Doxorubicin-loaded biodegradable self-assembly zein nanoparticle and its anti-cancer effect: Preparation, in vitro evaluation, and cellular uptake

Highlights

• The formation of doxorubicin-loaded zein nanoparticles (DOX-zein-NPs) is facile.

• The shell material zein is biodegradable and biocompatible.

• DOX-zein-NPs have a uniformly spherical shape with a particle size around 200 nm.

• DOX-zein-NPs have better anti-cancer effect compared to free DOX.

• DOX-zein-NPs can enter into both cell cytoplasm and nucleus.

Abstract

Cancer is one top leading cause of the deaths worldwide. Various anticancer drugs, which can effectively kill cancer cells, have been developed in the last decade. However, the problem is still about the low therapeutic index of the drugs, which means that the effective dose of drugs will cause cytotoxicity to normal cells. A strategy based on drug nano-encapsulation is applied to achieve an effective anti-cancer therapy. In this study, we use zein, which is an amphiphilic protein, to make the anti-cancer drug nano-encapsulation. Doxorubicin (DOX), a popular anti-cancer drug, is selected as the core drug. The results show that DOX could be successfully encapsulated into zein to form spherical nanoparticles. The encapsulation efficiency and loading efficiency could reach as high as 90.06% and 15.01 mg/g, respectively. The cumulative release result showed a desired pH-responsible release behavior: DOX could be released faster in acidic buffer solutions (pH 5.0 and 6.5) than neutral one (pH 7.4). The effects of the nano-encapsulation on the anti-proliferation of HeLa cells were also examined. It indicated that, compared with free DOX, the DOX-loaded zein nanoparticles (DOX-zein-NPs) had a better effect on cancer cell killing at low DOX concentrations. We also investigated the cellular uptake of DOX-zein-NPs using confocal laser scanning microscopy (CLSM), flow cytometry, and transmission electron microscopy (TEM). And the endocytosis mechanism of DOX-zein-NPs entering into HeLa cells was studied using various endocytosis pathway inhibitors.

Introduction

Cancer counts for one in four deaths in the 21th century [1]. Currently, tons of anti-cancer drugs have been developed. However, the general problem of these drugs is that they lack efficient selectivity toward tumor cells. They kill both normal and cancer cells and result in toxicity to normal tissues. Because of the weak selectivity, drugs reach the target site in concentrations much less than effective [2]. In this case, the drug dose has to be further increased to make it effective, and thus, more toxicity will be given to the normal tissues. Severe side effects decrease the life quality of the patients and can be fatal at times. Site-specific delivery, which protects drugs from release until the target site is reached, is applied to increase the drug concentration at the target site, and thus lower the dose used and reduces incidence of the side effects. The site-specific delivery can be achieved using a drug delivery system. A drug delivery system is often associated with particulate carriers, such as polymeric micelles, emulsions, liposomes [3], nanotubes, dendrimers [4], and nanoparticles (NPs) [4], [5], [6], [7], [8], which are designed to localize drugs at the target sites [9].

Nano-encapsulation is a superior method that nano-carriers are designed for drug delivery systems [9], [10]. Drugs are wrapped and protected by ultrafine vehicles or capsules to form nano-sized core–shell structures. In nano-encapsulation, there is one kind of NP carrier used for encapsulating drugs. This kind of NP-mediated delivery provides numerous advantages. Most drugs formulated with organic solvents have poor solubility and low bioavailability. The use of polymeric NPs allows for the preparation of hydrophobic cancer medications which have improved bioavailability [11]. Because of their small particle size and large surface, NPs can improve the drug solubility and stability, and enter the tumor cells through the enhanced permeability and retention (EPR) effect [12]. Besides, as a protection to the drugs, NP-mediated delivery can achieve a controlled slow release over a required time frame [13]. It also allows increased intracellular accumulation of the therapeutic agent and limits the uptake by healthy tissues. NP-mediated delivery could also circumvent the multidrug resistance (MDR) problem in cancer therapies. For example, paclitaxel-loaded NPs showed 10-fold lower IC₅₀ values than free paclitaxel in MDR cells [14].

NPs that are used in NP-mediated deliveries are produced from a wide variety of materials including carbon, heavy metals, semiconductors, and polymers, which have their own advantages and disadvantages. The material selection for the drug delivery agents is important and it will determine the selectivity and specificity as well as the toxicity of the agent. It is hard to ignore that many potential candidates of the polymeric micelles fail in preclinical studies because of high toxicity. To surmount the dilemma, biodegradable polymeric materials with low immunogenicity, controlled degradation, and good mechanical properties are becoming one of the most powerful candidates. The biocompatibility, toxicity, and biodegradability are always the first concerns about the materials used for drug delivery. So far, site-specific delivery is still a problem because of the selection of the carrier materials, and a balance between the biocompatibility and functionality of the carrier materials is difficult to achieve. The best choices could be the natural materials derived from plant based materials, which are non-toxic, and even edible. However, the interiors of general plant derived natural materials are their hydrophilicity, easy water absorption, and high digestibility [15], [16].

Zein, a major corn protein, unlike most natural materials, is amphiphilic, having both hydrophobic and hydrophilic compounds in its molecule. Zein is insoluble in water but readily disperses in ethanol–water mixture [17]. It is inexpensive, abundant, biodegradable, and biocompatible [18]. Zein is well applied in food and pharmaceutical industries because of its good biocompatibility and bioavailability. Zein has an amphiphilic molecular structure where over 50% of the amino acid in zein is hydrophobic. An amphiphile, such as zein, can self-assemble into various structures, including spheres, sponges and films [6], [17], [19], [20], [21]. Zein can self-assemble to form biobased films, hydrophobic surfaces, and encapsulations, and have potential applications in food packaging, electronic devices, and for oral administration enhancement [22]. Because of its self-assembly ability, zein has been extensively studied to encapsulate bioactive compounds, such as essential oil [23], flax oil [24], vitamin D3 [25], α-tocopherol [18], and citral and lime [19]. Nano-scaled drug carriers prepared by amphiphilic molecules are of great interest to scientists because of their unique core–shell structures, and the core provides an ideal compartment for drug loading, especially hydrophobic drugs [26], [27]. In addition, amphiphilic particles have the ability to transfer through cell membranes. They have better stability than hydrophobic or hydrophilic particles because of the unique surface properties.

There are many FDA approved drugs for cancer treatments. Doxorubicin (DOX), a water-soluble anti-cancer drug, is one of them and has been proved effective in a series of cancers [28]. However, the clinical use of DOX is limited because it is harmful to healthy tissues and has significant side effects, such as cardio toxicity, myelosuppression, weight loss, and alopecia. The objective of this work is to apply zein NPs to encapsulate DOX for anti-cancer therapies. The formation of the DOX-loaded zein NPs (DOX-zein-NPs) and their in vitro release profiles were studied. The anti-proliferative effects on HeLa cells and the cellular uptake were also investigated.