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Nanoparticles as a Novel Tool to Inhibit Inflammatory Cytokines in Human Lymphocytes and Macrophages of Coronary Artery Disease

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  • Shifa tameer-e-Millat university

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TNFα and NF-kB contribute in activation of pro-inflammatory signaling pathways and complications of coronary artery diseases (CAD). Current study highlights novel properties of Au (15 ± 2nm), ZnO (77± 45nm) and MgO (11± 4nm) nanoparticles (NPs) as possible anti-inflammatory agents with greater efficacy and lower toxicity. Decrease in TNFα and NF-kB levels in Single Vessel Disease (SVD), Double Vessel Disease (DVD) and Triple-Vessel coronary artery disease (TVD) macrophage and lymphocyte cultures at varying concentrations of NPs has been studied to find an effective therapeutic concentration (ETC). Au and MgO NPs exhibits 5µg/ml ETC compared to 1µg/ml ZnO in all three CAD categories with negligible toxicity. ZnO remains most statistically significant (p<0.001) in SVD and TVD cultures whereas MgO shows efficacy in DVD and TVD cultures with more than 50% reduction in TNFα and NF-kB levels at their respective ETCs. Au NPs exhibit prominent effect in DVD cultures. The mRNA expression results support the down-regulation of TNFα and NF-kB after NPs exposure in respective cultures. Findings of this prospective observational cohort study suggest use of NPs as an alternate anti-inflammatory agent in coronary artery and other diseases.
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Nanoparticles as a novel tool to inhibit inflammatory cytokines in
human lymphocytes and macrophages of coronary artery disease
Rabia Shabbir , Abida Raza , Afrose Liaquat , Saeed Ullah Shah ,
Sidra Saeed , Usama Sarwar , Muhammad Hamza ,
Fayyaz Chudhary , Zajif Hussain , N.M. Butt
PII: S0022-3549(22)00003-X
DOI: https://doi.org/10.1016/j.xphs.2022.01.001
Reference: XPHS 2652
To appear in: Journal of Pharmaceutical Sciences
Received date: 5 August 2021
Revised date: 28 December 2021
Accepted date: 4 January 2022
Please cite this article as: Rabia Shabbir , Abida Raza , Afrose Liaquat , Saeed Ullah Shah ,
Sidra Saeed , Usama Sarwar , Muhammad Hamza , Fayyaz Chudhary , Zajif Hussain , N.M. Butt ,
Nanoparticles as a novel tool to inhibit inflammatory cytokines in human lymphocytes and
macrophages of coronary artery disease, Journal of Pharmaceutical Sciences (2022), doi:
https://doi.org/10.1016/j.xphs.2022.01.001
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©2022 Published by Elsevier Inc. on behalf of American Pharmacists Association.
Nanoparticles as a novel tool to inhibit inflammatory cytokines in
human lymphocytes and macrophages of coronary artery disease
Rabia Shabbir1, Abida Raza*2, Afrose Liaquat3, Saeed Ullah Shah4, Sidra Saeed2,
Usama Sarwar2, Muhammad Hamza1, Fayyaz Chudhary1, Zajif Hussain5, N. M. Butt**1
1Preston Institute of Nanoscience and Technology (PINSAT), Preston University Kohat,
Islamabad campus, Islamabad and Pakistan Academy of Sciences, Islamabad
2NILOP Nanomedicine Research Laboratories, National Institute of Lasers and
Optronics College, PIEAS, Nilore, Islamabad.
3Shifa College of Medicine, Islamabad
4Shifa International Hospital, Islamabad
5Department of Chemistry, LUMS, Lahore.
*Corresponding author:
Dr Abida Raza
Principal Scientist, Group Head Nanotheranostic Research, NILOP Nanomedicine
Research Laboratories, National Institute of Lasers and Optronics College (NILOP),
Pakistan Institute of Engineering and Applied Sciences (PIEAS), Nilore, Islamabad
45650, Pakistan.
Email: abida_rao@yahoo.com
Phone: +923457713910
**Corresponding author:
Prof Dr.N.M. Butt
Chairman Preston Institute of Nanoscience and Technology (PINSAT), Preston
University Kohat, Islamabad campus H-8, Islamabad & Pakistan Academy of Sciences,
3-Constitution Avenue, Sector G-5/2, Islamabad, Pakistan.
Email: nmbutt36@gmail.com
Phone: +923005000513
Abstract
TNFα and NF-kB contribute in activation of pro-inflammatory signaling pathways and complications of
coronary artery diseases (CAD). Current study highlights novel properties of Au (15 ± 2nm), ZnO (77±
45nm) and MgO (11± 4nm) nanoparticles (NPs) as possible anti-inflammatory agents with greater efficacy
and lower toxicity. Decrease in TNFα and NF-kB levels in Single Vessel Disease (SVD), Double Vessel
Disease (DVD) and Triple-Vessel coronary artery disease (TVD) macrophage and lymphocyte cultures at
varying concentrations of NPs has been studied to find an effective therapeutic concentration (ETC). Au
and MgO NPs exhibits 5µg/ml ETC compared to 1µg/ml ZnO in all three CAD categories with negligible
toxicity. ZnO remains most statistically significant (p<0.001) in SVD and TVD cultures whereas MgO
shows efficacy in DVD and TVD cultures with more than 50% reduction in TNFα and NF-kB levels at their
respective ETCs. Au NPs exhibit prominent effect in DVD cultures. The mRNA expression results support
the down-regulation of TNFα and NF-kB after NPs exposure in respective cultures. Findings of this
prospective observational cohort study suggest use of NPs as an alternate anti-inflammatory agent in
coronary artery and other diseases.
Key Words: Tumor Necrosis Factor-alpha (TNFα), Coronary artery disease, NF-kB, Lymphocytes,
Macrophages, ZnO, MgO and Au NPs, Effective therapeutic concentrations (ETC).
