The uptake of PLGA micro or nanoparticles by macrophages ... · The uptake of PLGA micro or...

7
The uptake of PLGA micro or nanoparticles by macrophages provokes distinct in vitro inammatory response Roberto Nicolete a, b, , Daiane F. dos Santos a , Lúcia H. Faccioli a a Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café s/n, 14040-903, Ribeirão Preto, São Paulo, Brazil b Laboratório de Imunotecnologia, Pró-Reitoria de Pós-Graduação, Pesquisa e Extensão, Centro Universitário do Maranhão (UNICEUMA), Rua Josué Montello, 1, 65075-120, São Luís, Maranhão, Brazil abstract article info Article history: Received 18 March 2011 Received in revised form 5 May 2011 Accepted 13 May 2011 Available online 27 May 2011 Keywords: Micro and nanoparticles Poly (lactic-co-glycolic acid) Uptake process NF-κB translocation Inammatory cytokines Biodegradable micro/nanoparticles generated from PLGA have recently attracted attention due to their clinically proven biocompatibility, especially for immunization purposes. These polymeric particulate delivery systems are able to present antigens and activate both humoral and cellular responses. Many studies have discussed the ideal size of these particles in contributing to the generation of the different types of immune response. However, these studies do not demonstrate the effect of micro or nanoparticles, without any encapsulated bioactive, on phagocytic cells after the uptake process. In this context, the aim of this study was to analyze the in vitro inammatory behavior of J774 murine macrophages after particles' uptake, since nano/microparticles per se can differently activate phagocytic cells, using or not appropriate receptors, inducing distinct inammatory responses. An o/w emulsion solvent extractionevaporation method was chosen to prepare the particles. We determined their diameters, zeta potential and morphology. Fluorescent particles' uptake by J774 murine macrophage-likecells was also analyzed. To evaluate the in vitro inammatory prole of these cells after micro or nanoparticles' uptake, we conducted NF-κB translocation assay by confocal microscopy and also determined the pro-inammatory cytokines production provoked by the particles. © 2011 Elsevier B.V. 1. Introduction Particles made from poly (lactic-co-glycolic acid) (PLGA) can be used as a delivery system and also provide adjuvant activity [1,2] during immunization protocols. Biodegradable micro/nanoparticles generated from PLGA have recently attracted attention due to their clinically proven biocompatibility [3]. In addition to facilitating the uptake of encapsulated materials, PLGA particles also potentially protect different molecules, such as nucleic acids, peptides and protein antigens, increasing delivery efciency [4]. These polymeric particulate delivery systems are able to present antigens and activate both humoral and cellular responses [2,5,6]. Many studies have discussed the ideal size of these particles in contributing to the generation of immune response [79]. Polymer-entrapped antigens reveal that micron-sized range particles promote humoral response whereas nanoparticles promote cellular one [10,11]. In this context, it is difcult to predict the behavior of phagocytes after particles' uptake, since nano/microparticles per se can differently activate phagocytic cells, using or not appropriate receptors, inducing distinct inamma- tory responses. The presence of the biomaterial component inside the cell may increase the immune response against the biological component through an adjuvant effect, and also lead to unexpected inammatory prole. The link between the activation of NF-κB and inammation has been shown in various human and animal models of disease. In most cases, resident cells or those from infected tissue initiate the inammatory response by triggering pro-inammatory pathways through NF-κB in response to inammatory stimuli [12]. In canonical NF-κB signaling pathways, the pro-inammatory cytokine tumor necrosis factor (TNF) and interleukin-1 (IL-1), and also microbial products (lipopolysaccharide (LPS), i.e.) activate IKKβ, mediating the classical attributes of inammation [13]. Regarding PLGA-based nanoparticles, they are extensively taken up by non-phagocytic eukaryotic cells, macrophages and dendritic cells [14]. However, such clear uptake studies with large-sized polymer particles have not been shown conclusively. It is quite possible that bigger-sized particles such as microparticles are localized mainly in the cell membrane. This fact demands more time to the particles be engulfed and can lead to a physical disturbance in the membrane, involving or not the participation of specic receptors, and provoking an inammatory response by different mechanisms. In this study we produced PLGA nano and microparticles without any encapsulated bioactive to be examined with respect to their International Immunopharmacology 11 (2011) 15571563 Corresponding author at: Pró-Reitoria de Pós-Graduação, Pesquisa e Extensão, Centro Universitário do Maranhão (UNICEUMA), Rua Josué Montello, 1, 65075-120, São Luís, Maranhão, Brazil. Tel.: +55 98 3214 4336; fax: +55 98 3235 8600. E-mail address: [email protected] (R. Nicolete). 1567-5769 © 2011 Elsevier B.V. doi:10.1016/j.intimp.2011.05.014 Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp Open access under the Elsevier OA license. Open access under the Elsevier OA license.

