doi: 10.1002/adfm.200700416 in vivo cellular uptake ...bgdgeest/bruno/de koker, s...wim e. hennink,...

DOI: 10.1002/adfm.200700416

In Vivo Cellular Uptake, Degradation, and Biocompatibilityof Polyelectrolyte Microcapsules**

By Stefaan De Koker, Bruno G. De Geest, Claude Cuvelier, Liesbeth Ferdinande, Wies Deckers,Wim E. Hennink, Stefaan De Smedt,* and Nico Mertens

1. Introduction

There has been a growing interest in the design and develop-ment of polymeric microspheres as controlled release systemsfor drugs and antigens. Different types of polymers have beenused to design biodegradable polymeric microparticles.[1]

Poly(lactic-co-glycolic) acid (PLGA) microspheres have beenextensively investigated because of their biocompatibility andbiodegradability.[2] PLGA has been approved by the FDA, andGMP grade PLGA is commercially available. However, prepa-ration of PLGA microspheres requires the use of organic sol-

vents and involves large shear forces, both of which can ser-iously affect protein stability.[3] Protein encapsulationefficiency and loading capacity of PLGA microspheres areoften low.[3] Moreover, PLGA leads to acidification[4] of the in-jection site, because degradation of PLGA produces lactic acid,which can potentially denature encapsulated proteins. Newtypes of injectable microspheres that can avoid these problemsare needed.

A new type of biodegradable microcapsules[5–7] was recentlydeveloped using the layer-by-layer (LbL) technique.[8] Thistechnique is based on the sequential adsorption of oppositelycharged molecules, such as polyelectrolytes, onto a charged sa-crificial template (Fig. 1). The template is then dissolved, result-ing in hollow capsules with a wall thickness in the nanometer

range. Degradable polyelectrolyte microcapsules made of poly-peptides and/-or polysaccharides were reported recently.[9,10] Aspolyelecrolyte multilayers on planar substrates start to emergeas multifunctional delivery platforms for implants, e.g., drugeluting stents or dental implants,[11–20] the potential of polyelec-trolyte microcapsules as drug delivery systems could in-crease.[6,21] Polyelectrolyte microcapsules have the advantagesof mild preparation conditions, multifunctionality, and thecapacity to encapsulate large amounts of material.[22–24]

3754 © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Funct. Mater. 2007, 17, 3754–3763

–[*] Prof. S. De Smedt

Department of PharmaceuticsGhent UniversityHarelbekestraat 72, 9000 Ghent (Belgium)E-mail: [email protected]. De Koker, Dr. N. Mertens, W. DeckersDepartment for Molecular Biomedical Research, VIBTechnologiepark 927, 9052 Ghent (Belgium)S. De Koker, Dr. N. Mertens, W. DeckersDepartment of Molecular BiologyGhent UniversityTechnologiepark 927, 9052 Ghent (Belgium)Dr. B. G. De Geest, Prof. W. E. HenninkDepartment of PharmaceuticsUtrecht Institute of Pharmaceutical Sciences (UIPS)Utrecht UniversityPO Box 80082, 3508 TB, Utrecht (The Netherlands)Prof. Dr. C. Cuvelier, L. FerdinandeDepartment of PathologyGhent UniversityDe Pintelaan 185, 9000 Ghent (Belgium)

[**] S.D.K. thanks the Stichting Emmanuel van der Schueren (VlaamseLiga tegen Kanker) for funding this research. B.G.D.G. thanks TiPhar-ma for a postdoctoral fellowship. Supporting Information is availableonline from Wiley InterScience or from the authors.

Polyelectrolyte microcapsules are made by layer-by-layer (LbL) coating of a sacrificial template, followed by decomposition ofthe template, to produce hollow microcapsules. In this paper, we report on the in vivo cellular uptake, degradation and biocom-patibility of polyelectrolyte microcapsules produced from alternating dextran sulphate and poly-L-arginine layers on a templateof calcium carbonate microparticles. We show that a moderate tissue reaction is observed after subcutaneous injection of poly-electrolyte microcapsules in mice. Within sixteen days after subcutaneous injection, most of the microcapsules are internalizedby the cells and start to get degraded. The number of polyelectrolyte layers determines the stability of the microcapsules aftercellular uptake.

