Journal of Microencapsulation, December 2008; 25(8): 584–592

An approach to the design of a particulate system for oral protein delivery. I.

In vitro stability of various poly (a-hydroxy acids)-microspheres in simulated

gastrointestinal fluids

NASTARAN NAFISSI-VARCHEH1, MOHAMMAD ERFAN2, & REZA ABOOFAZELI1,2

1Pharmaceutical Sciences Research Center (PSRC), 2Department of Pharmaceutics, School of Pharmacy,

Shaheed Beheshti University of Medical Sciences, Tehran, Iran

(Received 31 May 2007; accepted 22 October 2007)

AbstractThe stability of various biodegradable polyester polymers with different molecular weights and lactic/glycolic acids ratios wereevaluated in simulated gastrointestinal fluids as an approach to apply microparticles for oral protein delivery on the basis ofparticle uptake mechanism. A common w/o/w emulsion solvent evaporation technique using dichloromethane for dissolving thepolymer and polyvinyl alcohol as the stabilizer was used for encapsulation. Microspheres were incubated at 37�C in USPsimulated fluids with a concentration of 20mgmL�1 and also in the literature, which suggested fed or fasted simulated intestinalfluids for different times up to 24 h, while shaking at 75 rpm. The stability assessment was done by detecting pH alterations of themedia, enzymatic assay of L-lactic acid, performing differential scanning calorimetric studies and observing the size andmorphology of particles. Results showed that the three polymers, namely Resomers� R207, RG756 and RG505, could besuitable for the preparation of protein-loaded microspheres.

Keywords: Poly (lactide co-glycolide), poly (lactic acid), simulated gastrointestinal fluids, microspheres, encapsulation, emulsionsolvent evaporation

Introduction

Recent rapid developments in biotechnology and

genetic engineering have led to the availability of a

large number of natural peptides and proteins as

therapeutic or prophylactic tools. For chronic treat-

ment, the oral route is the most desirable method of

drug administration. However, specific characteristics

of peptides and proteins (e.g. hydrophilic nature,

complex structure and insufficient stability in gastro-

intestinal tract [GIT]) are the major obstacles

(Allemann et al. 1998; Sinko et al. 2002). Therefore,

the design of particulate systems has received increasing

attention for oral delivery of these biomolecules (Dai

et al. 2005). Incorporation of protein drugs in particles

should at least protect them against degradation

induced by the substances and harsh conditions of

GIT and, in some cases, also improve their absorption

(Chen and Langer 1998).

Uptake of particulate materials across the intact GITand their passage into the blood and lymph were firstreported in 1844 by observing starch granules (3–50mm)in the blood and lymph vessels of dogs, 3 h afteradministration (Lavelle et al. 1995; Hussain et al. 2001).Intestinal uptake of particles is now a widely acceptedphenomenon, but there is still controversy about theuptake mechanism and extent (Delie and Blanco-Prieto2005). which could occur transcellularly and toa lesser extent through paracellular pathways orpersorption (Florence 1997; Chen and Langer 1998;Delie 1998; McClean et al. 1998; Noris et al. 1998;Yeh et al. 1998).Particle size is reported as an important factor in the

absorption of particles, but the optimum particle sizerange is still a matter for conjecture (Jani et al. 1990;Desai et al. 1997; Florence 1997; Noris et al. 1998;Yeh et al. 1998; Delie and Blanco-Prieto 2005).Depending on the gut tissues and the involved

Correspondence: Reza Aboofazeli, PhD, Department of Pharmaceutics, School of Pharmacy, Shaheed Beheshti University of Medical Sciences and Health Services,

Tehran, Iran. Fax: þ98 21 8879 5008. E-mail: raboofazeli@sbmu.ac.ir

ISSN 0265–2048 print/ISSN 1464–5246 online � 2008 Informa UK Ltd.

