Abstract
This article discusses stimuli-responsive poly(ethylene glycol) (PEG)-coated (PEGylated) nanogels and their biomedical applications. Preparation and characterization of stimuli-responsive PEGylated nanogels composed of a crosslinked poly(2-[N,N-diethylamino]ethyl methacrylate) (PEAMA) core and PEG tethered chains are initially described. Stimuli-responsive PEGylated nanogels show unique properties and functions in synchronizing with the reversible volume phase transition of the PEAMA core in response to the extracellular pH (7–6.5) of a tumor environment as well as endosomal/lysosomal pH (6.5–5.0) and temperature. We list several biomedical applications of stimuli-responsive PEGylated nanogels, including 19F magnetic resonance spectroscopic imaging (MRS/I) probe to visualize acidosis (tumor tissue), intracellular drug and siRNA delivery, antennas for cancer photothermal therapy and apoptosis probe for monitoring response to cancer therapy. Thus, stimuli-responsive PEGylated nanogels can be utilized as smart nanomedicines for cancer diagnostics and therapy.
Bibliography
- 1 Kabanov AV, Vinogradov SV: Nanogels as pharmaceutical carriers: finite networks of infinite capabilities. Angew. Chem. Int. Ed.48(30),5418–5429 (2009).Google Scholar
- 2 Yallapu MM, Reddy MK, Labhasetwar V: Nanogels: chemistry to drug delivery. In: Biomedical Applications Nanotechnology. Labhasetwar V, Diandra LL-P (Eds). John Wiley & Sons, Inc., New York, USA, 131–171 (2007).Google Scholar
- 3 Morimoto N, Hasegawa U, Sugawara A, Yamane S, Akiyoshi K: Polysaccharide nanogel engineering: design of nanostructured hydrogel materials and application to biotechnology and medicine. In: Nanotechnology in Carbohydrate Chemistry. Yuasa H (Ed.). Transworld Research Network, 67–87 (2006).Google Scholar
- 4 Vinogradov SV, Bronich TK, Kabanov AV: Nanosized cationic hydrogels for drug delivery: preparation, properties and interactions with cells. Adv. Drug Deliv. Rev.54(1),135–147 (2002).Google Scholar
- 5 Bronich TK, Bontha S, Shlyakhtenko LS, Bromberg L, Hatton TA, Kabanov AV: Template-assisted synthesis of nanogels from Pluronic-modified poly(acrylic acid). J. Drug Target.14(6),357–366 (2006).Google Scholar
- 6 Bontha S, Kabanov AV, Bronich TK: Polymer micelles with cross-linked ionic cores for delivery of anticancer drugs. J. Control. Release114(2),163–174 (2006).Google Scholar
- 7 Bronich TK, Keifer PA, Shlyakhtenko LS, Kabanov AV: Polymer micelle with cross-linked ionic core. J. Am. Chem. Soc.127(23),8236–8237 (2005).Google Scholar
- 8 Matsumura Y, Maeda H: A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res.46(12),6387–6392 (1986).Google Scholar
- 9 Gerweck LE, Seetharaman K: Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer. Cancer Res.56(6),1194–1198 (1996).Google Scholar
- 10 Wike-Hooley JL, Haveman J, Reinhold HS: The relevance of tumour pH to the treatment of malignant disease. Radiother. Oncol.2(4),343–366 (1984).Google Scholar
- 11 Gruenberg J: The endocytic pathway: a mosaic of domains. Nat. Rev. Mol. Cell Biol.2(10),721–730 (2001).Google Scholar
- 12 Clague MJ: Molecular aspects of the endocytic pathway. Biochem. J.336(2),271–282 (1998).Google Scholar
- 13 Mukherjee S, Ghosh RN, Maxfield FR: Endocytosis. Physiol. Rev.77(3),759–803 (1997).Google Scholar
- 14 Duncan R: Drug-polymer conjugates: potential for improved chemotherapy. Anti-Cancer Drugs3(3),175–210 (1992).Google Scholar
