June 2016 Volume 3, Issue 4

A Potential Treatment for Alzheimer's Disease: Encapsulation of Curcumin in Polymeric PLGA-PEG Nanoparticles Rescues Neuro2A cells from Beta-Amyloid Induced Cytotoxicity and Caspase Induced Apoptosis

Nikash Shankar 1*, Chris Spenner 2, Ibtisam Khalaf 3, and Keith Vossel 4
Student1, Teacher2: The Harker School, San Jose, CA
Mentor3: Schmahl Science, San Jose, CA
Mentor4: UCSF Gladstone Institute of Neurological Disease, San Francisco, CA
*Corresponding author: nikashshankar@gmail.com

Abstract

There is an increased need for a versatile drug with multifunctional properties to treat Alzheimer’s disease (AD). Curcumin, a principal curcuminoid of turmeric, has anti-amyloid, anti-apoptotic, and antioxidant activities; however, its water insolubility and poor bioavailability limit its efficacy in AD. Nanoparticle-based drug delivery circumvents the pitfalls of curcumin’s poor solubility. The goal of this project is to use a polymeric nanoparticle (PEG-PLGA) encapsulated curcumin (nanocurcumin) as an effective vehicle for curcumin delivery to an in vitro AD neuronal cell model and to study its anti-apoptotic and anti-amyloid effects. The results from this study demonstrated that nanocurcumin reduced the βA levels by around 30%, exhibited an antioxidant activity of 90%, and increased cell viability by over 20% when compared to curcumin. Moreover, the 24-hour in vitro release kinetics of nanocurcumin demonstrated a 70% curcumin release from nanoparticles, and the fluorescence microscopy images showed that nanocurcumin had a higher cellular uptake than did curcumin. This study is the first to use a polymeric nanoparticle vehicle for curcumin delivery in a cell model of AD. By effectively encapsulating curcumin in PEG-PLGA nanoparticles without destroying curcumin’s inherent properties, nanocurcumin with its multiple functions, may thus be a viable potential treatment for AD.

Introduction

Alzheimer’s disease (AD) affects more than five million Americans and this number is expected to quadruple by 2050. Today, one in three seniors dies with Alzheimer’s or another dementia, making it the sixth leading cause of death in the US. Alzheimer’s disease is histologically characterized by plaque lesions in the brain that contain beta-amyloid (Aβ) peptides and neurofibrillary tangles containing tau [1]. Aβ is thought to play a major role in the pathogenesis of AD [2]. A high level of Aβ within the brain leads to synaptic loss and apoptosis mediated by Cysteine-Aspartyl Specific Proteases (caspases) (Figure 1). Activation of caspase is an early event that has been shown to contribute to progression of AD [10].

Figure 1. The role of caspases in promoting the pathology associated with AD. (1) Aβ is generated from the cleavage of APP by β and γ secretases (2) Aβ forms plaques in the extracellular compartment (3) Extracellular and intracellular Aβ activates caspases through activation of cell surface death receptors, or mitochondrial stress respectively (4) Caspase activation leads to phosphorylation of Tau, cell membrane-stabilizing protein and subsequent cell death.  Reprinted from Caspases as Targets in AD by Rohn T. et al, Int J Clin Exp Pathol, 2009.

Much effort has been dedicated to preventing the overexpression of Aβ peptide and subsequent plaque formation [4, 5]. Currently available AD therapies only abate the symptom [6]. Regardless of when AD is diagnosed in the course of the disease, conventional medical approaches do not seem to be too optimistic in bringing about a complete cessation or reversal of disease progression [7, 8].

