In vitro and in vivo Evaluation of a Novel Estrogen-Targeted PEGylated Oxaliplatin Liposome for Gastric Cancer

Yuxin Sun; Yizhuo Xie; Huan Tang; Zhihui Ren; Xue Luan; Yan Zhang; Ming Zhu; Zhe Lv; Han Bao; Yan Li; Rui Liu; Yujia Shen; Yucui Zheng; Jin Pei

doi:10.2147/IJN.S340180

. 2021 Dec 23;16:8279–8303. doi: 10.2147/IJN.S340180

In vitro and in vivo Evaluation of a Novel Estrogen-Targeted PEGylated Oxaliplatin Liposome for Gastric Cancer

Yuxin Sun ^1,^*, Yizhuo Xie ^1,^*, Huan Tang ¹, Zhihui Ren ¹, Xue Luan ¹, Yan Zhang ¹, Ming Zhu ¹, Zhe Lv ¹, Han Bao ¹, Yan Li ¹, Rui Liu ¹, Yujia Shen ¹, Yucui Zheng ¹, Jin Pei ^1,^✉

PMCID: PMC8712509 PMID: 34992365

Abstract

Background

Chemotherapy is still the main first-line treatment for advanced metastatic gastric cancer, but it has the limitations of serious side effects and drug resistance. Conventional liposome has been substantially used as drug carriers, but they lack targeting character with lower drug bioavailability in tumor tissues. Based on the above problems, a novel estrogen-targeted PEGylated liposome loaded with oxaliplatin (ES-SSL-OXA) was prepared to further improve the metabolic behavior, the safety profile, and the anti-tumor efficacy of oxaliplatin.

Methods

Four kinds of oxaliplatin (OXA) liposomes were prepared by film hydration method. The obtained formulations were characterized in terms of entrapment efficiency (EE), particle size, and so on by HPLC and DLS (dynamic light scanning). The morphology of ES-SSL-OXA was detected by transmission electron microscope (TEM). The in vitro and in vivo targeting effect of ES-SSL-OXA was verified by fluorescence microscopy and in vivo imaging system in gastric cancer cells (SGC-7901) and tumor-bearing athymic mice. The in vitro and in vivo antitumor efficacies of ES-SSL-OXA were investigated on SGC-7901 cells and athymic tumor-bearing mice. Pharmacokinetic, biodistribution, and acute toxicity tests of ES-SSL-OXA were performed on ICR mice.

Results

The ES-SSL-OXA exhibited an average particle size of about 153.37 nm with an encapsulation efficiency of 46.20% and low leakage rates at 4°C and 25°C. In vivo and in vitro targeting study confirmed that ES-SSL-OXA could effectively target the tumor site. The antitumor activity demonstrated the strongest inhibition in tumor growth of ES-SSL-OXA. Pharmacokinetics and acute toxicity study showed that ES-SSL-OXA could significantly improve the metabolic behavior and toxicity profile of oxaliplatin.

Conclusion

In this study, a novel estrogen-targeted long-acting liposomal formulation of OXA was successfully prepared. ES fragment effectively targeted the delivery system to tumor tissues which highly express estrogen receptor, providing a promising therapeutic method for gastric cancer in clinic.

Keywords: estrogen receptors, chemotherapy, long-acting liposomes, targeted drug delivery, pharmacokinetics, antitumor activity

Graphical Abstract

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Introduction

Gastric cancer (GC) is the fifth most common cancer in the world.¹ It is the third leading cause of cancer-related deaths worldwide with the highest mortality rate in eastern Asia.² The diagnosis of GC in most patients is commonly in the advanced stage, and the prognosis of GC treatment remains poor. Despite the advances that have been made in GC therapy such as chemotherapy, the 5-year overall survival (OS) rate of metastatic gastric adenocarcinoma is estimated to be only around 5–20%.³ At present, chemotherapy is still the main first-line treatment for advanced metastatic gastric cancer.⁴

