiation in C6 and PC12 Cells

Viability assay for C6 glioblastoma and PC12 neuroblastoma cultured in increasing concentrations (0.0125–16 µM) of δ-T3 did not reveal any significant effect (Figure 1A). Cells were then treated with a 5 µM nontoxic concentration for 2–4 weeks. As shown in Figure 1B, δ-T3-treated cultures showed differentiation, as evidenced by changes in cell morphology, in both glial and neuronal cell lines. In C6 cells, vehicle (DMSO) treated controls showed very few differentiated cells (5.68 ± 3.68%), whereas 5 µM δ-T3 treatment showed a dramatic increase in differentiated cells (100.68 ± 2.68%), as evidenced by neurite-like extensions and branching. In PC12 cells, the DMSO control exhibited low differentiation (3.35 ± 2.56% to 7.51 ± 1.15%), while δ-T3-treated cells showed complete differentiation (100.63 ± 5.43%). Collectively, these data suggested the differentiation potential of δ-T3, which was further validated by molecular analyses and gene expression at the transcriptional level. RT-PCR analysis of control and δ-T3-treated cells revealed significant upregulation of GAP43 and GFAP in C6, but not in PC12 cells. PSD-95, NeuF-H and MAP2 showed an increase in both C6 and PC12 cells. On the other hand, downregulation of Vimentin, Nestin and Mortalin mRNA levels was detected in treated C6 and PC12 cells compared to the control. Survivin expression remained unaffected (Figures 2A–2C).

Figure 2. Effect of Delta-Tocotrienol (δ-T3) on Markers of Differentiation at the Transcriptional Level in C6 and PC12 Cells. (A) Representative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Gel Images of mRNA Expression of Neuronal Markers in Control (Dimethylsulfoxide [DMSO]) and δ-T3-treated C6 and PC12 Cells. Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) was Used as an Internal Standard. (B, C) Quantitation of RT-PCR Analysis in C6 Cells (B) and PC12 Cells (C) Exposed to DMSO or δ-T3 (5 µM). Gene Expression is Indicated as Relative mRNA Levels to GAPDH, Normalised and Expressed as a Percentage Relative to Control Cells (Assigned 100%). Bars Represent Mean ± SEM of Three Independent Experiments. *p < .05, **p < .01, ***p < .001; ns = Not Significant.

To assess the effect of δ-T3 on neural lineage commitment and survival pathways, expression of neuron-specific protein markers (MAP2, PSD-95, NeuF-H and NeuN) was analysed in C6 and PC12 cells upon treatment with δ-T3 (5 µM for 2, 4 and 6 weeks). Immunoblotting also revealed upregulation of neuron-specific proteins- MAP2 (75-kDa), PSD-95 (95-kDa), NeuF-H (200-kDa) and NeuN (48-kDa) in a time-dependent manner after δ-T3 treatment in the two cell lines. Induction was more pronounced in PC12 cells, especially at week 6, reflected by stronger band intensities and densitometric quantification (Figures 3A and 3B). Immunofluorescence staining (Figures 4A and 4B) revealed that treatment with δ-T3 (5 µM) caused a significant increase in the expression of neuron-specific markers, including MAP2, PSD-95, NeuF-H and NeuN, in both cell lines, with a more pronounced effect in PC12 cells.

Figure 3. Effect of Delta-Tocotrienol (δ-T3) on Proteins Involved in C6 and PC12 Cell Differentiation. (A) Representative Western Blots for Neuronal Markers (MAP2, PSD-95, NeuF-H, NeuN) in Control (Dimethylsulfoxide [DMSO]) and 5 µM δ-T3-treated C6 and PC12 Cells for 2, 4 and 6 Weeks (w2, w4, w6) Were Examined. β-actin Was Used as a Loading Control. (B, C) Relative Band Intensities Representing the Expression Levels of Proteins in Control and Treated C6 (B) and PC12 (C) Cells. All the Experiments Were Performed More Than Three Times. One-way Analysis of Variance (ANOVA) With Post Hoc Test; *p < .05, **p < .01, ***p < .001, ns = Not Significant. Treatment With δ-T3 Results in Time-dependent Upregulation of Neuronal and Survival Markers, Accompanied by a Concomitant Decrease in Astrocytic Markers, Reflective of Neuronal Differentiation.

