Degraded Hyaluronic Acid-Modified Magnetic Nanoparticles

Superparamagnetic iron oxide nanoparticles (SPIONs) conjugated with hyaluronic acid (HA) functional groups have potential applications as cell targeting materials. However, SPIONs incubated with high-molecular weight HA can result in severe agglomeration. In this work, we found that when modified with degraded HA (hyaluronan oligosaccharides (oHAs)), the nanoparticles were uniformly dispersed with small hydrodynamic sizes, and the oHA-modified SPIONs exerted minimal cytotoxicity. With the same functional groups as HA, the oHA-modified SPIONs may have various biomedical applications.


Introduction
Superparamagnetic iron oxide nanoparticles (SPIONs) have many uses in biomedical research, such as for drug delivery [1], magnetic resonance imaging [2], cancer cell targeting [3], size-related uses [4], magnetism [5], and optical performance [6]. Several SPIONs have been approved by U.S. Food and Drug Administration (FDA) to be the potential treatment of diagnosis and treatment [7]. Surface coating affects the application potential of the SPIONs, and the molecular structure, modification methods, and modification agent proportions lead to significantly different properties [8,9]. Hydrophilic polymer coating can guarantee the colloidal stability of nanoparticles with electrostatic or steric repulsion, reduce the uptake regulation effect of reticuloendothelial cells, and extend the duration of action of nanoparticles in vitro. As modification of polyethylene glycol (PEG) helps nanoparticles to escape the uptake of the system and then to reach cells and start drug release [10], glutathione (GSH) can lower cytotoxicity and enhance T1 MRI characteristics [11,12].
Hyaluronic acid (HA) plays an important role in cell proliferation, embryonic development, tumor cell migration, and wound repair [13,14]. Currently known cell surface HA receptors include CD44 [15], receptor for hyaluronanmediated motility (RHAMM) [16], lymphatic vessel endothelial HA receptor (LYVE-1) [17], layilin [18], and hyaluronan receptor for endocytosis (HARE) [19]. Special RHAMM receptors are distributed on the cell surface, cytoskeleton, and mitochondria. When the cells are stimulated accordingly, the RHAMM receptors stored in the cells are transported to the cell membrane [19]. Therefore, HA-modified SPIONs are expected to have great potential in cell targeting applications; little research work about the modification of HA on SPIONs was reported.
In this work, we found that HA-modified nanoparticles became heavily aggregated; however, degrading highmolecular weight HA via chemical methods [20][21][22] produced hyaluronan oligosaccharides (oHAs), and the oHA-modified SPIONs were uniformly dispersed with small hydrodynamic sizes. oHA is reported to increase the flexibility of the extracellular matrix and increase cell growth and mobility during division and differentiation [23,24]. Therefore, oHA-SPIONs with low toxicity may have applications in various biomedical fields.

SPION Preparation and Characterization.
The raw materials used and their synthesis were reported in our previously published work [25]. In brief, SPIONs were synthesized by decomposing 0.7 g of Fe(acac) 3 (Tokyo Chemical Industry, Japan) in 15.0 g of polyethylene glycol (PEG; Aladdin, China) mixed with 0.3 g of polyetherimide (PEI; Aladdin) at 260°C for 1 h in an argon atmosphere. The reactants were cooled to 60°C, then washed three times successively with toluene and acetone. The SPIONs were then collected using a magnet placed under the container.
2.2. HA Degradation. HA (80 mg) was dissolved in 10 mL of water, then fully dissolved at 4°C overnight. Next, 1 mL of 16% sodium hypochlorite was added every 6 hours for the next 24 hours; then, the pH was adjusted to approximately 7.0 using 0.1 M HCl.

2.3.
Modification of HA and oHA on the SPIONs. HA (20 mg, undegraded) and 10, 20, and 40 mg oHA (hereafter referred to as "m(SPIONs) : m(oHA)") at ratios of 2 : 1, 1 : 1, and 1 : 2 were mixed with 20 mL of 1 mg/mL of the SPION aqueous dispersions (Scheme 1). The reaction was carried out for 5 h in a shaker at 60 rpm and 4°C. After incubating overnight at 4°C in a refrigerator, the mixture was dialyzed against deionized water for 120 h (MWCO 100,000 dialysis bag, SpectrumLabs, USA). Samples were kept in a refrigerator at 4°C for later use.

