Reducible Polyamidoamine-magnetic Iron Oxide Self-assembled Nanoparticles for Doxorubicin Delivery

Wan Wenbing (Department of Mechanical Engineering, University of Manitoba, Winnipeg MB R3T 2N2, Canada)
Xing Malcolm M.Q. (Department of Mechanical Engineering, University of Manitoba, Winnipeg MB R3T 2N2, Canada)
Jun Chen (u of Manitoba)

Introduction

Magnetic iron oxide (IO) nanoparticles are extensively studied as a promising theranostic candidate for magnetically targeted drug delivery and molecular magnetic resonance imaging (MRI) diagnosis. However, these nanoparticles have limits in the stability because of hydrophobic coating. The amphiphilic copolymers coated oleic acid(OA) on magnetic nanoparticles(OA-SPIONS) were reported with both good biodegradability and MRI contrast effect but not for a controlled release or environmental response. To address this issue, we introduce reducible polyamidoamine (rPAA) to coat on the superparamagnetic iron oxide nanoparticles (SPIONs) and to form rPAA-SPIONs, which is a reducible copolymer self-assembled nanoparticles and can provide efficient drug loading, reduction-responsive drug release,and imaging capability especially for doxorubicin (DOX) delivering for cancer therapy. We employed two-photon excited fluorescence (TPEF) images and coherent anti-Stokes Raman (CARS) images to investigate intracellular delivery in live cells, and used Olympus Vivaview to show cancerous cell realtime inhibition. Finally, the OA-SPIONs were tested in vivo model with MRI.

Materials and Methods

Synthesis and characterization of copolymers with cystamine bisacrylamide, dodecylamine and PEG amine and synthesis magnetite nanocrystals coated witholeic acid, dispersed in chloroform using a modified peptization technique.OA-SPIONs (1 mg) and rPAA-DD-PEG (10 mg) were dissolved in chloroform(1 ml) to prepare reducible polyamidoamine(rPAA) SPIONs. In vitro part, we do tests such as drug loading and release profile, DOX release from DOX/rPAA-SPIONs, Cytotoxicity of DOX/rPAA-SPIONs, Cellular uptake of DOX/rPAA-SPIONs. In vivo part, we do the breast cancer tumor model and treatment, prussian blue and hematoxylin%26 eosin (H%26E) staining, MRI experiments.

Results

TEM images showed that the remained well dispersed and free of aggregation in water solutions. Dynamic light scattering measurements indicated that the hydrodynamic size of naoparticle-DOX in PBS (0.1 M, pH ¼ 7.4) and DMEM/10% FBS was w150 nm initially, and no significant changes in size were observed for weeks, indicating the rPAA-SPION retained good stability. In vitro part,MCF-7 cells incubated with nanoparticles, red dots with enhanced fluorescence intensity were clearly visible within cytoplasm around the nuclei(Fig.1). In vitro tests, DOX/rPAA-SPIONs present efficiently drug loading with reducible responsibility. The results showed that less than 10% of DOX was released without DTT in the first 3 h,and less than 20% of DOX was released after 48 h. Approximately 30% DOX loaded within the DOX/rPAA-SPION was released in 48 h at 1 mM DTT. DOX/rPAA-SPION nanoparticals were tested in mice with xenograft MDA-MB-231 breast tumor though i.v. injection and inhibited tumor growth efficiently(Fig.2).And histology and Prussian blue on kidney, liver, and heart in mice indicated that DOX/rPAA-SPIONs showed no significant toxicity for mice organs after 24 days treatment.MRI was used to monitor nanoparticles aggregation in tumor site.MRI has very high sensitivity to visualize iron oxides which is beneficial to the spinecho T2 weighted pulse sequence due to its inherent high magnetic susceptibility(Fig.3).

Discussion and Conclusion

In this study, we report a reducible copolymer self-assembled with superparamagnetic iron oxide nanoparticles (SPIONs) to deliver doxorubicin (DOX) for cancer therapy. These nanoparticles can provide efficient drug loading, reduction-responsive drug release,and imaging capability.This rPAA-SPIONs can be used as an efficient magnetic nanovector for delivering various therapeutic drugs into cells for in vitro assay and solid tumors in mice. The DOX/rPAA-SPION was administrated systemically into mice bearing breast cancers, and the in vivo biodistribution of the transported doxorubicin was evaluated and antitumor effects were estimated by the weight of the tumor growth.Cell and animal studies revealed the promising potential of the facile design in drug delivery applications.

Fig.1 The cell uptake and drug distribution investigated by multimodal nonlinear optical imaging microscopy. Two-photon excited fluorescence (TPEF) images showed thedoxorubicin fluorescence and coherent anti-Stokes Raman (CARS) images showed clear intra-cellular structures. Each image has a depth of

Fig.2 The therapeutic effect of DOX/rPAA@SPIONs. A :tumor growth curve of DOX and DOX/rPAA@SPION; B: the tumor volume change on the 24th day after treatment of DOX andDOX/rPAA@SPION.

Fig.3The short arrows without tail pointed at the tumor site while thearrows with tail pointed at the liver. A: before injection and B/C are 12th /24th h after injection. D: the axial T2 one before injection and E at 12th h after injection,F for 24th h after injection. G: mice with xenograft for MRI

Acknowledgements

NSERC Discovery Grant and Manitoba Institute of Child Health.

References

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