Introduction
Coronary artery disease (CAD) is the most common ailment of cardiovascular diseases and is accounting
for almost 17 million deaths every year worldwide. Four out of five deaths occurred due to strokes and
heart attacks and responsible for an estimated 31% of all deaths globally1. An epidemiological shift of
increase in mortality in developing and under-developed countries has necessitated the exploration of
effective therapeutic strategies. Nanoparticles have been reported in various novel applications in
biological properties and medical health care such as anti-angiogenesis, anti-cancer, and anti-
inflammation2.
CAD, being a multigenic and complex multifactorial disease, can be categorized into single, double and
triple vessel coronary artery disease depending upon the stenosis in major epicardial coronary arteries,
cardiac events and severity of inflammation. CAD severity has been defined according to the number of
obstructive coronary arteries corresponding to 50% or more narrowing of vessel. According to SCCT
grading scale for stenosis severity assessment 0% is no visible stenosis, 1-24% corresponds to minimal
stenosis, 25-49% describes mild stenosis, 50-69% is moderate stenosis, 70-99% is called severe
stenosis while100% is described as occlusion3. The term single-vessel is usually referred to as the
presence of ≥70% stenosis of a major coronary artery (left circumflex, right coronary arteries or left
anterior descending). Whereas, triple vessel disease develops when major blood supplying vessels to the
heart become diseased or damaged and also considered as a severe form of CAD4,5. The two major
causes of CAD are inflammation and plaque formation (deposits of cholesterol).
Scientists have investigated numerous genes as predisposing factors for CAD development including
inflammation, coagulation pathways in the renin-angiotensin and lipid metabolism6. The Tumor Necrosis
Factor-alpha (TNFα) is a potential therapeutic target, extensively studied in many pathological processes
that cause irreversible damage to the heart, the post-myocardial Infarction7. TNFα is considered as one of
the pro-inflammatory cytokines which is produced by inflammatory cells i.e., macrophages and
monocytes8. Evidence suggests TNFα as a key contributor in the development, progression and
complications of atherosclerosis9. Moreover, the duration and amount of TNFα expression is directly
related to its net effect on cardiac function. Stress could be a reason for short-term expression of TNFα in
the heart, whereas cardiac decompensation can lead to long-term expression10. The excessive release of
TNFα can cause cardiomyopathy, left ventricular dysfunction11, and heart failure12. TNFα also influences
the secondary systemic inflammatory response to burns, infection, hemorrhagic shock, trauma and
pancreatitis. It has been involved in other infectious processes such as ischemia-reperfusion injury,
allograft rejection, development of granuloma and delayed-type hypersensitivity13. Moreover, TNFα can
potentiate adverse effects in rheumatoid arthritis and cardiovascular disease and its overexpression may
lead to organ dysfunction and death14.
NF-kB has been considered a new target and strategy for the development of drugs to combat
inflammation because of its role in the expression of other genes including chemokines, adhesion
molecules and pro-inflammatory cytokines. NF-kB is responsible for the activation of pro-inflammatory
signaling pathways activated by pro-inflammatory cytokines such as TNFα and Interleukin-115. Currently,
two types of biological therapeutics are available as TNFα inhibitors. It is accomplished either by using
soluble recombinant fusion protein containing a human IgG1 fragment (FC region) and extracellular
ligand-binding domain of TNFR2 or monoclonal antibodies (adalimumab, infliximab, golimumab,
certolizumab). Although these therapeutic strategies are valuable for the treatment of psoriasis,
inflammatory arthritis and ankylosing spondylitis but their long-term use can have adverse effects16.
Safety considerations here include new-onset autoimmune diseases, congestive heart failure and
malignancies, an increased incidence of pulmonary and skin infections, as a result of an obstruction in
immunomodulatory effects of TNFα17.
Due to the limitations of the available anti-inflammatory drugs, nano therapy is gaining interest as anti-
inflammatory drugs and for targeted delivery of these therapeutic agents to improve clinical outcomes.
FDA has approved the use of nanotherapeutics for the treatment of cancer and infectious diseases18. It
has already been reported that various metal and metal oxide NPs like silver19, gold20, copper21, zinc
oxide22, magnesium oxide, iron oxide and titanium dioxide23, exhibit anti-inflammatory properties. Gold
and silver nanoparticles have demonstrated anti-microbial24, anti-inflammatory25, anti-angiogenesis
properties and anti-proliferative properties in the leukemic cell cultures26. Successful in-vivo anti-
inflammatory effect of Au NPs via NF-kB pathways in rheumatoid arthritis has been reported27. The
activation of NF-kB in lymphocytes and macrophages results in up-regulation of the TNFα gene28.
Therefore, it is assumed, if TNFα and NF-kB activation are targeted it must lead to a decline in TNFα
blood levels which can limit the pathogenesis of CAD, ultimately. We have not found any study on the
effective role of NPs on TNFα and NF-kB levels in lymphocytes and macrophages in CAD. Taking this
into consideration, the current study has been designed to investigate the anti-inflammatory effects of Au,
ZnO and MgO NPs by targeting the pro-inflammatory cytokines, TNFα and NF-kB in lymphocytes and
macrophages cultures (Thematic diagram, Figure 1). We have used a variety of nanoparticles to reduce
the TNFα and NF-kB levels and block mRNA expression in lymphocytes and macrophages isolated from
blood specimens of SVD, TVD and DVD patients. We have used the term, effective therapeutic
concentration (ETC), at which nanoparticles have shown greater therapeutic effect. This is the first study
ever conducted to evaluate the therapeutic effects of NPs for CAD treatment.
