Mediating the potent ROS toxicity of acrolein in neurons

Purdue UniversityPurdue e-Pubs

Birck and NCN Publications Birck Nanotechnology Center

2014

Mediating the potent ROS toxicity of acrolein inneurons with silica nanoparticles and a naturalproduct approachDesiree White-SchenkPurdue University, Birck Nanotechnology Center, [email protected]

Riyi ShiPurdue University, Birck Nanotechnology Center, [email protected]

James F. LearyPurdue University, Birck Nanotechnology Center, [email protected]

Follow this and additional works at: http://docs.lib.purdue.edu/nanopub

Part of the Nanoscience and Nanotechnology Commons

This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] foradditional information.

White-Schenk, Desiree; Shi, Riyi; and Leary, James F., "Mediating the potent ROS toxicity of acrolein in neurons with silicananoparticles and a natural product approach" (2014). Birck and NCN Publications. Paper 1595.http://dx.doi.org/10.1117/12.2040190

http://docs.lib.purdue.edu?utm_source=docs.lib.purdue.edu%2Fnanopub%2F1595&utm_medium=PDF&utm_campaign=PDFCoverPages

http://docs.lib.purdue.edu/nanopub?utm_source=docs.lib.purdue.edu%2Fnanopub%2F1595&utm_medium=PDF&utm_campaign=PDFCoverPages

http://docs.lib.purdue.edu/nano?utm_source=docs.lib.purdue.edu%2Fnanopub%2F1595&utm_medium=PDF&utm_campaign=PDFCoverPages

http://docs.lib.purdue.edu/nanopub?utm_source=docs.lib.purdue.edu%2Fnanopub%2F1595&utm_medium=PDF&utm_campaign=PDFCoverPages

http://network.bepress.com/hgg/discipline/313?utm_source=docs.lib.purdue.edu%2Fnanopub%2F1595&utm_medium=PDF&utm_campaign=PDFCoverPages

Mediating the potent ROS toxicity of acrolein in neurons with silica nanoparticles and a natural product approach

Désirée White-Schenka,c, Riyi Shib,c, James F. Learya,b,c

Interdisciplinary Biomedical Sciences Program; aBirck Nanotechnology Center; bDepartment of Basic Medical Sciences, School of Veterinary Medicine; cWeldon School of Biomedical

Engineering Purdue University, West Lafayette, Indiana

ABSTRACT

Acrolein, a very reactive aldehyde, is a culprit in the biochemical cascade after primary, mechanical spinal cord injury (SCI), which leads to the destruction of tissue initially unharmed, referred to as “secondary injury”. Additionally, in models of multiple sclerosis (MS) and some clinical research, acrolein levels are significantly increased. Due to its ability to make more copies of itself in the presence of tissue via lipid peroxidation, researchers believe that acrolein plays a role in the increased destruction of the central nervous system in both SCI and MS. Hydralazine, an FDA-approved hypotensive drug, has been shown to scavenge acrolein, but its side effects and short half life at the appropriate dose for acrolein scavenging must be improved for beneficial clinical translation. Therefore, a nanomedical approach has been designed using silica nanoparticles as a porous delivery vehicle hydralazine. The silica particles are formed in a one-step method that incorporates poly(ethylene) glycol (PEG), a stealth molecule, directly onto the nanoparticles. As an additional avenue for study, a natural product in green tea, epigallocatechin gallate (EGCG), has been explored for its ability to react with acrolein, disabling its reactive capabilities. Upon demonstration of attenuating acrolein, EGCG's delivery may also be improved using the nanomedical approach. The current work exposes the potential of using silica nanoparticles as a delivery vehicle and EGCG's antioxidant capabilities in B35 neuroblastoma cells exposed to acrolein. We also measure nanotoxicity to individual rat neurons using high-throughput image scanning cytometry. Keywords: Mesoporous silica nanoparticles, acrolein, EGCG, nanomedicine, nanotoxicity

1. INTRODUCTION 1.1 Acrolein's role in neurodegeneration

Acrolein, or 2-propenal, is a very reactive aldehyde formed endogenously in the breakdown of lipids. In the body, the formation is precisely controlled. Unchecked, acrolein will react with nucleophilic amino acid residues (lysine, histidine) and the lipids of the cell.1,2 Protein modification likely affects folding and function. In the central nervous system (CNS), not only the cell membrane's rich with lipids but the myelin sheaths are lipid-rich. When acrolein reacts with lipids, because it is a by-product of lipid breakdown, more acrolein forms in a cascade of acrolein formation and lipid peroxidation.

