Neuroprotective Effects of Curcumin-Loaded Emulsomes on Laser Axotomy-Induced CNS Injury Model

doi:10.21203/rs.3.rs-38217/v1

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Neuroprotective Effects of Curcumin-Loaded Emulsomes on Laser Axotomy-Induced CNS Injury Model

https://doi.org/10.21203/rs.3.rs-38217/v1

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Background

Curcumin, a polyphenol isolated from the rhizomes of turmeric, holds a great potential as a neuroprotective agent along with its anti-inflammatory and antioxidant characteristics. Its poor bioavailability and low stability in water lie as foremost restraints against the clinical use. This study aims at investigating the neuroprotective effect of curcumin on axonal injury by delivering the lipophilic polyphenol to primary hippocampal neuron by means of a lipid-based drug delivery system, named emulsomes.

Methods

To study the neuroregeneration on ex vivo, an injury model was established through single-cell laser axotomy on hippocampal neurites. Upon treatment with curcumin-loaded emulsomes (CurcuEmulsomes), curcumin and CurcuEmulsome uptake into neurons were verified by 3-dimensional z-stack images acquired with confocal microscopy. Neuron survival after axonal injury were tracked by PI and Hoechst staining. Alterations in expression levels of physiological markers such as anti-apoptotic marker Bcl-2, apoptotic marker cleaved caspase 3, neuroprotective marker Wnt3a and the neuronal survival marker mTOR were investigated by immunocytochemistry analyses.

Results

Results indicated significant improvement in the survival rates of injured neurons upon CurcuEmulsome treatment. Bcl-2 expression became significantly higher for injured neurons treated with curcumin or CurcuEmulsome. Caspase 3 expressions decreased in both curcumin- and CurcuEmulsome-treatments, whereas Wnt3a and mTOR expressions did not alter significantly.

Conclusions

The established laser-axotomy model was exposed as a reliable methodology to study neurodegenerative models ex vivo. CurcuEmulsomes delivered curcumin to primary hippocampal neurons successfully. Treated with CurcuEmulsomes, injured hippocampal neurons benefit from neuroprotective effects of curcumin in terms of higher survival rate and increased anti-apoptotic marker levels.

Cellular & Molecular Neuroscience

Curcumin

lipid-based drug delivery systems

neurodegeneration

hippocampus

axon injury

While the demographics in developed countries shift towards an aging population, neurodegenerative diseases (NDs) including Alzheimer’s Disease (AD), amyotrophic lateral sclerosis (ALS) and Parkinson’s Disease (PD) are predicted by World Health Organization (WHO) to be the second cause of death following the cardiovascular disease by 2040 [1]. NDs are characterized by progressive loss of function and structure of neurons where the physiological phenomenon ends up with the neuronal death [2, 3]. Axon degeneration, one of the common hallmarks of both nerve injuries and neurodegenerative diseases, causes permanent disabilities [4, 5]. Axons are the first damaged neuronal compartments, where the degeneration of axons may begin many years before the eventual death of the cell [6, 7]. Although axon regeneration is necessary for neuronal survival, there is no effective treatment for inhibiting axonal degeneration [5].

Axotomy is an experimental model of neuronal injury and sequent cell death. Axotomy is widely applied in research to study biological response of neurons and axons to an injury established via transection of the neurons’ axon at in vitro. Activation of the neuroprotective pathways and sustainment of the endogenous brain neurogenesis are regarded as therapeutic approaches to convert neurodegeneration to neuroregeneration [8].

Current challenges with neuroprotective therapeutic approaches lie on their high cost, lack of efficacy, drug resistance and certain hazardous side effects [9, 10]. As an alternative, natural biomolecules hold a renewed interest in recent years. For instance, curcumin, a polyphenol isolated from the rhizomes Curcuma Longa, is a biologically very active component shown to play a role as a neuroprotective agent in AD, PD, ischemic stroke and alcohol-induced neurodegeneration models [11–18]. Curcumin’s diverse biological functions include anti-oxidation, anti-inflammation, neuroprotection, support to mitochondrial and blood-brain barrier integrity [11, 19, 28, 20–27]. Thus, alone or combined with other neuroprotective agents, the use of curcumin may benefit current therapies for neurodegenerative diseases [29, 30].

However, the medical use of curcumin is limited due to its poor bioavailability and low stability. Curcumin exhibits very low water solubility (i.e. 11 ng/ml[31]) [32–36], poor absorption [36], rapid metabolism [32, 37, 38] and fast systemic elimination [18].

Drug delivery systems are considered as the prominent solution to enhance solubility, stability, cellular uptake efficacy of curcumin [39]. Delivery of curcumin within various nanocarriers were shown to increase the amount of curcumin penetrating through the blood-brain barrier (BBB) and sustain curcumin release inside the CNS [40–42].

In this study, a recently developed lipid-based nanoparticular system, curcumin-loaded emulsomes, or so-named CurcuEmulsomes [43], are used to enable the delivery of curcumin to primary hippocampal neuron culture. To study the neuroregenerative effect of the formulation, an axonal injury model was successfully established ex vivo through laser axotomy on the neurites. Curcumin-loaded emulsomes are investigated herewith for the first time as a neuroprotective nanomedicine on a novel, repetitive and robust neuron injury model.

Animals

Hippocampal neurons for in vitro studies were isolated from newborn BALB/c mice (Regenerative and Restorative Medicine Research Center (REMER), Istanbul, Turkey) on post-natal day zero (P0). Animal experiments were conducted in accordance with the European Community guidelines and approved by Istanbul Medipol University Ethics Committee for Animal Experimentation (Date:07/03/2016; Approval number: 38828770-604.01.01-E.3722).

Chemicals and Reagents

Curcumin (purity ≥ 95%), glyceryl tripalmitate (tripalmitin purity ≥ 99%), 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (DPPC, 99%), cholesterol (≥ 99%), sodium azide, acetone (99.5%), ethanol (99.8%), chloroform (≥ 99%), antibiotic-antimycotic solution, papain, L-15 medium (Leibovitz), poly-L-lysine, glutaraldehyde solution and propidium iodide were purchased from Sigma-Aldrich (Germany). Tween® 20 and DMSO (≥ 99.7%) were purchased from Fisher BioReagents, United States. B-27 supplement (50X), Hibernate-A medium, Neurobasal-A medium and glutamax-I were purchased from Gibco/Invitrogen. 3-(4,5-di-methyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS)-assay (CellTiter96 AqueousOne Solution) was purchased from Promega (Southampton, UK). Phosphate Buffer Solution (PBS, Multicell, Wisent Inc.), Fetal Bovine Serum Advanced, Heat Inactivated (FBS, Capricorn Scientific GmbH), DNAse I (Biomatik), Hoechst 33342, Blocker BSA in TBS (Cat^# 37520), antibody diluent (Cat^# 003118) were purchased from ThermoFisher Scientific (USA). Primary antibodies, including anti-Wnt3a (Cat^# ab28472), anti-mTOR (Cat^# ab87540) and secondary antibodies goat anti-mouse IgG Alexa Fluor 594 (Cat^# ab150116) and goat anti-rabbit IgG Alexa Fluor 633 secondary antibodies were purchased from Abcam (Cambridge, UK). Anti-Bcl-2 (Cat^# sc7382) was purchased from Santa Cruz Biotechnology (USA). Anti-cleaved-caspase-3 monoclonal antibody (Cat^# 9664) was purchased from Cell Signaling Technology, Inc (USA).