2. Materials and Methods
Materials
Histopaque (cat# 10771), DMSO (cat# 60153), FBS (cat# F 9665), Triton X-100 (cat# 30632) from Sigma-
Aldrich (USA), Phosphate Buffer Saline (PBS-cat# 41620016-2), HEPES Powder (cat# 40820000-3) from
Bio world (USA), Hanks Balanced Salt Solution (cat# L0606-500) from Bio West (France), DMEM Powder
(cat# DMPO8) from Cassion Labs (USA), Phytohemagglutinin (cat# 10576015) from Gibco by life
technologies (USA), TNFα and NF-kB ELISA kits Elab Sciences (USA). Zinc chloride (cat# 10379) from
BDH analaR (UAE), Magnesium nitrate (cat# MA0048) from Scharlau Chemies (Spain), Gold (III) chloride
trihydrate (≥99.9%) (cat# 520918-5G), Trisodium citrate dihydrate (cat# 1064480500) from Sigma-Aldrich
(USA).
Synthesis and characterization of MgO, ZnO and Au nanoparticles
ZnO NPs were synthesized by following the method described by Wang et al29. Briefly, 5.5 grams of zinc
chloride was dissolved in 200ml of 1,2 ethanediol under constant stirring. To this, 16ml of sodium
hydroxide (20% w/v in distilled water) was added drop wise until it turned white without any precipitation.
The remaining sodium hydroxide solution was added to complete the reaction. The ZnO NPs were
washed and dried at 160˚C for 20 minutes.
MgO nanoparticles were synthesized by wet chemical method described by Lio, D., et al30. Briefly, 12.83
g of magnesium nitrate (0.1 M) was added to starch solution (0.1%) and kept under constant stirring till
complete dissolution of contents. To this, 4ml of sodium hydroxide solution (0.2M) was added in drops
and stirred for 2 hours. The solution was centrifuged for 10 minutes (10,000 rpm at 25°C) and placed at
300°C for 2h to get MgO nano powders.
Au NPs were prepared after a little modification of Turkevich method31. Briefly, 25mL of 1 mM HAuCl4
solution was refluxed in a round bottom flask and heated till the start of reflux. Then 5 mL, 20 mM,
solution of sodium citrate tribasic dihydrate, pre-heated at 60°C, was added to the above refluxing
solution and it was allowed to stir for 1 h under continuing reflux to end up in ruby red-colored
nanoparticles of gold (Au NPs).
Size measurements of particles
Nanoparticles were initially characterized by UV-Spectroscopy. Hydrodynamic size was determined by
diluting the sample (10:990ul) for dynamic light scattering (Nanotrac Wave II, Microtrac USA). The diluted
sample of nanoparticles were drop costed on copper grid and air dried. Afterward, it was subjected to
perform Scanning Electron Microscopy (SEM) to observe the surface morphology and population
distribution using NOVA NanoSEM-450, operating at 17.5kV adjusted at a working distance of 5mm.
Study design and specimen collection
In current study, blood samples (n=10) were collected randomly from each category of SVD, DVD and
TVD) patient and RNA was extracted from each 30 specimens to investigate TNFα and NF-kB expression
of lymphocytes and macrophages. All the samples were collected after disease confirmation through
medical investigations (angiographic sever stenosis of 70-90%) by consultant cardiologist, from Shifa
International Hospital, Islamabad, Pakistan. This prospective observational cohort study was duly
approved by Institute Research Ethics Committee (IRB letter no. 1198-474-2018). After written informed
consent in accordance with the Helsinki Declaration, 5cc of blood in heparin (for isolation and culturing of
lymphocytes and macrophages), 2.5cc of blood in EDTA (for RNA extraction) and 2cc was collected for
serology. Macrophages and lymphocytes isolated from diseased patients were used to culture the cells
for studying the anti-inflammatory effect of Au, ZnO and MgO NPs on TNFα and NF-kB levels. Blood
collected from non-SVD/DVD/TVD individuals was used as control. Baseline serum TNFα and NF-kB
levels were estimated before conduct of any experiments.
Isolation and culturing of lymphocytes and macrophages
Fresh blood was diluted with an equal volume of phosphate buffer saline (PBS, pH 7.4) and slowly
layered on histopaque. The tubes were centrifuged at 400 x g for 30 min at 18°C32. The mononuclear cells
from the interface of plasma, were aspirated, washed twice with 1M PBS (pH 7.4) and counted through
the trypan blue exclusion method. Cells were seeded in 24-well plate at a density of 3 × 106 cells/well in
RPMI-1640 containing 2 mM L-glutamine, 10% FBS, 100 µg/mL streptomycin sulfate and 100 unit/mL
penicillin. The lymphocytes and macrophages were cultured in humidified condition at 37°C with 5% CO2.
The lymphocytes in medium were transferred into a new cell culture flask having fresh media for further
culturing32. Attached macrophages were trypsinized with trypsin EDTA and transferred into cell culture
flask containing fresh growth media33. Both flasks were incubated in CO2 incubator for 24h at 37°C.
Biocompatibility testing of nanoparticles
To explore the safe concentration of nanoparticles, the cytotoxicity of nanoparticles was evaluated by
hemolysis followed by MTT assay against macrophages. For hemolysis assay, fresh blood was
centrifuged at 500 x g for 5 minutes to separate the RBCs. RBCs were washed with PBS 3 times. 100µl
of RBCs suspension in PBS (10% in volume) was placed in each well of 96-well plates. Nano-formulation
(100µl) was added to each well in varying concentrations of 1 to100µg/ml. The plate was incubated for 1
hour at 37°C. Cell suspension was centrifuged at 1000 g for 5 minutes. The supernatant was studied for
hemolytic content at 576 nm (Microplate reader, Model FL ×800: Biotek, Winooski, VA, USA) 34.