Transcript of The uptake of PLGA micro or nanoparticles by macrophages ... · The uptake of PLGA micro or...

Page 1: The uptake of PLGA micro or nanoparticles by macrophages ... · The uptake of PLGA micro or nanoparticles by macrophages provokes distinct in vitro inflammatory response Roberto

International Immunopharmacology 11 (2011) 1557–1563

Contents lists available at ScienceDirect

International Immunopharmacology

j ourna l homepage: www.e lsev ie r.com/ locate / in t imp

The uptake of PLGA micro or nanoparticles by macrophages provokes distinct in vitroinflammatory response

Roberto Nicolete a,b,⁎, Daiane F. dos Santos a, Lúcia H. Faccioli a

a Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café s/n, 14040-903,Ribeirão Preto, São Paulo, Brazilb Laboratório de Imunotecnologia, Pró-Reitoria de Pós-Graduação, Pesquisa e Extensão, Centro Universitário do Maranhão (UNICEUMA), Rua Josué Montello, 1, 65075-120,São Luís, Maranhão, Brazil

⁎ Corresponding author at: Pró-Reitoria de Pós-GraCentro Universitário doMaranhão (UNICEUMA), Rua JosLuís, Maranhão, Brazil. Tel.: +55 98 3214 4336; fax: +

E-mail address: [email protected] (R. Nicolete).

1567-5769 © 2011 Elsevier B.V.doi:10.1016/j.intimp.2011.05.014

Open access under the Else

a b s t r a c t

a r t i c l e i n f o

Article history:Received 18 March 2011Received in revised form 5 May 2011Accepted 13 May 2011Available online 27 May 2011

Keywords:Micro and nanoparticlesPoly (lactic-co-glycolic acid)Uptake processNF-κB translocationInflammatory cytokines

Biodegradablemicro/nanoparticles generated from PLGA have recently attracted attention due to their clinicallyproven biocompatibility, especially for immunization purposes. These polymeric particulate delivery systems areable to present antigens and activate both humoral and cellular responses. Many studies have discussed the idealsize of these particles in contributing to the generationof the different types of immune response.However, thesestudies do not demonstrate the effect of micro or nanoparticles, without any encapsulated bioactive, onphagocytic cells after the uptake process. In this context, the aim of this study was to analyze the in vitroinflammatory behavior of J774murine macrophages after particles' uptake, since nano/microparticles per se candifferently activate phagocytic cells, using or not appropriate receptors, inducing distinct inflammatoryresponses. An o/w emulsion solvent extraction–evaporation method was chosen to prepare the particles. Wedetermined their diameters, zeta potential and morphology. Fluorescent particles' uptake by J774 murine“macrophage-like” cells was also analyzed. To evaluate the in vitro inflammatory profile of these cells after microor nanoparticles' uptake, we conducted NF-κB translocation assay by confocal microscopy and also determinedthe pro-inflammatory cytokines production provoked by the particles.

duação, Pesquisa e Extensão,ué Montello, 1, 65075-120, São55 98 3235 8600.

vier OA license.

© 2011 Elsevier B.V. Open access under the Elsevier OA license.

1. Introduction

Particles made from poly (lactic-co-glycolic acid) (PLGA) can beused as a delivery system and also provide adjuvant activity [1,2]during immunization protocols. Biodegradable micro/nanoparticlesgenerated from PLGA have recently attracted attention due to theirclinically proven biocompatibility [3]. In addition to facilitating theuptake of encapsulated materials, PLGA particles also potentiallyprotect different molecules, such as nucleic acids, peptides andprotein antigens, increasing delivery efficiency [4]. These polymericparticulate delivery systems are able to present antigens and activateboth humoral and cellular responses [2,5,6]. Many studies havediscussed the ideal size of these particles in contributing to thegeneration of immune response [7–9]. Polymer-entrapped antigensreveal that micron-sized range particles promote humoral responsewhereas nanoparticles promote cellular one [10,11]. In this context, itis difficult to predict the behavior of phagocytes after particles' uptake,since nano/microparticles per se can differently activate phagocyticcells, using or not appropriate receptors, inducing distinct inflamma-

tory responses. The presence of the biomaterial component inside thecell may increase the immune response against the biologicalcomponent through an adjuvant effect, and also lead to unexpectedinflammatory profile.