Production of CurcuEmulsomes

CurcuEmulsome was synthesized applying the procedure described by Bolat and the colleagues [44]. Briefly, lipids including tripalmitin, dipalmitoyl phosphatidylcholine and cholesterol together with curcumin were first dissolved in an organic solvent, i.e. chloroform. The solvent was completely removed in a rotary evaporator at 40^oC and under 474 mbar. A dry lipid film was established on the bottom of the rotary flask. Double-distilled water was added to the flask and the system was set to rotation at 80^oC and under atmospheric pressure for 4 hours to obtain emulsomes. To homogenize the particle size, emulsomes were applied to an ultrasonication bath at 70^oC for 1 hour. To spin down unincorporated curcumin within the solution, preparations were centrifuged at 13,200 rpm (16,100 g) for 10 minutes. The supernatant including the CurcuEmulsomes was stored at 4 °C until further studies. Empty emulsomes were prepared following the same procedure without the addition of curcumin.

Physicochemical Analysis

The mean particle size and zeta potential of emulsomes were determined by dynamic light scattering using Zetasizer instrument (Nano ZS, Malvern Instruments Ltd, UK). Accordingly, samples were diluted in 1 mM KCl solution to suitable concentrations. All analyses were performed in the auto-measuring mode at 25 °C. The average of triplicate analysis was recorded for each sample.

Stability

Particular stability of the formulations was assessed based on changes in size, zeta potential and polydispersity in time. Average size, zeta potential and polydispersity index (PDI) of the samples were analyzed periodically upto 11 months using Zetasizer instrument (Nano ZS, Malvern Instruments Ltd, UK).

Morphology: Particle Size and Shape

Particular size of the emulsomes were further investigated with scanning electron microscopy (SEM, Zeiss EVO-HD-15, Germany) together with the shape and the morphology. Pretreatment procedure implies a short-term fixation where samples were placed onto an aluminum holder and left at 4 °C overnight for drying. Dried samples were then treated with PBS buffer containing 2.5% glutaraldehyde for 15 minutes. Samples were washed 3 times with distilled water for 10 minutes. After sputtered by gold (EM ACE200, Leica, Germany), samples were investigated under the electron microscope.

Quantification of Curcumin Encapsulated inside CurcuEmulsomes

Amount of curcumin encapsulated inside CurcuEmulsomes was estimated as described elsewhere [43, 44]. A 1 mg/ml stock solution of curcumin was prepared in DMSO. A standard curve, generated by successive dilution of the stock solution (5, 10, 20, 50, 100 µg/ml) in a 96-well microplate (NEST Scientific, cat #701001, China), was used to determine curcumin concentrations in samples prepared by 1:10 dilution of CurcuEmulsome suspension in DMSO. Sample absorbance was measured at 430 nm on UV-vis spectrophotometer (Spectramax i3 Multi-Mode Microplate Reader Detection Platform, Molecular Device, Sunnyvale, CA, USA). A standard curve was prepared from the values of standards. Curcumin concentration of CurcuEmulsomes was estimated by the read-out of the absorbance intensity and corresponding concentration on the standard curve.

In Vitro Drug Release Analysis

The direct-dispersion method described by Bisht et al. (2007) and Anitha et al. (2011) was applied for the in vitro drug release profile of the CurcuEmulsomes as described elsewhere [45, 46]. Briefly, 4 ml of CurcuEmulsome solution was divided into 10 microcentrifuge tubes (400 µl each) and kept in a thermostable shaker at 37 °C and 300 rpm. At time zero and each time intervals (½, 1, 2, 3, 6, 12, 24, 48, 72 hours) three tubes were taken and centrifuged at 3000 g for 10 minutes. The supernatant containing the emulsomes was removed. The pellet containing the released curcumin was dissolved in 400 µl DMSO. The protocol described in the previous section was followed to quantify the amount of curcumin corresponding the amount of curcumin released from CurcuEmulsomes. The procedure was repeated three times.

Isolation of Primary Hippocampal Neurons

The Balb-c mice on postnatal day 0 (P0) were euthanized by decapitation, brain was removed. Hippocampi were dissected out, transferred into the 1 ml of L-15 medium (Leibovitz) (Sigma) containing 1% antibiotic-antimycotic (penicillin-streptomycin) (Sigma), 1% glutamax (Gibco), 2% B-27 (Gibco) and 20 µl of Papain (Sigma), agitated for 45 minutes at 4 °C. After the addition of DNAse, the cell suspension triturated with the help of a fire-polished glass pipette. To stop the enzymatic activity, homogeneous solution was taken into the L-15 medium (Leibovitz) (Sigma) containing 1% antibiotic-antimycotic (penicillin-streptomycin) (Sigma), 1% glutamax (Gibco) and 2% B-27 (Gibco) and 10% of fetal bovine serum, incubated at room temperature for 10 minutes. To spin-down the cells, the solution was centrifuged at 1200 rpm for 5 minutes. The supernatant was removed and 1 ml of Neurobasal-A medium (NBA) containing 1% antibiotic-antimycotic (penicillin-streptomycin) (Sigma), 1% glutamax (Gibco) and 2% B-27 (Gibco) was directly added to the cells in the pellet. Isolated hippocampal neurons were seeded into 96-well plates as 20.000 cells per well, into the 12-well plates and the 35-mm cell culture dishes as 100,000 cells per well for each experiment. Hippocampal neurons were cultured at 37 °C and 5% CO₂ for 2 days to allow the axon growth to reach to a certain length.

Treatment of Neurons with Curcumin and CurcuEmulsomes

After 2 days in culture, primary hippocampal neurons showed optimum neurite outgrowth. Cells supplemented with 1 ml of Neurobasal-A (NBA) medium containing 1% antibiotic-antimycotic (penicillin-streptomycin) (Sigma), 1% glutamax(Gibco) and 2% B-27 (Gibco) were grouped for the MTS assay as the following: (1) Control group including the untreated cells, (2) Vehicle control group including 0.01% DMSO, (3) Vehicle control group including blank emulsomes (4) 2 µM free curcumin (i.e. curcumin dissolved in DMSO), (5) 5 µM free curcumin, (6) 10 µM free curcumin (7) 2 µM CurcuEmulsome, (8) 5 µM CurcuEmulsome, and (9) 10 µM CurcuEmulsome. DMSO content in total cell medium of free curcumin treatment groups was kept below 0.01% to avoid any influence of DMSO to the neurons. The vehicle control group (2) was included with 0.01% DMSO content to monitor the influence of DMSO on neuron culture. At the succeeding axatomy and ICC studies, the experimental groups were kept limited with (1) control group (untreated cells), (2) 5 µM free curcumin treatment group, and (3) 5 µM CurcuEmulsome treatment group.