In spinal cord injury, primary injury involves the initial injury. The initial injury contains a concentrated area of necrotic cell death. In turn, the body responds with the inflammatory response to control the cellular debris and death. Therefore, the only treatment currently used after spinal cord injury is an immunosuppressant steroid, methylprednisone.3 The drug is meant to stem the immune response, as the immune response and cell death leads to the formulation of reactive oxygen species (ROS) and oxidative stress. Such stress leads to an increase in the injury area, damaging surrounding cells that were not damaged in the primary injury. Figure 1 briefly highlights how primary injury induces the biological cascade of secondary injury.

Invited Paper

Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications VI, edited by Samuel Achilefu, Ramesh Raghavachari, Proc. of SPIE Vol. 8956, 89560C · © 2014

SPIE · CCC code: 1605-7422/14/$18 · doi: 10.1117/12.2040190

Proc. of SPIE Vol. 8956 89560C-1

Downloaded From: http://proceedings.spiedigitallibrary.org/ on 06/27/2014 Terms of Use: http://spiedl.org/terms

Figure 1. Schematic of the biological responses of primary injury followed by secondary injury.

Specifically, acrolein has been elucidated as a major source of oxidative stress in secondary injury.4 With the large amount of stress the cells undergo during primary and secondary injury, the natural antioxidant system, glutathione, becomes overwhelmed with ROS. Therefore, a therapy that could help the cells to decrease to fight the large amount of acrolein and ROS being produced may be clinically relevant. Hydralazine, an FDA-approved hypertension drug, has been shown to prevent acrolein damage.5 Acrolein's carbonyl is free even after reacting with proteins; therefore, hydralazine can react with both free and conjugated acrolein. Unfortunately, though, hydralazine's short half-life hinders its clinical translation to spinal cord injury.

1.2 Catechins: Nature's natural antioxidants

Catechins are complex, natural flavanoid molecules found in various plant products. The most abundant source of catechins is extracted from green tea leaves, but they are also found in wines and fruits in lower concentrations. Catechins are related via two adjoining rings connected to a separate benzyl alcohol, but differ in the hydroxylation on the B ring and substitution on the C ring. (Figure 2)

OOH

OH

R1

OH

R2

R3

Figure 2. Basic structure of catechin (left) and table of common catechin derivatives. Chirality not shown.

Various works have looked at the properties of catechins, mainly of EGCG, as it is the most abundant catechin in green tea extracts. The major focus has been to study their strong antioxidant and anti-inflammatory properties. Specifically, the heavily substituted C ring can be oxidized, forming quinone-like molecules, thus preventing the oxidation of critical proteins and structures in a cell.6-9 Along with their promising antioxidant ability, they exhibit extremely low toxicity and are classified as GRAS products. A major limitation of using catechins includes their low bioavailability.10 Again, a therapeutic that could effectively deliver such molecules may increase their functionality in disease characterized by oxidative stress.

Necrosis releases cellular contents (primary)

Inflammation and ROS/Acrolein

Lipid peroxidation

Membrane and myelin damage

Loss of function and homeostasis

Name Abbrev. R1 (C ring) R2 (B ring) R3

Catechin C -OH -H -OH Epicatechin EC -OH -OH -H

Epigallocatechin EGC -OH -OH -OH Epicatechin gallate ECG Gallic ester -OH -OH

Epigallocatechin gallate EGCG Gallic ester -OH -OH



1.3 Nanomedicine

The potential of nanoparticles for use as therapeutic delivery vehicles is under constant exploration. Silica nanoparticles are particularly interesting based on the porous structure that forms during synthesis reactions.11 Ideally, a drug with extensively damaging side effects, such as chemotherapeutics, or a short half life, could become less dangerous or more bioavailable with use of a delivery vehicle. The drugs can adsorb onto the pores of the silica surface and, particularly with targeting molecules, slowly desorb to both keep the overall dosage low and keep the drug near the tissue of interest. If a drug is protected by interacting with the silica surface, it is less likely to be metabolized, which increases its chance of arriving at the site of interest.

1.4 Using silica-PEG nanoparticles for targeting SCI

The current study highlights the early steps of using PEGylated silica nanoparticles for their use in therapeutic delivery. Not only were the nanoparticles studied, but a potential new therapy in a natural product has been explored. Combining the two may be beneficial in attenuating oxidative stress from acrolein in neurological injury such as that after spinal cord injury or during multiple sclerosis.

