Nanopartículas Magnéticas com Dextrano e Ácido Protocatecuico para Terapia de Inflamação Vascular - Artigo Científico

Article Development of DextranCoated Magnetic Nanoparticles Loaded with Protocatechuic Acid for Vascular Inflammation Therapy Maria Anghelache 1 Mihaela Turtoi 1 Anca Roxana Petrovici 2 Adrian Fifere 2 Mariana Pinteala 2 and Manuela Calin 1 1 Medical and Pharmaceutical Bionanotechnologies Laboratory Institute of Cellular Biology and Pathology Nicolae Simionescu of the Romanian Academy BP Hasdeu 8 050568 Bucharest Romania mariaanghelacheicbpro MA mihaelacarnutaicbpro MT 2 Centre of Advanced Research in Bionanoconjugates and Biopolymers Department Petru Poni Institute of Macromolecular Chemistry 41A Grigore GhicaVoda Alley 700487 Iasi Romania petroviciancaicmppro ARP pintealaicmppro MP Correspondence fifereicmppro AF manuelacalinicbpro MC Abstract Vascular inflammation plays a crucial role in the progression of various pathologies including atherosclerosis AS and thus it has become an attractive therapeutic target The protocatechuic acid PCA one of the main metabolites of complex polyphenols is endowed with antiinflammatory activity but its formulation into nanocarriers may increase its bioavailability In this study we developed and characterized dextran shelliron oxide core nanoparticles loaded with PCA MNPDexPCA and assessed their cytotoxicity and antiinflammatory potential on cells acting as key players in the onset and progression of AS namely endothelial cells EC and monocytesmacrophages The results showed that MNPDexPCA exert an antiinflammatory activity at noncytotoxic and therapeutically relevant concentrations of PCA 350 µM as supported by the reduced levels of inflammatory molecules such as MCP1 IL1β TNFα IL6 and CCR2 in activated EC and M1type macrophages and functional monocyte adhesion assay The antiinflammatory effect of MNPDexPCA was associated with the reduction in the levels of ERK12 and p38α mitogenactivated protein kinases MAPKs and NFkB transcription factor Our data support the further development of dextran shellmagnetic core nanoparticles as theranostic nanoparticles for guidance imaging and therapy of vascular inflammation using PCA or other antiinflammatory compounds Keywords magnetic nanoparticles dextran protocatechuic acid antiinflammatory activity endothelial cells macrophages 1 Introduction Cardiovascular diseases CVD are the leading cause of global mortality and contribute significantly to a reduction in quality of life 1 Until recently cholesterol accumulation in the arterial wall were considered the primary cause of atherosclerosis AS 2 Both preclinical and clinical research have shown that chronic inflammatory processes and lipid abnormalities interact to cause AS 3 Although vascular endothelial cells EC normally impede the adhesion of circulating immune cells 4 inflammation and hyperlipidemia can stimulate the endothelial expression of cell adhesion molecules and increase the permeability of blood vessels walls as well as the neutrophil and monocyte recruitment with the subsequent overproduction of different inflammatory molecules 5 Many immune cells participate in AS development but monocytederived macrophages play a central role by phagocytosing lipids which triggers their transformation into foam cells 6 As chronic inflammation underlies the emergence and evolution of various pathologies it has become Citation Anghelache M Turtoi M Petrovici AR Fifere A Pinteala M Calin M Development of DextranCoated Magnetic Nanoparticles Loaded with Protocatechuic Acid for Vascular Inflammation Therapy Pharmaceutics 2021 13 1414 httpsdoiorg 103390pharmaceutics13091414 Academic Editor Xiangyang Shi Received 2 August 2021 Accepted 3 September 2021 Published 7 September 2021 Publishers Note MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations Copyright 2021 by the authors Licensee MDPI Basel Switzerland This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution CC BY license httpscreativecommonsorglicensesby40 Pharmaceutics 2021 13 1414 httpsdoiorg103390pharmaceutics13091414 httpswwwmdpicomjournalpharmaceutics Pharmaceutics 2021 13 1414 2 of 19 an attractive therapeutic target 7 The primary signaling molecules regulating chronic inflammatory responses within EC and macrophages are MitogenActivated Protein Ki nases MAPKs and Nuclear Factorkappa B NFkB 8 MAPKs are a family of enzymes more specifically serinethreonine protein kinases implicated in the synthesis regulation of several transcription factors that mediate the inflammation at a transcriptional level 9 Similarly the transcription factor NFkB is considered a master regulator of inflammation which once activated in response to different types of stimuli including inflammatory signals leads to the production of proinflammatory cytokines such as interleukin IL1β IL6 and TNFα and chemokines such as monocyte chemoattractant protein MCP1 10 The use of natural bioactive compounds including polyphenols in the development of new antiinflammatory therapies has become an emerging trend in recent years 11 Al though documented evidence exists of polyphenols exhibiting antiinflammatory activities their reduced bioavailability and stability represent a major drawback that restricts their use as therapeutic agents 12 Since nanoparticles can achieve controlled drug targeting and release a nanotherapeutic approach can overcome these obstacles as nanotherapy was successfully introduced in cancer treatment and has recently gained prominence in CVD therapeutic strategies 1314 Magnetic nanoparticles MNP specifically iron oxide nanoparticles have been widely used for diagnostic purposes for instance for visualization of tumors and metastases in the liver spleen and lymph nodes as well as inflammatory lesions such as atherosclerotic plaques 15 Based on their magnetic properties MNP have been extensively studied in the last decade for their potential use in drug target ing 1617 The effects of plain MNP on EC are still an area of research that is somewhat underdeveloped Considering their importance in regulating immune responses and their location at the interface between blood and the vessel wall more knowledge of MNPs intrinsic activity on EC will improve theranostic applications 18 Protocatechuic acid 34dihydroxybenzoic acid PCA a phenolic acid found in medicinal plants such as Roselle Hibiscus sabdariffa L or Japanese ginkgo Ginkgo biloba L has several therapeutic effects associated with antioxidant antibacterial antiaging antifibrotic and antiinflammatory activity or even anticancer properties at the appropriate concentrations 19 Human studies reported that PCA produced as a degradation metabolite by the intestinal mi crobiota from flavonoids such as anthocyanins or procyanidins is bioavailable in serum after flavonoids intake 20 After absorption through the intestinal epithelium PCA may reach systemic blood circulation delivered to the liver where it may undergo structural modifications resulting in sulfated and glucuronidated forms that can be distributed to the tissues or excreted in the urine