Figure 1. Schematic representation of the synthesis of hollow polyelectro-lyte capsules, using CaCO3 particles as a template. A) Precipitation of cal-cium carbonate particles (grey) during the mixing of calcium chloride andsodium carbonate solutions. B) LbL coating of the calcium carbonate par-ticles (red = polyanion, blue = polycation). C) Polyelectrolyte capsules ob-tained after dissolving the CaCO3 core with EDTA.

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Polyelectrolyte microcapsules are produced at room temper-ature using simple bench-top equipment, without organic sol-vents or harsh reaction conditions. Due to the versatility ofelectrostatic interaction, the physicochemical properties of themicrocapsules’ shells can be precisely tuned. Moreover, thecapsule surface can be furnished with a wide variety of func-tionalities, such as lipids,[25] polyethylene glycol,[26] antibodies,or[27] viruses.[28]

In a previous paper we reported on the cellular uptake anddegradation of polyelectrolyte microcapsules by an in vitro cul-tured cancer cell line.[9] In this paper we aim to assess the invivo cellular uptake, degradation and tissue reaction of poly-electrolyte microcapsules. To assess in vivo biocompatibility,skin sections were made at different time points after subcuta-neous injection of microcapsules in mice, and tissue reactionand cellular infiltration were examined. Furthermore, phagocy-tosis and in vivo degradation of fluorescently labeled microcap-sules were evaluated. Because subcutaneously injected foreignparticles in the 0.1–10 lm range are preferentially taken up byphagocytes, the in vitro cytotoxicity of the polyelectrolyte cap-sules was determined using bone marrow derived dendriticcells. Our data pave the way for further in vivo research onpolyelectrolyte microcapsules.

2. Results and Discussion

2.1. Fabrication of Hollow Polyelectrolyte Capsules

CaCO3 microparticles were LbL coated with several bilayersof dextran sulfate and poly-L-arginine.[24] The adsorption pro-cess was monitored by measuring electrophoretic mobilityafter deposition of each layer. Starting from a CaCO3 micro-particle with a zeta-potential of +12 mV, the zeta-potentialalternated between –50 mV and 50 mV during the depositionof 8 polyelectrolyte layers (Fig. 2A).

Hollow particles were produced by dissolving the LbL coat-ed CaCO3 microparticles in a buffered EDTA solution (pH5.4). The Ca2+ ions are complexed by the EDTA and can easilypermeate trough the polyelectrolyte membrane. Polyelectro-

lyte microcapsules consisting of 1, 2, and 4 bilayers of dextransulphate/poly-L-arginine were produced. Figure 2 shows confo-cal microscopy (2B) and scanning electron microscopy (2C)images of the capsules (see Supporting Information for DICimages of microcapsules consisting of 1, 2, and 4 bilayers). Themicrocapsules show a rather monodisperse size distributionwith an average diameter of 3 lm. The capsules were fluores-cently stained by using one layer of RITC labeled poly-L-argi-nine during the LbL coating process. The scanning electron mi-croscopy image shows the microcapsules in a collapsed statebecause the sample was dried for imaging.

2.2. Uptake of Polyelectrolyte Microcapsules by Bone MarrowDerived Dendritic Cells

Uptake of polyelectrolyte microcapsules by cells was investi-gated by incubating cultured dendritic cells with fluorescentlylabeled (RITC) microcapsules, followed by confocal microsco-py based on the procedures reported by Parak et al.[29–31] InFigure 3A, cells were stained with CellTrackerGreen, a fluores-cent dye that accumulates in the cytoplasm of living cells. In

Adv. Funct. Mater. 2007, 17, 3754–3763 © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.afm-journal.de 3755

Figure 2. A) Evolution of the zeta-potential during LbL coating of calcium carbonate microparticles with alternating dextran sulfate/poly-L-argininebilayers. Confocal (B) and scanning electron (C) microscopy images of polyelectrolyte microcapsules consisting of 4 dextran sulfate/poly-L-argininebilayers. The red colour in the confocal image is due to the poly-L-arginine, which was fluorescently labeled with RITC.

Figure 3. Confocal microscopy images showing the intracellular uptake ofmicrocapsules consisting of four bilayers of dextran sulphate/poly-L-argen-tine by bone marrow derived dendritic cells. In both images, the microcap-sules were fluorescently labeled red with RITC labeled poly-L-arginine.A) Bone marrow derived dendritic cells were stained with CellTrackerGreen,which stains the cytoplasm fluorescent green. B) Bone marrow derived den-dritic cells were stained with CD11c-FITC, which stains the cellular mem-brane fluorescent green. The overlay with the DIC image is shown.

