Antitumor Effect and Toxicity of an Albumin-Paclitaxel Nanocarrier

Mar 15, 2019 - Institute of Pathogenic Microbiology, Jiangsu Provincial Center for Disease Prevention and Control, Nanjing , Jiangsu 210009 , China...
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Antitumor effect and toxicity of an albumin-paclitaxel nanocarrier system constructed via controllable alkali-induced conformational changes Guangming Gong, Yongjun Jiao, Qinqin Pan, Hao Tang, Yanli An, Ahu Yuan, Kaikai Wang, Canping Huang, Weimin Dai, Ying Lu, Shudong Wang, Jian Zhang, and Hua Su ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00312 • Publication Date (Web): 15 Mar 2019 Downloaded from http://pubs.acs.org on March 17, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Antitumor effect and toxicity of an albuminpaclitaxel nanocarrier system constructed via controllable alkali-induced conformational changes Guangming Gonga, Yongjun Jiaob, Qinqin Panc, Hao Tanga, Yanli And, Ahu Yuane, Kaikai Wangf, Canping Huangg, Weimin Daih, Ying Lui, Shudong Wanga*, Jian Zhangj* and Hua Sua* a Department

of Pharmaceutics, Jinling Hospital, Nanjing University School of Medicine,

Nanjing 210002, China. b Institute

of Pathogenic Microbiology, Jiangsu Provincial Center for Disease Prevention and

Control, Nanjing 210009, China. c

HLA Laboratory, the First Affiliated Hospital with Nanjing Medical University, Nanjing,

People’s Republic of China d Jiangsu

Key Laboratory of Molecular and Functional Imaging, Department of Radiology,

Zhongda Hospital, Medical School, Southeast University, Nanjing 210009, China. e State

Key Laboratory of Pharmaceutical Biotechnology, Medical School of Nanjing University,

Nanjing 210093, China. f State

Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing,

210009, P.R. China. g Research

Department, Ringpu Bio-Tech, Tianjin, China.

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h Administrative i Department j

Committee of Taizhou Medical Hi-tech Zone, China Medical City, P.R. China.

of Pharmacy, Nanjing University of Chinese Medicine, Nanjing210023, China.

National Laboratory of Solid State, Microstructure and Department of Physics, Nanjing

University, Nanjing 210093, China. Guangming Gonga, Yongjun Jiaob, Qinqin Panc and Hao Tanga contributed equally to this work. *Correspondence: sdwangpharm@126.com; gonggm@126.com.

Keywords:Alkali, albumin-paclitaxel nanoparticles, toxicity, antitumor effect, bone marrow inhibition.

Abstract: Various strategies have been developed to construct albumin nanomaterials via biophysical or chemical changes. In this work, a compound comprising albumin-paclitaxel nanoparticles (NPs-PTX) with a drug loading efficiency of 21% was constructed via manipulation of alkali induced conformation changes and hydrophilic-hydrophobicity transition. The toxicity of two PTX formulations (Taxol® and NPs-PTX) in human umbilical vein endothelial cells (HUVECs); RAW264.7, K562, and HepG2 cells; and rats were determined. The IC50 of Taxol® was remarkably lower than that of NPs-PTX. Both PTX formulations promoted cell apoptosis, possibly via mitochondria-dependent (intrinsic) and mitochondria-independent pathways. The effect of PTX formulations (0.5 to 1 mg mL-1) on hemolysis and the LD50 values of the PTX formulations were significantly different (p < 0.01). Reductions in the number of white blood cells (WBCs) and monocytes (MNCs) and obvious pathological changes in the spleen, thymus, and mesenteric lymph nodes were observed and may have been related to the bone marrow inhibition effect of PTX. The tumor inhibition rate of NPs-PTX (60.8%) was higher than that of Taxol® (31.2%) (p < 0.05) when the dose of NPs-PTX (equivalent PTX) was

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2.5 times as that of Taxol® (30 mg kg-1 vs 12 mg kg-1). Taxol® is highly toxic, whereas NPs-PTX is moderately toxic. Thus, NPs-PTX has advantages over the commercially available Taxol® formulation in terms of low toxicity and increased dosage, indicating NPs-PTX is a better option for safe and effective PTX delivery.

Introduction Human serum albumin (HSA) and its associated nanomaterials exhibit three main advantages: i) active binding to the corresponding receptor overexpressed in tumor cells; 1ii) no competitive inhibition of the binding of HSA nanomaterials to tumor receptors by free albumin; and iii) suitability for large-scale extraction and industrial production. Albumin-based nanomaterials have gained increasing attention for their use in biomedical applications.2-3 Various strategies have been developed to produce and manipulate albumin nanomaterials for use in drug delivery, magnetic resonance imaging (MRI), fluorescence imaging, and photodynamic therapy.4-5 Furthermore, certain secondary structural changes are accompanied by the formation of aberrant special structures and free reduced-state sulfhydrols, which are indispensable for successfully utilizing albumin and its nanomaterials in nanomedicine.6-9 Albumin domain II can be unfolded, with significant lose of secondary structure, at approximately pH 12.5, and protein fluorescence intensity at 340 nm decreases significantly under extreme alkalinity.10 Alkali modifications induce increased exposure of hydrophobic groups in meat proteins, and increase he hydrophobicity of egg albumin proteins which aids in protein absorption to an interface.11 These characteristics have strong significance in utilizing HSA as a vehicle for therapeutic drugs and fluorescent probes through changes in physicochemical properties. Paclitaxel (PTX), a widely used hydrophobic chemotherapeutic, currently formulated as Taxol® containing Cremophor EL and dehydrated alcohol (1:1, v/v), can be used effectively for

