Development of a nanoliposomal formulation of erlotinib for lung cancer and in vitro/in vivo antitumoral evaluation.

The aim of this study was to develop PEGylation liposomes formulations of erlotinib and evaluate their characteristics, stability, and release characteristics. The average particle sizes and entrapment efficiency of PEGylation erlotinib liposomes are 102.4±3.1 nm and 85.3%±1.8%, respectively. Transmission electron microscopy images showed that the liposomes dispersed well with a uniform shape and no changes during the storage. The in vitro drug-release kinetic model of erlotinib release from the PEGylation liposomes in phosphate-buffered saline fit well with the Higuchi equation. In vitro anticancer activity assay showed that the blank liposomes had lower cellular cytotoxicity and that the cellular cytotoxicity of erlotinib liposomes increased significantly under the same incubation condition, which should contribute to the increase in intracellular drug concentration by the transportation of liposomes. The two liposomes of erlotinib (with and without PEGylation) exhibited similar cellular cytotoxicity with no significantly different concentrations. Pharmacokinetic results indicated that erlotinib-loaded PEGylation liposomes can significantly change the pharmacokinetic behavior of drugs and improve the drug bioavailability by nearly 2 times compared to ordinary liposomes. No sign of damages such as the appearance of epithelial necrosis or sloughing of epithelial cells was detected in histological studies.

Introduction

Erlotinib is a potent and selective tyrosine kinase inhibitor (TKI). It reversibly binds the binding site of ATP in the epidermal growth factor receptor tyrosine kinase domain to inhibit the automatic phosphorylation of tyrosine kinases.1,2 Inhibition of tyrosine kinase can promote apoptosis, inhibit angiogenesis, and ultimately prevent excessive cell proliferation.3–5 Erlotinib in treatment of various solid tumors such as non-small-cell lung cancer is available in oral form.6,7 Oral bioavailability of erlotinib in healthy volunteers and cancer patients was 59% and 76%, respectively.8,9 There are no data on other forms of such drugs. The new erlotinib delivery system is limited due to its poor solubility. Nevertheless, solid lipid nanoparticles containing erlotinib were produced by Bakhtiary et al10 recently. Moreover, Dora et al11 found that cyclodextrin nanosponge containing erlotinib could enhance oral bioavailability with less cytotoxicity. In another study conducted by Barghi et al,12 erlotinib-loaded functional material nanoparticles were introduced.

Although erlotinib is orally active and has acceptable bioavailability,13 we hypothesized that drug delivery approaches could improve the efficacy of combination therapies with EGFR inhibitors. Erlotinib accumulates in tumor, skin, kidney, and liver at levels significantly higher than in plasma,14 most likely because of its hydrophobicity. Encapsulation in carriers such as liposomes would reduce concentrations of free drug in the blood and thereby limit deposition in normal tissues such as bone marrow.15 Accumulation of erlotinib in tumors is just 2 times greater than in skin, and previous studies have shown that liposome encapsulation can result in higher tumor drug concentration compared with that in skin.16

Over the past 20 years, liposomal drug delivery systems have held extraordinary potential for the delivery of therapeutics to tumors, and various strategies have been used to improve their targeting specificity and cellular uptake. Earlier studies attempted to load TKI into liposomes at higher percentages, which led to rapid leakage/precipitation during storage or immediately after liposome preparation.17 Moreover, PEGylation of liposomes aimed to extend the blood half-life of loaded erlotinib served as a factor triggering the leakage of loaded erlotinib.18 PEGylation has been widely adopted to improve the accumulation of liposomes in tumor tissues through enhanced permeability and retention effects, which is a passive form of targeting.19

The primary objective of this work is to develop liposomal formulations of erlotinib and evaluate their characteristics, stability, and release characteristics. The targeting and antitumor efficiency in lung cancer were also evaluated in vitro and in vivo.

Materials and methods

Materials

Erlotinib was purchased from Sun Pharma Ltd., Co. (Mumbai, India). Soybean phosphatidylcholine (SPC), DSPE-PEG2000, DSPE, and cholesterol (CHOL) were obtained from Sinopharm Chemical Reagent (Shanghai, People’s Republic of China). A549 cell was purchased from Cobioer Biomedical Ltd., Co. (Nanjing, People’s Republic of China). All other reagents were obtained from Sinopharm Chemical Reagent (Shanghai, People’s Republic of China). Methanol and acetonitrile (chromatographic grade) were obtained from Sigma Aldrich (St Louis, MO, USA). Water for high-performance liquid chromatography was double distilled, and all other reagents were of analytical grade.

