Korean J Radiol.  2014 Aug;15(4):411-422. 10.3348/kjr.2014.15.4.411.

Trastuzumab-Conjugated Liposome-Coated Fluorescent Magnetic Nanoparticles to Target Breast Cancer

Affiliations
  • 1Department of Radiology, Seoul National University Bundang Hospital, Seongnam 463-707, Korea. kimsmlms@daum.net
  • 2Nanoimaging and Therapy Research Center, Institute of Nanoconvergence, Advanced Institutes of Convergence Technology, Seoul National University, Suwon 443-270, Korea.
  • 3NanoBio Materials Chemistry Lab., Department of Applied Bioscience, CHA University, Pocheon 487-010, Korea.
  • 4Program in Nano Science and Technology, Department of Transdisciplinary Studies, Seoul National University Graduate School of Convergence Science and Technology, Suwon 443-270, Korea.

Abstract


OBJECTIVE
To synthesize mesoporous silica-core-shell magnetic nanoparticles (MNPs) encapsulated by liposomes (Lipo [MNP@m-SiO2]) in order to enhance their stability, allow them to be used in any buffer solution, and to produce trastuzumab-conjugated (Lipo[MNP@m-SiO2]-Her2Ab) nanoparticles to be utilized in vitro for the targeting of breast cancer.
MATERIALS AND METHODS
The physiochemical characteristics of Lipo[MNP@m-SiO2] were assessed in terms of size, morphological features, and in vitro safety. The multimodal imaging properties of the organic dye incorporated into Lipo[MNP@m-SiO2] were assessed with both in vitro fluorescence and MR imaging. The specific targeting ability of trastuzumab (Her2/neu antibody, Herceptin(R))-conjugated Lipo[MNP@m-SiO2] for Her2/neu-positive breast cancer cells was also evaluated with fluorescence and MR imaging.
RESULTS
We obtained uniformly-sized and evenly distributed Lipo[MNP@m-SiO2] that demonstrated biological stability, while not disrupting cell viability. Her2/neu-positive breast cancer cell targeting by trastuzumab-conjugated Lipo[MNP@m-SiO2] was observed by in vitro fluorescence and MR imaging.
CONCLUSION
Trastuzumab-conjugated Lipo[MNP@m-SiO2] is a potential treatment tool for targeted drug delivery in Her2/neu-positive breast cancer.

Keyword

Breast cancer; Drug delivery; Iron oxide nanoparticles; Magnetic resonance imaging; Trastuzumab

MeSH Terms

3T3 Cells
Animals
Antibodies, Monoclonal, Humanized/*administration & dosage
Antineoplastic Agents/*administration & dosage
Breast Neoplasms/chemistry/*drug therapy
Cell Line, Tumor
Drug Delivery Systems/methods
Female
Ferric Compounds/chemistry
Humans
Liposomes
Magnetite Nanoparticles/administration & dosage/*chemistry
Mice
Molecular Targeted Therapy/methods
Nanoconjugates/administration & dosage/*chemistry
Nanoparticles/chemistry
*Receptor, erbB-2/immunology
Silicon Dioxide/administration & dosage/*chemical synthesis/chemistry
Antibodies, Monoclonal, Humanized
Antineoplastic Agents
Ferric Compounds
Liposomes
Magnetite Nanoparticles
Nanoconjugates
Silicon Dioxide
Receptor, erbB-2

Figure

  • Fig. 1 Structural and magnetic characterization of prepared Fe3O4 magnetic nanoparticles (MNPs).A. Magnetic nanoparticles obtained by seed-mediated growth had narrow size distribution and consisted of single domain. B. X-ray powder diffractogram of 16-nm MNP. MNPs exhibited similar pattern, confirming their ferrite structure and crystal sizes, measured by fitting major peak (311), were in agreement with transmission electron microscopy estimation. C. Magnetic properties of MNP determined with vibrating sample magnetometer. Particles were superparamagnetic at 27℃. Relaxivity (r2) was measured at 0.47 T and 40℃. 16-nm MNP showed high r2 of 320 s-1·mM-1 (metal).

  • Fig. 2 Synthesis and characterization of Lipo[MNP@m-SiO2] particle.A. Transmission electron microscopy image of MNP@m-SiO2 particles. Black core dot represents 16-nm Fe3O4 magnetic nanoparticle and wrinkled shell in grey represents m-SiO2. B. Scanning electron microscopy image of Lipo[MNP@m-SiO2] showing monodispersed size and shape. C. Simple synthetic scheme for Lipo[MNP@m-SiO2]. D. Size distribution and surface charge of prepared MNP@m-SiO2, liposome, and Lipo[MNP@m-SiO2] were investigated by dynamic light scattering.

  • Fig. 3 Synthetic scheme for Her2/neu antibody conjugation onto prepared Lipo(TR)[MNP@m-SiO2(FITC)].Synthesized Lipo(TR) [MNP@m-SiO2(FITC)] contained active amine site for DPPE lipid during preparatory steps. To terminate maleimide functional group on particle, commercially available Sulfo-SMCC was used and reacted with Lipo(TR)[MNP@m-SiO2(FITC)] by substitution reaction. Maleimide functional group could then be easily attached to thiol group of Her2/neu antibody after inducing thiolation of antibody with Traut's reagent. DPPC = 1,2-dipalmitory-sn-glycero-3-phosphatidycholine, DPPE = 1,2-dipalmitory-sn-glycero-3-phosphoethanolamine, Sulfo-SMCC = Sulfo-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate

  • Fig. 4 Magnetic nanoparticle cytotoxicity assay.Normal (3T3 fibroblast, white bar) and cancer (SKBR-3, black bar) cells were incubated for 24 hours in presence of varying amounts of particles. Cellular viability was then measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide assay using [MNP@m-SiO2] (A) and Lipo[MNP@m-SiO2]MNP@ (B).

  • Fig. 5 Specific targeting of breast cancer cells using Her2/neu antibody conjugated magnetic nanoparticles (MNPs).Dual fluorescent imaging using confocal laser scanning microscopy (CLSM) was performed on SKBR-3 breast cancer cells after treatment with bare Lipo(TR)[MNP@m-SiO2(FITC)] without antibody conjugation and Lipo(TR)[MNP@m-SiO2(FITC)]-Her2Ab (A); negative control Lipo(TR)[MNP@m-SiO2(FITC)]-cetuximab (B) carrying green and red organic dyes were subjected to fluorescence imaging by using CLSM. Uptake of MNPs through endocytosis was hindered by decreasing incubation temperature for SKBR-3 cells from 37℃ to 4℃ (C). Green = FITC, Red = hydrophilic regions, Blue = DAPI/nuclei

  • Fig. 6 MR imaging of breast cancer cells after targeting with Lipo[MNP@m-SiO2]-Her2Ab.T2 weighted images of SKBR-3 breast cancer cells after treatment with bare (Lipo[MNP@m-SiO2]) and trastuzumab conjugated nanoparticles (Lipo[MNP@m-SiO2]-Her2Ab) (A) T2 relaxometry. T2 weighted images of Lipo[MNP@m-SiO2] with and without trastuzumab conjugation. When regions of interest (ROI) was placed in cell pellet, T2 value can be automatically calculated (B). (C) Single-exponential fit of mean signal intensity versus echo time (TE) from Lipo[MNP@m-SiO2] with and without trastuzumab conjugation in different axial slices. Data as mean intensity with ROI with standard deviation in intensities of pixel.


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