Tuning payload delivery in tumour cylindroids using gold nanoparticles

Journal name:
Nature Nanotechnology
Year published:
Published online


Nanoparticles have great potential as controllable drug delivery vehicles because of their size and modular functionality. Timing and location are important parameters when optimizing nanoparticles for delivery of chemotherapeutics. Here, we show that gold nanoparticles carrying either fluorescein or doxorubicin molecules move and localize differently in an in vitro three-dimensional model of tumour tissue, depending on whether the nanoparticles are positively or negatively charged. Fluorescence microscopy and mathematical modelling show that uptake, not diffusion, is the dominant mechanism in particle delivery. Our results indicate that positive particles may be more effective for drug delivery because they are taken up to a greater extent by proliferating cells. Negative particles, which diffuse more quickly, may perform better when delivering drugs deep into tissues. An understanding of how surface charge can control tissue penetration and drug release may overcome some of the current limitations in drug delivery.

At a glance


  1. Schematic showing the delivery of payload by gold nanoparticles.
    Figure 1: Schematic showing the delivery of payload by gold nanoparticles.

    a, Delivery of payload (green ovals) into tumour cylindroids by gold nanoparticles (gold circles). Cells containing released FITC–SH are in green. Viable cells are shown with smooth, solid boundaries, and necrotic cells have irregular, dashed boundaries. Dashed arrows indicate diffusion and cellular uptake. In cylindroids, nanoparticles are present in the medium outside the boundary of the cell mass. b, Intratumoral delivery by gold nanoparticles following extravasation from the vessel lumen (red circles). c, Mixed monolayer-protected gold nanoparticles loaded with thioalkylated FITC or doxorubicin (DOX). d, Cellular uptake and FITC–SH release by thiol-mediated replacement reactions.

  2. Fluorescence calibration and cellular uptake and release of FITC-AuNPs.
    Figure 2: Fluorescence calibration and cellular uptake and release of FITC–AuNPs.

    a, Normalized fluorescence intensities plotted against nanoparticle concentration. Dotted lines represent linear least-squares fitting results. For all components, fluorescence intensity was linearly proportional to concentration. b,c, Differential interference contrast (DIC) and confocal green fluorescent images of cells in monolayer culture incubated with cationic (b) and anionic (c) gold nanoparticles. Scale bars, 50 μm.

  3. Release of FITC-SH from AuNPs in tumour cylindroids.
    Figure 3: Release of FITC–SH from AuNPs in tumour cylindroids.

    a–f, Green fluorescence images (a,c,e) and corresponding intensity profiles (b,d,f) of cylindroids treated with nanoparticles. Fluorescence images were acquired after incubation with p-FITC–AuNP (a), n-FITC–AuNP (c) and FITC (e) for 11, 21 and 46 h. Dotted circles indicate cylindroid edges. Scale bar, 300 µm. Fluorescence intensity profiles are shown at 11, 21, 38 and 46 h (b,d). g,h, Change of average fluorescence intensities over time in the inner and outer regions of the cylindroids (indicated by grey bars in b and d) after incubation with p-FITC–AuNP (g) and n-FITC–AuNP (h). Errors are standard error of the mean (n = 3). i, Blue fluorescence image of a cylindroid incubated with OPA for 18 h. Scale bar, 200 µm. j, Green confocal fluorescence microscope image of the outer region of the cylindroid treated with p-FITC–AuNP. Scale bar, 20 µm.

  4. Doxorubicin release in cylindroids.
    Figure 4: Doxorubicin release in cylindroids.

    a–c, Red fluorescence images and the corresponding radial intensity profiles of cylindroids treated with positive DOX–AuNP (a), negative DOX–AuNP (b) and free DOX (c). Dotted circles indicate cylindroid edges. Scale bars, 200 µm. d,e, Ratio of average fluorescence intensities in the outer region to those in the inner region of the cylindroids, 30 h after treatment (d) and as a function of time (e). Errors are standard error of the mean, and significance was determined using Student's t-test (*P < 0.05; n = 3).