For MTT assay, the macrophages were seeded (1.0×104/well) in 96-well plate (Corning Incorporated) and
placed in 85% humidity incubator (Model NU 5700; Nuaire, Plymouth, MA, USA) to grow at 37°C for 24h
in 5% CO2. The culture medium was decanted after 24h to calculate IC50. Three wells of cells were
incubated with concentrations ranging from 1µg/ml, 5µg/ml, 25µg/ml and 50µg/ml for 24h and 48h at
37°C. The nanoparticle solution was removed and MTT was added to each well. The cells were placed in
an incubator at 37°C for 4h. The solution was aspirated, and the formazan crystals were dissolved in
dimethyl sulfoxide. The absorbance was measured at 485nm by using a microplate reader (Microplate
reader, Model FL ×800: Biotek, Winooski, VA, USA). Cytotoxicity on cells was expressed as a
percentage of cell viability as compared to untreated cells (control). IC50 was calculated using the
regression analysis already available in GraphPad Prism Ver 7.3335.
Estimation of TNFα and NF-kB levels
Lymphocytes and macrophages were seeded in 24-well cell culture plate (8 × 104 cells/well). The cells
were exposed to varying concentrations of nanoparticles (1-50µg/ml) in triplicate for 24 h and incubated at
37°C in 5% CO2. Cells without NPs exposure were taken as positive control. Non SVD/DVD/TVD cells
isolated were used to compare the data. Cells were treated with lysis buffer (0.075% triton x-100) and
incubated at 37°C for 30 minutes followed by centrifugation at 1000xg at 2-8°C for 20 minutes. The
supernatant was collected and TNFα and NF-kB levels were measured by ELISA Kit (ELAB Science,
USA) as per manufacturer’s instructions by using microplate reader (Microplate reader, Model FL ×800:
Biotek, Winooski, VA, USA). Baseline serum TNFα and NF-kB levels were also estimated before
lymphocyte and macrophage isolation and culturing.
Gene expression analysis
Lymphocyte and macrophage cultures were exposed to varying concentrations of nanoparticles (Au,
ZnO, MgO) for 24h. RNA was extracted and cDNA was synthesized by using Revert Aid First kit (Thermo-
scientific, USA). Polymerase Chain Reaction (PCR) was performed by following an already published
protocol36. Briefly, initial denaturation for a single cycle at 95 ºC for 05 min, then 30 cycles of denaturation
at 95 ºC for 30 s, annealing temperature 60 ºC for TNF-α, 58 ºC for GAPDH and 54 ºC for NF-kB followed
by elongation at 72 ºC for 45s and final elongation at 72 ºC for 10 min using thermal cycler Bio RAD T100
(USA). Following sets of primers were used: TNF-α, F-5’- AAGAATTCAAACTGGGGCCT-3’ and R-5’-
GAGGAAGGCCTAAGGTCCAC-3, NF-kB, F-5-CTCCACAAGGCAGCAAATAGA-3’ and R-5’-
ACTGGTCAGAGACTCGGTAAA-3’, GAPDH F- 5’-CAAGGTCATCCATGACAACTTTG-3’ and R-5’
GTCCACCACCCTGTTGCTGTAG-3’. Gene expression analysis of TNF alpha and NFKB was
documented after data normalization of treated vs untreated cells whereby using GAPDH as endogenous
control. The relative expression was calculated by comparative CT method (2-Ct method)37,38.
Statistical analysis
Statistical analysis was performed using Graph Pad Prism 8.0.2. Descriptive statistics are given as mean
± standard error of mean. The results were analyzed by using ANOVA and Tukey’s test.
3. Results
Characterization of Au, ZnO and MgO NPs
UV visible spectroscopy was used for initial confirmation of synthesis of nanoparticles. Synthesis of ZnO
NPs was indicated by its absorption peak at 356 nm, at 305 nm for MgO and a characteristic surface
plasmon resonance peak appearing at ~520 nm for Au (Figure 2A-C). The hydrodynamic size measured
with DLS technique was found 80 ± 3, 17 ± 2, 11 ± 4 for ZnO, Au and MgO respectively. The
morphology, as well as population distribution of all three types of nanoparticles, was determined by
scanning electron microscopy (SEM). The SEM analysis confirmed the DLS size measurements. The
SEM size of ZnO NPs was found 77± 45 nm, round in shape with population distribution range of 21-
120nm. Au NPs exhibited 15 ± 2 nm size, round shape with 15-16nm densely populated distribution. The
size of MgO NPs was 11 ± 5 nm, amorphous in shape with size distribution of 9-16nm (Figure 2).
Toxicity of NPs was found dose-dependent
All three NPs (Au, MgO, ZnO) inhibited the proliferation of macrophages and lymphocytes in a
concentration-dependent manner. Cell viability at 1, 5, 25, 50, 75, 100 µg/mL nanoparticle exposure was
found 100, 98, 95, 90, 79, 52, 3% for Au NPs and 100, 100, 98, 94, 81, 54, 0% for MgO NPs. The cell
viability for ZnO NPs was observed as100,100,100,100, 80, 67, 54, 27, 0% at 0.25,0.5, 0.75, 1.5, 5, 25,
50,75,100µg/ml. IC50 values for Au, MgO and ZnO NPs were 74µg/ml, 72µg/ml and 50µg/ml respectively
(Figure 3A-C). Au NPs indicated more cytotoxic effect at lower concentrations after 24 h to macrophages
as compared to ZnO and MgO NPs. All the cells were viable even after 25 µg/ml of ZnO NPs exposure
and showed toxicity at 50 µg/ml. Microscopy also confirmed less toxic effect on macrophages and
lymphocytes morphology when images were captured at 50µg/ml nanoparticles for MgO and Au and at
1µg/ml concentration for ZnO after 24 hrs of exposure (Figure 4). However, effective treatment
concentration (ETC) ranged from 1 to 5 µg/mL for all three types of nanoparticles.