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S. De Koker et al./Polyelectrolyte Microcapsules In Vivo

Figure 3B, the cell surface was stained with CD11c, a markerfor bone marrow derived dendritic cells. The fact that both thecellular membrane and the microcapsules are seen in the sameconfocal plane indicates that the microcapsules are effectivelytaken up by cells, and not merely sticking to the cell surface byelectrostatic interactions. These observations are consistentwith published data on cellular uptake of polyelectrolyte mi-crocapsules by cancer cell lines.[9,27,29,30,32,33,34] Figure 3 clearlyshows that dendritic cells can take up very large amounts ofcapsules.

Microcapsules in the range of 0.1–10 lm can be easily phago-cytosed by macrophages and dendritic cells. Moreover, rapidlydegradable PLGA microspheres that deliver antigens intracel-lularly to dendritic cells have been shown to enhance antigenpresentation and immune responses. As polyelectrolyte micro-capsules can encapsulate large amounts of proteins,[22,23] theycan be useful for intracellular delivery of antigen to antigenpresenting cells (APC), providing that they do not affect theirviability.

2.3. In vitro Cytotoxicity

The in vitro cytotoxicity of the polyelectrolyte microcapsuleswas assessed by a MTT assay, because the uptake of largeamounts of polyelectrolyte capsules by APC could be toxic. In-deed, it has been reported that polycationic polymers reducecell viability.[35–38] Fisher et al. [38] showed that cytotoxicity ofpolycations is determined by their molecular weight, their cat-ionic charge density and the flexibility of their structure. Asthe polyelectrolyte microcapsules in this study have a positive,poly-L-arginine based outer layer, incubating them with cellscould disturb their membranes and cause cytotoxicity. Asshown in Figure 4, cell viability was reduced by 80 % whencapsule-to-dendritic cell ratio was ∼ 103. This large impact was

probably due to formation of a continuous film of microcap-sules on the dendritic cells. At moderate microcapsule/cell ra-tios, more than 60 % of the cells survive (for capsule/cell ratiosof 125 and below). At a ratio of 30 microspheres/cell, cytotox-icity was low and approximately 80 % of the dendritic cells re-mained viable. This experiment shows that it is possible to ap-ply rather large amounts of the microcapsules to dendritic cellswithout severely affecting viability. Similar cytotoxicity levelswere observed by Kirchner et al. using PSS/PAH based micro-capsules on a fibroblast cell line.[31]

2.4. In vivo Biocompatibility

To assess the in vivo biocompatibility of polyelectrolyte mi-croparticles, they were injected subcutaneously and tissue reac-tions were examined at different time intervals. Subcutaneousinjection of foreign material may disturb tissue architectureand initiate inflammation, wound healing and foreign body re-sponses. The extent of such responses determines the severityof the host reaction and thus the biocompatibility of the mate-rial. The bright field microscopy images in Figure 5 show thetissue reaction in mice after subcutaneous injection of micro-capsules consisting of four bilayers of dextran sulfate/poly-L-ar-ginine. (see Supporting Information for enlarged and addi-tional images) The top left image of each block shows anoverview of the injection site, while R1, R2, and R3 are de-tailed images of the regions marked in the overview image. APBS group was included as a negative control. As positive con-trol groups for inflammation, Complete Freund’s adjuvant(CFA) and Al(OH)3, two frequently used adjuvants were used.CFA consists of a water-in-oil emulsion containing heat-killedmycobacteria or mycobacterial cell wall components and isknown to cause extensive tissue inflammation, with possibleside effects like skin ulceration, local abscess formation and tis-sue necrosis. Because it causes very intense inflammatory reac-tions, CFA use is restricted to research purposes in laboratoryanimals. Al(OH)3 on the other hand, causes only mild inflam-matory reactions and is by large the most often used adjuvantin vaccines for human use. Table 1 gives an overview of the na-ture and extent of the tissue response at different time intervalsafter injection of the polyelectrolyte capsules, when comparedto CFA and Al(OH)3.