Recent advances in the study of AD pathogenesis have led many scientists to consider that it is a multifactorial disease with various pathogenic mechanisms and pathways that includes but is not limited to Aβ oligomers, oxidative stress, and inflammation [8, 9, 10, 11]. Thus, potential treatments for AD require multi-interventional strategies to prevent or to slow down disease progression [12, 13]. Also, the multifactorial nature of the disease warrants compounds with multiple properties for treatment. Curcumin is a natural polyphenolic compound present in the spice turmeric, a rhizomatous herbaceous perennial plant endemic to South Asia [14]. Curcumin is a versatile compound that has anti-inflammatory, antioxidant, and anti-amyloid properties [2]. It acts as an antioxidant by scavenging the free radicals and neutralizes them by donating a hydrogen ion from its functional phenolic group to the free radicals [15]. Firstly, curcumin reduces amyloid plaque induced cytotoxicity by reducing amyloid synthesis and by reducing plaque formation. Similarities in the structures of curcumin and beta-amyloid—symmetrical polar charges separated by a hydrophobic bridge—allow curcumin to bind to the protein, preventing beta-amyloid from binding to any other peptides [16]. Curcumin is also able to disassemble existing beta-amyloid plaques in a similar fashion. Secondly, it reduces amyloid synthesis through its inhibitory effect on caspase pathway [16]. Animal studies have shown that curcumin significantly decreases the size and number of senile plaques in diseased brains without significant effects on normal cell function [17]. Epidemiological studies have suggested that those who consume curcumin perform better in cognitive function than those who never or rarely ate curcumin [1,2].

However, curcumin’s optimal potential is limited by lack of water solubility and poor oral bioavailability [14]. Curcumin exhibits extremely poor gastrointestinal absorption and undergoes rapid metabolism to form several inactive compounds, which lack the same therapeutic potential as the parent compound [18]. Furthermore, to observe significant beneficial effects, one must consume up to 10g of curcumin per day [15, 18]. Increasing the water solubility of curcumin has shown to increase its biodistribution and bioavailability, and also to slow down the rapid metabolism and systemic elimination [19]. One approach to increasing water solubility in natural products like curcumin is to use polymer-based nanoparticles. Cancer related in vivo studies demonstrate that the encapsulation of curcumin in nanoparticles enhances the cellular uptake and bioavailability of the drug in comparison to non-encapsulated curcumin [4, 20]. Neuronal cells treated with polymeric nanoparticle encapsulated curcumin demonstrated protection from oxidative stress. In addition, intraperitoneal injection of nanoparticle-encapsulated curcumin in mice resulted in significant curcumin levels in the brain, and decreased levels of caspase activities in the brain [21]. However, polymeric nanoparticles as a drug delivery system and therapeutic option have not been well studied in AD [22]. Studying the effects of a nanoparticle drug delivery system for curcumin will help in understanding its beneficial effects in AD pathology.

The purpose of this investigation was to use a polymeric nanoparticle vehicle for curcumin delivery in an in vitro neuronal AD cell model to inhibit the caspase-mediated apoptosis, and to study the anti-amyloid and anti-inflammatory effects of nanoparticle encapsulated curcumin (nanocurcumin). The polymeric nanoparticle vehicle utilized in this study is an FDA-approved PEGylated poly (lactic-co-glycolic acid) (PEG-PLGA). PEG-PLGA nanoparticles are water-soluble, biocompatible, non-toxic, and biodegradable polymers that break down into lactic acid and glycolic acid, which are byproducts of various metabolic pathways in the body [23]. 

Based on curcumin’s role in reducing the Aβ levels and apoptosis through the caspase pathway and the nanoparticle encapsulated curcumin’s effect in enhancing bioavailability, it is possible that nanocurcumin might possible have a beneficial effect on the pathological findings in AD [15, 16, 18, 21]. Thus, it was hypothesized that nanocurcumin would be more effective than non-encapsulated curcumin at reducing Aβ levels, Aβ-induced caspase activation, and Aβ-induced cytotoxicity in Neuro-2A neuroblastoma cells.

Materials and Methods

Neuro-2A neuroblastoma cells were used as they are a common model system for Alzheimer’s disease research. The cells were cultured in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum, Penicillin/ Streptomycin antibiotic at 37°C in 5% CO2 in a humidified incubator.