- 15 Oishi M, Nakamura T, Jinji Y, Matsuishi K, Nagasaki Y: Multi-stimuli-triggered release of charged dye from smart PEGylated nanogels containing gold nanoparticles to regulate fluorescence signals. J. Mater. Chem.19(33),5909–5912 (2009).Google Scholar
- 16 Oishi M, Nagasaki Y: Synthesis, characterization, and biomedical applications of core-shell-type stimuli-responsive nanogels – nanogel composed of poly[2-(N,N-diethylamino)ethyl methacrylate] core and PEG tethered chains. React. Funct. Polym.67(11),1311–1329 (2007).Google Scholar
- 17 Oishi M, Hayashi H, Itaka K, Kataoka K, Nagasaki Y: pH-responsive PEGylated nanogels as targetable and low invasive endosomolytic agents to induce the enhanced transfection efficiency of nonviral gene vectors. Colloid Polym. Sci.285(9),1055–1060 (2007).Google Scholar
- 18 Oishi M, Miyagawa N, Sakura T, Nagasaki Y: pH-responsive PEGylated nanogel containing platinum nanoparticles: application to on-off regulation of catalytic activity for reactive oxygen species. React. Funct. Polym.67(7),662–668 (2007).Google Scholar
- 19 Hayashi H, Iijima M, Kataoka K, Nagasaki Y: pH-sensitive nanogel possessing reactive PEG tethered chains on the surface. Macromolecules37(14),5389–5396 (2004).Google Scholar
- 20 Oishi M, Sumitani S, Bronich TK, Kabanov AV, Boska MD, Nagasaki Y: Novel tumor-specific 19F-MRS/I nanoprobe based on pH-responsive PEGylated nanogel: pH-dependent 19F-magnetic resonance studies. Chem. Lett.38(2),128–129 (2009).Google Scholar
- 21 Oishi M, Sumitani S, Nagasaki Y: On-off regulation of 19F magnetic resonance signals based on pH-sensitive PEGylated nanogels for potential tumor-specific smart 19F MRI probes. Bioconj. Chem.18(5),1379–1382 (2007).Google Scholar
- 22 Oishi M, Hayashi H, Iijima M, Nagasaki Y: Endosomal release and intracellular delivery of anticancer drugs using pH-sensitive PEGylated nanogels. J. Mater. Chem.17(35),3720–3725 (2007).Google Scholar
- 23 Tamura A, Oishi M, Nagasaki Y: Enhanced cytoplasmic delivery of siRNA using the stabilized polyion complexes based on PEGylated nanogels bearing cross-linked polyamine structure. Biomacromolecules10(7),1818–1827 (2009).Google Scholar
- 24 Oishi M, Tamura A, Nakamura T, Nagasaki Y: A smart nanoprobe based on fluorescence-quenching PEGylated nanogel containing gold nanoparticles for monitoring the cancer response to therapy. Adv. Funct. Mater.19(6),827–834 (2009).Google Scholar
- 25 Poehlein GW: Emulsion polymerization. In: Encyclopedia of Polymer Science and Engineering Second Edition (Volume 6). Mark HF, Bikales NM, Overberger CG, Menges G, Kroschwitz JI (Eds). John Wiley & Sons, Inc., NY, USA, 1–51 (1986).Google Scholar
- 26 Westby MJ: Non-ionic emulsion polymerisation. Colloid Polym. Sci.266(1),46–51 (1998).Google Scholar
- 27 Hoshino F, Sasaki M, Kawaguchi H, Ohtsuka Y: Soap-free latices of polyoxyethylene chain-binding particles. Polym. J.19(4),383–389 (1998).Google Scholar
- 28 Lee AS, Gast AP, Bütün V, Armes SP: Characterizing the structure of pH dependent polyelectrolyte block copolymer micelles. Macromolecules32(13),4302–4310 (1999).Google Scholar
- 29 Kobayashi H, Brechbiel MW: Nano-sized MRI contrast agents with dendrimer cores. Adv. Drug Deliv. Rev.57(15),2271–2286 (2005).Google Scholar
- 30 Caravan P, Ellison JJ, McMurry TJ, Lauffer RB: Gadolinium (III) chelates as MRI contrast agents: structure, dynamics, and applications. Chem. Rev.99(9),2293–2352 (1999).Google Scholar
- 31 Yu J-X, Kodibagkar VD, Cui W, Mason RP: 19F: a versatile reporter for non-invasive physiology and pharmacology using magnetic resonance. Curr. Med. Chem.12(7),819–848 (2005).Google Scholar