  5. Effect of surface charge on diffusivity through extracellular matrix material.
    Figure 5: Effect of surface charge on diffusivity through extracellular matrix material.

    a, Schematic representation of loading module filled with Matrigel. b, Green fluorescence images acquired from the bottom of the loading module 4 and 24 h after adding particles. Dotted lines at both ends indicate the Matrigel edges. Scale bar, 500 µm. c, Fluorescence intensity profiles in Matrigel along the x-axis as a function of time (symbols) and intensity profiles generated by fitting to a Fickian diffusion model (solid lines). d, Comparison of diffusion coefficients of FITC, cationic and anionic gold nanoparticles in Matrigel (*P < 0.005; n = 3).

  6. Rate constants of cellular uptake and predictions of particle and ligand distribution in tumours.
    Figure 6: Rate constants of cellular uptake and predictions of particle and ligand distribution in tumours.

    a,b, Fluorescence intensity profiles measured in fluorescence images of cylindroids (symbols) and those predicted by the mathematical model (solid lines). Concentrations were normalized by the maximum concentration of FITC–AuNP in the cylindroids. c, Independent concentration profiles of fluorophore bound to nanoparticles (FITC–AuNP) and released fluorophore ligand (FITC–SH) predicted by the computation model. d, Comparison of forward (k1) and reverse (k2) rate constants in different regions. Errors are standard error of the mean, and significance was determined using Student's t-test (*P < 0.05). e,f, Modelled concentrations of FITC–AuNPs and released FITC–SH as a function of distance from blood vessels of a hypothetical tumour. Clearance half-life of FITC–AuNPs was assumed to be 3 h. g, Average concentrations of FITC–AuNPs in the regions 10–70 µm (proliferating) and 75–120 µm (quiescent) from the blood vessel wall as a function of time. h, Average concentrations of released FITC in deep tumour regions (1.0–1.5 mm) as a function of time.