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the treatment of metastatic breast, lung, and ovarian malignancies,and for tumor targeting therapy.12-13 To improve the solubility and targeting properties of PTX and diminish accessory toxicity, low tolerance, and anaphylaxis caused by Cremophor EL, researchers have applied various methods to construct HSA-PTX nanoparticles (NPs-PTX) as nanocarrier systems; of the developed composites, Abraxane® (nab-PTX) was approved by the FDA in 2005, 2012 and 2013 for the treatment of breast cancer, non-small cell lung cancer (NSCLC) and advanced prostate cancer.6, 14-15 PTX affects cytosolic Ca2+ signaling by opening mitochondrial permeability transition pores,16and stimulates the release of calcium into the cytosol, attenuating Bcl-2 resistance and leading to the induction of apoptosis.17-18 Mitochondrial stress occurs through activation of both the JNK and p38 pathways, and thus, PTX induces ROS accumulation and apoptosis.22 Ag@PEI@PTX activates cell apoptosis via ROS generation through the AKT, MAPK, and p53 signaling pathways.19 In toxicity assessment, morphological observation is a simple and effective method to observe direct toxicity to cells by nanomaterials. Intravenous injection or medium culture can significantly alter cytokine, chemokine, and adhesion molecule levels, circulation system status, and immune cell viability via varying mechanisms.20-21 Gold and polystyrene can jeopardize blood cells and cause blood clotting, whereas anionic particles are biocompatible.22-23 In terms of particle design, we aimed to use a very simple and effective approach through reducing the disulfide bonds in proteins or other methods to gradually expose the hydrophobic region of proteins and form stable nanoparticles through a noncovalent synergistic effect of hydrophobic drug molecules. In this work, an albumin-based PTX nanocarrier system was constructed via alkali induced conformational changes and exposure of hydrophobic patches; the compound had a drug loading efficiency of 21%, which is higher than that of Abraxane® (10%

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loading efficiency), and exhibited clinical trial potential. To establish clear safety protocols for the use of plasma proteins as nanocarrier systems, a systematic assay was conducted to assess the toxicity of Taxol® and NPs-PTX to human umbilical vein endothelial cells (HUVECs), mouse macrophages (RAW264.7), and human tumor cells. Blood cell alterations and pathological changes in rats administered the PTX formulations (NPs-PTX and Taxol®) were characterized. Hemolysis, median lethal dose (50% mortality, LD50), maximum tolerated dose (MTD), and antitumor effects in vivo were also characterized in mice administered the formulations. Materials and methods Materials HSA, rat serum albumin (RSA), 1-(anilinon)aphthalene-8-sulfonic acid (ANS), and PTX were purchased from Sigma-Aldrich (St. Louis, MO), Hanyun Biotechnology (Nanjing, China), Aladin (Shanghai, China) and Xi-An-Xuan-Hao (Xian, China), respectively. Taxol® (Cremophor-based PTX, Cremophor EL:dehydrated alcohol=1:1) was obtained from Qili Group (Haikou, China). The annexin-V FITC/PI apoptosis (KGA105), mitochondrial membrane potential (KGA602), and ROS (KGA010-1) detection kits were purchased from Keygen Biotechnology (Nanjing, China). The Fluo-3 AM kit (S1056) was purchased from Beyotime (China), and -(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was purchased from Guoyao Group (China). Animals Male Sprague-Dawley (SD) rats weighing 200–220 g were purchased from Nantong University (P. R. China). Animal experiments were performed with ethical approval from the Institutional Animal Experimentation Ethics Committee of Jinling Hospital. (Instruction No.20160524) Preparation of NPs-PTX

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Briefly, 100 mg of HSA was dissolved in 50 mL of ultrapure water at 25 °C. Alkali (sodium hydroxide, NaOH, 1M) was added at a final concentration of 10 µM, and the mixture was stirred for 10 min. To develop NPs-PTX, 28 mg of PTX (10 mg mL−1 in ethanol solution) was add. Fluorescence spectrum A 20 μL aliquot of NaOH (1 M) was added to 10 mL of HSA solution (2 mg mL−1) at 25 °C. Then, a certain volume of solution was taken to obtain the fluorescence spectra. The process for obtaining fluorescence spectra was repeated three times. Then, 40 μL of PTX solution (15 mg mL−1 in ethanol) was added. Fluorescence spectra were recorded on an F-7000 spectrofluorometer (HITACHI, Japan). The excitation wavelength was 295 nm, and emission was monitored from 300 to 450 nm. The slit width was 2.5 nm. The samples were measured in triplicate. Hydrophobic region of HSA after denaturation by NaOH Fluorescence measurements were performed on an RF-5301PC spectrofluorometer (Shimadzu, Japan), using a 1.0 cm quartz cell. Tests were carried out at 25 °C after the sample was equilibrated for 18 h. The concentration of HSA was 1 mg mL-1, and the concentration of ANS was 10 µM. The final concentration of NaOH ranged from 1 μM to 20 μM. The fluorescence intensity was evaluated after excitation at 390 nm excitation. The emission intensity was assessed at 400 – 650 nm. The measurements were performed three times. The fluorescence values of in the three groups were measured at 469 nm where the of ANS peaks. Mechanism of apoptosis induction by Taxol® and NPs-PTX HUVECs were incubated with varying concentrations of NPs-PTX and Taxol® (2 and 20 ng mL1)

for 4 and 16 h. The drug-treated cells were collected, and total protein concentrations were

quantified. Bax, Bcl-2, caspase-3 (Cas 3), caspase-8 (Cas 8), caspase-9 (Cas 9), and cytochrome

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C (CYC), were isolated via SDS-PAGE, and the proteins were then transferred to nitrocellulose membranes. The membranes were incubated with a Bax antibody for 24 h and then washed with PBS (+ 0.1% Triton) three times. An appropriate horseradish peroxidase (HRP)-conjugated secondary antibody was then added for 24 h. The membranes were washed, and a developer solution was added to assess differences in Bax protein expression between the NPs-PTX and Taxol® groups. After semiquantification of the Bax protein, Bcl-2, CYC, Cas 3, Cas 8, and Cas 9 were quantified similarly. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal standard for quantification of apoptosis-related proteins. Mouse blood hemolysis induced by the PTX formulations Blood samples were collected from mouse orbital veins and then centrifuged at 1000 rpm for 10 mins (2 times) to harvest red blood cells (RBCs). Then, 2% RBC suspensions were prepared in PBS, and 2 mL of the RBC suspensions was mixed with 2 mL of the PTX formulations (NPsPTX, Abraxane® and Taxol®) at varying concentrations (1, 0.5, 0.25, 0.1, 0.01, or 0.001 mg mL1)

for 30 minutes at 37 °C. The cells were then harvested via centrifugation, and released

hemoglobin in the supernatants was analyzed with a spectrophotometer (SAFIRE Tecan, Switzerland) at 570 nm. The hemolysis rates of RBCs incubated with equal volumes of distilled water or PBS were set as the 100% (Abs100) and 0% (Abs0) controls, respectively. The hemolysis rates induced by NPs-PTX and Taxol® were calculated according to Equation (1), where Abs0 and Abs100 were the detected absorbances of the 0% and 100% hemolysis controls, respectively. Because NPs-PTX absorbs at 570 nm, its absorbance value was calculated by subtracting the absorbance at 570 nm when incubated with RBCs from that of NPs-PTX. Hemolysis %= (Abs-Abs0) / (Abs100-Abs0) ×100% Acute toxicities of NPs-PTX and Taxol®