DOI: 10.1080/02652040802485485

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uptake mechanism, the actual upper size limits

are different. Some investigators have reported a limit

of 10 mm for transcellular uptake (Rafati et al. 1997;

McClean et al. 1998).Numerous papers reported the complexity of param-

eters involved in particle uptake. The importance of

nature and physicochemical properties of the

particles have been demonstrated (Noris et al. 1998;

Delie et al. 2005). In this regard, the favoured

polymers for oral peptide and protein delivery on the

basis of particle uptake mechanism are presently

aliphatic polyesters. Various poly (lactic acid)

(PLA) and its copolymers with glycolic acid (PLGA)

with different characteristics are produced by altering

a number of parameters such as the molecular weight

or the monomers ratio (Anderson and Shive 1997).

These polymers are biodegradable, biocompatible

and also approved by the US Food and Drug

Administration. which make them desirable polymers

for use in drug delivery applications (Vert et al. 1998).

However, more work should be done in order

to make the practical utility of the polymeric

particles for oral protein delivery possible. Obtaining

therapeutically worthwhile levels of a drug following

the administration of an oral particulate system

is restricted by two major obstacles including the

instability of the delivery systems in the GIT and

the low intestinal uptake of particles (Reich 1997;

Tobio 2000).The main aim of the current research was to design

and evaluate a particulate system for oral protein

delivery. As the preliminary step, the in vitro stability of

microspheres prepared with various PLA and PLGA

polymers were monitored and compared in different

simulated gastrointestinal fluids.

Materials

PLGA, 50 : 50 (Resomer� RG502H [inherent viscosity

(I.V.)¼ 0.2 dl g�1], RG504 [I.V.¼ 0.5 dl g�1], RG505

[I.V.¼ 0.7 dl g�1]); PLGA, 75 : 25 (Resomer� RG756,

I.V.¼ 0.8 dl g�1) and PLA (Resomer� R207

I.V.¼ 1.5 dl g�1) were obtained from Boehringer

Ingelheim Co. (Germany). It is noticeable that all

polymers contained a racemic mixture of D- and

L- lactic acid in their structures. Polyvinyl alcohol

(Mowiol�, 8–88) was a gift from Kuray Co. (Germany).

Pepsin and pancreatin were purchased from Sigma Co.

(USA). Lecithin was provided from Lucas Meyer Co.

(Germany). Lactate diagnostic kit and sodium taur-

ocholate were obtained from Randox laboratories

(UK) and Difco laboratories (USA), respectively. All

other chemicals were obtained from Merck Co.

(Germany). Water was purified with a Millipore

system (Millipore Corp., USA).

Methods

Preparation of blank microspheres

PLGA or PLA microspheres were all prepared by anoptimized double emulsion method developed in thelaboratory (data not published). Briefly, 100mg ofpolymer was dissolved in 2ml dichloromethane and200 ml water was then added to this solution. The twophases were emulsified together to make a primarywater-in-oil (w/o) emulsion through homogenization at26 000 rpm for 60 s using a Diax 900 homogenizer(Heidolph Co., Germany). The primary emulsion wasthen poured at once into 60ml of 5% w/v aqueouspolyvinyl alcohol solution (cooled to 15�C), underconstant stirring (1300 rpm) by a propeller stirrer(RZR50, Heidolph Co., Germany), to make a water-in-oil-in-water (w/o/w) emulsion. After 2min agitation,the resultant double emulsion was gradually transferred(within 2–3min) into 600ml of 1%w/v pre-chilled (4�C)aqueous polyvinyl alcohol solution. The mixture wasshaken magnetically (at 300 rpm) for 7 h, while thetemperature was maintained at 4–8�C to allow thesolvent to be evaporated. The resulting microsphereswere harvested and cleaned by filtration and washedwith 500ml distilled water. Finally, the microsphereswere dried under reduced pressure within 24 h andstored in a desiccator.

Preparation of simulated gastrointestinal fluids

Six different media were selected for evaluating thein vitro stability of polymeric microspheres in GIT(Table I). All media were prepared freshly before use.NaOH or HCl was applied for the pH adjustment, asneeded.

Incubation of microspheres in simulated fluids

Pre-weighted microspheres (20mg) were transferred to1.5-ml microcentrifuge tubes and incubated in 1ml ofthe simulated medium at 37�C using an orbitalincubator (SI 150, Stuart Scientific Co., UK), whileshaking at 75 rpm.

