A high-loading drug delivery system based on magnetic nanomaterials modified by hyperbranched phenylboronic acid for tumor-targeting treatment with pH response

Highlights

• A novel magnetic nano-DDS modified by hyperbranched phenylboronic acid was prepared.

• The nano-DDS has significantly high drug loading capacity which means lower doses.

• The nano-DDS simultaneously achieves cell targeting and pH response release.

Abstract

To overcome the limited drug loading capacity of magnetic nanopharmaceuticals arising from the relatively large mass of the metal core, a high-loading drug delivery system based on amino-functionalized Fe₃O₄ magnetic nanospheres modified by hyperbranched phenylboronic acid (HPBA-Fe₃O₄) were prepared for the first time. The obtained nanomaterials were characterized by transmission electron microscopy, Fourier transform infrared, zeta potential, elemental analysis, vibrating sample magnetometry and X-ray diffraction analysis, and the results showed that hyperbranched phenylboronic acid (HPBA) were successfully grafted onto the surface of the magnetic nanospheres. The polymerization conditions, adsorption and desorption performance, and tumor-targeting ability of HPBA-Fe₃O₄ was investigated in detail through chemical and biological experiments. The drug loading amount and capacity of HPBA-Fe₃O₄ are 271.3 mg/g and 27.13%, respectively, which are 2.26 and 3.27 times greater than those of PBA-Fe₃O₄ and PEI-Fe₃O₄, and the thermodynamic fitting results further demonstrate the high drug loading ability of HPBA-Fe₃O₄. In vitro studies performed in U-87 MG malignant glioma cells and astrocytes via light and fluorescence microscopy analyses, cell counting kit-8 assays, and HPLC tests confirm the pH-sensitive release and tumor-targeted drug delivery capabilities of HPBA-Fe₃O₄. The facile fabrication of hyperbranched magnetic nanomaterials could be an alternative solution for designing high-loading, tumor cell-targeting and pH-responsive DDSs with high-mass cores.

Introduction

Compared with traditional tumor treatments, drug delivery systems (DDSs) designed with nanomaterials can greatly augment the cumulative amount of drugs at the tumor site through targeted delivery [[1], [2], [3]]. In addition, the DDSs can also response to the internal or external stimuli [4,5], increase the water solubility of drugs [6,7], and enhance therapeutic effect by changing the tumor microenvironment [8,9], which tremendously improve drug efficacy and reduce toxic side effect. Recently, various nanopharmaceutical DDSs have been developed, including liposomes, metal organic frameworks, polymer nanoparticles and inorganic nanoparticles [[10], [11], [12], [13], [14], [15], [16]]. Among these DDSs, iron oxide nanoparticles, a kind of magnetic nanoparticle that has already been approved for clinical use by the US Food and Drug Administration, have attracted much attention nowadays [17].

Iron oxide nanoparticles have shown broad application prospects in the biomedical field, including drug delivery, hyperthermia therapy and diagnostics, for their excellent biosafety, biodegradability, high delivery efficiency, potential targeting function and magnetic resonance imaging enhancement ability [[18], [19], [20], [21], [22]]. However, due to the large mass of iron oxide nanoparticles, they suffer from low drug loading per unit mass, and large doses of them may even be harmful to humans. Ding et al. reported a remotely triggered DDS based on magnetic iron oxide nanocubes with a loading capacity of 3.7% [23]. Li et al. constructed an enzyme-responsive DDS based on magnetic iron oxide nanoparticles as the core, and the loading capacity is 12.2% [24]. Yang et al. used Fe₃O₄@mSiO₂ core-shell NPs as a host to design a pH-sensitive release system with a loading capacity of 2.7% [25]. These iron oxide-based drug carriers are generally limited by their low drug loading capacity, which not only seriously affect life quality and treatment effect of patients but also reduce the utilization ratio of the materials, and may even induce unnecessary side effects. Therefore, the realization of a high-drug-loading system with magnetic iron oxide as the core is imperative to the future use of magnetic nanoparticles in DDSs.

