Copyright copy 2011 American Scientific PublishersAll rights reservedPrinted in the United States of America
Science ofAdvanced Materials
Vol 3 687ndash694 2011
Immunomodulatory Effects of Poly(ethylene glycol)Microspheres Adsorbed with Nanofractions of
Momordica charantia L on DiabeticHuman Blood Phagocytes
Edson Fredulin Scherer1 Adenilda Cristina Honorio-Franccedila2 Cristiane de Castro Pernet Hara3Ana Paula Barcelos Reinaque3 Mayra Aparecida Cocircrtes3 and Eduardo Luziacutea Franccedila3lowast
1Post Graduate Program in Material Science Institute of Biological and Health ScienceFederal University of Mato Grosso ndash Pontal do Araguaia ndash MT 78698-000 Brazil
2Institute of Biological and Health Science Federal University of Mato Grosso ndash Pontal do Araguaia ndash MT 78698-000 Brazil3Post Graduate Program in Material Science Institute of Biological and Health Science
Federal University of Mato Grosso ndash Barra do Garccedilas ndash MT 78600-000 Brazil
Polyethylene glycol (PEG) based microspheres have the capacity to absorb organic compounds andare a promising strategy for the delivery of therapeutics In Brazilian ethnopharmacology and exper-imental studies on individuals with diabetes the use of Momordica charantia L has been shown toimprove the immune system The aim of this study was to evaluate the adsorption of Momordicacharantia L onto PEG microspheres and to verify the in vitro immunomodulatory effect of this nano-material on blood phagocytes from diabetic patients Blood samples were collected according toglycemic status normal glycemia (N = 120) and clinical diabetes (N = 120) We explored the useof PEG microspheres fabricated via thermal adsorption of plant extract We determined the effectsof PEG microspheres adsorbed with plant extract on the viability superoxide release phagocytosisand microbicidal activity of blood phagocytes In the presence of the PEG microspheres superoxiderelease increased in the phagocytes collected from the clinical diabetes patients and phagocy-tosis increased significantly in the control group Phagocytes had low bactericidal activity againstEPEC in the diabetic group These data suggest that PEG microspheres adsorbed with nanofrac-tions of Momordica charantia L represent a potential nanomaterial for future clinical applications ofdiabetes
Keywords Diabetes PEG Microsphere Momordica charantia L Phagocytosis BloodPhagocytes
1 INTRODUCTION
The therapeutic properties of compounds isolated fromplants and their incorporation into controlled release sys-tems offer an important strategy for developing drugs withintelligent properties Microparticles are spherical polymermatrix particles (microspheres) or reservoirs (microcap-sules) that promote the controlled release of drugs or bio-logically active hydrophilic or hydrophobic substances1
Thus the polymers are essential components for the directand controlled release of drugsPoly(ethylene glycol) (PEG) microspheres are poly-
meric particles that have the capacity to absorb organic
lowastAuthor to whom correspondence should be addressed
compounds and are considered a major carrier2 Themicrospheres adsorption capacity for organic compoundscan be modified to improve their biological function3 Syn-thetic polymers allow for control over recognition andcommunication to trigger the modulation of the immuneresponse cell adhesion or signal transduction45 and areimportant in diseases processesOn the other hand plants are considered to be an unlim-
ited source of potential molecules for the treatment ofdisease There is growing interest in the investigation ofdifferent plant species to identify their potential for ther-apeutic application because of the large historical legacyof medicinal plants6 with fewer side effects lower costand minimal toxicity There is a general consensus that the
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Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
adverse effects of herbal medicines are less frequent whencompared to synthetic drugs7
Literature reports the effectiveness of medicinal plantsthat are prospects for new drugs8 Several medicinal plantshave shown the ability to induce immune system activa-tion and display beneficial effects against disease mainlychronic degenerative diseases such as diabetes9ndash11 Diabetesmellitus is a metabolic disease characterized by elevatedblood glucose levels It results from the absence or inade-quate pancreatic insulin secretion with or without concur-rent impairment of insulin action12
Experimental studies have shown that plants exhibit theability to reduce glucose levels11 Brazilian ethnopharma-cology studies with Momordica charantia L showed thatit had hypoglycemic properties and it can be useful forimproving the immune system However the incorporationof nanofractions of Momordica charantia L onto micro-spheres and their effect on cellular functional activity havenot been studied13ndash14
Several studies have attempted to elucidate the antimi-crobial action of phagocytes In this respect it wasassigned an important role for oxygen-derived radicalsand their possible modulators15ndash19 The multiple cascadescaused by free radicals may be associated with phagocyto-sis and microbicidal activity designed to eliminate poten-tially pathogenic agents1720ndash21
Phagocytosis represents an important defense mecha-nism against bacterial infections During phagocytosis themassive activation of cellular oxidative metabolism andthe generation of potent active oxygen metabolites occurOxygen-derived radicals are involved in many importantprocesses such as immune reactions longevity and per-oxidation of cellular lipids proteins carbohydrates andDNA1622ndash23
On the other hand the literature has indicated that manyplants can stimulate immune cells which constitutes realpromise for the treatment of infections24 Plants are apotential source of traditional medicines and chemical sub-stances with useful properties in the modulation of theimmune system25 Immunostimulatory or immunosuppres-sive activities have been investigated using a large numberof plants and their isolated constituents26ndash27 These stud-ies have attracted attention worldwide as an alternative totraditional treatment methods28 In this paper we describethe adsorption of nanofractions of Momordica charantia Lonto microspheres of poly(ethylene glycol) and verify thein vitro immunomodulatory effects of this nanomaterial onhuman blood phagocytes from diabetic patients
2 MATERIALS AND METHODS
21 Subjects
Samples of 10 mL of blood were collected from120 volunteers confirmed to be normoglycemic and
120 volunteers diagnosed with diabetes by the GlycemiaTest The volunteers signed an informed consent formthat was approved by the local Ethics Committee beforeentering the study The mean and standard deviation forglycemia were 7556plusmn1136 for the normoglycemic groupand 16339plusmn5125 for the hyperglycemic groupThe controlled variables were smoking status (yesno)
arterial hypertension (yesno) and glycemic index (GI)whose mean plasma glucose level GI was classifiedas adequate (GI lt 120 mgdL) or inadequate (GI ge120 mgdL29
22 Separation of Blood Cells
Blood samples were collected into heparinized (25 UmL)tubes Cells were separated by a Ficoll-Paque gradient(Pharmacia Upsala Sweden) to produce preparations ofmononuclear cells with 98 purity as analyzed by lightmicroscopy Purified macrophages were resuspended inde-pendently in serum-free medium 199 to a final concentra-tion of 2times106 cellsmL
23 E coli Strain
The enteropathogenic Escherichia coli (EPEC) used wasisolated from the stool of an infant with acute diarrhea(serotype 0111H2 LA1 eae1 EAF1 bfp1) This materialwas prepared and adjusted to 107 bacteriamL as previ-ously described17
24 Preparation of Momordica charantia L
The plants were collected and deposited in the herbariumat the Institute of Biological and Health Science-FederalUniversity of Mato GrossondashPontal do AraguaiandashMTBrazil located at Lat 15 55rsquo08rdquo S Long 52 16rsquo44rdquo Wat an altitude of 365 m Preparation of the plants involveda mixing process followed by maceration according to theBrazilian pharmaceutical code30 The total plant parts weremacerated by placing 200 g of the plant into one literof distillated water After the preparation was filtered theMomordica charantia L extract was stored at 4 C
The microspheres were obtained from poly(ethylene gly-col) 6000 using a modification of the method describedby Scott et al31 Briefly 20 g of PEG 6000 was sus-pended in phosphate-buffered saline (PBS) diluted witha solution of sodium sulfate to 2 PBS and incubatedat 37 C for 45 min After incubation the PEG micro-spheres were diluted 31 in PBS and washed twice in PBS(500timesg 5 min) PEG microspheres were resuspended inPBS PEG microspheres with low density (LDPEG) or
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Scherer et al Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L
high density (HDPEG) were obtained by incubating thesuspension at 95 C for 3 min or 95 C for 10 min respec-tively PEG microspheres were fluorescence labeling withsolution of Dylight-488 (10 g mLminus1-Pierce) overnight atroom temperature in dimethylformamide at a 1001 molarratio of PEGDylight
26 Flow Cytometry
We performed immunofluorescence staining with phyco-erythrin (PE ndash Sigma) to assess the ability of PEG micro-sphere to biding with fluorescent markers to comparewith the microsphere polymethylmethacrylate (CaliBRITEBD) The PEG microsphere was incubated with 5 L ofPE (01 mgmL) for 30 min at 37 C After the incuba-tion the PEG microspheres were washed twice in PBScontaining BSA (5 mgmL 500timesg 10 min 4 C) In allexperiments the PEG microspheres were analyzed by flowcytometry The study was performed on FACS Calibur(BD San Jose USA) The PEG microsphere size was com-pared with the BD microsphere size (6 m-CaliBRITE3 Beads-BD Cat No 340486 USA) binding or not withPE The ratio of fluorescence intensity of PEG microspherewas express in geometric mean fluorescence intensity andthe size calculated according to geometric mean of For-ward Scatter (FSC) Experiments were repeated on severaloccasions the data presented in the figures are from singleexperiments performed
27 Release of Superoxide Anion
Superoxide release was determined by cytochrome C(Sigma) reduction1732 Briefly mononuclear phagocytesand bacteria mononuclear phagocytes and bacteria inthe presence or not of PEG microsphere adsorbed withMomordica charantia L were mixed and incubated for 30min for phagocytosis Cells were then resuspended in PBScontaining 26 mM CaCl2 2 mM MgCl2 and cytochromeC (2 mgmL) The suspensions (100 L) were incubatedfor 60 min at 37 C on culture plates The reaction rateswere measured by absorbance at 550 nm and the resultswere expressed as nmolO2minus All the experiments wereperformed in duplicate or triplicate
28 Bactericidal Assay
Microbicidal activity and phagocytosis were evaluated bythe acridine orange method described by Bellinati-Pireset al33 Equal volumes of bacteria and cell suspensionsin the presence or not of PEG microsphere adsorbedwith Momordica charantia L were mixed and incubatedat 37 C for 30 min with continuous shaking Phago-cytosis was stopped by incubation in ice To eliminateextracellular bacteria the suspensions were centrifugedtwice (160timesg 10 min 4 C) Cells were resuspended in
serum-free medium 199 and centrifuged The supernatantwas discarded and the sediment was dyed with 200 Lof acridine orange (144 gL) for 1 min The sedimentwas resuspended in cold culture 199 washed twice andobserved under an immunofluorescence microscope at400times and 1000times magnification The phagocytosis indexwas calculated by counting the number of cells ingestingat least 3 bacteria within a pool of 100 cells To deter-mine the bactericidal index we stained the slides withacridine orange and counted 100 cells with phagocytizedbacteria The bactericidal index is calculated as the ratiobetween orange- stained (dead) and green- stained (alive)bacteriatimes 10033 All the experiments were performed induplicate or triplicate
29 Statistical Analysis
Analysis of variance (ANOVA) was used to evaluate super-oxide anion release phagocytosis bactericidal index in thepresence or absence of opsonized EPEC Statistical sig-nificance was considered for a p-value lower than 005[p lt 005]
3 RESULTS
31 Characterization of PEG Microspheres
The Figure 1(A) shows the relationship between the PEGmicrosphere and BD microsphere (standard) by fluores-cence intensity Both microspheres had similar geomet-ric mean of fluorescence intensity When compared tosize the PEG microsphere was similar to standard micro-sphere (Fig 1(B)) The fluorescence microscopy image(Fig 2(A)) shows that the PEG microspheres producedin PBS have a narrow size distribution This result con-firms that our method produces differents sizes micro-spheres and shows that both the high and low-densitymicrospheres easily separate in the suspension The micro-spheres produced were highly stable and retained theirspherical structure without deformation (Fig 2(A)) Flu-orescence microscopy showed that PEG microspheres areable to absorb the extract of plant (Fig 2(B)) Fluores-cence microscopy showed that blood phagocytes are ableto internalize the microspheres (Fig 2(C))
32 The Effect of PEG Microspheres ContainingPlant Extract on Blood Mononuclear Phagocytes
Retrieval and viability of blood phagocytes from boththe normoglycemic and hyperglycemic groups were notaffected by the PEG microspheres orMomordica charantiaL extracts (p lt 005ndashTable I)
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Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
33 The Effect of PEG Microspheres Adsorbed withPlant Extract on Superoxide Release
The hyperglycemic and normoglycemic groups had sim-ilar spontaneous superoxide release by blood phago-cytes When exposed to PEG microspheres independentof density the cells showed no difference between thegroups Phagocytes incubated with plant extract had highersuperoxide release than untreated phagocytes (p lt 005)irrespective of their glycemic status Additionally thephagocytes in the presence of PEG microspheres adsorbedwith plant extract regardless of glycemic status dis-played an increased release of superoxide (Table II) Whenexposed to EPEC superoxide release increased (p lt 005)in the hyperglycemic group (p lt 005) in the absence ofthe PEG microspheres or plant extract Normoglycemic
Fig 1 PEG Microspheres were stained directly with phycoerythrin(PE) as described in Materials and Methods PolymethylmethacrylateMicrosphere PE-labeled (BD Microsphere-Becton Dickinson USA) wasused as standard Immunofluorescence analysis and size were then carriedout by flow cytometry (FACScalibur Becton Dickinson USA)
(A)
(B)
(C)
Fig 2 Fluorescence microscopy image of the PEG microspheresstained with Dylight-488 (100times - panels-A) The PEG microspheres wereincubated with Momordica charantia L (100times ndash panel-B) on a shaker for30 min at 37 C and stained with Dylight-488 The phagocytes were incu-bated with PEG microsphere (100times - panels-C) on a shaker for 30 minat 37 C After washing at 4 C cells were stained with acridine orangeand analyzed by fluorescent microscopy Experiments were repeated fivetimes and the results were comparable
group phagocytes incubated with EPEC in the presence ofhigh density PEG microspheres loaded with plant extracthad higher superoxide release than phagocytes exposedonly to bacteria (p lt 005ndashTable II)