Oxaliplatin (OXA), (1R-trans)-(1,2-cyclohexanediamine-N,N’)[oxalic acid (2-)-O,O’] platinum, is third-generation platinum compound with significant activity against advanced or metastatic digestive tumors⁵ through the cross-linking of platinum atoms with DNA strands that blocks the replication and transcription of DNA.⁶^,⁷ Oxaliplatin is considered a standard of GC treatment by National Comprehensive Cancer Network Guidelines for gastric cancer.⁸ However, oxaliplatin, like other chemo-drug, has serious side effects, not only gastrointestinal toxicity including nausea, vomiting, diarrhea, and mucosal inflammation, but also serious neurological toxicity and system toxicity.^9–11 Unlike other platinum-based drugs, the main side effect of OXA is polyneuropathy, which is ascribed to its pharmacokinetics properties, such as highly irreversible binding to plasmatic proteins and tissue proteins.⁵^,¹² In addition, because traditional chemotherapy has no targeting property to maintain high drug concentration in the tumor, the antitumor effect does not achieve the optimization and meanwhile the drug in normal tissues causes numerous adverse effects. In order to solve these problems, the encapsulation of chemo-drugs in liposome represents a strategy to overcome these limitations.¹³^,¹⁴

Liposomes have been widely used as drug carriers due to their biocompatible and biodegradable characteristics.¹⁵ However, liposomes have low stability in physiological media and mainly accumulate in reticuloendothelial system (RES) like liver and kidney.¹⁶ Since PEGylation can significantly reduce recognition by RES, liposomes have been modified with polyethylene glycol (PEG) to improve biological stability and bioavailability.^17–19 Therefore, chemo-drugs loaded in PEGylated liposome could be delivered to cancer cells more efficiently and stably.

Even though conventional and PEGylated liposomes have been widely used as a drug delivery system, the deficiency of the carrier is that it has no targeting property in tumor tissues. Recently, researchers have focused on tumor-targeted liposomal delivery system. Lactoferrin,²⁰ galactose²¹ and glycyrrhetinic acid,²²^,²³ and so on were used to modify liposomes to target drugs into the tumor tissue more effectively. Estrogen receptors belong to the nuclear receptor protein family²⁴ involved in various physiological processes such as cell growth, reproduction, development, and differentiation.²⁵^,²⁶ However, their over-expression has been reported in various malignant tumors, such as breast,²⁷ ovarian,²⁸ cervical, lung, and gastric cancer.²⁹ Studies have shown that 40–50% of gastric cancer tissues expressed estrogen receptor.^30–32 Estrogen receptors have been isolated in many gastric adenocarcinomas.³³ Based on the above reports, we believe that estrogen receptor could be an effective target spot for the treatment of gastric cancer.

In our previous study, we had successfully synthesized DSPE-PEG₂₀₀₀-ES fragment as a targeting ligand for tumor cells which highly express estrogen receptor.³⁴ The in vitro and in vivo results indicated that ES can effectively target the delivery system to tumor tissues which highly express estrogen receptor. In this study, a novel ES-targeted PEGylated liposome (termed as ES-SSL) loaded with OXA (ES-SSL-OXA) was prepared for gastric cancer which highly express estrogen receptor. The morphology and physical and chemical properties of ES-SSL-OXA were determined. The drug release behavior and stability of ES-SSL-OXA were studied. The in vitro and in vivo targeting efficiency of ES-SSL-OXA were detected. The uptake mechanism of SGC-7901 for ES-SSL-OXA was investigated. The ability of the ES-SSL-OXA against the cancer cell proliferation was studied in SGC-7901 cells. The in vivo antitumor efficacy of ES-SSL-OXA was evaluated in SGC-7901 tumor-bearing athymic mice. Moreover, pharmacokinetics, biodistribution, and acute toxicity tests were determined in ICR mice.