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(1)

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Nontumor-bearing mice (25-28 g) were administered NPs-PTX (2-10 animals per dose group) or Taxol® (2-10 animals per dose group) at 15.6, 22, 30, 42, 57 or 80 mg/kg/d (1.4 spacing). The drugs were administered continuously through the tail vein for 5 days, and the mice were observed continuously for 14 days after the last administration. LD50 values were calculated using fitted mortality curves (GraphPad Prism 5.0), and the MTD was defined as the dose resulting in < 10% mortality. In vivo therapeutic study Tumor inhibition efficiency against an S180 tumor model was evaluated using male ICR mice. ICR mice were subcutaneously injected with 0.2 mL of S180 cell suspension containing 1×107 cells in the right forelimb axilla. All tumor-bearing mice were randomly divided into four groups (n=6). Treatment was started when the tumor volume in the mice reached 150 – 200 mm3. Each group was treated through tail vein injection on days 1, 3, 5, and 7 with saline, Taxol® (12 mg kg−1), or NPs-PTX (30 mg kg−1). Tumor sizes were measured every two days to evaluate antitumor efficiency. At the end of the experiment, all mice were sacrificed, and the tumors were removed and weighed. Toxicity in SD rats Administration regimen and blood sample collection To diminish the influence of NPs-PTX as a heterologous protein, NPs-PTX (human serum albumin as carrier) was replaced with NPs-PTX(R) (rat serum albumin as carrier) for evaluation of in vivo toxicity in SD rats, and the size of NPs-PTX(R) was approximately 180 nm (data not shown). Twelve rats were randomly divided into four groups based on the drug administered (n = 3): Taxol® (10 mg kg−1), NPs-PTX(R) (equal to 10 mg kg−1 PTX) and PBS (control). The PTX formulations and associated carrier systems were intravenously administered on days 1, 3, and 7, and retro-orbital sinus blood samples were collected on days 1, 7, and 14. Complete blood counts

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Blood samples were collected in heparinized tubes, and the following parameters were analyzed on days 1, 7, and 14: white blood cells (WBCs), RBCs, monocytes (MNCs), hemoglobin (HGB), and platelets (PLTs). All samples were analyzed using a hematology blood autoanalyzer (XS800i, SYSMEX, Japan). Pathological observations After blood sample collection on day 14, the heart, liver, spleen, lungs, kidneys, thymus, and mesenteric lymph nodes were harvested from each of the animals and fixed in PBS solution containing 10% formaldehyde to prepare tissue sections using histopathological methods. The tissue samples were further processed and stained with hematoxylin and eosin (H&E) for histopathological examination and observed under an optical microscope (Nikon). Statistical analysis All experiments were performed at least three times, and the results are expressed as the mean ± standard derivation (SD). Two populations were compared using Student’s t-tests, and *p < 0.05 was considered statistically significant. Results and discussion PTX is one of the most effective first-line chemotherapeutic agents against a variety of cancers. In this work, a full toxicity analysis of PTX formulations and their nanocarrier system was conducted in vitro, in situ, and in vivo. Preparation and characterization of NPs-PTX After the addition of PTX, the solution turned blue, indicating that the interaction between HSA and PTX resulted in the formation of NPs-PTX, constructed via NaOH-triggered unfolding, and hydrophobic PTX acted as a bridge to bring the molecules closer. The NPs-PTX particle size was approximately 180 nm (Figure 1A), and the PTX loading efficiency (weight of PTX in

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NPs/weight of PTX and albumin in NPs-PTX) was approximately 21% (the concentration of PTX was determined via high pressure-performance chromatography (HPLC), Supporting Information, Equation 2), and that of HSA was determined using a Bradford kit for protein quantification (Nanjing Jiancheng Bioengineering Institute). To confirm the role of PTX in the self-assembly process, we prepared HSA-PTX nanoparticles with different concentrations of HSA and paclitaxel. When the concentration of HSA increases from 0.5 to 5 mg mL-1 and the concentration of PTX increases from 5 to 25% (w/w), the hydrodynamic diameter of HSA-PTX nanoparticles increases from 10 to 220 nm (Table S1, supporting information). When the concentration of albumin was 2 mg mL-1, the NPs-PTX with the size of 151 nm could be obtained with the additional PTX (15% of albumin, w/w). When the dosage was less than 15%, nanoparticles smaller than 150 nm could be obtained. The taxane related nanoparticles with the size from 50-150 nm could be fabricated.6, 24 The encapsulation efficiency of NPs-PTX was approximately 92.5% (Supplementary Table. S1). After the addition of NaOH, the endogenous fluorescence of albumin gradually weakened, indicating that the protein and protein molecules are gradually approaching (Figure. 1B). Protein denaturation and exposure of tryptophan to the aqueous environment leads to a decrease in tryptophan fluorescence and exposure of the interior hydrophobic residues of the protein, increasing the hydrophobicity.25 The chromophores are more exposed to the solvent, and fluorescence decreases when they interacts with quenching agents either in the solvent or in the protein itself. Therefore, with the addition of PTX, the gradual decrease in fluorescence indicates the formation of NPs-PTX. However, the fluorescence intensity of ANS, a hydrophobic probe to examine the hydrophobic interface,26-27 was gradually increased, which showed that the hydrophobic areas were gradually exposed and brought close to each other. As shown in Figure 1C, the addition of PTX gradually decreased the ANS