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Scherer et al Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L
Table I General characteristics of the experimental groups The results represent the mean and SD of ten experiments
Parameters Control Diabetes Statistics
Glycemia (mgdL) 7556plusmn1136 16339plusmn5125a F = 20 517 p = 00001 (comparing groups)Leukocytes number (times106 cellsmL) 218plusmn074 240plusmn064 F = 1 354 p = 06352 (comparing groups)Leukocytes viabilityb 866plusmn628 892plusmn354 F = 3 139 p = 00532 (comparing groups)
Data represented by meanplusmn standard deviation (SD) aStatistically significant differences between normoglycemic and hyperglycemic groups considering the samplesbViability in the presence of Momordica charantia L and Polyethyleneglycol (PEG)
34 The Effects of PEG Microsphere Adsorbed withPlant Extract on the Phagocytosis of BloodMononuclear Cells
Blood phagocytes from the diabetic group had somephagocytic activity in response to EPEC Phagocytosisincreased significantly in the presence of microsphere PEGadsorbed with plant extract for the normoglycemic groupA comparison of the groups showed that EPEC phagocy-tosis in general was similar for the diabetic and normo-glycemic groups (Fig 3(A))
35 The Effects of Microsphere PEG Adsorbed withPlant Extract on the Bacterial Activity of BloodPhagocytes
In general blood mononuclear phagocytes from the dia-betic group had low bactericidal activity against EPECMononuclear phagocytes from the normoglycemic groupincubated with plant extract or low density microspheresPEG adsorbed with or without plant extract had increasedmicrobicidal activity in response to EPEC (Fig 3(B))
Table II Superoxide release by blood mononuclear phagocytes(meanplusmnSD N = 10 in each treatment)
Superoxide release (nmol)
MN phagocytes Bacteria Control Diabetes
PBS No 59plusmn12 40plusmn14Yes 13plusmn004 113plusmn11d
Momordica charantia L No 125plusmn33c 104plusmn38c
Yes 46 plusmn 05c 138plusmn09d
HDPEGa No 61plusmn26 54plusmn12Yes 37plusmn15c 116plusmn25d
LDPEGb No 62plusmn23 44plusmn18Yes 19plusmn03 118plusmn32d
Momordica charantia L No 98plusmn27c 76plusmn24plus HDPEG
Yes 44plusmn03c 125plusmn07d
Momordica charantia L No 88plusmn17c 87plusmn15c
plus LDPEGYes 16plusmn02 131plusmn09d
aHigh Density Polyethylene Glycol bLow Density Polyethylene Glycol Bacteriawere treated with PEG (High or Low density) andor Momordica charantia LIn controls assays mononuclear cells were pre-incubated with PBS cIndicatesdifferences between each bacteria treatment and the control treatment within agroup (ANOVA P lt 005) d indicates intergroup differences within each treatment(ANOVA P lt 005)
4 DISCUSSION
In the present study we produced PEG microspheres ofdifferent densities adsorbed with extract from Momordicacharantia L and studied their in vitro effect on bacterial-killing activity and superoxide production in blood phago-cytes from diabetic individualsPolymer microspheres can be employed to deliver medi-
cation in a rate-controlled and sometimes targeted mannerMedication is released from a microsphere by drug leach-ing from the polymer or by degradation of the polymermatrix Because the rate of drug release is controlled bythese two factors it is important to understand the physi-cal and chemical properties of the releasing medium35 Inthis study analysis by fluorescence microscopy and flowcytometry showed PEG microspheres to be a regular sizeregardless of density and easily separated from the sus-pension The ability to form gradients in microspheres mayprove to be useful to achieve better control over the kinet-ics of protein release36 The literature have reported theuse of ow cytometry as an alternative method for analy-ses and visualization of particles37ndash38 Here we showed byflow cytometry that the PEG microsphere have a size ofabout 58 mPEG Microspheres are a kind of copolymer used in
clinical administration of drugs due to their incorporationcapacity36 Density gradients of signaling are importantin immunity39 In particular chemotaxis the preferentialmovement of cells up a density gradient of singlingmolecules is dependent on the gradient as opposed tothe average concentration of the molecule40 Here wehad found that the PEG microsphere independent ofthe density gradient adsorbed with plant extract pre-sented a potent immunostimulatory effect on the func-tional activity of blood phagocytes Cells obtained fromthe blood of normoglycemic individuals are known tokill enteropathogenic Escherichia coli in the presence ofherbal plants associated with peptides This interactionmay represent an alternative mechanism of defense againstinfection27
In this study we observed that the interaction ofPEG microspheres with blood phagocytes regardless ofglycemic status did not affect the viability of the cellsFurthermore superoxide release was higher when thephagocytes were in the presence of the PEG microspheresadsorbed with plant extract irrespective of glycemic statusThe phagocytes incubated with EPEC in the presence of
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Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
(A)
(B)
Fig 3 Bacterial phagocytosis (A) and bactericidal activity index (B) by blood mononuclear cells (meanplusmnSD N = 10 in each treatment) Bacterialphagocytosis and elimation index by mononuclear cells from blood was determined with the acridine orange method lowastindicates differences betweentreatment (PEG orand plant) and 199 medium (ANOVA P lt 005) indicates intergroup differences for each treatment (ANOVA P lt 005) (HDPEG-Hight Density Polyethylene Glicol LDPEG-Low Density Polyethylene Glicol MOR ndash Momordica charantia L)
PEG microspheres adsorbed with plant extract had highersuperoxide release than phagocytes exposed only to thebacteria The activation ability of these cells indicated bysuperoxide release remained at the same level for both thehyperglycemic and control groupsA great variety of mechanisms have been suggested
for the formation of these free reactive oxygen radicals41
Glucose oxidation is believed to be the main source of freeradical production in diabetes42 Hyperglycemia has alsobeen found to promote lipid peroxidation by a superoxide-dependent pathway resulting in free radical generation andrelease43ndash44 Another important source of free radicals in
diabetes is the interaction of glucose with proteins lead-ing to the formation of advanced glycation end-productsthat contribute to an excessive burst in mitochondrial freeradicals45 The role of free radicals is an extremely impor-tant phenomenon during immune responses and inflam-matory reactions whereas the microbicidal activity ofphagocytes has been linked to the activation of cellularoxidative metabolism and release of large amounts of freeradicals46
On the other hand the high glucose levels of diabeticpatients can impair their antioxidant systems by decreasingimportant responses such as phagocytic and microbicidal
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Scherer et al Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L
activity47ndash49 In this work the PEG microspheres adsorbedwith plant extract increased phagocytosis Similar resultswith respect to the functional activity of leukocytes wereobserved in phagocytes from patients with diabetes45 andfrom animals with induced diabetes50ndash52 Mononuclearphagocytes play an important role in host defense Theyproduce phagocytic NADPH oxidase which forms super-oxide and is imperative for killing bacterial53 and crucialfor the success of immune responses and inflammatoryreactions46
In our studies the elimination of EPEC by blood phago-cytes was stimulated only in the normoglycemic groupwhen these cells were incubated with plant extract or lowdensity PEG microspheres adsorbed with plant extractThe scientific literature has already reported the elimi-nation of bacteria by mononuclear phagocytes and theirmicrobicidal activity was stimulated in the presence ofimmunomodulatory agents17ndash5455
The high glucose levels impair a series of physiolog-ical processes and the functional activity seems to beaffected Although the PEG microspheres adsorbed withMomordica charantia L did not increase the microbici-dal activity of blood phagocytes from diabetes patientsthis polymer can be activated inside cells This studyoutlined the immunomodulatory effects of microspherepoly(ethylene glycol) adsorbed with Momordica charan-tia L on blood phagocytes This system may be usefulfor the delivery of a variety of therapeutic plants and wasshown to provide additional protection against infectionsin normoglycemic and hyperglycemic individuals Thesedata suggest that microspheres of poly(ethylene glycol)adsorbed with nanofractions of Momordica charantia Lrepresent a potential nanomaterial for future clinical appli-cations of diabetes
Acknowledgments This research received grants fromFundaccedilatildeo de Amparo agrave Pesquisa de Mato Grosso (FAPE-MAT No 7382642008 FAPEMAT No 2990322010 andNo 3016102010)
References and Notes
1 R M Ribeiro-Costa E C G Pereira J A Alves N P Santos S CNascimento N K Honda N H Silva and N S Santos-MagalhatildeesJ Microencap 21 371 (2004)
2 J Park M Ye and K Park Molecules 10 146 (2005)3 E A Scott M D Nichols R Kuntz-Willits and D L Elbert Acta
Biomat 6 29 (2010)4 R Duncan Nat Rev Drug Discov 2 346 (2003)5 K L Kiick Science 317 1182 (2007)6 T J A Recircgo Phytogeography of Medicinal Plants in the Maranhatildeo
2nd edn Edufma Satildeo Luiacutes MA Brazil (1995) pp 108ndash1097 J B Calixto Braz J Med Biol Res 32 179 (2000)8 S Hasani-Ranjbar B Larijani and M A Abdollah Arch Med Sci
4 285 (2008)9 R W Bussmann A G K Meyer A Kuhlma and A Townesmith
J Ethnobiol Ethnomed 6 2 (2010)
10 S Asgary S Parkhideh A Solhpour H Madani P Mahzouni andP Rahimi J Med Food 11 533 (2008)
11 A C Honorio-Franccedila C Marins F Boldrini and E L Franccedila ActaCir Braz 23 504 (2008)
12 American Dietetic Association J Am Diet Assoc 109 1926(2009)
13 T Miura T Itoh N Iwamoto M Kato and T Ishida Biol PharmaBull 27 248 (2004)
14 G V K Senanayake M Maruyama KShibuya M SakonoN Fukuda T Morishita C Yukizaki M Kawano and H OhtaJ Ethnopharmacol 91 257 (2004)
15 H Mundi B Bjoumlrksten C Svanborg L Ohman and C DahlgrenInfect Immun 59 4168 (1991)
16 N R Asad L M B O Assad C E B Almeida and A C LeitatildeoBr J Med Biol Res 27 2551 (1994)
17 A C Honorio-Franccedila M P S M Carvalho L Isaac L R Tra-bulsi and M M S Carneiro-Sampaio Scand J Immunol 46 59(1997)
18 A C Honorio-Franccedila P Launay M M S Carneiro-Sampaio andR C Monteiro J Leuk Biol 69 280 (2001)
19 E L Franccedila Jr A Pereira S L Oliveira and A C Honorio-Franccedila Intern J Microbiol 6 1 (2009)
20 J M McCord Am J Med 108 652 (2000)21 C Vorbach R Harrison and M R Capecchi Trend Immunol
24 512 (2003)22 B N Ames M K Shinnenaga and T M Hagen Proc Nat
Acad Sci 90 7915 (1993)23 C K B Ferrari and E A F S Torres Biomed Pharmacother
57 251 (2003)24 U Tiwari B Rastogi P Singh D K Saraf and S P Vyas
J Ethnopharmacol 92 113 (2004)25 C Viegas Jr V S Bolzani and E J Barreiro Quim Nova 29 326
(2006)26 B Patwardhan and G Manish Drug Discov Today 10 495
(2005)27 E L Franccedila J C Maynie V S C Correa U C R Pereira
C Batalini C K B Ferrari and A C Honorio-Franccedila Int J Phy-tomed 2 345 (2010)
28 S Mehrotra K P Mishra R Maurya R C Srimal V SYadav R Pandey and V K Singh Int Immunopharmacol 3 53(2003)
29 G Morceli E L Franccedila V B Magalhatildees D C Damasceno I MP Calderon and A C H Franccedila Acta Paediatr 100 550 (2011)
30 V S C Correcirca J C Maynieacute E L Franccedila and A C Honorio-Franccedila Braz J Med Plants 8 26 (2006)
31 E A Scott M D Nichols R Kuntz-Willits and D L Elbert ActaBiomat 6 29 (2010)
32 E Pick and D Mizel J Immunol Methods 46 211 (1981)33 R Bellinati-Pires M M Salgado I P Hypolito A S Grumach
and M M S Carneiro-Sampaio J Investig Allergol Clin Immunol5 337 (1995)
34 J L Roam H Xu P K Nguyen and D L Elbert Biomaterials31 8642 (2010)
35 S Freiberg and X X Zhu Int J Pharmaceut 282 1 (2004)36 R Gang and F Si-Shen Biomaterials 24 5037 (2003)37 H Ai J J Pink X T Shuai D A Boothman J M Gao J Biomed
Mater Res 73A 303 (2005)38 B Staumldler A D Price and A N Zelikin Adv Funct Mater 21 14
(2011)39 X Q Wang E Wenk X H Zhang L Mennel G Vunjak-
Novakovic and D L Kaplan J Control Releas 134 81 (2009)40 K Moore M Macsween and M Shoichet Tissue Eng 12 267
(2006)41 C K B Ferrari E L Franccedila and A C Honorio-Franccedila Applied
Biomed 7 163 (2009)42 Z Y Jiang A C Woollard and S P Wolff FEBS Lett 268 69
(1990)
Sci Adv Mater 3 687ndash694 2011 693
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Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
43 E C Tsai I B Hirsch J D Brunzell and A Chait Diabetes43 1010 (1994)
44 M Kawamura J W Heinecke and A Chait J Clin Invest 94 771(1994)
45 A C Maritim R A Sanders and B Watkins J Biochem Mol Tox-icol 17 24 (2003)
46 M Dizdaroglu P Jaruga M Birincioglu and H Rodriguez FreeRad Biol Med 32 1102 (2002)
47 A J Rassias C A Marrin J Arruda P K Whalen M Beach andM P Yeager Anesth Analg 88 1011 (1999)
48 J Jakelic S Kokic I Hozo J Maras and D Fabijanic Med Arh49 9 (1995)
49 B Larijani P Shooshtarizadeh N Mosaffa and R Heshmat Br JBiomed Sci 64 13 (2007)
50 N Seyrek W Marcinkowski M Smogorzewski T M Demerdashand S G Massry Nephrol Dial Transplant 12 265 (1997)
51 E L Franccedila N D Feliciano K A Silva C K B Ferrari andA C Honorio-Franccedila Bratisl Med J 110 517 (2009)
52 A C Honorio-Franccedila K A Silva N D Feliciano I MP Calderon M V C Rudge and E L Franccedila Int J DiabetesMetabol 17 87 (2009)
53 M Djaldetti H Salman M Bergman Djaldetti and H BesslerMicro Res Techn 57 421 (2002)
54 E L Franccedila R V Bitencourt M Fujimori T C Morais I M PCalderon and A C Honorio-Franccedila J Microbiol Immunol Infect44 1 (2011)
55 E L Franccedila G Morceli D L G Fagundes M V C Rugde I MP Calderon and A C Honorio-Franccedila APMIS 119 710 (2011)
Received 1 August 2011 Accepted 18 September 2011
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Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
adverse effects of herbal medicines are less frequent whencompared to synthetic drugs7
Literature reports the effectiveness of medicinal plantsthat are prospects for new drugs8 Several medicinal plantshave shown the ability to induce immune system activa-tion and display beneficial effects against disease mainlychronic degenerative diseases such as diabetes9ndash11 Diabetesmellitus is a metabolic disease characterized by elevatedblood glucose levels It results from the absence or inade-quate pancreatic insulin secretion with or without concur-rent impairment of insulin action12
Experimental studies have shown that plants exhibit theability to reduce glucose levels11 Brazilian ethnopharma-cology studies with Momordica charantia L showed thatit had hypoglycemic properties and it can be useful forimproving the immune system However the incorporationof nanofractions of Momordica charantia L onto micro-spheres and their effect on cellular functional activity havenot been studied13ndash14
Several studies have attempted to elucidate the antimi-crobial action of phagocytes In this respect it wasassigned an important role for oxygen-derived radicalsand their possible modulators15ndash19 The multiple cascadescaused by free radicals may be associated with phagocyto-sis and microbicidal activity designed to eliminate poten-tially pathogenic agents1720ndash21