Materials and Methods

Materials

Oxaliplatin (OXA) was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Soya phosphatidylcholine was purchased from Guangfu Fine Chemical Research Institute (Tianjin, China). Cholesterol was bought from Huishi Biochemical Reagent Co., Ltd. (Shanghai, China). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DSPE-PEG₂₀₀₀-NH₂) was purchased from Nanocs Inc. (New York, USA). Estrone was purchased from TCI Development Co., Ltd. (Shanghai, China). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol) 2000] (DSPE-mPEG₂₀₀₀) was obtained from Seebio Biotech Co., Ltd. (Shanghai, China). 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) is the product of MYM Biochemical Reagent Co., Ltd. (Beijing, China). Rhodamine B (RhB) was purchased from Tianjin Guangfu Fine Chemical Research Institute. DiR was purchased from Meilun Biotechnology (Dalian, China). All the reagents were of analytical or HPLC grade.

Cells

Human gastric cancer cell (SGC-7901) was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and authenticated by Short Tandem Repeat (STR) profiling. Cells were cultured in RPMI-1640 medium,³⁵ supplemented with 10% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin at 37°C in an environment containing 5% CO_2.

Animals

5–6 weeks old (20.0 ± 2.0 g) BALB/c nude mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and the animal license number is SCXK Beijing 2012-0001. ICR mice of specific pathogen free (SPF) were purchased from the Experimental Animal Center of the Basic Medical College of Jilin University (Changchun, China). The animals were fed a standard diet and allowed water ad libitum. All animal procedures complied with the China National Institute’s Guidelines on the Care and Use of Laboratory and approved by the Ethics Committee of the School of Pharmaceutical Sciences, Jilin University (Approval No. 20150012, Approval No. 20170019). All experimental procedures were performed in accordance with the ethics and regulations of general animal experiments at Jilin University.

Preparation of Liposomes

For the optimal conditions for the preparation of the final product, the liposomes in our study were composed of phospholipids and cholesterol, which formed the shell-core structure with hydrophilic core and the phospholipid bilayer. For the phospholipid selection, the effects of soybean lecithin and egg yolk lecithin on the encapsulation efficiency were investigated, and the results showed that soybean lecithin has a better encapsulation efficiency on OXA. For the ratio of phospholipid to cholesterol, the effect of different ratios of phospholipid to cholesterol on the encapsulation efficiency was explored through preliminary experiments. The results showed that the encapsulation efficiency of OXA was the highest when the mass ratio of phospholipid to cholesterol was 5:1. Besides that, several key preparation conditions, such as ratio of OXA to phospholipid, hydration volume, hydration time, and hydration temperature, were studied through preliminary experiments. Thereby, a preparation method with higher encapsulation efficiency and drug loading was obtained.

Based on the above studies, the final product of the ES-targeted PEGylated OXA liposomes (ES-SSL-OXA) was prepared employing the film hydration method.¹² Briefly, 40.0 mg of soya phosphatidylcholine (SPC), 8.0 mg of cholesterol (CHO), and 2.7 mg of DSPE-mPEG₂₀₀₀ were dissolved in 8.0 mL of chloroform in a round-bottomed flask. The organic solvent was removed to form the thin film by evaporation in a rotary evaporator (Rotary Evaporator R201D; Yukang Science and Equipment Co., Ltd, Shanghai, China) at 37°C for 1 h. Then, the lipid film was hydrated with 4.0 mL of 5.0% dextrose solution³⁶ containing 4.0 mg of oxaliplatin at 37°C for 2 h, followed by sonication for 5 min at 100 W with an ultrasound probe at 4°C to form the PEGylated OXA liposomes (SSL-OXA). Finally, 0.5 mg of DSPE-PEG₂₀₀₀-ES (DSPE-PEG₂₀₀₀-ES, produced as described previously³⁴) was added to form the ES-targeted PEGylated OXA liposome (ES-SSL-OXA). The formulations were then sequentially extruded through 450 nm and 220 nm polycarbonate membrane three times. In addition, the ES-targeted non-PEGylated liposome (ES-L-OXA) was prepared without DSPE-mPEG₂₀₀₀, the non-targeted PEGylated liposome (SSL-OXA) was prepared without DSPE-PEG₂₀₀₀-ES, and the non-targeted and non-PEGylated liposome (L-OXA) was prepared without DSPE-mPEG₂₀₀₀ and DSPE-PEG₂₀₀₀-ES. The formulation composition of OXA liposomes is shown in Table 1.