- 32 Buetuen V, Billingham NC, Armes SP: Unusual aggregation behavior of a novel tertiary amine methacrylate-based diblock copolymer: formation of micelles and reverse micelles in aqueous solution. J. Am. Chem. Soc.120(45),11818–11819 (1998).Google Scholar
- 33 Duncan R: The dawning era of polymer therapeutics. Nat. Rev. Drug Discov.2(5),347–360 (2003).Google Scholar
- 34 Kopecek J: Smart and genetically engineered biomaterials and drug delivery systems. Eur. J. Pharm. Sci.20(1),1–16 (2003).Google Scholar
- 35 Maeda H: SMANCS and polymer-conjugated macromolecular drugs: advantages in cancer chemotherapy. Adv. Drug Deliv. Rev.46(1–3) 169–185 (2001).Google Scholar
- 36 Avichechter D, Schechter B, Arnon R: Functional polymers in drug delivery: carrier-supported CDDP (cis-platin) complexes of polycarboxylates – effect on human ovarian carcinoma. React. Funct. Polym.36(1),59–69 (1998).Google Scholar
- 37 Bogdanov A Jr, Wright SC, Marecos EM et al.: A long-circulating co-polymer in “passive targeting” to solid tumors. J. Drug Target.4(5),321–330 (1997).Google Scholar
- 38 Lee CM, Tanaka T, Murai T et al.: Novel chondroitin sulfate-binding cationic liposomes loaded with cisplatin efficiently suppress the local growth and liver metastasis of tumor cells in vivo. Cancer Res.62(15),4282–4288 (2002).Google Scholar
- 39 Gordon AN, Fleagle JT, Guthrie D, Parkin DE, Gore ME, Lacave A: Recurrent epithelial ovarian carcinoma: a randomized phase III study of pegylated liposomal doxorubicin versus topotecan. J. Clin. Oncol.19(14),3312–3322 (2001).Google Scholar
- 40 Newman MS, Colbern GT, Working PK, Engbers C, Amntea MA: Comparative pharmacokinetics, tissue distribution, and therapeutic effectiveness of cisplatin encapsulated in long-circulating, pegylated liposomes (SPI-077) in tumor-bearing mice. Cancer Chemother. Pharmacol.43(1),1–7 (1999).Google Scholar
- 41 Nakanishi T, Fukushima S, Okano K et al.: Development of the polymer micelle carrier system for doxorubicin. J. Control. Release74(1–3),295–302 (2001).Google Scholar
- 42 Yokoyama M, Okano T, Sakurai Y, Suwa S, Kataoka K: Introduction of cisplatin into polymeric micelle. J. Control. Release39(2–3) 351–356 (1996).Google Scholar
- 43 Maeda H, Sawa T, Konno T: Mechanism of tumor-targeted delivery of macromolecular drugs, including the EPR effect in solid tumor and clinical overview of the prototype polymeric drug SMANCS. J. Control. Release74(1–3),47–61 (2001).Google Scholar
- 44 Lian T, Ho RJY: Trends and developments in liposome drug delivery systems. J. Pharm. Sci.90(6),667–680 (2001).Google Scholar
- 45 Vasey PA, Kaye SB, Morrison R et al.: Phase I clinical and pharmacokinetic study of PK1 [N-(2-hydroxypropyl)methacrylamide copolymer doxorubicin]: first member of a new class of chemotherapeutic agents-drug-polymer conjugates. Clin. Cancer Res.5(1),83–94 (1999).Google Scholar
- 46 Tsukioka Y, Matsumura Y, Hamaguchi T, Koike H, Moriyasu F, Kakizone T: Pharmaceutical and biomedical differences between micellar doxorubicin (NK911) and liposomal doxorubicin (Doxil). Cancer Sci.93(10),1145–1153 (2002).Google Scholar
- 47 Kwon G, Naito M, Yokoyama M, Okano T, Sakurai Y, Kataoka K: Block copolymer micelles for drug delivery: loading and release of doxorubicin. J. Control. Release48(2–3),195–201 (1997).Google Scholar
- 48 Gillies ER, Frechet JM: pH-responsive copolymer assemblies for controlled release of doxorubicin. Bioconj. Chem.16(2),361–368 (2005).Google Scholar