Phagocytosis represents an important defense mecha-nism against bacterial infections During phagocytosis themassive activation of cellular oxidative metabolism andthe generation of potent active oxygen metabolites occurOxygen-derived radicals are involved in many importantprocesses such as immune reactions longevity and per-oxidation of cellular lipids proteins carbohydrates andDNA1622ndash23
On the other hand the literature has indicated that manyplants can stimulate immune cells which constitutes realpromise for the treatment of infections24 Plants are apotential source of traditional medicines and chemical sub-stances with useful properties in the modulation of theimmune system25 Immunostimulatory or immunosuppres-sive activities have been investigated using a large numberof plants and their isolated constituents26ndash27 These stud-ies have attracted attention worldwide as an alternative totraditional treatment methods28 In this paper we describethe adsorption of nanofractions of Momordica charantia Lonto microspheres of poly(ethylene glycol) and verify thein vitro immunomodulatory effects of this nanomaterial onhuman blood phagocytes from diabetic patients
2 MATERIALS AND METHODS
21 Subjects
Samples of 10 mL of blood were collected from120 volunteers confirmed to be normoglycemic and
120 volunteers diagnosed with diabetes by the GlycemiaTest The volunteers signed an informed consent formthat was approved by the local Ethics Committee beforeentering the study The mean and standard deviation forglycemia were 7556plusmn1136 for the normoglycemic groupand 16339plusmn5125 for the hyperglycemic groupThe controlled variables were smoking status (yesno)
arterial hypertension (yesno) and glycemic index (GI)whose mean plasma glucose level GI was classifiedas adequate (GI lt 120 mgdL) or inadequate (GI ge120 mgdL29
22 Separation of Blood Cells
Blood samples were collected into heparinized (25 UmL)tubes Cells were separated by a Ficoll-Paque gradient(Pharmacia Upsala Sweden) to produce preparations ofmononuclear cells with 98 purity as analyzed by lightmicroscopy Purified macrophages were resuspended inde-pendently in serum-free medium 199 to a final concentra-tion of 2times106 cellsmL
23 E coli Strain
The enteropathogenic Escherichia coli (EPEC) used wasisolated from the stool of an infant with acute diarrhea(serotype 0111H2 LA1 eae1 EAF1 bfp1) This materialwas prepared and adjusted to 107 bacteriamL as previ-ously described17
24 Preparation of Momordica charantia L
The plants were collected and deposited in the herbariumat the Institute of Biological and Health Science-FederalUniversity of Mato GrossondashPontal do AraguaiandashMTBrazil located at Lat 15 55rsquo08rdquo S Long 52 16rsquo44rdquo Wat an altitude of 365 m Preparation of the plants involveda mixing process followed by maceration according to theBrazilian pharmaceutical code30 The total plant parts weremacerated by placing 200 g of the plant into one literof distillated water After the preparation was filtered theMomordica charantia L extract was stored at 4 C
The microspheres were obtained from poly(ethylene gly-col) 6000 using a modification of the method describedby Scott et al31 Briefly 20 g of PEG 6000 was sus-pended in phosphate-buffered saline (PBS) diluted witha solution of sodium sulfate to 2 PBS and incubatedat 37 C for 45 min After incubation the PEG micro-spheres were diluted 31 in PBS and washed twice in PBS(500timesg 5 min) PEG microspheres were resuspended inPBS PEG microspheres with low density (LDPEG) or
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Scherer et al Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L
high density (HDPEG) were obtained by incubating thesuspension at 95 C for 3 min or 95 C for 10 min respec-tively PEG microspheres were fluorescence labeling withsolution of Dylight-488 (10 g mLminus1-Pierce) overnight atroom temperature in dimethylformamide at a 1001 molarratio of PEGDylight
26 Flow Cytometry
We performed immunofluorescence staining with phyco-erythrin (PE ndash Sigma) to assess the ability of PEG micro-sphere to biding with fluorescent markers to comparewith the microsphere polymethylmethacrylate (CaliBRITEBD) The PEG microsphere was incubated with 5 L ofPE (01 mgmL) for 30 min at 37 C After the incuba-tion the PEG microspheres were washed twice in PBScontaining BSA (5 mgmL 500timesg 10 min 4 C) In allexperiments the PEG microspheres were analyzed by flowcytometry The study was performed on FACS Calibur(BD San Jose USA) The PEG microsphere size was com-pared with the BD microsphere size (6 m-CaliBRITE3 Beads-BD Cat No 340486 USA) binding or not withPE The ratio of fluorescence intensity of PEG microspherewas express in geometric mean fluorescence intensity andthe size calculated according to geometric mean of For-ward Scatter (FSC) Experiments were repeated on severaloccasions the data presented in the figures are from singleexperiments performed
27 Release of Superoxide Anion
Superoxide release was determined by cytochrome C(Sigma) reduction1732 Briefly mononuclear phagocytesand bacteria mononuclear phagocytes and bacteria inthe presence or not of PEG microsphere adsorbed withMomordica charantia L were mixed and incubated for 30min for phagocytosis Cells were then resuspended in PBScontaining 26 mM CaCl2 2 mM MgCl2 and cytochromeC (2 mgmL) The suspensions (100 L) were incubatedfor 60 min at 37 C on culture plates The reaction rateswere measured by absorbance at 550 nm and the resultswere expressed as nmolO2minus All the experiments wereperformed in duplicate or triplicate
28 Bactericidal Assay
Microbicidal activity and phagocytosis were evaluated bythe acridine orange method described by Bellinati-Pireset al33 Equal volumes of bacteria and cell suspensionsin the presence or not of PEG microsphere adsorbedwith Momordica charantia L were mixed and incubatedat 37 C for 30 min with continuous shaking Phago-cytosis was stopped by incubation in ice To eliminateextracellular bacteria the suspensions were centrifugedtwice (160timesg 10 min 4 C) Cells were resuspended in
serum-free medium 199 and centrifuged The supernatantwas discarded and the sediment was dyed with 200 Lof acridine orange (144 gL) for 1 min The sedimentwas resuspended in cold culture 199 washed twice andobserved under an immunofluorescence microscope at400times and 1000times magnification The phagocytosis indexwas calculated by counting the number of cells ingestingat least 3 bacteria within a pool of 100 cells To deter-mine the bactericidal index we stained the slides withacridine orange and counted 100 cells with phagocytizedbacteria The bactericidal index is calculated as the ratiobetween orange- stained (dead) and green- stained (alive)bacteriatimes 10033 All the experiments were performed induplicate or triplicate
29 Statistical Analysis
Analysis of variance (ANOVA) was used to evaluate super-oxide anion release phagocytosis bactericidal index in thepresence or absence of opsonized EPEC Statistical sig-nificance was considered for a p-value lower than 005[p lt 005]
3 RESULTS
31 Characterization of PEG Microspheres
The Figure 1(A) shows the relationship between the PEGmicrosphere and BD microsphere (standard) by fluores-cence intensity Both microspheres had similar geomet-ric mean of fluorescence intensity When compared tosize the PEG microsphere was similar to standard micro-sphere (Fig 1(B)) The fluorescence microscopy image(Fig 2(A)) shows that the PEG microspheres producedin PBS have a narrow size distribution This result con-firms that our method produces differents sizes micro-spheres and shows that both the high and low-densitymicrospheres easily separate in the suspension The micro-spheres produced were highly stable and retained theirspherical structure without deformation (Fig 2(A)) Flu-orescence microscopy showed that PEG microspheres areable to absorb the extract of plant (Fig 2(B)) Fluores-cence microscopy showed that blood phagocytes are ableto internalize the microspheres (Fig 2(C))
32 The Effect of PEG Microspheres ContainingPlant Extract on Blood Mononuclear Phagocytes
Retrieval and viability of blood phagocytes from boththe normoglycemic and hyperglycemic groups were notaffected by the PEG microspheres orMomordica charantiaL extracts (p lt 005ndashTable I)
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Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
33 The Effect of PEG Microspheres Adsorbed withPlant Extract on Superoxide Release
The hyperglycemic and normoglycemic groups had sim-ilar spontaneous superoxide release by blood phago-cytes When exposed to PEG microspheres independentof density the cells showed no difference between thegroups Phagocytes incubated with plant extract had highersuperoxide release than untreated phagocytes (p lt 005)irrespective of their glycemic status Additionally thephagocytes in the presence of PEG microspheres adsorbedwith plant extract regardless of glycemic status dis-played an increased release of superoxide (Table II) Whenexposed to EPEC superoxide release increased (p lt 005)in the hyperglycemic group (p lt 005) in the absence ofthe PEG microspheres or plant extract Normoglycemic
Fig 1 PEG Microspheres were stained directly with phycoerythrin(PE) as described in Materials and Methods PolymethylmethacrylateMicrosphere PE-labeled (BD Microsphere-Becton Dickinson USA) wasused as standard Immunofluorescence analysis and size were then carriedout by flow cytometry (FACScalibur Becton Dickinson USA)
(A)
(B)
(C)
Fig 2 Fluorescence microscopy image of the PEG microspheresstained with Dylight-488 (100times - panels-A) The PEG microspheres wereincubated with Momordica charantia L (100times ndash panel-B) on a shaker for30 min at 37 C and stained with Dylight-488 The phagocytes were incu-bated with PEG microsphere (100times - panels-C) on a shaker for 30 minat 37 C After washing at 4 C cells were stained with acridine orangeand analyzed by fluorescent microscopy Experiments were repeated fivetimes and the results were comparable
group phagocytes incubated with EPEC in the presence ofhigh density PEG microspheres loaded with plant extracthad higher superoxide release than phagocytes exposedonly to bacteria (p lt 005ndashTable II)
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Scherer et al Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L
Table I General characteristics of the experimental groups The results represent the mean and SD of ten experiments
Parameters Control Diabetes Statistics
Glycemia (mgdL) 7556plusmn1136 16339plusmn5125a F = 20 517 p = 00001 (comparing groups)Leukocytes number (times106 cellsmL) 218plusmn074 240plusmn064 F = 1 354 p = 06352 (comparing groups)Leukocytes viabilityb 866plusmn628 892plusmn354 F = 3 139 p = 00532 (comparing groups)
Data represented by meanplusmn standard deviation (SD) aStatistically significant differences between normoglycemic and hyperglycemic groups considering the samplesbViability in the presence of Momordica charantia L and Polyethyleneglycol (PEG)
34 The Effects of PEG Microsphere Adsorbed withPlant Extract on the Phagocytosis of BloodMononuclear Cells
Blood phagocytes from the diabetic group had somephagocytic activity in response to EPEC Phagocytosisincreased significantly in the presence of microsphere PEGadsorbed with plant extract for the normoglycemic groupA comparison of the groups showed that EPEC phagocy-tosis in general was similar for the diabetic and normo-glycemic groups (Fig 3(A))
35 The Effects of Microsphere PEG Adsorbed withPlant Extract on the Bacterial Activity of BloodPhagocytes
In general blood mononuclear phagocytes from the dia-betic group had low bactericidal activity against EPECMononuclear phagocytes from the normoglycemic groupincubated with plant extract or low density microspheresPEG adsorbed with or without plant extract had increasedmicrobicidal activity in response to EPEC (Fig 3(B))
Table II Superoxide release by blood mononuclear phagocytes(meanplusmnSD N = 10 in each treatment)
Superoxide release (nmol)
MN phagocytes Bacteria Control Diabetes
PBS No 59plusmn12 40plusmn14Yes 13plusmn004 113plusmn11d
Momordica charantia L No 125plusmn33c 104plusmn38c
Yes 46 plusmn 05c 138plusmn09d
HDPEGa No 61plusmn26 54plusmn12Yes 37plusmn15c 116plusmn25d
LDPEGb No 62plusmn23 44plusmn18Yes 19plusmn03 118plusmn32d
Momordica charantia L No 98plusmn27c 76plusmn24plus HDPEG
Yes 44plusmn03c 125plusmn07d
Momordica charantia L No 88plusmn17c 87plusmn15c
plus LDPEGYes 16plusmn02 131plusmn09d
aHigh Density Polyethylene Glycol bLow Density Polyethylene Glycol Bacteriawere treated with PEG (High or Low density) andor Momordica charantia LIn controls assays mononuclear cells were pre-incubated with PBS cIndicatesdifferences between each bacteria treatment and the control treatment within agroup (ANOVA P lt 005) d indicates intergroup differences within each treatment(ANOVA P lt 005)
4 DISCUSSION
In the present study we produced PEG microspheres ofdifferent densities adsorbed with extract from Momordicacharantia L and studied their in vitro effect on bacterial-killing activity and superoxide production in blood phago-cytes from diabetic individualsPolymer microspheres can be employed to deliver medi-
cation in a rate-controlled and sometimes targeted mannerMedication is released from a microsphere by drug leach-ing from the polymer or by degradation of the polymermatrix Because the rate of drug release is controlled bythese two factors it is important to understand the physi-cal and chemical properties of the releasing medium35 Inthis study analysis by fluorescence microscopy and flowcytometry showed PEG microspheres to be a regular sizeregardless of density and easily separated from the sus-pension The ability to form gradients in microspheres mayprove to be useful to achieve better control over the kinet-ics of protein release36 The literature have reported theuse of ow cytometry as an alternative method for analy-ses and visualization of particles37ndash38 Here we showed byflow cytometry that the PEG microsphere have a size ofabout 58 mPEG Microspheres are a kind of copolymer used in
clinical administration of drugs due to their incorporationcapacity36 Density gradients of signaling are importantin immunity39 In particular chemotaxis the preferentialmovement of cells up a density gradient of singlingmolecules is dependent on the gradient as opposed tothe average concentration of the molecule40 Here wehad found that the PEG microsphere independent ofthe density gradient adsorbed with plant extract pre-sented a potent immunostimulatory effect on the func-tional activity of blood phagocytes Cells obtained fromthe blood of normoglycemic individuals are known tokill enteropathogenic Escherichia coli in the presence ofherbal plants associated with peptides This interactionmay represent an alternative mechanism of defense againstinfection27
In this study we observed that the interaction ofPEG microspheres with blood phagocytes regardless ofglycemic status did not affect the viability of the cellsFurthermore superoxide release was higher when thephagocytes were in the presence of the PEG microspheresadsorbed with plant extract irrespective of glycemic statusThe phagocytes incubated with EPEC in the presence of
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Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
(A)
(B)
Fig 3 Bacterial phagocytosis (A) and bactericidal activity index (B) by blood mononuclear cells (meanplusmnSD N = 10 in each treatment) Bacterialphagocytosis and elimation index by mononuclear cells from blood was determined with the acridine orange method lowastindicates differences betweentreatment (PEG orand plant) and 199 medium (ANOVA P lt 005) indicates intergroup differences for each treatment (ANOVA P lt 005) (HDPEG-Hight Density Polyethylene Glicol LDPEG-Low Density Polyethylene Glicol MOR ndash Momordica charantia L)
