Gold Nanoparticles Affect Pericyte Biology and Capillary Tube Formation

pharmaceutics

Article Gold Nanoparticles Affect Pericyte Biology and Capillary Tube Formation

Sasikarn Looprasertkul 1, Amornpun Sereemaspun 1,2, Nakarin Kitkumthorn 3 , Kanidta Sooklert 1,2, Tewarit Sarachana 4,5 and Depicha Jindatip 1,6,*

1 Department of Anatomy, Faculty of Medicine, Chulalongkorn University, 1873 Rama 4 Rd., Wangmai, Pathumwan, Bangkok 10330, Thailand; [email protected] (S.L.); [email protected] (A.S.); [email protected] (K.S.) 2 Nanomedicine Research Unit, Department of Anatomy, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand 3 Department of Oral Biology, Faculty of Dentistry, Mahidol University, Payathai Rd., Ratchathewi, Bangkok 10400, Thailand; [email protected] 4 Age-Related Inflammation and Degeneration Research Unit, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, 154 Rama 1 Rd., Wangmai, Pathumwan, Bangkok 10330, Thailand; [email protected] 5 SYstems Neuroscience of Autism and PSychiatric Disorders (SYNAPS) Research Unit, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand 6 Division of Histology and Cell Biology, Department of Anatomy, School of Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan * Correspondence: [email protected]; Tel.: +66-2-256-4281 Abstract: Gold nanoparticles (AuNPs) are used for diagnostic and therapeutic purposes, especially Citation: Looprasertkul, S.; antiangiogenesis, which are accomplished via inhibition of endothelial cell proliferation, migration, Sereemaspun, A.; Kitkumthorn, N.; and tube formation. However, no research has been performed on the effects of AuNPs in pericytes, Sooklert, K.; Sarachana, T.; Jindatip, which play vital roles in endothelial cell functions and capillary tube formation during physiological D. Gold Nanoparticles Affect Pericyte and pathological processes. Therefore, the effects of AuNPs on the morphology and functions of Biology and Capillary Tube pericytes need to be elucidated. This study treated human placental pericytes in monoculture with Formation. Pharmaceutics 2021, 13, 20 nm AuNPs at a concentration of 30 ppm. Ki-67 and platelet-derived growth factor receptor- 738. https://doi.org/10.3390/ β (PDGFR-β) mRNA expression was measured using real-time reverse transcription-quantitative pharmaceutics13050738 polymerase chain reaction (RT-qPCR). Cell migration was assessed by Transwell migration assay. The fine structures of pericytes were observed by transmission electron microscopy. In addition, Academic Editors: Khair Alhareth and Karine Andrieux 30 ppm AuNP-treated pericytes and intact human umbilical vein endothelial cells were cocultured on Matrigel to form three-dimensional (3D) capillary tubes. The results demonstrated that AuNPs Received: 23 April 2021 significantly inhibited proliferation, reduced PDGFR-β mRNA expression, and decreased migration Accepted: 11 May 2021 in pericytes. Ultrastructural analysis of pericytes revealed AuNPs in late endosomes, autolysosomes, Published: 17 May 2021 and mitochondria. Remarkably, many mitochondria were swollen or damaged. Additionally, capillary tube formation was reduced. We found that numerous pericytes on 3D capillary tubes Publisher’s Note: MDPI stays neutral were round and did not extend their processes along the tubes, which resulted in more incomplete with regard to jurisdictional claims in tube formation in the treatment group compared with the control group. In summary, AuNPs can published maps and institutional affil- affect pericyte proliferation, PDGFR-β mRNA expression, migration, morphology, and capillary tube iations. formation. The findings highlight the possible application of AuNPs in pericyte-targeted therapy for antiangiogenesis.

Keywords: angiogenesis; capillary tube formation; endothelial cells; gold nanoparticles; pericytes Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and 1. Introduction conditions of the Creative Commons Recently, nanoparticles have been developed for innovative therapeutic strategies Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ involving nanotechnology. Nanoparticles are small particles with diameters of diverse 4.0/). sizes from 1 to 100 nm. Notably, gold nanoparticles (AuNPs), which are some of the most

Pharmaceutics 2021, 13, 738. https://doi.org/10.3390/pharmaceutics13050738 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2021, 13, 738 2 of 13