The link between the activation of NF-κB and inflammation hasbeen shown in various human and animal models of disease. In mostcases, resident cells or those from infected tissue initiate theinflammatory response by triggering pro-inflammatory pathwaysthrough NF-κB in response to inflammatory stimuli [12]. In canonicalNF-κB signaling pathways, the pro-inflammatory cytokine tumornecrosis factor (TNF) and interleukin-1 (IL-1), and also microbialproducts (lipopolysaccharide (LPS), i.e.) activate IKKβ, mediating theclassical attributes of inflammation [13].

Regarding PLGA-based nanoparticles, they are extensively takenup by non-phagocytic eukaryotic cells, macrophages and dendriticcells [14]. However, such clear uptake studies with large-sizedpolymer particles have not been shown conclusively. It is quitepossible that bigger-sized particles such as microparticles arelocalized mainly in the cell membrane. This fact demands more timeto the particles be engulfed and can lead to a physical disturbance inthe membrane, involving or not the participation of specific receptors,and provoking an inflammatory response by different mechanisms.

In this study we produced PLGA nano and microparticles withoutany encapsulated bioactive to be examined with respect to their

Page 2: The uptake of PLGA micro or nanoparticles by macrophages ... · The uptake of PLGA micro or nanoparticles by macrophages provokes distinct in vitro inflammatory response Roberto

1558 R. Nicolete et al. / International Immunopharmacology 11 (2011) 1557–1563

uptake by J774 macrophages and further in vitro inflammatorycytokine production.We analyzed by confocal images the interactionsof different sized polymer particles with these cells for correlating thatwith NF-κB translocation to the cell nucleus after the uptake processand the production of pro-inflammatory cytokines TNF-α and IL-1-βby these cells.

2. Materials and methods

2.1. Materials

For the purposes of this study, Poly (D,L-lactide-co-glycolide)(PLGA) with a co-monomer ratio of 50:50 (lactic/glycolic acid) andmolecular weight of 78 kDa was obtained from Boehringer Ingelheim(Ingelheim, Germany). Poly (vinyl-alcohol) (Mowiols 40–88) wasobtained from Aldrich Chemicals (Wankee, WI, USA). Methylenedichloride was purchased from Merck (Dietikon, Switzerland).Lipopolysaccharide (LPS), the dye 6-coumarin and saponin werefrom Sigma Chemical Co. (St. Louis, MO, USA). Acqua Poly/Mount wasobtained from Polysciences, Inc. (Warrington, PA). Lyso Tracker™ Redwas from Molecular Probes. The Limulus Amoebocyte Lysate test (LALtest, QCL-1000, Bio Whittaker, CAMBREX Company, Walkersville, MD,USA) was used for the detection of endotoxin in the particles. RPMI-1640medium, fetal bovine serum (FBS) and antibiotics (penicillin andgentamicin) were from Gibco (Grand Island, NY, USA). For cellcultures were used non-tissue culture treated plates from BectonDickinson (BD Falcon, Franklin Lakes, NJ, USA).

2.2. Micro and nanoparticles preparation

Microparticles were prepared using an oil-in-water (O/W)emulsion solvent extraction–evaporation process as previouslydescribed [15,16]. In brief, 10 ml of methylene dichloride containing30 mg of PLGA were homogenized. This phase was poured into anexternal aqueous phase (40 ml of polyvinyl alcohol (Sigma ChemicalCo.) solution at 3% w/v) and stirred mechanically (RW20; IKALabortechnik, Germany) at 600 rpm for 4 h to extract the organicsolvent. For nanoparticles preparation, the O/W emulsion solutionwas homogenized at 24,000 rpm for 5 min by using a T25 Ultraturraxhomogenizer (IKA, Labortechnik, Germany). The solution was thenplaced under a magnetic stirring condition for 4 h to evaporate thesolvent. Finally, both micro/nanoparticles formed were washed threetimes with doubly distilled water, centrifuged and then freeze-dried.Fluorescent-labeled particles were prepared by adding 6-coumarin(green fluorescence; Sigma Aldrich) to the organic phase. The amountof 6-coumarin/mg polymer was 0.15 μg [17].

2.3. Micro and nanoparticle characterization

Particle diameterswere characterized using a particle size analyzer(LS 13 320 Laser Diffraction Particle Size Analyzer; Beckman Coulter,USA). Zeta potential analysis of the particles was performed using aNano Zeta Sizer (Malvern instruments, England). To determinewhether the particles and media were contaminated by LPS, a LALtest was performed. The shape and surface of the dried micro andnanoparticles were observed by scanning electron microscopy (SEM),using a ZEISS scanning microscope (ZEISS, Evo 50, Cambridge,England).