  1. Hicks, K. O., Pruijn, F. B., Sturman, J. R., Denny, W. A. & Wilson, W. R. Multicellular resistance to tirapazamine is due to restricted extravascular transport: a pharmacokinetic/pharmacodynamic study in HT29 multicellular layer cultures. Cancer Res. 63, 59705977 (2003).
  2. Lankelma, J. et al. Doxorubicin gradients in human breast cancer. Clin. Cancer Res. 5, 17031707 (1999).
  3. Minchinton, A. I. & Tannock, I. F. Drug penetration in solid tumours. Nature Rev. Cancer 6, 583592 (2006).
  4. Jain, R. K. Transport of molecules, particles and cells in solid tumors. Annu. Rev. Biomed. Eng. 1, 241263 (1999).
  5. Primeau, A. J., Rendon, A., Hedley, D., Lilge, L. & Tannock, I. F. The distribution of the anticancer drug doxorubicin in relation to blood vessels in solid tumors. Clin. Cancer Res. 11, 87828788 (2005).
  6. Vaupel, P., Kallinowski, F. & Okunieff, P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res. 49, 64496465 (1989).
  7. Tredan, O., Galmarini, C. M., Patel, K. & Tannock, I. F. Drug resistance and the solid tumor microenvironment. J. Natl Cancer Inst. 99, 14411454 (2007).
  8. Grantab, R., Sivananthan, S. & Tannock, I. F. The penetration of anticancer drugs through tumor tissue as a function of cellular adhesion and packing density of tumor cells. Cancer Res. 66, 10331039 (2006).
  9. Goldacre, R. J. & Sylven, B. On the access of blood-borne dyes to various tumour regions. Br. J. Cancer 16, 306322 (1962).
  10. You, C. C., Verma, A. & Rotello, V. M. Engineering the nanoparticle–biomacromolecule interface. Soft Matter 2, 190204 (2006).
  11. You, C. C., De, M., Han, G. & Rotello, V. M. Tunable inhibition and denaturation of alpha-chymotrypsin with amino acid-functionalized gold nanoparticles. J. Am. Chem. Soc. 127, 1287312881 (2005).
  12. Goodman, C. M. et al. DNA-binding by functionalized gold nanoparticles: mechanism and structural requirements. Chem. Biol. Drug. Des. 67, 297304 (2006).
  13. McIntosh, C. M. et al. Inhibition of DNA transcription using cationic mixed monolayer protected gold clusters. J. Am. Chem. Soc. 123, 76267629 (2001).
  14. You, C. C., De, M., Han, G. & Rotello, V. M. Tunable inhibition and denaturation of alpha-chymotrypsin with amino acid-functionalized gold nanoparticles. J. Am. Chem. Soc. 127, 1287312881 (2005).
  15. Thomas, M. & Klibanov, A. M. Conjugation to gold nanoparticles enhances polyethylenimine's transfer of plasmid DNA into mammalian cells. Proc. Natl Acad. Sci. USA 100, 91389143 (2003).
  16. Rosi, N. L. et al. Oligonucleotide-modified gold nanoparticles for intracellular gene regulation. Science 312, 10271030 (2006).
  17. Jain, P. K., El-Sayed, I. H. & El-Sayed, M. A. Au nanoparticles target cancer. Nano Today 2, 1829 (2007).
  18. Chithrani, B. D. & Chan, W. C. Elucidating the mechanism of cellular uptake and removal of protein-coated gold nanoparticles of different sizes and shapes. Nano Lett. 7, 15421550 (2007).
  19. Kasinskas, R. W. & Forbes, N. S. Salmonella typhimurium specifically chemotax and proliferate in heterogeneous tumor tissue in vitro . Biotechnol. Bioeng. 94, 710721 (2006).
  20. Kim, B. J. & Forbes, N. S. Flux analysis shows that hypoxia-inducible-factor-1-alpha minimally affects intracellular metabolism in tumor spheroids. Biotechnol. Bioeng. 96, 11671182 (2007).
  21. Freyer, J. P. & Sutherland, R. M. Selective dissociation and characterization of cells from different regions of multicell tumor spheroids. Cancer Res. 40, 39563965 (1980).
  22. Kasinskas, R. W. & Forbes, N. S. Salmonella typhimurium lacking ribose chemoreceptors localize in tumor quiescence and induce apoptosis. Cancer Res. 67, 32013209 (2007).
  23. Sutherland, R. M. Cell and environment interactions in tumor microregions—the multicell spheroid model. Science 240, 177184 (1988).
  24. Kim, B. J. & Forbes, N. S. Single-cell analysis demonstrates how nutrient deprivation creates apoptotic and quiescent cell populations in tumor cylindroids. Biotechnol. Bioeng. 101, 797810 (2008).
  25. Helmlinger, G., Yuan, F., Dellian, M. & Jain, R. K. Interstitial pH and pO2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation. Nature Med. 3, 177182 (1997).
  26. De, M., You, C. C., Srivastava, S. & Rotello, V. M. Biomimetic interactions of proteins with functionalized nanoparticies: a thermodynamic study. J. Am. Chem. Soc. 129, 1074710753 (2007).
  27. Chompoosor, A., Han, G. & Rotello, V. M. Charge dependence of ligand release and monolayer stability of gold nanoparticles by biogenic thiols. Bioconjug. Chem. 19, 13421345 (2008).