One day after injection of the polyelectrolyte capsules(Fig. 5, day 1) inflammatory cells, mostly polymorphonuclearleukocytes (R3, ), are recruited to the injection site. As themass of injected microcapsules resembles a porous implant,there is a lag time between infiltration of the edge of the injec-tion site, and infiltration of the centre. One day after injectiononly the edge of the injected site is infiltrated (R1). The micro-capsules retain their spherical shape and appear as white discs(R1, R2, ). Also lymphocytes (R1, ) and apoptotic cells(R1, ), probably dying short-lived polymorphonuclear cells,are visible at the edge. No inflammation can be seen in thePBS control group (Fig. 6, day 1), while a massive inflamma-tory response is observed in the CFA group (Fig. 7, day 1). In-jection of Al(OH)3 particles (Fig. 8, day 1, ) results in a fastrecruitment of polymorphonuclear cells to the injection site.

3756 www.afm-journal.de © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Funct. Mater. 2007, 17, 3754–3763

Figure 4. Viability of bone marrow derived dendritic cells incubated withincreasing amounts of polyelectrolyte microcapsules. The amount of mi-crocapsules is expressed as the number of capsules per number of seededdendritic cells. The inset shows a confocal microscopy image of a bonemarrow derived dendritic cell that has phagocytosed several tens of micro-capsules. For visualisation, the microcapsules were labeled fluorescentgreen by incorporation of FITC-dextran while the dendritic cells were la-beled fluorescent red with LysoTracker, which selectively stains lysosomes.

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Figure 5. Bright field microscopy images of tissue sections of the injection spot at several time points after subcutaneous injection of mice with micro-capsules consisting of four bilayers of dextran sulphate/poly-L-arginine. Tissue sections were assessed for the recruitment of inflammatory cells to the mi-crocapsule mass. At early time points, only the edge of the microcapsule mass has been infiltrated, mostly by polymorphonuclear cells ( ), predominantlyeosinophils( ). Individual microcapsules ( ) can still be distinguished. Fibroblasts ( ) start to surround the injection mass, even on day 4. After 18 days,macrophages ( ) appear to have phagocytosed the microspheres, which seem to have lost their spherical shape. On day 30 the entire injected mass hasbeen infiltrated by cells, and encapsulated by 5–10 layers of fibroblasts, which can also be seen scattered throughout the injection mass. At the edgemainly macrophages are present, while at the centre the infiltrate consists of a mixture of macrophages and polymorphonuclear cells. Many new bloodvessels ( ) are being formed.

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As for the microparticles, infiltration starts at the edge andconsists predominantly of polymorphonuclear cells.

Four days after injection (Fig. 5, day 4), inflammatory cellsare still being recruited to the injection site. R1 shows leuko-cytes adhering and crossing the endothelial wall, and eosino-phils (R1, ) moving towards the microcapsule mass. At thistime, fibroblasts start to surround the implant site (R2, ). Atthe edge of the microcapsule mass, many eosinophils (R2, )and some macrophages (R2, ) are seen. The microcapsules,which still appear spherical, have not been phagocytosed yet(R3, ). At this early time points, the tissue response is clearlydominated by polymorphonuclear cells, which is characteristicof acute phases of inflammation.

Eight days after injection (Fig. 5, day 8), inflammatory cellshave infiltrated a large part of the injected mass, but in its centre,few cells are seen and the microcapsules remain intact (R2, ).Eosinophils (R2, ) become scarcer, and many macrophages ap-pear at the edge (R2, R3, ) where microcapsules are becoming

deformed and difficult to distinguish (R3). Inthe CFA control group (Fig. 7, day 8), a severeinflammatory response is observed, with theinflux of predominantly polymorphonuclearcells, but also lymphocytes and fibroblasts.Macrophages start to appear at this stage. In-filtration is much less predominant in theAl(OH)3 control group, as few cells are scat-tered between the particles. The injection siteis surrounded by 5–10 layers of fibroblasts,and infiltrated by eosinophils, other polymor-phonulear cells and some macrophages(Fig. 8, day 8). In general, the tissue responseto the large, inert Al(OH)3 particles is less ex-tensive than to the biodegradable, phagocyta-ble microcapsules.