common metal nanoparticles, have been widely used in nanomedicine. The remarkable properties of AuNPs are their low cytotoxicity, good biocompatibility, and ease of conjuga- tion to a variety of biomolecules [1,2]. At present, AuNPs are used for cell imaging, targeted drug delivery, thermal therapy, antiangiogenesis, and anticancer applications [3–5]. Recent studies have reported that AuNPs ranging from 15 to 30 nm in size can aid in antiangiogenesis by inhibiting the activity of human umbilical vein endothelial cells (HUVECs) [1,6,7]. For example, 15 nm AuNPs inhibit migration and tube formation [6]. In addition, 20 nm AuNPs inhibit proliferation and tube formation and reduce phosphorylation-mediated activation of the extracellular signal-regulated kinase 1/2 (ERK 1/2), protein kinase B (Akt), and focal adhesion kinase (FAK) signaling pathways in vascular endothelial growth factor (VEGF)165-induced HUVECs [1,6]. Similarly, 26 nm AuNPs reduce cell viability, cell proliferation, cell migration, and tube formation and increase the expression of autophagy genes, including autophagy related 5 (ATG5), beclin1 (an autophagosome formation marker), p62 (a degradation marker), and microtubule-associated protein 1 light chain 3 (LC3)-II (an autophagy activation marker), in HUVECs [7]. Apart from inhibiting HUVEC activity, AuNPs can suppress angiogenesis in choroidal neovascularization-induced age-related macular degeneration in vivo [1]. Moreover, AuNPs have been found to inhibit oxygen- induced retinopathy by reducing both neovascular and avascular areas in an in vivo retinal neovascularization study [7]. The formation, maintenance, and remodeling of capillaries are accomplished by co- operative activity between endothelial cells and pericytes. Pericytes are perivascular cells located at the abluminal surfaces of small, thin vessels, i.e., capillary and postcapillary venules, and share a basement membrane with endothelial cells [8]. Pericytes are responsi- ble for regulating vascular maturation, stabilizing blood vessels, and controlling blood ﬂow, capillary diameter, and hydrostatic balance [9–11]. In addition, pericytes can promote endothelial cell survival and migration through paracrine signaling, such as platelet-derived growth factor-BB (PDGF-BB)-platelet-derived growth factor receptor-β (PDGFR-β) signaling [12]. This signaling is essential to endothelial cells and is also crucial for pericyte survival, proliferation, migration, recruitment, attachment, and differentiation [13]. Previ- ous reports have indicated that a lack of PDGF or PDGFR-β expression prevents pericyte recruitment to vessels [14,15]. Recently, pericytes have been discovered to be targets in antiangiogenesis mediated by blockade of PDGFR-β [16]. As mentioned above, there have been studies on the use of AuNPs for antiangiogenesis via endothelial cell targeting. However, the capillary networks formed during angiogenesis require two cell types: endothelial cells and pericytes. Therefore, we aimed to determine whether AuNPs might also affect pericyte properties. In this study, the effects of AuNPs on pericyte morphology, proliferation, migration, and PDGFR-β mRNA expression were elucidated. Additionally, AuNP-treated pericytes were cocultured with intact endothelial cells to discover the characteristics of three-dimensional (3D) capillary tube formation.

2. Materials and Methods 2.1. AuNP Characterization Stable suspensions of 20 nm-diameter AuNPs (Cat. no. 741965, Sigma-Aldrich, St. Louis, MO, USA) in citrate buffer were used in this study. The shapes and sizes of the AuNPs were conﬁrmed by transmission electron microscopy (TEM) (JEM-1400PLUS; JEOL, Tokyo, Japan). In addition, the ultraviolet–visible (UV-Vis) absorption of the AuNPs was evaluated using a microplate spectrophotometer (Multiskan GO; Thermo Scientiﬁc, Vantaa, Finland).

2.2. Cell Culture Human placental pericytes (hPC-PLs; Cat. no. C-12980, PromoCell, Heidelberg, Ger- many) were cultured in pericyte growth medium (Cat. no. C-28040, PromoCell, Heidelberg, Germany) supplemented with 10% SupplementMix (Cat. no. C-39840, PromoCell, Heidel- berg, Germany) and 1% antibiotic-antimycotic solution (Cat. no. 15240062, Gibco, New Pharmaceutics 2021, 13, 738 3 of 13

York, NY, USA). HUVECs (Cat. no. C2519A, Lonza, Basel, Switzerland) were cultured in endothelial cell growth medium supplemented with EGM-2MV SingleQuots (Cat. no. CC- 4147, Lonza, Basel, Switzerland) and 1% antibiotic-antimycotic solution (Cat. no. 15240062, Gibco, New York, NY, USA). Both cell types were maintained at 37 ◦C in a humidiﬁed 5% CO2 atmosphere.

2.3. Cell Viability Assay Pericytes (1 × 104 cells/well) were seeded into 96-well plates and incubated overnight. To determine the optimal dose for the induction of cytotoxicity, pericytes were treated with 20 nm AuNPs at various concentrations, i.e., 10, 20, 30, 40, and 50 ppm, for 24 h. PrestoBlue (Cat. no. A13262, Invitrogen, New York, NY, USA) was used according to the manufacturer’s protocol. After adding the reagent and incubating for 4 h, pericyte viability was determined by measuring the UV absorbance using a microplate reader at 570/600 nm.