2. MATERIALS AND METHODS 2.1 Synthesis of PEGylated silica nanoparticles

The silica nanoparticles were synthesized using a modified Stöber method.12,13 20 mg of hydrazide- modified methoxy poly(ethylene) glycol (mPEG-Hz) MW5000 (0.004 mmol) (Laysan Bio LLC, Auburn, AL) was dissolved in 6 mL of methanol. Upon dissolution, 750 uL of 2 M NaOH (1.5 mmol) was added to the mixture and vortexed. After 1 hr of mixing, 64 uL of tetramethylorthosilicate (0.434 mmol) (TMOS, Sigma, St. Louis) was added slowly. The solution was vortexed at 25°C for an additional hour. To remove most of the solvent, the milky solution was added to a 30 kDa Amicon membrane filter (Millipore, Billerica, MA) and centrifuged for 15 minutes at 2800 g. The filtrate was removed, and 5 mL of nanograde water was added to the filter and spun again for 10 minutes. The solution was aged for 15 days before 3 mL of nanograde water was added to the filter and spun for 15 minutes. Residual methanol was removed via evaporation. Concentration of the nanoparticle solution was obtained gravimetrically by solvent evaporation under vacuum. For use in cell culture, the nanoparticle samples were sterile filtered using a 0.2 µm syringe filter and diluted to the appropriate concentrations in phosphate buffered saline (PBS).

2.2 Near-infrared fluorescent labeling

As synthesized particles were vortexed with 1:25 molar ratio of CF 680:PEG (Sigma, St. Louis) for 2 hours in the dark before undergoing dialysis overnight to remove unreacted dye.

2.3 Cell culture of B35 neuroblastoma cells

B35 rat neuroblastoma cells were obtained from American Tissue Culture Collection (ATCC, Manassas, Virginia) and cultured in T-25 or T-75 flasks with DMEM (ATCC) media containing 10% fetal bovine serum (FBS). The cells were co-cultured twice per week and seeded in concentrations from 1:5 to 1:8.

2.4 Lactate dehydrogenase (LDH) cell permeability assay

LDH activity gives a measure of cell's membrane integrity. As the membrane is damaged, cytosolic LDH is released from the cell. The LDH Toxicology Kit was purchased from Sigma and used per the manufacturer's instructions. B35 cells were seeded in triplicates at 5 x 105 cells/mL in complete growth media overnight. The cells were incubated with nanoparticles in complete growth media for 24 hours before analyzing the growth medium for LDH activity. After incubation, the LDH activity was measured using a VersaMax microplate reader (Molecular Devices, LLC, Sunnyvale, CA). The data is reported as a change of LDH activity compared to the vehicle control, calculated as follows:

Percent change % =(Test490-690- ControlAverage 490-690 )

ControlAverage(490-690)∗ 100

2.5 Measuring oxidative stress with dihyroethidium (DHE)

Dihydroethidium (Invitrogen) is a compound that, once activated by reactive oxygen species, integrates with DNA and fluoresces red. To determine oxidative stress or DNA damage caused by the nanoparticles, B35 cells were seeded



overnight in a Cyntellect LEAP (Laser Enabled Analysis and Processing) plate at 5 x 105 cells/mL in DMEM with 10% FBS. Various concentrations of nanoparticles were added to the cells and incubated for 24 hours. 150 uM acrolein and 150 uM hydrogen peroxide were used as positive controls for oxidative stress. Before running the experiment, the optimal dye concentrations were determined using untreated cells and cells treated with the positive controls. After 24 hours, the cells were washed with DPBS three times, and they were incubated in 10 uM DHE for 30 minutes. The excess dye was washed away and the cells were analyzed in DMEM with 10% FBS and 5 ug/mL Hoechst 33342 (Invitrogen) using LEAP. The analysis settings were optimized using the positive and negative controls before obtaining data from all samples. The data was exported for post-processing in Excel.

2.6 Cell viability

B35 cells were seeded overnight in a Cyntellect LEAP plate at 5 x 105 cells/mL in DMEM with 10% FBS. Vehicle or various concentrations of nanoparticles in vehicle were added to the cells and incubated for 24 hours. 150 uM acrolein was used as positive control. Before running the experiment, the optimal dye concentrations were determined using untreated cells and cells treated with the positive control. After 24 hours, the cells were washed with DPBS three times, and they were incubated in 2 uM calcein am (Invitrogen) for 30 minutes. The excess dye was washed away and the cells were analyzed in DMEM with 10% FBS and 5 ug/mL Hoechst 33342 using LEAP. The analysis settings were optimized using the positive and negative controls before obtaining data from all samples. The data was exported for post-processing in Excel.

2.7 Nanoparticle uptake via confocal microscopy

B35 cells were seeded overnight at 5 x 105 cells/mL in 35 mm Fluorodishes. Vehicle or 350 ug/mL nanoparticles in DPBS was added to the cells and incubated for 24 hours. After the incubation period, the cells were washed three times with DPBS, and the cells were fixed in 4% paraformaldehyde (PFA) for 25 minutes and subsequently washed three times. They were permeabilized with 0.1% Triton X-100 for 5 minutes and washed before staining with 5 uL of phalloidin Alexa Fluor 488 (Invitrogen) in 1% blocking buffer for 30 minutes. In the last ten minutes, 300 ng/mL propidium iodide (PI, Invitrogen) was added as a counterstain. The excess dye solution was removed, and Vectashield was added to the dishes.