PEG microspheres adsorbed with plant extract had highersuperoxide release than phagocytes exposed only to thebacteria The activation ability of these cells indicated bysuperoxide release remained at the same level for both thehyperglycemic and control groupsA great variety of mechanisms have been suggested
for the formation of these free reactive oxygen radicals41
Glucose oxidation is believed to be the main source of freeradical production in diabetes42 Hyperglycemia has alsobeen found to promote lipid peroxidation by a superoxide-dependent pathway resulting in free radical generation andrelease43ndash44 Another important source of free radicals in
diabetes is the interaction of glucose with proteins lead-ing to the formation of advanced glycation end-productsthat contribute to an excessive burst in mitochondrial freeradicals45 The role of free radicals is an extremely impor-tant phenomenon during immune responses and inflam-matory reactions whereas the microbicidal activity ofphagocytes has been linked to the activation of cellularoxidative metabolism and release of large amounts of freeradicals46
On the other hand the high glucose levels of diabeticpatients can impair their antioxidant systems by decreasingimportant responses such as phagocytic and microbicidal
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Scherer et al Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L
activity47ndash49 In this work the PEG microspheres adsorbedwith plant extract increased phagocytosis Similar resultswith respect to the functional activity of leukocytes wereobserved in phagocytes from patients with diabetes45 andfrom animals with induced diabetes50ndash52 Mononuclearphagocytes play an important role in host defense Theyproduce phagocytic NADPH oxidase which forms super-oxide and is imperative for killing bacterial53 and crucialfor the success of immune responses and inflammatoryreactions46
In our studies the elimination of EPEC by blood phago-cytes was stimulated only in the normoglycemic groupwhen these cells were incubated with plant extract or lowdensity PEG microspheres adsorbed with plant extractThe scientific literature has already reported the elimi-nation of bacteria by mononuclear phagocytes and theirmicrobicidal activity was stimulated in the presence ofimmunomodulatory agents17ndash5455
The high glucose levels impair a series of physiolog-ical processes and the functional activity seems to beaffected Although the PEG microspheres adsorbed withMomordica charantia L did not increase the microbici-dal activity of blood phagocytes from diabetes patientsthis polymer can be activated inside cells This studyoutlined the immunomodulatory effects of microspherepoly(ethylene glycol) adsorbed with Momordica charan-tia L on blood phagocytes This system may be usefulfor the delivery of a variety of therapeutic plants and wasshown to provide additional protection against infectionsin normoglycemic and hyperglycemic individuals Thesedata suggest that microspheres of poly(ethylene glycol)adsorbed with nanofractions of Momordica charantia Lrepresent a potential nanomaterial for future clinical appli-cations of diabetes
Acknowledgments This research received grants fromFundaccedilatildeo de Amparo agrave Pesquisa de Mato Grosso (FAPE-MAT No 7382642008 FAPEMAT No 2990322010 andNo 3016102010)
References and Notes
1 R M Ribeiro-Costa E C G Pereira J A Alves N P Santos S CNascimento N K Honda N H Silva and N S Santos-MagalhatildeesJ Microencap 21 371 (2004)
2 J Park M Ye and K Park Molecules 10 146 (2005)3 E A Scott M D Nichols R Kuntz-Willits and D L Elbert Acta
Biomat 6 29 (2010)4 R Duncan Nat Rev Drug Discov 2 346 (2003)5 K L Kiick Science 317 1182 (2007)6 T J A Recircgo Phytogeography of Medicinal Plants in the Maranhatildeo
2nd edn Edufma Satildeo Luiacutes MA Brazil (1995) pp 108ndash1097 J B Calixto Braz J Med Biol Res 32 179 (2000)8 S Hasani-Ranjbar B Larijani and M A Abdollah Arch Med Sci
4 285 (2008)9 R W Bussmann A G K Meyer A Kuhlma and A Townesmith
J Ethnobiol Ethnomed 6 2 (2010)
10 S Asgary S Parkhideh A Solhpour H Madani P Mahzouni andP Rahimi J Med Food 11 533 (2008)
11 A C Honorio-Franccedila C Marins F Boldrini and E L Franccedila ActaCir Braz 23 504 (2008)
12 American Dietetic Association J Am Diet Assoc 109 1926(2009)
13 T Miura T Itoh N Iwamoto M Kato and T Ishida Biol PharmaBull 27 248 (2004)
14 G V K Senanayake M Maruyama KShibuya M SakonoN Fukuda T Morishita C Yukizaki M Kawano and H OhtaJ Ethnopharmacol 91 257 (2004)
15 H Mundi B Bjoumlrksten C Svanborg L Ohman and C DahlgrenInfect Immun 59 4168 (1991)
16 N R Asad L M B O Assad C E B Almeida and A C LeitatildeoBr J Med Biol Res 27 2551 (1994)
17 A C Honorio-Franccedila M P S M Carvalho L Isaac L R Tra-bulsi and M M S Carneiro-Sampaio Scand J Immunol 46 59(1997)
18 A C Honorio-Franccedila P Launay M M S Carneiro-Sampaio andR C Monteiro J Leuk Biol 69 280 (2001)
19 E L Franccedila Jr A Pereira S L Oliveira and A C Honorio-Franccedila Intern J Microbiol 6 1 (2009)
20 J M McCord Am J Med 108 652 (2000)21 C Vorbach R Harrison and M R Capecchi Trend Immunol
24 512 (2003)22 B N Ames M K Shinnenaga and T M Hagen Proc Nat
Acad Sci 90 7915 (1993)23 C K B Ferrari and E A F S Torres Biomed Pharmacother
57 251 (2003)24 U Tiwari B Rastogi P Singh D K Saraf and S P Vyas
J Ethnopharmacol 92 113 (2004)25 C Viegas Jr V S Bolzani and E J Barreiro Quim Nova 29 326
(2006)26 B Patwardhan and G Manish Drug Discov Today 10 495
(2005)27 E L Franccedila J C Maynie V S C Correa U C R Pereira
C Batalini C K B Ferrari and A C Honorio-Franccedila Int J Phy-tomed 2 345 (2010)
28 S Mehrotra K P Mishra R Maurya R C Srimal V SYadav R Pandey and V K Singh Int Immunopharmacol 3 53(2003)
29 G Morceli E L Franccedila V B Magalhatildees D C Damasceno I MP Calderon and A C H Franccedila Acta Paediatr 100 550 (2011)
30 V S C Correcirca J C Maynieacute E L Franccedila and A C Honorio-Franccedila Braz J Med Plants 8 26 (2006)
31 E A Scott M D Nichols R Kuntz-Willits and D L Elbert ActaBiomat 6 29 (2010)
32 E Pick and D Mizel J Immunol Methods 46 211 (1981)33 R Bellinati-Pires M M Salgado I P Hypolito A S Grumach
and M M S Carneiro-Sampaio J Investig Allergol Clin Immunol5 337 (1995)
34 J L Roam H Xu P K Nguyen and D L Elbert Biomaterials31 8642 (2010)
35 S Freiberg and X X Zhu Int J Pharmaceut 282 1 (2004)36 R Gang and F Si-Shen Biomaterials 24 5037 (2003)37 H Ai J J Pink X T Shuai D A Boothman J M Gao J Biomed
Mater Res 73A 303 (2005)38 B Staumldler A D Price and A N Zelikin Adv Funct Mater 21 14
(2011)39 X Q Wang E Wenk X H Zhang L Mennel G Vunjak-
Novakovic and D L Kaplan J Control Releas 134 81 (2009)40 K Moore M Macsween and M Shoichet Tissue Eng 12 267
(2006)41 C K B Ferrari E L Franccedila and A C Honorio-Franccedila Applied
Biomed 7 163 (2009)42 Z Y Jiang A C Woollard and S P Wolff FEBS Lett 268 69
(1990)
Sci Adv Mater 3 687ndash694 2011 693
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Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
43 E C Tsai I B Hirsch J D Brunzell and A Chait Diabetes43 1010 (1994)
44 M Kawamura J W Heinecke and A Chait J Clin Invest 94 771(1994)
45 A C Maritim R A Sanders and B Watkins J Biochem Mol Tox-icol 17 24 (2003)
46 M Dizdaroglu P Jaruga M Birincioglu and H Rodriguez FreeRad Biol Med 32 1102 (2002)
47 A J Rassias C A Marrin J Arruda P K Whalen M Beach andM P Yeager Anesth Analg 88 1011 (1999)
48 J Jakelic S Kokic I Hozo J Maras and D Fabijanic Med Arh49 9 (1995)
49 B Larijani P Shooshtarizadeh N Mosaffa and R Heshmat Br JBiomed Sci 64 13 (2007)
50 N Seyrek W Marcinkowski M Smogorzewski T M Demerdashand S G Massry Nephrol Dial Transplant 12 265 (1997)
51 E L Franccedila N D Feliciano K A Silva C K B Ferrari andA C Honorio-Franccedila Bratisl Med J 110 517 (2009)
52 A C Honorio-Franccedila K A Silva N D Feliciano I MP Calderon M V C Rudge and E L Franccedila Int J DiabetesMetabol 17 87 (2009)
53 M Djaldetti H Salman M Bergman Djaldetti and H BesslerMicro Res Techn 57 421 (2002)
54 E L Franccedila R V Bitencourt M Fujimori T C Morais I M PCalderon and A C Honorio-Franccedila J Microbiol Immunol Infect44 1 (2011)
55 E L Franccedila G Morceli D L G Fagundes M V C Rugde I MP Calderon and A C Honorio-Franccedila APMIS 119 710 (2011)
Received 1 August 2011 Accepted 18 September 2011
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Scherer et al Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L
high density (HDPEG) were obtained by incubating thesuspension at 95 C for 3 min or 95 C for 10 min respec-tively PEG microspheres were fluorescence labeling withsolution of Dylight-488 (10 g mLminus1-Pierce) overnight atroom temperature in dimethylformamide at a 1001 molarratio of PEGDylight
26 Flow Cytometry
We performed immunofluorescence staining with phyco-erythrin (PE ndash Sigma) to assess the ability of PEG micro-sphere to biding with fluorescent markers to comparewith the microsphere polymethylmethacrylate (CaliBRITEBD) The PEG microsphere was incubated with 5 L ofPE (01 mgmL) for 30 min at 37 C After the incuba-tion the PEG microspheres were washed twice in PBScontaining BSA (5 mgmL 500timesg 10 min 4 C) In allexperiments the PEG microspheres were analyzed by flowcytometry The study was performed on FACS Calibur(BD San Jose USA) The PEG microsphere size was com-pared with the BD microsphere size (6 m-CaliBRITE3 Beads-BD Cat No 340486 USA) binding or not withPE The ratio of fluorescence intensity of PEG microspherewas express in geometric mean fluorescence intensity andthe size calculated according to geometric mean of For-ward Scatter (FSC) Experiments were repeated on severaloccasions the data presented in the figures are from singleexperiments performed
27 Release of Superoxide Anion
Superoxide release was determined by cytochrome C(Sigma) reduction1732 Briefly mononuclear phagocytesand bacteria mononuclear phagocytes and bacteria inthe presence or not of PEG microsphere adsorbed withMomordica charantia L were mixed and incubated for 30min for phagocytosis Cells were then resuspended in PBScontaining 26 mM CaCl2 2 mM MgCl2 and cytochromeC (2 mgmL) The suspensions (100 L) were incubatedfor 60 min at 37 C on culture plates The reaction rateswere measured by absorbance at 550 nm and the resultswere expressed as nmolO2minus All the experiments wereperformed in duplicate or triplicate
28 Bactericidal Assay
Microbicidal activity and phagocytosis were evaluated bythe acridine orange method described by Bellinati-Pireset al33 Equal volumes of bacteria and cell suspensionsin the presence or not of PEG microsphere adsorbedwith Momordica charantia L were mixed and incubatedat 37 C for 30 min with continuous shaking Phago-cytosis was stopped by incubation in ice To eliminateextracellular bacteria the suspensions were centrifugedtwice (160timesg 10 min 4 C) Cells were resuspended in
serum-free medium 199 and centrifuged The supernatantwas discarded and the sediment was dyed with 200 Lof acridine orange (144 gL) for 1 min The sedimentwas resuspended in cold culture 199 washed twice andobserved under an immunofluorescence microscope at400times and 1000times magnification The phagocytosis indexwas calculated by counting the number of cells ingestingat least 3 bacteria within a pool of 100 cells To deter-mine the bactericidal index we stained the slides withacridine orange and counted 100 cells with phagocytizedbacteria The bactericidal index is calculated as the ratiobetween orange- stained (dead) and green- stained (alive)bacteriatimes 10033 All the experiments were performed induplicate or triplicate
29 Statistical Analysis
Analysis of variance (ANOVA) was used to evaluate super-oxide anion release phagocytosis bactericidal index in thepresence or absence of opsonized EPEC Statistical sig-nificance was considered for a p-value lower than 005[p lt 005]
3 RESULTS
31 Characterization of PEG Microspheres
The Figure 1(A) shows the relationship between the PEGmicrosphere and BD microsphere (standard) by fluores-cence intensity Both microspheres had similar geomet-ric mean of fluorescence intensity When compared tosize the PEG microsphere was similar to standard micro-sphere (Fig 1(B)) The fluorescence microscopy image(Fig 2(A)) shows that the PEG microspheres producedin PBS have a narrow size distribution This result con-firms that our method produces differents sizes micro-spheres and shows that both the high and low-densitymicrospheres easily separate in the suspension The micro-spheres produced were highly stable and retained theirspherical structure without deformation (Fig 2(A)) Flu-orescence microscopy showed that PEG microspheres areable to absorb the extract of plant (Fig 2(B)) Fluores-cence microscopy showed that blood phagocytes are ableto internalize the microspheres (Fig 2(C))
32 The Effect of PEG Microspheres ContainingPlant Extract on Blood Mononuclear Phagocytes
Retrieval and viability of blood phagocytes from boththe normoglycemic and hyperglycemic groups were notaffected by the PEG microspheres orMomordica charantiaL extracts (p lt 005ndashTable I)
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RESEARCH
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Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
33 The Effect of PEG Microspheres Adsorbed withPlant Extract on Superoxide Release
The hyperglycemic and normoglycemic groups had sim-ilar spontaneous superoxide release by blood phago-cytes When exposed to PEG microspheres independentof density the cells showed no difference between thegroups Phagocytes incubated with plant extract had highersuperoxide release than untreated phagocytes (p lt 005)irrespective of their glycemic status Additionally thephagocytes in the presence of PEG microspheres adsorbedwith plant extract regardless of glycemic status dis-played an increased release of superoxide (Table II) Whenexposed to EPEC superoxide release increased (p lt 005)in the hyperglycemic group (p lt 005) in the absence ofthe PEG microspheres or plant extract Normoglycemic
Fig 1 PEG Microspheres were stained directly with phycoerythrin(PE) as described in Materials and Methods PolymethylmethacrylateMicrosphere PE-labeled (BD Microsphere-Becton Dickinson USA) wasused as standard Immunofluorescence analysis and size were then carriedout by flow cytometry (FACScalibur Becton Dickinson USA)
(A)
(B)
(C)
Fig 2 Fluorescence microscopy image of the PEG microspheresstained with Dylight-488 (100times - panels-A) The PEG microspheres wereincubated with Momordica charantia L (100times ndash panel-B) on a shaker for30 min at 37 C and stained with Dylight-488 The phagocytes were incu-bated with PEG microsphere (100times - panels-C) on a shaker for 30 minat 37 C After washing at 4 C cells were stained with acridine orangeand analyzed by fluorescent microscopy Experiments were repeated fivetimes and the results were comparable
group phagocytes incubated with EPEC in the presence ofhigh density PEG microspheres loaded with plant extracthad higher superoxide release than phagocytes exposedonly to bacteria (p lt 005ndashTable II)
690 Sci Adv Mater 3 687ndash694 2011
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RESEARCH
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Scherer et al Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L
Table I General characteristics of the experimental groups The results represent the mean and SD of ten experiments
Parameters Control Diabetes Statistics
