as model drugs, and phosphate buffer [3]. Crosslinking was completed 30 minafter the ATPS has been exposed to UV-light (366 nm, 3 mW/cm2). Tocharacterize the hydrogel network regarding the influence of DS and molecularweight on the release profile fluorescence labelled lysozyme and FITC-dextranwith varying molecular size (20 kDa, 70 kDa und 500 kDa) were incorporatedand analysed regarding their release behaviour. The release studieswere carriedout under sink conditions at pH7 and at a temperature of 37 °C in a shaking bath.

Results and discussion

The produced spherical microparticles show a monomodal particle sizedistribution with an average size of about 10 µm (Fig. 2a, b).

As expected, increasing the network density reduced the release rate. Inaddition, smaller molecules are retained to a lower extent than substances withhigher molecular weight. This can be explained by the different degree ofunhindered diffusion (Figs. 3 and 4). The data of the initial release suggests thathydrogelswithDS0.05and0.22differmostly in theamountof pores in the rangeof 6.6 nm (~70 kDa) (Fig. 4). Moleculeswhich are bigger than that sizewere onlyreleased by degradation of the hydrogel, which was very slow in PBS at pH 7. Toaccelerate the biodegradation of the hydrogel and thus the release of FITC-dextran, the loadedmicrosphereswere incubated in sodium carbonate buffer atpH 9.6 and human serum respectively. In case of sodium carbonate buffer, thehydrolytically labile carbonate group of HES-HEMA is cleaved rapidly. Adependency on molecular weight and DS can be seen (Fig. 4). Incubating inhuman serum leads to a degradation of the hydrogel network due to thepresence of the enzyme α-amylase, an endoamylase, which is able to degradethe HES-backbone at positions 1–4 and thereby accelerate the release (Fig. 5).

Conclusion

These studies show that the described method is well suited for thepreparation of drug loaded hydrogel microspheres. The release of FITC-

dextranwas bi-phasic, with a rapid release of predominantly small moleculesby diffusion and a slow release after hydrolytic degradation of the hydrogel.Both phases are influenced by the degree of substitution of the polymer,which allows to tailor the release profile from HES-HEMA hydrogels.

Acknowledgements

This project was founded by Deutsche Forschungsgemeinschaft (SFB578-D1).

We would like to thank Fresenius Kabi and BASF for substances.

References

[1] W.N.E. Van Dijk-Wolthuis, Polymer 38 (25) (1997) 6235–6242.[2] E. Heim, et al., J. Magn. Magn. Mater. 311 (2007) 150–154.[3] P. Lawin, et al., Thieme, Stuttgart, 1989.[4] M.C. Manning, et al., Pharm. Res. 6 (1989) 903–918.[5] A. Schwoerer, Oral presentation, CRS, Freiburg, 2007.

doi:10.1016/j.jconrel.2008.09.050

The use of endocytic inhibitors to study uptake of gene carriers

D.N.E. Vercauteren⁎, R.E. Vandenbroucke, J. Demeester, S.C. De Smedt,K. Braeckmans, N.N. SandersLaboratory of General Biochemistry and Physical Pharmacy, Ghent University,Harelbekestraat 72, B-9000 Ghent, BelgiumE-mail: dries.vercauteren@ugent.be

Abstract summary

A mechanistic understanding of the different uptake mechanisms in thecellular internalization of non-viral nucleic acid containing nanoparticles

Fig. 3. Effect of the degree of substitution of the HES-HEMA on the release offluorescence labelled lysozyme from microspheres. Symbols: DS 0.05 ○, DS 0.07 ▲,DS 0.14 ■ and DS 0.17 □.

Fig. 4. Release of FITC-dextran (FD20 — 20 kDa ●○, FD70 — 70 kDa □■, FD500 —

500 kDa Δ▲) from microspheres with DS 0.05 or DS 0.22 (filled symbols).

Fig. 5. (a) Release of FITC-dextran from microspheres with DS 0.05 and 0.22 at pH 9.6(b) Release of FITC-dextran 70 kDa from microspheres with DS 0.14 in human serum incomparison to sodium phosphate buffer.

Abstracts/Journal of Controlled Release 132 (2008) e1–e18 e17

should open up possibilities to correlate uptake to intracellular processingand subsequently to transfection efficiency. One method to quantitativelyassess contributions of different endocytic pathways is to inhibit themthrough the application of endocytic inhibitors. However, one should takeextra care when using these chemicals as their cytotoxicity, effectivity andspecificity is clearly cell dependent and not always as stated in literature.Therefore, these inhibitors should only be used after doing the appropriatecontrol experiments and preferably in combination with complementaryinhibition methods.

Introduction

Although non-viral gene carriers, such as cationic liposomes or polymers,are a safe(r) alternative to viral carriers, their lower transfection efficiencylimits their use in clinical trials. Several types of endocytosis have beendemonstrated to be involved in the uptake of these nucleic acid containingnanoparticles, varying according to cell type, type of carrier and particle size[1]. As the intracellular processing can differ strongly depending on theprecise uptake mechanism, unravelling this uptake mechanism of individualnon-viral gene carriers in a specific cell line model can open up possibilitiesto correlate uptake to intracellular processing and subsequently to transfec-tion efficiency. Consequently, several research groups have tried toquantitatively assess the contribution of each endocytic pathway to theuptake of non-viral gene delivery vehicles through the use of endocytosisinhibitors [2–4]. These chemicals are easy to apply and are in some casespresumed to inhibit endocytic pathways in a specific way.