Confocal images were obtained using and Olympus Fluoview at 20X set with the appropriate filters for Alexa Fluor 488 using a 488 nm laser, PI using a 543 nm laser, and Cy5.5 using a 633 nm laser. Bright field images were obtained using the 543 nm laser. Images were exported for analysis using ImageJ.

2.8 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging

To evaluate free radical scavenging capability, effectiveness against 2,2-diphenyl-1-picrylhydrazyl (DPPH), a stable free radical, was measured. DPPH forms a dark purple solution with a peak absorbance at 517 nm. A stock solution of DPPH was prepared in ethanol. Catechin hydrate, epigallocatechin gallate (EGCG), and hydralazine were all purchased from Sigma and used as received. Solutions of each compound were prepared in bubbled PBS. For each reaction, done in three replicates, 50 µL of DPPH in ethanol was added to 50 µL of catechin, EGCG ,or hydralazine for a final DPPH concentration of 200 µM. The reactions were shaken at room temperature in the dark for 45 minutes before the absorbance was read at 517 nm and 690 nm using a VersaMax microplate reader (Molecular Devices, LLC, Sunnyvale, CA). The concentration of each reaction was calculated using a standard curve of DPPH in 1:1 (v/v) of ethanol/PBS. The scavenged percentage was calculated as follows:

Percent % DPPH reacted = [Initial DPPH concentration] -[DPPH concentration remaining]

[Initial DPPH concentration]*100

2.9 LDH to evaluate acrolein scavenging of EGCG

A stock solution of EGCG was prepared and bubbled in sterile ethanol. Directly before use, the stock was diluted to the appropriate concentrations in DPBS. To determine if EGCG is an effective scavenger of acrolein, the LDH Toxicology Kit was purchased from Sigma and used per the manufacturer's instructions. B35 cells were seeded in triplicates at 5 x 105 cells/mL in DMEM with 10% FBS overnight. A freshly-prepared solution of acrolein in DPBS was added to the cells and incubated for 30 minutes before the addition of EGCG. The vehicle used for the controls contained the highest EtOH concentration based on the dilution of EGCG, which was less than 1.5%. After 4 hours with EGCG, the LDH activity



was measured using a VersaMax microplate reader. To determine the total LDH amount, a set of untreated cells were lysed using 0.5% Triton-X. The data is reported as a change of LDH activity compared to the lysed control wells, calculated as follows:

Percent released % =Test490-690

ControlAverage(490-690)∗ 100

2.10 Statistical analysis

To determine statistical significance, ANOVA was performed on each data set. For pair wise comparisons, a test using the Least Significant Difference (LSD) was used. P-values less than 0.01 were considered significant.

3. RESULTS AND DISCUSSION 3.1 Characterization of mPEG-Am modified nanoparticles

The silica nanoparticles were prepared to incorporate the polymer into the silica network (Figure 3), which eliminates an additional PEGylation step that is common in many other synthesis schemes.14–18 In the current study, the PEG serves a dual purpose. PEG is nanomedicine's "stealth" molecule, as it has been shown to prevent opsonization.19,20 Specifically for spinal cord injury, PEG serves as a target for injured neurons and associates with them, aiding in membrane repair.21

Figure 3. Synthesis scheme of Si-mPEG NP's. The silica precursor, TMOS, is hydrolyzed and condensed with methoxy-PEG and TMOS. Condensation with TMOS allows the nanoparticle network to grow whereas condensation with mPEG stops the growth of the particle.

In the one-step synthesis, the PEGylation is a function of its hydrolysis and condensation with the siloxane. Furthermore, the Stöber method creates water and alcohol as by-products, eliminating the need to remove the organic solvents and surfactants in emulsion techniques. Volatile alcohols are fast and easy to remove. Inorganic surfactants used to synthesize silica particles require harsh, acidic environments to remove them from the surface of the silica particles.22–24 Ma et al. recently reported a synthesis technique using a combination of the Stöber and emulsification methods.11 The synthesis and purification was performed at 30°C and utilized a surfactant with an alcohol and water solvent system. Especially for use in biological systems and scaling procedures, eliminating side products and steps may make production much faster, easier, and cheaper.

3.2 Evaluating nanotoxicity

An additional important factor in using a nanoparticle-based delivery system is the potential toxicity of the delivery system. Because a neuron's membrane is essential to its function, the lactate dehydrogenase activity assay was chosen to measure toxicity. As the membrane integrity of the cells are compromised, the protein escapes from the cytoplasm. Therefore, higher activity indicates a higher level of membrane damage in the cell population. Triton X-100 was chosen as the positive control, and the cells within those wells had lysed after the incubation period. The LDH activity is measured relative to the vehicle control as described in the methods, and none of the chosen nanoparticle concentrations showed significant cytotoxic effects. (Figure 4) Upon microscopic examination, the cells maintained their morphology, including dendrite-like processes (data not shown), rather than rescinding their processes and changing cell body morphology. Because the LDH assay is limited to looking at the entire cell population rather than single cells, though, it is limited in its scope. Therefore, it is imperative to corroborate its results with other, more sensitive assays.