Glycemia (mgdL) 7556plusmn1136 16339plusmn5125a F = 20 517 p = 00001 (comparing groups)Leukocytes number (times106 cellsmL) 218plusmn074 240plusmn064 F = 1 354 p = 06352 (comparing groups)Leukocytes viabilityb 866plusmn628 892plusmn354 F = 3 139 p = 00532 (comparing groups)
Data represented by meanplusmn standard deviation (SD) aStatistically significant differences between normoglycemic and hyperglycemic groups considering the samplesbViability in the presence of Momordica charantia L and Polyethyleneglycol (PEG)
34 The Effects of PEG Microsphere Adsorbed withPlant Extract on the Phagocytosis of BloodMononuclear Cells
Blood phagocytes from the diabetic group had somephagocytic activity in response to EPEC Phagocytosisincreased significantly in the presence of microsphere PEGadsorbed with plant extract for the normoglycemic groupA comparison of the groups showed that EPEC phagocy-tosis in general was similar for the diabetic and normo-glycemic groups (Fig 3(A))
35 The Effects of Microsphere PEG Adsorbed withPlant Extract on the Bacterial Activity of BloodPhagocytes
In general blood mononuclear phagocytes from the dia-betic group had low bactericidal activity against EPECMononuclear phagocytes from the normoglycemic groupincubated with plant extract or low density microspheresPEG adsorbed with or without plant extract had increasedmicrobicidal activity in response to EPEC (Fig 3(B))
Table II Superoxide release by blood mononuclear phagocytes(meanplusmnSD N = 10 in each treatment)
Superoxide release (nmol)
MN phagocytes Bacteria Control Diabetes
PBS No 59plusmn12 40plusmn14Yes 13plusmn004 113plusmn11d
Momordica charantia L No 125plusmn33c 104plusmn38c
Yes 46 plusmn 05c 138plusmn09d
HDPEGa No 61plusmn26 54plusmn12Yes 37plusmn15c 116plusmn25d
LDPEGb No 62plusmn23 44plusmn18Yes 19plusmn03 118plusmn32d
Momordica charantia L No 98plusmn27c 76plusmn24plus HDPEG
Yes 44plusmn03c 125plusmn07d
Momordica charantia L No 88plusmn17c 87plusmn15c
plus LDPEGYes 16plusmn02 131plusmn09d
aHigh Density Polyethylene Glycol bLow Density Polyethylene Glycol Bacteriawere treated with PEG (High or Low density) andor Momordica charantia LIn controls assays mononuclear cells were pre-incubated with PBS cIndicatesdifferences between each bacteria treatment and the control treatment within agroup (ANOVA P lt 005) d indicates intergroup differences within each treatment(ANOVA P lt 005)
4 DISCUSSION
In the present study we produced PEG microspheres ofdifferent densities adsorbed with extract from Momordicacharantia L and studied their in vitro effect on bacterial-killing activity and superoxide production in blood phago-cytes from diabetic individualsPolymer microspheres can be employed to deliver medi-
cation in a rate-controlled and sometimes targeted mannerMedication is released from a microsphere by drug leach-ing from the polymer or by degradation of the polymermatrix Because the rate of drug release is controlled bythese two factors it is important to understand the physi-cal and chemical properties of the releasing medium35 Inthis study analysis by fluorescence microscopy and flowcytometry showed PEG microspheres to be a regular sizeregardless of density and easily separated from the sus-pension The ability to form gradients in microspheres mayprove to be useful to achieve better control over the kinet-ics of protein release36 The literature have reported theuse of ow cytometry as an alternative method for analy-ses and visualization of particles37ndash38 Here we showed byflow cytometry that the PEG microsphere have a size ofabout 58 mPEG Microspheres are a kind of copolymer used in
clinical administration of drugs due to their incorporationcapacity36 Density gradients of signaling are importantin immunity39 In particular chemotaxis the preferentialmovement of cells up a density gradient of singlingmolecules is dependent on the gradient as opposed tothe average concentration of the molecule40 Here wehad found that the PEG microsphere independent ofthe density gradient adsorbed with plant extract pre-sented a potent immunostimulatory effect on the func-tional activity of blood phagocytes Cells obtained fromthe blood of normoglycemic individuals are known tokill enteropathogenic Escherichia coli in the presence ofherbal plants associated with peptides This interactionmay represent an alternative mechanism of defense againstinfection27
In this study we observed that the interaction ofPEG microspheres with blood phagocytes regardless ofglycemic status did not affect the viability of the cellsFurthermore superoxide release was higher when thephagocytes were in the presence of the PEG microspheresadsorbed with plant extract irrespective of glycemic statusThe phagocytes incubated with EPEC in the presence of
Sci Adv Mater 3 687ndash694 2011 691
Delivered by Ingenta toUniversity of Wollongong
IP 1301303785Mon 27 Feb 2012 185227
RESEARCH
ARTIC
LE
Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
(A)
(B)
Fig 3 Bacterial phagocytosis (A) and bactericidal activity index (B) by blood mononuclear cells (meanplusmnSD N = 10 in each treatment) Bacterialphagocytosis and elimation index by mononuclear cells from blood was determined with the acridine orange method lowastindicates differences betweentreatment (PEG orand plant) and 199 medium (ANOVA P lt 005) indicates intergroup differences for each treatment (ANOVA P lt 005) (HDPEG-Hight Density Polyethylene Glicol LDPEG-Low Density Polyethylene Glicol MOR ndash Momordica charantia L)
PEG microspheres adsorbed with plant extract had highersuperoxide release than phagocytes exposed only to thebacteria The activation ability of these cells indicated bysuperoxide release remained at the same level for both thehyperglycemic and control groupsA great variety of mechanisms have been suggested
for the formation of these free reactive oxygen radicals41
Glucose oxidation is believed to be the main source of freeradical production in diabetes42 Hyperglycemia has alsobeen found to promote lipid peroxidation by a superoxide-dependent pathway resulting in free radical generation andrelease43ndash44 Another important source of free radicals in
diabetes is the interaction of glucose with proteins lead-ing to the formation of advanced glycation end-productsthat contribute to an excessive burst in mitochondrial freeradicals45 The role of free radicals is an extremely impor-tant phenomenon during immune responses and inflam-matory reactions whereas the microbicidal activity ofphagocytes has been linked to the activation of cellularoxidative metabolism and release of large amounts of freeradicals46
On the other hand the high glucose levels of diabeticpatients can impair their antioxidant systems by decreasingimportant responses such as phagocytic and microbicidal
692 Sci Adv Mater 3 687ndash694 2011
Delivered by Ingenta toUniversity of Wollongong
IP 1301303785Mon 27 Feb 2012 185227
RESEARCH
ARTIC
LE
Scherer et al Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L
activity47ndash49 In this work the PEG microspheres adsorbedwith plant extract increased phagocytosis Similar resultswith respect to the functional activity of leukocytes wereobserved in phagocytes from patients with diabetes45 andfrom animals with induced diabetes50ndash52 Mononuclearphagocytes play an important role in host defense Theyproduce phagocytic NADPH oxidase which forms super-oxide and is imperative for killing bacterial53 and crucialfor the success of immune responses and inflammatoryreactions46
In our studies the elimination of EPEC by blood phago-cytes was stimulated only in the normoglycemic groupwhen these cells were incubated with plant extract or lowdensity PEG microspheres adsorbed with plant extractThe scientific literature has already reported the elimi-nation of bacteria by mononuclear phagocytes and theirmicrobicidal activity was stimulated in the presence ofimmunomodulatory agents17ndash5455
The high glucose levels impair a series of physiolog-ical processes and the functional activity seems to beaffected Although the PEG microspheres adsorbed withMomordica charantia L did not increase the microbici-dal activity of blood phagocytes from diabetes patientsthis polymer can be activated inside cells This studyoutlined the immunomodulatory effects of microspherepoly(ethylene glycol) adsorbed with Momordica charan-tia L on blood phagocytes This system may be usefulfor the delivery of a variety of therapeutic plants and wasshown to provide additional protection against infectionsin normoglycemic and hyperglycemic individuals Thesedata suggest that microspheres of poly(ethylene glycol)adsorbed with nanofractions of Momordica charantia Lrepresent a potential nanomaterial for future clinical appli-cations of diabetes
Acknowledgments This research received grants fromFundaccedilatildeo de Amparo agrave Pesquisa de Mato Grosso (FAPE-MAT No 7382642008 FAPEMAT No 2990322010 andNo 3016102010)
References and Notes
1 R M Ribeiro-Costa E C G Pereira J A Alves N P Santos S CNascimento N K Honda N H Silva and N S Santos-MagalhatildeesJ Microencap 21 371 (2004)
2 J Park M Ye and K Park Molecules 10 146 (2005)3 E A Scott M D Nichols R Kuntz-Willits and D L Elbert Acta
Biomat 6 29 (2010)4 R Duncan Nat Rev Drug Discov 2 346 (2003)5 K L Kiick Science 317 1182 (2007)6 T J A Recircgo Phytogeography of Medicinal Plants in the Maranhatildeo
2nd edn Edufma Satildeo Luiacutes MA Brazil (1995) pp 108ndash1097 J B Calixto Braz J Med Biol Res 32 179 (2000)8 S Hasani-Ranjbar B Larijani and M A Abdollah Arch Med Sci
4 285 (2008)9 R W Bussmann A G K Meyer A Kuhlma and A Townesmith
J Ethnobiol Ethnomed 6 2 (2010)
10 S Asgary S Parkhideh A Solhpour H Madani P Mahzouni andP Rahimi J Med Food 11 533 (2008)
11 A C Honorio-Franccedila C Marins F Boldrini and E L Franccedila ActaCir Braz 23 504 (2008)
12 American Dietetic Association J Am Diet Assoc 109 1926(2009)
13 T Miura T Itoh N Iwamoto M Kato and T Ishida Biol PharmaBull 27 248 (2004)
14 G V K Senanayake M Maruyama KShibuya M SakonoN Fukuda T Morishita C Yukizaki M Kawano and H OhtaJ Ethnopharmacol 91 257 (2004)
15 H Mundi B Bjoumlrksten C Svanborg L Ohman and C DahlgrenInfect Immun 59 4168 (1991)
16 N R Asad L M B O Assad C E B Almeida and A C LeitatildeoBr J Med Biol Res 27 2551 (1994)
17 A C Honorio-Franccedila M P S M Carvalho L Isaac L R Tra-bulsi and M M S Carneiro-Sampaio Scand J Immunol 46 59(1997)
18 A C Honorio-Franccedila P Launay M M S Carneiro-Sampaio andR C Monteiro J Leuk Biol 69 280 (2001)
19 E L Franccedila Jr A Pereira S L Oliveira and A C Honorio-Franccedila Intern J Microbiol 6 1 (2009)
20 J M McCord Am J Med 108 652 (2000)21 C Vorbach R Harrison and M R Capecchi Trend Immunol
24 512 (2003)22 B N Ames M K Shinnenaga and T M Hagen Proc Nat
Acad Sci 90 7915 (1993)23 C K B Ferrari and E A F S Torres Biomed Pharmacother
57 251 (2003)24 U Tiwari B Rastogi P Singh D K Saraf and S P Vyas
J Ethnopharmacol 92 113 (2004)25 C Viegas Jr V S Bolzani and E J Barreiro Quim Nova 29 326
(2006)26 B Patwardhan and G Manish Drug Discov Today 10 495
(2005)27 E L Franccedila J C Maynie V S C Correa U C R Pereira
C Batalini C K B Ferrari and A C Honorio-Franccedila Int J Phy-tomed 2 345 (2010)
28 S Mehrotra K P Mishra R Maurya R C Srimal V SYadav R Pandey and V K Singh Int Immunopharmacol 3 53(2003)
29 G Morceli E L Franccedila V B Magalhatildees D C Damasceno I MP Calderon and A C H Franccedila Acta Paediatr 100 550 (2011)
30 V S C Correcirca J C Maynieacute E L Franccedila and A C Honorio-Franccedila Braz J Med Plants 8 26 (2006)
31 E A Scott M D Nichols R Kuntz-Willits and D L Elbert ActaBiomat 6 29 (2010)
32 E Pick and D Mizel J Immunol Methods 46 211 (1981)33 R Bellinati-Pires M M Salgado I P Hypolito A S Grumach
and M M S Carneiro-Sampaio J Investig Allergol Clin Immunol5 337 (1995)
34 J L Roam H Xu P K Nguyen and D L Elbert Biomaterials31 8642 (2010)
35 S Freiberg and X X Zhu Int J Pharmaceut 282 1 (2004)36 R Gang and F Si-Shen Biomaterials 24 5037 (2003)37 H Ai J J Pink X T Shuai D A Boothman J M Gao J Biomed
Mater Res 73A 303 (2005)38 B Staumldler A D Price and A N Zelikin Adv Funct Mater 21 14
(2011)39 X Q Wang E Wenk X H Zhang L Mennel G Vunjak-
Novakovic and D L Kaplan J Control Releas 134 81 (2009)40 K Moore M Macsween and M Shoichet Tissue Eng 12 267
(2006)41 C K B Ferrari E L Franccedila and A C Honorio-Franccedila Applied
Biomed 7 163 (2009)42 Z Y Jiang A C Woollard and S P Wolff FEBS Lett 268 69
(1990)
Sci Adv Mater 3 687ndash694 2011 693
Delivered by Ingenta toUniversity of Wollongong
IP 1301303785Mon 27 Feb 2012 185227
RESEARCH
ARTIC
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Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
43 E C Tsai I B Hirsch J D Brunzell and A Chait Diabetes43 1010 (1994)
44 M Kawamura J W Heinecke and A Chait J Clin Invest 94 771(1994)
45 A C Maritim R A Sanders and B Watkins J Biochem Mol Tox-icol 17 24 (2003)
46 M Dizdaroglu P Jaruga M Birincioglu and H Rodriguez FreeRad Biol Med 32 1102 (2002)
47 A J Rassias C A Marrin J Arruda P K Whalen M Beach andM P Yeager Anesth Analg 88 1011 (1999)
48 J Jakelic S Kokic I Hozo J Maras and D Fabijanic Med Arh49 9 (1995)
49 B Larijani P Shooshtarizadeh N Mosaffa and R Heshmat Br JBiomed Sci 64 13 (2007)
50 N Seyrek W Marcinkowski M Smogorzewski T M Demerdashand S G Massry Nephrol Dial Transplant 12 265 (1997)
51 E L Franccedila N D Feliciano K A Silva C K B Ferrari andA C Honorio-Franccedila Bratisl Med J 110 517 (2009)
52 A C Honorio-Franccedila K A Silva N D Feliciano I MP Calderon M V C Rudge and E L Franccedila Int J DiabetesMetabol 17 87 (2009)
53 M Djaldetti H Salman M Bergman Djaldetti and H BesslerMicro Res Techn 57 421 (2002)
54 E L Franccedila R V Bitencourt M Fujimori T C Morais I M PCalderon and A C Honorio-Franccedila J Microbiol Immunol Infect44 1 (2011)
55 E L Franccedila G Morceli D L G Fagundes M V C Rugde I MP Calderon and A C Honorio-Franccedila APMIS 119 710 (2011)
Received 1 August 2011 Accepted 18 September 2011
694 Sci Adv Mater 3 687ndash694 2011
Delivered by Ingenta toUniversity of Wollongong
IP 1301303785Mon 27 Feb 2012 185227
RESEARCH
ARTIC
LE
Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
33 The Effect of PEG Microspheres Adsorbed withPlant Extract on Superoxide Release
The hyperglycemic and normoglycemic groups had sim-ilar spontaneous superoxide release by blood phago-cytes When exposed to PEG microspheres independentof density the cells showed no difference between thegroups Phagocytes incubated with plant extract had highersuperoxide release than untreated phagocytes (p lt 005)irrespective of their glycemic status Additionally thephagocytes in the presence of PEG microspheres adsorbedwith plant extract regardless of glycemic status dis-played an increased release of superoxide (Table II) Whenexposed to EPEC superoxide release increased (p lt 005)in the hyperglycemic group (p lt 005) in the absence ofthe PEG microspheres or plant extract Normoglycemic
Fig 1 PEG Microspheres were stained directly with phycoerythrin(PE) as described in Materials and Methods PolymethylmethacrylateMicrosphere PE-labeled (BD Microsphere-Becton Dickinson USA) wasused as standard Immunofluorescence analysis and size were then carriedout by flow cytometry (FACScalibur Becton Dickinson USA)
(A)
(B)
(C)
Fig 2 Fluorescence microscopy image of the PEG microspheresstained with Dylight-488 (100times - panels-A) The PEG microspheres wereincubated with Momordica charantia L (100times ndash panel-B) on a shaker for30 min at 37 C and stained with Dylight-488 The phagocytes were incu-bated with PEG microsphere (100times - panels-C) on a shaker for 30 minat 37 C After washing at 4 C cells were stained with acridine orangeand analyzed by fluorescent microscopy Experiments were repeated fivetimes and the results were comparable