In Vitro Cellular Uptake

Following the treatment of neurons with curcumin and CurcuEmulsome, the neurons were investigated under the confocal laser scanning microscope (CLSM) periodically at 24, 48 and 72 hours. Uptake of curcumin and CurcuEmulsomes by the neurons were tracked and cell morphology was studied for presence of any alterations. Accordingly, at DIV2 (Day in vitro 2) following the cell seeding, cells were treated with 5 µM free curcumin (curcumin dissolved in DMSO) and 5 µM CurcuEmulsomes. Curcumin and CurcuEmulsome uptakes were visualized by with the help of autofluorescent property of curcumin for 72 hours.

Cellular uptake was furhter studied by the 3-dimensional z-stack imaging via addition of certain fluorescent agents at DIV2 after cell seeding. Accordingly, the red Vybrant Tongue (V22885-ThermoFisher Scientific Orange Red) was used for staining the entire cell and the Hoechst 33342 (H3570, Thermo Fischer Scientific) (3:1000) was used to stain the nucleus. Following 10 minutes of staining, CurcuEmulsomes were added into the culture and images were taken in series for 15 minutes from different Z-stacks with 1-µm intervals. When the imaging was completed, images were merged to realize the 3D structuring using ZEN program (Zeiss).

MTS Assay

A commercially available one-solution cell proliferation assay containing a novel tetrazolium compound (MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)) and an electron coupling reagent (phenazine ethosulfate; PES) (The CellTiter 96® Aqueous, Promega) was used to test the effects of free curcumin and CurcuEmulsomes on the viability of primary hippocampal neurons. NADPH or NADH produced by metabolically active cells cause reducement of the MTS tetrazolium compound (Owen’s Reagent) into a colored formazan product that can be detected at 490 nm. At certain time intervals (DIV1, DIV2, and DIV3) after the treatments, the culture media was discarded and the cells were washed with PBS once. 100 µl Neurobasal-A medium (minus phenol red and without additives) containing 20 µl of the MTS solution was added to the culture. Cells were incubated for 3 hours at 37 °C, the absorbance of formazan was read at 490 nm utilizing a microplate reader (Spectramax i3 Multi-Mode Microplate Reader Detection Platform, Molecular Device, Sunnyvale, CA, USA).

Formation of Laser Beam-Induced Injury: Axotomy

To be able to track the injured neurons throughout the experiment easily, a gridline pattern has been plotted on the surface of the dishes prior seeding the cells. A laser microdissection system (PALM CombiSystem, ZEISS), operating with a 355-nm UV laser (1-100 pulse production per second with an energy release equivalent to 90 µJ), was used for the induction of neurite injury. Neurons that have no bleb, vacuoles, beaded axons or damaged membrane on their surface were selected and marked using ZEN software. The axotomy procedure was carried on PALMRobo software using a 40X dry phase contrast objective. The laser was applied to the selected neurites, each time with a distance of 25–30 µm from the neuronal body.

Axotomy was applied after the neurons (DIV2) were exposed to curcumin or CurcuEmulsome treatment for 6 hours. Axotomy study involves six different experimental groups (3 test groups and 3 control groups), which were there after assessed through both cell viability and immunocytochemistry analysis:

1. Untreated control group: no treatment, no injury.

2. Sham control group: the laser focus was adjusted to an empty spot 25–30 µm away from the neurons to question whether the laser causes any effect on cell viability or degeneration via heating or radiation (Applied only at cell viability analysis).

3. Untreated injured group: no treatment applied in this group following the axotomy to investigate the effect of injury when the cells are not treated.

4. Curcumin-treated control group: neurons (DIV2) were treated with 5 µM curcumin for 6 hours; no axotomy was applied (Only for immunocytochemistry analysis).

5. Curcumin-treated injured group: neurons (DIV2) were treated with 5 µM curcumin for 6 hours; axotomy was applied.

6. CurcuEmulsome-treated injured group: neurons (DIV2) were treated with 5 µM CurcuEmulsomes for 6 hours; axotomy was applied.

Axotomy procedure involved axotomy on 50 neurites for each group, which was repeated four times for both cell viability and immunocytochemistry study corresponding altogether to total number of 200 neuron injury for each study.

Cell Viability After Injury

Cell survival upon neurite injury was assessed by propidium iodide (PI) and Hoechst 33342 staining 24 hours after axotomy procedure. A PALM microdissection microscopy (Zeiss, Turkey) equipped with an integrated stage-top incubator was regulating the cell environment at 37 °C and 5% CO₂ throughout this experiment. Tile images were taken using 20X lens as the gridline patterns on the surface of the culture dishes were adjusted in the center of the screen. Per each of the six experimental groups, 50 healthy neurons were selected and marked in total for the axotomy. 24 hours after the laser-induced neurite injury, the media was replaced with a fresh medium containing Hoechst 33342 (1:1000). Cells were incubated again at 37 °C, 5% CO₂ for 10 more minutes. Then, PI (1:1000) was added to the medium and cells were incubated at same conditions for 5 more minutes. Fluorescent images of the marked locations acquired on PALM microdissection microscope. Fluoresencent signals of Hoechst and PI were detected at wavelengths 350/461 nm and 535/617 nm (excitation/emission) with a 20x objective, respectively. Viable (Hoechst-positive; PI-negative) and dead neurons (Hoechst-positive; PI-positive) were manually counted using ImageJ software.

Immunocytochemistry

24 hours after the laser-induced neurite injury, primary hippocampal neurons were fixed by incubating in 4% paraformaldehyde in PBS for 15 minutes at room temperature. Then, cells were washed twice with PBS and incubated in a blocker solution (Blocker BSA in TBS, Thermofisher Scientific) in PBS for 90 minutes. Thereafter the blocking solution was discarded and neurons were washed twice with PBS at room temperature. Subsequently, neurons were treated with the primary antibodies with different dilution ratios for 4 hours at room temperature as the following: rabbit polyclonal anti-Wnt3a antibody [1:200] (Abcam ab28472), mouse monoclonal [53E11] to anti-mTOR antibody [1:200] (Abcam ab87540), mouse monoclonal IgG₁ anti-Bcl-2 antibody [1:200] (Santa Cruz Biotechnology, sc7382), rabbit monoclonal caspase-3 (Cell Signaling Technology, 8G10) diluted in antibody diluent (ThermoFisher Scientific). Cells were washed with PBS and incubated at room temperature with 1:200 diluted goat anti-mouse IgG Alexa Fluor 594 or goat anti-rabbit IgG Alexa Fluor 633 secondary antibodies for 1.5 hours. Cells were washed 3 times with PBS containing 0.2% Tween-20 for overall 12 minutes. Then, cells were incubated in DAPI solution [1:1000] diluted in PBS for 3 more minutes at room temperature. Finally, cells were washed with PBS once and incubated in PBS containing 0.1% sodium azide. The marked neurons on the gridline pattern were positioned using 10X objective lens. After localization of the injured neurons, images were captured with 40X oil-immersion lens using confocal microscope (LSM 800, ZEISS). DAPI was detected at wavelengths 358/461 nm, Alexa Fluor 594 at 590/617 nm, Alexa Fluor 633 at 632/642 nm (excitation/emission).