Table 1.

The Formulation Composition of OXA Liposomes

Sample	Composition
Sample	SPC (mg)	CHO (mg)	OXA (mg)	DSPE-mPEG₂₀₀₀ (mg)	DSPE-PEG₂₀₀₀-ES (mg)	Chloroform (mL)	5% Dextrose (mL)
L-OXA	40.0	8.0	4.0	–	–	8.0	4.0
SSL-OXA	40.0	8.0	4.0	2.7	–	8.0	4.0
ES-L-OXA	40.0	8.0	4.0	–	0.5	8.0	4.0
ES-SSL-OXA	40.0	8.0	4.0	2.7	0.5	8.0	4.0

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The rhodamine B liposome (L-RhB) (SPC: CHO = 11:9, molar ratio), the PEGylated rhodamine B liposome (SSL-RhB) (SPC: CHO: DSPE-mPEG₂₀₀₀ = 11:9:1, molar ratio), the ES-targeted rhodamine B liposome (ES-L-RhB) (SPC: CHO: DSPE-PEG₂₀₀₀-ES = 11:9:0.1, molar ratio), and the ES-targeted PEGylated rhodamine B liposome (ES-SSL-RhB) (SPC: CHO: DSPE-mPEG₂₀₀₀: DSPE-PEG₂₀₀₀-ES = 11:9:1:0.1, molar ratio) were prepared by the film hydration method. The molar ratio of rhodamine B to lipids was 1:30. The SPC, CHO, and DSPE-mPEG₂₀₀₀ were dissolved in chloroform in a round-bottomed flask. The film was formed by rotary evaporation at 37°C for 1 h. Then, the lipid film was hydrated with PBS containing RhB at room temperature for 2 h, followed by sonication for 10 min. The formulations were then dialyzed in a dialysis bag (MWCO = 8–10 kDa) for 4 h in 1.0 L PBS to remove the un-encapsulated rhodamine B.

The PEGylated DiR liposome (SSL-DiR) (SPC: CHO: DSPE-mPEG₂₀₀₀ = 11:9:1, molar ratio) and the ES-targeted PEGylated DiR liposome (ES-SSL-DiR) (SPC: CHO: DSPE-mPEG₂₀₀₀: DSPE-PEG₂₀₀₀-ES = 11:9:1:0.1, molar ratio) were prepared by the film hydration method. The molar ratio of DiR to lipids was 1:1000. The SPC, CHO, DSPE-mPEG₂₀₀₀, and DiR were dissolved in chloroform in a round-bottomed flask to form a lipid film, and then the lipid film was hydrated with PBS. The liposome suspension preparation was performed as described in section of preparation of the rhodamine B liposomes.

Encapsulation Efficiency and Drug Loading

The encapsulation efficiency (EE) and drug loading (DL) of OXA in different formulations were quantified by the high-performance liquid chromatography (HPLC, Shimadzu, Japan). 0.1 mL of oxaliplatin liposome solution without dialysis was taken and demulsified with 0.9 mL sodium dodecyl sulfate (SDS) solution. The total content of oxaliplatin was determined by HPLC, which was recorded as W₀. Un-encapsulated free OXA was removed by dialysis in 500 mL, 5.0% dextrose solution for 3 h, and then 0.1 mL of liposomal solution was added with 0.9 mL SDS solution for sonicating 10 min to break liposomes. DETC was added into demulsified liposome solution at a molar ratio of 5:1 to OXA. The samples were incubated at 37°C for 30 min. Then isopyknic chloroform was added into the sample following vortexing for 1 min. The chloroform layer that contains OXA was collected by 10,000 rpm centrifugation for 10 min at 4°C. The content of oxaliplatin was determined by HPLC at 254 nm, which was recorded as W.