Au, ZnO and MgO NPs showed <5% hemolytic activity
Percentage hemolysis at 5, 10, 20, 40, 60, 80 µg/mL nanoparticle concentration after 1h exposure was
found 0, 0.4, 1.2, 1.4, 1.4, 1.7% for Au NPs, 0.5, 1.9, 2, 2.3, 3.9, 4.5 % for MgO and 0.5, 1.9, 2.5, 2.6, 2.9,
4% for ZnO. Less than 5% values declare these NPs safe for use at 80 µg/mL as per ASTM E2524-08
standard 39 (Figure 3D-F).
Therapeutic effects on lymphocytes TNFα and NF-kB levels depend upon CAD
category, NP type and concentration
Varying effects on decrease in TNFα and NF-kB levels were observed in nanoparticles treated
lymphocyte cultures when compared with untreated ones (Figure 5). ZnO NPs treatment resulted in
substantial decrease in TNFα (69%) and NF-kB (53%) levels at an ETC of 1µg/ml in SVD cultures
(p≤0.001) (Figure 5 A & B). ZnO NPs also exhibited significant (p≤0.001) effect in TVD cultures by
reducing 52% TNF level at only 1µg/m. ZnO NPs reduced only 31% level in DVD cultures. A two-fold
decrease in TNFα (56%) and NF-kB (54%) was observed (p≤0.001) in DVD cultures at 5µg/ml of MgO
NPs (Figure 5 C &, D). MgO also reduced 40% TNF level in TVD cultures at 5µg/ml whereas they were
less effective in SVD cultures (Figure 5E). Au NPs showed significant (p≤0.001) reduction of 46% and
42% TNFα level in SVD and DVD cultures at an ETC value of 5µg/ml. Au NP showed non-significant
effect in TVD cultures. A significant (p≤0.001) decrease of 50% and 63% in NF-kB level was observed in
TVD cultures when exposed with 5µg/ml of Au and MgO NPs respectively (Figure 5 E &, F).
ZnO were most effective in SVD and TVD cultures with more than 50% reduction whereas MgO showed
greater efficacy in DVD cultures with 56% reduction in TNFα level. ZnO NPs were found highly effective
in SVD cultures with 3-fold reduction whereas moderate effective in TVD cultures with 2-fold reduction in
TNFα. ZnO showed minimum efficacy in DVD cultures with 1.7-fold reduction in TNF level. MgO showed
higher efficacy in SVD cultures with 3-fold reduction and moderate (2-fold) in DVD and TVD cultures in
TNFα level. AuNPs exhibited moderate efficacy of 2-fold reduction in all three types of cultures (Table 1).
Au NPs were found highly effective (3-fold reduction) in SVD, DVD and TVD cultures against NF-kB level.
ZnO NPs showed 3-fold reduction in DVD cultures and moderate effective (2-fold reduction) in SVD and
TVD cultures. MgO NPs exhibited moderate effect (2.5-fold reduction) in all three disease types against
NF-kB level (Table 1).
Nanoparticles exhibit good efficacy against TNFα and NF-kB in CAD macrophage
cultures
Significant reduction in TNFα and NF-kB levels were observed in SVD, DVD and TVD macrophage
cultures after nanoparticles exposure. It was observed that ZnO NPs treatment resulted in considerable
decrease in TNFα level (48%) and NF-kB (34%) at a minimal ETC value of 1µg/ml in SVD cultures with
p≤0.001 (Figure 6 A &B). Also, a three-fold (77%) decrease (p≤0.001) in TNFα and 34% decrease
(p≤0.001) in NF-kB level was observed in DVD culture when treated with 1µg/ml ZnO NPs. At this ETC,
ZnO NPs also reduced the 42% TNFα and 41% NF-kB level in TVD cultures. MgO decreased 42% and
35% TNFα level in TVD and DVD cultures at and ETC value of 5µg/ml whereas they showed non-
significant effect in SVD cultures of macrophages. Au NPs significantly (p≤0.001) reduced the TNFα
(55%) in DVD cultures (Figure 6 C&D). Overall, Au NPs did not show a promising effect in TVD and SVD
cultures in lowering TNFα level while they showed greater efficacy in DVD.
A 4-fold reduction in TNFα level was found when DVD macrophage culture was treated with ZnO NPs.
Also, ZnO was found effective in TVD and SVD cultures with reduction of 1.5-fold TNFα level. Lower
effect (1.5-fold) was noted on TNFα in SVD, DVD and TVD cultures when treated with Au and MgO NPs
(Table 1). Whereas, all three NPs exhibited 1-1.5-fold reduction of NF-kB level in SVD, DVD and TVD
macrophage cultures (Table 1).
Effect of NPs on gene expression of TNFα and NF-kB in SVD, DVD and TVD
lymphocyte and macrophage cultures
Gene expression analysis of TNF alpha and NFKB was documented after data normalization, using
GAPDH as endogenous control. The TNF alpha and NFKB expression data was collected after exposure
to different concentrations of nanoparticles. Percentage difference was generated by the comparative
analysis of the effect on TNF alpha and NFKB expression on different concentrations of individual
nanoparticles. The results indicated that reduction in expression of TNFα and NF-kB at transcriptional
level contributed to the inhibitory effect of nanoparticles on cytokine production.
AuNPs showed (p<0.01) with 57% reduction in TNFα expression in SVD, 77% in DVD and 36% in TVD
lymphocyte cultures at 5µg/ml (Figure 7A). However, MgO NPs resulted in substantial effect (p<0.001) of
85% in DVD and 35% in TVD lymphocyte cultures at g/ml (Figure 7C). In case of ZnO NPs, the most
noticeable effect with 71% reduction in mRNA expression was observed in DVD culture and 64% in TVD
culture at 1µg/ml (Figure 7E). Moreover, AuNPs showed greater effect (p<0.001) with 54% reduction in
NF-kB expression of SVD, 73% in DVD and 45% in TVD lymphocyte cultures at 1µg/ml (Figure 7B).
