Hyperbranched lipopolymer-folate-stabilized manganese ferrite nanoparticles for the water-soluble targeted MRI contrast agent

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Abstract

A powerful T2-weighted magnetic resonance imaging (MRI) contrast agent with targetability has been fabricated by encapsulating manganese ferrite (MnFe2O4) nanoparticles (NPs) into biocompatible L-α-phosphatidylethanolamine-hyperbranched polyglycidol (PE–HBPG) bioconjugates covalently functionalized with folic acid (FA). The PE–HBPG is synthesized by anionic ring-opening multibranching polymerization of glycidol using soy-based PE as an initiator. FA is then conjugated onto the periphery of PE–HBPG for targeting tumor cells which have over-expressed the folate receptor. The highly monodisperse MnFe2O4 NPs with uniform size (7–10 nm) stabilized by oleylamine are labeled with the PE–HBPG–FA by ligand exchange reaction to fabricate the water-soluble and biocompatible hybrid NPs, MnFe2O4@ PE–HBPG–FA, showing targetability through receptor-mediated cellular binding and uptake. The transverse relaxivity value of MnFe2O4@PE–HBPG–FA is 140.56 mM−1 s−1, higher than those of conventional superparamagnetic iron oxide particle contrast agents.

Graphical abstract

A powerful T2-weighted magnetic resonance imaging contrast agent with targetability is fabricated by encapsulating MnFe2O4 nanoparticles into biocompatible phospholipid–hyperbranched polyglycidol bioconjugates covalently functionalized with folic acid. The highly monodisperse MnFe2O4 nanoparticles stabilized by water-soluble bioconjugate show high transverse relaxivity with targetability through receptor-mediated cellular binding and uptake.

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Introduction

The applications of magnetic nanoparticles (MNPs) as magnetic resonance imaging (MRI) contrast agents have been investigated as one of the important topics in biotechnology [[1], [2], [3], [4]]. due to their unique physical properties and ability to function at the cellular and molecular level of biological interactions. MRI contrasting agents are either paramagnetic ion complexes bearing lanthanide elements like gadolinium (Gd3+) or transition metal manganese (Mn2+) or superparamagnetic magnetite particles. The elements in paramagnetic ion complexes increase signal intensity on T1-weighted images while dysprosium, superparamagnetic agents and ferromagnetic agents, negative contrast agents, appear darker on T2-weighted images. Compared with conventional paramagnetic gadolinium complex-based contrast agents such as Magnevist® (gadopentetate dimeglumine), ProHance® (Gadoteridol) injection and Dotarem® (gadoterate meglumine), superparamagnetic MNPs are emerging as the next generation magnetic probes for MRI because of their excellent magnetism and long circulating times [[5], [6], [7]]. In recent years, many researches have tried to design MNPs with controllable nanoscale properties, such as size, morphologies, magnetism, and surface states not only for improving the contrast quality of MR images but also for excellent colloidal-stability and targeting capabilities [[8], [9], [10], [11]].

Especially, spinel-type manganese ferrite (MnFe2O4) MNPs have been proved that they have outstanding properties such as nanometer size, large surface area to volume ratio, superparamagnetic behavior, high saturation magnetization, and large relaxivity [12]. For such reasons the MnFe2O4 MNPs have been vigorously investigated as MRI contrast agents and have identified as efficient T2-weighted contrast agents, because their transverse relaxivity coefficients (r2), spin-spin relaxation rate (1/T2) per metal oxide concentration, is higher than that of conventional superparamagnetic iron oxide particle (SPIO) like Feridex I.V.® (ferumoxides injectable solution). Variety of synthetic techniques such as co-precipitation method and microemulsions, sol-gel syntheses, sonochemical reactions, hydrothermal reactions, and thermal decomposition of precursors have been reported [13]. Among these methods, thermal decomposition of organometallic and coordination compounds in the presence of a surfactant is the most efficient approach in preparing the monodisperse MnFe2O4 MNPs with uniform size and defined morphology [14]. However, these MNPs were soluble only in organic solvents due to the presence of hydrophobic ligands like oleylamine (OA) on the MNPs surfaces. Therefore, the surface modification of the MNPs is essential to improve their solubility in aqueous media and to guarantee their biocompatibility by employing micellar encapsulation or ligand exchange [[15], [16], [17], [18], [19]].

Setting our sights on the improvement of MR contrast image, biocompatibility, and targetability toward cancer cell, we have designed a new protocol to fabricate MnFe2O4 MNPs as a T2-weighted contrast agent by combining the thermal decomposition method with the ligand exchange (Fig. 1). Lipopolymers were firstly prepared by an anionic ring-opening multibranching polymerization (ROMBP) of glycidol using soy-based L-α-phosphatidylethanolamine (PE) as an initiator. Folic acid (FA) molecules were then cojugated to the resultant biocompatible [19] PE-block-hyperbranched polyglycidol lipopolymers with multiple terminal hydroxyl groups to carry targetability since the folate receptor is over expressed on many human epithelial cancer cell surfaces including cancers of breast, ovary, uterus, colon and lung [[20], [21], [22], [23], [24]]. Then the MnFe2O4 MNPs passivated by OA molecules were subjected to ligand exchange with the PE–HBPG–FA lipopolymers to yield MnFe2O4@PE–HBPG–FA MNPs. The resulting hybrid MNPs displayed water solubility and biocompatibility as well as excellent contrast property in T2-weighted MR imaging. Cell culture and viability studies were performed to evaluate folate receptor mediated intracellular uptake with and without folate receptor by using confocal laser scanning microscopic images.

Section snippets

Materials

PE was extracted from soybean lecithin (Daejung Chemicals, South Korea) by solvent extraction according to the procedures developed by our group [25]. Potassium methoxide solution (CH3OK, 25% in methanol), N-hydroxysuccinimide (NHS, 98%), OA (technical grade, 70%) and 1,4-dioxane (≥99%) were purchased from Sigma-Aldrich and were used without further purifications. Glycidol (96%) and folic acid (96–102% pure) from Acros Organics, and iron (III) acetylacetonate, manganese (II) acetylacetonate,

Synthesis and characterization of PE–HBPG–FA conjugates

PE is one of the most important lipids that can be used for various applications in the pharmaceutical industry such as drug and gene deliveries. Above all things, amine end group of PE can be used for the chemical conjugation for various applications. PE was firstly extracted by using flash column chromatography from commercial soy lecithin [26]. The PE–HBPG conjugate was then synthesized by anionic ring-opening multibranching polymerization of glycidol using the resultant PE as an initiator (

Conclusions

The PE-HBPG-FA lipopolymer bearing cancer cell moiety was successfully synthesized by means of anionic ring-opening polymerization of glycidols using PE as an initiator. The uniform and OA-stabilized MnFe2O4 nanoparticles (6–8 nm) were solvothermally synthesized in organic phase. The OA molecules in the resultant MnFe2O4@OA MNPs were easily exchanged to PE-HBPG-FA molecules to form water-soluble and biocompatible MnFe2O4@PE–HBPG–FA MNPs. The dispersed aqueous solutions of MnFe2O4@PE–HBPG–FA

Acknowledgements

This work was supported by the Basic Science Research Program of the National Research Foundation of Korea (2018R1D1A1A09081809). Rimesh Augustine and Hye Ri Lee contributed equally to this work.

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