or feces However the available concentrations of PCA and its conjugates may be too low to exert their therapeutic benefit at a defined place To achieve efficient antiinflammatory activity PCA has to be concentrated at the inflammatory sites and intracellular delivery has to be promoted With this in mind we aimed to develop and characterize coreshell magnetic nanoparti cles covered with dextran MNPDex and loaded with protocatechuic acid MNPDexPCA as well as to assess their cytotoxicity and antiinflammatory potential on cells playing a central role in the onset and progression of AS namely EC and macrophages The antiinflammatory activity of MNPDexPCA was investigated by assessing the adhesion capacity of THP1 monocytes to tumor necrosis factor TNFα activated EC EAhy926 cells and treated with MNPDexPCA In addition the effect of MNPDexPCA on the protein expression of MAPKs ERK12 and p38α isoform as well as the transcription factor NFkB and levels of MCP1 chemokine in activated EC and proinflammatory factors IL1β TNFα IL6 and CCR2 in THP1derived M1 type inflammatory macrophages were measured To the best of our knowledge this is the first study showing that coreshell magnetic nanoparticles covered with dextran and loaded with PCA have an antiinflammatory ac tivity at noncytotoxic concentrations by reducing the levels of inflammatory molecules such as MCP1 IL1β TNFα IL6 and CCR2 in activated EC and M1type inflamma tory macrophages The antiinflammatory effect of MNPDexPCA was associated with diminution in the levels of MAPKs ERK12 p38α and NFkB transcription factor Pharmaceutics 2021 13 1414 3 of 19 The data support the use and further development of dextrancoated MNP loaded with PCA as a new theranostic system for guidance and therapeutic action of PCA in various chronic inflammatory illnesses including AS The formulation of PCA into magnetic nanoparticles may assure a targeted delivery at the affected site due to the magnetic properties of the nanocarrier and the augmented efficiency of PCA as an antiinflammatory agent by improving its cellular internalization 2 Materials and Methods 21 Materials Commercial sources of the primary reagents and consumables used in this study were as follows ferric chloride FeCl36H2O ferrous chloride FeCl24H2O 25 ammonium solution 25 PCA ammonium persulfate APS 27bis2carboxyethyl56carboxyfluorescein ace toxymethyl ester BCECFAM dexamethasone Dexa Dulbeccos modified Eagles medium ethylenediaminetetraacetic acid tetrasodium salt dihydrate EDTA glycerol hydrochloric acid HCl lipopolysaccharides from Escherichia coli serotype O111B 2mercaptoethanol NNNNTetramethyl ethylenediamine TEMED phenylmethylsulphonyl fluoride PMSF paraformaldehyde PFA Ponceau S solution potassium hexacyanoferrateII trihydrate RPMI1640 medium sodium dodecyl sulphate SDS TritonX100 and TrisHCl which were purchased from SIGMAAldrich Merck KGaA Darmstadt Germany fetal bovine serum and penicillinstreptomycin which were purchased from Thermo Fisher Scientific Waltham MA USA recombinant human tumor necrosis factoralpha TNFα from RD Systems Inc Abingdon UK cell culture dishes which were purchased from TPP Trasadingen Switzerland the transparentblack 96well micro test plates Fbottom which were purchased from Ratiolab Ratiolab GmbH Dreieich Germany and UV 96well micro test plates Fbottom which were purchased from Corning Inc New York NY USA Dextran was biosynthesized as exopolysaccharides from Weissella confusa a yogurtisolated lactic acid bacterium as previously reported 20 A culture medium consisting of DeManRogosa Sharpe MRS agar 553 gL and sucrose 80 gL dissolved in UHT milk was inoculated with Weissella confusa and after 48 h of fermentation the dextran was extracted and purified as detailed in 21 22 Loading of PCA on Magnetic Nanoparticles 221 MNP Synthesis The MNP were synthesized by the coprecipitation method and coated with dextran as reported previously 22 Briefly a solution of 0871 g FeCl24H2O and 223 g FeCl36H2O dissolved in 40 mL of deionized water was deoxygenated by bubbling with nitrogen and heated to 70 C with mechanical stirring The pH of the solution was raised to 14 by the dropwise addition of 10 mL of ammonium hydroxide solution 30 NH4OH under a stirring rate of 1000 rpm The reaction was maintained at room temperature under nitrogen for 30 min after which the black precipitate formed was extracted by magnetic decantation and washed by centrifugation until it reached neutral pH MNP were stored in alcohol at 4 C in the refrigerator and washed followed by redispersion into water before use 222 Synthesis of DextranCoated MNP MNPDex MNP were coated with dextran by a method reported in our previous work 22 A solution formed by suspending 100 mg MNP in 50 mL of 2 dextran solution was heated at 80 C for 3 h under vigorous mechanical stirring The nanoparticles were magnetically decanted and washed five times with deionized water to remove free polymers 223 Synthesis of PCALoaded DextranCoated MNP MNPDexPCA MNPDex were loaded with PCA by our previously reported method applied for polyethyleneiminecoated MNP with minor modifications 23 Over 1 mL of a 10 mgmL PCA solution was added to 1 mL of MNPDex solution 10 mgmL in water and sonicated for 3 min The aqueous formulations were further mixed and shaken for 20 min at 23 C Pharmaceutics 2021 13 1414 4 of 19 900 rpm Subsequently the PCAfunctionalized nanoparticles were separated from the final mixture by centrifugation at 10000 rpm for 15 min followed by two washing steps and redispersion in sterile water 23 PhysicoChemical Characterization of PCALoaded Magnetic Nanoparticles 231 Morphological Analysis of MNPDexPCA The MNP MNPDex and MNPDexPCA dispersed in water were deposited on carboncoated copper grids 300mesh sizes and further investigated by transmission electron microscopy TEM using a Hitachi HighTech HT7700 Transmission Electron Microscope Hitachi HighTechnologies Corporation Tokyo Japan operated at a 100 kV accelerating voltage in highcontrast fashion 232 Size and Zeta Potential The hydrodynamic diameter and Zeta potential of waterdispersed MNP MNPDex and MNPDexPCA were evaluated at room temperature using the Delsa Nano C Submi cron Particle Size Analyzer Beckman Coulter Inc Fullerton CA USA equipped with a laser operating at 658 nm as previously described 23 233 PCA Loading on MNPDex The MNPDexPCA suspensions were decanted by centrifugation a volume of 10 µL of the supernatant was injected into a Zorbax SBC8 column 5 µm 150 mm 46 mm and the volume of the mobile phase was 12 mLmin The mobile gradient phase consisted of A 01 HCOOH in water pH 2 and B acetonitrile H2O at a ratio of 955 Using the PCA calibration curve the loading efficiency of PCA on MNPDex was calculated with the formula Loading efficiency total amount of PCA free amount of PCA total amount of PCA where the total amount of PCA is the quantity of PCA used for sample preparation and the free amount of PCA is the amount of PCA determined from the supernatant resulting from the centrifugation of the nanoparticle suspension 24 In Vitro Evaluation of PCALoaded Magnetic