SiO

OO O

nSiOSi O O NH

O

NH2+

nO O NH

O

NH2

NaOH

Methanol, 1 hr.



Figure 4. Lactate Dehydrogenase (LDH) assay in B35 cells. The nanoparticles were incubated with B35 cells for 24 hours, and the cell population was assayed. The nanoparticles did not show significant toxicity (P < 0.01) when compared to the vehicle control (DMEM). The error bars show the standard error for each sample (n=3).

To overcome the weakness of the LDH assay, dihydroethidium (DHE) and calcein, a live-cell dye, were used to monitor cells that were incubated with nanoparticles. After incubation, the nanoparticles were removed, and the cells were stained with DHE, calcein, and Hoechst. DHE reacts with ROS and upon intercalation with DNA, it has a red emission spectra. Calcein indicates living cells through their esterase activity. Laser-scanning cytometry via LEAP counts and records the characteristic for each cell, giving a detailed view of each cell's behavior, which can be compiled.

Figure 5. ROS production from mPEG-Hydrazide nanoparticles measured via dihydroethidium (DHE) fluorescence and analysis by LEAP. Nanoparticles were incubated for 24 hours. None of the concentrations showed significant damage from the vehicle control (n=3; mean + SE; P > 0.01).

Similar to the LDH assay, the results using DHE show very little oxidative stress induced by the mPEG-Hydrazide nanoparticles. (Figure 5) More than likely, their small size and the outer layer of PEG prevents an adverse reaction to the nanoparticles. The cell viability does not decrease significantly in cells incubated with mPEG-hydrazide nanoparticles. (Figure 6) Interestingly, though, the cell number decreased significantly in the samples with the highest concentrations. The cell growth may be stifled from the large quantity of nanoparticles, or, in a more likely

0

10

20

30

40

50

60

70

80

90

100

110

120

Vehicle 0.5% Triton X-100

50 ug/mL 100 ug/mL 200 ug/mL 300 ug/mL 400 ug/mL 500 ug/mL

Perc

ent L

DH

rele

ase

com

pare

d to

pos

itive

cont

rol

Nanoparticle Concentration

LDH Assay of mPEG-Hz nanoparticle toxicity in B35 cells after 24 h

Controls

0

5

10

15

20

25

30

35

40

45

50

Vehicle 50 ug/mL 100 ug/mL 200 ug/mL 300 ug/mL 400 ug/mL 500 ug/mL

Perc

ent (

%) R

OS-

posi

tive

Treatment

ROS-production: mPEG-Hz nanoparticles



case, they are dying. If they undergo apoptosis, their contents will be packaged and exported, or death via necrosis may cause the cells to essentially "explode"; both cases would account for a decrease in cell number without adversely affecting the overall viability, as one cannot count what is not present. The drop becomes significant in the cells incubated with both 400 ug/mL and 500 ug/mL of nanoparticles. In those samples, the cell population is healthy, but its population was stifled.

The high viability of the 500 ug/mL sample exemplifies seeing a high viability number merely from the hardiest cells surviving, and demonstrates the importance of single-cell analysis techniques. Oftentimes, a high viability may be seen in a whole cell population, but likely these cells have undergone "survival of the fittest" since missing cells may simply disappear through apoptosis or violent, explosive necrosis. It is crucial to ensure that the cell population is not only healthy but also still present to prevent artifacts in similar experiments. Although some toxicity was seen in this experiment, the relevant concentration of nanoparticles that cells will be exposed to is on the lower end of the tested concentrations.

Figure 6. Cell viability and cell number of B35 cells after being exposed to mPEG-Hydrazide nanoparticles for 24 hours. The samples with particles do not show a significant decrease in viability, but their cell numbers had decreased significantly. (n=3; mean ± SE; *P < 0.01)

3.3 Nanoparticle uptake via confocal microscopy

For the purpose of delivery, it is important that the nanoparticles affiliate with the cells of interest. In an in vitro setting, the cells are incubated with the nanoparticles for a period of time and the cells are explored for the presence of nanoparticles. In this study, nanoparticles were labeled with a near-infrared fluorescent (NIRF) dye similar to Cy5.5, CF 680, and incubated with nanoparticles for 24 hours. The labeling of the nanoparticles requires easy "click" chemistry without the introduction of other reagents. Furthermore, upon seeing success in vitro, the same dye is often used in vivo for studying nanoparticle distribution.25