Characterization of nanoparticles: PEG-PLGA nanoparticles and PEG-PLGA encapsulated curcumin were formulated using nanoprecipitation technique by the provider of the polymeric microspheres and nanoparticles (Figure 2) [4]. The entrapment efficacy (E%) of nanocurcumin was determined by separating the amount of curcumin in the supernatant of the centrifuged nanoparticle sample and the amount of encapsulated curcumin by using spectrophotometer at 430 nm [4, 5]. The in vitro release kinetics of curcumin from nanocurcumin was obtained by centrifuging nanoparticle samples at 5 and 24 hours after solubilization and measuring the absorbance through spectrophotometric analysis at 450 nm [4, 5]. The average size and size distribution of PEG-PLGA nanoparticles were determined using dynamic light scattering techniques [3, 4]. The results for entrapment efficacy and in vitro release kinetics were a contribution from the provider of polymeric microspheres and nanoparticles. Shape of PEG-PLGA encapsulated curcumin was studied using scanning electron microscopy [5]. The cellular uptake of PEG-PLGA encapsulated curcumin was compared with that of curcumin and analyzed using a fluorescence microscope and ImageJ [5].

Figure 2. Formulation of PEG-PLGA Nanoparticle-Encapsulated Curcumin (Nanocurcumin). Reprinted from Immunosuppressive Activity of Size-Controlled PEG-PLGA Nanoparticles Containing Encapsulated Cyclosporine A by Li Tang et al, Journal of Transplantation, 2012.

Determining optimal dosage of curcumin: The cells were initially treated with 20 µM of oligomeric rAβ42 for 1 hour [17]. Following this procedure, the cells were incubated with different concentrations of curcumin (5, 10, 15, 20, 25 µM) in serum-free medium at 37°C for 24 hours. Cellular viability was calculated using an MTS assay and Aβ concentrations using an ELISA assay [5]. Based on statistical analysis using Student’s t-tests and trends in the data, it was concluded that doses of 15 and 20 µM were the most optimal.

Effectiveness of nanocurcumin vs. non-encapsulated curcumin in AD cell model: The cells were initially treated with 20 µM of oligomeric rAβ42 for 1 hour. Following this procedure, the cells were incubated with the optimal concentrations of curcumin (15, 20 µM), PEG-PLGA encapsulated curcumin (15, 20 µM), and PEG-PLGA nanoparticles (15, 20 µM) in serum-free medium at 37°C for 24 hours [5, 17].

To assess cell viability profile of PEG-PLGA nanoparticles and PEG-PLGA encapsulated curcumin, the MTS assay was used [5]. Cells were cultured overnight prior to treatment with PEG-PLGA nanoparticles, PEG-PLGA encapsulated curcumin, and curcumin. 20 μl of the MTS reagent CellTiter 96 Aqueous One Solution was added directly to the cells and incubated for 4 hours at 37°C in humidified 5% CO2. In MTS assay, the viable cells reduced the yellow tetrazolium reagent to purple formazan crystals using dehydrogenase enzymes secreted by the mitochondria of metabolically active cells. Thus the number of formazan crystals formed was proportional to the number of viable cells. The absorbance was then measured at 490 nm with a reference wavelength at 540 nm.

Assaying Beta-Amyloid Concentrations: The anti-amyloid effect of curcumin and nanocurcumin was established by ELISA assay. Supernatants of the treated cell samples were assayed for Aβ levels in triplicate using the BetaMark x-42. 

Assaying Caspase-3 Activity: To study the apoptotic effect of curcumin and nanocurcumin, a caspase assay was utilized [5]. Following 24 hours of treatment with curcumin and nanocurcumin, cell lysates were exposed to the substrate DEVD-p-NA and the absorbance of cleaved p-NA was measured using a spectrophotometer 400. DEVD is an amino acid sequence within a DNA repair enzyme, which is cleaved by caspase 3 during apoptosis.

