**1. Introduction**

Arsenic trioxide (ATO) is the main active ingredient of traditional Chinese medicine (TCM) Arsenic. In the 1970s, it was first applied to acute promyelocytic leukemia (APL) with significant efficacy [1] and was approved by the National Medical Products Administration (NMPA) and Food and Drug Administration (FDA) as a first-line treatment for APL in 1999 and 2000, respectively [2,3]. ATO can induce cell di fferentiation, inhibit apoptosis, and exert anti-tumor e ffect [4]. In recent years, research studies have confirmed the significant growth inhibition and apoptosis induction e ffect of ATO in solid tumors, such as liver cancer, breast cancer, stomach cancer, glioma and lung cancer [5–11]. At present, ATO injection has been employed clinically in the treatment of APL and advanced primary liver cancer. However, the unique physicochemical properties of ATO allow it to be rapidly cleared from blood, and it requires daily administration during clinical treatment. At the same time, the uptake of the reticuloendothelial system (RES) makes only a slight amount of ATO reach the tumor site. Nevertheless, considering the potent toxicity of ATO, increasing the dose of ATO will increase the systemic toxicity and cause damage to the liver, kidney, heart, and peripheral nerve [12–14].

Based on the size advantage, nanoparticles (NPs) can exude through the tumor vasculature and effectively delivery the drugs to cells through enhanced permeability and retention (EPR) e ffects [15]. Therefore, it is considered to be a kind of formulation with low toxicity and high stability. Di fferent ATO delivery systems (DDS) have been developed, including magnetic nanoparticles [16], chitosan nanoparticles [17], microspheres [18], liposomes [19], and mesoporous silica nanoparticles [10,20]. These formulations can achieve sustained release of ATO, which can reduce the transient plasma concentration and toxicity of drugs to a certain extent. However, they are still deficient in biocompatibility, and the safety of these systems needs to be verified [21].

Sodium alginate (SA) is a sort of polyanionic polysaccharide alginic acid salt found in brown algae that is water-soluble and has the advantages of anti-tumor e ffect, immune regulation, non-toxic, biodegradability, and excellent biocompatibility [22,23]. It has been approved by FDA for the pharmaceutical industry as an excipient [24,25]. For the past few years, NPs prepared from SA as drug carrier systems have also attracted more attention [26,27]. Red blood cells membrane (RBCM) will be formed into vesicles (RVs) using extrusion or sonication methods [28]. As a drug carrier, it can be attached to the surface of NPs to sustain the release of drugs, avoid elimination by the immune system, increase drug stability, improve biocompatibility and thus prolong drug circulation in vivo [29,30].

Moreover, RBCM coating nanotechnology already has excellent precedents. Che-Ming J et.al [31] demonstrated the synthesis of an RBCM coated polymeric nanoparticle for long-circulating cargo delivery, Jinghan Su et al. [32–34] extensively studied the e ffectiveness of RBCM-camouflaged NPs for treating metastatic breast cancer. In addition, if the non-toxic SA nanoparticles can be encapsulated by natural RBCM and combine the superiorities of sustained release and prolonging residence time, the nano-system can achieve the purpose of maintaining e fficacy and reducing toxicity. In addition, this can provide a new possibility for safe application of ATO.

In this study, RBCM-camouflaged ATO-loaded sodium alginate nanoparticles (RBCM-SA-ATO-NPs, RSANs) were prepared as shown in Figure 1. ATO-loaded sodium alginate nanoparticles (SA-ATO-NPs, SANs) were prepared by the ion crosslinking method, followed by coating of RBCM to obtain RSANs. It was then systematically characterized and evaluated for its efficacy and toxicity. The results indicated that the system might become a promising delivery system for the safe, e ffective, and sustained release of ATO.

**Figure 1.** Preparation and Characterization of RSANs.

### **2. Materials and Methods**