2.4. Real-Time Reverse Transcription-Quantitative Polymerase Chain Reaction (Real-Time RT-qPCR) According to the cell viability assay, the 30 ppm concentration was selected for the following experiments because it was the highest dose at which pericytes remained viable (over 90%). Pericytes were seeded into 24-well plates at a density of 1 × 105 cells/well. The cells were treated with 30 ppm AuNPs for 24 h. Total RNA was isolated from the pericytes with TRIzol reagent (Cat. no. 15596026, Invitrogen, Carlsbad, CA, USA) and reverse-transcribed into cDNA with a RevertAid First-Strand cDNA Synthesis Kit (Cat. no. K-1622, Thermo Fisher Scientiﬁc Inc., Rockford, IL, USA). Next, real-time RT-qPCR was carried out using AccuPower 2X Greenstar qPCR Master Mix (Cat. no. K-6254, Bioneer, Daejeon, Korea) in a volume of 12.5 µL with a CFX 96 thermocycler (Bio-Rad Laboratories, Hercules, CA, USA). The mRNA expression of PDGFR-β, a known pericyte marker, was examined with forward (50-ACTGCCCAGACCTAGCAGTG-30) and reverse (50-CAGGGAAGTAAGGTGCCAAC-30) primers. Moreover, Ki-67 mRNA expression was analyzed to detect cell proliferation using forward (50-GATCGTCCCAGTGGAGTT-30) and reverse (50-CCCCTTCCAAACAAGCAGGT-30) primers. GAPDH was used as a house- keeping gene.

2.5. Transwell Migration Assay A cell migration assay was performed using 8 µm pore size Transwell inserts (Corning Inc., New York, NY, USA). After pretreatment with 30 ppm AuNPs for 24 h, pericytes (200 µL, 2.5 × 104 cells-treated/well) were plated on the upper chamber and cultured in serum-free culture medium. In the lower chamber, culture medium containing PDGF-BB (Cat. no. 220-BB, R&D Systems, Minneapolis, MN, USA) and 10% FBS (Cat. no. 1600044, Gibco, Carlsbad, CA, USA) was used as a chemoattractant. The cells were incubated for 48 h. Migrating cells that moved to the lower surface of the membrane were ﬁxed with 4% paraformaldehyde (Cat. no. 818715.1000, Merck Millipore, Darmstadt, Germany) for 10 min and then stained with 1% crystal (Cat. no. 03050–25, Loba Chemie, Mumbai, India) violet in 2% ethanol (Cat. no. 1.000983.2500, Merck Millipore, Darmstadt, Germany) for 20 min. Nonmigrating cells in the upper chamber were removed with a cotton swab and then washed with distilled water. The migrating cells were imaged by inverted light microscopy (CKX53, Olympus, Tokyo, Japan) and counted with ImageJ software version 1.52a (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).

2.6. Transmission Electron Microscopy AuNP-treated pericytes were fixed with 2.5% glutaraldehyde (Cat. no. 16220, EMS, Hatfield, PA, USA) at 4 ◦C for 2 h and postfixed with 1% osmium tetroxide (Cat. no. 19110, EMS, Hatfield, PA, USA) at 4 ◦C for 1.5 h. The cell pellets were dehydrated with an alcohol series (Cat. no. 1.000983.2500, Merck Millipore, Darmstadt, Germany) and cleared by gradual addition of propylene oxide (Cat. no. 8.07027.1001, Merck Millipore, Darmstadt, Germany). Then, the pellets were embedded in epoxy resin and cut into slices of 70 nm Pharmaceutics 2021, 13, 738 4 of 13

thickness with an ultramicrotome (EM UC7, Leica, Vienna, Austria). The ultrathin sections were stained with uranyl acetate and lead citrate and observed by transmission electron microscopy (JEM-1400PLUS; JEOL, Tokyo, Japan).

2.7. Capillary Tube Formation Assay To coculture pericytes and endothelial cells for capillary tube formation, 100 µL of Matrigel (Cat. no. 356231, Corning, Bedford, MA, USA) was added to an 8-chamber slide, and the slide was incubated at 37 ◦C for 30 min for Matrigel solidiﬁcation. AuNP-treated pericytes and intact endothelial cells were seeded and cocultured at a ratio of 1:20 on the 8-chamber slide. All tubes in the control and 30 ppm AuNP-treated groups were quantiﬁed by manual cell counting with the aid of ImageJ software version 1.52a under an inverted light microscope (CKX53, Olympus, Tokyo, Japan).

