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Biomimetic Delivery Strategies at the Urothelium: Targeted Cytoinvasion in Bladder Cancer Cells via Lectin Bioconjugates

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ABSTRACT

Purpose

Urothelial cells, including bladder cancer (BCa) cells, represent a highly valuable but challenging target for localized antineoplastic therapy. This study describes a novel, biomimetic approach to improve intravesical drug delivery, based on glycan-specific targeting. In direct analogy to the invasion mechanism used by uropathogenic bacteria, we evaluate the potential of lectin bioconjugates to facilitate binding and uptake of large payload molecules at this penetration-hostile barrier.

Methods

Wheat germ agglutinin (WGA) served as a targeting ligand and was covalently coupled to fluorescein-labeled bovine serum albumin (fBSA), yielding multivalent protein bioconjugates. Cytoadhesion, uptake and intracellular processing were characterized on a panel of urothelial cell lines of non-malignant and malignant origin.

Results

Conjugation to WGA rendered the fBSA payload protein strongly cytoadhesive, with a clear preference in binding to cancerous cells. The highly specific, lectin-mediated recognition process was followed by rapid internalization, and extensive but non-exclusive accumulation in acid and LAMP-2-positive compartments. Stage of malignancy and mechano-structural cell configuration were important determinants for the sorting between different processing pathways.

Conclusion

Lectin-bioconjugates allow for triggering endogenous uptake routes and influencing the intracellular distribution in BCa cells. They hold considerable promise for enhancing the delivery of small molecule drugs and complex biomolecules in intravesical therapy.

UPEC bacteria invade urothelial cells by FimH-mediated binding to mannosylated membrane components. This process can be mimicked with bioconjugates, consisting of a payload protein and a plant lectin as a carbohydrate-specific targeter. Using model bioconjugates of bovine serum albumin (BSA) and wheat germ agglutinin (WGA), we show that such strategies may be exploited for the design of drug delivery vectors with higher binding and uptake in cancerous bladder cells, and an enhanced control over the sorting to disease-modulated processing pathways

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Abbreviations

aWGA:

Alexa Fluor® 594-labeled wheat germ agglutinin

BCa:

Bladder cancer

bt-fBSA:

Biotinylated fluorescein-labeled bovine serum albumin

DFV:

Discoidal fusiform vesicles

fBSA:

Fluorescein-labeled bovine serum albumin

GEMMA:

Gasphase electrophoretic mobility macromolecular analysis

LAMP:

Lysosome-associated membrane protein

M1/M2 :

Mander’s co-localization coefficients

PCC:

Pearson’s correlation coefficients

RFI:

Relative cell-associated fluorescence intensity

RPE:

R-phycoerythrin

SEM:

Standard error of the mean

UP:

Uroplakin

WGA:

Wheat germ agglutinin

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ASSOCIATED CONTENT

Supplemental data and experimental methods on the physicochemical and initial biological characterization of the conjugate fractions from SEC with a calculation of the normalized cytoadhesive capacity (Table SI) are available in the Supplementary Material. Figure S1 shows a more detailed time-lapse study of bioconjugate uptake in 5637 single cells. Co-localization of fBSA/WGA with the free targeting ligand (aWGA) in SV-HUC-1 monolayers prior and after internalization can be taken from Figure S2.

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Althanstr. 14, Vienna, 1090, Austria

    Lukas Neutsch, Britta Eggenreich, Franz Gabor & Michael Wirth

  2. Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9, Vienna, 1060, Austria

    Ela Herwig, Martina Marchetti-Deschmann & Günter Allmaier

Authors
  1. Lukas Neutsch
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  2. Britta Eggenreich
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  6. Franz Gabor
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  7. Michael Wirth
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Corresponding author

Correspondence to Michael Wirth.

Electronic Supplementary Material

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ESM 1

Supplemental data and experimental methods on the physicochemical and initial biological characterization of the conjugate fractions from SEC with a calculation of the normalized cytoadhesive capacity (Table SI1) are available in the supporting information. Figure SI1 shows a more detailed time-lapse study of bioconjugate uptake in 5637 single cells. Co-localization of fBSA/WGA with the free targeting ligand (aWGA) in SV-HUC-1 monolayers prior and after internalization can be taken from Figure SI2. (DOCX 55074 kb)

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Neutsch, L., Eggenreich, B., Herwig, E. et al. Biomimetic Delivery Strategies at the Urothelium: Targeted Cytoinvasion in Bladder Cancer Cells via Lectin Bioconjugates. Pharm Res 31, 819–832 (2014). https://doi.org/10.1007/s11095-013-1204-3

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