- 49 Bae Y, Fukushima S, Harada A, Kataoka K: Design of environment-sensitive supramolecular assemblies for intracellular drug delivery: polymeric micelles that are responsive to intracellular pH change. Angew. Chem. Int. Ed.42(38),4640–4643 (2003).Google Scholar
- 50 Wirth T, Kuhnel F, Fleischmann-Mundt B et al.: Telomerase-dependent virotherapy overcomes resistance of hepatocellular carcinomas against chemotherapy and tumor necrosis factor–related apoptosis-inducing ligand by elimination of Mcl-1. Cancer Res.65(16),7393–7402 (2005).Google Scholar
- 51 Huesker M, Folmer Y, Schneider M, Fulda C, Blum HE, Hafkemeyer P: Reversal of drug resistance of hepatocellular carcinoma cells by adenoviral delivery of anti-MDR1 ribozymes. Hepatology36(4),874–884 (2002).Google Scholar
- 52 Omelyanenko V, Kopecková P, Kopecek J: Targetable HPMA copolymer–adriamycin conjugates. recognition, internalization, and subcellular fate. J. Control. Release53(1–3),25–37 (1998).Google Scholar
- 53 Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T: Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature411(6836),494–498 (2001).Google Scholar
- 54 Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC: Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature391(6669),806 (1998).Google Scholar
- 55 Paroo Z, Corey DR: Challenges for RNAi in vivo. Trends Biotechnol.22(8),390–394 (2004).Google Scholar
- 56 Itaka K, Kanayama N, Nishiyama N et al.: Supramolecular nanocarrier of siRNA from PEG-based block catiomer carrying diamine side chain with distinctive pKa directed to enhance intracellular gene silencing. J. Am. Chem. Soc.126(42),13612–13613 (2004).Google Scholar
- 57 Schiffelers RM, Ansari A, Xu J et al.: Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic Acids Res.32(19),E149 (2004).Google Scholar
- 58 Oishi M, Nagasaki Y, Itaka K, Nishiyama N, Kataoka K: Lactosylated poly(ethylene glycol)–siRNA conjugate through acid-labile β-thiopropionate linkage to construct pH-sensitive polyion complex micelles achieving enhanced gene silencing in hepatoma cells. J. Am. Chem. Soc.127(6),1624–1625 (2005).Google Scholar
- 59 Bartlett DW, Davis ME: Physicochemical and biological characterization of targeted, nucleic acid-containing nanoparticles. Bioconj. Chem.18(2),456–468 (2007).Google Scholar
- 60 de Wolf HK, Snel CJ, Verbaan FJ, Schiffelers RM, Hennink WE, Storm G: Effect of cationic carriers on the pharmacokinetics and tumor localization of nucleic acids after intravenous administration. Int. J. Pharm.331,167–175 (2007).Google Scholar
- 61 Sonawane ND, Szoka FC Jr, Verkman AS: Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes. J. Biol. Chem.278(45),44826–44831 (2003).Google Scholar
- 62 Boussif O, Lezoualc’h F, Zanta MA et al.: A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl Acad. Sci. USA92(16),7297–7301 (1995).Google Scholar
- 63 Vamvakaki M, Billingham NC, Armes SP: Synthesis of controlled structure water-soluble diblock copolymers via oxyanionic polymerization. Macromolecules32(6),2088–2090 (1999).Google Scholar
- 64 Nagasaki Y, Sato Y, Kato M: A novel synthesis of semitelechelic functional poly(methacrylate)s through an alcoholate initiated polymerization. Synthesis of poly[2-(N,N-diethylaminoethyl) methacrylate] macromonomer. Macromol. Rapid Cornmun.18(9),827–835 (1997).Google Scholar
- 65 Chittimalla C, Zammut-Italiano L, Zuber G, Behr J-P: Monomolecular DNA nanoparticles for intravenous delivery of genes. J. Am. Chem. Soc.127(32),11436–11441 (2005).Google Scholar
- 66 El-Sayed IH, Huang X, El-Sayed MA: Selective laser photo-thermal therapy of epithelial carcinoma using anti-EGFR antibody conjugated gold nanoparticles. Cancer Lett.239(1),129–135 (2006).Google Scholar