The main constraint on drug loading is the very limited functional groups on the surface of carriers which can’t provide enough binding sites for drugs. Thus, one of the feasible methods is to enrich the functional groups and make carriers with high-density binding sites. Hyperbranched polyethyleneimine (PEI) will be an ideal choice for its dendritic network structure and abundant amino groups at the end of the branched chain. Recent reports on the application of PEI to tumor therapy are generally limited to the use of its cationic polymer property to achieve siRNA delivery; or served as hydrophilic chain of the block copolymer to form micelles [[26], [27], [28], [29]]. The hyperbranched property of PEI to realize better tumor therapy have not been studied yet. Additionally, in our previous study, using PEI as functional monomer to prepare molecularly imprinted polymers (MIPs), we verify that PEI can create high-density recognition sites and improve MIPs’ binding capacity [30]. Thus, employing the characteristics of hyperbranched amino groups of PEI, in this work, we decorated PEI onto amino-functionalized Fe₃O₄ (NH₂-Fe₃O₄) nanoparticles to obtain a hyperbranched amino-functionalized magnetic material. Furthermore, the hyperbranched modified material can serve as an ideal basis for the preparation of any desired functional hyperbranched material by grafting small molecules with specific functional groups.

As we all know, phenylboronic acid (PBA) can selectively and reversibly combine with polyhydroxylated compounds with vicinal diol or meta-diol structure to form covalent complexes [31]. This unique property allows PBA to be used as a functional group in a variety of applications, for example glycoprotein separation, glucose sensors, and RNA affinity columns [[32], [33], [34], [35], [36]]. The commonly used antitumor drug doxorubicin (DOX), which has a vicinal diol moiety, can stably combine with PBA under physiological and alkaline conditions, and the resulting boronate ester bonds can be broken in the acidic tumor microenvironment, thereby achieving effective release of DOX. What’s more, PBA can also be served as a targeting ligand to specifically bind to the sialylated epitope which is overexpressed on the surface of various tumor cells. After the nanovectors reach the tumor region through the enhanced permeability and retention effect, they can be targeted to tumor cells and introduced into the cells by receptor-mediated endocytosis. Thus the DDSs modified by PBA can achieve pH-responsive release and sialic acid targeting in tumor-specific microenvironments. This intelligent response can reduce toxic side effects on normal tissues and has been fully confirmed by the relevant literatures, which makes PBA an ideal component for DDSs.

Based on the above considerations, we combined PEI and PBA to subtly modify the heavy metal oxides to develop a novel DDS with high drug loading and intelligent response. First, PEI was covalently bond onto the surface of NH₂-Fe₃O₄ by reducing the imine bond to enrich the amino groups, and then PBA reacted with the amino groups of PEI to obtain the hyperbranched PBA modified magnetic nanomaterial (HPBA-Fe₃O₄) for loading DOX. The synthesis and adsorption conditions of the material were investigated carefully, and the experimental results show that the maximum adsorption amount of HPBA-Fe₃O₄ is 2.26 and 3.27 times greater than that of the PBA-Fe₃O₄ and PEI-Fe₃O₄. In vitro drug release studies show that the DOX release amount of HPBA-Fe₃O₄ is significantly increased with a decrease in the pH value, demonstrating the pH-responsive release ability of the materials, which will improve the drug utilization ratio and enhance the therapeutic effect in tumor tissues. Moreover, through cell fluorescence assays and DOX content detection tests in the cells culture supernatant by HPLC, we observe that HPBA-Fe₃O₄ have an obvious tumor cell-targeted recognition function and its cell uptake ability is also improved. Finally, the biosafety of HPBA-Fe₃O₄ and the tumor killing effect of DOX-loaded HPBA-Fe₃O₄ were verified by cell counting kit-8 (CCK-8) assays.