Figure 4. Effect of Delta-Tocotrienol (δ-T3) on Proteins Involved in C6 and PC12 Cell Differentiation. (A) Representative Immunofluorescence Images of Control (Dimethylsulfoxide [DMSO]) or δ-T3 (5 µM) Treated C6 and PC12 cells Illustrating Expression of Neuronal (MAP2, PSD-95, NeuF-H and NeuN) Markers. (B) Relative Quantitative Fluorescence Intensity Measurement of Individual Markers, Given as a Percentage of the Respective Control (Mean ± SEM; n > 3). Statistical Significance Using Unpaired t-test; *p < .05, **p < .01, ***p < .001, ns = Not Significant.

Delta-tocotrienol and Delta-tocotrienol–γCyclodextrin Restored Memory Deficits With Concomitant Modulation of Molecular Markers

The NOR test was used to evaluate recognition memory consolidation, based on rodents’ innate tendency to explore a novel object more than a familiar one. Time spent with familiar and novel objects and DI were assessed to quantify the behavioural observations (Figure 5A). Vehicle-treated control animals spent more time with novel objects than familiar ones (Saline—Familiar-41.79%, Novel-62.69%; DMSO—Familiar-40.1%, Novel-67.04%). As anticipated, SC-treated mice could not distinguish between the two objects and spent equal time with both (Familiar-52%, Novel-48%). Amnesic mice treated with δ-T3 spent significantly more time with the novel object (81%) as compared to the familiar object (36 %), indicating drastic restoration of memory impairment (Figure 5B). Of note, in SC-treated amnesic mice, the ability to discriminate between the familiar and novel object was significantly enhanced in both δ-T3 and δ-T3–γCD treated mice, although δ-T3–γCD contained only 20.7 % δ-T3 (Figure 5C).

Figure 5. Effect of Delta-Tocotrienol (δ-T3) and δ-T3–γCyclodextrin (γCD) on Object Recognition Memory in Scopolamine (SC)-induced Amnesic Mice. (A) Representative Tracking Traces of Mice During the Novel Object Recognition (NOR) Test. (B) Familiar Versus Novel Object Time Spent Quantification is Expressed as White (Familiar Objects) and Black (Novel Objects) Bars, Respectively. The Data Were Expressed as Mean ± SD. *p < .05, Significant Difference Between Novel and Familiar Objects of Respective Groups. (C) Discrimination Index of Object Exploration. The Data Were Expressed as Mean ± SD. ^#p < .05 and *p < .05 Significant Difference as Compared With Saline Control and SC, Respectively. (D) Relative mRNA Expression of BDNF, a Memory Marker; (E) Nestin, an Indicator of Neural Progenitors; and (F) NeuN, a Marker for Mature Neurons, in the Cerebral Cortex and Hippocampus. The Histogram Represents the Mean of the Data (±SEM). Experiments Were Performed With n = 9 Animals Per Group. Statistical Analyses Were Performed Using One-way Analysis of Variance (ANOVA) Followed by Post Hoc Student–Newman–Keuls Test. ^#p < .05 and *p < .05: Significant Difference Compared With Saline Control and SC, Respectively. ^$p < .05 Represents a Significant Difference Between δ-T3 and δ-toco–γCD.

