Subscriber access provided by Nottingham Trent University
Article
Ferric-Tannic Nanoparticles Increase Neuronal Cellular Clearance Isara Phiwchai, Watchareeporn Chariyarangsitham, Thipjutha Phatruengdet, and Chalermchai Pilapong ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00345 • Publication Date (Web): 29 Jul 2019 Downloaded from pubs.acs.org on July 29, 2019
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
Ferric-Tannic Nanoparticles Increase Neuronal Cellular Clearance Isara Phiwchai, Watchareeporn Chariyarangsitham, Thipjutha Phatruengdet and Chalermchai Pilapong* Abstract: Targeting cellular clearance function in brain cells provides new opportunities for the prevention of dementia by clearance of potentially dangerous molecules. Herein, we present a new approach to enhancing neuroactive and neuroprotective activities in neuronal cell line using ferric-tannic nanoparticles (FTs). Major biological function mediated by FTs was clearly found to promote neuronal tube growth through activation of axon guidance pathways. A number of neuronal tubes were found to increase under stimulation of amyloid beta peptides, oxidative stress, and serum deprivation. The generated neuronal tubes play a role in clearing debris and amyloid beta peptides. Another key function in cellular clearance mediated by FTs was their capabilities of inducing autophagy with activation of lysosomes. Therefore, FTs are a promising new strategy for brain cell protection through activation of cellular clearance function. Hopefully, our findings will pave the way for the development of new methods for the prevention and therapy of dementia.
KEYWORDS:
Dementia, Nanoparticles, Autophagy, Amyloid beta-peptides
Lysosome,
INTRODUCTION According to The United Nations (UN) reports, the elderly population has dramatically increased in recent years. Between 2015 and 2030, the number of elderly people is projected to grow by 56%, from 901 million to 1.4 billion 1, 2. Therefore, there is no doubt that this will create an ever-increasingly aging population. As a consequence of this tendency, this aging population will become one of the most significant social and financial transformations of the twenty-first century. Age-related diseases, especially for non-communicable diseases, will become a major problem for health systems that must strive for maintaining the well-being of elderly people 3. Currently, several diseases found in elderly people cannot easily be cured because the treatments are usually performed in the late stage of the diseases. Dementia is one of the most common diseases found in elderly people. It causes cognition function impairments in old people. In 2015, approximately 50 million people worldwide are living with dementia, and this number will double every 20 years 4. Although dementia is caused by various parameters including genetic and environmental factors, age is the strongest risk factor for dementia 3, 5. Various aging mechanisms such as inflammation and senescence, oxidative stress, proteasome, and lysosome activities have found to be related to the initiation and progression of dementia 6. Recently, several works demonstrated that cognition function (i.e. not simply memory) can occur in a healthy person as early as in the late 20s and can continue over the lifespan 7, 8. This indicates that aging processes in the brain can start from an early age. Thus, younger people can be affected by dementia, as well.
Thanks to our quality-control system of brain cells, not all people are easily affected by dementia 9-11. However, once our quality-control system is in a state of imbalance, the initiation and progression of dementia can occur. For example, loss of protein homeostasis and lysosome dysfunction leads to accumulation of neurotoxic protein, causing cognitive impairment 12. Therefore, targeting cellular clearance function is promising for the prevention and treatment of dementia. By using this approach, the autophagy-lysosomal pathways play a vital role in degradation or detoxification of potentially dangerous molecules 13-15. Targeting such pathways is increasingly being tested for their beneficial effects on the prevention and treatment of dementia 16-19. In addition, intercellular structures, especially long-distance neuronal tubes (e.g. tunneling nanotube) play a crucial role in cellular clearance mechanisms by spreading or secreting various molecules including tau and prion-like proteins 20-22. Thus, modulating cellular degradation activity along with the promotion of neuronal tubes could be an effective strategy for prevention of initiation and progression of dementia. For the past decade, nanomedicine has delivered promising methods for prevention and treatment for dementia 23-25. Different strategies involving the use of nanoparticles have been developed. Various polymeric nanoparticles were utilized as delivery systems of neuroprotective agents or drugs into the brain 26. Ultrasound-sensitive nanoparticles enabled localized drug delivery to the brain in order to modulate brain activity 27. Magnetic nanoparticles could also be used as transducers for remote and wireless activation of neural cells by converting magnetic fields into different stimuli 28. Optical nanomaterials such as upconversion nanoparticles can serve as optogenetic actuators for modulating brain functions by taking advantage of emitting visible light after absorption of near-infrared (NIR) light 29. Previously, we have developed the ferric-tannic molecular nanoparticles (FTs) using a green chemistry approach 30. The FTs were successfully synthesized at room temperature within a few minutes without using any toxic agents or expensive equipment. Additionally, FTs exhibited good physico-chemical properties including very small size, high stability against transchelation and transmetallation, and excellent water solubility and colloidal stability 31. The integration of imaging capability of paramagnetic ferric ions (MRI) and the protective potential of tannic acid (antioxidant, inhibition of Aβ aggregation and etc 32.) allows it to be used for both MR imaging and prevention (or treatment). Additionally, the FTs are capable of activating lysosome function and inducing autophagy 31. Therefore, they are potentially considered beneficial for modulating cellular clearance functions in order to enhance neuroprotective and neuroactive properties. Therefore, in this study, we aimed to investigate the biological effects of FT in the neuroblastoma cell line (SH-SY5Y) in order to explore key neuroprotective and neuroactive properties.