Assaying Antioxidant Potentials of Curcumin and Nanocurcumin: To analyze the effect of nanocurcumin on antioxidant activity, free-radical scavenging capacity of nanoparticles was tested. 2, 2-diphenyl-2-picrylhydrazyl (DPPH) was the source of free-radicals [5]. 4ml of nanoparticles was mixed with methanolic solution of DPPH in the dark at room temperature for 5 hours. The DPPH scavenging activity was determined through spectrophotometric analysis at 520 nm against DPPH solution as control. Antioxidant activity was calculated using the following equation: % Antioxidant Activity = ((Absorbance Control – Absorbance Sample)/ Absorbance Control) * 100.

Statistical Analysis: All data were expressed as means ± SD from three independent experiments performed in triplicate. Statistical analysis was performed using two-tailed Student’s t-test. The null hypothesis was rejected when p < 0.05.

Results

Nanocurcumin exhibited increased water solubility and cellular uptake compared to curcumin
Nanocurcumin was completely soluble in water, and varied in size between 100-150 nm (mean diameter by volume: 192.2 ± 52.16 nm) as shown in Figure 3A. The entrapment efficiency of curcumin in nanoparticles was 30.98%. Scanning Electron Microscopy (SEM) images confirmed the spherical nature of the nanoparticles (Figure 3B). In Figure 3C, the in vitro release kinetics demonstrated ~70% curcumin release from PLGA nanoparticles at 24 hours.

Figure 3. Characterization of Nanocurcumin. A) Dynamic light scattering demonstrates a mean diameter by volume of 192.2 ± 52.16 nm. B) SEM image of nanocurcumin demonstrates nanoparticles are spherical.  C) Release kinetics of curcumin from nanocurcumin demonstrates a burst release of 60% over the first 5 hours and a subsequent release of 10% over the next 20 hours.

Figure 4 demonstrates the relative absorption of curcumin and nanocurcumin by neuronal cells. Fluorescence was detected under a fluorescence microscope with a GFP filter due to curcumin’s auto-fluorescent property [5]. All representative images (Figure 4A, 4B, 4C) had similar numbers of cells. Cells treated with nanocurcumin showed an increase in fluorescence (green) and thus cellular uptake (Figure 4A), when compared cells that were treated with non-encapsulated curcumin—cells treated with curcumin demonstrated space fluorescence and thus minimal cellular uptake of the compound.

Figure 4. Uptake of Curcumin in Neuro2A Cells. A) Increased cellular uptake (green) seen under fluorescent microscope (GFP). B) Sparse cellular uptake (green) seen under fluorescence microscope (GFP). C) Neuro2a cells as a control.

Optimal doses of Curcumin to use for encapsulation was determined to be 15 and 20 µM
As shown in Figure 5, optimal doses of curcumin were determined to be 15 and 20 µM, because cells treated with these doses of curcumin had significantly more cell viability (p< 0.05), by almost 50-60%, than those treated with Aβ alone (Figure 5A). In addition curcumin at 15, 20, and 25 µM demonstrated least amount of Aβ levels (Figure 5B). This optimal dose of curcumin was subsequently used for the formulation of nanocurcumin.

Figure 5. Determination of Optimal Dose of Curcumin. A) Neuro-2A cells treated with varying curcumin concentration and cell viability measured using MTS assay. Cells exposed to Aβ served as positive control and Neuro-2A cells without Aβ served as negative control. B) Neuro-2A cells were treated with varying curcumin concentration and the effect on Aβ levels were analyzed using ELISA assay. Cells exposed to Aβ served as positive control.

Nanocurcumin exhibited greater cell viability than curcumin
Results on cell toxicity demonstrated that curcumin and nanocurcumin showed significantly greater cell viability (p<0.05) as early as 7 hours with continued effect till 24 hours, compared to the cells exposed to Aβ alone (positive control). Also at 24 hours, cells treated with nanocurcumin (15 and 20 µM) exhibited greater cell viability than those treated with curcumin (15 and 20 µM). Cells treated with PEG-PLGA nanoparticles (15 and 20 µM) did not possess any toxicity in vitro (Figure 6A).