The EE and DL of OXA were calculated using the following formulas:

W_L is the total mass of OXA liposomes, which is the sum of phospholipid, cholesterol, DSPE-mPEG₂₀₀₀, DSPE-PEG₂₀₀₀-ES, and OXA.

The HPLC method³⁷^,³⁸ was established as follows: Column: Shimadzu C18 reverse phase column (150.0 mm × 4.6 mm, 5.0 μm), column temperature: 25°C, mobile phase: methanol-water (78:22, v/v), detection wavelength: 254 nm, flow rate: 1.0 mL/min, injection volume: 20.0 μL. The chromatographic method was validated according to the requirement of Chinese Pharmacopoeia, including limit of detection, limit of quantitation, intra- and inter-day precision, and so on. The standard curve formula of OXA was y=13355x-4406, R²=0.9999. All those above indicated that establishment of HPLC method for determination of encapsulation efficiency and drug loading was feasible and repeatable.

The Morphology of Liposomes

The morphology of liposomes was analyzed by transmission electron microscopy (TEM) (JEM-2100F; JEOL Ltd., Tokyo, Japan). For TEM sample preparation, ES-SSL-OXA was dropped onto a pure carbon film copper mesh and stained with 2.0% phosphotungstic acid.³⁹

The Particle Size, PDI, and Zeta Potential

The particle size, polydispersity index (PDI), and zeta potential of different liposomes containing OXA (L-OXA, ES-L-OXA, SSL-OXA, and ES-SSL-OXA) were measured using the dynamic light scanning (DLS) (Zetasizer Nano ZS90; Malvern Instruments, Worcestershire, UK). Formulations were diluted 1:100 (v/v) in deionized water and measured at 25°C.

Drug Release Study

The release behaviors of OXA from different liposomes (L-OXA, SSL-OXA, ES-L-OXA, and ES-SSL-OXA) were analyzed by dialysis method. Briefly, the dialysis bag (MWCO = 8–10 kDa) containing 2.0 mL of liposomal solution was transferred to a beaker containing 100.0 mL of release medium (0.01 M PBS, pH 7.4)⁶^,⁴⁰ by maintaining the temperature at 37°C, with continuous stirring at 100 rpm for 48 h. At different time points of 1, 2, 4, 8, 12, 24, and 48 h, 1.0 mL of release medium was withdrawn and replaced with an equal volume of fresh medium. The amount of OXA released into the collected media was analyzed by HPLC method as described above.

Stability Study

Stability studies of the liposomes were carried out at 25°C ± 2°C and 4°C ± 1°C in 5.0% dextrose solution.⁴¹^,⁴² At different time points of 0, 1, 2, 4, 8, 24, and 48 h, the free OXA was removed as mentioned above. The concentrations of OXA inside the liposomes (L-OXA, SSL-OXA, ES-L-OXA, and ES-SSL-OXA) were determined by HPLC method and recorded as C_n (n = 0, 1, 2, 4, 8, 24 or 48 h). The drug leakage rate was calculated using the following formula:

In vitro Targeting Evaluation

Fluorescence microscopy (Olympus IX71, Japan) was used to observe targeting capacity of rhodamine B liposomes into ER-positive SGC-7901 cells. The SGC-7901 cells were seeded into 24-well plates with a density of 1.0  ×  10⁵ cells/well and incubated in a humidified atmosphere of 5% CO₂ at 37°C. After 12 h incubation, media containing L-RhB, ES-L-RhB, SSL-RhB, or ES-SSL-RhB were separately added to the cells. The cells were incubated continuously for 1, 2, 3, and 4 h. Then, the medium was aspirated and the cells were washed 3 times with cold PBS. Hoechst 33342 solution as a nuclear dye was added to each well for 30 min at a temperature of 37°C. Fluorescence intensity of different rhodamine B formulations was observed under an inverted fluorescence microscope.