ACS Paragon Plus Environment
ACS Chemical Neuroscience
RESULTS AND DISCUSSION The FTs were prepared according to our previous report 31. They were easily obtained by self-assembly reaction between ferric chloride and tannic acid in a PBS buffer (pH 7.4) at room temperature for a few minutes in ambient air. Schematic illustrations of FTs preparation and typical physico-chemical properties are summarized in Fig. S1. It has a spherical shape with diameters in the range of ~10 nm. The large negative value of zeta potential (-21.6 mV) indicates good colloidal stability of the FTs in the aqueous medium 33. Next, we investigated various biological effects of different concentrations of FTs against SHSY5Y cells in order to assess the optimum dose for further experiments regarding their neuroprotective potentials. Cellular toxicity was initially determined by counting viable cells. As the results show in Fig.1A, the number of viable cells increased as the concentration of FT increased. Similar results are found when measuring metabolic activity of mitochondria enzyme or MTT assay (Fig.1B). The consistent results indicate that FTs are non-toxic, having a capability of inducing cell proliferation without alteration of metabolic activity of mitochondria. Analysis of cell cycle distribution is one of the crucial factors for cytotoxic evaluation. Cell cycle analysis of FTs treated cells was carried out and number of cells in different phases was determined. As shown in Fig. 1C, the percentages of the cells in each phase are not significantly different between untreated and treated cells, indicating that the cell cycle is not disrupted by FTs. As is known, cell morphology is a crucial biophysical property representing particular changes in the molecular levels of cells 34. Thus, we tried to investigate the change in cell shape and find out the intercellular structure induced by the FTs. Fig. S2A shows microscopic images of the cells after being treated with different concentrations of FTs for different lengths of time.
A.
MTT viability (%)
Cell count (%)
200 150 100 24h 48h 72h
50 0
1
10
20
30
[FT] M
Treatment with 20-50 µM of FTs was found to induce morphological changes, but there were no significant changes observed in the cells treated with lower concentrations of FTs (5, 10 µM). Obviously, treatment with lower concentrations of FTs was found to induce formation of neuronal cell tubes (Fig.S2B). To confirm whether FTs can enter the cell in order to exert its neuroactive and neuroprotective activities, cellular accumulation of FTs was also investigated. As seen in Fig. S3C, the FTs had been taken up by the cells in a time and concentrationdependent manner. From the results mentioned above, we selected 10 µM of FT for further investigations. In order to gain insight into neuroactive and neuroprotective activities of FTs, RNA sequencing (RNA-Seq) was performed on Illumina HiSeq PE150 platform. In RNA-seq experiment, FPKM (fragments per kilo base per million mapped reads) was used to estimate the level of gene expression. The list of FPKM values (adjustable read count) of untreated and FTs-treated cells is summarized in Table S1. Based on differential expression data, FTs were found to alter gene expressions consisting of 98 upregulated genes and 12 down-regulated genes (Fig.1D). Through enrichment analysis (e.g. gene ontology (GO) enrichment and DO Enrichment, KEGG) of the differential expressed genes, we could visualize the functional profiles of genomic coordinates and gene and gene clusters. According to gene ontology (GO) enrichment analysis, we can find out which cellular components, biological functions, or pathways were significantly associated with differentially expressed genes. A GO enrichment list of two comparison groups (FTs treated and control cells) is shown in Table S2. Fig.1E shows the top 20 enriched in the GO enrichment analysis. Unfortunately, no significant enriched GO term is observed. However, KEGG analysis shows that 3 significant KEGG pathways are markedly observed including axon guidance, a T-cell receptor signaling
D.
