PEGylated and zwitterated silica nanoparticles as doxorubicin carriers applied in a breast cancer cell line: Effects on protein corona formation

Highlights

• Silica nanoparticles exposed to biological fluids form protein coronas on the surface, changing their original properties.

• Poly (ethylene glycol) and zwitterionic-based surface modifications of SiNPs were able to prevent protein corona formation.

• PEG and ZWT-SiNP increased the biocompatibility in MCF-7 cells when compared with bare SiNPs.

• PEG and ZWT-SiNP improved doxorubicin (DOX) delivery in MCF-7 cells.

• In higher salt concentration at 37 °C: SiNP-PEG severe aggregation state; this was not observed for SiNP-ZWT.

Abstract

Nanomaterials exposed to biological fluids form protein coronas on their surfaces, changing their original properties. Surface functionalization with nonfouling moieties is a strategy to synthesize silica nanoparticles (SiNPs) with the ability to hinder protein corona formation. We report a simple poly(ethylene glycol) (PEG) and zwitterionic (ZWT)-based surface modification method to prepare doxorubicin-loaded SiNPs to avoid protein corona formation in breast cancer cells. Drug-loaded SiNPs were characterized in terms of particle size, charge, and drug loading content. A comparison of the stability between PEG and ZWT on SiNP surfaces was performed at 25 °C and 37 °C in a high salt solution using turbidimetry. At 37 °C, the PEGylated SiNPs showed aggregation. In contrast, under the same conditions, zwitterated SiNPs did not show aggregation. Both PEG and ZWTSiNP savoided protein adsorption in the presence of increasing concentrations of bovine serum albumin (BSA). In vitro cell experiments demonstrated that both functionalization approaches (SiNPs-PEG and SiNPs-ZWT) improved biocompatibility in the breast cancer cell line MCF-7compared to SiNPs. The antitumour effects of doxorubicin-loaded SiNPs were evaluated via red neutral cell uptake and annexin V assays, and the results demonstrated that surface functionalization plays an important role in the cytotoxic profile of these systems, with SiNPs-DOX-PEG and SiNPs-DOX-ZWT exhibiting a superior cytotoxic effect compared to SiNPs-DOX.

Introduction

Silica nanoparticles (SiNPs) have emerged as promising platforms for drug delivery, biological sensing, imaging, gene therapy, and diagnosis due to their advantages, such as their biocompatibility, biodegradability, large surface area, and easily functionalized surface [1].

Surface functionalization of nanoparticles has been used as a strategy to improve colloidal stability, biocompatibility, circulation time in the body, and target specificity [2,3]. Functionalization through chemical groups on the nanoparticle surface allows for specific interactions with cells, improving the nanoparticle therapeutic effect, decreasing the toxicity, and inhibiting protein corona formation [4,5]. Protein corona is the term used for a set of proteins and biomolecules associated with the nanomaterial surface after exposure to biological fluids, which completely changes their fate, decreases blood circulation time, and increases phagocytosis [6,7].

The primary strategy for synthesizing an effective nanocarrier is to ensure a nontoxic surface, which can also inhibit protein corona formation [8]. Many studies have reported that SiNPs cause toxicity due to the hydroxyl groups (OH•) on their surface [[9], [10], [11]]. Thus, surface modifications may mitigate the cytotoxic effects of SiNPs. In addition, active targeting and effective drug delivery are achieved when there is a lack of nanomaterial recognition by plasma proteins [8], which may be achieved by surface modification. The most common compound used for avoiding protein corona formation is poly(ethylene glycol) (PEG) due to the hydration layer formed on the nanomaterial surface, which improves the blood circulation time, colloidal stability, and biocompatibility of several types of nanoparticles [12]. However, in recent years, reports have shown that PEGylated nanoparticles induce immunogenicity and are susceptible to oxidative damage in the presence of oxygen and transition metal ions, changing the nanoparticle identity and reducing its biological effects [13]. An alternative to circumvent this limitation to hindering corona formation is functionalization with zwitterionic (ZWT) compounds.