Nanoparticles 241 Cell Culture The cell types used for the in vitro studies are human endothelial cell EC line EAhy926 and the human monocytic cell line THP1 purchased from American Type Culture Collection ATCC Manassas VA USA EC were grown as monolayers in T25 culture flasks TPP Tissue Culture Plates Trasadingen Switzerland while monocytes were cultured in 60 mm lowadherent cell culture dishes Eppendorf Hamburg Germany Both cell types were maintained under standard culture conditions 37 C 95 humidi fied atmosphere and 5 CO2 in Dulbeccos Modified Eagle Medium DMEM with 01 glucose or RPMI1640 medium respectively supplemented with 10 fetal bovine serum FBS heatinactivated in the case of monocytes and 1 antibiotic solution containing a mixture of penicillin and streptomycin 100 unitsmL each The cultures were periodically subcultivated using a TrypsinEDTA solution according to the ATCC protocols The antiinflammatory effect of MNPDexPCA was assessed on EAhy926 and M1polarized THP1derived macrophages To induce monocytetomacrophage differentiation THP1 cells were cultured for 48 h in the presence of 100 nM phorbol 12myristate 13acetate PMA obtaining resting macrophages M0 Next M0 macrophages were further incu bated for 24 h with 20 ngmL lipopolysaccharides LPS to obtain M1macrophages with a proinflammatory phenotype Pharmaceutics 2021 13 1414 5 of 19 242 Cell Cytotoxicity Assays Colorimetric Assay The cytotoxicity of different concentrations of MNPDexPCA MNPDex and free PCA was assessed by the XTT assay as previously described 24 Briefly EC were seeded for 24 h at a density of 15 105 cellscm2 on flatbottom 96well plates and incubated for an additional 24 h with increasing concentrations of MNPDexPCA 1880 µgmL nanoparticles corresponding to 10 450 µM of loaded PCA Unfunctionalized nanoparti cles MNPDex and free PCA were used as controls at the corresponding concentrations At the end of the incubation period a mixture of XTT reagent and phenazine methosulfate PMS was added to each well for 2 h at 37 C Next the absorbance was measured at a wavelength of 450 nm using the Tecan Infinite M200Pro Spectrophotometer microplate reader Cytotoxicity was normalized to untreated EC control and expressed as fold change relative to control Bioluminescent Assay The cytotoxicity of various concentrations of MNPDexPCA as well as of plain MNP Dex and PCA was assessed on M1 macrophages after 24 and 48 h and EC after 48 h of incubation using the ToxiLightTM BioAssay Kit Lonza Bioscience Basel Switzerland 25 The release of adenylate kinase in the cell medium indicates a loss of cell integrity which can be quantified by bioluminescent detection To remove possible interference from uninter nalized MNP and MNPDexPCA supernatants were centrifuged at 10000 rpm for 10 min Measurements were made with Mithras LB 940 Multimode Microplate Reader Berthold Technologies Oak Ridge TN USA at 1s integrated reading as per the manufacturers instructions Cell cytotoxicity was normalized to control cells M0 macrophages and untreated EC and results were expressed as foldchange relative to M0 and EC respectively 243 Magnetic Nanoparticles Internalization The uptake of magnetic nanoparticles was assessed by Prussian Blue staining Both cell types were cultured at a density of 03 105 cellscm2 in 48well plates EAhy926 cells were left to adhere for 24 h after which they were stimulated for another 24 h with 20 ngmL TNFα To evaluate the internalization of magnetic nanoparticles by M1 macrophages THP1 cells were activated to macrophages as above mentioned Both cell types were treated for 24 h with magnetic nanoparticles at an iron concentration of 62 µgmL representing the iron content found in MNPDexPCA for 350 µM PCA loaded onto the system Cells were washed with PBS fixed in 4 paraformaldehyde for 30 min and incubated for another 15 min with a fresh mixture of 10 potassium ferricyanide K3FeCN6 and 20 HCl 11 volume ratio at room temperature As a result of iron oxide dissolution by hydrochloric acid treatment ferric ions were released from MNP forming a waterinsoluble complex with potassium ferrocyanide known as Prussian Blue Therefore the cellular compartments containing MNP were stained in a dark bluepurple color 26 The cytoplasm was counterstained at RT for 10 min with eosin Brightfield microscopy Olympus CKX41 inverted microscope was used to examine the cells with eight areas per well being acquired and processed using ImageJ software version 180 National Institutes of Health NIH Bethesda MD USA 244 Monocyte Adhesion Assay The functional role of MNPDexPCA in reducing endothelium inflammation was investigated by monocyte adhesion assay performed as previously reported 27 EAhy926 cells were cultured in 24well plates at a density of 05 105 cellscm2 and stimulated with 20 ngmL TNFα After 18 h the media was replaced and the cells were incubated for 24 h with MNPDexPCA at an iron concentration of 62 µgmL corresponding to 350 µM of polyphenol loaded on the surface of MNPDex nanoparticles Plain MNPDex and free PCA were used as controls at the appropriate concentrations The monocytes were fluorescently labelled with 1 µg of BCECFAM per 106 cells and incubated at 21 ratio for Pharmaceutics 2021 13 1414 6 of 19 30 min with EC that were previously treated with MNPDexPCA MNPDex and free PCA at 37 C in RPMI 1640 medium containing 005 FBS After the incubation period nonadherent monocytes were removed by washing three times with warm PBS 1 The adherent monocytes were examined using an Olympus IX81 Olympus Corporation Tokyo Japan inverted microscope with a 10 objective and FITC filter and four areas were acquired per well which were later processed with ImageJ software 245 Immunological Detection of Proteins Involved in the Inflammatory Process Immunoblotting Assay In order to evaluate the protein expression of the different key molecules involved in the inflammatory response EAhy926 and THP1 cells were seeded in 6well culture plates at a density of 035 105 cellscm2 and activated accordingly EC were stimulated with 20 ngmL TNFα while monocytetoM1 phenotype macrophage activation was achieved using 100 nM PMA and 20 ngmL LPS Both cell types were incubated for 48 h with MNPDexPCA at a concentration corresponding to 350 µM of polyphenol captured on the surface of magnetic nanoparticles plain MNPDex 62 µgmL and PCA 350 µM as well as dexamethasone Dexa 25 µM After the incubation period cells were lysed in radio immuneprecipitation assay RIPA buffer 28 and subjected to total protein concentration determination by the bicinchoninic acid protein assay BCA The protein extracts were loaded at 30 µglane and separated by 520 SDSPAGE followed by transfer onto a 045 µm pore diameter nitrocellulose membrane using the TransBlot SemiDry system In order to detect proteins of interest the following primary antibodies were used for the membrane probing rabbit antip38α 11000 RD Systems cat no AF8691 mouse antiERK12 11000 Abcam cat no ab36991 rabbit antiBax 11000 Thermo Fisher Scientific cat no MA532031 rabbit antiNFκB 11000 Abcam cat no ab16502 rabbit antiMCP1 12500 Abcam