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fluorescence, indicating decrease and enclosure of the hydrophobic region of unfolded albumin. If 15% ethanol (v/v) is added to NPs-PTX solution, NPs-PTX precipitates because ethanol binds the hydrophobic site in albumin to compete with the binding of PTX within NPs-PTX.8 Ethanol can replace ANS molecules and occupy the binding sites of fatty acids in HSA at the hydrophobic region.30,6 Gong et al. supposed that the hydrophobic interaction was necessary for the formation of HSA-PTX nanoparticles because albumin-PTX nanoparticles easily form after chemical modification of albumin with ctyl.31 Therefore, the hydrophobic force between PTX and albumin is decreased. There was significant change on the hydrophobic region of HSA between solution S1 (1 mg mL-1 HSA +1 μM NaOH) and S2 (1 mg mL-1 HSA + 5 μM NaOH, p = 0.013), S1 and S3 (1 mg mL-1 HSA + 20 μM NaOH, p = 0.0045), and S3 and S5 (1 mg mL-1 HSA + 20 μM NaOH + 60 μg mL-1 PTX, p = 0.03) (Figure 1D). After the NaOH was added, the hydrophobic area of albumin is gradually exposed, but it is insufficient to form nanoparticles. The addition of PTX leads to a decrease in hydrophobicity and the formation of a stable nanosystem. In self-assembly process in which albumin was used as carrier system, albumin was unfolded to expose hydrophobic regions and interacted with hydrophobic drugs or probes which is inserted into hydrophobic pocket of albumin to form nanoparticles fabricated through hydrophobic interaction.24, 28-29 During this process, the hydrophobic regions and inner fluorescent intensity of albumin gradually decreased, which had been deduced in our previous work.30,6 The formation of albumin-hydrophobic drugs because of the physiochemical changes is illustrated in Scheme 1. The size of NPs-PTX didn’t changed within 24 h (Supporting Information, Figure S1), and the cumulative release of PTX from NPs-PTX was 20% at a with drug loading content of 21% (Supporting Information, Figure S2). In vitro cytotoxicity

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The effects of Taxol® and NPs-PTX on HUVECs and macrophage viability were compared. Supporting Information, Figure S3A demonstrates the morphologies of HUVECs incubated with different concentrations of the PTX formulations (0.1, 1, and 10 nM) for 72 h. Loss of cell-cell contact between neighboring cells was observed in PTX-treated cells in a dose-dependent manner. As shown in Supporting Information, Figure S3B, an inverse relationship was observed existed between the PTX concentration and the number of RAW264.7 cells, which became rounded and detached, indicating a proliferation inhibition effect of the PTX formulations. Figure 2A and 2B show the survival rates of HUVECs and RAW264.7 cells treated with the PTX formulations, determined by the percentage of living cells treated with nanoparticles versus that of cells treated with saline; the highest concentrations of Taxol® and NPs-PTX led to serious toxicity after 72 h, and only 10%−15% of the cells remained alive relative to the total number of viable saline-treated cells. Furthermore, 2.85 ng mL-1 Taxol® inhibited HUVEC proliferation by 50% at 72 h post exposure, reflecting serious toxicity (Figure 2A). Significant differences in the IC50 of NPs-PTX and Taxol® in HUVECs (4.2 ± 0.1 vs 2.85 ± 0.3 (ng mL-1), p = 0.037) and RAW264.7 cells (87.5 ± 183.9 vs 20.4 ± 10.5 (ng mL-1), p = 0.0011) were observed. An increase in IC50 was observed for NPs-PTX, and Taxol® was more toxic than NPs-PTX. Because the PTX IC50 concentration in HUVECs was close to 2 ng mL-1, and apoptotic cell death followed PTX treatment at this concentration, 2 and 20 ng mL-1 was selected for apoptosis and western blot analyses. In the PTX-treated K562 group, the IC50 values of NPS-PTX (0.702 µg mL−1) and Abraxane® (0.666 µg mL−1) were significantly higher than that of Taxol® (0.188 µg mL−1) (p < 0.01) (Figure 2C). No significant difference was found in the IC50 among NPs-PTX, NPs-PTX (R), and Abraxane in K562 cells (p > 0.05). The PTX concentration was lower than 0.01 µg

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mL−1 and presented almost no toxicity toward K562 cells in the Taxol® and NPs-PTX groups. This finding indicates an absence of toxicity of albumin constructed via alkali-induced conformational changes. In the PTX-treated HepG2 cells, a significant difference was observed between the IC50 of Taxol® and that of NPs-PTX (p = 0.03) and Abraxane® (p = 0.025) (Figure 2D). However, the toxicity of NPs-PTX (R) was similar to that of Taxol® in HepG2 cells (p = 0.119). No significant difference was found in the IC50 among NPs-PTX, NPs-PTX (R), and Abraxane®. Change in the Ca2+ content, apoptosis, ROS level, and Δψm The fluorescent probe Fluo-3 AM was applied to detect cellular Ca2+ content, and the results revealed that the cytosolic level of Ca2+ was augmented in a concentration-dependent manner. The results were visualized using fluorescence microscopy and analyzed via FCS (Supporting Information, Figure S4). Taxol® increased the Ca2+ content to a greater extent than NPs-PTX at the same concentration (Figure 3A). The increased Ca2+ content, indicated cell mitochondrial dysfunction and the disruption of Ca2+ equilibrium state were destructed, resulting in augmented Ca2+ levels. The Annexin-V FITC/PI double staining was utilized to monitor apoptosis. The green and red fluorescence intensities of cells cultured with the PTX formulations increased, indicating that apoptosis occurred (Figure 3B). Taxol® induced apoptosis to a greater extent than NPs-PTX at the same concentration. The percentages of cells that underwent early apoptosis in the NPs-PTX and Taxol® groups were less than 5%, nearly equivalent to that in the control group (Supporting Information, Figure S5). Ca2+ overloaded is a pathological stimulator that opens the permeability transition pore (PTP), leading to mitochondrial inflammation, decreased mitochondrial transmembrane potential (Δψm), and release of apoptosis factors, such as CYC, ultimately inducing apoptosis.31-32 Mitochondria are

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hypothesized to be the major cellular source of ROS.34 Green fluorescence intensity increased in cells treated with the PTX formulations, indicating increased ROS levels (Supporting Information, Figure S6). Cells treated with Taxol® exhibited greater ROS production than cells treated with NPs-PTX at the same concentration (Figure 3C). NPs-PTX and Taxol® produced excessive ROS, which could not be eliminated by the antioxidant system, disrupting the Δψm and inducing apoptosis.33 Elevated intracellular ROS and oxidative stress levels have been shown to play roles in mitochondrial dysfunction and plasma membrane and cellular damage.3435