Endocytosis Mechanisms

Endocytosis can be inhibited with 10.0 μg/mL sucrose, 150.0 μM genistein, or 50.0 μM 5-(N-ethyl-N-isopropyl) amiloride, which are inhibitors of clathrin-mediated endocytosis, caveolae-mediated endocytosis⁴³ or macropinocytosis,⁴⁴ respectively. Besides, estrogen was used as a competitive inhibitor to block the estrogen receptors on the surface of SGC-7901 cells.⁴⁵^,⁴⁶ Briefly, SGC-7901 cells were seeded in 24-well plates at a density of 1.0×10⁵ cells/well. The cells were cultured with 1.0 mL/well of RPMI 1640 complete medium containing 10% FBS. After cells grew to confluency, estrogen or the three endocytosis inhibitors including sucrose, genistein, and amiloride were added to the cells, respectively, for an incubation time of 30 min. Then, ES-SSL-RhB medium solution was added to each well for an incubation time of 2 h. After incubation, the medium was removed, and the cells were washed 3 times with cold PBS.⁴¹ The fluorescence intensity of the cells was observed under an Olympus IX71 fluorescence microscope.

In vivo Targeting Study

BALB/c nude mice weighing 20.0 ± 2.0 g were inoculated subcutaneously on the back of mouse with 1.0×10⁷ cells/0.2 mL of SGC-7901. When the tumor volume reached about 200 mm³, the mice were randomly divided into two groups, including SSL-DiR group and ES-SSL-DiR group. Each group received a single dose of 500.0 ng DiR/mouse by tail vein injection.⁴⁷ At the time point of 1, 2, 6, 12, and 24 h (n=3 for each time point) after injection, mice were anesthetized with 10% chloral hydrate. The tissue biodistribution of the drug in anesthetized mice was photographed using a live imaging system (IVIS SPECTRUM, USA). Then euthanasia was performed on animals, and the tumor, heart, liver, spleen, lungs, and kidneys of the mice were collected for detection of fluorescence intensity.

Cytotoxicity Assay

The cytotoxicity of different OXA formulations on SGC-7901 cells was determined using MTT assay.⁴⁸ Briefly, SGC-7901 cells in the logarithmic growth phase were seeded triplicated into 96-well culture plates at a density of 1.0×10⁴ cells/well overnight in a humidified atmosphere of 5% CO₂ at 37°C. Then, cells were incubated with OXA, blank liposome (Blank-L), L-OXA, ES-L-OXA, SSL-OXA, and ES-SSL-OXA for 24, 48 and 72 h, respectively, in which the OXA concentration was 1.0–100.0 µg/mL. Subsequently, each well was incubated with 5.0 mg/mL, 20.0 μL MTT for 4 h. The medium was carefully removed, and 150.0 μL DMSO was added to each well. The optical density (OD) value of each well was measured at 490 nm with a microplate reader. The cell viability was calculated by the following equation, and the 50% inhibitory concentration (IC₅₀) was calculated according to the cell viability:

In vivo Antitumor Activity

SGC-7901 tumor-bearing BALB/c nude mice models were used to evaluate the therapeutic efficacy of ES-SSL-OXA. When the tumor volume reached about 100 mm³, the mice were randomly divided into five groups, with each group of 6 mice treated with normal saline, blank liposome (Blank-L), free OXA, SSL-OXA, and ES-SSL-OXA. Each preparation was administered via the tail vein at a dose of 2.5 mg OXA/kg body weight every 2 days. Tumor volume and body weight were measured twice a week. When the diameter of the tumor in the Normal Saline group reached 17.0 mm, animals were sacrificed and dissected. By the end of the experiment, all animals were sacrificed and dissected. Tumors, heart, liver, spleen, lungs, and kidneys were collected and weighed. The tumor volumes were calculated to assess the anti-tumor efficacy. The tumor weight was measured to calculate the tumor inhibition rate. Tumor volumes, tumor inhibition rate, and organ coefficient were calculated by the following equations:

“a” is the major axis length, and “b” is the minor axis length.⁴⁹

“W_{Normal Saline}” is the tumor weight of Normal Saline group, and “W_Test” is the tumor weight of Blank-L, Free OXA, SSL-OXA, or ES-SSL-OXA group.

