An organic solvent-free technology for the fabrication of albumin-based paclitaxel nanoparticles for effective cancer therapy

Highlights

• Novel Lip-PTX/BSA NPs were fabricated via modified thin-film method.

• The fabrication process avoided direct contact of BSA with toxic organic solvents.

• The fabrication process eliminated organic solvent residual.

• The NPs exhibited excellent colloidal stability and sustained release manner.

• The NPs showed effective in vitro/vivo antitumor efficacy.

Abstract

Organic solvents have been reported to exert certain influence on the structure and drug loading efficiency of albumin. It is urgent to develop organic solvent-free albumin-based paclitaxel nanoparticles for effective anticancer therapy. In this study, novel PTX liposome-albumin composite nanoparticles (Lip-PTX/BSA NPs) aimed at avoiding the direct contact of albumin with toxic organic solvents and enhancing the colloidal stability of the formulation were prepared. To methodically evaluate the impacts of multifarious factors on the critical characteristics of the nanoparticles, Box-Behnken design was applied in the formulation optimized process. Ratio of drug-phosphatidylcholine (EPC), ratio of drug-BSA and pH of the media were chosen as the independent variables, while particle size and drug-loading content (DLC) loss rate were applied as the selected response variables. A quadratic model fitted best to describe the data with maximal lack-of-fit p-value and minimum sequential p-value. Three-dimension surface figures were utilized to describe the correlation of independent variables with response variables. Optimized formulation of the nanoparticles with size of 116.2 ± 2.0 nm and zeta potential of -18.4 ± 1.01 mV were obtained with a high encapsulation efficiency of 99.8%. PTX was involved physical interaction with the excipient during the preparation process of the nanoparticles. The release of PTX from Lip-PTX/BSA NPs exhibited a sustained release manner compared to albumin-bound PTX (nab-PTX) and Taxol. Besides, Lip-PTX/BSA NPs presented enhanced in vitro cytotoxicity against 4T1 cells due to highly nonspecific internalization in the cytoplasm. Simultaneously, Lip-PTX/BSA NPs showed effective in vivo antitumor efficacy against 4T1 bearing BALB/c mice, while no apparent adverse effect was observed by histological section and blood biochemical analysis. In conclusion, the novel Lip-PTX/BSA NPs could be applied as a promising drug delivery system for PTX to exert efficient cancer curative effects in clinic.

Introduction

Cancer is reported as one of the major death causes that seriously affect human health and threaten human life all around the world [1]. The three major treatments for cancer, including surgery, radiotherapy and chemotherapy, are commonly utilized to extend the survival time of the patients and improve their life quality. Chemotherapy, a systemic treatment, is typically preferred at advanced stages of cancer for its superior effectiveness [2,3]. Various chemotherapeutic agents have been discovered over the past century for anticancer therapy through specific targeted pathways [4].

Paclitaxel (PTX), a natural diterpenoid ester isolated from Pacific Taxus and other Taxus plants, is one of the most popular chemotherapeutic drugs after doxorubicin. PTX exerts its prompt clinical curative effects via accelerating tubulin aggregation and microtubule stabilization, which finally leads to programmed cell death at the G₂-M phase [[5], [6], [7], [8], [9]]. However, on account of the extremely poor solubility of PTX in water (less than 1 μg/mL), the therapeutic potential of PTX in the natural form has been inhibited [[10], [11], [12], [13]]. To overcome the drawbacks, PTX injection (Taxol®), utilized Cremophor EL and absolute ethanol (1:1, v/v) as co-solvent, has been developed for the usage of parenteral administration in 1992 [14]. Although the application of Cremophor EL promotes the dissolution and absorption of PTX, it also brings various adverse effects, including hypersensitivity reactions, nephrotoxicity, and neurotoxicity [12,15]. In order to avoid the adverse eff ; ;ects of Cremophor EL and enhance the therapeutic efficacy of PTX, Abraxane®, the classical Cremophor EL-free human serum albumin-bound PTX (nab-PTX), was approved by the US Food and Drug Administration (FDA) for cancer treatment in 2006 [16] and has gained heightened attention for researches in human serum albumin (HSA)-based nanomedicine from then on owing to the desired properties of HSA, such as chemical stability, biodegradability, and biocompatibility [17,18].

Bovine serum albumin (BSA), a major soluble protein with a structure similar to human serum albumin, has been typically utilized as alternative nanocarrier to HSA in virtue of its low cost [19,20]. Several bovine serum albumin-based PTX nanoparticles [11,[21], [22], [23]] have been reported to accumulate in tumor tissue due to the enhanced permeability and retention (EPR) effect arisen from the peculiar tumor characteristics of leaky vasculature and defective lymphatic drainage systems [24]. However, most of the preparation processes for the above albumin-based nanoparticles, including Abraxane®, involve organic solvents [20,25], which have been reported to exert certain influence on the structure and the drug load efficiency of albumin [26], and eventually bring apparent in vivo toxicity due to residual solvent [27]. Besides, the poor colloidal stability of albumin-based nanoparticles will also lead to their rapid elimination in the blood circulation [10,28].

Long-circulating liposomes with modifying hydrophilic polymers, such as distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), have been utilized as versatile drug delivery systems for the last few decades contributed to their prolonged blood circulation time [29]. Nevertheless, the challenging problems of fusion, aggregation and leakage for liposomes still remain unsolved in clinic [30]. In the current study, in order to avoid direct contact of albumin with organic solvents and enhance the colloidal stability of the formulation, a novel PTX liposome-albumin composite nanoparticles (Lip-PTX/BSA NPs) (Fig.1) were prepared without further chemical modification for the sake of developing a simple fabrication method. Two critical factors including particle size and drug-loading content loss of the nanoparticles were selected for DOE study through Box-Benhnken design-response surface (BBD) to optimize the preparation process. The binding of liposome to albumin during the self-assembly process were verified through spectroscopic studies. Furthermore, characterization of the nanoparticles, including size, morphology, and cumulative release behavior in vitro, were evaluated as well. Eventually, in vitro cytotoxicity and in vivo antitumor efficacy of the nanoparticles were carried out.