2.8. Immunofluorescence Histochemistry of Pericytes during Tube Formation Capillary tubes from pericyte/endothelial cell coculture were fixed with 4% paraformaldehyde (Cat. no. 818715.1000, Merck Millipore, Darmstadt, Germany) at 4 ◦C for 20 min. Nonspecific binding on the tubes was blocked with 2% normal goat serum (Cat. no. S-1000, Vector Laboratories, Burlingame, CA, USA) in PBS at 30 ◦C for 20 min. The tubes were then incubated with a monoclonal rabbit IgG anti-PDGFR-β antibody (diluted 1:100, 28E1, Cat. no. 3169S, Cell Signaling, Danvers, MA, USA) overnight at 4 ◦C followed by an Alexa Fluor 488 goat anti-rabbit IgG conjugated secondary antibody (diluted 1:200, Cat. no. A-11008, Invitrogen, Paisley, UK) at 30 ◦C for 30 min. The nuclei were stained with Prolong Diamond Antifade Mountant with DAPI (Cat. no. P36962, Invitrogen, Eugene, OR, USA). The morphology and location of pericytes were observed with a confocal laser scanning microscope (LSM900, ZEISS, Oberkochen, Germany) and analyzed with Imaris software version 9.3.1 (Bitplane, Belfast, UK).

2.9. Statistical Analysis The data are expressed as the mean ± SEM from three independent experiments. Student’s t-test was conducted to compare two groups, whereas one-way ANOVA and Tukey’s post hoc test were used to compare multiple groups. Statistical analyses were carried out using GraphPad Prism 5.01 (GraphPad Software, Inc., San Diego, CA, USA) and SPSS 23.0 (SPSS, Inc., Armonk, NY, USA). Differences with a value of p < 0.05 were considered statistically signiﬁcant.

3. Results 3.1. Characterization of the AuNP

Pharmaceutics 2021, 13, x FOR PEER REVIEWThe AuNPs observed under TEM revealed a spherical morphology with an average5 of 13 diameter of 20.04 ± 0.13 nm (Figure1A). The UV-Vis spectrum of 20 nm-diameter AuNPs showed a single wavelength peak at approximately 522 nm (Figure1B).

Figure 1. CharacterizationCharacterization of of AuNPs AuNPs by by transmission transmission electron electron microscopy microscopy (A) ( Aand) and UV UV-Vis-Vis spec- spec- troscopy (B). Bar: 100 nm.nm.

3.2. Pericyte Viability To select the optimal concentration of 20 nm AuNPs in the present study, the effects of AuNPs on pericyte viability were assessed. The morphological changes upon the treatment of the cells with 20 nm AuNPs are shown in Figure 2A–F. The quantitative results showed that no difference was detectable between the control pericytes and the pericytes treated with 10, 20, and 30 ppm AuNPs, but pericytes treated with AuNPs at concentrations higher than 40 ppm exhibited significantly reduced cell viability (Figure 2G, *** p < 0.001). Notably, the sizes and shapes of AuNP-treated pericytes started to change when an AuNP concentration of 30 ppm was used, as observed under microscopy. Therefore, we selected a concentration of 30 ppm for the next experiments.

Figure 2. Cell viability of control pericytes (A) and pericytes treated with AuNPs at 10 (B), 20 (C), 30 (D), 40 (E), and 50 ppm (F) concentrations. The graph shows the quantitative data for pericyte viability (G). *** p < 0.001. Bar: 100 μm.

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Pharmaceutics 2021, 13, 738 5 of 13 Figure 1. Characterization of AuNPs by transmission electron microscopy (A) and UV-Vis spec- troscopy (B). Bar: 100 nm.

3.2. Pericyte Viability To selectselect thethe optimaloptimal concentrationconcentration of of 20 20 nm nm AuNPs AuNPs in in the the present present study, study, the the effects effects of AuNPsof AuNPs on on pericyte pericyte viability viability were were assessed. assessed. The The morphological morphological changes changes upon upon the treatmentthe treat- ofment the of cells the with cells 20 with nm 20 AuNPs nm AuNPs are shown are shown in Figure in2 FigureA–F. The 2A quantitative–F. The quantitative results showed results thatshowed no difference that no difference was detectable was detectable between between the control the pericytescontrol pericytes and the and pericytes the pericytes treated withtreated 10, with 20, and10, 20, 30 and ppm 30 AuNPs, ppm AuNPs, but pericytes but pericytes treated treated with with AuNPs AuNPs at concentrations at concentra- highertions higher than 40than ppm 40 exhibitedppm exhibited signiﬁcantly significantly reduced reduced cell viability cell viability (Figure (Figure2G, *** 2G,p < 0.001).*** p < Notably,0.001). Notably, the sizes the and sizes shapes and of shapes AuNP-treated of AuNP pericytes-treated pericytes started to started change to when change an AuNPwhen concentrationan AuNP concentration of 30 ppm wasof 30 used, ppm as was observed used, as under observed microscopy. under Therefore,microscopy. we Therefore, selected a concentrationwe selected a concentration of 30 ppm for of the 30 next ppm experiments. for the next experiments.