- 67 Zharov VP, Galitovsky V, Viegas M: Photothermal detection of local thermal effects during selective nanophotothermolysis. Appl. Phys. Lett.83(24),4897–4899 (2003).Google Scholar
- 68 Loo C, Lowery A, Halas N, West J, Drezek R: Immuno targeted nanoshells for integrated cancer imaging and therapy. Nano Lett.5(4),709–711 (2005).Google Scholar
- 69 Hirsch LR, Stafford RJ, Bankson JA et al.: Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance. Proc. Natl Acad. Sci. USA100(23),13549–13554 (2003).Google Scholar
- 70 Kawano T, Niidome Y, Mori T, Katayama Y, Niidome T: PNIPAM gel-coated gold nanorods for targeted delivery responding to a near-infrared laser. Bioconj. Chem.20(2),209–210 (2009).Google Scholar
- 71 Huang X, El-Sayed IH, Qian W, El-Sayed MA: Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. J. Am. Chem. Soc.12(6),2115–2120 (2006).Google Scholar
- 72 Niidome T, Yamagata M, Okamoto Y et al.: PEG-modified gold nanorods with a stealth character for in vivo applications. J. Control. Release114(3),343–347 (2006).Google Scholar
- 73 Link S, El-Sayed MA: Spectral properties and relaxation dynamics of surface plasmon electronic oscillations in gold and silver nanodots and nanorods. J. Phys. Chem. B103(4),8410–8426 (1999).Google Scholar
- 74 Boyer D, Tamarat P, Maali A, Lounis B, Orrit M: Phtothermal imaging of nanometer-sized metal particles among scatteres. Science297(5584),1160–1163 (2002).Google Scholar
- 75 Oishi M, Nakaogami J, Ishii T, Nagasaki Y: Smart PEGylated gold nanoparticles for the cytoplasmic delivery of siRNA to induce enhanced gene silencing. Chem. Lett.35(9),1046–1047 (2006).Google Scholar
- 76 Hong R, Han G, Fernandez JM, Kim B-J, Forbes NS, Rotello VM: Glutathione-mediated delivery and release using monolayer protected nanoparticle carriers. J. Am. Chem. Soc.128(4),1078–1079 (2006).Google Scholar
- 77 Verma A, Simard JM, Worrall JW, Rotello VM: Tunable reactivation of nanoparticle-inhibited β-galactosidase by glutathione at intracellular concentrations. J. Am. Chem. Soc.126(43),13987–13991 (2004).Google Scholar
- 78 Meister A, Anderson ME: Glutathione. Annu. Rev. Biochem.52,711–760 (1983).Google Scholar
- 79 Oishi M, Hayashi H, Uno T, Ishii T, Iijima M, Nagasaki Y: One-pot synthesis of pH-responsive PEGylated nanogels containing gold nanoparticles by autoreduction of chloroaurate ions within nanoreactors. Macromol. Chem. Phys.208(11),1176–1182 (2007).Google Scholar
- 80 Oishi M, Nakamura T, Jinji Y, Matsuishi K, Nagasaki Y: Multi-stimuli-triggered release of charged dye from smart PEGylated nanogels containing gold nanoparticles to regulate fluorescence signals. J. Mater. Chem.19(33),5909–5912 (2009).Google Scholar
- 81 Haba Y, Kojima C, Harada A, Ura T, Horinaka H, Kono K: Preparation of poly(ethylene glycol)-modified poly(amido amine) dendrimers encapsulating gold nanoparticles and their heat-generating ability. Langmuir23(10),5243–5246 (2007).Google Scholar
- 82 Schoenberger J, Bauer J, Moosbauer J, Eilles C, Grimm D: Innovative strategies in in vivo apoptosis imaging. Curr. Med. Chem.15(2),187–194 (2008).Google Scholar
- 83 Thornberry NA, Lazebnik Y: Caspases: enemies within. Science281(5381),1312–1316 (1998).Google Scholar
- 84 Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier GG, Earnshaw WC: Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature371(6495),346–347 (1994).Google Scholar
- 85 Wyllie AH: Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature284(5756),555–556 (1980).Google Scholar