Eighteen days after injection (Fig. 5, day18) the whole injection site has become infil-trated by inflammatory cells and surroundedby a fibrous capsule consisting of approxi-mately five layers of fibroblasts (R1, ). Theenlarged image R3 shows that the cytoplasmof the infiltrated macrophages (R3, ) exhib-its a remarkably granular structure that ismost likely due to phagocytosed microcap-

sules (R3, ). In the CFA control group, inflammation is muchmore widespread, and tissue necrosis is observed (Fig. 7, day18, R1) Granulomatous structures are formed, consisting of adense infiltrate of inflammatory cells (R2). Lots of macro-phages and polymorphonuclear cells are present, as well as sig-nificant numbers of lymphocytes (R2, ).

After one month (Fig. 5, day 30), most microcapsules havebeen phagocytosed, and debris of degraded/deformed micro-capsules is observed in the cytoplasm of phagocytes (R3, ).The injected mass is now entirely infiltrated by cells, and sur-rounded by five to ten layers of fibroblasts (R2, ), which arealso scattered throughout the injected volume.

Some lymphocytes are present too (R1, ), but their numbersremain much lower than in the CFA group. The injection site isbeing vascularized, and many newly formed blood vessels (R1,R2, ) are now visible throughout. At the edge of the implantsite, the tissue response is dominated by macrophages and fewpolymorphonuclear cells can be seen, while at the centre theyare still present in significant numbers. By this, the microsphereinjection spot resembles a porous implant, with infiltrationstarting at the edge by polymorphonuclear cells followed byphagocytes and gradually proceeding to the centre of the mi-crosphere mass over time. No giant cells could be observed atany time. In contrast to the CFA control group, inflammationremains locally and no necrosis can be seen. In the CFA group,the entire subcutaneous tissue is heavily inflamed, with forma-tion of granulomatous structures, lots of macrophages, lympho-cytes and polymorphonuclear cells (Fig. 7, day 30, R2). Inflam-mation remains mild in the Al(OH)3 control group: besidesmacrophages, granulocytes, especially eosinophils (Fig. 8, day30, R2), are still present throughout the injection side which issurrounded by fibroblasts.


Table 1. Overview of the inflammatory tissue response to polyelectrolyte microcapsules, PBS,CFA and Al(OH)3 after subcutaneous injection. The response was rated according to the follow-ing scoring system: – = not present to +++++ = extensive; n.a. = not assessed.

Lymphocytes PMN Macrophages Fibroblasts Vascularisation

Day 1 PBS – – – – –

Capsules + + –/+ – –

Al(OH)3 + + – + –

CFA ++ ++++ –/+ + -

Day 4 PBS – – – – –

Capsules + ++ + + +

Al(OH)3 n.a. n.a. n.a n.a. n.a.

CFA n.a. n.a. n.a n.a. n.a.

Day 8 PBS – – – – –

Capsules + ++ ++ ++ ++

Al(OH)3 + + + ++ +

CFA ++ ++++ + ++ +

Day 18 PBS – – – – –

Capsules + +++ +++ +++ +++

Al(OH)3 n.a. n.a. n.a. n.a. n.a.

CFA ++ ++++ ++ +++ ++

Day 30 PBS – – – – –

Capsules + +++ +++ +++ +++

Al(OH)3 + + + ++ +

CFA ++++ ++++ +++ ++ +

Figure 6. Bright field microscopy images of tissue sections after injectionof PBS. No inflammation can be observed.

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2.5. In vivo Cellular Uptake and Structural Integrity

Confocal microscopy was used to investigate the fate of thepolyelectrolyte capsules after subcutaneous injection. Fig-ure 6 shows confocal microscopy images of the injection site atdifferent time intervals after injection of capsules consisting oftwo bilayers dextran sulfate/poly-L-arginine, produced usingRITC labeled poly-LL-arginine to make them fluorescent red.The images show the overlay of the DIC image, the nuclearstaining and the red fluorescence signal of the microcapsules.In line with the bright field images of Figure 5, cells infiltratethe edge of the injection spot within two days of injection, butit takes them longer to infiltrate the centre of the injection site(Fig. 9). Two days after injection, the microcapsules more orless retain their spherical shape and are scattered between thecells as seen in the DIC images (Fig. 9, third column, day 2).