Figure 2. Cell viability of of control control pericytes pericytes ( (AA)) and and pericytes pericytes trea treatedted with with AuNPs AuNPs at at 10 10 (B (B), ),20 20 (C (C), ), 30 ( D), 40 ( (E),), and and 50 50 ppm ppm ( (FF)) concentrations. concentrations. The The graph graph shows shows the the quantitative quantitative data data for for pericyte pericyte viability (G).). ****** p < 0.001. Bar: 100 100 μmµm..

3.3. Ki-67 and PDGFR-β mRNA Expression The relative mRNA expression of Ki-67, a marker of cell proliferation, was signiﬁcantly decreased after treatment with 30 ppm AuNPs (fold change of control = 0.955 ± 0.034; 30 ppm AuNPs = 0.037 ± 0.004). In addition, the relative mRNA expression of PDGFR-β, a marker of pericytes, was also decreased in the treatment group compared with the control group (fold change of control = 0.987 ± 0.033; 30 ppm AuNPs = 0.363 ± 0.028) (Figure3, *** p < 0.001). Pharmaceutics 2021, 13, x FOR PEER REVIEW 6 of 13

3.3. Ki-67 and PDGFR-β mRNA Expression The relative mRNA expression of Ki-67, a marker of cell proliferation, was significantly decreased after treatment with 30 ppm AuNPs (fold change of control = 0.955 ± 0.034; 30 ppm AuNPs = 0.037 ± 0.004). In addition, the relative mRNA expression of PDGFR-β, a marker of pericytes, was also decreased in the treatment group compared Pharmaceuticswith2021 , the13, 738 control group (fold change of control = 0.987 ± 0.033; 30 ppm AuNPs = 0.3636 ± of 13 0.028) (Figure 3, *** p < 0.001).

FigureFigure 3. Pericyte 3. Pericyte proliferation proliferation and PDGFR- andβ mRNA PDGFR expression-β mRNA after treatment expression with after 30 ppm treatment AuNPs for with 24 h. Representative 30 ppm imagesAuNPs are shown for 24 for h. the Representative control group (A) images and the 30are ppm shown AuNP-treated for the control pericyte group ( B().A The) and graphs the 30 show ppm the Ki-67 β (C) andAuNP PDGFR--treated(D) mRNApericyte expression group ( levels.B). The *** graphsp < 0.001. show Bar: 100 the nm. Ki-67 (C) and PDGFR-β (D) mRNA expression levels. *** p3.4. < 0.001. Pericyte Bar: Migration 100 nm. Migrating cells of both the control and 30 ppm AuNP-treated groups passed through 3.4. Pericyte Migrationthe membrane pores and expanded their cytoplasm onto the lower surface of the Tran- Migrating cellsswell of membrane.both the control The results and showed 30 ppm that AuNP AuNPs-treated markedly groups reduced passed the number through of cells moving through the membrane (365 ± 10 cells) from the number in the control group the membrane pores(3022 ± and80 cells) expanded (Figure4, *** theirp < 0.001). cytoplasm onto the lower surface of the Transwell membrane. The results showed that AuNPs markedly reduced the number of cells moving through the membrane (365 ± 10 cells) from the number in the control group (3022 ± 80 cells) (Figure 4, *** p < 0.001).

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FigureFigure 4.4. CellCell migrationmigration of pericytes after treatment treatment with with 30 30 ppm ppm AuNPs. AuNPs. Representative Representative images images areare shownshown for for the the control control group group ( A(A,C,C)) magniﬁed magnified view view of of (A (A) and) and the the 30 30 ppm ppm AuNP-treated AuNP-treated pericyte peri- groupcyte group (B,D) ( magniﬁedB,D) magnified view ofview (B). of The (B). graph The graph shows shows the numbers the numbers of migrating of migrating cells (E ).cells *** (pE<). 0.001.*** p < 0.001. Bars: 100 μm. Bars: 100 µm.

3.5.3.5. TransmissionTransmission ElectronElectron Microscopy Microscopy ToTo studystudy thethe intracellularintracellular uptakeuptake ofof AuNPs,AuNPs, transmissiontransmission electronelectronmicroscopy microscopy waswas used.used. InIn the the control control group, group, pericytes pericytes presented presented no no disruption disruption of the of cytoplasmicthe cytoplasmic and nuclearand nu- membranes.clear membranes. The mitochondria The mitochondria and endoplasmic and endoplasmic reticulum reticulum were were normal normal (Figure (Figure5). The 5). plasmaThe plasma membrane, membrane, nucleus, nucleus, and nuclear and nuclear membrane membrane structures structures of the 30 of ppmthe 30 AuNP-treated ppm AuNP- pericytestreated pericytes were similar were to similar those ofto thethose control of the pericytes control pericytes (Figure6A). (Figure AuNPs 6A). were AuNPs found were in latefound endosomes in late endosomes (Figure6B), (Figureautolysosomes 6B), autolysosomes (Figure6C,D), (Figure and mitochondria 6C,D), and ( mitochondriaFigure6E,F ). Interestingly,(Figure 6E,F). mitochondria Interestingly, affectedmitochondria by AuNPs affected were by swollen AuNPs andwere damaged swollen (andFigure damaged6E,F ). Free(Figure AuNPs 6E,F). in Free the AuNPs cytosol in were the cytosol not observed. were not In observed. addition, In we addition, treated we pericytes treated withperi- 50cytes ppm with AuNPs, 50 ppm and AuNPs, the results and the revealed results that revealed pericytes that were pericytes severely were damaged severely anddamaged died (Supplementaland died (Supplemental Figures S1 Figure and S2).s S1 and S2).