cat no ab9669 rabbit antiIL1β 11000 Abcam cat no ab9722 rabbit antiTNFα 11000 Abcam cat no ab215188 mouse antiCCR2 1600 RD Systems cat no MAB150 and mouse antiβ actin 12000 BIORAD cat no MCA5775GA This step was carried out overnight at 4 C under constant agitation After washing three times with salineTris buffer TBS to which 01 Tween 20 detergent TBST was added the membranes were incubated for 1 h at room temperature RT with the corresponding secondary antibody goat antirabbit IgG or goat antimouse IgG conjugated with horseradish peroxidase HRP 15000 Thermo Fisher Scientific Waltham MA USA cat no 32460 and 32430 respectively Immunological detection was achieved by using SuperSignalTM West Dura chemiluminescent substrate Thermo Fisher Scientific cat no 34076 and G BOX Chemi XX6 image analyzer Band densitometry corresponding to the proteins of interest was performed using the TotalLab TL120 v2009 software Nonlinear Dynamics Ltd Durham NC USA and the values obtained were subsequently reported to the βactin housekeeping protein Flow CytometryBased Immunoassay To evaluate the antiinflammatory potential of MNPDexPCA M1 polarized macropha ges were incubated for 48 h with MNPDexPCA containing 350 µM PCA and relevant controls plain MNPDex free PCA and Dexa Then the IL6 cytokine level was measured in culture medium using a beadbased flow cytometry technique Human IL6 Flex Set Kit BD Biosciences San Jose CA USA as previously described 29 Samples were centrifuged at 10000 rpm for 10 min and analyzed using the GalliosTM flow cytometer Beckman Coulter Life Sciences Brea CA USA Instrument setup was achieved using the beads from the Human Soluble Protein Master Buffer Kit BD Biosciences USA according to the manufacturers instructions Data processing was performed using the Kaluza 15 software Beckman Coulter Life Sciences Brea CA USA IL6 concentration pgmL values were calculated using the calibration curve supplied by the kit Pharmaceutics 2021 13 1414 7 of 19 25 Statistical Analysis Statistical data analysis was performed by oneway ANOVA with Dunnetts or Tukey post hoc test using GraphPadTM Prism software version 703 GraphPad Software Inc San Diego CA USA Statistically significant differences were considered for p values 005 The results were expressed as mean standard deviation SD of at least two independent experiments and replicates 3 Results 31 PhysicoChemical Characterization of PCALoaded Magnetic Nanoparticles 311 Morphological Analysis of Nanoparticles by TEM The analysis of TEM images reveals nanoparticles of approximately spherical shape for MNP MNPDex and MNPDexPCA having an aggregation tendency Figure 1A Following the preparation processes MNP have approximately the same shape and size as the resulting products MNPDex and MNPDexPCA having a diameter in the range of 710 nm Obviously the morphology and the similar dimensions for all types of nanopar ticles are due to the fact that the metal oxide core does not change after the preparation processes and in our case at this resolution the TEM shows mainly the inorganic core 312 Size and Zeta Potential The average hydrodynamic diameter and ζpotential of magnetic nanoparticles un coated MNP MNPDex and MNPDexPCA were measured after 11000 dilution in filtered distilled water Figure 1BD Dynamic light scattering revealed that the hydrody namic diameter of nanoparticles increased after dextran coating from 90 nm for uncoated MNP to 670 nm for MNPDex A possible explanation may be the expansion of dextran due to the solvation of the polymer chains in water which leads to a substantial increase in the hydrodynamic diameter On the other hand the increases in the hydrodynamic diameter may be due to the aggregation phenomena as observed in the analysis of TEM micrographs These results are in agreement with previous results reported for MNP coated with dextran 22 and polyethyleneimine 2330 Similarly the size of MNPDexPCA increased by 70 nm when compared to MNPDex indicating that PCA was loaded on the surface of dextrancoated MNP Figure 1BD The measurements of ζpotential show that it changes as a result of MNP function alization Figure 1CD Thus in water at neutral pH MNP has a relatively low negative ζpotential 1 mV pointing out the propensity of MNP for aggregation The covering of MNP with dextran leads to an increase in the negative value of the ζpotential which reaches a value of about 20 mV indicative of the nanoparticles stability nanoparticles are less likely to aggregate The addition of PCA determines a decrease in the negative value of ζpotential up to 7 mV The inclusion of PCA in the dextran layer may conceivably cause the masking of the negative charges of dextran This process can lead to a slight intensification in aggregation phenomena as observed by the increase in the size which in turn amplifies the decrease in the negative value of the zeta potential Figure 1D 313 PCA Loading Efficiency Following HPLC analysis it was established that the loading efficiency of PCA is 87 87 mg PCA are captured on the surface of 10 mg of MNP Pharmaceutics 2021 13 1414 8 of 19 Pharmaceutics 2021 13 x FOR PEER REVIEW 8 of 21 Figure 1 The schematic representation of MNPDexPCA synthesis A Electron micrographs B representative meas urements of average hydrodynamic diameter C and ζpotential D of MNP MNPDex and MNPDexPCA Results reported as mean SD for 3 individual measurements E Figure 1 The schematic representation of MNPDexPCA synthesis A Electron micrographs B representative mea surements of average hydrodynamic diameter C and ζpotential D of MNP MNPDex and MNPDexPCA Results reported as mean SD for 3 individual measurements E Pharmaceutics 2021 13 1414 9 of 19 32 In Vitro Evaluation of PCALoaded Magnetic Nanoparticles Cytotoxicity The cytotoxic effect of MNPDexPCA was assessed in two cell types EAhy926 cells and THP1derived macrophages by means of two cell cytotoxicity tests XTT Figure 2A and ToxiLight Figure 2BD assays Cells were treated with increasing doses of MNPDexPCA corresponding to polyphenol concentrations of 10 50 100 150 250 350 and 450 µM in the case of EAhy926 cells or 250 300 350 400 and 450 µM in the case of THP1 derived M1 macrophages After 24 h of treatment it can be observed that the PCAfunctionalized magnetic nanoparticles did not have a cytotoxic effect on EAhy926 cells at any concentration tested An increased cytotoxic effect can be observed on M1 macrophages at higher concentrations of captured PCA 400 and 450 µM Figure 2AC Concentrations equal to or exceeding 70 µgmL plain MNPDex the concentration corresponding to MNP loaded with 400 µM PCA proved to be cytotoxic for both cell types after 24 h of incubation Figure 2AC The free polyphenol did not have a cytotoxic effect on the two cell types at any concentration investigated On the contrary as PCA concentration increased M1 phenotype macrophages released less quantifiable adenylate kinase in the cell medium implying that the polyphenol could reverse the cytotoxicity induced by cell treatment with PMA and LPS used in THP1 monocytes differentiation A similar effect can be observed when M1 