Stability assessment

Following incubation, samples were centrifuged at10 000 rpm for 10min by a microcentrifuge (5415C,Eppendorf Co., Germany). The supernatant was thenremoved and analysed directly for the pH determina-tion or lactate detection. The precipitant part was driedin a vacuum desiccator and used for morphologicalstudies or DSC measurements.

pH determination

Any pH changes of the media were monitored intriplicate after 24 h incubation of microspheres.

An approach to the design of a particulate system for oral protein delivery 585

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L-lactic acid assay

Polymer degradation, following 0.5, 6, 12 or 24 hincubation in various media was determined by lactate

detection in bulk solution using an enzymatic kit.Briefly, 2 ml of each standard solution or test samplewas transferred to a microplate well. Water orsimulated fluids were selected as blank samples, as

necessary. Kit reagent (200ml) was rapidly added to allwells and after shaking and incubation for 10min atroom temperature, the absorbance was measured at550 nm using a microplate reader (Thermo Spectra

Rainbow, Tecan Co., Austria).

Morphological studies

The size and morphological characteristics of micro-spheres were examined by light or scanning electron

microscopy (SEM) before and after incubation. ForSEM studies, the microspheres were mounted ontoaluminum stubs, vacuum-coated with a gold film usinga sputter coater (SCDOOS, Bal-TEC Co., Switzerland)

and directly analysed by scanning electron microscope(XL30, Philips Co., Netherlands).

Thermal analysis

The glass transition temperature (Tg) of the rawpolymer and microspheres before and after 24 h

incubation were measured using a differential scanningcalorimeter (DSC-60, Shimadzu Co., Japan). Thesamples (�5mg) were loaded in aluminium crimpcells. The scanning speed was 10�Cmin�1 within the

temperature rage of 20–100�C. In order to consider anyprobable interference, thermal analyses were similarlyperformed for each individual organic material appliedfor microsphere fabrication or simulated fluids pre-

paration. The DSC instrument was calibrated dailyusing the melt of indium while heating from 20 to 200�Cat 20�C min�1.

Results and discussion

The aim of the present investigation was to study the

stability of some polyester polymers to assess and

compare their suitability for the design of a protein

delivery system. Blank macrospheres were prepared by

an emulsification-solvent evaporation technique using

various types of Resomers� including R207, RG502H,

RG504, RG505 and RG756. Considering the size effect

on the GI particle uptake, the applied method for

microspheres fabrication was optimized to prepare

particles less than 5 mm.The obtained microspheres were incubated in several

simulated gastric or intestinal fluids to mimic the

normal conditions of GIT. The potential of an

enzymatic degradation (due to the presence of pancrea-

tin or pepsin) vs a plain hydrolytic process was studied

using USP simulated fluids prepared with and without

these enzymes. Fasted state simulated intestinal fluid

(FaSSIF) and fed state simulated intestinal fluid

(FeSSIF) were applied to investigate the bile salts

effects and evaluate the influence of simulated fasted or

fed states on the microspheres stability.The microspheres stability studies were performed

for various time periods up to 24 h. Although forparticles less than 1mm, the maximum reported gastric

residence time (GRT) and small intestinal transit time

(SITT) were reported �2.5 and 5 h, respectively (Qiu

and Zhang 2000), in the present work more longer

incubation times were selected, taking into account the

possible in vivo transit delay.

Table I. Simulated media for in vitro stability studies of polymeric microspheres in GIT.

Medium code Description pH Ref.

SGF-a Simulated gastric fluid with pepsin 1.2 (United States Pharmacopeia 27 2005)

SGF-b Simulated gastric fluid without pepsin 1.2 (United States Pharmacopeia 27 2005)

SIF-a Simulated intestinal fluid with pancreatin 6.8 (United States Pharmacopeia 27 2005)

SIF-b Simulated intestinal fluid without pancreatin 6.8 (United States Pharmacopeia 27 2005)

FaSSIF� Fasted state simulated intestinal fluid 6.5 (Charman et al. 1997)

FeSSIFy Fed state simulated intestinal fluid 5 (Charman et al. 1997)

*For FaSSIF, 78mg monobasic potassium phosphate, 26mg sodium taurocholate, 10mg lecithin, 154mg potassiumchloride and 180ml of 1N sodium hydroxide solution were mixed and the volume was then adjusted to 20ml withdistilled water.yEach 20ml of FeSSIF was prepared by dissolving 166ml glacial acetic acid, 240mg sodium taurocholate,52mg lecithin, 304mg potassium chloride and 2.25ml of 1N sodium hydroxide solution in sufficientdistilled water.