  28. Hong, R. et al. Glutathione-mediated delivery and release using monolayer protected nanoparticle carriers. J. Am. Chem. Soc. 128, 10781079 (2006).
  29. Jones, D. P. et al. Glutathione measurement in human plasma evaluation of sample collection, storage and derivatization conditions for analysis of dansyl derivatives by HPLC. Clin. Chim. Acta 275, 175184 (1998).
  30. Hassan, S. S. M. & Rechnitz, G. A. Determination of glutathione and glutathione-reductase with a silver sulfide membrane-electrode. Anal. Chem. 54, 19721976 (1982).
  31. Baldwin, A. L., Wu, N. Z. & Stein, D. L. Endothelial surface-charge of intestinal mucosal capillaries and its modulation by dextran. Microvasc. Res. 42, 160178 (1991).
  32. Ghitescu, L. & Fixman, A. Surface-charge distribution on the endothelial-cell of liver sinusoids. J. Cell Biol. 99, 639647 (1984).
  33. Chen, A. M. et al. Oligodeoxynucleotide nanostructure formation in the presence of polypropyleneimine dendrimers and their uptake in breast cancer cells. Nanotechnology 17, 54495460 (2006).
  34. Holzapfel, V., Musyanovych, A., Landfester, K., Lorenz, M. R. & Mailander, V. Preparation of fluorescent carboxyl and amino functionalized polystyrene particles by miniemulsion polymerization as markers for cells. Macromol. Chem. Phys. 206, 24402449 (2005).
  35. Mislick, K. A. & Baldeschwieler, J. D. Evidence for the role of proteoglycans in cation-mediated gene transfer. Proc. Natl Acad. Sci. USA 93, 1234912354 (1996).
  36. Tauskela, J. S. et al. Evaluation of glutathione-sensitive fluorescent dyes in cortical culture. Glia 30, 329341 (2000).
  37. Orwar, O., Fishman, H. A., Ziv, N. E., Scheller, R. H. & Zare, R. N. Use of 2,3-naphthalenedicarboxaldehyde derivatization for single-cell analysis of glutathione by capillary electrophoresis and histochemical-localization ion by fluorescence microscopy. Anal. Chem. 67, 42614268 (1995).
  38. Davies, C. D., Berk, D. A., Pluen, A. & Jain, R. K. Comparison of IgG diffusion and extracellular matrix composition in rhabdomyosarcomas grown in mice versus in vitro as spheroids reveals the role of host stromal cells. Br. J. Cancer 86, 16391644 (2002).
  39. Pluen, A. et al. Role of tumor–host interactions in interstitial diffusion of macromolecules: cranial vs. subcutaneous tumors. Proc. Natl Acad. Sci. USA 98, 46284633 (2001).
  40. Ramanujan, S. et al. Diffusion and convection in collagen gels: implications for transport in the tumor interstitium. Biophys. J. 83, 16501660 (2002).
  41. Kleinman, H. K. et al. Isolation and characterization of type-IV procollagen, laminin, and heparan-sulfate proteoglycan from the Ehs sarcoma. Biochemistry (Mosc) 21, 61886193 (1982).
  42. Boucher, Y., Baxter, L. T. & Jain, R. K. Interstitial pressure gradients in tissue-isolated and subcutaneous tumors: implications for therapy. Cancer Res. 50, 44784484 (1990).
  43. Jain, R. K. Transport of molecules in the tumor interstitium: a review. Cancer Res. 47, 30393051 (1987).
  44. Young, J. S., Lumsden, C. E. & Stalker, A. L. The significance of the tissue pressure of normal testicular and of neoplastic (Brown–Pearce carcinoma) tissue in the rabbit. J. Pathol. Bacteriol. 62, 313333 (1950).
  45. You, C. C., De, M. & Rotello, V. M. Contrasting effects of exterior and interior hydrophobic moieties in the complexation of amino acid functionalized gold clusters with alpha-chymotrypsin. Org. Lett. 7, 56855688 (2005).
  46. Paciotti, G. F. et al. Colloidal gold: a novel nanoparticle vector for tumor directed drug delivery. Drug Deliv. 11, 169183 (2004).

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Author information


  1. Department of Chemical Engineering, University of Massachusetts, Amherst, Massachusetts 01003-9303, USA

    • Byoungjin Kim,
    • Bhushan J. Toley &
    • Neil S. Forbes
  2. Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003-9303, USA

    • Gang Han,
    • Chae-kyu Kim &
    • Vincent M. Rotello


N.F. and V.R. conceived and designed the experiments. B.K., G.H., B.T. and C.K. performed the experiments. G.H. synthesized the FITC nanoparticles. C.K. synthesized the DOX nanoparticles. B.K. performed all cell and cylindroid experiments with FITC nanoparticles and wrote all mathematical models. B.T. performed all cylindroid and cell experiments with DOX nanoparticles. B.K., B.T. and N.F. analysed the data. V.R. contributed materials. B.K. and N.F. co-wrote the paper. All authors discussed the results and commented on the manuscript.

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