This indicates that the microcapsules have not yet been phago-cytosed to an appreciable extent. Eight days after injection,most of the microcapsules are still scattered between the cells(Fig. 9, day 8), but at the edge of the injection site, microcap-sules have been phagocytosed (Supporting Information, day 8,R2). These phagocytosed particles (day 8, R2) start to shrinkand become deformed, while the extracellular capsules (day 8,R1) remain intact. The same observations can be made at day16: particles that have been phagocytosed have lost their spher-ical shape and are much smaller (Fig. 9 & Supporting Informa-tion, day 16, R2), while those that have not been taken up re-main intact. No particle debris can be observed outside thecells. This indicates that most of the capsule deformation anddegradation occurs inside the cells. These observations are alsoin agreement with the bright field images in Figure 5: the gran-ularity of the cytoplasm of the infiltrating cells observed from


Figure 7. Bright field microscopy images of tissue sections at different time intervals after subcutaneous injection of CFA. Injection of CFA results in awidespread inflammation, with the influx of large numbers of polymorphonuclear cells, lymphocytes and macrophages, formation of granulomatousstructures and tissue necrosis.

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day 18 is due to phagocytosed microcapsules. Thirty days afterinjection (Fig. 9) shows shapeless microcapsule debris in thecell cytoplasm, indicating severe capsule degradation, mostlikely due to a combination of enzymatic attack and mechani-cal deformation.

These results were obtained with microcapsules consisting oftwo bilayers of dextran sulfate/poly-L-arginine. As we were alsointerested in the influence of the number of polyelectrolytelayers on the fate of the microcapsules after in vivo cellular up-take, we injected mice subcutaneously with microcapsules con-sisting of 1, 2, and 4 bilayers of dextran sulfate/poly-L-arginine.Figure 10 shows confocal microscopy images of sections of theinjection site 16 days after subcutaneous injection of the micro-capsules. In all cases the infiltrated inflammatory cells havephagocytosed the microcapsules, but a clear influence of thenumber of polyelectrolyte layers on the microcapsules’ degra-dation and destruction is observed. Intact microcapsules con-sisting of one bilayer can no longer be distinguished within thecells, only debris of degraded microcapsules. This contrastswith microcapsules consisting of four bilayers, which can bedistinguished individually, with some having retained theirspherical shape after in vivo cellular uptake. For microcapsulesconsisting of two bilayers, an intermediate situation is ob-served, with most of the microcapsules reduced to unidentifi-able debris, but some retaining an intact appearance. These ob-

servations lead us to conclude that incorporating morepolyelectrolyte layers increases in vivo intracellular stability.Microcapsules consisting of too few layers have a lower me-chanical strength and are degraded faster by enzymatic action.

3. Conclusions

We first studied tissue reaction following subcutaneous injec-tion of microcapsules in mice. A moderate immune reactionwas observed, with an acute phase characterized by recruit-ment of polymorphonuclear cells and a more chronic phase inwhich microspheres are phagocytosed by macrophages and theinjection site is surrounded by fibroblasts. Similar tissue reac-tions have been reported for other types of biodegradable mi-crospheres, such as PLGA,[2] methacrylated dextran,[39] andpoly(ether-ester)[40] microspheres. Secondly, we demonstratedthe in vivo uptake and intracellular degradation of the micro-capsules. We also observed that the number of bilayers fromwhich the microcapsules are constituted determines their sta-bility after intracellular uptake. These combined observationsindicate that polyelectrolyte microcapsules made from degrad-able polyelectrolytes are suitable for drug delivery. Thesepolyelectrolyte capsules can be used to deliver[41] DNA,[42,43]

proteins,[44–46] nucleic acids,[47] and other materials inside pha-


Figure 8. Bright field microscopy images of tissue sections after subcutaneous injection of Al(OH)3. Injection of the large Al(OH)3 particles results in amild inflammatory response with recruitment of polymorphonuclear cells, predominantly eosinophils, macrophages and lymphocytes. The entire injec-tion site gets encapsulated by fibroblasts.

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gocytic cells,[48] which are key players in immunity and toler-ance. Thus, they may be useful tools in the field of vaccination.

4. Experimental

Materials: Rhodamine isothiocyanate (RITC), FITC-dextran(Mw ∼ 2000 kDa), dextran sulphate (DEXS; Mw ∼ 10 kDa) and poly-L-arginine hydrochloride (pARG; Mw > 70 kDa) were purchased formSigma–Aldrich–Fluka. Calcium chloride (CaCl2) and sodium carbonate(Na2CO3) were purchased from Merck. RITC labeled pARG was

synthesized by mixing 100 mg pARG and 4 mgRITC in 40 ml 0.1 M borate buffer, pH 8.5.After overnight reaction the mixture was dia-lyzed for several days against pure water usingSpectra Por dialysis bags with a molecularweight cut off of 25 kDa to remove residualRITC.