2.4. Uptake assay with fluorescent particles

J774 murine “macrophage-like” cells (5×105/24-wells) wereobtained from European Collection of Animal Cell Cultures (Salisbury,UK). Cells were plated on wells containing coverslips and co-incubatedwith 1 mg/ml of fluorescent particles during 2 and 4 h. After thisincubation period, the cells were washed twice with PBS, fixed for

15min with 2% paraformaldehyde in PBS and rinsed in PBS. Coverslipswere mounted using Acqua Poly/Mount (Polysciences) and examinedby confocal microscopy (Leica TCS SP5 AOBS — Leitz, Manheim,Germany).

2.5. Confocal laser scanning microscopy (CLSM) analyses

J774 murine macrophages (5×105 cells/well) were grown oncoverslips inside sterile 24-well tissue culture plates and incubatedwith fluorescent nano or microparticles (1 mg/ml) at 37 °C, 5% CO2 for4 h. In order to analyze intracellular localization of particles, cells werelater incubated with organelle-specific contrasting fluorescent dyeLyso Tracker™ Red (Molecular Probes) 160 nM, for 30 min at 37 °Cand washed three times with sterile 50 mM phosphate-bufferedsaline (PBS). The endolysosomes appeared red in color when labeledwith Lyso Tracker™ Red and visualized under RITC filter. Series of z-sections were performed using CLSM following which the colocaliza-tion of the fluorescent particles within the endolysosomal compart-ments at different sections of the cells could be visualized under bothFITC and RITC filters. The extent of colocalization between fluorescentparticles (green) and endolysosomal compartments (Lyso Tracker™Red) was quantified using the Jacop plug-in for Image J [18]. Thebackground of the collected images was corrected by the Image Jalgorithm plug-in. The specific algorithm used was based on theMander's overlap coefficient [18]. This coefficient varies from 0 to 1,the former corresponding to non-overlapping images and the latterreflecting 100% colocalization between both images.

For NF-κB translocation assay, cells were 4 h incubated in culturemedium, with LPS (0.5 μg/ml), used as positive control, and with1 mg/ml of non-fluorescent nano or microparticles. They were washedtwice with PBS and fixed for 15 min with 2% paraformaldehyde in PBSand rinsed in PBS with 0.1 M glycine for 5 min. Then, cells werepermeabilizedwith 0.01% of saponin in PBS for 20 min and blockedwith3% donkey serum (Jackson ImmunoResearch Laboratories) in PBS for40 min at room temperature. J774murinemacrophageswere incubated1 h with rabbit polyclonal antibodies against NF-κB p65 subunit(Santa Cruz Biotechnology). After PBS wash, secondary antibodies(rabbit anti-IgG labeled with Alexa-Fluor 594 (Invitrogen MolecularProbes))were added for 30 min. DAPI (Calbiochem)was used for nucleistaining during 15 min. After the described staining steps, coverslipswere mounted using Acqua Poly/Mount (Polysciences) and examinedfor intracellular localization of the particles and NF-κB translocation byconfocal microscopy (Leica TCS SP5 AOBS— Leitz, Manheim, Germany).Densitometric analyses of the images were performed using Image J.For each cell nucleus, thepixel density I.O.D. (integrated optical density)was determined by selecting a circle of identical surface for alldeterminations, designed to entirely cover the area of interest.

2.6. Pro-inflammatory cytokines production by particles-stimulated J774macrophages

In order to evaluate the in vitro inflammatory cytokines produc-tion, J774 macrophages (5×105cells/well) were incubated for 4 hwith micro or nanoparticles at 37 °C, 5% of CO2. At the end of theexperiment, cell-free supernatants were collected for IL-1β and TNF-αdetection assays. Cytokine levels were measured by ELISA, usingcommercially available antibodies with reference standard curves.The optical density of samples was determined at 450 nm in amicroplate reader. Sensitivities were N8.0 pg/ml.

2.7. Statistical analysis

The data are presented as mean±S.E.M. and analyzed usingunpaired t-test. Values of Pb0.05 were considered statisticallysignificant.