Figure 7 shows representative images obtained from confocal microscopy. Actin was stained with phalloidin Alexa Fluor 488 (green) while the nuclei were stained with propidium iodide (blue). As a comparison, the bright field images and NIRF-channel (magenta) images are shown for comparison for both the control and the cells incubated with nanoparticles. Nanoparticles were seen in various planes and were seen inside the cells, as opposed to sticking to the outside. The bright field images are qualitative and show that the overall morphology of the cells is maintained, even after incubation with the nanoparticles. The current resolution of such microscopy will not distinguish single nanoparticles, but signals from aggregates within a cell population can be found. This limitation is inherent in studying nanoparticles, and silica-based nanoparticles are difficult in their detection without a probe.

0

1000

2000

3000

4000

5000

6000

7000

0

10

20

30

40

50

60

70

80

90

100

Vehicle 50 ug/mL 100 ug/mL 200 ug/mL 300 ug/mL 400 ug/mL 500 ug/mL

Tota

l Cel

l Num

ber

Perc

ent (

%) o

f cel

l pop

ulat

ion

Treatment

Nanotoxicity: mPEG-Hz nanoparticles

Viability Total Cell Number

**



Figure 7. nuclei arethe cells.

3.4 EGCG f

In order to evwas measurequenched, whevaluate the c

Figure 8 showDPPH. Only amount of DPconcentrationand hydralazcapabilities wthe aromatic

20X magnificae blue, and nano

for acrolein at

valuate the eacd in various rehere it turns yecompounds ag

ws the free radat the two high

PPH after 45 mns, with the excine had similar

with an increasrings to form q

ation of cells incoparticle aggrega

ttenuation

h therapeutic ceactions with eaellow. Becauseainst a larger a

dical quenchinghest concentrat

minutes. EGCGception of 40 µr performancesing number of quinone-like pr

cubated with PBates within the c

compound's abach compounde the reaction wamount of DPP

g of each comptions of 20 and

G removed signµM, where hyds. Compared tof hydroxyl grouroducts.

S and 350 ug/mcells are shown

ility to quenchd. In solution, Dwith EGCG occPH.

pound. The insed 40 µM were anificantly moredralazine and Eo the other two ups that can don

mL of nanoparticin magenta. Nan

h free radicals, DPPH remains curs very rapid

et shows a typiall three compoe DPPH than hyEGCG were no

molecules, EGnate electrons

cles for 24 hournoparticles are s

the change in Da dark purple c

dly, low molar r

ical standard coounds able to rydralazine andt significantly

GCG has higheand be stabiliz

s. Actin is seen seen in various r

DPPH concentcolor until beinratios were cho

oncentration curemove a signifd catechin at alldifferent. Cate

er nucleophilic zed with π-bond

in green, regions of

tration ng osen to

urve for ficant l echin

ds from



Figure 8. DPPH Scavenging with catechin, EGCG, and hydralazine. Different concentrations of catechin, EGCG, and hydralazine were reacted with 200 µM of DPPH for 45 minutes. The absorbance of DPPH at 517 nm was measured. The inset shows a representative standard curve used to calculate the remaining DPPH concentration. Displayed is the mean of three reactions + SE. There were no significant differences between hydralazine and catechin.

Especially for acrolein scavenging, maintaining membrane integrity is vital for keeping cells healthy. To determine if EGCG could prevent damage by acrolein, cells were incubated with acrolein for 30 minutes before the addition of EGCG, which was left for four hours. All of the samples containing EGCG were similar to the vehicle control whereas the untreated cells exposed to acrolein had significant damage. The results indicate that EGCG was able to prevent damage to the cells. (Figure 9) A similar experiment with catechin added both before and after application of the same concentration of hydrogen peroxide did not improve LDH results (data not shown). Such results are likely due to the high scavenging ability of EGCG compared to other catechins, which has been shown previously.9,26

Figure 9. Acrolein scavenging with EGCG. 50 uM of acrolein was added to B35 cells for 30 minutes before the application of EGCG for 4 hours. All concentrations were able to prevent acrolein from causing intense damage and cell death. Displayed is the mean + SE.