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FigureFigure 5 5. .5 Transmission.Transmission Transmission electron electronelectron microscopy microscopymicroscopy of pericytes ofof pericytes in the in controlthe control group group group at low at at ( lowA low) and ( (AA )high) and and high high magniﬁcationsmagnificationsmagnifications (B ((B).B). ).The TheThe pericytes pericytespericytes showed showed normal normal normal ultrastructure. ultrastructure. ultrastructure. Note Note Notethe healthy the the healthy healthy mitochondria mitochondria mitochondria (arrowhead).(arrowhead).(arrowhead). Bars: Bars: Bars: 5000 5000 5000 nm nm nm (A ( A(),A ),1000), 1000 1000 nm nm nm (B () B.( B).).

Figure 6 6.. TransmissionTransmission electron electron microsco microscopypy of ofthe the 30 30ppm ppm AuNP AuNP-treated-treated pericyte pericyte group group at low at ( lowA) (A) and high magnifications (B–F). Representative images are shown for late endosomes containing and high magniﬁcations (B–F). Representative images are shown for late endosomes containing AuNPsFigure and6. Transmission multivesicular electron bodies (microscoB), autolysosomespy of the containing30 ppm AuNP only- treatedAuNPs (pericyteC), and autolyso-group at low (A) B C AuNPssomesand high containing and magnifications multivesicular AuNPs and ( bodiesB– intracellularF). Representative ( ), autolysosomes debris (imagesD). Affected containing are shown mitochondria only for AuNPs late showedendosomes ( ), and mild autolysosomes containing to se- containingvereAuNPs swelling and AuNPs multivesicular(E,F). andNote intracellular the AuNPs bodies debris(electron(B), autolysosomes (D-).dense Affected dots), mitochondriacontaining multivesicular only showed bodies AuNPs mild (black (C to), andarrows), severe autolyso- swelling (intracellularEsomes,F). Note containing thedebris AuNPs (doubleAuNPs (electron-dense blackand intracellular arrows), dots),unaffected debris multivesicular (mitochondriaD). Affected bodies mitochondria(black (black arrowhead arrows), showed), and intracellular mildswol- to se- debrislenvere or swellingdamaged (double black( mitochondriaE,F). arrows),Note the (white unaffectedAuNPs arrowhead). (electron mitochondria- denseBars: 1000 dots), (black nm multivesicular arrowhead),(A), 100 nm ( andB bodies–D swollen), 500 (black nm or ( Earrows), damaged,F). mitochondriaintracellular debris (white (double arrowhead). blackBars: arrows), 1000 unaffected nm (A), 100 mitochondria nm (B–D), 500 (black nm arrowhead (E,F). ), and swollen or damaged mitochondria (white arrowhead). Bars: 1000 nm (A), 100 nm (B–D), 500 nm (E,F).

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3.6.3.6. CapillaryCapillary TubeTube FormationFormation TheThe tubestubes thatthat formedformed inin thethe controlcontrol groupgroup werewere completecomplete withwith thinthin andand elongatedelongated walls,walls, similarsimilar toto capillaries in vivo. vivo. I Inn the the AuNP AuNP-treated-treated group, group, pericytes pericytes and and endothelial endothe- lialcells cells did didnot migrate not migrate to form to form tubes. tubes. Completed Completed tubes were tubes rarely were found, rarely found,and some and of some these oftubes these presented tubes presented thicker walls thicker in the walls AuNP in the-treated AuNP-treated group than group in the than control in the group. control To group.quantify To tube quantify formation, tube formation, all completed all completed tubes in tubes the control in the control and AuNP and- AuNP-treatedtreated groups groupswere counted were counted after culture after culturefor 4 h. forThe 4 results h. The resultsshowed showed that the that number the number of tubes of formed tubes formedsignificantly signiﬁcantly decreased decreased from 467.70 from 467.70± 26.76± tubes26.76 to tubes 290.80 to 290.80± 19.56± tubes19.56 (Figure tubes (Figure 7, *** p7 ,< ***0.001).p < 0.001).