Experimental methods

Chlorpromazine, methyl-β-cyclodextrin (mBCD), nystatin, genistein, andfilipin III were purchased from Sigma Aldrich (Bornem, Belgium) andcytochalasin D (cytoD) from Biosource (Nivelles, Belgium). In case ofpotassium depletion, cells were washed once with potassium-free buffer(140 mM NaCl, 20 mM Hepes, 1 mM CaCl2, 1 mM MgCl2, 1 mg/ml d-glucose,pH 7.4), then with hypotonic buffer (1:1 potassium-free buffer and H2O)during 5 min and again 3 times with potassium-free buffer. Humantransferrin-AlexaFluor633 (hTF) and BODIPY FL C5-lactosylceramide (LacCer)were purchased from Molecular Probes (Merelbeke, Belgium).

The used cell lines were COS-7 cells (African green monkey kidneyfibroblast cell line; ATCC number CRL-1651), Vero cells (African greenmonkey kidney epithelial cell line; ATCC number CCL-81), HuH-7 cells(human hepatocellular carcinoma cell line; HSRRB number JCRB 0403) andthe D407 RPE (retinal pigment epithelium) cell line [5], which was a kind giftfrom Dr Richard Hunt (University of South Carolina, Medical School,Columbia, USA).

The cytotoxicity of the different inhibition protocols was evaluated usingthe non-radioactive EZ4U cell proliferation and cytotoxicity assay kit(Biomedica, Vienna, Austria).

For the inhibition and specificity studies, cells were seeded at a celldensity of 2.5×104 cells per cm2 on sterile MatTek coverslip (1.5)-bottomdishes (MatTek corporation, MA, USA) 24 h prior to the experiment.

The cells were subsequently incubated with the appropriate concentra-tion of inhibitor in OptiMEM or depleted of potassium for the appropriatetime. Next, cells were incubated with 25 µg/ml hTf or 1.35 µM LacCer for10 min at 37 °C in the presence of the appropriate inhibitor or buffer. hTF andLacCer uptake, respectively markers for clathrin dependent endocytosis [6]and clathrin independent endocytosis [7], was then qualitatively evaluatedfor the different conditions by the use of confocal laser scanning microscopy(CLSM).

Results and discussion

The results from this work clearly show that one should take extra carewhen using endocytic inhibitors to perturb endocytic pathways. Theinhibitory efficiency appears to be strongly cell type dependent, and so isthe concentration threshold of cellular toxicity. Additionally, we found thatthe inhibition is not always as specific as has been suggested in literature.Finally, we also demonstrated that the chemical compounds can exhibit sideeffects such as dramatic changes in cellular morphology.

Conclusion

Despite the difficulties as mentioned above, it is our opinion that the useof endocytotic inhibitors is still a very attractive and useful approach forstudying the uptake mechanisms of nucleic acid containing particles, butonly when the appropriate control experiments are performed to assessinhibitor toxicity, efficacy and specificity. Additionally, combining theseinhibitors with complementary tools, such as fluorescence dual colour co-localization studies or suppression of specific endocytic pathways throughthe introduction of dominant negative mutants or RNAi, is a necessity todraw reliable conclusions.

Acknowledgements

Dries Vercauteren is a doctoral fellow of the Institute for the Promotion ofInnovation through Science and Technology in Flanders (IWT), Belgium. NiekN. Sanders is supported by the Fund for Scientific Research— Flanders (FWO).Kevin Braeckmans is a Postdoctoral Fellow of the Fund for Scientific Research,Flanders (Belgium).The European Commission is thanked for fundingthrough the Integrated 6th Framework Programme MediTrans.

References

[1] R. Wattiaux, N. Laurent, S. Wattiaux-De Coninck, M. Jadot, Endosomes, lysosomes:their implication in gene transfer. Adv. Drug Deliv. Rev. 41 (2000) 201–208.

[2] J. Rejman, A. Bragonzi, M. Conese, Role of clathrin- and caveolae-mediatedendocytosis in gene transfer mediated by lipo- and polyplexes, Molec. Ther. 12(2005) 468–474.

[3] M. Mano, C. Teodosio, A. Paiva, S. Simoes, M.C.P. de Lima, On the mechanisms ofthe internalization of S4(13)-PV cell-penetrating peptide, Biochem. J. 390 (2005)603–612.

[4] K. von Gersdorff, N.N. Sanders, R. Vandenbroucke, S.C. De Smedt, E. Wagner, M.Ogris, The internalization route resulting in successful gene expression depends onpolyethylenimine both cell line and polyplex type, Molec. Ther. 14 (2006) 745–753.

[5] A.A. Davis, P.S. Bernstein, D. Bok, J. Turner, M. Nachtigal, R.C. Hunt, A humanretinal-pigment epithelial-cell line that retains epithelial characteristics afterprolonged culture, Investig. Ophthalmol. Vis. Sci. 36 (1995) 955–964.

[6] S.K. Rodal, G. Skretting, O. Garred, F. Vilhardt, B. van Deurs, K. Sandvig,Extraction of cholesterol with methyl-beta -cyclodextrin perturbs formation ofclathrin-coated endocytic vesicles, Mol. Biol. Cell. 10 (1999) 961–974.

[7] V. Puri, R. Watanabe, R.D. Singh, M. Dominguez, J.C. Brown, C.L. Wheatley, D.L.Marks, R.E. Pagano, Clathrin-dependent and -independent internalization ofplasma membrane sphingolipids initiates two golgi targeting pathways, J. Cell Biol.154 (2001) 535–547.

doi:10.1016/j.jconrel.2008.09.051

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