Figure 6. A) Neuro-2A cells treated with curcumin, nanocurcumin, PEG-PLGA at 15 and 20 µM. The light and dark bars correspond to treatment exposure to at 7 and 24 hours. Student T-test two-tailed statistical analysis. * p < 0.05 statistical significance.

Cells treated with nanocurcumin showed significantly less Aβ levels than curcumin
Aβ ELISA results demonstrated that cells treated with 15 and 20 µM nanocurcumin showed significantly less Aβ levels (p<0.05) than those exposed to Aβ alone (positive control). Nanocurcumin at 20 µM showed the least level of Aβ. Comparing the effect between nanocurcumin and curcumin, cells treated with 20 µM nanocurcumin showed significantly less Aβ level (p<0.05) than those treated with 20 µM curcumin(Figure 6B).

Figure 6. B) Neuro-2A cells treated with curcumin, nanocurcumin, PEG-PLGA at 15 and 20 µM. Student T-test two-tailed statistical analysis. * p < 0.05 statistical significance. The error bars represent standard deviation (SD).

Cells treated with nanocurcumin exhibited less caspase activity than those treated with curcumin
Results for the apoptosis of cells using caspase assay demonstrated that cells treated with 15 and 20 µM nanocurcumin and cells treated with 15 and 20 µM curcumin exhibited significantly less caspase activity (p<0.05) than those with Aβ (positive control). Cells treated with 20 µM nanocurcumin exhibited less caspase activity than those treated with 20 µM curcumin, and cells treated with 15 µM nanocurcumin exhibited significantly less caspase activity (p<0.05) than those treated with 15 µM curcumin (Figure 7).

Figure 7. Nanocurcumin and Curcumin reduce Caspase Activity. Neuro-2A cells treated with curcumin and nanocurcumin at 15 and 20 µM. Apoptotic effect was determined by caspase-3 activity. Student T-test two-tailed statistical analysis. * p < 0.05 statistical significance. The error bars represent standard deviation (SD).

Nanocurcumin exhibited comparable anti-oxidant activity to that of the curcumin
DPPH assay was used to study the free-radical scavenging capacity of nanocurcumin in comparison with curcumin and the results are shown in Figure 8. Curcumin at 15 and 20 µM showed more than 90% anti-oxidant activity. Nanocurcumin at 15 and 20 µM also exhibited comparable anti-oxidant activity (90%) to that of the curcumin.

Figure 8. Antioxidant Activity of Curcumin and Nanocurcumin. Neuro-2A cells treated with curcumin and nanocurcumin at 15 and 20 µM. The antioxidant activity was measured by DPPH assay. Student T-test two-tailed statistical analysis. * p < 0.05 statistical significance. The error bars represent standard deviation (SD).

Discussion

Although curcumin is a promising compound with numerous beneficial properties, its utility is greatly restricted by its insolubility in water and its low bioavailability. Water solubility is one of the important requirements for an ideal drug. In this project, water solubility was achieved by encapsulating curcumin in biodegradable PEG-PLGA nanoparticle. The encapsulation efficacy of curcumin in the nanoparticles was 40%. The optimal release rate of 70% of curcumin from the PEG-PLGA nanoparticles at 24 hours indicate that curcumin reached the model cells after delivery. PLGA nanoparticles are taken up by the cells mainly through clarithrin-mediated endocytosis [24]. The results indicate an increase in bioavailability of the curcumin within cells, when encapsulated with PEG-PLGA nanoparticles. The results of the characteristics of the nanoparticle-encapsulated curcumin in this current study are in agreement with that of the prior studies [4, 20]. Furthermore, the DPPH assay demonstrated that encapsulation of curcumin with the nanoparticles preserved the anti-oxidant properties and is able to scavenge the free radical source-DPPH. Cell viability results prove that PEG-PLGA nanoparticles and nanocurcumin were not toxic to the cells. Similar results showing the polymeric nanoparticle’s high biocompatibility, its non-toxic nature, and its ability to preserve curcumin’s natural properties such as its anti-oxidant property were seen in other in vitro studies and in vivo mice models  [4, 5, 20]