B. 250
250
0
150 100 24h 48h 72h
50 0
50
0
1
10
20
[FT] M
30
50
F. 24h
60 40 20 0
5
10
20
[FT] M
30
50
80 60 40 20 0
72h
100
No. of cells (%)
80
0
48h
100
No. of cells (%)
100
0
5
10
E.
200
C. No. of cells (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 11
20
[FT] M
30
50
G. Netrin-3
80
PAK
60
NFAT
40 20 0
Axon outgrowth
0
5
10
20
[FT] M
30
50
AP1
Cell proliferation
Figure 1. (A and B) Cell counting and MTT assays of cells after incubation with different concentrations of FT for different lengths of time (%). (C) Number of cells in different cell cycle phases (■ G1; ■ S; ■ G2/M) after incubation with different concentrations of FT for different lengths of time. (D) Volcano diagram of whole distribution of differential expression genes (the threshold of differential expression genes is: |log2(FoldChange)| > 1 and qvalue < 0.005.). (E) Top 20 enriched in the GO enrichment analysis (GO terms with Corrected_pValue < 0.05 are significant enrichment). (F) top 20 enriched in the KEGG enrichment analysis (KEGG terms with Corrected_pValue < 0.05 are significant enrichment). (G) Significant changes in genes expression of KEGG pathways; by upregulating genes such as Netrin-3, PAK and NFAT and downregulating gene like AP1, FT promotes Axon outgrowth and cell proliferation, respectively.
ACS Paragon Plus Environment
deprivation. From the above phenotypic analysis, it can be said that biological functions of FTs against potentially dangerous molecules (e.g. Aβ1-42 and H2O2) and serum deprivation are associated with the promotion of neuronal tubes. To show the neuroprotective potential of FTs against cell death, we conducted cell viability assays to assess the cytotoxicity of stimulators and ability of FTs to protect cell death induced by the stimulators. By measuring viable cells, the number of cells was clearly found to reduce when incubated and directed into oxidative stress and serum deprivation in a time-dependent manner, as compared with a control. In cases of Aβ1-42 treatment, no effect on cell viability was found when incubated with mAβ1-42 and aAβ1-42 for 24-48 hours, while those of 72 hours treatment were found to decrease cell viability, especially for aAβ1-42, indicating more toxicity in the aggregated form 43. In the case of the co-treatments, the addition of FTs (FT+) did not clearly show protective activity against cell death (Fig. 2B (top panel)). Similar trends were found in MTT assay, whereby hydrogen peroxide and serum deprivation were found to reduce cell viability, whereas Aβ1-42 did not. Likewise, FTs also did not clearly show the protective activity (Fig. 2B (lower panel)). From these results, it can be implied that FTs have no significant role in protecting against cell death induced by such stimulators. Next, we investigated other biological consequences of the FTs in terms of neuroactive and neuroprotective activities. It is well known that oxidative stress can alter biological functions of cells and mediate various pathological processes of dementia 44. Thus, we determined the change in intracellular ROS and lipid peroxidation which are important factors for monitoring cellular homeostasis of oxidative stress. Under stimulations, no significant changes in ROS level were observed between untreated cells (control) and serum-deprived cells, mAβ1-42, aAβ142, and H2O2 treated cells (Fig. 2C). Even though reduced ROS levels were found in serum-deprived cells and the cells treated with mAβ1-42 and H2O2 for 72 hours; when compared to the B. Cell count (%)
24h
150
200 -FT +FT
100 50 0
150 100 50 Serum mA aA deprivation
C.
Figure 2. (A) Cell morphology under stimulation of mis-folding peptide (Aβ1-42), Hydrogen peroxide (H2O2) and Serum deprivation with and without addition of FT for 24h. Red arrow indicates the observed neuronal fibers. (scale bar = 50 μm. (B) Cell counting (top panel) and MTT (below panel) assays of cells after incubation with different stimulators with and without addition of FT for 24 hours, 48 hours and 72 hours. (C) Change in intracellular ROS level. (D) Flow cytometric histograms of oxidized lipid in the cells under different treatments regimens at indicated time points (24 hours and 72 hours). * P