group phagocytes incubated with EPEC in the presence ofhigh density PEG microspheres loaded with plant extracthad higher superoxide release than phagocytes exposedonly to bacteria (p lt 005ndashTable II)
690 Sci Adv Mater 3 687ndash694 2011
Delivered by Ingenta toUniversity of Wollongong
IP 1301303785Mon 27 Feb 2012 185227
RESEARCH
ARTIC
LE
Scherer et al Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L
Table I General characteristics of the experimental groups The results represent the mean and SD of ten experiments
Parameters Control Diabetes Statistics
Glycemia (mgdL) 7556plusmn1136 16339plusmn5125a F = 20 517 p = 00001 (comparing groups)Leukocytes number (times106 cellsmL) 218plusmn074 240plusmn064 F = 1 354 p = 06352 (comparing groups)Leukocytes viabilityb 866plusmn628 892plusmn354 F = 3 139 p = 00532 (comparing groups)
Data represented by meanplusmn standard deviation (SD) aStatistically significant differences between normoglycemic and hyperglycemic groups considering the samplesbViability in the presence of Momordica charantia L and Polyethyleneglycol (PEG)
34 The Effects of PEG Microsphere Adsorbed withPlant Extract on the Phagocytosis of BloodMononuclear Cells
Blood phagocytes from the diabetic group had somephagocytic activity in response to EPEC Phagocytosisincreased significantly in the presence of microsphere PEGadsorbed with plant extract for the normoglycemic groupA comparison of the groups showed that EPEC phagocy-tosis in general was similar for the diabetic and normo-glycemic groups (Fig 3(A))
35 The Effects of Microsphere PEG Adsorbed withPlant Extract on the Bacterial Activity of BloodPhagocytes
In general blood mononuclear phagocytes from the dia-betic group had low bactericidal activity against EPECMononuclear phagocytes from the normoglycemic groupincubated with plant extract or low density microspheresPEG adsorbed with or without plant extract had increasedmicrobicidal activity in response to EPEC (Fig 3(B))
Table II Superoxide release by blood mononuclear phagocytes(meanplusmnSD N = 10 in each treatment)
Superoxide release (nmol)
MN phagocytes Bacteria Control Diabetes
PBS No 59plusmn12 40plusmn14Yes 13plusmn004 113plusmn11d
Momordica charantia L No 125plusmn33c 104plusmn38c
Yes 46 plusmn 05c 138plusmn09d
HDPEGa No 61plusmn26 54plusmn12Yes 37plusmn15c 116plusmn25d
LDPEGb No 62plusmn23 44plusmn18Yes 19plusmn03 118plusmn32d
Momordica charantia L No 98plusmn27c 76plusmn24plus HDPEG
Yes 44plusmn03c 125plusmn07d
Momordica charantia L No 88plusmn17c 87plusmn15c
plus LDPEGYes 16plusmn02 131plusmn09d
aHigh Density Polyethylene Glycol bLow Density Polyethylene Glycol Bacteriawere treated with PEG (High or Low density) andor Momordica charantia LIn controls assays mononuclear cells were pre-incubated with PBS cIndicatesdifferences between each bacteria treatment and the control treatment within agroup (ANOVA P lt 005) d indicates intergroup differences within each treatment(ANOVA P lt 005)
4 DISCUSSION
In the present study we produced PEG microspheres ofdifferent densities adsorbed with extract from Momordicacharantia L and studied their in vitro effect on bacterial-killing activity and superoxide production in blood phago-cytes from diabetic individualsPolymer microspheres can be employed to deliver medi-
cation in a rate-controlled and sometimes targeted mannerMedication is released from a microsphere by drug leach-ing from the polymer or by degradation of the polymermatrix Because the rate of drug release is controlled bythese two factors it is important to understand the physi-cal and chemical properties of the releasing medium35 Inthis study analysis by fluorescence microscopy and flowcytometry showed PEG microspheres to be a regular sizeregardless of density and easily separated from the sus-pension The ability to form gradients in microspheres mayprove to be useful to achieve better control over the kinet-ics of protein release36 The literature have reported theuse of ow cytometry as an alternative method for analy-ses and visualization of particles37ndash38 Here we showed byflow cytometry that the PEG microsphere have a size ofabout 58 mPEG Microspheres are a kind of copolymer used in
clinical administration of drugs due to their incorporationcapacity36 Density gradients of signaling are importantin immunity39 In particular chemotaxis the preferentialmovement of cells up a density gradient of singlingmolecules is dependent on the gradient as opposed tothe average concentration of the molecule40 Here wehad found that the PEG microsphere independent ofthe density gradient adsorbed with plant extract pre-sented a potent immunostimulatory effect on the func-tional activity of blood phagocytes Cells obtained fromthe blood of normoglycemic individuals are known tokill enteropathogenic Escherichia coli in the presence ofherbal plants associated with peptides This interactionmay represent an alternative mechanism of defense againstinfection27
In this study we observed that the interaction ofPEG microspheres with blood phagocytes regardless ofglycemic status did not affect the viability of the cellsFurthermore superoxide release was higher when thephagocytes were in the presence of the PEG microspheresadsorbed with plant extract irrespective of glycemic statusThe phagocytes incubated with EPEC in the presence of
Sci Adv Mater 3 687ndash694 2011 691
Delivered by Ingenta toUniversity of Wollongong
IP 1301303785Mon 27 Feb 2012 185227
RESEARCH
ARTIC
LE
Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
(A)
(B)
Fig 3 Bacterial phagocytosis (A) and bactericidal activity index (B) by blood mononuclear cells (meanplusmnSD N = 10 in each treatment) Bacterialphagocytosis and elimation index by mononuclear cells from blood was determined with the acridine orange method lowastindicates differences betweentreatment (PEG orand plant) and 199 medium (ANOVA P lt 005) indicates intergroup differences for each treatment (ANOVA P lt 005) (HDPEG-Hight Density Polyethylene Glicol LDPEG-Low Density Polyethylene Glicol MOR ndash Momordica charantia L)
PEG microspheres adsorbed with plant extract had highersuperoxide release than phagocytes exposed only to thebacteria The activation ability of these cells indicated bysuperoxide release remained at the same level for both thehyperglycemic and control groupsA great variety of mechanisms have been suggested
for the formation of these free reactive oxygen radicals41
Glucose oxidation is believed to be the main source of freeradical production in diabetes42 Hyperglycemia has alsobeen found to promote lipid peroxidation by a superoxide-dependent pathway resulting in free radical generation andrelease43ndash44 Another important source of free radicals in
diabetes is the interaction of glucose with proteins lead-ing to the formation of advanced glycation end-productsthat contribute to an excessive burst in mitochondrial freeradicals45 The role of free radicals is an extremely impor-tant phenomenon during immune responses and inflam-matory reactions whereas the microbicidal activity ofphagocytes has been linked to the activation of cellularoxidative metabolism and release of large amounts of freeradicals46
On the other hand the high glucose levels of diabeticpatients can impair their antioxidant systems by decreasingimportant responses such as phagocytic and microbicidal
692 Sci Adv Mater 3 687ndash694 2011
Delivered by Ingenta toUniversity of Wollongong
IP 1301303785Mon 27 Feb 2012 185227
RESEARCH
ARTIC
LE
Scherer et al Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L
activity47ndash49 In this work the PEG microspheres adsorbedwith plant extract increased phagocytosis Similar resultswith respect to the functional activity of leukocytes wereobserved in phagocytes from patients with diabetes45 andfrom animals with induced diabetes50ndash52 Mononuclearphagocytes play an important role in host defense Theyproduce phagocytic NADPH oxidase which forms super-oxide and is imperative for killing bacterial53 and crucialfor the success of immune responses and inflammatoryreactions46
In our studies the elimination of EPEC by blood phago-cytes was stimulated only in the normoglycemic groupwhen these cells were incubated with plant extract or lowdensity PEG microspheres adsorbed with plant extractThe scientific literature has already reported the elimi-nation of bacteria by mononuclear phagocytes and theirmicrobicidal activity was stimulated in the presence ofimmunomodulatory agents17ndash5455
The high glucose levels impair a series of physiolog-ical processes and the functional activity seems to beaffected Although the PEG microspheres adsorbed withMomordica charantia L did not increase the microbici-dal activity of blood phagocytes from diabetes patientsthis polymer can be activated inside cells This studyoutlined the immunomodulatory effects of microspherepoly(ethylene glycol) adsorbed with Momordica charan-tia L on blood phagocytes This system may be usefulfor the delivery of a variety of therapeutic plants and wasshown to provide additional protection against infectionsin normoglycemic and hyperglycemic individuals Thesedata suggest that microspheres of poly(ethylene glycol)adsorbed with nanofractions of Momordica charantia Lrepresent a potential nanomaterial for future clinical appli-cations of diabetes
Acknowledgments This research received grants fromFundaccedilatildeo de Amparo agrave Pesquisa de Mato Grosso (FAPE-MAT No 7382642008 FAPEMAT No 2990322010 andNo 3016102010)
References and Notes
1 R M Ribeiro-Costa E C G Pereira J A Alves N P Santos S CNascimento N K Honda N H Silva and N S Santos-MagalhatildeesJ Microencap 21 371 (2004)
2 J Park M Ye and K Park Molecules 10 146 (2005)3 E A Scott M D Nichols R Kuntz-Willits and D L Elbert Acta
Biomat 6 29 (2010)4 R Duncan Nat Rev Drug Discov 2 346 (2003)5 K L Kiick Science 317 1182 (2007)6 T J A Recircgo Phytogeography of Medicinal Plants in the Maranhatildeo
2nd edn Edufma Satildeo Luiacutes MA Brazil (1995) pp 108ndash1097 J B Calixto Braz J Med Biol Res 32 179 (2000)8 S Hasani-Ranjbar B Larijani and M A Abdollah Arch Med Sci
4 285 (2008)9 R W Bussmann A G K Meyer A Kuhlma and A Townesmith
J Ethnobiol Ethnomed 6 2 (2010)
10 S Asgary S Parkhideh A Solhpour H Madani P Mahzouni andP Rahimi J Med Food 11 533 (2008)
11 A C Honorio-Franccedila C Marins F Boldrini and E L Franccedila ActaCir Braz 23 504 (2008)
12 American Dietetic Association J Am Diet Assoc 109 1926(2009)
13 T Miura T Itoh N Iwamoto M Kato and T Ishida Biol PharmaBull 27 248 (2004)
14 G V K Senanayake M Maruyama KShibuya M SakonoN Fukuda T Morishita C Yukizaki M Kawano and H OhtaJ Ethnopharmacol 91 257 (2004)
15 H Mundi B Bjoumlrksten C Svanborg L Ohman and C DahlgrenInfect Immun 59 4168 (1991)
16 N R Asad L M B O Assad C E B Almeida and A C LeitatildeoBr J Med Biol Res 27 2551 (1994)
17 A C Honorio-Franccedila M P S M Carvalho L Isaac L R Tra-bulsi and M M S Carneiro-Sampaio Scand J Immunol 46 59(1997)
18 A C Honorio-Franccedila P Launay M M S Carneiro-Sampaio andR C Monteiro J Leuk Biol 69 280 (2001)
19 E L Franccedila Jr A Pereira S L Oliveira and A C Honorio-Franccedila Intern J Microbiol 6 1 (2009)
20 J M McCord Am J Med 108 652 (2000)21 C Vorbach R Harrison and M R Capecchi Trend Immunol
24 512 (2003)22 B N Ames M K Shinnenaga and T M Hagen Proc Nat
Acad Sci 90 7915 (1993)23 C K B Ferrari and E A F S Torres Biomed Pharmacother
57 251 (2003)24 U Tiwari B Rastogi P Singh D K Saraf and S P Vyas
J Ethnopharmacol 92 113 (2004)25 C Viegas Jr V S Bolzani and E J Barreiro Quim Nova 29 326
(2006)26 B Patwardhan and G Manish Drug Discov Today 10 495
(2005)27 E L Franccedila J C Maynie V S C Correa U C R Pereira
C Batalini C K B Ferrari and A C Honorio-Franccedila Int J Phy-tomed 2 345 (2010)
28 S Mehrotra K P Mishra R Maurya R C Srimal V SYadav R Pandey and V K Singh Int Immunopharmacol 3 53(2003)
29 G Morceli E L Franccedila V B Magalhatildees D C Damasceno I MP Calderon and A C H Franccedila Acta Paediatr 100 550 (2011)
30 V S C Correcirca J C Maynieacute E L Franccedila and A C Honorio-Franccedila Braz J Med Plants 8 26 (2006)
31 E A Scott M D Nichols R Kuntz-Willits and D L Elbert ActaBiomat 6 29 (2010)
32 E Pick and D Mizel J Immunol Methods 46 211 (1981)33 R Bellinati-Pires M M Salgado I P Hypolito A S Grumach
and M M S Carneiro-Sampaio J Investig Allergol Clin Immunol5 337 (1995)
34 J L Roam H Xu P K Nguyen and D L Elbert Biomaterials31 8642 (2010)
35 S Freiberg and X X Zhu Int J Pharmaceut 282 1 (2004)36 R Gang and F Si-Shen Biomaterials 24 5037 (2003)37 H Ai J J Pink X T Shuai D A Boothman J M Gao J Biomed
Mater Res 73A 303 (2005)38 B Staumldler A D Price and A N Zelikin Adv Funct Mater 21 14
(2011)39 X Q Wang E Wenk X H Zhang L Mennel G Vunjak-
Novakovic and D L Kaplan J Control Releas 134 81 (2009)40 K Moore M Macsween and M Shoichet Tissue Eng 12 267
(2006)41 C K B Ferrari E L Franccedila and A C Honorio-Franccedila Applied
Biomed 7 163 (2009)42 Z Y Jiang A C Woollard and S P Wolff FEBS Lett 268 69
(1990)
Sci Adv Mater 3 687ndash694 2011 693
Delivered by Ingenta toUniversity of Wollongong
IP 1301303785Mon 27 Feb 2012 185227
RESEARCH
ARTIC
LE
Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
43 E C Tsai I B Hirsch J D Brunzell and A Chait Diabetes43 1010 (1994)
44 M Kawamura J W Heinecke and A Chait J Clin Invest 94 771(1994)
45 A C Maritim R A Sanders and B Watkins J Biochem Mol Tox-icol 17 24 (2003)
46 M Dizdaroglu P Jaruga M Birincioglu and H Rodriguez FreeRad Biol Med 32 1102 (2002)
47 A J Rassias C A Marrin J Arruda P K Whalen M Beach andM P Yeager Anesth Analg 88 1011 (1999)
48 J Jakelic S Kokic I Hozo J Maras and D Fabijanic Med Arh49 9 (1995)
49 B Larijani P Shooshtarizadeh N Mosaffa and R Heshmat Br JBiomed Sci 64 13 (2007)
50 N Seyrek W Marcinkowski M Smogorzewski T M Demerdashand S G Massry Nephrol Dial Transplant 12 265 (1997)
51 E L Franccedila N D Feliciano K A Silva C K B Ferrari andA C Honorio-Franccedila Bratisl Med J 110 517 (2009)
52 A C Honorio-Franccedila K A Silva N D Feliciano I MP Calderon M V C Rudge and E L Franccedila Int J DiabetesMetabol 17 87 (2009)
53 M Djaldetti H Salman M Bergman Djaldetti and H BesslerMicro Res Techn 57 421 (2002)
54 E L Franccedila R V Bitencourt M Fujimori T C Morais I M PCalderon and A C Honorio-Franccedila J Microbiol Immunol Infect44 1 (2011)
55 E L Franccedila G Morceli D L G Fagundes M V C Rugde I MP Calderon and A C Honorio-Franccedila APMIS 119 710 (2011)
Received 1 August 2011 Accepted 18 September 2011
694 Sci Adv Mater 3 687ndash694 2011
Delivered by Ingenta toUniversity of Wollongong
IP 1301303785Mon 27 Feb 2012 185227
RESEARCH
ARTIC
LE
Scherer et al Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L
Table I General characteristics of the experimental groups The results represent the mean and SD of ten experiments
Parameters Control Diabetes Statistics
Glycemia (mgdL) 7556plusmn1136 16339plusmn5125a F = 20 517 p = 00001 (comparing groups)Leukocytes number (times106 cellsmL) 218plusmn074 240plusmn064 F = 1 354 p = 06352 (comparing groups)Leukocytes viabilityb 866plusmn628 892plusmn354 F = 3 139 p = 00532 (comparing groups)
Data represented by meanplusmn standard deviation (SD) aStatistically significant differences between normoglycemic and hyperglycemic groups considering the samplesbViability in the presence of Momordica charantia L and Polyethyleneglycol (PEG)