- 86 Myc A, Majoros IJ, Thomas TP, Baker JR Jr: Dendrimer-based targeted delivery of an apoptotic sensor in cancer cells. Biomacromolecules8(1),13–18 (2007).Google Scholar
- 87 Stefflova K, Chen J, Marotta D, Li H, Zheng G: Photodynamic therapy agent with a built-in apoptosis sensor for evaluating its own therapeutic outcome in situ. J. Med. Chem.49(13),3850–3856 (2006).Google Scholar
- 88 Wu Y, Xing D, Luo S, Tang Y, Chen Q: Detection of caspase-3 activation in single cells by fluorescence resonance energy transfer during photodynamic therapy induced apoptosis. Cancer Lett.235(2),239–247 (2006).Google Scholar
- 89 Kim K, Lee M, Park H et al.: Cell-permeable and biocompatible polymeric nanoparticles for apoptosis imaging. J. Am. Chem. Soc.128(11),3490–3495 (2006).Google Scholar
- 90 Tanford C, Nozaki Y, Rohde MF: Size and shape of globular micelles formed in aqueous solution by n-alkyl polyoxyethylene ethers. J. Phys. Chem.81(16),1555–1560 (1977).Google Scholar
- 91 Gueroui Z, Libchaber A: Single-molecule measurements of gold-quenched quantum dots. Phys. Rev. Lett.93(16),166108 (2004).Google Scholar
- 92 Zhang L, Torgerson TR, Liu X-Y et al.: Preparation of functionally active cell-permeable peptides by single-step ligation of two peptide modules. Proc. Natl Acad. Sci. USA95(16),9148–9189 (1998).Google Scholar
- 93 Agasti SS, You C-C, Arumugam P, Rotello VM: Structural control of the monolayer stability of water-soluble gold nanoparticles. J. Mater. Chem.18(1),70–73 (2008).Google Scholar
- 94 Isaacs SR, Cutler EC, Park J-S, Lee TR, Shon Y-S: Synthesis of tetraoctylammonium-protected gold nanoparticles with improved stability. Langmuir21(13),5689–5692 (2005).Google Scholar
- 95 Templeton AC, Hostetler MJ, Kraft CT, Murray RW: Reactivity of monolayer-protected gold cluster molecules: steric effects. J. Am. Chem. Soc.120(8),1906–1911 (1998).Google Scholar
- 96 Lee S, Cha E-J, Park K et al.: A near-infrared-fluorescence-quenched gold-nanoparticle imaging probe for in vivo drug screening and protease activity determination. Angwe. Chem. Int. Ed.47(15),2804–2807 (2008).Google Scholar
- 97 Oishi M, Nagasaki Y, Nishiyama N et al.: Enhanced growth inhibition of hepatic multicellular tumor spheroids by lactosylated poly(ethylene glycol)-siRNA conjugate formulated in PEGylated polyplexes. ChemMedChem2(9),1290–1297 (2007).Google Scholar
- 98 Thielecke H, Mack A, Robitzki A: A multicellular spheroid-based sensor for anti-cancer therapeutics. Biosens. Bioelectron.16(4–5),261–269 (2001).Google Scholar
- 99 Desoize B, Jardillier J-C: Multicellular resistance: a paradigm for clinical resistance? Crit. Rev. Oncol. Hemat.36(2–3),193–207 (2000).Google Scholar
- 100 Hamilton G: Multicellular spheroids as an in vitro tumor model. Cancer Lett.131(1),29–34 (1998).Google Scholar
- 101 Santini MT, Rainaldi G, Indovina PL: Multicellular tumour spheroids in radiation biology. Int. J. Radiat. Biol.75(6),787–799 (1999).Google Scholar
- 102 Sutherland RM: Cell and environment interactions in tumor microregions: the multicell spheroid model. Science240(4849),177–184 (1988).Google Scholar
- 103 Moscona A: The development in vitro chimeric aggdegates of dissociated embryonic chick and mouse cells. Proc. Natl Acad. Sci. USA43(1),184–194 (1957).Google Scholar
- 104 Falcieri E, Matelli AM, Bareggi R, Cataldi A, Cocco L: The protein kinase inhibitor staurosporine induces morphological changes typical of apoptosis in MOLT-4 cells without concomitant DNA fragmentation. Biochem. Biophys. Res. Commun.193(1),19–25 (1993).Google Scholar