Zwitterionic compounds are polymers that are biologically inspired by phosphatidylcholine (PC) headgroups, which are abundant in the phospholipid bilayer of cell membranes. These polymers are promising candidates for nanomaterial functionalization due to their antifouling properties [[14], [15], [16]]. The hydration layer formed with this compound is based on electrostatic interactions, which are stronger than the hydrogen bonds formed by PEG compounds. Several reports have explored the antifouling properties of ZWT compounds as an alternative to PEGylated nanomaterials [16]. As far as we know, the comparative behavior between PEGylated and ZWT-based nanomaterials has not been evaluated in the context of drug carriers in the treatment of breast cancer.

The progressively increasing incidence of breast cancer is a serious world health problem. It is the most common malignant disease affecting women, accounting for approximately 26% of all cancer cases [17]. In addition to surgery and radiation, chemotherapy is an essential tool in the treatment of cancer [17].

Doxorubicin (DOX) is an antitumoural used to treat breast cancer, but it is also used for many other cancer types, such as solid tumors in childhood, soft tissue sarcomas, osteosarcomas, malignant lymphoma carcinoma, and ovarian carcinoma [18,19]. Therefore, DOX is considered one of the most potent chemotherapeutic drugs approved by the Food and Drug Administration (FDA) [20]. Although effective at killing cancer cells, DOX also exerts cytotoxic effects on healthy cells in various organs, including the heart, brain, kidney, and liver [21]. Most of the side effects of DOX are associated with an increase in reactive oxygen species (ROS), as is the case in cardiotoxicity [22]. Thus, low selectivity to cancer cells is a problem in the use of DOX [23].

One strategy to overcome the limitations of chemotherapy drugs is the use of nanomaterials as carriers, mainly due to their ability to increase drug accumulation inside tumor cells, leading to a decrease in side effects [24,25]. DOX is the most common drug used for assessing drug delivery by nanomaterials to cancer cells, and it has been evaluated with several types of nanoparticles [[26], [27], [28], [29]]. In this context, SiNPs are strong candidates for drug delivery due to their robust nature, tunable surface area, biodegradability, uniform distribution of molecules on the porous space, and the possibility of surface functionalization [30]. It is worth mentioning that SiNPs with a modified surface recently received FDA approval for stage I human clinical trials for imaging, demonstrating the potential of these nanomaterials [31].

Several reports have explored the antifouling properties of PEG on the surface of nanoparticles applied to anticancer therapy; however, few studies have examined ZWT compounds. Pourjavadi et al. showed that SiNPs coated with PEG and polyvinyl pyridine were efficient for DOX delivery triggered by pH in solid tumors [32]. Nguyen et al. demonstrated that SiNPs functionalized with PEG and polylysine were able to codeliver DOX and small interfering RNA into a breast cancer cell line [33]. Salcedo et al. synthesized SiNPs coated with ZWT to avoid protein corona formation for daunorubicin delivery in ovarian cancer [34].

In the present paper, we comparatively discuss the viability of the use of PEG and ZWT compounds(Fig. 1) as strategies to obtain hybrid nanosilica particles produced by the Stöber method to hinder protein corona formation without toxicity.

Thus, six systems were synthesized: two nonfunctionalized systems (SiNPs and SiNPs-DOX), two systems functionalized with PEG (SiNPs-PEG and SiNPs-DOX-PEG), and two systems functionalized with ZWT (SiNPs-ZWT and SiNPs-DOX-ZWT). Nanoparticle characterization was performed by measuring size, colloidal stability, protein corona formation, drug release, cytotoxicity, and hemolytic effects. In vitro assays in a breast cancer cell line (MCF-7) were performed to evaluate the potential for doxorubicin delivery via SiNPs with a modified surface.