Table II. pH loss in USP simulated intestinal fluids after incubation

of microspheres for 24 h at 37�C while shaking at 75 rpm (n¼ 3).

Polymer applied for microsphere fabrication

Medium R207 RG756 RG505 RG504 RG502H

SIF-a 0 0.05 0.08 0.2 0.98

SIF-b 0 0.05 0.1 0.19 0.34

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Detection of pH alteration

Dramatic drop in the pH, which could be caused by theformation of lactic and glycolic acids as a result ofpolymer degradation, is suggested in the literature as amethod for the degradation study of these kinds ofpolymers (Kiss and Vargha-Butler 1999). In this study,the pH changes of various simulated gastrointestinalfluids after the completion of the 24 h incubation ofmicrospheres were applied as a criterion of polymerinstability.The results showed no significant differences in the

pH of simulated gastric fluid with pepsin (SGF-a) andsimulated gastric fluid without pepsin (SGF-b). Thestability of PLA after 8 h exposure to simulated gastricfluids has been reported previously (Landry et al. 1996).However, in these cases data interpretation is not easybecause of high inherent acidity of the gastric media.pH drop of FaSSIF medium was 0.03, 0.04 and 0.27units after 24 h incubation of RG505, RG504 andRG502H microspheres, respectively. No pH alterationof FaSSIF was observed following the incubation ofRG756 and R207 microspheres for the same time.It should be noted that pH of FeSSIF did not changewithin 24 h incubation of the microspheres.Significant pH alterations of simulated intestinal

fluids were observed after the 24 h contact with PLGA-microspheres (Table II). For all samples exceptRG502H microspheres, these changes were practicallysimilar in both Simulated intestinal fluid with pancrea-tin (SIF-a) and Simulated intestinal fluid withoutpancreatin (SIF-b) media. About RG502H, the pHdrop was more significant in pancreatin-rich fluid thanthe other one. Following 24 h incubation of R207microspheres in these two media, no pH change wasobserved.

DSC results

Differential scanning calorimetry was used to detect thechanges of thermal properties of the microspheresfollowing degradation. For this purpose, Tg of rawpolymers and three different batches of fabricatedmicrospheres were determined and compared.Calculated Tg of all microspheres (data not shown)was lower than those of the raw polymers, indicatingthe plasticizer effects of other present ingredients. Nosignificant change in thermal transition patterns ofmicrospheres was observed after 24 h incubation invarious media, suggesting their acceptable resistance tothese environments. Since the absolute stability ofmicrospheres in these conditions was not confirmedby the data obtained from the other methods applied inthis study, DSC results might be alternatively explainedby considering the fact that this technique is not preciseenough for the detection of minor changes possiblyoccurred in microspheres over the exposure period(24 h). Figure 1 shows DSC thermograms of driedRG504 microspheres before and after incubation invarious simulated gastrointestinal fluids for 24 h.

Lactate detection

Lactate production, as an indicator of polymerdegradation, was detected based upon an enzymaticmethod relying on the quantitative oxidation of lactateto pyruvate in the presence of lactate oxidase. In theliterature, several methods have been reported forlactate measurement on the basis of gas chromatogra-phy, liquid chromatography or colorimetry (Cheng andRao Gadder 1986; Kiss and Vargha-Butler 1999;Omole et al. 1999; Haschke-Becher et al. 2000; Huanget al. 2002). However, due to the complexity of the

Figure 1. DSC thermograms of dried RG504 microspheres before and after incubation in various simulated gastrointestinalfluids for 24 h.