Pharmacokinetics and Biodistribution Determination

One hundred and seventeen ICR mice were randomly divided into three groups of OXA, L-OXA, and ES-SSL-OXA. Each group of 39 mice was administered with a single dose of 10.0 mg/kg of free OXA, L-OXA, or ES-SSL-OXA via tail vein injection, respectively. At different time points (n=3)⁵⁰^,⁵¹ of 5, 10, 15, and 30 min, 1, 2, 4, 6, 8, 12, 24, 36, and 48 h after IV injection, 0.5 mL of blood sample of each mouse was collected into heparinized tubes. The animals were sacrificed and dissected to collect main organs of heart, liver, spleen, lungs, and kidneys. The collected blood was immediately processed for plasma by centrifugation at 3000 rpm for 10 min. Plasma samples were frozen at −20°C until analysis. The collected organs were washed with a normal saline solution to be stored at −20°C until analysis.

The concentration of OXA in plasma and organs was determined by a Shimadzu chromatograph (Shimadzu, Japan) using the Shimadzu C18 reverse phase column (150.0 mm × 4.6 mm, 5.0 μm) at room temperature. The mobile phase consisted of ultra-pure grade water and methanol (22:78 v/v). The flow rate was 1.0 mL/min. The effluent was monitored at 254 nm. Experimental scheme of pharmacokinetics was validated according to Technical Guidelines for Non-clinical Pharmacokinetic Research of Drugs by Center for Drug Evaluation of NMPA (National Medical Products Administration) in China, including specificity, limit of detection, limit of quantitation, intra- and inter-day precision, recovery rate, stability, and so on. All the R² values were >0.9990 in the standard curves for the detection of plasma, heart, liver, spleen, lungs, and kidneys. Briefly, 200.0 μL plasma or tissue homogenate was mixed with 400.0 μL acetonitrile, following the centrifugation at 10,000 rpm to obtain the supernatant. The supernatant was dried with nitrogen and added with 200.0 μL of 5.0% dextrose solution. The sample was extracted with 50.0 μL DETC (dissolved in NH₃-NH₄Cl buffer solution, pH 9.0) in water bath at 37°C for 30 min. Finally, 200.0 μL of chloroform was added to extract OXA, and the chloroform layer which contains OXA was collected by 10,000 rpm centrifugation for 10 min. The injection volume was 20.0 μL. Pharmacokinetic parameters were calculated using Drug and Statistics 2.0 (DAS 2.0) software.^52–54

Acute Toxicity Studies

One hundred and eighty healthy ICR mice, male and female, were assigned randomly into four groups, including the Normal Saline group, Free OXA group, L-OXA group, ES-SSL-OXA group. Administration dosages were designed based on a preliminary study. Each dose assigned 10 mice. OXA group received a dose of 10.0, 12.0, 14.0, 16.0, 18.0, or 20.0 mg/kg. L-OXA group received a dose of 10.0, 13.0, 16.0, 19.0, or 22.0 mg/kg. ES-SSL-OXA group received a dose of 10.0, 13.0, 16.0, 19.0, 22.0, or 25.0 mg/kg. The mice were administered a single dose via tail vein injection after 12 h of fasting. All animals were monitored daily to be recorded with their body sign, body weight, and mortality till 14 days. At the end of experiment, blood was taken from the eye socket of the remaining surviving mice after removing eyeball for a blood test by a hematology analyzer. Part of the blood was centrifuged at 3000 rpm for 10 min for creatinine and urea nitrogen determination by a blood biochemical analyzer. The mice were sacrificed for collection of heart, liver, spleen, lungs, and kidneys. The organs were weighed for calculating the organ coefficients.⁵⁵ The calculation formula of organ coefficients is consistent with the method of in vivo antitumor activity.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 5.0 software. One-way analysis of variance (ANOVA) was performed using the mean ± standard deviation (mean  ±  SD). The comparison of mortality was carried out by chi-square test using IBM SPSS Statistics 22. p < 0.05 was considered as significant different.