0

10

20

30

40

50

60

70

80

90

100

0 5 (1/40) 10 (1/20) 20 (1/10) 40 (1/5)

Perc

ent (

%) D

PPH

rem

oved

Concentration [uM] (scavenger/DPPH molar ratio)

DPPH Scavenging

Catechin EGCG Hydralazine

0.000

0.100

0.200

0.300

0.400

0.500

0.600

50 100 150 200A

bs a

t 517

nm

Concentration [uM]

DPPH Standard Curve

**

*0 uM# EGCG vs hydralazine& EGCG vs catechin

#&_______

*

**

*

**

#&_______

#&_______

&____

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

110.0

Vehicle 50 uM Acrolein

0.5% Triton X-

100

50 uM 100 uM 200 uM 300 uM 400 uM 500 uM

Perc

ent L

DH

Rel

ease

d

Cell Treatment

Acrolein scavenging with EGCG in B35 cells

*#*# *# *#

*# *#

αControl* Acrolein# Triton X-100

α #

α *



4. CONCLUSIONS Much of the current state of the project focuses on the characterization of the interactions between silica-based nanoparticles and neuronal cells. With the current study, toxicity was only found when using extremely high and unrealistic concentrations of nanoparticles. Additional studies to look at the mechanism of death, apoptosis or necrosis, are necessary for a deeper understanding. Furthermore, the nanoparticles were found within the cells after a 24-hour period. In the case of injured cells with compromised membranes, uptake would present less of an issue, but if the nanoparticles were to translate for other applications, crossing into cells would be important.

Additionally, this study has shown the first steps in exploring a natural product, EGCG, as a potent scavenger for acrolein. Not only does it perform better than hydralazine in scavenging free radicals, but it has also shown promise in an early in vitro study with acrolein. Further explorations will include determining how each compound may benefit via delivery with the silica nanoparticles. Particularly, the slow release and protection of EGCG in a nanoparticle would increase its bioavailability and antioxidant efficacy. On the other hand, hydralazine's deleterious side effects could be ameliorated in the delivery system by keeping the dosage low enough to keep it concentrated at a site of injury.

ACKNOWLEDGEMENTS This research was funded by the Christopher Columbus Foundation to JFL. Ms. White-Schenk is funded by an NSF graduate fellowship, grant DGE0833366.

REFERENCES [1] Luo, J., Uchida, K., and Shi, R., “Accumulation of Acrolein–Protein Adducts after Traumatic Spinal Cord

Injury,” Neurochemical Research 30(3), 291–295 (2005). [2] Uchida, K., Kanematsu, M., Morimitsu, Y., Osawa, T., Noguchi, N., and Niki, E., “Acrolein Is a Product of

Lipid Peroxidation Reaction,” Journal of Biological Chemistry 273(26), 16058 –16066 (1998). [3] Bracken, M.B., Shepard, M.J., Collins, W.F., Holford, T.R., Young, W., Baskin, D.S., Eisenberg, H.M., Flamm,

E., Leo-Summers, L., et al., “A randomized, controlled trial of methylprednisolone or naloxone in the treatment of acute spinal-cord injury,” New England Journal of Medicine 322(20), 1405–1411 (1990).

[4] Hamann, K., and Shi, R., “Acrolein scavenging: a potential novel mechanism of attenuating oxidative stress following spinal cord injury,” Journal of Neurochemistry 111, 1348–1356 (2009).

[5] Liu�Snyder, P., Borgens, R.B., and Shi, R., “Hydralazine rescues PC12 cells from acrolein�mediated death,” Journal of Neuroscience Research 84(1), 219–227 (2006).

[6] Harada, M., Kan, Y., Naoki, H., Fukui, Y., Kageyama, N., Nakai, M., Miki, W., and Kiso, Y., “Identification of the Major Antioxidative Metabolites in Biological Fluids of the Rat with Ingested (+)-Catechin and (-)-Epicatechin,” Bioscience, Biotechnology, and Biochemistry 63(6), 973–977 (1999).

[7] Raza, H., and John, A., “In Vitro Effects of Tea Polyphenols on Redox Metabolism, Oxidative Stress, and Apoptosis in PC12 Cells,” Annals of the New York Academy of Sciences 1138(1), 358–365 (2008).

[8] Zheng, L.T., Ryu, G.-M., Kwon, B.-M., Lee, W.-H., and Suk, K., “Anti-inflammatory effects of catechols in lipopolysaccharide-stimulated microglia cells: Inhibition of microglial neurotoxicity,” European Journal of Pharmacology 588(1), 106–113 (2008).

[9] Zhu, Q., Zheng, Z.-P., Cheng, K.-W., Wu, J.-J., Zhang, S., Tang, Y.S., Sze, K.-H., Chen, J., Chen, F., et al., “Natural Polyphenols as Direct Trapping Agents of Lipid Peroxidation-Derived Acrolein and 4-Hydroxy-trans-2-nonenal,” Chemical Research in Toxicology 22(10), 1721–1727 (2009).

[10] Ferruzzi, M.G., Lobo, J.K., Janle, E.M., Cooper, B., Simon, J.E., Wu, Q.-L., Welch, C., Ho, L., Weaver, C., et al., “Bioavailability of Gallic Acid and Catechins from Grape Seed Polyphenol Extract is Improved by Repeated Dosing in Rats: Implications for Treatment in Alzheimer’s Disease.,” Journal of Alzheimer’s Disease 18(1), 113–124 (2009).