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media and the presence of several interfering constitu-ents, enzymatic assay was the choice. It should benoticed that the enzymatic basis of detection not onlyresulted in a very high amount, but also this methodwas very fast and had an acceptable sensitivity.Linearity was studied over a range of 10–300mgml�1

at six different concentration levels of lactic acid in eachsimulated fluid and in water. The obtained calibrationcurves were all linear over the studied range (R24 0.98)and the equations intercepts were not statisticallydifferent from zero (regression analysis, p4 0.05).Since the slopes were not the same, it was necessaryto determine the lactate content in each medium usingits specific standard solution. Furthermore, because ofthe relationship between the incubation temperatureand time with absorbance reading, a standard solution(100mgml�1) of L-lactic acid in the medium was alsotested in each run.Results obtained with lactate assay kit are shown in

Table III. The reported percentage of polymer degrada-tion is estimated from the amount of lactate generatedwithin the experiment time in relation to the totalexpected amount in the sample. Results are comparableand in good agreement with the results obtained withthe pH determination method. Degradation percentageof all polymers in simulated gastric fluids even after 24 hincubation period were low and no significant differ-ence was observed between SGF-a and SGF-b media.

The results obtained from studying microspheres inSIF-a are depicted in Figure 2. Based on the literature,it is generally accepted that the polymer hydrolysismechanism is highly pH-dependent (Kiss and Vargha-Butler 1999). In acidic medium, a random scissionalong the polymeric chain results in the formation ofmostly insoluble oligomers but an alkaline mediumfavours a non-random cleavage at the end of the

Table III. Degradation percentage of various polymeric microspheres following incubation for various times, calculated on the basis of free

lactate production (n¼ 3).

SGF-a SGF-b SIF-a SIF-b FAASSIF

Polymers 6 h 12 h 24 h 6 h 12 h 24 h 6 h 12 h 24 h 6 h 12 h 24 h 6 h 12 h 24 h

R207 50.1 0.13 0.193 50.1 0.15 0.19 50.1 50.1 0.14 50.1 50.1 0.12 50.1 50.1 50.1

RG756 50.14 50.14 0.213 50.14 0.185 0.25 50.14 50.14 0.229 50.14 50.14 0.227 50.14 50.14 50.14

RG505 50.2 50.2 0.224 50.2 50.2 0.275 50.2 0.31 1.27 50.2 0.4 0.64 50.2 50.2 50.2

RG504 50.2 0.23 0.45 50.2 50.2 0.320 0.236 0.64 1.50 50.2 50.2 1.113 50.2 50.2 0.254

RG502H 50.2 50.2 50.2 50.2 50.2 50.2 2.70 4.08 7.5 50.2 0.473 1.56 50.2 50.2 0.216

The minimum detectable degradation percentage for PLA, PLGA 75 : 25 and PLGA 50 : 50 were 0.1, 0.14 and 0.2, respectively.For all polymers, no degradation was observed after 0.5 h incubation in the media.For FeSSIF, even after 24 h, the degradation percentage was less than limit of detection.

Figure 2. Comparison of polymer degradation following themicrospheres exposure to SIF-a (n¼ 3).

Figure 3. Scanning electron micrographs of microspheresprepared from RG756 polymer, immediately afterpreparation.

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polymeric chains with high production of solublederivatives like lactic acid (Berbella et al. 1996; Kissand Vargha-Butler 1999). Therefore, these results couldnot indicate that the polymer integrity was not affectedby the gastric environment, at all.The highest degradability was observed for RG502H

microspheres. The data also showed that a largerproportion of glycolic acid in RG502H, RG504,RG505 polymers increased their degradation riskwhen exposed to GI environment. By the samereason, RG756 and R207 microspheres were the moststable ones.

The role of enzyme in any PLGA biodegradation isunclear (Cai et al. 2003). Most of the literature indicatesthat the PLGA biodegradation does not involve anyenzymatic activity and is purely through hydrolysis.However some investigators have suggested an enzy-matic role in PLGA breakdown based upon thedifferences between in vitro and in vivo degradationrates (Jain 2000). The direct role of enzymes in polymerdegradation is doubtful. Normally, enzymes are highlysubstrate-specific. Therefore, enzymatic degradation ofa synthetic polymer is possible only if it could bind tothe active site of the enzyme (Landry et al. 1996). Non-parametric statistical test on lactate assay results of thepresent study showed that the degradation of RG502Hmicrospheres in SIF-a is significantly more than SIF-b(Mann-Withney U-test; p5 0.05). The blocked andunblocked polymers contain a hydrophobic alkyl and ahydrophilic free carboxylic end group, respectively.It seems that unblocked polymers are more susceptibleto enzymatic degradation. For the interpretation ofthese results, it is important to note that the increasedpolymer cleavage in the presence of enzymes could berelated to some indirect effects such as solubilizationof hydrophobic compounds (Landry et al. 1996; Caiet al. 2003).The highest stability of microspheres was observed in

FESSIF and by less extent in FASSIF. This may beexplained by possible stabilization effects of bile salts.