Δψm loss, a limiting factor in the apoptotic pathway, induces membrane damage and the

release of Bcl-2 and caspase activators to initiate apoptosis. To monitor Δψm in cells exposed to the PTX formulations and the associated carriers, the mitochondrial-specific dye JC-1 was used. The red fluorescence in cells cultured with the PTX formulations gradually decreased compared with that in control cells, indicating a loss of Δψm following exposure to the PTX formulations (Figure 3D, Supporting Information, Figure S7). A significant difference between the effect of NPs-PTX and Taxol® on the loss of Δψm was observed at the same concentration. In addition to the mitochondrial apoptosis pathway, ROS, Ca2+, and Δψm levels might also induce HUVEC apoptosis (Supporting Information, Figure S4-S7). Mechanism underlying apoptosis induction by Taxol® and NPs-PTX Protein expression analyses to assess metabolic activities in cells exposed to the PTX formulations indicated that Taxol® and NPs-PTX induced HUVEC apoptosis via Cas 3, Cas 8, and Cas 9 pathway as well as through mitochondrial damage. In the trend of increased expression levels of protein factors, Taxol® appeared to be stronger than nanoparticles (Figure 4A-4F, Supporting Information, Table S2). This result was positively correlated with the disturbance of mitochondrial activity that led to cell apoptosis (Figure 3B) and the inhibition of

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HUVEC proliferation (Supporting Information, Figure S3A) induced by Taxol®. In addition to targeting microtubules, PTX also targets Bcl-2.36-37 Compared with NPs-PTX, Taxol® induced a higher Bax/Bcl-2 ratio (Figure 4B) and higher Cas 3/GAPDH (Figure 4C) and Cas 8/GAPDH levels (Figure 4D), although these differences were not significant (p > 0.05). Notably, the Cas 9/GAPDH expression levels in cells treated with NPs-PTX and Taxol® (2 ng mL-1) were significantly altered at 24 h (Figure 4E). Changes in Bax and Bcl-2 and activation of Cas3 and 9 have been observed previously in Taxol®-treated human leukaemia cells.40 Differences in CYC levels in PTX-treated cells (2 and 20 ng mL-1) were also significant at 48 h (Figure 4F). At equal concentrations and treatment times, the expression levels of Bcl-2, Cas 3, and Cas 8 in the Taxol® group were lower than those in the NPs-PTX group; the Bcl-2 ratio between the Taxol® and control groups was 0.036, while that between the NPs-PTX and control groups was 0.008 at 2 ng mL-1. The Cas 3 ratio between the Taxol® and control groups was 0.021, while that between the NPs-PTX and control groups was 0.033, indirectly indicating that the toxicity of Taxol® was higher than that of NPs-PTX (Table S2). The apoptotic sensitivity of HUVECs to Taxol® and NPs-PTX might have contributed to the differential expression and increased Bax/Bcl-2 ratio. PTX might activate Cas 8 to evoke apoptosis, and Cas 8 may invoke effector caspases (Cas 3, Cas 6, and Cas 7), which play roles in the biochemical and morphological changes that occur during apoptosis. Furthermore, PTX can induce loss of Δψm (Supporting Information, Figure S7). Microtubule-damaged-related proteins regulate the balance of pro- and anti-apoptotic proteins by transferring Bax from the cytosol to mitochondria,36, 38 which induces CYC release via opening of the PTP.39-40 Cas 9 and Cas 3 are subsequently activated and commit the cell to undergoing apoptosis. Elevated ROS and Ca2+ levels lead to HUVECs apoptosis directly via mitochondrial-independent pathways.41-425 Together, these factors might underlie the release of

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CYC and induction of Cas 9, which activate executing caspases to trigger apoptosis and necrosis. A possible mechanism underlying the apoptosis induced by Taxol® and NPs-PTX is elucidated in Scheme 2. Hemolysis and acute mouse toxicity induced by the PTX formulations The percentage of hemolysis induced by NPs-PTX (1, 0.5, and 0.25 mg mL-1) was approximately 10 – 20 % (Figure 5, Supporting Information, Figure S8A-B, and Figure S8C-D), whereas that induced by Taxol® (0.25 mg mL-1) was 90% (Figure 5). Both 0.5 mg mL-1 and 1 mg mL-1 Taxol® induced 100% hemolysis (no RBCs at the bottom of the test tube. The released hemoglobin forms a brown substance after incubation with Taxol® at 1 mg mL-1, and led to decreased absorption at 570 nm compared with that after treatment with Taxol® at 0.5 mg mL-1 (Supporting Information, Figure S8E-F). The hemolysis rates induced by the two drugs at 1, 0.5, and 0.25 mg mL-1 were significantly different (p < 0.001), while the hemolysis rates induced by both drugs at 0.1 mg mL-1 were less than 10%, without a significant difference among the three drugs. In the Taxol® treated group, erythrocytes were reincubated with Taxol® solution for 30 min and centrifuged to separate the supernatant and precipitate (reconstituted with PBS). Red blood cells of the same volume were reincubated with the supernatant and PBS-reconstituted precipitate for another 30 min. The absorbance of the supernatant in the Taxol® and NPs-PTX group at 570 nm was determined after centrifugation. The results showed that the supernatant of Taxol® (1 mg mL−1)-incubated red blood cells underwent secondary hemolysis because the absorption value increased by 40%, whereas the precipitate showed no secondary hemolysis effect. Extensive hemolysis occurred in the Taxol® group but was scarcely observed in the NPs-PTX group. Taxol® is diluted to 0.3-1.2 mg mL-1 when administered to patients,43 and within this

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concentration range, NPs-PTX appear to be a much safer clinical treatment option than PTX in terms of hemolysis. Toxic responses such as dyspnea, severe prostration, tachycardia, circling, and piloerection, were observed in mice. The dose versus mortality relationship in mice administered the PTX formulations are shown in Figure 6. The LD50 values for NPs-PTX and Taxol® at the indicated dosages were 50.9 ± 8.9 and 30.4 ± 0.3 mg kg-1, respectively (p = 0.009, n = 3), while the MTD values were 42 mg kg-1 and 22 mg kg-1 for NPs-PTX and Taxol®, respectively. NPs-PTX significantly reduced the toxicity of the commercially available Taxol® preparation. The LD50 and MTD are proportional to the body weight of the mice. The low toxicity (high LD50 and MTD) of NPs-PTX provides a significant argument that its chemotherapeutic dosage can be increased. Meanwhile, the inhibition of cellular uptake caused by the hemolysis-prohibiting activity of castor oil in Taxol® can be avoided, which can enhance the therapeutic effect of PTX in tumor patients.14 Antitumor activity in vivo The in vivo antitumor efficiency of NPs-PTX was evaluated in tumor-bearing mice. Both NPsPTX and Taxol® exhibited tumor inhibition. NPs-PTX was more therapeutically efficient than Taxol®, and the tumor inhibition rates of Taxol® and NPs-PTX were 31.2% and 60.8% (p = 0.04), respectively (Figure 7A). For NPs-PTX, the dose reached as high as 30 mg/kg, with an inhibition rate of 60.8%, which was 1.9-fold higher than that of Taxol® (12 mg kg-1). This finding indicates that NPs-PTX exhibits advantages of decreased side effects and increased drug tolerance and is a promising and efficient cancer chemotherapy drug. The body weight of the injected mice was monitored every two days. As shown in Figure 7B, body weights of the mice