Preparation of Polyelectrolyte Microcapsules:Polyelectrolyte microcapsules were made usingcalcium carbonate (CaCO3) microparticles as asacrificial template. CaCO3 microparticles weresynthesized according to Volodkin et al. [23,24]by mixing CaCl2 and Na2CO3 solutions (0.33 M)with vigorous stirring for 30 s followed by ex-tensive washing with pure water to remove un-reacted reagents. Spherically shaped CaCO3

microparticles with an average diameter of3 lm were obtained.

The CaCO3 particles were coated using thelayer-by-layer (LbL) technique by dispersingthem in 5 ml of a 2-mg-ml–1 dextran sulphatesolution containing 0.5 M NaCl. After 10 minshaking the microparticles were collected bycentrifugation, and residual dextran sulphatewas removed by washing twice with 10 ml ofpure water. Thereafter the microparticles weresuspended in 5 ml of a 1-mg-ml–1 poly-L-argi-nine solution in 0.5 M NaCl and shaken for10 min, followed by centrifugation and twowashing steps. This procedure was repeated un-til the desired number of layers was deposited(2, 4, and 8 in this study).

Hollow capsules were obtained by removingthe CaCO3 core by incubating the coated mi-croparticles for 10 min in 10 ml of 0.2 M EDTAsolution (pH 5.2) to dissolve the CaCO3. Thedissolved ions were then removed by threecentrifugation and washings steps. Finally thecapsules were resuspended in 1 ml PBS. Thecapsule concentration wad determined by hae-mocytometry.

Characterization of Polyelectrolyte Microcap-sules: Electrophoretic Mobility: The electro-phoretic mobility of the polyelectrolyte micro-capsules was measured in deionized water atroom temperature using a Malvern Zetasizer2000 (Malvern Instruments). The zeta-potential(f-potential) was calculated from the electro-phoretic mobility (l) using the Smoluchowskifunction f = lg/e where g and e are the viscosityand permittivity of the solvent, respectively.

Electron Microscopy: A drop of capsule sus-pension was placed on a silicon wafer and driedunder a nitrogen stream, and then coated withgold. SEM images were recorded with a Quanta200 FEG FEI scanning electron microscope op-erated at 5 kV.

Optical Microscopy: The Olympus BX51 mi-croscope, equipped with 4x, 10x, 20x, 40x, 60x, and 100x lenses and aCoolsnap Color camera (from Roper Scientific – Tucson USA) wereused for bright field inspection and recording.

Fluorescence Microscopy: The RITC labeled microspheres in theskin of the mice were imaged with a Leica SP5 AOBS confocal micro-scope (63x 1.4 oil objective, HCX PL APO 63.0 x 1.40 OIL UV), usingthe 543 nm HeNe laser line for the RITC-labelled microspheres. Nucleiwere stained with DAPI included in the mounting medium Vectashield,and excited with the 405 nm line of a UV diode laser.

Generation of Bone-Marrow Derived Dendritic Cells: Dendritic cellswere generated using a modified Inaba protocol [49]. Two to sixmonths Balb/c mice were sacrificed and bone marrow was flushed from


Figure 9. Confocal microscopy images of tissue sections taken at different time points after subcu-taneous injection of microcapsules consisting of two bilayers of dextran sulphate/poly-L-arginine.The left column shows a large area of the injection site as an overlay of red fluorescence (due toRITC labeled poly-L-arginine) and blue fluorescence (due to staining of nuclei with DAPI). The mid-dle and right columns show a more detailed view of the injection spot. Middle: the red and bluefluorescence overlay at a higher magnification where individual cells and capsules can clearly bedistinguished. Right: an additional DIC overlay for the right column to visualize cellular contours.