Image Analysis Using ImageJ

The total dead-alive cells, total fluorescence per cell and corrected integrated density for each cell analysis were performed using image analysis platform Fiji (ImageJ) [47].

Statistical Analyses

Values were calculated using GraphPad Prism Software (version 6.01) and presented as mean ± standard error of the mean (mean ± SEM). Statistical analyses were performed using one-way ANOVA post-Hoc Tukey for the viability and ICC analysis. Chi-square test was performed for the viability following the neurite injury. The level of significance was set at P < 0.05.

Characterization Studies

Dispersity

Free curcumin is poorly soluble in aqueous media, with macroscopic undissolved flakes of the compound visible in particular on the surface of the solution (Fig. 1-A); while CurcuEmulsome is a clear, dispersed formulation, with its hue derived from the natural color of curcumin. The dispersity was observed to be stable for at least 11 months under storage conditions at 4 °C.

Physicochemical Properties of CurcuEmulsomes

DLS analysis with Zetasizer Nano ZS (Malvern Instruments Ltd, UK) determined the average diameter of ten distinct CurcuEmulsome formulations as 178.8 ± 13.7 nm (with an average polydispersity index of 0.18 ± 0.06) (Table 1) - where the plus-minus sign indicates the margin of average sizes of numerous CurcuEmulsome productions made of the same composition. Size distribution curve indicated a variation in particle size in a range of 75–450 nm (Fig. 2-A). Similarly, the average diameter of empty emulsomes was found as 204.3 ± 20.0 nm, with an average PDI value of 0.269 ± 0.050. With a moderate PDI value (between 0.01–0.04), both emulsomes and CurcuEmulsomes displayed slightly polydisperse characteristics (Table 1). In addition, loaded with negatively-charged curcumin inside, CurcuEmulsomes differed from empty emulsomes (-24.2 ± 6.3 mV) with a higher negative average zeta potential (-33.2 ± 6.7 mV) (Fig. 2-B).

Table 1

**ZetaSizer Analysis Data.** Average particle size, polydispersity index (PDI) and average zeta potential values of CurcuEmulsome and emulsomes
Formulation	Particle Diameter (nm)	PDI	Zeta Potential (mV)
Emulsome	204.3 ± 20.0	0.269 ± 0.050	-24.2 ± 6.3
CurcuEmulsome	176.8 ± 13.0	0.180 ± 0.060	− 33.2 ± 6.7

Stability

Protected from light and stored at 4^oC, both emulsomes and CurcuEmulsomes conserved their average size, zeta potential and PDI values for as long as 11 months (Table 2). The conserved physicochemical charateristics indicated no occurrence of significant loss of structural integrity of the formulations.

Table 2

**Stability Data of CurcuEmulsomes.** ZetaSizer analysis data of CurcuEmulsomes at the day of production and 1 month, 2 months, 4 months and 11 months after the production.
Time	Particle Diameter (nm)	PDI	Zeta Potential (mV)
Day of production	162.6	0.101	-28.0 ± 7.5
2 months later	164.4	0.096	-21.1 ± 6.7
4 months later	157.0	0.026	− 25.8 ± 5.0
11 months later	149.6	0.096	− 25.0 ± 7.3

Morphology

SEM micrographs verified that CurcuEmulsomes are spherical in shape and display a smooth, uniform surface characteristics as morphology (Fig. 1-B). In parallel to the DLS data, size of CurcuEmulsomes was seen to be around 200 nm in average, with a particle size distribution between 50–400 nm.

Curcumin Encapsulation

As estimated by absorbance measurements, CurcuEmulsomes achieved curcumin encapsulation of around 0.08 mg/ml in average (in range of 0.06–0.11 mg/ml), where the maximum value corresponds to a 10,000-fold increase in solubility of curcumin as exactly reported by Ucisik and the colleagues [43]. CLSM analysis further provided evidence not only for the presence of curcumin within each emulsome particle (Fig. 1-C), but also provided evidence for the lack of any liposomes as by-product which would be seen as hollow circles under fluorescent microscope. Besides, CLSM images also pointed out the highly dispersed character of CurcuEmulsomes in water.

In Vitro Drug Release

The release profile suggests that CurcuEmulsomes release approximately 20% of their curcumin content within the first 6 hours in vitro; which becomes nearly 35% at 12 hours and – reaching a maximum – remains constant (Fig. 1-D). Herewith, drug release data claims that no further release occurs between 12 and 72 hours, which is regarded as quenstionable. Due to the low solubility and poor stability of curcumin in water it is thought that the in vitro release data does not accurately reflect the actual curcumin release occurring from CurcuEmulsomes. It will be further elaborated in the Discussion.

Cell Culture Studies

Isolation of Primary Hippocampal Neurons

A neurite growth around 100 µm was set as a requirement to proceed to laser axotomy. Upon the isolation of neurons at post-natal 0, neurite growth was observed 3 days long. Two days of incubation was found to be sufficient for the required neurite elongation. Accordingly, depending on the time of day when the isolation process has been carried out (i.e. in the morning or in the evening), neurite lengths were observed to become equal or greater than 100 µm reproducibly each time at 2 and 3 days after the neuron seeding (Fig. 3-A).

Cell Uptake Studies of Curcumin and CurcuEmulsomes

Auto-fluorescence property of curcumin enabled monitoring the uptake of CurcuEmulsomes by the primary hippocampal neurons using CLSM. Three-dimensional z-stack imaging provided us evidence that the uptake mechanism works pretty fast for both free curcumin and CurcuEmulsomes, and within 15 minutes fluorescent signal of the compound could be detected largely not only in the membrane, but also inside the neuron body and the cytoplasm (Fig. 3-B). Merged image of membrane stain DiI (red) and curcumin (green) shows that fluorescent signals of both curcumin and DiI are overlapping throughout the cell body (Fig. 3-B; Additional file 1: Video S1), thereby indicating once again that curcumin with its lipophilic character largely prefer localization within the lipophilic compartments inside the cell, as the DiI membrane stain does. Moreover, no overlap occurs between the signals of DAPI (blue) and curcumin (green), indicating that CurcuEmulsomes do not enter into the nucleus. Although this study is the first to apply emulsomes as the nanocarrier system to the primary hippocampal neurons, inaccessibility of the system to the nucleus is not unexpected, as a previous study on HepG2 carcinoma cell line had monitored CurcuEmulsomes in the membrane and in the cytoplasm but not inside the nucleus [43]. Interestingly, free curcumin was also not observed to localize inside the nucleus (Fig. 3-B,C).