In AD, increased amyloid and formation of amyloid plaques result in neuronal apoptosis [6]. The current study not only demonstrated curcumin’s anti-amyloid abilities but also improved its capabilities by encapsulating the compound in polymeric nanoparticles. Cells treated with nanocurcumin at 20 µM had significantly less Aβ (p<0.05) than just those that were treated with curcumin at 20 µM. Furthermore, ample evidence connecting the presence of Aβ and increase activity of caspase pathways indicates that the activation of caspases and apoptosis are early events that contribute to the progression of AD [3]. Nanocurcumin significantly inhibited (p<0.05) the activation of caspases and apoptotic pathways in cells previously exposed to Aβ. Though previous studies have demonstrated curcumin and nanocurcumin’s effect in reducing caspace activity, this study is novel in that it looks at the drugs’ effects on caspace and other downstream proteins such as Aβ [21]. This project successfully achieved increased water solubility and cellular uptake of curcumin through the encapsulation of curcumin in PEG-PLGA nanoparticles of size 100-150 nm, factors that have been shown to be ideal for drug delivery across blood brain barrier [25]. The results indicate that our first hypothesis—that nanocurcumin was effective not only in reducing the Aβ significantly but also in significantly increasing cell viability—was supported; after 7 hours, nanocurcumin was able to increase cell viability much more than curcumin showing a sustained release effect of curcumin. Furthermore, the aforementioned results indicate that our second hypothesis is supported, as there was a greater inhibition of caspases when the cells were treated with nanocurcumin. One possible cause for higher activity of nanocurcumin could be due to higher cellular uptake of curcumin from the nanoparticles [4].

The results of this in vitro project suggest that curcumin encapsulated in PEG-PLGA nanoparticles could potentially be a therapeutic option in AD. In addition to biocompatible nature of nanocurcumin, the long-term safety and low cost of curcumin make it a promising candidate for treating AD.

Limitations and Future Directions

The aforementioned results indicate that encapsulating curcumin in nanoparticles improves the drug’s bioavailability and ability to reduce Aβ induced cytotoxicity; however, the present study has several limitations. First, we studied the effect of nanocurcumin using a neuronal cell culture model. So, it remains to be determined whether these results could be replicated in patients with AD. Although the model used in the study demonstrated that the encapsulation of curcumin in nanoparticles improves the drug’s bioavailability and ability to reduce Aβ induced cytotoxicity, the results need to be revalidated and confirmed in in vivo models. Second, while the use of PLGA PEG improve bioavailability of curcumin in neuronal cells, the question remains if nanocurcumin would be able to effectively deliver the drug to central nervous system in patients with AD. Targeting the central nervous system has always been a challenge due to its selectively permeable blood brain barrier and the presence of tight junctions within the capillary endothelium that severely restricts the delivery of therapeutics to the brain [26]. Tagging PLGA-PEG nanoparticles with a glycosylated heptapeptide to target the drug to central nervous system has been successfully studied in in vivo models [25, 27]. Recently retrograde axonal transport and effective delivery of the drug to the neurons has been well studied and established [28]. Tet-1, a 12 amino acid peptide, which has the binding characteristics of the tetanus toxin, can interact specifically with neurons and is capable of delivering drugs in a retrograde fashion into the neuronal cells [5].

These limitations open new possibilities to future research in this area. Replication of these in vitro results in an in vivo model would confirm the findings and further validate the results. Furthermore, improving the design of the nanoparticle could enhance its drug-delivery abilities; tagging the nanoparticles with polymers such as Tet-1 would improve the cell specific targeting nature of the treatment and its effectiveness in the model organism, altering the average nanoparticle size, shape, and charge could effect its uptake into the cells, and changing the material of the nanoparticles could improve the bioavailability of curcumin in the cells. Thus, while the conjugation of the Tet-1 peptide with curcumin nanoparticles could present a direction of future research in AD by increasing the bioavailability of the drug to select tissues, altering the properties of the nanoparticle, such as the size, shape, and charge, could improve cellular uptake and decrease the drug’s degradation.

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