macrophages are treated with MNPDexPCA in which polyphenol concentration was below 400 µM Figure 2C Pharmaceutics 2021 13 x FOR PEER REVIEW 9 of 21 313 PCA Loading Efficiency Following HPLC analysis it was established that the loading efficiency of PCA is 87 87 mg PCA are captured on the surface of 10 mg of MNP 32 In Vitro Evaluation of PCALoaded Magnetic Nanoparticles Cytotoxicity The cytotoxic effect of MNPDexPCA was assessed in two cell types EAhy926 cells and THP1derived macrophages by means of two cell cytotoxicity tests XTT Figure 2A and ToxiLight Figure 2BD assays Cells were treated with increasing doses of MNP DexPCA corresponding to polyphenol concentrations of 10 50 100 150 250 350 and 450 μM in the case of EAhy926 cells or 250 300 350 400 and 450 μM in the case of THP1 derived M1 macrophages After 24 h of treatment it can be observed that the PCA functionalized magnetic nanoparticles did not have a cytotoxic effect on EAhy926 cells at any concentration tested An increased cytotoxic effect can be observed on M1 macro phages at higher concentrations of captured PCA 400 and 450 μM Figure 2AC Con centrations equal to or exceeding 70 µgmL plain MNPDex the concentration corre sponding to MNP loaded with 400 μM PCA proved to be cytotoxic for both cell types after 24 h of incubation Figure 2AC The free polyphenol did not have a cytotoxic effect on the two cell types at any concentration investigated On the contrary as PCA concen tration increased M1 phenotype macrophages released less quantifiable adenylate kinase in the cell medium implying that the polyphenol could reverse the cytotoxicity induced by cell treatment with PMA and LPS used in THP1 monocytes differentiation A similar effect can be observed when M1 macrophages are treated with MNPDexPCA in which polyphenol concentration was below 400 μM Figure 2C Figure 2 Cell cytotoxicity after 24 h of incubation with increasing concentrations of MNPDex MNPDexPCA and PCA on human endothelial cells A and M1 type macrophages C and after 48 h of incubation of human endothelial cells in the absence or the presence of TNFα B and M1 macrophages D with the chosen pharmacologically relevant concentration of PCA 350 µM loaded on MNP MNPDexPCA plain MNPDex and free PCA p 005 p 001 p 0001 and p 00001 vs control cells EC or M0 macrophages cultured in the absence of any treatment The working concentration of 350 µM PCA loaded on MNPDex nanoparticles was chosen based on our results showing the absence of cytotoxicity and on therapeutic properties of the polyphenol at this concentration as previously reported 3133 Similarly Pharmaceutics 2021 13 1414 10 of 19 the corresponding iron concentration of MNPDex and MNPDexPCA ie 62 µgmL was found to be noncytotoxic on both cell types tested as stated above Cell cytotoxicity was also assessed after 48 h of treatment but only with the selected pharmacologically relevant concentration of PCAloaded MNP and the corresponding controls free PCA and plain MNPDex MNPDexPCA and free PCA did not have a cyto toxic effect on both cell types Figure 2BD while MNPDex treatment of M1 macrophages determined an increase in cytotoxicity of approximately twofold compared to control M0 macrophages which can also be observed in the case of untreated M1 macrophages Figure 2D This result could be explained by the fact that plain MNPDex unlike PCA and MNPDexPCA do not reverse the cytotoxic effect of the two inductors of monocyte to macrophage differentiation PMA and LPS Figure 2CD There were no significant differences in cell cytotoxicity between MNPDextreated and untreated EC as well as TNFαstimulated EC Figure 2B 33 Intracellular Localization of MNPDexPCA The intracellular localization of MNPDexPCA and plain MNPDex was assessed microscopically using the Prussian Blue staining which is based on the formation of a blue waterinsoluble complex between potassium ferrocyanide and the released ferric ions The data obtained revealed that MNPDexPCA as well as MNPDex were internalized by EAhy926 cells and THP1 derived M1 macrophages after 24 h of incubation blue spots Figure 3A MNPDex were internalized at similar rate by M1 macrophages and EC with a staining density of approximately 22 compared to untreated controls whereas MNP DexPCA was taken up nearly twofold higher in the case of EC compared to M1 macrophages p 0001 Figure 3B MNPDexPCA was also found to be internalized twofold higher by EC than plain MNPDex although both magnetic nanoparticles are similarly internalized by M1 macrophages Figure 3AB This could imply that EC internalize polyphenolfunctionalized MNP more efficiently by receptors that specifically recognize polyphenols for example estrogen receptors ER 34 which are absent on the surface of M1 macrophages 35 Pharmaceutics 2021 13 x FOR PEER REVIEW 11 of 21 Figure 3 Brightfield microscopy images showing the uptake of magnetic nanoparticles at 62 μgmL concentration in EAhy926 cells Endothelial cells and THP1 derived M1 macrophages after Prussian Blue staining A and the quantifi cation of ironlabelled cells using ImageJ software B The data are expressed as mean SD from two independent exper iments p 00001 Scale Bar 10 μM 34 AntiInflammatory Effect of MNPDexPCA 341 AntiInflammatory Activity of MNPDexPCA on Activated EC The antiinflammatory activity of MNPDexPCA on TNFα activated EC was vali dated at a functional level employing the monocyte adhesion assay In addition the pro tein expression of NFκB p65 subunit and MCP1 molecules involved in the inflammatory response was determined in EC exposed to TNFα and treated with MNPDexPCA MNPDex or free PCA An increase in the number of adherent monocytes to TNFα acti vated EC as compared to unstimulated control EC was measured Figure 4AB This re sult is a consequence of the increases in NFκB p65 and MCP1 protein levels in TNFα activated EC Figure 4CD We found that 48 h of treatment with both MNPDexPCA and free PCA reduced the adhesion of monocytes to TNFα activated EC 33 p 001 compared with that to ac tivated EC left untreated whereas plain MNPDex did not modify the number of adher ent monocytes Figure 4AB The antiinflammatory effect of the MNPDexPCA treat ment was further confirmed by a decrease in the protein expression of NFκB p65 subunit and MCP1 35 p 001 and 33 p 005 respectively while the incubation with MNPDex exhibited no significant differences in protein levels in comparison to TNFα Figure 3 Brightfield microscopy images showing the uptake of magnetic nanoparticles at 62 µgmL concentration in EAhy926 cells Endothelial cells and THP1 derived M1 macrophages after Prussian Blue staining A and the quantification of ironlabelled cells using ImageJ software B The data are expressed as mean SD from two independent experiments p 00001 Scale Bar 10 µM Pharmaceutics 2021 13 1414 11 of 19 34 AntiInflammatory Effect of MNPDexPCA 341 AntiInflammatory Activity of MNPDexPCA on Activated EC The antiinflammatory activity of MNPDexPCA on TNFα activated EC was val idated at a functional level employing the monocyte adhesion assay In addition the protein expression of NFκB p65 subunit and MCP1 