Page 3: The uptake of PLGA micro or nanoparticles by macrophages ... · The uptake of PLGA micro or nanoparticles by macrophages provokes distinct in vitro inflammatory response Roberto

1559R. Nicolete et al. / International Immunopharmacology 11 (2011) 1557–1563

3. Results and discussion

3.1. Size, zeta potential and surface morphology

Lyophilized particles were dispersed in distilled water and submit-ted to analysis of size distribution. For microparticles, the averagediameterwas6.5±3.9 μmand for nanoparticles, 389 nmwith PdI=0.2.The average zeta potential was −34.5 and −17.2±6.1 mV, for microand nanoparticles, respectively. Scanning electron micrographies ofmicro and nanoparticles are shown in Fig. 1. Both particles weresphericalwith smooth surface and irregularities were not observed. Theparticles (2 batches) and also the media were assayed for detection ofendotoxin activity using the Limulus Amoebocyte assay (LAL test).Endotoxin activity in all batches was lower than 0.1 EU/μg. According tothe European Pharmacopeia, the safety level for intravenous adminis-tration is 5 EU/kg/h.

Our results show that both micro and nanoparticles had typicalnegative zeta potential attributed to PLGA preparations due to carboxylgroups present in their structure. Also, Fig. 1 shows that micro andnanoparticles produced were intact for the use in the assays.

3.2. Analysis of particles' uptake

To visualize particles' uptake by J774 macrophages, cells wereincubated with both PLGA micro and nanoparticles for two differenttime points (2 and 4 h). These times were chosen in order to get anidea about the initial inflammatory behavior of phagocytic cells in thepresence of particles. Studies were conducted using fluorescentparticles. Fluorescence microscopy showed that most of the nano-particles (389 nm) were localized intracellularly and better uptake

Fig. 1. (A) Micro and (B) nanoparticles morphologies

was observed for 4 h incubation (Fig. 2). The interesting observationwas that PLGAmicroparticles of size 6.5 μmwere found to be attachedto the cells surface at 2 and 4 h incubation. In this context, fewmicroparticles could be seen inside the cells when compared tonanoparticles.

Based on our previous studies employing PLGA microparticles forphagocytosis purposes [15,19], in this study, we decided to use thesame diameter range (5–7 μm) achieved to compare with nanopar-ticles. The particles' diameters were adequate for cell phagocytosis,since J774 murine “macrophage-like” cells efficiently engulfed them.In vitro studies have shown that microparticles having less than 10 μmare efficiently internalized by macrophages [20]. Regarding thepossible toxic effect of particles at concentration of 1 mg/ml employedduring 2–4 h in our experiments, is well know that PLGA has an inertprofile, showing high biocompatibility [3]. Also, other studiesconducted with different polystyrene beads' size (20 and 200 nm)at low and high concentrations demonstrated limited cytotoxicity(assessed by MTT and LDH assays) over the 48 h period [21].

It has been documented that size and surface chemistry of particlesdo influence particle uptake [22,23] and in particular, hydrophobicpolymer particles have the tendency to adhere to the cell surface [24].Also, the nanoparticles used in this study presented less negativecharge on the surface of the polymer, as determined by zeta potential,when compared to microparticles (−17.2 vs.−34.5 mV). Since cellmembrane has negative polarity, this fact could contribute to betterinteraction with the nanoparticles, improving their uptake. Fromthese results, we decided to speculate whether micro or nanoparticlesper se, without any encapsulated bioactive could confer differentinflammatory profiles in J774 murine “macrophage-like” cells afterthe uptake process.

assessed by scanning electron microscopy (SEM).

Page 4: The uptake of PLGA micro or nanoparticles by macrophages ... · The uptake of PLGA micro or nanoparticles by macrophages provokes distinct in vitro inflammatory response Roberto

Fig. 2. Uptake assay in J774 murine “macrophage-like” cells using different fluorescent PLGA particles (micro and nanoparticles) at 2 and 4 h. Each bar represents 10 μm.

1560 R. Nicolete et al. / International Immunopharmacology 11 (2011) 1557–1563

3.3. Intracellular analyses of micro and nanoparticles after the uptake byJ774 macrophages

The colocalization of particles and lysosomes showed up as yellowfluorescence in the overlay between green fluorescent micro (Fig. 3A)or nanoparticles (Fig. 3B) and red fluorescent vesicles. No evidentcolocalization of the fluorescent microparticles was observed withinthe labeled cells after 4 h incubationwhen compared to nanoparticles.This fact suggests that theywere seen to be attached to the cell surfaceor they demand more time to be engulfed by the cells. On the otherhand, the colocalization of nanoparticles and lysosomes (measured bythe overlap coefficient) was statistically greater compared tomicroparticles (Fig. 3C). A great number of nanoparticles could beseen inside the vesicles, suggesting the prompt entrance of them tocell cytoplasm, as described by other studies [10].