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their femurs and tibias. After lysis of red blood cells with ACL lysisbuffer (BioWhittaker, Wakersville, MD), granulocytes and B cells weredepleted using Gr-1 (Pharmingen) and B220 (Pharmingen) antibodiesrespectively, and low-toxicity rabbit complement (Cedarlane Laborato-ries Ltd., Hornby, Ontario, Canada). Cells were seeded at a density of2 x 105 cells mL–1 in 175cm2 Falcon tubes (Becton Dickinson) in DCmedium (RPMI 1640 medium containing 5 % LPS-free FCS, 1 % pe-nicillin/streptomycin, 1 % L-glutamine and 50 lM b-mercaptoethanol)containing 10 ng mL–1 IL-4 and 10 ng ml–1 GM-CSF (both from Pepro-tech, Rock Hill, NJ). After two days and again after four days of cul-ture, the non-adherent cells were centrifuged, resuspended in fresh me-dium and replated to the same falcons. On the sixth day, non-adherentcells were removed and fresh medium containing 10 ng ml–1 GM-CSFand 5 ng ml–1 IL-4 was added. On day 8 of culture, non-adherent cellswere harvested and used for the experiments.

In vitro Cytotoxicity Assay: The cytotoxicity of dex/Parg polyelectro-lyte microcapsules was evaluated on bone marrow derived dendriticcells using a classical MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide). Day 8 dendritic cells were plated in a96-well plate at 2,5 x 103 cells/well and incubated with different concen-trations of microspheres for 6 h. Afterwards, medium was refreshedand cells were cultured for another 48 h. Medium was removed andMTT, a water-soluble tetrazolium salt, was added. MTT is convertedinto an insoluble purple formazan dye by mitochondrial dehydro-genases of living cells. After 3 h of incubation at 37 °C, cells were lysedand formazan was dissolved in dimethylsulfoxide (DMSO). Measuringthe absorbance at 570 nm assessed the relative viability of the cells.

Histological Examination: Dextran/pARG polyelectrolyte microcap-sules were prepared under endotoxin-free and sterile conditions.Female C57/BL6 mice (6–8 wks, Janvier) were housed under SPF con-ditions. After being anesthesized intraperitoneally with a mixture of10 % ketamine/5 % xylazine, mice were shaven at the right flank andgroups of three mice were injected with 50 million microcapsules resus-pended in a volume of 100 ll PBS. As a negative control for inflamma-tion, mice were injected with a 100 ll of sterile PBS. As positive con-trols, mice were injected with 50 ll of a water-in-oil emulsion of PBS incomplete Freund’s adjuvant (Sigma) at a ratio 1/10 (PBS/CFA) or with1 mg of Al(OH)3 (Sigma) suspended in 100 ll of PBS. Mice were sacri-ficed at different time intervals and the tissue surrounding the injectionsite was carefully dissected. For each time interval, samples from threedifferent mice were taken. Fixation was performed in 4 % paraformal-dehyde in PBS, and the samples were dehydrated in ethanol and em-bedded in paraffin. Sections of 5 lm were cut and stained with haema-toxylin and eosin, and examined by light microscopy. The tissueresponse was rated by at least two persons according to the followingscoring system: – = no infiltration to +++++ extensive infiltration oflymphocytes, polymorphonuclear cells, macrophages and fibroblasts.The same scoring system was applied to assess the degree of vasculari-zation.

In vivo Uptake and Structural Integrity of theMicrospheres: To assess in vivo the cellular up-take and intracellular degradation of the poly-electrolyte capsules, rhodamine isothiocyanate(RITC)-labelled capsules were prepared andinjected subcutaneously. Mice were sacrificedat different time intervals and the injection sitewas dissected. Tissue samples were fixed in 4 %paraformaldehyde, embedded in paraffin, and5-lm sections were cut. After deparaffinisation,sections were mounted with DAPI-containingVectashield mounting medium for fluorescence(Vector Laboratories, Inc.) to visualize nuclei.The integrity and cellular uptake of the poly-electrolyte capsules were evaluated using con-focal microscopy.

Received: April 12, 2007Revised: August 14, 2007

Published online: November 28, 2007

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Figure 10. Confocal microscopy images of tissue sections taken 16 days after injecting mice subcu-taneously with microcapsules consisting of 1 (R2), 2 (R4), and 4 (R8) bilayers of dextran sulfate/poly-L-arginine. The images compare the structural integrity of the microcapsules depending onthe number of dextran sulphate/poly-L-arginine bilayers. The microcapsules were labeled fluores-cent red with RITC-labelled poly-L-arginine. Nuclei were stained fluorescent blue with DAPI.

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doi: 10.1002/adfm.200700416 in vivo cellular uptake ...bgdgeest/bruno/de koker, s...wim e. hennink,...

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