molecules involved in the inflamma tory response was determined in EC exposed to TNFα and treated with MNPDexPCA MNPDex or free PCA An increase in the number of adherent monocytes to TNFα ac tivated EC as compared to unstimulated control EC was measured Figure 4AB This result is a consequence of the increases in NFκB p65 and MCP1 protein levels in TNFα activated EC Figure 4CD Pharmaceutics 2021 13 x FOR PEER REVIEW 12 of 21 stimulated EC Figure 4CD No significant difference in NFkB and MCP1 expression was observed when activated EC were treated with dexamethasone 25 µM Figure 4 Antiinflammatory effects of MNPDexPCA on activated EC Functional role of MNPDexPCA in reducing THP1 monocytes adhesion to TNFα 20 ngmLactivated EC Fluorescence microscopy images showing fluorescently labelled monocytes green A and quantification of adhered monocytes expressed as numberfield B Protein expression of NFkB p65 C and MCP1 D in quiescent and TNFα activated EC treated for 48 h with MNPDexPCA MNP Dex and free PCA at a pharmacologically relevant concentration of PCA 350 μM Dexa treatment of EC with dexame thasone 25 μM Protein expression was normalized to βactin The data are expressed as mean SD from 3 independent experiments p 005 p 001 and p 0001 342 AntiInflammatory Activity of MNPDexPCA on M1 Macrophages The expression of proinflammatory cytokines IL1β TNFα and that of the receptor for MCP1 chemokine CCR2 were evaluated by Western Blotting in M1 macrophages after incubation for 48 h with MNPDexPCA and the corresponding concentrations of plain MNPDex and free PCA As controls untreated and dexamethasonetreated M1 macrophages were used THP1derived M1 macrophages expressed significantly higher levels of IL1β TNFα and IL6 compared to M0 macrophages indicating the successful polarization of the cells towards a proinflammatory phenotype Figure 5 The data shows a similar decrease pattern in the levels of the four proinflammatory molecules in vestigated after incubating the cells with MNPDexPCA Thus MNPDexPCA treatment Figure 4 Antiinflammatory effects of MNPDexPCA on activated EC Functional role of MNPDexPCA in reducing THP1 monocytes adhesion to TNFα 20 ngmLactivated EC Fluorescence microscopy images showing fluorescently labelled monocytes green A and quantification of adhered monocytes expressed as numberfield B Protein expression of NFkB p65 C and MCP1 D in quiescent and TNFα activated EC treated for 48 h with MNPDexPCA MNPDex and free PCA at a pharmacologically relevant concentration of PCA 350 µM Dexa treatment of EC with dexamethasone 25 µM Protein expression was normalized to βactin The data are expressed as mean SD from 3 independent experiments p 005 p 001 and p 0001 Pharmaceutics 2021 13 1414 12 of 19 We found that 48 h of treatment with both MNPDexPCA and free PCA reduced the adhesion of monocytes to TNFα activated EC 33 p 001 compared with that to activated EC left untreated whereas plain MNPDex did not modify the number of adherent monocytes Figure 4AB The antiinflammatory effect of the MNPDexPCA treatment was further confirmed by a decrease in the protein expression of NFκB p65 subunit and MCP1 35 p 001 and 33 p 005 respectively while the incubation with MNPDex exhibited no significant differences in protein levels in comparison to TNF αstimulated EC Figure 4CD No significant difference in NFkB and MCP1 expression was observed when activated EC were treated with dexamethasone 25 µM 342 AntiInflammatory Activity of MNPDexPCA on M1 Macrophages The expression of proinflammatory cytokines IL1β TNFα and that of the receptor for MCP1 chemokine CCR2 were evaluated by Western Blotting in M1 macrophages after incubation for 48 h with MNPDexPCA and the corresponding concentrations of plain MNPDex and free PCA As controls untreated and dexamethasonetreated M1 macrophages were used THP1derived M1 macrophages expressed significantly higher levels of IL1β TNFα and IL6 compared to M0 macrophages indicating the successful polarization of the cells towards a proinflammatory phenotype Figure 5 The data shows a similar decrease pattern in the levels of the four proinflammatory molecules investigated after incubating the cells with MNPDexPCA Thus MNPDexPCA treatment reduced the protein expres sion of IL1β by 27 p 0001 CCR2 by 55 p 00001 TNFα by 40 p 001 and IL6 by 27 p 00001 in M1 macrophages in comparison to untreated ones Figure 5AD The free PCA also reduced the levels of the proinflammatory molecules and in a more drastic manner in the case of IL1β 83 p 00001 TNFα 80 p 0001 and IL6 90 p 00001 which is similar to the effect of Dexa Figure 5AD On the other hand plain MNPDextreated M1 macrophages had no significant changes in the protein expression of IL1β CCR2 and TNFα while IL6 concentration increased by 11 p 001 Dexamethasone was used as a gold standard antiinflammatory drug 343 The Effect of MNPDexPCA on Key Proteins of MAPKs Pathway The protein expression of proinflammatory molecules can be increased by the acti vation of specific signaling components of the MAPKs pathway To further investigate the antiinflammatory activity of MNPDexPCA we assessed their effect on two main molecules of the MAPK signaling pathway p38α and ERK 12 and compared it with that of MNPDex free PCA and the wellknown antiinflammatory agent dexamethasone Figure 6 At the concentration tested of 25 µM dexamethasone the data revealed little or no significant differences in the protein levels of p38α and ERK 12 when compared to TNFα activated EC and PMALPSactivated M1 macrophages On the other hand the MNPDexPCA treatment significantly decreased p38α protein expression in both cell types 32 in EAhy926 cells and 30 in M1 macrophages p 005 Figure 6AC compared to the stimulated control cells TNFα activated EAhy926 cells and PMALPS activated M1 macrophages The treatment with plain MNP increased the synthesis of p38α isoform 75 p 00001 in M1 macrophages Figure 6C ERK12 protein synthesis exhibited no significant differences when TNFαstimulated EAhy926 cells were treated with MNPDex but decreased when EC were treated with MNPDexPCA 22 p 001 and PCA 62 p 0001 compared to TNFαstimulated EAhy926 cells Figure 6B Interestingly we found that in the case of M1 macrophages the ERK12 protein expression was reduced by 57 p 0001 after 48 h of incubation with plain MNPDex Figure 6D Because ERK12 is a protein involved in cell survival and proliferation we further looked at the expression of the proapoptotic molecule Bcell lymphoma 2 Bcl2associated X protein Bax in M1 macrophages treated with MNP MNPDexPCA and free PCA compared to M0 and M1 untreated macrophages Figure 6E Thus we found that Bax level was significantly enhanced 31 p 001 upon MNPDex treatment while MNPDexPCA free PCA and Dexa reduced Bax level Pharmaceutics 2021 13 1414 13 of 19 by 32 p 001 62 p 00001 and 50 p 0001 respectively when com pared to untreated M1 macrophages suggesting an antiapoptotic effect of these three formulations This result may be a possible explanation of the decrease in ERK12 protein expression in M1 macrophages treated with plain MNPDex Pharmaceutics 2021 13 x FOR