However, MgO NPs resulted in substantial effect (p<0.001) of 85% in DVD and 28% in TVD cultures at
1µg/ml (Figure 7D). In case of ZnO NPs, the most noticeable effect with 95% reduction in mRNA
expression was observed in DVD culture and 95% in SVD culture at 1µg/ml (Figure 7F).
AuNPs showed statistically significant effect (p<0.01) with 56% reduction in TNFα expression in SVD,
71% in DVD and 30% in TVD macrophage cultures at 5µg/ml (Figure 8A). However, MgO NPs resulted in
a substantial effect (p<0.01) of 58% in SVD, 47% in DVD and 42% in TVD macrophage cultures at 5µg/ml
(Figure 8C). In case of ZnO NPs, the most noticeable effect with 89% reduction in mRNA expression was
observed in DVD culture and 80% in SVD culture at 1µg/ml (Figure 8E). Moreover, AuNPs showed a
greater effect (p<0.1) with 34% reduction in NF-kB expression of SVD, 30% in DVD and 26% in TVD
macrophage cultures at 5µg/ml (Figure 8B). However, MgO NPs resulted in a substantial effect (p<0.01)
of 40% in SVD and 72% in DVD cultures at 5µg/ml (Figure 8D). In case of ZnO NPs, the most noticeable
effect with 74% reduction in mRNA expression was observed in TVD culture and 57% in SVD culture at
1µg/ml (Figure 8F).
4. Discussion
The role of TNFα in coronary artery disease (CAD) severity has intrigued scientists to develop new
therapeutic strategies. Patients having Non-Steroid Anti-inflammatory Drugs (NSAIDs) may suffer from
cardiovascular complications, gastrointestinal tract complications and peptic ulcers40. In current study, we
have successfully reduced the TNFα and NF-kB levels in CAD cultures by using gold, zinc oxide and
magnesium oxide nanoparticles. Anti-inflammatory properties of nanoparticles like silver19, gold20,
copper21, zinc oxide22, magnesium oxide41, iron oxide42 and titanium dioxide23 are well documented.
These particles have shown good penetration capability in inflammatory and epithelial cells which
ultimately result in effective treatment. The anti-inflammatory effect of 10-20nm gold NPs(is well
documented in Rheumatoid Arthritis (RA) and type-I diabetes with significant reduction in cytokine levels
and suppression of COX-2 activity27,43 . Reports have been found stating a dose-dependent decrease in
TNFα release with 100nm MgO44. We were unable to find effect of ZnO particles in any disease model.
We hereby report the anti-inflammatory novel properties of Au (15nm), MgO (11nm) and ZnO (70nm) in
different coronary artery disease categories, for the first time.
Role of TNFα and NF-kB is multifaceted. Targeting the TNFα and NF-kB signaling pathway might be an
important approach for the development of effective anti-inflammatory therapies. We have investigated
the post-treatment effect of varying concentrations of Au, MgO and ZnO NPs on TNFα and NF-kB levels
in CAD lymphocytes and macrophage cultures. Our findings showed that ZnO and MgO NPs exhibited
noticeable effect (3.2-fold reduction, p≤0.001) against the TNFα level in lymphocytes and MgO and Au
NPs significantly (1.6-fold p≤0.001,) reduced the TNFα in macrophages of SVD culture. Au NPs showed
greater efficacy (p≤0.001) against NF-kB level in both lymphocytes (3-fold) and macrophages (1.8-fold) of
SVD culture (Table 1). Interestingly, our findings provided an insight that how diverse and specific is the
role of each nanoparticle to combat inflammation. Findings suggest that the effective concentration of
ZnO NPs is 1µg/ml against SVD, DVD and TVD in both lymphocyte and macrophage cultures with
various reduction levels. ZnO nanoparticles have shown dose-dependent increase in TNFα level from
5µg/ml to 50µg/ml (Figure 5 and 6). But this increase may be attributed towards its potential to induce
inflammation and cytotoxicity at higher concentrations. We have observed 20-50% cell death at these
concentrations (Figure 3). It has been reported that cytotoxicity is a result of subsequent release of Zn2+
ions and dissolution45. For Au and MgO NPs, the effective therapeutic concentrations in all three disease
categories have been observed from 5 to 25 µg/ml. Below this concentration both particles have been
found biocompatible with less than 3% hemolytic activity and 98% cell viability (Figure 3). The inhibition of
TNFα and NF-kB is unlikely to have resulted from nanoparticle-induced toxicity because the cells remain
viable at respective effective concentrations of each nanoparticle. Above 25µg/ml, an increase in toxicity
i.e., >20% has been observed making this concentration unsafe for therapeutic effect.
The use of gene regulation through nanoparticles has been hypothesized as preventive therapy for
atherosclerosis. Nanoparticle loaded small interfering RNAs have shown to downregulate chemokine
receptor expression which is a known culprit for inflammatory monocyte infiltration at atherosclerotic
plaque46. There have been various attempts in targeting the transformation of inflammatory monocytes
into macrophages. Nanoparticles conjugated drugs have demonstrated positive results in converting the
pro-inflammatory monocytes to non-inflammatory cells in mice models. Moreover, the use of anti-
inflammatory drugs like methotrexate and statins has been investigated in experimental models with
promising results47. In a recent study in Pakistani population, it is reported that statins (anti-inflammatory
drugs) down-regulate the expression of NF-kB and TNFα genes36. This potentiates our findings that anti-
inflammatory effects of nanoparticles can not only improve the disease cure process but also delay the
progression of this debilitating disease. The real-time PCR analysis of TNFα and NF-kB genes also
revealed that all three NPs down-regulate the expression of mRNA in treated cells.