34 The Effects of PEG Microsphere Adsorbed withPlant Extract on the Phagocytosis of BloodMononuclear Cells
Blood phagocytes from the diabetic group had somephagocytic activity in response to EPEC Phagocytosisincreased significantly in the presence of microsphere PEGadsorbed with plant extract for the normoglycemic groupA comparison of the groups showed that EPEC phagocy-tosis in general was similar for the diabetic and normo-glycemic groups (Fig 3(A))
35 The Effects of Microsphere PEG Adsorbed withPlant Extract on the Bacterial Activity of BloodPhagocytes
In general blood mononuclear phagocytes from the dia-betic group had low bactericidal activity against EPECMononuclear phagocytes from the normoglycemic groupincubated with plant extract or low density microspheresPEG adsorbed with or without plant extract had increasedmicrobicidal activity in response to EPEC (Fig 3(B))
Table II Superoxide release by blood mononuclear phagocytes(meanplusmnSD N = 10 in each treatment)
Superoxide release (nmol)
MN phagocytes Bacteria Control Diabetes
PBS No 59plusmn12 40plusmn14Yes 13plusmn004 113plusmn11d
Momordica charantia L No 125plusmn33c 104plusmn38c
Yes 46 plusmn 05c 138plusmn09d
HDPEGa No 61plusmn26 54plusmn12Yes 37plusmn15c 116plusmn25d
LDPEGb No 62plusmn23 44plusmn18Yes 19plusmn03 118plusmn32d
Momordica charantia L No 98plusmn27c 76plusmn24plus HDPEG
Yes 44plusmn03c 125plusmn07d
Momordica charantia L No 88plusmn17c 87plusmn15c
plus LDPEGYes 16plusmn02 131plusmn09d
aHigh Density Polyethylene Glycol bLow Density Polyethylene Glycol Bacteriawere treated with PEG (High or Low density) andor Momordica charantia LIn controls assays mononuclear cells were pre-incubated with PBS cIndicatesdifferences between each bacteria treatment and the control treatment within agroup (ANOVA P lt 005) d indicates intergroup differences within each treatment(ANOVA P lt 005)
4 DISCUSSION
In the present study we produced PEG microspheres ofdifferent densities adsorbed with extract from Momordicacharantia L and studied their in vitro effect on bacterial-killing activity and superoxide production in blood phago-cytes from diabetic individualsPolymer microspheres can be employed to deliver medi-
cation in a rate-controlled and sometimes targeted mannerMedication is released from a microsphere by drug leach-ing from the polymer or by degradation of the polymermatrix Because the rate of drug release is controlled bythese two factors it is important to understand the physi-cal and chemical properties of the releasing medium35 Inthis study analysis by fluorescence microscopy and flowcytometry showed PEG microspheres to be a regular sizeregardless of density and easily separated from the sus-pension The ability to form gradients in microspheres mayprove to be useful to achieve better control over the kinet-ics of protein release36 The literature have reported theuse of ow cytometry as an alternative method for analy-ses and visualization of particles37ndash38 Here we showed byflow cytometry that the PEG microsphere have a size ofabout 58 mPEG Microspheres are a kind of copolymer used in
clinical administration of drugs due to their incorporationcapacity36 Density gradients of signaling are importantin immunity39 In particular chemotaxis the preferentialmovement of cells up a density gradient of singlingmolecules is dependent on the gradient as opposed tothe average concentration of the molecule40 Here wehad found that the PEG microsphere independent ofthe density gradient adsorbed with plant extract pre-sented a potent immunostimulatory effect on the func-tional activity of blood phagocytes Cells obtained fromthe blood of normoglycemic individuals are known tokill enteropathogenic Escherichia coli in the presence ofherbal plants associated with peptides This interactionmay represent an alternative mechanism of defense againstinfection27
In this study we observed that the interaction ofPEG microspheres with blood phagocytes regardless ofglycemic status did not affect the viability of the cellsFurthermore superoxide release was higher when thephagocytes were in the presence of the PEG microspheresadsorbed with plant extract irrespective of glycemic statusThe phagocytes incubated with EPEC in the presence of
Sci Adv Mater 3 687ndash694 2011 691
Delivered by Ingenta toUniversity of Wollongong
IP 1301303785Mon 27 Feb 2012 185227
RESEARCH
ARTIC
LE
Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
(A)
(B)
Fig 3 Bacterial phagocytosis (A) and bactericidal activity index (B) by blood mononuclear cells (meanplusmnSD N = 10 in each treatment) Bacterialphagocytosis and elimation index by mononuclear cells from blood was determined with the acridine orange method lowastindicates differences betweentreatment (PEG orand plant) and 199 medium (ANOVA P lt 005) indicates intergroup differences for each treatment (ANOVA P lt 005) (HDPEG-Hight Density Polyethylene Glicol LDPEG-Low Density Polyethylene Glicol MOR ndash Momordica charantia L)
PEG microspheres adsorbed with plant extract had highersuperoxide release than phagocytes exposed only to thebacteria The activation ability of these cells indicated bysuperoxide release remained at the same level for both thehyperglycemic and control groupsA great variety of mechanisms have been suggested
for the formation of these free reactive oxygen radicals41
Glucose oxidation is believed to be the main source of freeradical production in diabetes42 Hyperglycemia has alsobeen found to promote lipid peroxidation by a superoxide-dependent pathway resulting in free radical generation andrelease43ndash44 Another important source of free radicals in
diabetes is the interaction of glucose with proteins lead-ing to the formation of advanced glycation end-productsthat contribute to an excessive burst in mitochondrial freeradicals45 The role of free radicals is an extremely impor-tant phenomenon during immune responses and inflam-matory reactions whereas the microbicidal activity ofphagocytes has been linked to the activation of cellularoxidative metabolism and release of large amounts of freeradicals46
On the other hand the high glucose levels of diabeticpatients can impair their antioxidant systems by decreasingimportant responses such as phagocytic and microbicidal
692 Sci Adv Mater 3 687ndash694 2011
Delivered by Ingenta toUniversity of Wollongong
IP 1301303785Mon 27 Feb 2012 185227
RESEARCH
ARTIC
LE
Scherer et al Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L
activity47ndash49 In this work the PEG microspheres adsorbedwith plant extract increased phagocytosis Similar resultswith respect to the functional activity of leukocytes wereobserved in phagocytes from patients with diabetes45 andfrom animals with induced diabetes50ndash52 Mononuclearphagocytes play an important role in host defense Theyproduce phagocytic NADPH oxidase which forms super-oxide and is imperative for killing bacterial53 and crucialfor the success of immune responses and inflammatoryreactions46
In our studies the elimination of EPEC by blood phago-cytes was stimulated only in the normoglycemic groupwhen these cells were incubated with plant extract or lowdensity PEG microspheres adsorbed with plant extractThe scientific literature has already reported the elimi-nation of bacteria by mononuclear phagocytes and theirmicrobicidal activity was stimulated in the presence ofimmunomodulatory agents17ndash5455
The high glucose levels impair a series of physiolog-ical processes and the functional activity seems to beaffected Although the PEG microspheres adsorbed withMomordica charantia L did not increase the microbici-dal activity of blood phagocytes from diabetes patientsthis polymer can be activated inside cells This studyoutlined the immunomodulatory effects of microspherepoly(ethylene glycol) adsorbed with Momordica charan-tia L on blood phagocytes This system may be usefulfor the delivery of a variety of therapeutic plants and wasshown to provide additional protection against infectionsin normoglycemic and hyperglycemic individuals Thesedata suggest that microspheres of poly(ethylene glycol)adsorbed with nanofractions of Momordica charantia Lrepresent a potential nanomaterial for future clinical appli-cations of diabetes
Acknowledgments This research received grants fromFundaccedilatildeo de Amparo agrave Pesquisa de Mato Grosso (FAPE-MAT No 7382642008 FAPEMAT No 2990322010 andNo 3016102010)
References and Notes
1 R M Ribeiro-Costa E C G Pereira J A Alves N P Santos S CNascimento N K Honda N H Silva and N S Santos-MagalhatildeesJ Microencap 21 371 (2004)
2 J Park M Ye and K Park Molecules 10 146 (2005)3 E A Scott M D Nichols R Kuntz-Willits and D L Elbert Acta
Biomat 6 29 (2010)4 R Duncan Nat Rev Drug Discov 2 346 (2003)5 K L Kiick Science 317 1182 (2007)6 T J A Recircgo Phytogeography of Medicinal Plants in the Maranhatildeo
2nd edn Edufma Satildeo Luiacutes MA Brazil (1995) pp 108ndash1097 J B Calixto Braz J Med Biol Res 32 179 (2000)8 S Hasani-Ranjbar B Larijani and M A Abdollah Arch Med Sci
4 285 (2008)9 R W Bussmann A G K Meyer A Kuhlma and A Townesmith
J Ethnobiol Ethnomed 6 2 (2010)
10 S Asgary S Parkhideh A Solhpour H Madani P Mahzouni andP Rahimi J Med Food 11 533 (2008)
11 A C Honorio-Franccedila C Marins F Boldrini and E L Franccedila ActaCir Braz 23 504 (2008)
12 American Dietetic Association J Am Diet Assoc 109 1926(2009)
13 T Miura T Itoh N Iwamoto M Kato and T Ishida Biol PharmaBull 27 248 (2004)
14 G V K Senanayake M Maruyama KShibuya M SakonoN Fukuda T Morishita C Yukizaki M Kawano and H OhtaJ Ethnopharmacol 91 257 (2004)
15 H Mundi B Bjoumlrksten C Svanborg L Ohman and C DahlgrenInfect Immun 59 4168 (1991)
16 N R Asad L M B O Assad C E B Almeida and A C LeitatildeoBr J Med Biol Res 27 2551 (1994)
17 A C Honorio-Franccedila M P S M Carvalho L Isaac L R Tra-bulsi and M M S Carneiro-Sampaio Scand J Immunol 46 59(1997)
18 A C Honorio-Franccedila P Launay M M S Carneiro-Sampaio andR C Monteiro J Leuk Biol 69 280 (2001)
19 E L Franccedila Jr A Pereira S L Oliveira and A C Honorio-Franccedila Intern J Microbiol 6 1 (2009)
20 J M McCord Am J Med 108 652 (2000)21 C Vorbach R Harrison and M R Capecchi Trend Immunol
24 512 (2003)22 B N Ames M K Shinnenaga and T M Hagen Proc Nat
Acad Sci 90 7915 (1993)23 C K B Ferrari and E A F S Torres Biomed Pharmacother
57 251 (2003)24 U Tiwari B Rastogi P Singh D K Saraf and S P Vyas
J Ethnopharmacol 92 113 (2004)25 C Viegas Jr V S Bolzani and E J Barreiro Quim Nova 29 326
(2006)26 B Patwardhan and G Manish Drug Discov Today 10 495
(2005)27 E L Franccedila J C Maynie V S C Correa U C R Pereira
C Batalini C K B Ferrari and A C Honorio-Franccedila Int J Phy-tomed 2 345 (2010)
28 S Mehrotra K P Mishra R Maurya R C Srimal V SYadav R Pandey and V K Singh Int Immunopharmacol 3 53(2003)
29 G Morceli E L Franccedila V B Magalhatildees D C Damasceno I MP Calderon and A C H Franccedila Acta Paediatr 100 550 (2011)
30 V S C Correcirca J C Maynieacute E L Franccedila and A C Honorio-Franccedila Braz J Med Plants 8 26 (2006)
31 E A Scott M D Nichols R Kuntz-Willits and D L Elbert ActaBiomat 6 29 (2010)
32 E Pick and D Mizel J Immunol Methods 46 211 (1981)33 R Bellinati-Pires M M Salgado I P Hypolito A S Grumach
and M M S Carneiro-Sampaio J Investig Allergol Clin Immunol5 337 (1995)
34 J L Roam H Xu P K Nguyen and D L Elbert Biomaterials31 8642 (2010)
35 S Freiberg and X X Zhu Int J Pharmaceut 282 1 (2004)36 R Gang and F Si-Shen Biomaterials 24 5037 (2003)37 H Ai J J Pink X T Shuai D A Boothman J M Gao J Biomed
Mater Res 73A 303 (2005)38 B Staumldler A D Price and A N Zelikin Adv Funct Mater 21 14
(2011)39 X Q Wang E Wenk X H Zhang L Mennel G Vunjak-
Novakovic and D L Kaplan J Control Releas 134 81 (2009)40 K Moore M Macsween and M Shoichet Tissue Eng 12 267
(2006)41 C K B Ferrari E L Franccedila and A C Honorio-Franccedila Applied
Biomed 7 163 (2009)42 Z Y Jiang A C Woollard and S P Wolff FEBS Lett 268 69
(1990)
Sci Adv Mater 3 687ndash694 2011 693
Delivered by Ingenta toUniversity of Wollongong
IP 1301303785Mon 27 Feb 2012 185227
RESEARCH
ARTIC
LE
Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
43 E C Tsai I B Hirsch J D Brunzell and A Chait Diabetes43 1010 (1994)
44 M Kawamura J W Heinecke and A Chait J Clin Invest 94 771(1994)
45 A C Maritim R A Sanders and B Watkins J Biochem Mol Tox-icol 17 24 (2003)
46 M Dizdaroglu P Jaruga M Birincioglu and H Rodriguez FreeRad Biol Med 32 1102 (2002)
47 A J Rassias C A Marrin J Arruda P K Whalen M Beach andM P Yeager Anesth Analg 88 1011 (1999)
48 J Jakelic S Kokic I Hozo J Maras and D Fabijanic Med Arh49 9 (1995)
49 B Larijani P Shooshtarizadeh N Mosaffa and R Heshmat Br JBiomed Sci 64 13 (2007)
50 N Seyrek W Marcinkowski M Smogorzewski T M Demerdashand S G Massry Nephrol Dial Transplant 12 265 (1997)
51 E L Franccedila N D Feliciano K A Silva C K B Ferrari andA C Honorio-Franccedila Bratisl Med J 110 517 (2009)
52 A C Honorio-Franccedila K A Silva N D Feliciano I MP Calderon M V C Rudge and E L Franccedila Int J DiabetesMetabol 17 87 (2009)
53 M Djaldetti H Salman M Bergman Djaldetti and H BesslerMicro Res Techn 57 421 (2002)
54 E L Franccedila R V Bitencourt M Fujimori T C Morais I M PCalderon and A C Honorio-Franccedila J Microbiol Immunol Infect44 1 (2011)
55 E L Franccedila G Morceli D L G Fagundes M V C Rugde I MP Calderon and A C Honorio-Franccedila APMIS 119 710 (2011)
Received 1 August 2011 Accepted 18 September 2011
694 Sci Adv Mater 3 687ndash694 2011
Delivered by Ingenta toUniversity of Wollongong
IP 1301303785Mon 27 Feb 2012 185227
RESEARCH
ARTIC
LE
Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
(A)
(B)
Fig 3 Bacterial phagocytosis (A) and bactericidal activity index (B) by blood mononuclear cells (meanplusmnSD N = 10 in each treatment) Bacterialphagocytosis and elimation index by mononuclear cells from blood was determined with the acridine orange method lowastindicates differences betweentreatment (PEG orand plant) and 199 medium (ANOVA P lt 005) indicates intergroup differences for each treatment (ANOVA P lt 005) (HDPEG-Hight Density Polyethylene Glicol LDPEG-Low Density Polyethylene Glicol MOR ndash Momordica charantia L)
PEG microspheres adsorbed with plant extract had highersuperoxide release than phagocytes exposed only to thebacteria The activation ability of these cells indicated bysuperoxide release remained at the same level for both thehyperglycemic and control groupsA great variety of mechanisms have been suggested
for the formation of these free reactive oxygen radicals41
Glucose oxidation is believed to be the main source of freeradical production in diabetes42 Hyperglycemia has alsobeen found to promote lipid peroxidation by a superoxide-dependent pathway resulting in free radical generation andrelease43ndash44 Another important source of free radicals in
diabetes is the interaction of glucose with proteins lead-ing to the formation of advanced glycation end-productsthat contribute to an excessive burst in mitochondrial freeradicals45 The role of free radicals is an extremely impor-tant phenomenon during immune responses and inflam-matory reactions whereas the microbicidal activity ofphagocytes has been linked to the activation of cellularoxidative metabolism and release of large amounts of freeradicals46
On the other hand the high glucose levels of diabeticpatients can impair their antioxidant systems by decreasingimportant responses such as phagocytic and microbicidal
692 Sci Adv Mater 3 687ndash694 2011
Delivered by Ingenta toUniversity of Wollongong
IP 1301303785Mon 27 Feb 2012 185227
RESEARCH
ARTIC
LE
Scherer et al Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L