Uptake and localization of free curcumin and CurcuEmulsomes in primary hippocampal neurons were further investigated with confocal miscroscopy for 72 hours upon treatment. Upon the cell uptake, compartmental localization of both free curcumin and CurcuEmulsomes inside the neuron were further investigated along 72 hours after treatment. Delivered as free or within the emuslome, curcumin was found to be accumulate in particular at the neuronal body and along the axons (Fig. 3-C). Neurons treated with free curcumin treatment (5 µM) distinguished from neurons treated with CurcuEmulsomes(5 µM) at DIV1 with a brighter fluorescent signal as indication of higher curcumin uptake into the neuron. At DIV2, indicating a similar or equal curcumin uptake, the fluorescence intensities of both neuron groups became comparable and the neurons appear very bright with the curcumin present inside. At DIV3, fluorescence signals weakened for both curcumin and CurcuEmulsome treated neurons. Yet, highly fluorescent spherical regions were detected inside the neurons treated with CurcuEmulsomes. In previous studies, the cellular uptake mechanism of emulsomes was discovered to follow endocytosis [43, 48]. The inset image in Fig. 3-C indicates a similar observation that upon the uptake CurcuEmulsomes localize inside the endosomes within the cell and sustain curcumin release from the inside of endosome with time. Therefore, the punctual bright spots inside the inset image are attributed to the endosomes carrying the CurcuEmulsomes.

MTS Assay

MTS cell viability tests indicated that both free curcumin and CurcuEmulsomes do not cause any toxicity on primary hippocampal neuron culture within a concentration range of 2–10 µM. No significant difference was observed between any treatment group and the untreated control (P > 0.05) (Fig. 3-D).

Cellular Viability After Neurite Injury

During laser axotomy, the laser was applied on the neurite each time at a distance of 25–30 µm from the neuronal body. By this means, a replicable neurite injury could be established. Microscopy images of neurites before and after the laser axotomy (black arrows) were visualized by microscope (Fig. 4-A; Additional file 2: Video S2). 24 hours after the injury, PI and Hoechst 33342 staining were applied on primary hippocampal neurons to quantify the survival rates of the injured neurons. Viable cells were labeled with Hoechst (blue), while dead cells were labeled by PI (red) (Supplementary Material, Figure S.3). Cell survival rates were quantitatively displayed by graphbar illustrations in Fig. 4-B. In case of no axotomy, around 83% of primary hippocampal neuron culture was found to be alive. Likewise, the sham control group showed approximately 82% viability. In other words, the laser beam has made no effect on the cell survival by means of heat or radiation when applied to the cell environment. Laser axotomy negatively affected on cell viability and cell survival rate became approximately 52% 24 hours after the injury. When treated with 5 µM free curcumin, as high as 67% of the neurons remained alive after the injury. The difference in cell survival rates was statistically found to be significant (P < 0.05). Similary, CurcuEmulsome treatment with equal curcumin concentration (i.e., 5 µM) improved cell survival rate up to 69%. This increase is not only found again significant when compared with the survival rate of the injured control group, but also found to surpassing the value of free curcumin treatment.

Immunocytochemistry Analysis

Immunostained with anti-Wnt3A, -Bcl2, -mTOR and -cleaved caspase 3, neurons were further investigated under confocal fluorescence microscope to quantitatively study the expression levels of these biomarkers (Fig. 5). Accordingly, the fluorescence intensities of 50 neurons were analyzed for each sample and the average expression levels were quantitatively defined for each study group. Control healthy neurons (ctrl), control injury group (injury), curcumin-treated healthy neurons (FC), curcumin-treated injury group (FC + injury) and CurcuEmulsome-treated injury group (CE + injury) were studied for their expression levels of selected biomarkers (Fig. 6).

Rabbit polyclonal anti-Wnt3a antibody, used to assess the expression of Wnt3a protein, is essential for the regulation and maintenance of hippocampal neurogenesis [24]. A significantly high Wnt3a expression was observed on healthy cells when treated with curcumin, compared to both not-treated healthy and injured control groups (Fig. 6-A). However, after axotomy was applied, the increase in Wnt3a expression of curcumin-treated neurons became less remarkably compared to the injury control. On the contrary, CurcuEmulsomes did not cause any significant change in the level of Wnt3a expression of neurons after injury.

Mouse monoclonal anti-mTOR antibody was used to assess the expression of the mammalian target of rapamycin (mTOR) protein, ie. a serine/threonine kinase. It has been previously shown that mTor participates in several processes in the brain such as neural development of axon and dendrites [49]. In hippocampal neurons, mTOR proteins were observed to exist largely within the cytoplasm of the neuronal body and along the axons (Fig. 5). As seen in Fig. 6-B, curcumin-treated injured neurons show a slight increase in the expression of mTOR compared to the injured control group. CurcuEmulsome treatment did not induce any significant change in mTOR expression.

Bcl-2 protein, a key regulator of endogenous apoptosis that also promotes neuronal survival in CNS [50], was found to be localized particularly in the cytoplasm as well as within the nucleus, whereas its localization, and hence its expression levels, along the axons, were found to be relatively lower (Fig. 5). The quantification of fluorescence intensities reveals that both curcumin- and CurcuEmulsome-treatment groups have significantly higher Bcl-2 expressions (Fig. 6-C) compared to control injury group (P-values: 0.011 and 0.009, respectively).

The cleaved caspase 3 apoptotic marker was observed to be present in the nucleus and cytoplasm (Fig. 5). As reported previously, the translocation of cleaved caspase 3 from cytoplasm into the nucleus might occur if apoptosis is induced [51]. Accordingly, Cleaved caspase 3 signal (green) was high in the nucleus of the neurons than within the cytoplasm for all groups except the curcumin-treated injury group (Fig. 5). In the curcumin-treated injury group, cleaved caspase 3 signal secreted by the cytoplasm was seen to be more intensive than the signal acquired in the nucleus. Indeed, when the fluorescence intensities were compared, cleaved caspase 3 expression of curcumin-treated injured neurons were found to be significantly lower (P-value: 0.026) compared to injured control neurons (Fig. 6-D). No significant difference was observed when the neurons were treated with CurcuEmulsomes after injury.

Characterization of CurcuEmulsomes

While curcumin’s low solubility (ie 11 ng/ml)[31] restricts further medicinal applications, nanocarrier systems stand for a prominent solution addressing this bioavailability limitation. Composed of a solid fat core surrounded by phospholipid layer, emulsomes offer an alternative approach to overcome this limitation. Encapsulating curcumin inside its lipid matrix, CurcuEmulsomes could enhance the solubility of curcumin up to 10,000 fold corresponding to 0.11 mg curcumin per milliliter as reported previously [43]. CurcuEmulsome formulations produced for this study showed an average concentration of 0.08 ± 0.02 mg/ml. SEM images presented the smooth morphology of the outermost phospholipid surface, thereby indicating once again the resemblance of emulsomes with its surface features to liposomes (Fig. 1-B). Accordingly, CurcuEmulsomes are roundly-shaped and have sizes in a range of 100–400 nm. Size is predominantly determined by phospholipid:tripalmitin ratio and can be further tuned through alteration of this ratio [48, 52]. Molecular physicochemical properties of lipid composition, as well as the production and homogenization conditions (e.g. synthesis/homogenization duration, temperature, mixing speed) are the other factors influencing on the final size of emulsomes [52]. Besides, physicochemical properties of the drug incorporated into the emulsomes seem to have an effect on both overall size and zeta potential of the formulation [44]. For instance, CurcuEmulsomes distinguished from emulsomes with a higher negative charge (-33.2 ± 6.7 mV), which is attributed to the contribution of molecular negative charge of curcumin to the blank formulation (-24.2 ± 6.3 mV) (Table 1). Owing a more negative zeta potential, CurcuEmulsomes seem to distinguish from blank emulsomes (204.3 ± 20.0 nm) also with a smaller average particle size (176.8 ± 13.0 nm) as reported elsewhere [44]. The higher negative surface charge of CurcuEmulsomes are ascribed as the reason for their smaller average size in comparison to blank emulsomes. The polydispersity index data shows a linearity between negative zeta potential and monodispersity (i.e. PDI values lower than 0.3) of the formulations (Table 1). Monodispersity of the formulation was further confirmed by confocal laser microscopy, as the autoflorescence of curcumin enabled the tracking of each particle (Fig. 1-C).