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in the PTX groups were similar to those of mice in the control group treated with saline (p > 0.05). Toxicity study in SD rats Hematological analysis was executed; the pivotal hematological parameters are shown in Figure 8. The PTX formulations did not induce a marked difference in RBC and HGB counts (Figure 8A and 8B). Treatment with Taxol® reduced WBC counts on days 7 and 14, and changes between days 1 and 7 (p < 0.05) and days 1 and 14 (p < 0.05) varied in the PTX group (Figure 8C). However, no obvious difference in WBC counts between days 1 and 7 (p > 0.05) and days 1 and 14 (p > 0.05) were observed in the NPs-PTX(R) group. The number of WBCs in the Taxol® group was lower than that in the NPs-PTX(R) group on day 7 (p < 0.05). In both the Taxol® and NPs-PTX(R) groups, MNCs appeared damaged on both days 7 and 14 (Figure 8D). Differences in MNC percentages between days 1 and 7 (p < 0.001) and days 1 and 14 (p < 0.01) in the Taxol® group and between days 1 and 7 (p < 0.01) and days 1 and 14 (p < 0.05) in the NPsPTX(R) group were significant; the toxicity of NPs-PTX(R) on toward MNCs was milder than that of Taxol®. Rats treated with the PTX formulations exhibited significantly reduced PLT counts on days 1 and 7 (p < 0.05) (Figure 8E). However, the PLT counts decreased continuously in the Taxol® group, with significant changes being observed between days 1 and 14 (p < 0.01). The PLT counts remained constant between days 1 and 14 (p < 0.05) in the NPs-PTX group. Like other antitumor agents, multiple doses of Taxol® and NPs-PTX(R) affect the immune function of the host, resulting in symptomatic toxicity.47 After performing cell counts and cytokine determination, the pathological effects of Taxol® and NPs-PTX(R) were compared. Rats intravenously administered the PTX formulations lost weight, whereas those in the control group gained weight (data not shown). Compared with NPs-PTX treatment, which caused PTX

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toxicity, Taxol® treatment clearly reduced the number of splenic lymphocytes (quadrangle region, spleen, row three) and augmented the number of thymic macrophages (denoted by the black arrow, thymus, row six) (Figure 9). These observations were consistent with the lymphocyte assay results and might be related to inhibition of immune function. The medulla (mesentery lymph nodes, row seven) demonstrated further lymph sinus dilation in the Taxol® group, while lymph sinus dilation in the NPs-PTX(R) was similar to that in the control group. No visible PTX-related inflammatory responses or lesions were observed in the heart, liver, lung, or kidneys, and these tissues appeared similar in the treatment and control groups. Conclusions Herein, a NPs-PTX nanocarrier system with a drug loading efficiency of 21% was constructed via alkali-induced conformational change and hydrophilic - hydrophobic transition. To different degrees, Taxol® and NPs-PTX altered HUVEC viability and apoptosis in a dose-dependent manner. The toxicity of Taxol® in HUVECs and RAW264.7, K562, and HepG2 cells was greater than that of NPs-PTX, and the mechanism underlying the apoptosis induced by NPs-PTX was similar to that induced by Taxol®. Compared with Taxol®, NPs-PTX induced hemolysis to a lesser degree. The LD50 and MTD values of NPs-PTX were higher than those of Taxol®, suggesting that the therapeutic dosage of NPs-PTX could be increased, leading to improved effects and avoidance of the highly toxic drug Cremophor EL. The antitumor effect of NPs-PTX was higher than that of Taxol® (p < 0.05) when the dose of NPs-PTX (equivalent PTX) was 2.5 times that of Taxol® (30 mg kg-1 vs 12 mg kg-1). Rats administered the PTX formulations exhibited significant bone marrow suppression toxicity. Overall, Taxol® is more toxic than NPsPTX. NPs-PTX is a better nanocarrier system for PTX delivery, and this study suggests suitable guidelines for future studies on NPs-PTX administration. This work provides a basis for

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understanding how to evaluate the toxicity of polymers, plasma proteins, peptides and their analogues for use as building blocks in the assembly of nanocarrier systems for MRI with imaging probes and for delivery of poorly soluble chemotherapy agents. Associated content The authors declare no competing financial interests. Supporting Information. Purification and drug loading efficiency of human serum albumin paclitaxel nanoparticles (NPs-PTX); endotoxin detection; stability of HSA–PTX nanoparticles in rat serum (Figure S1); in vitro release study (Figure S2); cells culture, morphological observation (Figure S3) and cytotoxicity analysis; cellular Ca2+ content with Fluo-3 AM (Figure S4); apoptosis detection via annexin-V FITC/PI dual staining (Figure S5); intracellular ROS content determined with DCFH-DA (Figure S6); Δψm assessed via JC-1 staining (Figure S7); hemolysis induced by NPs-PTX and Taxol® (Figure S8); characterization of NPs-PTX (Table. S1); expression of apoptosis-inducing proteins in PTX-treated and control cells (Table. S2). rate of hemolysis caused by NPs-PTX, Abraxane and Taxol® (Table S3). This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION *Correspondence: sdwangpharm@126.com; gonggm@126.com Author Contributions The manuscript was written through contributions from all authors. All authors have given approval to the final version of the manuscript. Acknowledgements