The memory-restoring potential of δ-T3 and δ-T3–γCD was corroborated at the molecular level. Molecular markers involved in neurogenesis, neuronal differentiation, neuronal maturation and synaptic plasticity, all integral to memory processes, were selected. In the cerebral cortex, SC reduced the expression of all the markers (BDNF 0.37-fold; Nestin 0.02-fold; NeuN 0.36-fold) (Figures 5D–5F). δ-T3 and δ-T3–γCD attenuated the reduction in BDNF, Nestin and NeuN, and even upregulated BDNF and NeuN above baseline levels (Figures 5D–5F). In the hippocampus, the SC-treated group showed downregulation of BDNF mRNA (0.31-fold), Nestin mRNA (0.05-fold) and NeuN mRNA (0.43-fold). The δ-T3 and δ-T3–γCD groups showed upregulation of all genes (BDNF, Nestin and NeuN) (Figures 5D–5F). Importantly, the δ-T3–γCD group showed significantly higher potency despite only 20.7% δ-T3 in the δ-T3–γCD complex (Figure 5 and Supplementary Figure 2, showing the effect normalised to the δ-T3 group at 100% tocotrienol). The normalisation process was employed to compare the biological efficacy and represent a true dose-for-dose comparison of δ-T3 containing 100% δ-T3 and its bioactive conjugate δ-T3–γCD (δ-T3 complexed with γCD), containing 20.7% active δ-T3 and 79.3% cyclodextrin. Here, we performed a mathematical normalisation (linear scaling) to simulate the theoretical response if an equivalent 100% δ-T3 had been delivered via the γCD complex.

As the δ-T3 group contains a 100% pure compound, whereas δ-T3–γCD is a complex powder containing 20.7% δ-T3 complexed with cyclodextrin, a mathematical normalisation was performed. Normalised DI and molecular markers for 20.7% δ-T3–γCD, relative to 100% δ-T3, are shown in Supplementary Figure 2.

Discussion

Here, we have identified the differentiation and neurocognitive potential of δ-T3 and its bioactive conjugate δ-T3–γCD beyond its classical antioxidant properties. Our findings suggest that δ-T3 promotes neuronal differentiation. This attests to both the neurotrophic and neuroprotective roles of δ-T3, with relevance to neural repair, neurodegenerative and neurodevelopmental disorders. Aside from being a free radical scavenger, δ-T3 may directly influence cell fate determination by modulating signalling cascades involved in proliferation and differentiation, such as the MAPK/ERK pathways, the PI3K/Akt pathway and PKC isoforms. Neo et al. ²⁵ showed that the tocotrienol-rich fraction increased the proliferation of HT-22, mouse hippocampal neuronal cells, by activating BDNF/TrkB pathways via the phosphorylation of TrkB, followed by activation of the MAPK/ERK and PI3K/Akt signalling cascades. Changes in neurodifferentiation marker expression, both in vitro and in the mouse brain at transcript and protein levels, suggested transcriptional regulation by δ-T3. Emerging evidence suggests that δ-T3 can influence gene expression through epigenetic modifications, particularly in cancer and inflammation-related pathways. It was shown to inhibit DNMT1 and DNMT3a, leading to global hypomethylation and re-expression of tumour suppressor p21, PTEN and E-cadherin in breast cancer MCF-7 and MDA-MB-231 cells. δ-T3 administration caused demethylation and activation of p16^INK4a in pancreatic cancer cells.^26–29 δ-T3 has also been reported to lower global methylation levels in HL-60 leukaemia cells, leading to the re-expression of silenced apoptotic and differentiation genes. δ-T3 induced histone acetylation by inhibiting HDAC in malignant mesothelial cells. ³⁰ However, epigenetic regulation of genes by δ-T3 in neural cells and its contribution to neurodifferentiation, synaptic plasticity and cognition remain largely unexplored.