Average size, zeta potential and PDI values were traced by regular DLS analysis to investigate the long-term stability of the formulation. Mean particle diameter and zeta potential values were remained steady (Table 2), indicating that CurcuEmulsomes preserve structural stability for at least 11 months. Solidity of the lipid core matrix that is additionally supported and stabilized with a negatively-charged phospholipid shell is ascribed as the reason behind the long-term stability of the formulation. Nonetheless, although CurcuEmulsomes were discovered to be structurally stable for that long period of time, considering that any release of even a small amount of curcumin from CurcuEmulsomes might influence on the reproducibility of the experimental data, CurcuEmulsomes no older than one month were used each time in cell culture experiments throughout the study.

The drug release studies of CurcuEmulsomes designates a prolonged release profile showing less than 40% curcumin release within the first 30 hours. This time point is particularly important in our study, as the viability and immunocytochemistry analysis were performed after 30 hours of CurcuEmulsome treatment, where axotomy was applied 6 hours after treatments and neurons were analyzed 24 hours after axotomy. Surprisingly the maximum curcumin release was achieved at around 35% after 12 hours and the value remained stable until 72 hours (Fig. 1-D). The incidence of low release profile might be attributed as result of two possible occurrences: Firstly, the solid phase of tripalmitin core of CurcuEmulsomes may result in a prolonged release of encapsulated curcumin at 37 °C – which is expected to occur [43]. Secondly, the rapid denaturation of curcumin in the water [53] may lead to underestimation of curcumin amount actually released. To express more explicitly, the curcumin released from the CurcuEmulsomes may have been undergoing denaturation repeatedly so that the curcumin quantity in the buffer solution detected each time might correspond to less than the actual release.

On the other hand, curcumin release within the first three hours was found to be around 20% of curcumin content. Although this relatively high early release percentage might be seen contrary to the solid feature of the emulsome’s solid core matrix, the reason behind shall be hidden again in the characteristic structure of CurcuEmulsomes, where curcumin is located both within the hydrophobic regions of the phospholipid multilayer as well as inside the solid core matrix [52]. Accordingly, curcumin loaded within the phospholipid layers of CurcuEmulsomes must have been released first, as its relatively more mobile nature within the fluidic lipid matrix would allow its diffusion to the buffer more easily, rather than the curcumin entrapped and immobilized within the solid fat core of CurcuEmulsomes (Fig. 1-D).

Cell Studies: Cell Isolation, Uptake and Toxicity

The isolation of hippocampus was set at postnatal day 0 throughout the study. To contribute to the reproducibility of the data, a neurite growth around 100 µm was set as a requirement to proceed to laser axotomy. Upon the isolation of neurons at post-natal 0, neurite growth was observed 3 days long and 2 days of incubation was found to be optimum for neurite growth (Fig. 3-A).

As this is study is the first report on the emulsomes tested on primary hippocampal neurons, the uptake of CurcuEmulsomes into the primary hippocampal neurons was examined separately first in 15 minutes period of time upon treatment, to investigate the necessary time for the delivery of curcumin into the neurons in vitro. CurcuEmulsomes were visible on the membrane and throughout the cytoplasm (Fig. 3-B). Cell uptake analysis pointed out that CurcuEmulsomes can enter the primary hippocampal neurons within 15 minutes and localize rapidly within the membrane and inside the cytoplasm. As this is the first study from our group reporting the use of emulsomes for delivery of neuroprotective agents into a primary neuron culture, it might be important to mention that the uptake of CurcuEmulsomes into the hippocampal neurons was observed to occur faster compared to the previously studied uptake rates of different emulsome formulations into the cancer cells such as HepG2 [43], HCT116 [44], LnCap (unpublished data) or MCF7 (unpublished data) in vitro.

Neurons were imaged each day during 72-hours treatment with curcumin and CurcuEmulsomes. The mean fluorescence intensity of free curcumin was found as highest on DIV1, while CurcuEmulsomes showed the brightest signal on DIV2 (Fig. 3-C) (Quantitative data not shown). Spherical local spots with higher fluorescence intensities were observed inside the cytoplasm of the neurons treated with CurcuEmulsomes. These spots were interpreted as endosomes, as previous studies suggest that the uptake of emulsomes proceeds through endocytosis [43, 48, 54]. These spots were not present at neurons treated with free curcumin where the uptake followed diffusion [55]. Cell uptake studies verified that CurcuEmulsomes achieve delivery of curcumin into the hippocampal neurons, where curcumin mainly localized inside the membrane and in the cytoplasm. On the other hand, it is important to state that neither treated with free curcumin nor CurcuEmulsomes no curcumin signal was detected inside the nucleus throughout the 72-hour examination.

Our study also revealed that curcumin is not toxic on primary hippocampal neurons for curcumin concentrations of 2, 5 and 10 µM (Fig. 3-D). Indicating the safety of the emulsome as the nanocarrier, CurcuEmulsomes did not alter the viability of the neurons significantly when applied with the doses corresponding to the prementioned curcumin concentrations.

The effective dose of curcumin was reported in various reports differently in a range of 250 nm to 27 µM, mainly depending on the type of neuron culture studied, day of cell isolation and the cell origin. For instance, So and the colleagues have previously reported that 500 nM curcumin was the most effective in stimulating proliferation of neural progenitor cells (NPC), whereas high concentrations were cytotoxic [56]. Ray et al. reported that the polymeric nanoparticle-encapsulated curcumin (NanoCurc™) treatment protects neuronally differentiated human SK-N-SH cells from ROS (H₂O₂) mediated insults in doses between 250 nM and 5 µM significantly [57]. In another study, high curcumin concentrations (ie 2.7–27 µM) were found to be effective against H₂O₂-induced renal epithelial (LLC-PK1) cell injury with comparable inhibition of lipid peroxidation and lipid degradation [58]. A study on primary cultures of cerebellar granule neurons (CGNs) demonstrated that curcumin (10–15 µM) provides also a neuroprotective effect against iodoacetate (IAA)-induced neuronal death [59]. Srivastava et al. have proven the protective function of curcumin in arsenic-induced N-methyl-D-aspartate (NMDA) receptor dysfunctions and PI3K/Akt/GSK3b signaling on in vitro primary hippocampal neuron model isolated from 6–8 day old Wistar rat [60]. In light of these findings, curcumin concentration was set as 5 µM for the proceeding experiments including laser axotomy. Safety concentration data of CurcuEmulsomes (ie. ≤10 µM) as well as the observed particle density inside the neuron culture had an influence on determination of the experimental concentration as 5 µM for the axotomy and further studies.