FigureFigure 7.7. TubeTube formation formation of of the the control control group group (in (in which which intact intact endothelial endothelial cells cells were were cocultured cocultured with intactwith intact pericytes) pericytes) (A) and (A) the and AuNP-treated the AuNP-treated group group (in which (in which intact intact endothelial endothelial cells cells and and pericytes peri- werecytes pretreatedwere pretreated withAuNPs) with AuNPs) (B). The (B). graph The graph shows shows the numbers the numbers of tubes of tu (Cbes). Note(C). Note the cluster the clus- of mixedter of mixed pericytes pericytes and endothelial and endothelial cells (arrow), cells (arrow), the thick the wall thick of wall tube of formation tube formation (arrowhead), (arrowhead), and the and the area of incomplete tubes (asterisk). *** p < 0.001. Bar: 500 µm. area of incomplete tubes (asterisk). *** p < 0.001. Bar: 500 µm.

3.7. Immunofluorescence Histochemistry of Pericytes during Tube Formation 3.7. ImmunoﬂuorescenceTo observe the morphology Histochemistry and oflocalization Pericytes during of pericytes Tube Formation during tube formation, im- munofluorescenceTo observe the histochemical morphology staining and localization for PDGFR of- pericytesβ, which is during a marker tube of formation, pericytes, immunoﬂuorescencewas conducted. In the histochemical control group, staining pericytes for showed PDGFR- intenseβ, which PDGFR is a marker-β-immunopositive of pericytes, wasstaining, conducted. had elongated In the control shapes, group, and pericyteswere located showed beside intense endothelial PDGFR- cellsβ-immunopositive along the tubes staining,(Figure 8A had,B). elongated On the other shapes, hand, and the were tube located-forming beside pericytes endothelial in the cellsAuNP along-treated the tubesgroup (Figureappeared8A,B). round On in the shape other and hand, did the not tube-forming extend processes pericytes (Figure in the 8C, AuNP-treatedD). In addition, group many appearedmixed cell round clusters in shapeof pericytes and did and not endothelial extend processes cells were (Figure observed8C,D). in Inthe addition, treated group. many mixed cell clusters of pericytes and endothelial cells were observed in the treated group.

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FigureFigure 8.8. ImmunoﬂuorescenceImmunofluorescence staining staining of of pericytes pericytes during during tube tube formation formation in in the the control control group group (A ,B) and(A,B the) and 30 ppmthe 30 AuNP-pretreated ppm AuNP-pretreated pericyte pe groupricyte (C group,D). PDGFR- (C,D). βPDGFR-immunopositive-β-immunopositive cells are pericytescells are (greenpericytes pseudocolor), (green pseudocolor), and unstained and unstained cells are endothelial cells are endothelial cells. In the cells. treated In the group, treated pericytes group, peri- were roundcytes were and didround not and extend did not processes. extend processes. Note that Note the endothelial that the endothelial cells and cells AuNP-treated and AuNP pericytes-treated pericytes formed thicker walls than the control cells. The nuclei are stained with DAPI (blue pseu- formed thicker walls than the control cells. The nuclei are stained with DAPI (blue pseudocolor). docolor). Bars: 100 µm. Bars: 100 µm.

4.4. DiscussionDiscussion TheThe objectiveobjective of of this this study study was was to to reveal reveal the the effects effects of of AuNPs AuNPs on on biological biological changes changes in pericytesin pericytes and and capillary capillary tube tube formation. formation. AuNPs AuNPs significantly significantly reduced reduced pericyte pericyte proliferation, prolifer- migrationation, migration and PDGFR- and PDGFRβ mRNA-β mRNA expression. expression. Transmission Transmission electron electron microscopy microscopy revealed re- AuNPvealed aggregation AuNP aggregation within mitochondria,within mitochondria, which caused which pleomorphiccaused pleomorphic features features and mild and to severemild to damage. severe damage. Moreover, Moreover, we found we that found pericytes that pericytes that were that treated were with treated AuNPs with formedAuNPs incompleteformed incomplete capillary capillary tubes even tubes though even they though were they cocultured were cocultured with intact with endothelial intact endo- cells. thelialPrevious cells. studies have reported that AuNPs larger than 5 nm have a chemically inert character,Previous resulting studies in lowhave cytotoxicity. reported that AuNPs AuNPs with larger a size than of 20 5 nm have are more a chemically effective thaninert 5character, and 10 nm resulting AuNPs in in low inhibiting cytotox HUVECicity. AuNPs proliferation with a size [17] andof 20 do nm not are induce more cytotoxi-effective citythan in 5 theand retinal 10 nm pigmentAuNPs in epithelium inhibiting [HUVEC1], retinal proliferation microvascular [17] endothelial and do not cellsinduce [18 cyto-], or nonmalignanttoxicity in the retinal NIH3T3 pigment fibroblast epithelium cells [19 ].[1], Moreover, retinal microvascular AuNPs of this endothelial size are frequently cells [18], appliedor nonmalignant in HUVEC NIH3T3 tube formation fibroblast and cells angiogenesis [19]. Moreover, studies AuNPs [1,17 of,20 this,21]. size Previous are frequently reports haveapplied mentioned in HUVEC that tube 20 nmformation AuNPs, and especially angiogenesis at relatively studies [1,17,20,21] low concentrations,. Previous can reports sig- nificantlyhave mentioned change that the 20 biological nm AuNPs, characteristics especially ofat relatively HUVECs; low for example,concentrations, they can can reduce signif- cellicantly proliferation, change the cell biological migration, characteristics and tube formation of HUVECs; [1,7 ].for In example, our study, they 20 can nm reduce AuNPs cell at