Material Characterization.
The samples were characterized using a Zetasizer Nano ZS90 (Malvern Instruments), a Quantum Design MPMS XL-7 superconducting quantum interference device, a PL-GPC 50 gel permeation chromatograph, a JEM-2100F transmission electron microscope (TEM), X-ray photoelectron spectroscopy (XPS), and a thermal gravimetric analyzer. The crystal structure of the nanoparticles was confirmed using a PANalytical X'Pert PRO powder X-ray diffractometer (XRD) with CuKα radiation (λ = 0:15406 nm) with a scanning step of 0.017°in the 2θ range of 20-60°at room temperature.
The X-ray photoelectron spectroscopy (XPS) of the dried sample was performed in vacuum on Thermo ESCALAB 250 to further characterize the coating materials. XPS is a technique which detects the organic surface elements of particles up to a 10 nm thickness or inorganic surface elements of particles up to 3 nm.

Results and Discussion
3.1. Molecular Weight Measurement. Figure 1 shows the molecular structure of hyaluronic acid. After degrading high-molecular weight HA in sodium hypochlorite, the molecular weight change of the HA was tested under the aqueous environment of PL-GPC 50 ( Figure 2). The molecular weight of HA before degradation was between 200 kDa and 2.5 MDa, the distribution was uneven, and the viscosity was thick. The molecular weight of oHA after HA degradation was concentrated around 26 kDa, and the viscosity was low with good fluidity.

Hydrodynamic Size Distribution Profiles and Zeta
Potentials. Figure 4 shows the hydrodynamic sizes of the SPIONs, HA-SPIONs, and oHA-SPIONs. The highmolecular weight HA-modified nanoparticles showed severe agglomeration (Figure 4(a)   3 Journal of Nanomaterials XPS. The spectrum decomposition was performed using the XPSPEAK41 program with Gaussian functions after subtraction of a Shirley background; the ratio between the Lorentzian and Gaussian functions is 60%. Figure 5(a) shows the XPS spectra of the oHA-SPIONs-2 : 1, oHA-SPIONs-1 : 1, and oHA-SPIONs-1 : 2, showing peaks at 711.0 eV, 530.0 eV, 400.0 eV, and 286.0 eV, corresponding to Fe 2p, O 1s, N 1s, and C 1s [29], respectively. Figure 5(b) shows the fitted peaks of N 1s for the oHA-SPIONs-2 : 1, oHA-SPIONs-1 : 1, and oHA-SPIONs-1 : 2. The peaks at 399.2 eV, 400.69 eV, and 401.1 eV correspond to the C-N, C=O-N, and C-NH 3 groups, respectively. The XPS results showed that the binding energies of oHA were the same as those of HA, indicating that their structures were not obviously changed after degradation. The C=O-N group indicated that the SPION surface was modified with oHA   Journal of Nanomaterials [30,31]. As the weight proportion of the oHA increased during modification, the C-N bond proportion decreased ( Figure 5(b)), and the C=O-N and C-NH 3 group proportions increased accordingly.

Conclusion
We synthesized superparamagnetic iron oxide nanoparticles conjugated with HA and oHA. Modifying high-molecular weight HA on the nanoparticle surface caused severe agglomeration, but the degraded HA did not affect the      Journal of Nanomaterials dispersibility of the nanoparticles. The nanoparticles were uniformly dispersed with high modification and low cytotoxicity, which satisfies the requirements for surface modification of the nanomaterials. This work provides a reference for modifying nanoparticles with functional groups of highly viscous materials, such as HA, while maintaining a small hydrodynamic size.

Data Availability
All data are provided in full in the results section of the manuscript. Authors can find all the data in the results section for further analyses.

Conflicts of Interest
The authors declare no competing financial interest. Statistical significance was evaluated using one-way analysis of variance followed by post hoc comparison of the means using Fisher's least significant difference test, n = 6. * P < 0:05, compared with the control group.