Increased levels of cytokines (TNFα and NF-kB) in atherosclerotic plague are one of the main causes of
conversion of monocyte into macrophages. These macrophages are involved in pathology of
atherosclerosis converted into lipid-filled foam cells by absorbing oxidized LDLs and start releasing
inflammatory cytokines and the cycle continues48. Hence reduction in pro-inflammatory cytokines might
be an effective approach to decrease the risk of arterial stenosis and atherosclerosis. Other than anti-
inflammatory properties, lowering of TNFα and NF-kB levels can be a valuable strategy to prevent the
complications of coronary artery disease. Abnormal lipid profile is listed among causative factors of CAD.
High levels of TNFα indirectly increase triglycerides levels through hepatic acid synthesis pathway49, 50.
Data collected from patients (n=50) after three months of statin therapy has revealed that lipid profile
(Cholesterol, Low Density Lipoproteins (LDL), Triglycerides) of CAD patients decreased from 283±10,
228± 20,150±10 mg/dL to 190±10, 124±20, 127±10 mg/dL respectively. High density lipoproteins (HDL)
level also increases from low (25±10) to high (34±10) after treatment. Moreover, TNFα directly interferes
with lipid metabolism pathways (Triglycerides, Cholesterol) and induces proatherogenic lipoprotein
changes. It has already been reported that patients with increased triglycerides, LDL and low HDL levels
have inflammation and a higher risk of disease36. Popa C et al., has reported an increase in HDL and
lower atherogenic index in RA by using anti-TNFα therapy51. High level of TNFα suppresses the
antithrombotic protein C pathway in endothelial cells and enhances the pro-coagulant activity (PAI-1, von
Willebrand factor) by interfering with thrombotic process that increases the risk of CAD52. It also
contributes to clinical problems related to autoimmune disorders53. The canonical NF-kB pathway,
prototypical pro-inflammatory cytokines have an important role in the pathogenesis of inflammatory
diseases like asthma, inflammatory bowel disease (IBD), chronic obstructive pulmonary disease (COPD)
and rheumatoid arthritis15.
To conclude, nanoparticles can be used as a novel approach for the development of targeted preventive
therapies in reducing the inflammation in different coronary artery disease conditions. Lowering of
cytokine levels may reduce the risk of atherosclerosis and myocardial infarction by lowering the
inflammation and improving the lipid profile of CAD patients. Au, ZnO and MgO NPs showed good down-
regulation capability against TNFα and NF-kB levels, depending on the disease condition and particle
concentration. The effective therapeutic concentration for Au and MgO NPs is 5µg/ml and 1µg/ml for ZnO
NPs in all CAD conditions. ZnO NPs remain most statistically significant (p>0.001) in SVD and TVD
cultures whereas MgO show efficacy in DVD and TVD cultures with more than 50% reduction in TNFα
and NF-kB level. Au NPs exhibit prominent effect in DVD cultures. The substantial experimental evidence
of reduction on inflammatory TNFα with the use of nanoparticles of ZnO, MgO and Au with low toxic
concentrations encourages the study application against inflammatory diseases like inflammatory bowel
disease and rheumatoid arthritis and where the inflammation is found to be more prominent.
Acknowledgments
We heartily thank Pakistan Academy of Sciences for financial support and Dr. Abdul Basit, Chancellor of
the Preston University for providing all facilities of the University at the disposal of the project. We thank
Dr. Irshad Hussain from LUMS and Dr. M. Yakoob from NCP for providing analytical facilities for particle
characterization.
Funding
This work was supported by Pakistan Academy of Sciences [Grant No.5-9/PAS/261].
Declaration of interest
The authors have no conflict of interest.
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Figure 1. Thematic Diagram of Anti-Inflammatory Properties of ZnO, MgO and Au Nanoparticles as a
Novel Tool to Down-Regulate TNFα and NF-kB in Human Lymphocytes and Macrophages of Coronary
Artery Disease.
ZnO
MgO
Au
A
B
C
D
E
F
G
J
H
K
I
L
A
D
B
C
E
F
Figure 3. Au, ZnO and MgO nanoparticles biocompatibility studies: Cell viabilities (A-C) of A) Au NPs, B)
MgO NPs, C) ZnO NPs. Rectangular area corresponds to effective therapeutic concentration (ETC) range
where cell viability is almost 100%; Hemolytic activities (D-F) of D) Au NPs, E) MgO NPs, F) ZnO NPs. All
three nanoparticles show <1% hemolysis within ETC.
A
B
C
D
E
F
G
H
Figure 4. Microscopic confirmation of less toxic effect of nanoparticles on lymphocyte and
macrophage cultures. A-D) Lymphocyte culture; The cells remain alive and intact in shape after 50µg/ml
exposure of Au NPs (A), MgO NPs (B) ZnO NPs at 1µg/ml (C), when compared with lymphocytes without
NPs exposure (D). Similarly, Macrophage culture; The cells remain alive and intact in shape after 50µg/ml
exposure of Au NPs (E), MgO NPs (F) ZnO NPs at 1µg/ml (G), when compared with macrophages without
NPs exposure (H). The exposure time of nanoparticles is 24h. Images were taken by using EVOS FL®
Thermo scientific Fisher, UK at 10x.
C
D
B
A
E
F
Figure 5. Effect of Au, ZnO and MgO nanoparticles on TNFα and NF-kB level levels in lymphocyte
cultures. Coronary artery disease culture categories A-B) Single Vessel Disease (SVD); C-D) Double
Vessel Disease (DVD); E-F); Triple Vessel Disease (TVD). Lymphocytes were exposed from 1 to 50
µg/ml of NPs for 24h. Non-SVD/TVD/DVD C: A non-SVD/TVD/DVD lymphocyte culture control. Positive
control: Lymphocytes without NPs exposure. Bars correspond both inter-individual differences and
experimental errors. Statistical analysis was carried out with Two-way ANOVA followed by the Tukey
multiple comparison test (*P<0.05 **P < 0.01, ***P < 0.001, ns Non significant).