[11] Ma, K., Sai, H., and Wiesner, U., “Ultrasmall Sub-10 nm Near-Infrared Fluorescent Mesoporous Silica Nanoparticles,” Journal of the American Chemical Society 134(32), 13180–13183 (2012).

[12] Stöber, W., Fink, A., and Bohn, E., “Controlled growth of monodisperse silica spheres in the micron size range,” Journal of Colloid and Interface Science 26(1), 62–69 (1968).

[13] Xu, H., Yan, F., Monson, E.E., and Kopelman, R., “Room-temperature preparation and characterization of poly (ethylene glycol)-coated silica nanoparticles for biomedical applications,” Journal of Biomedical Materials Research Part A 66A(4), 870–879 (2003).



[14] Cho, Y., Shi, R., Borgens, R.B., and Ivanisevic, A., “Functionalized mesoporous silica nanoparticle-based drug delivery system to rescue acrolein-mediated cell death,” Nanomedicine 3, 507–519 (2008).

[15] He, Q., Zhang, J., Shi, J., Zhu, Z., Zhang, L., Bu, W., Guo, L., and Chen, Y., “The effect of PEGylation of mesoporous silica nanoparticles on nonspecific binding of serum proteins and cellular responses,” Biomaterials 31(6), 1085–1092 (2010).

[16] Zhang, Z., Berns, A.E., Willbold, S., and Buitenhuis, J., “Synthesis of poly(ethylene glycol) (PEG)-grafted colloidal silica particles with improved stability in aqueous solvents,” Journal of Colloid and Interface Science 310(2), 446–455 (2007).

[17] Bagwe, R.P., Hilliard, L.R., and Tan, W., “Surface Modification of Silica Nanoparticles to Reduce Aggregation and Nonspecific Binding,” Langmuir 22, 4357–4362 (2006).

[18] Feng, L., Wang, Y., Wang, N., and Ma, Y., “Preparation of poly(ethylene glycol)-grafted silica nanoparticles using a facile esterification condensation method,” Polymer Bulletin 63(3), 313–327 (2009).

[19] Niidome, T., Yamagata, M., Okamoto, Y., Akiyama, Y., Takahashi, H., Kawano, T., Katayama, Y., and Niidome, Y., “PEG-modified gold nanorods with a stealth character for in vivo applications,” Journal of Controlled Release: Official Journal of the Controlled Release Society 114(3), 343–347 (2006).

[20] Peracchia, M.., Fattal, E., Desmaële, D., Besnard, M., Noël, J.., Gomis, J.., Appel, M., d’ Angelo, J., and Couvreur, P., “Stealth® PEGylated polycyanoacrylate nanoparticles for intravenous administration and splenic targeting,” Journal of Controlled Release 60(1), 121–128 (1999).

[21] Liu-Snyder, P., Logan, M.P., Shi, R., Smith, D.T., and Borgens, R.B., “Neuroprotection from secondary injury by polyethylene glycol requires its internalization,” Journal of Experimental Biology 210, 1455–1462 (2007).

[22] Berriozabal, G., and de Miguel, Y.R., “Synthesis and characterisation of silica nanoparticles bearing different functional groups obtained via a two-stage method,” physica status solidi (c) 7(11-12), 2692–2696 (2010).

[23] Cai, Q., Luo, Z.-S., Pang, W.-Q., Fan, Y.-W., Chen, X.-H., and Cui, F.-Z., “Dilute Solution Routes to Various Controllable Morphologies of MCM-41 Silica with a Basic Medium,” Chemistry of Materials 13(2), 258–263 (2001).

[24] Bagwe, R.P., Yang, C., Hilliard, L.R., and Tan, W., “Optimization of Dye-Doped Silica Nanoparticles Prepared Using a Reverse Microemulsion Method,” Langmuir 20(19), 8336–8342 (2004).

[25] Key, J., Cooper, C., Kim, A.Y., Dhawan, D., Knapp, D.W., Kim, K., Park, J.H., Choi, K., Kwon, I.C., et al., “In vivo NIRF and MR dual-modality imaging using glycol chitosan nanoparticles,” Journal of Controlled Release 163(2), 249–255 (2012).

[26] Zhu, Q., Liang, C.-P., Cheng, K.-W., Peng, X., Lo, C.-Y., Shahidi, F., Chen, F., Ho, C.-T., and Wang, M., “Trapping Effects of Green and Black Tea Extracts on Peroxidation-Derived Carbonyl Substances of Seal Blubber Oil,” Journal of Agricultural and Food Chemistry 57(3), 1065–1069 (2009).



Documents

Mediating the potent ROS toxicity of acrolein in neurons