Hollow Manganese Phosphate Nanoparticles as a Bifunctional Agent for Cancer Cell Targeted Magnetic Resonance Imaging and Drug Delivery

摘要

Hollow nanoparticles (NPs) with sizes of less than 100 nm have shown great potential in bio-imaging and drug delivery applications. However, most of as-synthetized NPs are hydrophobic. Various approaches has been reported to turn hydrophobic NPs into hydrophilic, among of which, coating silane on NPs is one of the most common ways. There has been emerging numerous of biomedical imaging technologies in recent years, and magnetic resonance imaging (MRI) is one of the most powerful and non-invasive diagnostic techniques based on the interaction of protons with the surrounding molecules. Recently, hollow Mn_3O_4 NPs were found to improve the MRI contrast effect. In addition, multifunctional agents for diagnosis and therapeutics were also rapidly developed in recent decades. However, manganese-based positive contrast agents were only limited in some manganese oxides, manganese phosphate NPs (MNPs) with high relaxivity have rarely been reported. In this work, we designed a controlled ion transfer process to prepare hollow manganese phosphate NPs as previously reported. XRD and TEM results confirmed the crystalline phase and particle size to be 15 nm with a 5 nm hollow structure. To transfer the NPs into water, PEGylation 3-aminopropyltrimethoxysilane (APS-PEG) was synthesized through DCC/NHS reaction and then then linked to MNPs. TGA and FTIR confirmed the formation of APS-PEG. To promote the binding to cancer cells, folic acid (FA) was bind to MNPs as a target molecular to form Mn_3(PO_4)_2@APS- PEG-FA NPs (MNPs-FA). TEM characterization indicated the morphology of the NPs remained and their size to be 20 nm. DLS analysis is in agreement with this size. The formed MNPs-FA were well dispersed in water and examined their relaxivity at 1.5T MR system. T_1 and T_2 relaxation (r_1 and r_2) were 4.03 mM~(-1)s~(-1) and 37.97 mM~(-1)s~(-1) respectively. Anti-cancer drug Doxorubicin (DOX) was loaded into the MNPs by mixing MNPs-FA with DOX in water to form Mn_3(PO_4)_2@APS-PEG-FA-DOX NPs (MNPs-FA-DOX) and dispersed into water in various pH. The drug release capacities of MNPs-FA-DOX at pH=5.4 and 7.4 were investigated by UV-Vis spectrometer at λ～470nm. DOX released at pH=5.4 is five times greater than that of pH=7.4, which indicated that the NPs are pH sensitive. The cytotoxicity of MNPs-FA-DOX at the DOX concentration varied from 86.2 μM to 2.7 μM was evaluated on Hela cells, compared with free DOX and MNPs-FA using an MTT assay. The results showed MNPs-FA are biocompatible even at Mn concentration of 100 μg/ml, MNPs-FA-DOX, however, showed a dose-dependent cytotoxic, which was smaller than that of equivalent amount of free DOX. To validate the targeting effect of the NPs for targeting delivery of DOX, cytotoxicity of MNPs-FA-DOX in KB cells (strong overexpress FA receptors), Hela cells (medium express FA receptors) and A549 cells (FA receptors negative) was investigated by MTT assay. Greater cytotoxicity was observed in KB cells compared with Hela cells, while for A549 cells, the least cytotoxicity it performed, which demonstrated the targeting effect of the MNPs-FA-DOX through the receptor-mediated delivery pathway. DOX in MNPs-FA-DOX NPs allowed further confocal microscopic imaging of the cellular uptake of the NPs. After incubation with KB, Hela and A549 cells for 2 h, a strong fluorescence was observed in the cytoplasm of the KB cells and mediate fluorescence in Hela cells, compared with that of A549 cells, which showed almost no fluorescence signal under similar condition. In comparison with the non-targeted MNPs-DOX, targeting NPs MNPs-FA-DOX treated KB cells were more fluorescent, indicating higher cellular uptake of the NPs. Flow cytometry assay also confirmed the results. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was used for further investigating the cellular uptake of the NPs by measure Mn concentration in each cell after incubation with NPs with various concentrations of Mn. The result showed that KB cells uptake the most NPs, while A549 cells the least. To evaluate the feasibility by using MRI to detect cancer cells, MR signal intensity of KB, Hela and A549 cells incubated with MNPs-FA-DOX with different Mn concentrations were measured using a 1.5 T MRI system. It is clear that the MR signal decreased in order of KB, Hela and A549 cells. In summary, we demonstrated a dual-modality agent for targeted cancer cell MRI imaging and drug delivery based on silane-coated Mn_3(PO_4)_2 NPs. The in vitro MTT assay, flow cytometry, confocal microscopic imaging, ICP and MRI studies showed that the MNPs-FA-DOX can specifically target cancer cells overexpressing FA receptors. It provides a new approach for developing multi-modality platforms combining diagnosing with targeted drug delivery.