Regarding external stimuli and further inflammatory cytokinesproduction by many cells, previous studies have shown thatperturbations in the cell cycle can activate the NF-κB pathway [25].In this context, statistically, our results from I.O.D. quantifications(Fig. 4G) show that microparticles (Fig. 4E) are more potentinflammatory stimulus than nanoparticles (Fig. 4F) and also LPS(Fig. 4D) in activating NF-κB translocation to cell nucleus.

3.4. Microparticles-stimulated J774 macrophages produce moreinflammatory cytokines

It is a fact that macrophages can produce inflammatory cytokinesduringphagocytosis process and cell activation [26]. In order to getmoreconclusive data about the acute inflammation response (2–4 h) elicitedby J774 macrophages after particles' uptake, cytokines production wasdetermined by ELISA. Microparticles induced significant release of

TNF-α compared with medium and especially with nanoparticles(Fig. 5A). The same profile was observed for IL-1β production by thecells (Fig. 5B). Correlating these quantitative results with thoseobtained from NF-κB translocation assay, we can suggest thatPLGA microparticles are more inflammatory than nanoparticleswhen J774 murine macrophages were used as test cells. Manystudies have shown a large number of encapsulated substancesand their role in modulating the immune response. However, fewdata are known about the difference between micro andnanoparticles per se, without encapsulated active compound, inacting directly on a nuclear transcription factor, such as NF-κB, andthe consequences for cytokines production after the particles'uptake possibly affecting the cellular phenotype (e.g. expression ofsurface receptors, adhesion and costimulatory molecules).

In order to confirm the need of the preformed NF-κB in the cellcytoplasm to be further requested to nucleus compartment under thepresence of a stimulus (LPS,micro or nanoparticles), we conducted a setof experiments employing the NF-κB inhibitor 6-amino-4-(4-phenoxyphenylethylamino) quinazoline. This drug completely abol-ished TNF-α production by the cells during 1 h pre-incubation. Theresults showed thatmicro or nanoparticles were not able to restore anylevel of this cytokine production (2–4 h) when NF-κB pathway wasinhibited. Interestingly, when IL-1β production was assessed at sameconditions, the results showed that bothmicro (60.8±20.2 pg/ml) andnanoparticles (42.6±2.47 pg/ml) could restore this cytokine levelsunder the effect of the NF-κB inhibitor. However, the values were notstatistically significant between them. We can speculate that thecytokines TNF-α and IL-1β present different times of induction whencells were stimulated with the particles and also, pre-formed NF-κBis needed to confer the distinct inflammatory responses elicited bymicro or nanoparticles. Other authors also demonstrate that PLG

Page 5: The uptake of PLGA micro or nanoparticles by macrophages ... · The uptake of PLGA micro or nanoparticles by macrophages provokes distinct in vitro inflammatory response Roberto

C

0.0

0.5

1.0

1.5Microparticles

Nanoparticles

**

Ove

rlap

Co

effic

ien

t

Fig. 3. Localization of fluorescent (A) micro and (B) nanoparticles in J774 murine macrophages. Representative cells after treatment with dyes were selected at random andseries of optical sections (z-sections) were taken in the dual filter mode. Images captured in FITC (6-coumarin), RITC (Lysotracker) and dual mode were overlaid todetermine localization and colocalization of the particles inside the lysosomes (Merge). Each bar represents 10 μm. (C) Quantification of fluorescent particles/endolysosomal compartments colocalization was determined as described in the Materials and methods. Measurements of overlap coefficient show data from threeseparated experiments (mean±S.E.M). **Pb0.01.

1561R. Nicolete et al. / International Immunopharmacology 11 (2011) 1557–1563

microparticles (1–10 μm) promote the activation of nucleotide-bindingoligomerization domain-like receptor (NLR), specifically NALP3 inflam-masome, in dendritic cells [27]. Taking those results together with ourfindings, another point to be discussed is the increase of adhesion(CD11b) and/or costimulatory (CD86) molecules expression in phago-cytic cells after PLG microparticles' uptake. In this context, we can inferthat although microparticles demand more time to be engulfed by themacrophages they further activate intracellular inflammatory events ina more potent profile compared to nanoparticles. Also, their previousand intense interaction with the membrane ruffles present on themacrophages surface can increase the expression of the moleculesdescribed above leading to NF-κB translocation to cell nucleus andfurther pro-inflammatory cytokines production, as assessed.