PEER REVIEW 13 of 21 reduced the protein expression of IL1β by 27 p 0001 CCR2 by 55 p 00001 TNFα by 40 p 001 and IL6 by 27 p 00001 in M1 macrophages in comparison to untreated ones Figure 5AD The free PCA also reduced the levels of the proinflam matory molecules and in a more drastic manner in the case of IL1β 83 p 00001 TNFα 80 p 0001 and IL6 90 p 00001 which is similar to the effect of Dexa Figure 5AD On the other hand plain MNPDextreated M1 macrophages had no sig nificant changes in the protein expression of IL1β CCR2 and TNFα while IL6 concen tration increased by 11 p 001 Dexamethasone was used as a gold standard anti inflammatory drug Figure 5 The antiinflammatory effect of MNPDexPCA on THP1derived M1 macrophages was determined by evalu ating the expression of intracellular proinflammatory molecules IL1β A CCR2 B TNFα C and the levels of released IL6 D after 48 h of treatment Protein expression was normalized to βactin For comparison the levels of proinflam matory molecules in M0 macrophages were shown The data are expressed as mean SD from at least three independent experiments performed in duplicates p 001 p 0001 and p 00001 The protein levels of cytokines in M0 macrophages are also shown Dexatreatment of M1 macrophages with dexamethasone 25 µM Figure 5 The antiinflammatory effect of MNPDexPCA on THP1derived M1 macrophages was determined by evaluating the expression of intracellular proinflammatory molecules IL1β A CCR2 B TNFα C and the levels of released IL6 D after 48 h of treatment Protein expression was normalized to βactin For comparison the levels of proinflammatory molecules in M0 macrophages were shown The data are expressed as mean SD from at least three independent experiments performed in duplicates p 001 p 0001 and p 00001 The protein levels of cytokines in M0 macrophages are also shown Dexatreatment of M1 macrophages with dexamethasone 25 µM Pharmaceutics 2021 13 1414 14 of 19 Pharmaceutics 2021 13 x FOR PEER REVIEW 15 of 21 Figure 6 Protein expression of p38α AC and total ERK 12 BD in quiescent and TNFα 20 ngmLactivated EAhy926 cells and M1 macrophages after 48 h of incubation with MNPDexPCA 62 µgmL MNP 350 µM PCA as well as the protein expression of antiapoptotic molecule Bax in M1 macrophages E Plain MNPDex free PCA and dexame thasone Dexa were used as controls Protein expression was normalized to βactin The data are expressed as mean SD from three independent experiments p 005 p 001 p 0001 and p 00001 Figure 6 Protein expression of p38α AC and total ERK 12 BD in quiescent and TNFα 20 ngmLactivated EAhy926 cells and M1 macrophages after 48 h of incubation with MNPDexPCA 62 µgmL MNP 350 µM PCA as well as the protein expression of antiapoptotic molecule Bax in M1 macrophages E Plain MNPDex free PCA and dexamethasone Dexa were used as controls Protein expression was normalized to βactin The data are expressed as mean SD from three independent experiments p 005 p 001 p 0001 and p 00001 Pharmaceutics 2021 13 1414 15 of 19 4 Discussion Inflammation is a process involved in the inception and progression of atherosclerosis AS triggered by endothelial activation and characterized by an increase in the expression of cell adhesion molecules CAM and chemokines facilitating the recruitment and trans migration of immune cells 3637 The key role played by the inflammatory molecules such as cytokines and chemokines in the initiation and progression of AS has been well es tablished Many drug therapies have been developed to prevent or reduce the formation of atherosclerotic plaques however the mortality rate from CVD is increasing alarmingly 1 Thus targeting chronic inflammation with potent inhibitors of chemokines and cytokines expression is critical requiring new and innovative nanotherapeutic strategies Because many healthbeneficial properties of polyphenols have been described including antiinflammatory activities attempts have been made to introduce them as novel comple mentary drugs in the prevention andor treatment of noncommunicable diseases having an associated inflammatory process including CVD diabetes and cancer 38 Although in vitro studies have confirmed protocatechuic acid PCAs therapeutic potential in alleviating exac erbated inflammatory processes 323339 the reduced bioavailability prevents the exercising of the therapeutic properties in vivo 4041 Thus the formulation into nanoparticles may represent a good strategy to increase its concentration at the affected sites Since EC and macrophages are key players in the onset and progression of chronic vascular inflammation the focus of this paper was to synthesize characterize and further investigate the antiinflammatory potential of novel PCAfunctionalized dextrancoated magnetic nanoparticles MNPDexPCA on these cells by examining the protein expres sion of various proinflammatory molecules eg IL1β TNFα IL6 MCP1 and CCR2 and signaling molecules involved in inflammation eg NFkB p38α and ERK 12 More over the functional role of MNPDexPCA in inhibiting monocyte adhesion to activated EC was investigated We developed a nanocarrier with a core shelllike structure consisting of an iron oxide hydrophobic core and a hydrophilic shell represented by the polysaccharide dextran MNPDex which serves as a stabilizer The tendency to aggregation of uncoated MNP decreases after their coating with dextran as suggested by the higher negative value of the ζpotential whereas the higher hydrodynamic diameter is due to the expansion of the coating polymer in water The loading of PCA on the surface of MNPDex results in an increase of the hydrodynamic diameter and a decrease in the ζpotential negative value The dextran coating and PCA loading of magnetic nanoparticles cause minor changes from a morphological point of view as proved by the TEM investigation According to in vivo studies iron oxide nanoparticles are relatively safe because they do not accumulate in vital organs and are quickly removed from the body 42 Dextran is a polymer used in various applications where the surface of material needs to be modified to improve its properties 43 The presence of dextran on its surface has been shown to attenuate iron oxide toxicity in EC and monocytes 4445 In vitro cytotoxicity evaluation after 24 h showed that MNPDexPCA possess good biocompatibility at iron concentrations less than 100 µgmL Our findings are consistent with previously published data indicating that nanoparticles with negative or slightly negative zeta potential are less likely to be cytotoxic and show good internalization 4648 The polyphenol used in this study PCA was also found to be noncytotoxic at all concentrations tested We further investigated the cytotoxicity of MNPDexPCA after 48 h of incubation with the pharmacologically relevant concentration of PCA on EC and M1 type macrophages as well as the corresponding controls While MNPDexPCA and free PCA 350 µM had no cytotoxic effect on both cell types plain MNPDex increased the cell cytotoxicity of M1 macrophages by almost twofold which could be attributed to LPS stimulation and a similar cytotoxicity was also observed in