Injury and Immunostainings

The previous studies on dorsal root ganglions (DRGs) reported an increase in neuron death with increasing proximity of the injury to the cell body, which is attributed to the increased influx of extracellular Ca⁺⁺, generating a stronger injury current [61]. This explanation on DRGs’ cell death upon axotomy seems to be prevalent also for the primary hippocampal model, where 52% of the injured neurons remained alive after induced injury at close proximity (25–30 µm) to the cell body (ie data for the untreated injured group). Reducing the hazards of the injury, Curcumin and CurcuEmulsome treatments significantly increased the survival rate of neurons up to 67% and 69%, respectively, compared to the untreated injured group (P-values: 0.03 and 0.01, respectively) (Fig. 4-B). These findings provide evidence for the neuroprotective role of both curcumin and CurcuEmulsomes on hippocampal cells.

Wnts, lipid-modified secreted glycoproteins, signal through different pathways including the planar cell polarity, the Wnt/Ca^+ 2 and the canonical Wnt signaling pathway. The canonical Wnt signaling pathway regulates hippocampal neurogenesis, axonal remodeling and patterning. Among the members of Wnt family, Wnt3a is essential for the regulation and the maintenance of hippocampal neurogenesis [62]. To date, various studies have shown that use of neuroprotective agents resulted in up-regulation of the canonical Wnt3a signaling, and thus, further enhances the neurogenesis [24, 63–65]. Following an axonal injury, Wnt signaling could promote the neuronal survival and regeneration of CNS axons on retinal ganglion cells [66]. The neuroprotective effects of Wnt signaling includes prevention of excitotoxicity and promotion of axonal growth within the adult spinal cord and sensory neurons following the injury [66–71]. In our study, curcumin treatment significantly upregulated the Wnt3a on control cells as expected (p-value: 0.00 when compared to untreated control cells). However, the observed increase in Wnt3a expression levels of injured cells upon treatment with curcumin or CurcuEmulsomes was statistically found as insignificant (Fig. 6-A). Earlier, curcumin has been reported to activate Wnt/β-catenin pathway and increase Wnt3 expression and protein levels, leading to enhanced neurogenesis [19, 24]. It is important to emphasize that significant increase in neurogenesis was observed at 0.5 µM curcumin concentration, while higher doses (≥ 5 µM) significantly reduced the proliferation/viability of hippocampal derived neural stem cells [24], indicating that curcumin may display neuroregenerative effect in a concentration-dependent manner. These findings might be attributed to the efficacy of curcumin in regulating multiple targets in different pathways. In our study, it is yet uncertain if the insignificant increases in Wnt3a expressions upon the curcumin and CurcuEmulsome treatment play a role in the improvement of survival rates of neurons after injury, however the lower increase in Wnt3a after the CurcuEmulsome treatment – relative to the curcumin treatment – might be attributed to the prolonged release profile of the nanocarrier system.

The mammalian target of rapamycin (mTOR), a master kinase, has a pivotal role in both dendritic, axonal growth, synaptic plasticity and metabolic regulation [49]. It has been reported that mTOR signaling pathway has a crucial function in neuronal survival [72]. As being a pleiotropic molecule, curcumin affects numerous targets including mTORC1 and mTORC2 signaling pathways in cancer cells making curcumin as a chemotherapeutic/chemopreventive agent against many types of cancer [73]. According to our findings, curcumin treatment increased the level of mTOR expression compared to untreated control cells (Fig. 6-B). Curcumin-treated injured neurons exhibited the highest expression level of mTOR among all groups, suggesting that curcumin has a significant role in the survival of injured neurons. In contrast, no significant alteration was observed in mTOR expression level of CurcuEmulsome-treated injured neurons. In other words, despite CurcuEmulsomes achieve the delivery of curcumin into the cytoplasm successfully (Fig. 3-B,C), they do not cause the same effect on mTOR expression as curcumin itself. This incidence might be attributed to the fact that CurcuEmulsomes are taken up by the neurons via endocytosis, then from the endosome curcumin was released slowly but sustained as the release profile points out (Fig. 1-D). Ultimately, CurcuEmulsomes might not be enabling curcumin to achieve its effective concentrations within the cytoplasm in terms of mTOR expression.

Bcl2 is known as an anti-apoptotic gene. The members of the Bcl2 family are widely accepted as the regulators of apoptosis. Furthermore, they also play a key role in normal cell physiology [74]. It has been shown that Bcl2 could block cell death in different cell types including glia and neurons [75, 76]. Inhibition of the release of cytochrome c from mitochondria is an action of mechanism of Bcl2, which is particularly critical for the activation of caspases, and hence, the apoptosis [76]. It has been previously disclosed that curcumin treatment has increased the mRNA expression level of Bcl2 [77]. Likewise, in our study, curcumin treatment on untreated control neurons upregulated the Bcl2 expression with a modest difference between the free curcumin- and CurcuEmulsome-treated axotomy injured neurons (p-values:0,453 and 0,592, respectively) (Fig. 6-C). Curcumin and CurcuEmulsome treatment groups on injured neurons significantly increased the level of Bcl2 (p-values: 0.011 and 0.009, respectively) when compared to the injured control group. The findings of Bcl2 immunostaining study stressed the anti-apoptotic activity of both curcumin and CurcuEmulsomes on the axotomy-injured hippocampal model.

Cleaved caspase 3 has been regarded as a marker in apoptosis [78]. Neurodegeneration and neuron death upon mechanical hazard are known to occur through induced apoptosis [79]. In parallel, immunocytochemistry analysis revealed an increase in expression of cleaved caspase 3 upon neuron injury. Counteracting the induction of apoptosis, curcumin decreased cleaved caspase 3 expression compared to control cells significantly (Fig. 6-D). CurcuEmulsome treatment also decreased the cleaved caspase expression level compared to the axotomy-injured group; however, the decrease remained limited compared to free curcumin treatment, which – like in mTOR analysis – is again attributed to the sustained release profile of CurcuEmulsomes resulting a lower curcumin concentration available and active inside the cytoplasm. Consistent with the data of an ischemia-induced delayed neuronal death study showing that inhibition of mitochondrial-mediated apoptotic signaling cascade occurs through dietary supplementation of curcumin [80], our findings suggested that curcumin treatment inhibited the apoptotic signaling on axotomized cells. However, CurcuEmulsomes’ anti-apoptotic effect remained low compared to its free form applied at the same concentration, which is attributed to the slow release profile of the emulsomes.

Sustained release is widely accepted as advantageous in in vivo and clinical studies due to offering less frequent dose administration and minimizing the side effects of the system as the drug is first being released upon its uptake to the inflammatory organ or the tissue. In our in vitro example; however, where curcumin is controlled-release to the system by CurcuEmulsomes, neuroprotective effect remained lower than the free curcumin’s effect in the same concentration, since the concentration of the curcumin released to the cytoplasm from the CurcuEmulsomes remained less than the total amount of curcumin encapsulated inside the cytoplasm for the period of time of the study. Higher concentrations of CurcuEmulsomes might be considered for future studies in vivo to further improve formulation’s efficacy in neuroprotection against neural injury.