This work suggests that although other studies had demonstratedsignificant differences in the modulation of immune responses byemploying micro or nano-structured biodegradable polymers, especiallyfor immunization purposes, is extremely important to evaluate these

preparations without the encapsulated bioactive compound. Takentogether, our results show that PLGA microparticles (5–7 μm) are notable to be phagocytosed by J774 macrophages with the same aviditydemonstrated for nanoparticles (389 nm) but instead, they attach to cellmembrane and constitute more potent inflammatory stimulus afterthe uptake process. This fact leads to NF-κB translocation to cellnucleus and further pro-inflammatory cytokines production bymicroparticles-stimulated cells.

Acknowledgments

The authors thank José O. Del Ciampo, Rodrigo Silva and Lenaldo B.Rocha for technical assistance. Fundação de Amparo à Pesquisa do Estadode São Paulo (FAPESP) and Conselho Nacional de DesenvolvimentoCientífico e Tecnológico (CNPq) (proc. 151678/2008-1) supported thisstudy.

Page 6: The uptake of PLGA micro or nanoparticles by macrophages ... · The uptake of PLGA micro or nanoparticles by macrophages provokes distinct in vitro inflammatory response Roberto

G

0

10

20

30

40

50

60

70Medium

LPS

Microparticles

Nanoparticles

*

***

&&

#

Inte

gra

ted

Op

tica

lD

ensi

ty (

cell

nu

cleu

s)

Fig. 4. NF-κB translocation assay in murine macrophage cell line J774 after micro or nanoparticles' uptake. Representative cells after 4 h incubation were selected at random andseries of optical sections (z-sections) were taken in the dual filter mode. Images were captured separated for DAPI (A), NF-κB (B, showed by white arrows) and dual mode (Merge).(C) represents cells incubated only in culture medium (negative control); (D) cells stimulated with LPS (positive control); (E) and (F) cells incubated with 1 mg/ml of micro andnanoparticles, respectively. (G) Densitometric measurements show data from three separated experiments (mean±S.E.M). *Pb0.05 and ***Pb0.001, values compared to medium;#Pb0.05, microparticles compared to LPS; &&Pb0.01, microparticles compared to nanoparticles.

1562 R. Nicolete et al. / International Immunopharmacology 11 (2011) 1557–1563

Page 7: The uptake of PLGA micro or nanoparticles by macrophages ... · The uptake of PLGA micro or nanoparticles by macrophages provokes distinct in vitro inflammatory response Roberto

Mediu

m

Micropar

ticles

Nanopar

ticles

0

10

20

30

40

50

60

70

80 **

##

AT

NF

-α (

pg

/ml)

Mediu

m

Micropar

ticles

Nanopar

ticles

0

50

100

150

200

250

300**

###

B

IL-1

β (p

g/m

l)

Fig. 5. (A) TNF-α and (B) IL-1β production by J774 macrophages after 4 h incubation inculture medium and with 1 mg/ml of micro or nanoparticles. The concentrations in thesupernatants were quantified by ELISA. Results are expressed as mean±S.E.M. (n=4);**Pb0.01, values compared to medium. ##Pb0.01 and ###Pb0.001, microparticlescompared to nanoparticles.

1563R. Nicolete et al. / International Immunopharmacology 11 (2011) 1557–1563

References

[1] Jiang W, Gupta RK, Deshpande MC, Schwendeman SP. Biodegradable poly(lactic-co-glycolic acid) microparticles for injectable delivery of vaccine antigens. AdvDrug Deliv Rev 2005;57:391–410.

[2] O'Hagan DT, Singh M. Microparticles as vaccine adjuvants and delivery systems.Expert Rev Vaccines 2003;2:269–83.

[3] Mundargi RC, Babu VR, Rangaswamy V, Patel P, Aminabhavi TM. Nano/microtechnologies for delivering macromolecular therapeutics using poly (D,L lactide-co-glycolide) and its derivatives. J Control Release 2008;125:193.

[4] Panyam J, Labhasetwar V. Biodegradable nanoparticles for drug and gene deliveryto cells and tissue. Adv Drug Deliv Rev 2002;55:329–47.

[5] Men Y, Audran R, Thomasin C, Eberl G, Demotz S, Merkle HP, et al. MHC class I- andclass II-restricted processing and presentation of microencapsulated antigens.Vaccine 1999;17:1047–56.

[6] Carcaboso AM, Hernandez RM, Igartua M, Rosas JE, Patarroyo ME, Pedraz JL.Potent, long lasting systemic antibody levels and mixed Th1/Th2 immuneresponse after nasal immunization with malaria antigen loaded PLGA micropar-ticles. Vaccine 2004;2:1423–32.

[7] Tabata Y, Ikada Y. Macrophage phagocytosis of biodegradable microspherescomposed of L-lactic acid/glycolic acid homo- and copolymers. J BiomedMater Res1988;22:837–58.