Our in vivo findings demonstrated that δ-T3–γCD had improved efficacy compared to unencapsulated δ-T3 in restoring memory. Several studies have highlighted that cyclodextrin complexation significantly increases the stability of natural bioactives while preserving their biological activity. Owing to its relatively large hydrophobic cavity, γCD can form inclusion complexes with diverse phytochemicals, carotenoids, polyphenols, vitamins and other antioxidants, thereby protecting them from degradation caused by light, heat, oxygen, moisture and gastric juices upon ingestion. Encapsulation within γCD reduces molecular mobility and shields reactive functional groups from external environmental stress, resulting in improved chemical stability, longer shelf life, enhanced aqueous dispersibility and often improved bioavailability. γCD complexation of vitamin E (α-tocopherol) and some other bioactive compounds, including curcumin, resveratrol, quercetin, fucoxanthin, β-carotene and lycopene that exhibit poor aqueous solubility and are highly susceptible to photodegradation and oxidation, has been shown to significantly improve their stability, protect against light-induced degradation, and enhance bioactivity^31–33 making γCD particularly attractive as a molecular encapsulation system for nutraceutical, pharmaceutical and functional food applications enhancing the physicochemical stability of bioactive compounds. For CNS-targeted lipophilic drugs, CDs offer a promising strategy to bypass the challenge of the BBB. Yokoyama et al. ³⁴ showed that lactose-appended β-cyclodextrin (Lac-β-CD) is a promising novel carrier for the transportation of drugs across the BBB. Human brain microvascular endothelial cells and mouse brain in vivo exhibited potent Lac-β-CD uptake, absorption, permeation and brain accumulation. Carbamazepine, an anti-epileptic drug, when conjugated with βCD and hydroxypropyl-β-CD (HP-βCD), showed improved solubility and bioavailability. ³⁵ Nishida et al. ³⁶ showed that lactose-appended HP-βCD resulted in a reduction in cholesterol accumulation and alleviated motor dysfunction in Niemann–Pick type C (NPC) disease model mice. Bioactive compounds of natural origin, such as curcumin, when encapsulated with βCD, showed improved water solubility and reduced toxicity, with potential application in neurodegenerative diseases. ³⁷

Preclinical studies have suggested that CDs may be useful therapeutic agents for AD, both by alleviating symptoms and by targeting its pathophysiology.^{38, 39} CDs were also suggested to prevent AD progression by inhibiting amyloid plaque formation or accelerating the clearance of preformed aggregates, thereby addressing one of the disease’s main hallmarks. ⁴⁰ Oxyresveratrol βCD demonstrated potent antioxidant activity and inhibited histone deacetylase activity in rat models of AD and was further associated with a restoration of cognitive deficits. ⁴¹ Animal models of Parkinson’s disease showed that subcutaneous administration of HP-βCD for 2 weeks in mice reduced dopaminergic neuron loss. ⁴² Although less explored than βCD, γCD has also been shown to enhance the stability and solubility of symptomatic drugs such as acetylcholinesterase inhibitors widely used in AD symptom management. NPC disease is a rare genetic disorder caused by cholesterol and other lipid accumulation in brain cells, leading to progressive neuronal atrophy. HP-βCD is considered a therapeutic agent for NPC and has received orphan drug status from the FDA and the EMA based on promising early-phase trials. ⁴³ HP-βCD works by extracting and clearing cholesterol accumulated in neuronal cells, as supported by clinical trials. ⁴³ Long-term intrathecal administration successfully stabilised patients aged 1.5–20 years. ⁴⁴ The above-mentioned studies have endorsed CD as a molecular shield that protects the labile functional groups of active molecules from intrinsic and extrinsic stressors, including light, oxygen and humidity, thereby inhibiting photolysis and oxidation. The enhanced functionality of δ-T3–γCD may hence be attributed to improved stability of δ-T3 in the complex. The study endorses further exploration in biomanufacturing for applications in the management of stress and age-related brain/memory dysfunction.

Conclusion

δ-T3 showed marked neurodifferentiation and anti-amnesic potential with improved efficacy when complexed with γCyclodextrin (-T3-γCD) in both in vitro and mouse model systems. The study endorses further exploration of Cyclodextrin-enabled δ-T3 in biomanufacturing for applications in the management of stress and age-related brain/memory dysfunction.

Authors’ Contribution

Investigation, methodology, data curation, formal analysis, validation, visualisation and the original draft were contributed by Arpita Konar, Priyanshu Bhargava and Kazumi Hirano. Conceptualisation, funding acquisition, project administration, validation, data curation, manuscript review and editing were contributed by Yoshiyuki Ishida, Daisuke Nakata, Keiji Terao, Sunil C. Kaul and Renu Wadhwa. All authors read and approved the submitted version.

Consent for Publication

All the authors have consented to the submission of the manuscript.

Data Availability

The data sets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Statement of Ethics

Animal handling and experiments were conducted in accordance with the guidelines of the Institutional Animal Ethics Committee of the CSIR-Institute of Genomics and Integrative Biology (IGIB), New Delhi, India, and AIST, Japan.