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concentrations of 40 and 50 ppm caused cell death. Thus, 30 ppm, which was the highest concentration that maintained high cell survival, was chosen for further experiments (Figure2 ). However, it is worth noting that cellular changes are dependent not only on the size and concentration of nanoparticles but also on the shape, surface charge, dispersion in solvent, exposure duration, molecule coating, and type of cell [4,7,19,22–24]. AuNPs can inhibit several endothelial activities and cell survival by changing the conformation of VEGF or VEGF receptor in HUVECs [1,6]. For pericytes, the PDGF-BB- PDGFR-β signaling pathway is essential for proliferation, migration, maturation, differentiation, and survival during capillary tube formation [15,25,26]. PDGF-BB is released from endothelial cells and interacts with PDGFR-β, a receptor on the surfaces of pericytes. This interaction results in autophosphorylation and noncovalent dimerization of speciﬁc tyrosine residues in the pericyte cytoplasm. Cell proliferation is a process that occurs through the phosphoinositide-3-kinase (PI3K) pathway, and cell migration is mediated by the Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP-2)-mediated mitogen- activated protein kinase (MAPK) pathway [13,26]. Therefore, in the present study, we hypothesized that AuNPs could reduce PDGFR-β mRNA expression, leading to decreases in proliferation and migration (Figures3 and4) via these pathways. In our nanostructural study, as shown in Figure6, AuNPs at 30 ppm did not cause cell death or destroy the plasma membrane, nucleus, or nuclear membrane, but they did cause swelling and damage to mitochondria. AuNPs were mainly engulfed into endosomes that fused with lysosomes containing digestive enzymes (Figure6A–D) in the typical endocytic pathway [27]. However, a number of AuNPs were observed in mitochondria (Figure6A,E,F ). These nanoparticles were transferred from endosomes through the endosomal escape pathway [28] and might subsequently have been transferred to autolysosomes for degradation. Mitochondrial dysfunction disrupts ATP production, apoptosis regulation, metabolism integration, and calcium homeostasis in cells [29]. The rest of the AuNPs that lysosomes could not digest might have been eliminated by cellular exocytosis [30]. Studies by Roh et al. (2016) and Shen et al. (2018) have shown that AuNPs can inhibit 3D capillary tube formation [1,7]. However, both studies performed capillary tube formation in monocultures of a single cell type: endothelial cells. In contrast, our present study performed tube formation using a 3D coculture method with both endothelial cells and pericytes to imitate the capillary vasculature found in almost all tissues. Moreover, the results shown in Figures7 and8 reveal that although endothelial cells were intact, defects in pericytes induced incomplete capillary tube formation. Furthermore, the cell aggregation found in the 3D culture system indicated that a number of cells did not migrate to form tubes, consistent with the results of the migration assay shown in Figure4. We presume that the tube-guiding property of pericytes [9] is impaired after administration of AuNPs.

5. Conclusions This is the ﬁrst study on the effects of AuNPs focusing on changes in pericyte characteristics and properties. Moreover, the results from the 3D coculture, which included pericytes in capillary formation, suggest that this cell type could be considered an antiangiogenic target in nanoparticle therapy. Finally, future directions for this research include evaluating the elimination of AuNPs on pericytes in vitro, as well as studying the cytotoxicity and clearance rate of AuNPs in vivo, both of which are essential for developing novel antiangiogenic therapies.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/pharmaceutics13050738/s1, Figures S1 and S2: Transmission electron microscopy of the 50 ppm AuNP-treated pericyte group. Author Contributions: Conceptualization, D.J.; methodology, D.J.; validation, T.S.; formal analysis, S.L., T.S. and D.J.; investigation, S.L., T.S. and D.J.; resources, K.S., A.S. and N.K.; writing—original Pharmaceutics 2021, 13, 738 12 of 13

draft preparation, S.L.; writing—review and editing, D.J.; visualization, K.S.; supervision, A.S. and D.J. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Ratchadapiseksompotch Fund, Faculty of Medicine, Chulalongkorn University, grant number RA61/100. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Acknowledgments: We are grateful to the Research Affairs, Faculty of Medicine, Chulalongkorn University for assistance regarding the transmission electron microscope. We thank Trairak Pisitkun for supporting the confocal laser scanning microscope. We thank Anucha Sacharoen (Faculty of Dentistry, Mahidol University) for his transmission electron microscopic experiences. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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