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This work was financially supported by NFSC grant no. 31671026. The authors thank Yuxi Wang and Jihua Fu for their constructive suggestions and Lei Liang for help with blood dynamic analysis. References 1. Petros, R. A.; DeSimone, J. M., Strategies in the design of nanoparticles for therapeutic applications. Nature reviews. Drug discovery 2010, 9 (8), 615-27. DOI: 10.1038/nrd2591. 2. Blanco, E.; Shen, H.; Ferrari, M., Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat Biotechnol 2015, 33 (9), 941-51. DOI: 10.1038/nbt.3330. 3. Jin, Q.; Liu, J.; Zhu, W.; Dong, Z.; Liu, Z.; Cheng, L., Albumin-Assisted Synthesis of Ultrasmall FeS2 Nanodots for Imaging-Guided Photothermal Enhanced Photodynamic Therapy. ACS Appl Mater Interfaces 2018, 10 (1), 332-340. DOI: 10.1021/acsami.7b16890. 4. Bensimhon, L.; Metaye, T.; Guilhot, J.; Perdrisot, R., Influence of temperature on the radiochemical purity of 99mTc-colloidal rhenium sulfide for use in sentinel node localization. Nucl Med Commun 2008, 29 (11), 1015-20. DOI: 10.1097/MNM.0b013e32830ebd13. 5. Sheng, Z.; Hu, D.; Zheng, M.; Zhao, P.; Liu, H.; Gao, D.; Gong, P.; Gao, G.; Zhang, P.; Ma, Y.; Cai, L., Smart human serum albumin-indocyanine green nanoparticles generated by programmed assembly for dual-modal imaging-guided cancer synergistic phototherapy. ACS Nano 2014, 8 (12), 12310-22. DOI: 10.1021/nn5062386. 6. Gong, G.; Xu, Y.; Zhou, Y.; Meng, Z.; Ren, G.; Zhao, Y.; Zhang, X.; Wu, J.; Hu, Y., Molecular switch for the assembly of lipophilic drug incorporated plasma protein nanoparticles and in vivo image. Biomacromolecules 2012, 13 (1), 23-8. DOI: 10.1021/bm201401s. 7. Wang, W.; Huang, Y.; Zhao, S.; Shao, T.; Cheng, Y., Human serum albumin (HSA) nanoparticles stabilized with intermolecular disulfide bonds. Chem Commun (Camb) 2013, 49 (22), 2234-6. DOI: 10.1039/c3cc38397k. 8. Kinoshita, R.; Ishima, Y.; Chuang, V. T. G.; Nakamura, H.; Fang, J.; Watanabe, H.; Shimizu, T.; Okuhira, K.; Ishida, T.; Maeda, H.; Otagiri, M.; Maruyama, T., Improved anticancer effects of albumin-bound paclitaxel nanoparticle via augmentation of EPR effect and albuminprotein interactions using S-nitrosated human serum albumin dimer. Biomaterials 2017, 140, 162-169. DOI: 10.1016/j.biomaterials.2017.06.021. 9. Yuan, A.; Wu, J.; Song, C.; Tang, X.; Qiao, Q.; Zhao, L.; Gong, G.; Hu, Y., A novel selfassembly albumin nanocarrier for reducing doxorubicin-mediated cardiotoxicity. Journal of pharmaceutical sciences 2013, 102 (5), 1626-35. DOI: 10.1002/jps.23455. 10. Ahmad, B.; Kamal, M. Z.; Khan, R. H., Alkali-induced conformational transition in different domains of bovine serum albumin. Protein Pept Lett 2004, 11 (4), 307-15. 11. Moayedi, V.; Omana, D. A.; Chan, J.; Xu, Y.; Betti, M., Alkali-aided protein extraction of chicken dark meat: composition and stability to lipid oxidation of the recovered proteins. Poult Sci 2010, 89 (4), 766-75. DOI: 10.3382/ps.2009-00494. 12. Desai, N.; Trieu, V.; Yao, Z.; Louie, L.; Ci, S.; Yang, A.; Tao, C.; De, T.; Beals, B.; Dykes, D.; Noker, P.; Yao, R.; Labao, E.; Hawkins, M.; Soon-Shiong, P., Increased antitumor activity, intratumor paclitaxel concentrations, and endothelial cell transport of cremophor-free, albumin-bound paclitaxel, ABI-007, compared with cremophor-based paclitaxel. Clin Cancer Res 2006, 12 (4), 1317-24. DOI: 10.1158/1078-0432.ccr-05-1634.

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Figures and Tables

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Figure 1. Characterization of NPs-PTX. A) HRTEM images depicting the size, shape, and morphology of NPs-PTX. B) HSA fluorescence changes of after addition NaOH or PTX at 25 ° C, in the free albumin (1 mg mL-1 HSA), S1 (1 mg mL-1 HSA +1 μM NaOH), S2 (1 mg mL-1 HSA + 5 μM NaOH),S3 (1 mg mL-1 HSA + 20 μM NaOH), S4 (1 mg mL-1 HSA + 20 μM NaOH + 30 μg mL-1 PTX), S5 (1 mg mL-1 HSA + 20 μM NaOH + 60 μg mL-1 PTX) groups. The peak of fluorescence decreased as the concentration of NaOH increased in S1-S3. In S4-S5, the HAS fluorescence decreased when the concentration of NaOH increased and PTX was added. C) Fluorescence spectra of ANS in the S0, S1, S2, S3, S4, and S5 groups with the addition 10 μM ANS at 25 ° C; free ANS (10μM ANS) was used as the control, respectively. D) Comparison of the peak value of ANS (469 nm) in each group (SO, S1, S2, S3, S4, S5) in which the concentration of ANS was 10 μM. In S1-S3, the intensity of ANS increased as the concentration of NaOH increased. The intensity of ANS decreased with the addition of PTX in S4-S5.

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Figure 2. In vitro toxicity of PTX formulations. The final concentrations of NPs-PTX, and Taxol® ranged from 0.1–6000 ng mL-1 and 0.1–200 ng mL-1, respectively. Viabilities of A) HUVECs and B)RAW264.7 cells treated with Taxol® (0.1–6000 nM), or NPs-PTX (equal to PTX) at 37 °C for 72 h. Viability of C) K562 and D) HepG2 cells treated with Taxol®, NPs–PTX, NPs-PTX (R), or Abraxane® ranging from 0.001-10 μg mL-1, respectively. Cell viability was calculated by dividing the viability of the treated samples by that of the control. The results were derived from three to five independent experiments and are expressed as the mean ± SD. The inset photograph is a comparison of NPs-PTX formualations and Taxol® IC50 values (n = 3) Taxol® NPs–PTX NPs–PTX (R) Abraxane®. *p < 0.05, **p < 0.01 compared between PTX formulations and Taxol® injection at the same concentration.