Figure 4. Scanning electron micrographs of RG502Hmicrospheres after incubation at 37�C in (a) SGF-a for 6 h,(b) SIF-a for 6 h and (c) SIF-a for 12 h.

Figure 5. Scanning electron micrographs of RG756 micro-spheres after incubation at 37�C in (a) SGF-a for 3 h and (b)SGF-a for 6 h.

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It is noticeable that bile salts content of FESSIF ishigher than FASSIF. It is also important to know thattaurocholate salts have been applied as emulsifiers inmicrosphere fabrication (Bibby et al. 2000).

Morphological studies

Morphology and degradation of microspheres werestudied using optical or scanning electron micro-scopes. Figure 3 is a typical scanning electronmicrograph of microspheres prepared from RG756polymer, immediately after preparation. Particles hadspherical shape and smooth surface structure. Nopores and asperities could be detected. Similaraspects were observed for microspheres fabricatedfrom other polymers.

As observed by light microscopy, RG756 and R207microspheres showed acceptable stability in simulatedfluids, even after 24 h of incubation. Similar results werealso observed for RG505 and RG504 microspheresafter at least 12 h of incubation. RG502H microspheresshowed some changes in size and morphology afterincubation for more than 6 h in USP simulated fluids,specially SIF-a and SIF-b. The results were approvedby SEM experiments, which showed that these condi-tions led to an appreciable erosion of RG502H(Figure 4). The degradation is more apparent in thecase of SIF and is visible in the forms of pores, cavitiesand significant changes on the sample surface. The poresize increased with time such that, after 12 h, themicrospheres were highly destroyed (Figure 4c).Figures 5–7 show RG756 microspheres after incuba-

tion in various simulated media. The surfaces ofmicrospheres appeared smooth and no morphologicaldamage or visible pinholes, cracks or pores wereobserved following the incubation.

Conclusion

The objective of the current study was to selectpolymers from the PLGA or PLA family withsuitable in vitro stability in simulated gastrointestinal

Figure 6. Scanning electron micrographs of RG756 micro-spheres after incubation at 37�C in (a) SIF-a for 6 h and (b, c)SIF-a for 12 h.

Figure 7. Scanning electron micrographs of RG756 micro-spheres after incubation for 12 h at 37�C in (a) FaSSIF and(b) FeSSIF.

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fluids. The results obtained provide clear in vitroevidence that the surface characteristics of the

microspheres prepared by polymers with lowermolecular weight and higher content of glycolic acid(like RG502H) changed a lot when exposed toincubation and the data demonstrated their instabilityin a gastrointestinal environment. In contrast, RG756and R207 microspheres seemed to be more promising

carriers of oral delivery of drugs.Since drug loading may alter the microsphere

structure and characteristics, it is critical to evaluatethe stability pattern of microspheres containing drugs incomparison to blank microspheres and the raw poly-mer. The results of this study on protein-loadedmicrospheres designed as an approach for oral delivery

will be presented in a forthcoming paper.

Acknowledgements

The authors would like to thank Professor BrunoGander, Institute of Chemistry and AppliedBiosciences, ETH, Zurich, Switzerland and ProfessorJennifer B. Dressman, Institute of PharmaceuticalTechnology, Johann Wolfgang Goethe University,Frankfurt on Main, Germany for their valuable

advice and Mr Rezaee, Tarbiyat Modarres University,Tehran, for his assistance in taking SEM pictures. Wewould also like to express our appreciation to theResearch Deputy of Shaheed Beheshti University ofMedical Sciences and Health Services for financialsupport of this research.

Declaration of interest: The authors report no conflictsof interest. The authors alone are responsible for thecontent and writing of the paper.

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