untreated M1 macrophages The qualitative Prussian Blue staining confirms the internalization of nanoparticles by both EC and M1 macrophages While plain MNPDex and MNPDexPCA are taken up at the same rate by M1 macrophages the data showed higher uptake in the case of EC when PCA is loaded on the surface of MNPDex which is consistent with recent Pharmaceutics 2021 13 1414 16 of 19 findings highlighting polyphenols ability to interact with the lipid bilayer as well as specific membrane receptors which could ultimately facilitate nanoparticleuptake 4950 As a result we expect that the increased uptake of MNPDexPCA by EC after 24 h would determine maximum targeting efficacy when targeting the vascular wall It was also revealed that in the case of dietary iron polyphenol addition increases uptake in human colorectal adenocarcinoma Caco2 epithelial cells 51 Nevertheless there is little information about the role of transporters in cellular accumulation and evidence for individual phenolic compounds is limited Previous studies demonstrated the ability of PCA to inhibit monocyte adhesion to the mouse aortic endothelial cells in vitro and in vivo by reducing the expression of CAM such as ICAM1 and VCAM1 both as a consequence of inhibiting the activity of the transcription factor NFkB 52 There was also evidence of PCAs antiinflammatory effect on different types of cultured macrophages 5354 as well as isolated from in vivo sources 55 In the current study we found that MNPDexPCA exerts its antiinflammatory activity on human EC and THP1derived M1 macrophages by lowering the levels of essential proinflammatory and signaling molecules involved in the cellular inflammatory process eg IL1β TNFα IL6 MCP1 CCR2 and NFκB p38α and ERK 12 respectively As a result unlike the plain MNP the treatment of EC with MNPDexPCA treatment significantly reduced the number of adhered monocytes on EC as well as NFkB and MCP1 protein expression In the case of M1 macrophages MNPDexPCA determined not only a reduction in IL1β TNFα and CCR2 protein expression but also a decrease in the concentration of IL6 released in the culture medium In contrast the data suggest that plain MNPDex have slightly proinflammatory properties on macrophages aggravating the already LPSstimulated cells This action is countered by the presence of PCA on the surface of MNP which is consistent with published data highlighting the protective effect of PCA 5657 Interestingly while the wellknown antiinflammatory drug dexamethasone which was used as a positive control had no significant antiinflammatory effect on activated EC at the tested concentration 25 µM it significantly reduces the expression of IL1β and TNFα as well as the extracellular releasing of IL6 by M1 macrophages This result could imply that a concentration higher than 25 µM is required to produce an effect on EC Similarly Nehme and Edelman 2008 found that Dexa did not suppress IL1β and TNFα in retinal EC pointing to a possible mechanism of resistance 58 The ability of cells to induce the overexpression of inflammatory mediators is a primary biological response to the activation of NFkB and MAPK pathways NFkB is a recognized regulator of inflammation that controls in EC the expression of MCP1 a chemokine involved in monocytes recruitment and activation and in macrophages the production of cytokines such as IL1β TNFα and IL6 910 The isoform p38αMAPK in particular is expressed in both EC and macrophages and plays a vital role in regulating cellular processes involved in inflammation 59 ERK 12 also regulates cellular differentia tion proliferation and survival as well as inflammatory processes 60 We were interested in how MNPDexPCA affected the protein expression of these signaling molecules given that it is known to be responsible for the overexpression of inflammatory molecules We found that MNPDexPCA treatment reduced NFkB expression in EC as well as p38α synthesis in both cell types supporting the antiinflammatory activity reported for PCA In contrast plain MNP did not change the level of p38α in TNFαactivated EC but increased p38α protein synthesis in M1 macrophages Others have thoroughly documented MNPs proinflammatory effect which has been attributed to consistent macrophage reactive oxygen species ROS production as well as an iron overload both of which have been shown to aggravate inflammation in LPSstimulated mice 6162 MNPDexPCA inhib ited ERK protein synthesis in both cell types similar to the effect seen with p38α and the reduction was significantly higher when M1 macrophages were treated with plain MNP The discrepancy could be attributed to ERKs role in cellular proliferation and survival with its expression being reduced as a result of the increase in cell cytotoxicity and increase Pharmaceutics 2021 13 1414 17 of 19 in proapoptotic Bax protein expression observed after 48 h of M1 macrophages incubation with plain MNP 5 Conclusions In this study new biocompatible and noncytotoxic dextrancoated iron oxide nanopar ticles for the delivery of the protocatechuic acid were synthesized We demonstrated that i MNPDexPCA reduce the adhesion of monocytes to TNFαactivated EC ii MNP DexPCA have antiinflammatory activity at noncytotoxic concentrations by reducing the levels of proinflammatory mediators such as MCP1 IL1β TNFα IL6 and CCR2 in activated EC and M1 phenotype macrophages and iii the antiinflammatory effect of MNPDexPCA was associated with the reduction in the levels of p38α and ERK12 MAPKs and NFkB transcription factor The benefit to formulate PCA into MNPDex nanoparticles is that we developed a theranostic system able to guide the therapeutic agent to the target due to the magnetic properties of the nanocarrier and to potentiate the efficiency of PCA as an antiinflammatory agent by improving its cellular internalization According to our findings the newly developed dextran shelliron oxide core nanocar riers could be used as theranostic tools for guidance imaging and therapy of various inflammatory diseases including atherosclerosis Author Contributions Conceptualization MA MT and MC Methodology MA MT and MC Formal analysis MA and ARP Validation MA MT and AF Investigation MA MT ARP and AF Writingoriginal draft preparation MA MT and AF Writingreview and editing MA MT ARP AF MP and MC supervision MC funding acquisition MC All authors have read and agreed to the published version of the manuscript Funding This work was supported by the Romanian Ministry of Research Innovation and Digitiza tion CNCSCCCDIUEFISCDI project number PNIIIP112PCCDI20170697 INTERA contract no 13PCCDI2018 within PNCDI III and by the Romanian Academy Institutional Review Board Statement Not applicable Informed Consent Statement Not applicable Conflicts of Interest The authors declare no conflict of interest References 1 McAloon CJ Osman F Glennon P Lim PB Hayat SA Global Epidemiology and Incidence of Cardiovascular Disease In Cardiovascular Diseases Elsevier Amsterdam The Netherlands 2016 pp 5796 ISBN 9780128033135 2 Sako Y Effects of Turbulent Blood Flow and 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