Results and Discussion

Encapsulation Efficiency and Drug Loading

The four formulations of liposomes named L-OXA, ES-L-OXA, SSL-OXA, and ES-SSL-OXA were prepared using the film hydration method. The encapsulation efficiency and drug loading were determined by dialysis and HPLC method. As shown in Table 2, the encapsulation efficiency of the four liposomes formulations was 45.40–49.90%, and the drug loading was 2.98–3.84%. There is no significant difference in encapsulation efficiency and drug loading efficacy among the four preparations, which indicated that the addition of DSPE-PEG₂₀₀₀-ES molecule had little influence on the encapsulation efficiency and drug loading of oxaliplatin. This was mainly because the targeting fragment of DSPE-PEG₂₀₀₀-ES was used at a very low concentration of 0.125 mg/mL in formulations, and the ES molecule was located on the surface of the liposome particle, while DSPE-mPEG₂₀₀₀ molecule was a commonly used lipid composition in the liposome delivery system. It is worth noting that the encapsulation efficiency of the four liposome formulations was higher than that of the previous reports.⁹^,¹²

Table 2.

The Characterization Properties of OXA Liposomes (n=3)

Sample	Particle Size (nm)	PDI	Zeta Potential (mV)	Encapsulation Efficiency (%)	Drug Loading (%)
L-OXA	135.83 ± 1.25	0.162 ± 0.010	−38.97 ± 0.62	49.90 ± 1.79	3.84 ± 0.13
ES-L-OXA	142.07 ± 1.43	0.172 ± 0.012	−38.57 ± 0.79	47.30 ± 2.41	3.58 ± 0.16
SSL-OXA	149.40 ± 0.50	0.124 ± 0.021	−30.20 ± 1.00	45.40 ± 3.74	2.98 ± 0.24
ES-SSL-OXA	153.37 ± 1.21	0.162 ± 0.007	−28.13 ± 0.21	46.20 ± 3.62	3.03 ± 0.23

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The Morphology of ES-SSL-OXA

The morphology of the ES-SSL-OXA was observed by TEM. As shown in Figure 1, most liposomes present spherical shapes in a uniform distribution without aggregation.

Transmission electron microscope photographs of ES-targeted PEGylated liposome loaded with OXA (n=3). The nanoparticles were stained with 2.0% phosphotungstic acid.

The Particle Size, PDI, and Zeta Potential

As the results determined by dynamic light scattering (DLS) analysis shown in Table 2 and Figure 2, the particle sizes of the four preparations of L-OXA, ES-L-OXA, SSL-OXA, ES-SSL-OXA uniformly distributed and showed a normal distribution curve, in which 60% of the nanoparticles were between 130 and 140 nm. Compared to L-OXA, the particle size of ES-SSL-OXA (153.37 ± 1.21 nm) was slightly increased due to the introduction of PEG ligand and ES targeting ligand. The PDI of the four liposomes ranged from 0.12 to 0.18, indicating that the particle size distribution of the OXA liposomes was uniformly dispersed. The four liposomes were shown to be negatively charged, wherein L-OXA zeta potential was −38.97 ± 0.62 mV, and ES-L-OXA was −38.57 ± 0.79 mV. The zeta potential of SSL-OXA and ES-SSL-OXA was −30.20 ± 1.00 mV and −28.13 ± 0.21 mV, respectively, which was higher than that of L-OXA and ES-L-OXA, most likely due to the introduced PEG molecule. In addition, it was found that the targeting fragment DSPE-PEG₂₀₀₀-ES did not have a significant effect on the zeta potential. Like the references reported,⁵⁶^,⁵⁷ when the absolute value of zeta potential exceeded 30 mV, the liposomes were regarded as highly stable because the relatively high charge could prevent the particles from flocculating to make the liposomes become more stable, indicating that the ES-SSL-OXA prepared was with a good stability.

Particle size distribution of OXA liposomes. Liposomes particle sizes of (A) L-OXA, (B) ES-L-OXA, (C) SSL-OXA and (D) ES-SSL-OXA were measured using dynamic light scattering (DLS) analysis by Malvern Nano-ZS90 particle size analyzer.