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A)

C)

B)

D)

Figure 3. Ca2+, apoptosis, ROS, and Δψm levels in HUVECs treated for 24 h at 37 °C. Cells were exposed to Taxol® (2, 20, 200 ng mL-1) or NPs-PTX (equivalent to ng mL-1) for 24 h. A)Exposed cells were treated with annexin Fluo-3 AM according to the manufacturer’s protocol and analyzed via FCS for the Ca2+ assay. B)Cells were treated with annexin V-FITC/PI and analyzed via FCS for the apoptosis assay. C)Cells were treated with DCFH-DA according to the manufacturer’s protocol and analyzed via FCS for ROS quantification. D)Cells were treated with JC-1 and analyzed via FCS to detect changes in Δψm. *p < 0.05, **p< 0.01 compared between NPs-PTX and Taxol® at the same concentration.

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Taxol®

NPs-PTX

A) (ng mL-1) Control

4h 2

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Taxol®

NPs-PTX 16 h

2

20

2

20

2

B)

20

Bax Bcl-2 Caspase-3 Caspase-8 Caspase-9 Cytc GAPDH

C)

E)

D)

F)

Figure 4. Western blot analysis to quantify the expression of apoptosis-inducing proteins in HUVECs in response to Taxol® and NPs-PTX exposure. A)Cells were cultured in the presence of Taxol® and NPs-PTX for 4 and 16 h, and control cells (medium did not contain PTX) were treated for 4 h. Bax, Bcl-2, Cas 3 (17 kD), Cas 8, (18 kD), Cas 9 (37 kD), and CYC levels were determined by western blot analysis. GAPDH served as the control. Expression levels of apoptosis-inducing proteins in HUVECs after treatment with PTX formulations at varying concentrations for 4 and 16 h. B) Bax/Bcl-2. C) Cas 3/GAPDH. D) Cas 8/GAPDH. E) Cas 9/GAPDH. F) CYC/GAPDH. Differences in Cas 9/GAPDH and CYC/GAPDH ratios between the groups treated with NPs-PTX and Taxol® at 2 ng mL-1 (Cas 9/GAPDH, p = 0.044; CYC/GAPDH, p = 0.003) and 20 ng mL-1 (CYC/GAPDH, p = 0.008) at 4 h were significant. *p < 0.05, **p < 0.01 compared between NPs-PTX and Taxol® at the same concentration. ACS Paragon Plus Environment

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Figure 5. Rates of hemolysis induced by the PTX formulations. Each data point represents the mean ± SD (n=3). *p < 0.05, ** p < 0.01 compared among NPs-PTX, Abraxane and Taxol® injection at the same concentration.

Figure 6. Dose acute toxicity evaluation (LD50) of NPs-PTX and market Taxol®. Nontumor-bearing mice (25-28 g) were administered NPs-PTX (2-10 animals per dose group) or Taxol® (2-10 animals per dose group) at 15.6, 22, 30, 42, 57 or 80 mg/kg/d. The drugs were administered continuously through the tail vein for 5 days. Mortality data from tumor-free mice were plotted against dosage and Taxol®; NPs-PTX. The inset shows a comparison curve-fit using GraphaPad Prism software. ® ** of NPs-PTX and Taxol LD50 values ( p < 0.01).

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A)

B) Saline

Taxol® NPs-PTX

Figure 7. Antitumor effect of NPs–PTX and Taxol® in S180 tumor-bearing mice. Time course of tumor volume after administration of different PTX formulations on the indicated days (shown by arrow) after the tumor volume reached 150–200 mm3. The inset shows representative tumors from different groups (A); body weight changes in mice treated with NPs–PTX and Taxol® (B). Saline group; Taxol® (12 mg kg−1); NPs-PTX (30 mg kg−1 in PTX equivalents). Each point represents the mean ± S.D of six mice. Student’s t-test was employed to compare NPs–PTX and Taxol®. *p < 0.05 compared between NPs–PTX and Taxol®.

A)

B)

D)

C)

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Figure 8. Whole blood cell analysis of rats injected with different formulations on days 1, 3, and 7. A) RBCs, B) HGB, C) WBCs, D) MNCs, and E) PLTs were quantified on days 1, 7, 14. Significant differences between days 1 and 7 and between days 1 and 14 among the Control, NPs-PTX(R) and Taxol® groups were determined with Student’s t-tests (*p < 0.05 was considered significant).

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Control

NPs-PTX

Taxol®

Heart

Liver

Spleen

Lung

Kidney

Thymus Mesenteric lymph node

Figure 9. Histological observation of organs from rats on day 14 after continuous injection of Taxol®, NPs-PTX(R), or saline (control) on days 1, 3, and 7. All slices were stained with H&E. Scale bar: 50 μm. The quadrangle region and black arrows represent lymphocyte and macrophage abnormalities in the Taxol® and NPs-PTX(R) groups; rats administered NPs-PTX(R) exhibited less tissue damage.

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Native state albumin

PTX

Scheme. 1. The formation of NPs-PTX nanoparticles. The red region represents hydrophobic amino acids. When the solution pH is increased, the protein structure changes, gradually unfolding the hydrophilic albumin protein to expose hydrophobic patches. When paclitaxel is added, multiple albumin molecules aggregate together to form a stable albumin-PTX nanosuspension.

Scheme 2. Mechanism of Taxol®/NPs-PTX-induced HUVEC apoptosis via mitochondria-dependent (intrinsic) and mitochondria-independent pathways Microtubule damage-related proteins regulate the balance of apoptosis by transferring Bax from the cytosol to mitochondria, which induces CYC release. Thus, Cas 9 and Cas 3 are subsequently activated and commit the cell to undergoing apoptosis. Elevated ROS and Ca2+ levels lead to the apoptosis of HUVEC apoptosis directly via mitochondrial-independent pathways.

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TOC

Antitumor effect and toxicity of an albumin-paclitaxel nanocarrier system constructed via controllable alkali-induced conformational changes Guangming Gonga, Yongjun Jiaob, Qinqin Panc, Hao Tanga, Yanli And, Ahu Yuane, Kaikai Wangf, Canping Huangg, Weimin Daih, Ying Lui, Shudong Wanga*, Jian Zhangj* and Hua Sua*

Released

Albumin-paclitaxel nanoparticles (NPs-PTX) was constructed via alkali-induced conformational changes. PTX formulations promoted cell apoptosis possibly via mitochondria-dependent (intrinsic) and mitochondria-independent ®

pathways. NPs-PTX has advantages over the commercially available Taxol formulation in terms of low toxicity and increased dosage, indicating NPs-PTX is a better option for safe and effective PTX delivery.

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