activity47ndash49 In this work the PEG microspheres adsorbedwith plant extract increased phagocytosis Similar resultswith respect to the functional activity of leukocytes wereobserved in phagocytes from patients with diabetes45 andfrom animals with induced diabetes50ndash52 Mononuclearphagocytes play an important role in host defense Theyproduce phagocytic NADPH oxidase which forms super-oxide and is imperative for killing bacterial53 and crucialfor the success of immune responses and inflammatoryreactions46
In our studies the elimination of EPEC by blood phago-cytes was stimulated only in the normoglycemic groupwhen these cells were incubated with plant extract or lowdensity PEG microspheres adsorbed with plant extractThe scientific literature has already reported the elimi-nation of bacteria by mononuclear phagocytes and theirmicrobicidal activity was stimulated in the presence ofimmunomodulatory agents17ndash5455
The high glucose levels impair a series of physiolog-ical processes and the functional activity seems to beaffected Although the PEG microspheres adsorbed withMomordica charantia L did not increase the microbici-dal activity of blood phagocytes from diabetes patientsthis polymer can be activated inside cells This studyoutlined the immunomodulatory effects of microspherepoly(ethylene glycol) adsorbed with Momordica charan-tia L on blood phagocytes This system may be usefulfor the delivery of a variety of therapeutic plants and wasshown to provide additional protection against infectionsin normoglycemic and hyperglycemic individuals Thesedata suggest that microspheres of poly(ethylene glycol)adsorbed with nanofractions of Momordica charantia Lrepresent a potential nanomaterial for future clinical appli-cations of diabetes
Acknowledgments This research received grants fromFundaccedilatildeo de Amparo agrave Pesquisa de Mato Grosso (FAPE-MAT No 7382642008 FAPEMAT No 2990322010 andNo 3016102010)
References and Notes
1 R M Ribeiro-Costa E C G Pereira J A Alves N P Santos S CNascimento N K Honda N H Silva and N S Santos-MagalhatildeesJ Microencap 21 371 (2004)
2 J Park M Ye and K Park Molecules 10 146 (2005)3 E A Scott M D Nichols R Kuntz-Willits and D L Elbert Acta
Biomat 6 29 (2010)4 R Duncan Nat Rev Drug Discov 2 346 (2003)5 K L Kiick Science 317 1182 (2007)6 T J A Recircgo Phytogeography of Medicinal Plants in the Maranhatildeo
2nd edn Edufma Satildeo Luiacutes MA Brazil (1995) pp 108ndash1097 J B Calixto Braz J Med Biol Res 32 179 (2000)8 S Hasani-Ranjbar B Larijani and M A Abdollah Arch Med Sci
4 285 (2008)9 R W Bussmann A G K Meyer A Kuhlma and A Townesmith
J Ethnobiol Ethnomed 6 2 (2010)
10 S Asgary S Parkhideh A Solhpour H Madani P Mahzouni andP Rahimi J Med Food 11 533 (2008)
11 A C Honorio-Franccedila C Marins F Boldrini and E L Franccedila ActaCir Braz 23 504 (2008)
12 American Dietetic Association J Am Diet Assoc 109 1926(2009)
13 T Miura T Itoh N Iwamoto M Kato and T Ishida Biol PharmaBull 27 248 (2004)
14 G V K Senanayake M Maruyama KShibuya M SakonoN Fukuda T Morishita C Yukizaki M Kawano and H OhtaJ Ethnopharmacol 91 257 (2004)
15 H Mundi B Bjoumlrksten C Svanborg L Ohman and C DahlgrenInfect Immun 59 4168 (1991)
16 N R Asad L M B O Assad C E B Almeida and A C LeitatildeoBr J Med Biol Res 27 2551 (1994)
17 A C Honorio-Franccedila M P S M Carvalho L Isaac L R Tra-bulsi and M M S Carneiro-Sampaio Scand J Immunol 46 59(1997)
18 A C Honorio-Franccedila P Launay M M S Carneiro-Sampaio andR C Monteiro J Leuk Biol 69 280 (2001)
19 E L Franccedila Jr A Pereira S L Oliveira and A C Honorio-Franccedila Intern J Microbiol 6 1 (2009)
20 J M McCord Am J Med 108 652 (2000)21 C Vorbach R Harrison and M R Capecchi Trend Immunol
24 512 (2003)22 B N Ames M K Shinnenaga and T M Hagen Proc Nat
Acad Sci 90 7915 (1993)23 C K B Ferrari and E A F S Torres Biomed Pharmacother
57 251 (2003)24 U Tiwari B Rastogi P Singh D K Saraf and S P Vyas
J Ethnopharmacol 92 113 (2004)25 C Viegas Jr V S Bolzani and E J Barreiro Quim Nova 29 326
(2006)26 B Patwardhan and G Manish Drug Discov Today 10 495
(2005)27 E L Franccedila J C Maynie V S C Correa U C R Pereira
C Batalini C K B Ferrari and A C Honorio-Franccedila Int J Phy-tomed 2 345 (2010)
28 S Mehrotra K P Mishra R Maurya R C Srimal V SYadav R Pandey and V K Singh Int Immunopharmacol 3 53(2003)
29 G Morceli E L Franccedila V B Magalhatildees D C Damasceno I MP Calderon and A C H Franccedila Acta Paediatr 100 550 (2011)
30 V S C Correcirca J C Maynieacute E L Franccedila and A C Honorio-Franccedila Braz J Med Plants 8 26 (2006)
31 E A Scott M D Nichols R Kuntz-Willits and D L Elbert ActaBiomat 6 29 (2010)
32 E Pick and D Mizel J Immunol Methods 46 211 (1981)33 R Bellinati-Pires M M Salgado I P Hypolito A S Grumach
and M M S Carneiro-Sampaio J Investig Allergol Clin Immunol5 337 (1995)
34 J L Roam H Xu P K Nguyen and D L Elbert Biomaterials31 8642 (2010)
35 S Freiberg and X X Zhu Int J Pharmaceut 282 1 (2004)36 R Gang and F Si-Shen Biomaterials 24 5037 (2003)37 H Ai J J Pink X T Shuai D A Boothman J M Gao J Biomed
Mater Res 73A 303 (2005)38 B Staumldler A D Price and A N Zelikin Adv Funct Mater 21 14
(2011)39 X Q Wang E Wenk X H Zhang L Mennel G Vunjak-
Novakovic and D L Kaplan J Control Releas 134 81 (2009)40 K Moore M Macsween and M Shoichet Tissue Eng 12 267
(2006)41 C K B Ferrari E L Franccedila and A C Honorio-Franccedila Applied
Biomed 7 163 (2009)42 Z Y Jiang A C Woollard and S P Wolff FEBS Lett 268 69
(1990)
Sci Adv Mater 3 687ndash694 2011 693
Delivered by Ingenta toUniversity of Wollongong
IP 1301303785Mon 27 Feb 2012 185227
RESEARCH
ARTIC
LE
Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
43 E C Tsai I B Hirsch J D Brunzell and A Chait Diabetes43 1010 (1994)
44 M Kawamura J W Heinecke and A Chait J Clin Invest 94 771(1994)
45 A C Maritim R A Sanders and B Watkins J Biochem Mol Tox-icol 17 24 (2003)
46 M Dizdaroglu P Jaruga M Birincioglu and H Rodriguez FreeRad Biol Med 32 1102 (2002)
47 A J Rassias C A Marrin J Arruda P K Whalen M Beach andM P Yeager Anesth Analg 88 1011 (1999)
48 J Jakelic S Kokic I Hozo J Maras and D Fabijanic Med Arh49 9 (1995)
49 B Larijani P Shooshtarizadeh N Mosaffa and R Heshmat Br JBiomed Sci 64 13 (2007)
50 N Seyrek W Marcinkowski M Smogorzewski T M Demerdashand S G Massry Nephrol Dial Transplant 12 265 (1997)
51 E L Franccedila N D Feliciano K A Silva C K B Ferrari andA C Honorio-Franccedila Bratisl Med J 110 517 (2009)
52 A C Honorio-Franccedila K A Silva N D Feliciano I MP Calderon M V C Rudge and E L Franccedila Int J DiabetesMetabol 17 87 (2009)
53 M Djaldetti H Salman M Bergman Djaldetti and H BesslerMicro Res Techn 57 421 (2002)
54 E L Franccedila R V Bitencourt M Fujimori T C Morais I M PCalderon and A C Honorio-Franccedila J Microbiol Immunol Infect44 1 (2011)
55 E L Franccedila G Morceli D L G Fagundes M V C Rugde I MP Calderon and A C Honorio-Franccedila APMIS 119 710 (2011)
Received 1 August 2011 Accepted 18 September 2011
694 Sci Adv Mater 3 687ndash694 2011
Delivered by Ingenta toUniversity of Wollongong
IP 1301303785Mon 27 Feb 2012 185227
RESEARCH
ARTIC
LE
Scherer et al Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L
activity47ndash49 In this work the PEG microspheres adsorbedwith plant extract increased phagocytosis Similar resultswith respect to the functional activity of leukocytes wereobserved in phagocytes from patients with diabetes45 andfrom animals with induced diabetes50ndash52 Mononuclearphagocytes play an important role in host defense Theyproduce phagocytic NADPH oxidase which forms super-oxide and is imperative for killing bacterial53 and crucialfor the success of immune responses and inflammatoryreactions46
In our studies the elimination of EPEC by blood phago-cytes was stimulated only in the normoglycemic groupwhen these cells were incubated with plant extract or lowdensity PEG microspheres adsorbed with plant extractThe scientific literature has already reported the elimi-nation of bacteria by mononuclear phagocytes and theirmicrobicidal activity was stimulated in the presence ofimmunomodulatory agents17ndash5455
The high glucose levels impair a series of physiolog-ical processes and the functional activity seems to beaffected Although the PEG microspheres adsorbed withMomordica charantia L did not increase the microbici-dal activity of blood phagocytes from diabetes patientsthis polymer can be activated inside cells This studyoutlined the immunomodulatory effects of microspherepoly(ethylene glycol) adsorbed with Momordica charan-tia L on blood phagocytes This system may be usefulfor the delivery of a variety of therapeutic plants and wasshown to provide additional protection against infectionsin normoglycemic and hyperglycemic individuals Thesedata suggest that microspheres of poly(ethylene glycol)adsorbed with nanofractions of Momordica charantia Lrepresent a potential nanomaterial for future clinical appli-cations of diabetes
Acknowledgments This research received grants fromFundaccedilatildeo de Amparo agrave Pesquisa de Mato Grosso (FAPE-MAT No 7382642008 FAPEMAT No 2990322010 andNo 3016102010)
References and Notes
1 R M Ribeiro-Costa E C G Pereira J A Alves N P Santos S CNascimento N K Honda N H Silva and N S Santos-MagalhatildeesJ Microencap 21 371 (2004)
2 J Park M Ye and K Park Molecules 10 146 (2005)3 E A Scott M D Nichols R Kuntz-Willits and D L Elbert Acta
Biomat 6 29 (2010)4 R Duncan Nat Rev Drug Discov 2 346 (2003)5 K L Kiick Science 317 1182 (2007)6 T J A Recircgo Phytogeography of Medicinal Plants in the Maranhatildeo
2nd edn Edufma Satildeo Luiacutes MA Brazil (1995) pp 108ndash1097 J B Calixto Braz J Med Biol Res 32 179 (2000)8 S Hasani-Ranjbar B Larijani and M A Abdollah Arch Med Sci
4 285 (2008)9 R W Bussmann A G K Meyer A Kuhlma and A Townesmith
J Ethnobiol Ethnomed 6 2 (2010)
10 S Asgary S Parkhideh A Solhpour H Madani P Mahzouni andP Rahimi J Med Food 11 533 (2008)
11 A C Honorio-Franccedila C Marins F Boldrini and E L Franccedila ActaCir Braz 23 504 (2008)
12 American Dietetic Association J Am Diet Assoc 109 1926(2009)
13 T Miura T Itoh N Iwamoto M Kato and T Ishida Biol PharmaBull 27 248 (2004)
14 G V K Senanayake M Maruyama KShibuya M SakonoN Fukuda T Morishita C Yukizaki M Kawano and H OhtaJ Ethnopharmacol 91 257 (2004)
15 H Mundi B Bjoumlrksten C Svanborg L Ohman and C DahlgrenInfect Immun 59 4168 (1991)
16 N R Asad L M B O Assad C E B Almeida and A C LeitatildeoBr J Med Biol Res 27 2551 (1994)
17 A C Honorio-Franccedila M P S M Carvalho L Isaac L R Tra-bulsi and M M S Carneiro-Sampaio Scand J Immunol 46 59(1997)
18 A C Honorio-Franccedila P Launay M M S Carneiro-Sampaio andR C Monteiro J Leuk Biol 69 280 (2001)
19 E L Franccedila Jr A Pereira S L Oliveira and A C Honorio-Franccedila Intern J Microbiol 6 1 (2009)
20 J M McCord Am J Med 108 652 (2000)21 C Vorbach R Harrison and M R Capecchi Trend Immunol
24 512 (2003)22 B N Ames M K Shinnenaga and T M Hagen Proc Nat
Acad Sci 90 7915 (1993)23 C K B Ferrari and E A F S Torres Biomed Pharmacother
57 251 (2003)24 U Tiwari B Rastogi P Singh D K Saraf and S P Vyas
J Ethnopharmacol 92 113 (2004)25 C Viegas Jr V S Bolzani and E J Barreiro Quim Nova 29 326
(2006)26 B Patwardhan and G Manish Drug Discov Today 10 495
(2005)27 E L Franccedila J C Maynie V S C Correa U C R Pereira
C Batalini C K B Ferrari and A C Honorio-Franccedila Int J Phy-tomed 2 345 (2010)
28 S Mehrotra K P Mishra R Maurya R C Srimal V SYadav R Pandey and V K Singh Int Immunopharmacol 3 53(2003)
29 G Morceli E L Franccedila V B Magalhatildees D C Damasceno I MP Calderon and A C H Franccedila Acta Paediatr 100 550 (2011)
30 V S C Correcirca J C Maynieacute E L Franccedila and A C Honorio-Franccedila Braz J Med Plants 8 26 (2006)
31 E A Scott M D Nichols R Kuntz-Willits and D L Elbert ActaBiomat 6 29 (2010)
32 E Pick and D Mizel J Immunol Methods 46 211 (1981)33 R Bellinati-Pires M M Salgado I P Hypolito A S Grumach
and M M S Carneiro-Sampaio J Investig Allergol Clin Immunol5 337 (1995)
34 J L Roam H Xu P K Nguyen and D L Elbert Biomaterials31 8642 (2010)
35 S Freiberg and X X Zhu Int J Pharmaceut 282 1 (2004)36 R Gang and F Si-Shen Biomaterials 24 5037 (2003)37 H Ai J J Pink X T Shuai D A Boothman J M Gao J Biomed
Mater Res 73A 303 (2005)38 B Staumldler A D Price and A N Zelikin Adv Funct Mater 21 14
(2011)39 X Q Wang E Wenk X H Zhang L Mennel G Vunjak-
Novakovic and D L Kaplan J Control Releas 134 81 (2009)40 K Moore M Macsween and M Shoichet Tissue Eng 12 267
(2006)41 C K B Ferrari E L Franccedila and A C Honorio-Franccedila Applied
Biomed 7 163 (2009)42 Z Y Jiang A C Woollard and S P Wolff FEBS Lett 268 69
(1990)
Sci Adv Mater 3 687ndash694 2011 693
Delivered by Ingenta toUniversity of Wollongong
IP 1301303785Mon 27 Feb 2012 185227
RESEARCH
ARTIC
LE
Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
43 E C Tsai I B Hirsch J D Brunzell and A Chait Diabetes43 1010 (1994)
44 M Kawamura J W Heinecke and A Chait J Clin Invest 94 771(1994)
45 A C Maritim R A Sanders and B Watkins J Biochem Mol Tox-icol 17 24 (2003)
46 M Dizdaroglu P Jaruga M Birincioglu and H Rodriguez FreeRad Biol Med 32 1102 (2002)
47 A J Rassias C A Marrin J Arruda P K Whalen M Beach andM P Yeager Anesth Analg 88 1011 (1999)
48 J Jakelic S Kokic I Hozo J Maras and D Fabijanic Med Arh49 9 (1995)
49 B Larijani P Shooshtarizadeh N Mosaffa and R Heshmat Br JBiomed Sci 64 13 (2007)
50 N Seyrek W Marcinkowski M Smogorzewski T M Demerdashand S G Massry Nephrol Dial Transplant 12 265 (1997)
51 E L Franccedila N D Feliciano K A Silva C K B Ferrari andA C Honorio-Franccedila Bratisl Med J 110 517 (2009)
52 A C Honorio-Franccedila K A Silva N D Feliciano I MP Calderon M V C Rudge and E L Franccedila Int J DiabetesMetabol 17 87 (2009)
53 M Djaldetti H Salman M Bergman Djaldetti and H BesslerMicro Res Techn 57 421 (2002)
54 E L Franccedila R V Bitencourt M Fujimori T C Morais I M PCalderon and A C Honorio-Franccedila J Microbiol Immunol Infect44 1 (2011)
55 E L Franccedila G Morceli D L G Fagundes M V C Rugde I MP Calderon and A C Honorio-Franccedila APMIS 119 710 (2011)
Received 1 August 2011 Accepted 18 September 2011
694 Sci Adv Mater 3 687ndash694 2011
Delivered by Ingenta toUniversity of Wollongong
IP 1301303785Mon 27 Feb 2012 185227
RESEARCH
ARTIC
LE
Immunomodulatory Effects of PEG Microspheres Adsorbed with Nanofractions of Momordica charantia L Scherer et al
43 E C Tsai I B Hirsch J D Brunzell and A Chait Diabetes43 1010 (1994)
44 M Kawamura J W Heinecke and A Chait J Clin Invest 94 771(1994)
45 A C Maritim R A Sanders and B Watkins J Biochem Mol Tox-icol 17 24 (2003)
46 M Dizdaroglu P Jaruga M Birincioglu and H Rodriguez FreeRad Biol Med 32 1102 (2002)
47 A J Rassias C A Marrin J Arruda P K Whalen M Beach andM P Yeager Anesth Analg 88 1011 (1999)
48 J Jakelic S Kokic I Hozo J Maras and D Fabijanic Med Arh49 9 (1995)
49 B Larijani P Shooshtarizadeh N Mosaffa and R Heshmat Br JBiomed Sci 64 13 (2007)
50 N Seyrek W Marcinkowski M Smogorzewski T M Demerdashand S G Massry Nephrol Dial Transplant 12 265 (1997)
51 E L Franccedila N D Feliciano K A Silva C K B Ferrari andA C Honorio-Franccedila Bratisl Med J 110 517 (2009)
52 A C Honorio-Franccedila K A Silva N D Feliciano I MP Calderon M V C Rudge and E L Franccedila Int J DiabetesMetabol 17 87 (2009)
53 M Djaldetti H Salman M Bergman Djaldetti and H BesslerMicro Res Techn 57 421 (2002)
54 E L Franccedila R V Bitencourt M Fujimori T C Morais I M PCalderon and A C Honorio-Franccedila J Microbiol Immunol Infect44 1 (2011)
55 E L Franccedila G Morceli D L G Fagundes M V C Rugde I MP Calderon and A C Honorio-Franccedila APMIS 119 710 (2011)