E
F
Figure 6. Effect of Au, ZnO and MgO nanoparticles on TNFα and NF-kB level levels in macrophage
cultures. Coronary artery disease culture categories A-B) Single Vessel Disease (SVD); C-D) Double
Vessel Disease (DVD); E-F); Triple Vessel Disease (TVD). Macrophages were exposed from 1 to 50
µg/ml of NPs for 24h. Non-SVD/TVD/DVD C: A non-SVD/TVD/DVD macrophage culture control. Positive
control: Macrophage without NPs exposure. Bars correspond both inter-individual differences and
experimental errors. Statistical analysis was carried out with Two-way ANOVA followed by the Tukey
B
D
A
multiple comparison test (*P<0.05 **P < 0.01, ***P < 0.001, ns Non significant).
C
D
E
F
A
C
B
Figure 7. Relative expression of TNFα and NF-kB gene after NPs exposure in lymphocytes. A & B:
Au NPs effect on TNFα and NF-kB; C& D: MgO NPs effect on TNFα and NF-kB; E & F; ZnO NPs effect on
TNFα and NF-kB. Lymphocytes were exposed are exposed to varying concentrations of NPs for 24h. The
relative expression was calculated by comparative CT method (2-Ct method). Bars correspond both inter-
individual differences and experimental errors. Statistical analysis was carried out with Two-way ANOVA
followed by the Tukey multiple comparison tests (*P<0.05 **P < 0.01, ***P < 0.001, ns non-significant).
C
D
E
F
Figure 8. Relative expression of TNFα and NF-kB gene after NPs exposure in macrophages. A & B:
Au NPs effect on TNFα and NF-kB; C& D: MgO NPs effect on TNFα and NF-kB; E & F; ZnO NPs effect
on TNFα and NF-kB. Macrophages are exposed to varying concentrations of NPs for 24h. The relative
expression was calculated by comparative CT method (2-Ct method). Bars correspond both inter-
individual differences and experimental errors. Statistical analysis was carried out with Two-way ANOVA
followed by the Tukey multiple comparison tests (*P<0.05 **P < 0.01, ***P < 0.001, ns non-significant).
A
B
Table1. Fold reduction in TNFα and NF-kB level in coronary artery disease macrophage and lymphocyte
culture after nanoparticle exposure
TNFα level after NPs exposure
NPs concentrations
1
5
25
50
1
5
25
50
Disease
Nanoparticle
type
Macrophage Culture
Lymphocyte Culture
Au NPs
0
1.3
1.4
1.6
1.6
1.85
2.2
2.6
ZnO NPs
1.8
1.6
1.58
1.55
3.25
2.85
2.7
1.08
MgO NPs
0
1.2
1.25
1.6
1.29
1.35
1.9
3.25
Au NPs
1.88
2
2.1
2.2
1.62
1.66
1.77
2.2
ZnO NPs
4.1
4
2.3
2.1
1.7
1.39
1.35
1.25
MgO NPs
1.5
1.6
1.7
1.8
2
2.1
2.22
2.3
Au NPs
1.1
1.22
1.32
1.39
1.1
1.53
1.90
2.06
ZnO NPs
1.70
1.70
1.6
1.3
2.1
1.90
1.68
1.5
MgO NPs
1.42
1.78
1.85
1.87
1.61
1.82
1.97
2.21
NF-kB level after NPs exposure
Disease
Nanoparticle
type
Macrophages Culture
Lymphocyte Culture
Au NPs
0.08
1.2
1.55
1.8
1.91
2.1
2.4
3.0
ZnO NPs
1.45
1.25
1.21
1.12
2.54
2.45
2.24
2.13
MgO NPs
1.07
1.25
1.66
1.73
1.88
2
2.2
2.7
Au NPs
0
0.5
0.97
0.8
2.3
2.45
2.88
3.12
ZnO NPs
1.35
1.25
0.82
0.76
3
2.94
2.9
2.7
MgO NPs
1.58
1.63
1.82
1.86
2.08
2.17
2.34
2.41
Au NPs
0.91
1.15
1.31
1.59
2.02
2.10
2.50
2.76
ZnO NPs
1.67
1.35
1.15
1.04
2
1.81
1.61
1.59
MgO NPs
1.0
1.1
1.20
1.21
2.5
2.7
2.7
2.9
Lymphocyte and macrophage cells from coronary artery disease patients were treated with Au, ZnO and
MgO nanoparticles at various concentrations of 1, 5, 25, and 50µg/ml. The NPs significantly reduced the
TNF and NF-kB level which is mentioned in term of fold reduction. Single Vessel Disease (SVD), Double
Vessel Disease (DVD) and Triple-Vessel coronary artery disease (TVD)
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Nanomedicine is one of the most promising therapeutic modalities researchers are working on. It involves development of drugs and devices that work at the nanoscale (10–9 m). Coronary artery disease (CAD) is responsible for more than a third of all deaths in age group >35 years. With such a huge burden of mortality, CAD is one of the diseases where nanomedicine is being employed for preventive and therapeutic interventions. Nanomedicine can effectively deliver focused drug payload at sites of local plaque formation. Non-invasive strategies include thwarting angiogenesis, intra-arterial thrombosis and local inflammation. Invasive strategies following percutaneous coronary intervention (PCI) include anti-restenosis and healing enhancement. However, before practical application becomes widespread, many challenges need to be dealt with. These include manufacturing at the nanoscale, direct nanomaterial cellular toxicity and visualization.
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