As verified for the first time on neurons, CurcuEmulsomes offer a prominent and safe approach for the delivery of curcumin to axotomy-injured primary hippocampal cell model. Displaying stable, dispersed characteristics, CurcuEmulsomes achieved the delivery of curcumin to the hippocampal neurons. The formulation exhibited no toxicity against primary hippocampal neuron culture in the tested range of 2–10 µM curcumin content. The neuroprotective activity of CurcuEmulsomes was studied on a reproducible laser-axotomy model that is successfully established on primary hippocampal neurons isolated from newborn Balb-c mice. Delivered within CurcuEmulsomes, curcumin promoted cell survival after the laser injury. Immunocytochemistry analyses revealed significantly higher expression levels for Bcl-2 when injured neurons are treated with CurcuEmulsome- or curcumin-injured neurons compared to the untreated injured control group (P values ≤ 0.05). Caspase 3 expression levels decreased when treated with CurcuEmulsome or curcumin. However, the neuroprotective effect of CurcuEmulsomes remained limited, which is attributed to the sustained release profile of the emulsomes yielding lower concentrations of curcumin actively present within the cytoplasm. Minor changes in expression levels of Wnt3a and mTOR were observed for both CurcuEmulsome- and curcumin-treated injured neurons compared to the values for the untreated injured control group.

Concisely, with their safety profile and prementioned biological effects on the studied in vitro axotomy model of primary hippocampal neurons, CurcuEmulsomes highlights the potential to achieve neuroprotection against neuronal injuries in the CNS. Our future research will include in vivo studies where not only the neuroprotective effect of the formulation, but also its permeability through the blood-brain barrier will be the focus and uppermost challenge in front of the clinical applications.

Alzheimer’s Disease

AKT

Protein Kinase B (PKB)

ALS

Amyotrophic Lateral Sclerosis

BBB

Blood-Brain Barrier

Bcl-2

B-Cell Lymphoma 2

BSA

Bovine Serum Albumin

CGN

Cerebellar Granule Neuron

CLSM

Confocal Laser Scanning Microscope

CNS

Central Nervous System

DAPI

4′,6-Diamidino-2-Phenylindole

DiI

(1,1'-Dioctadecyl-3,3,3 ', 3'-Tetramethylindocarbocyanine Perchlorate

DIV

Day In Vitro

DLS

Dynamic Light Scattering

DMSO

Dimethyl Sulfoxide

DNAse I

Deoxyribonuclease I

DPPC

1,2-Dipalmitoyl-rac-Glycero-3-Phosphocholine

DRG

Dorsal Root Ganglion

FBS

Fetal Bovine Serum

GSK3b

Glycogen Synthase Kinase 3 Beta

HCT116

A human colon cancer cell line

HepG2

A human liver cancer cell line

IAA

Iodoacetamide

ICC

Immunocytochemistry

IgG

Immunoglobulin G

LLC-PK1

Epithelial-like Pig Kidney Cell Line

LNCaP

Cell line derived from a metastatic lymph node lesion of human prostate cancer

MCF7

A human breast adenocarcinoma cell line

mTOR

Mammalian Target of Rapamycin

mTORC

Mammalian Target of Rapamycin Complex

MTS

(3-(4,5-Dimethylthiazol-2-yl)-5-(3-Carboxymethoxyphenyl)-2-(4-Sulfophenyl)-2H-Tetrazolium)

NADH

Reduced Nicotinamide Adenine Dinucleotide

NADPH

Reduced Nicotinamide Adenine Dinucleotide Phosphate

NanoCurc™

Nanoparticle-Encapsulated Curcumin

NBA

Neurobasal-A

Neurodegenerative Disease

NMDA

N-Methyl-D-Aspartate

Post-natal Day Zero

PBS

Phosphate Buffer Saline

Parkinson’s Disease

PDI

Polydispersity Index

PES

Phenazine Ethosulfate

Propidium Iodide

PI3K

Phosphoinositide 3-Kinase

ROS

Reactive Oxygen Species

SEM

Scanning Electron Microscopy

SEM

Standard Error of the Mean

TBS

Tris-buffered Saline

WHO

World Health Organization

Wnt3a

Wingless-Type MMTV Integration Site Family, Member 3A

Availability of data and materials

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Ethics approval and consent to participate

Animal experiments were conducted in accordance with the European Community guidelines and approved by Istanbul Medipol University Ethics Committee for Animal Experimentation (Date:07/03/2016; Approval number: 38828770-604.01.01-E.3722).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

Financial support from the Scientific and Technological Research Council of Turkey (TÜBİTAK) under project number 116Z347 is gratefully acknowledged.

Authors’ Contributions

EY synthesized CurcuEmulsomes and performed the related characterization experiments. EY and SB isolated hippocampal neurons from newborn Balb-c mice. EY and SB performed primary cell culture studies including cellular uptake, cell viability, laser axotomy and confocal microscopy analysis. EY executed immunocytochemistry analysis, produced the quantitative data from the fluorescence images and interpreted the data. MU and GO designed the framework of the study, guided the conduct of studies, and supervised data analysis. EY and MU were actively involved in writing and revising the manuscript. All authors have read and approved the final manuscript.

Acknowledgements

Financial support from the Scientific and Technological Research Council of Turkey (TÜBİTAK) under project number 116Z347 is gratefully acknowledged. The authors would like to thank to Dr. Bilal Kerman from Istanbul Medipol University and Dr. Aysegul Yildiz from Mugla Sitki Kocman University for their important remarks and feedbacks on experimental framework.

Author details

1 Regenerative and Restorative Medicine Research Center (REMER), Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Ekinciler Cad. 19, 34810 Beykoz, Istanbul, Turkey;

2 Graduate School of Engineering and Natural Sciences, Istanbul Medipol University, Ekinciler Cad. 19, 34810 Beykoz, Istanbul, Turkey;

3 Department of Physiology, International School of Medicine, Istanbul Medipol University, Ekinciler Cad. 19, 34810 Beykoz, İstanbul, Turkey;

4 Department of Biomedical Engineering, School of Engineering and Natural Sciences, Istanbul Medipol University, Ekinciler Cad. 19, 34810 Beykoz, Istanbul, Turkey.

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Neuroprotective Effects of Curcumin-Loaded Emulsomes on Laser Axotomy-Induced CNS Injury Model

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Animals

Chemicals and Reagents

Results

Characterization Studies

Dispersity

Physicochemical Properties of CurcuEmulsomes

Stability

Morphology

Curcumin Encapsulation

Cell Culture Studies

Isolation of Primary Hippocampal Neurons

Cell Uptake Studies of Curcumin and CurcuEmulsomes

MTS Assay

Cellular Viability After Neurite Injury

Immunocytochemistry Analysis

Discussion

Characterization of CurcuEmulsomes

Cell Studies: Cell Isolation, Uptake and Toxicity

Injury and Immunostainings

Conclusion

Abbreviations

Declarations

Availability of data and materials

Funding

Authors’ Contributions

Acknowledgements

References

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