[8] Eldridge JH, Staas JK, Meulbroek JA, McGhee JR, Tice TR, Gilley RM. Biodegradablemicrospheres as a vaccine delivery system. Mol Immunol 1991;28:287–94.

[9] Thiele L, Rothen-Rutishauser B, Jilek S, Wunderli-Allenspach H, Merkle HP,Walter E. Evaluation of particle uptake in human blood monocyte-derived cellsin vitro. Does phagocytosis activity of dendritic cells measure upwithmacrophages?J Control Release 2001;76:59–71.

[10] Gutierro I, Hernandez RM, Igartua M, Gascon AR, Pedraz JL. Size dependentimmune response after subcutaneous, oral and intranasal administration of BSAloaded nanospheres. Vaccine 2002;21:67–77.

[11] Chong CS, Cao M, Wong WW, Fischer KP, Addison WR, Kwon GS, et al.Enhancement of T helper type 1 immune responses against hepatitis B viruscore antigen by PLGA nanoparticle vaccine delivery. J Control Release 2005;102:85–99.

[12] Hayden MS, West AP, Ghosh S. NF κB and the immune response. Oncogene2006;25:6758–80.

[13] Hayden MS, Ghosh S. Shared principles in NF κB signaling. Cell 2008;132:344–62.[14] Lutsiak ME, Robinson DR, Coester C, Kwon GS, Samuel J. Analysis of poly(D, L-

lactic-co-glycolic acid) nanosphere uptake by human dendritic cells andmacrophages in vitro. Pharm Res 2002;19:1480–7.

[15] Nicolete R, Lima KM, Rodrigues Júnior JM, BaruffiMD, Medeiros AI, Bentley MVLB,et al. In vitro and in vivo activities of leukotriene B4-loaded biodegradablemicrospheres. Prostaglandins Other Lipid Mediat 2007;83:121–9.

[16] dos Santos DF, Nicolete R, de Souza PRM, Bitencourt CS, dos Santos Junior RR,Bonato VLD, et al. Characterization and in vitro activities of cell-free antigens fromHistoplasma capsulatum-loaded biodegradable microspheres. Eur J Pharm Sci2009;38:548–55.

[17] Trombone APF, Silva CL, Almeida LP, Rosada RS, Lima KM, Oliver C, et al. Tissuedistribution of DNA-Hsp65/TDM-loaded PLGA microspheres and uptake byphagocytic cells. Genet Vaccin Ther 2007;5:1–8.

[18] Bolte S, Cordelieres FP. A guided tour into subcellular colocalization analysis inlight microscopy. J Microsc 2006;224:213–32.

[19] Nicolete R, Lima KM, Rodrigues Júnior JM, Jose PJ, Sanz MJ, Faccioli LH.Prostaglandin E2-loaded microspheres as strategy to inhibit phagocytosis andmodulate inflammatory mediators release. Eur J Pharm Biopharm 2008;70:784–90.

[20] Champion JA, Walker A, Mitragotri S. Role of particle size in phagocytosis ofpolymeric microspheres. Pharm Res 2008;25:1815–21.

[21] Clift MJD, Bhattacharjeea S, Browna DM, Stonea V. The effects of serum on thetoxicity of manufactured nanoparticles. Toxicol Lett 2010;198:358–65.

[22] Tomazic-Jezic VJ, Merritt K, Umbreit TH. Significance of the type and the size ofbiomaterial particles on phagocytosis and tissue distribution. J Biomed Mater Res2001;55:523–9.

[23] Foged C, Brodin B, Frokjaer S, Sundblad A. Particle size and surface charge affectparticle uptake by human dendritic cells in an in vitro model. Int J Pharm2005;298:315–22.

[24] Muller RH, Ruhl D, Luck M, Paulke BR. Influence of fluorescent labelling ofpolystyrene particles on phagocytic uptake, surface hydrophobicity, and plasmaprotein adsorption. Pharm Res 1997;14:18–24.

[25] Mistry P, Deacon K, Mistry S, Blank J, Pate R. NF-kappaB promotes survival duringmitotic cell cycle arrest. J Biol Chem 2004;279:1482–90.

[26] Underhill DM, Ozinsky A. Phagocytosis of microbes: complexity in action. AnnuRev Immunol 2002;20:825–52.

[27] Sharp FA, Ruane D, Claass B, Creagh E, Harris J, Malyala P, et al. Uptake ofparticulate vaccine adjuvants by dendritic cells activates the NALP3 inflamma-some. PNAS 2009;106:870–5.