Preparation of erlotinib liposomes

The PEGylation liposomes containing erlotinib were prepared by the thin-film hydration method as described previously.20 Briefly, erlotinib, SPC, CHOL, and PEG-DSPE were mixed, with the molar ratio of PEG-DSPE:CHOL:SPC being 3:10:22 and the weight ratio of lipid:erlotinib being 17:1. The solvent was then evaporated using an RE52 rotary evaporator (Shanghai Yarong Biochemistry Instrument Company, Shanghai, People’s Republic of China) in a round-bottomed flask at 40°C for about 40 minutes to obtain a solid film. This film was then flushed with nitrogen for 30 minutes and stored overnight in a desiccator to remove any traces of chloroform. After that, the thin film was hydrated by sonication in 5% glucose solution and placed in water bath for 10 minutes to produce a suspension of liposomes. And then the liposomes were frozen for 72 hours. The dry powder was rehydrated and sonicated for 3 minutes before application. For the preparation of erlotinib liposomes without PEG, a similar procedure was carried out except that the PEG-DSPE was replaced by DSPE.

Characterization of liposomes

The mean particle size and polydispersity index (PDI) of liposome dispersions were determined by dynamic light-scattering method using fiber-optics particle analyzer. The system was set in the auto-measuring mode. Particle size analysis data were evaluated using volume distribution to detect large droplets.

The entrapment efficiency (% EE) was determined by measuring the concentration of unentrapped drug in the lipid dispersion. Briefly, erlotinib liposomes were placed in a sedimentation centrifuge for 10 minutes at 4°C and 12,000 rpm, and the amount of erlotinib in supernatant was determined by dissolving the supernatant in an acetone and ethanol (1:2) mixture at 332 nm by high-performance liquid chromatography. The drug encapsulation efficiency was calculated by the following equation: where Werlotinib represents the amount of erlotinib loaded in the liposomes and Wliposomes represents the weight of erlotinib liposomes.

The stability of erlotinib liposomes was evaluated by monitoring the changes in mean particle size, PDI, and EE% during storage at 4°C or room temperature. The morphology of erlotinib liposomes by the negative staining transmission electron microscopy (TEM) method was also evaluated. The samples were added to the surface of copper grids and stained with phosphotungstic acid (1%, w/v). The accelerating voltage was 120 kV. The samples were magnified by 100,000 times.

In vitro release study

The release test was performed using a Franz-type vertical diffusion cell.21 Approximately 10 mg erlotinib liposomes was applied into the donor compartment of each cell, and the receptor chamber was filled with 17 mL of phosphate buffer solution (pH 7.4). Phosphate buffer solution was used to solubilize the leaked erlotinib as a hydrotropic agent. Magnetic stirring speed and temperature were set at 600 rpm and 37°C respectively. Dialysis membrane (molecular weight cutoff 12,000–14,000) was placed between the donor compartment cell and receptor chamber. An aliquot of the sample (2 mL for each time point) was taken from the dissolution medium at different time intervals (0.5, 1, 2, 4, 6, 8, 12, 24, 36, 72, and 96 hours) and analyzed for its drug concentration at 332 nm. The receptor chamber was replaced by an equal volume of phosphate buffer solution.

Ethics in animal experiments

All in vivo experimental protocols were approved by the animal care committee of the Faculty of Medicine, Anhui Medical University animal center, and were carried out in accordance with the guideline of experimental animals of Anhui Medical University. All Sprague–Dawley rats in this study were purchased from Anhui Medical University animal center (Hefei, People’s Republic of China).

In vitro anticancer activity assay

In vitro anticancer activity of PEGylation erlotinib liposomes on human lung carcinoma A549 cells was evaluated by MTT assay. Control experiments were carried out using a complete growth culture medium as a nontoxic control. A549 cells (1×105 cells/well in 1 mL medium) were seeded into 96-well plate. Twenty-four hours later, cells were incubated with varied concentrations of PEGylation erlotinib liposomes, erlotinib liposomes, free drugs, or blank liposomes. After 48 hours of incubation under cell culture conditions, the growth and viability of cells were determined by using MTT assay. Then 1 mL complete growth culture medium and 60 μL MTT solution (5 mg/mL in phosphate-buffered saline [PBS]) were added to each well and incubated for 4 hours. The absorbance was determined using a microplate reader at 540 nm. The results were expressed as percentages relative to those obtained from the group with a nontoxic control.

Pharmacokinetic studies

Sprague–Dawley rats weighing from 200 to 220 g were used for pharmacokinetic study. Before the experiment, the rats were kept in a state of fasting for 6 hours. In the experiment, the rats were divided into three groups (8 each). One group was given erlotinib injection (free drug, as a control), and the other two were given erlotinib liposomes and PEGylation liposomes (dose =10 mg/kg), respectively. Then all formulations were introduced into rats via intravenous injection. Blood samples (2 mL) were collected from the tail vein into heparinized 5 mL polythene tubes just before administration and 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 hours after dosing. The plasma obtained was stored at −20°C until analysis.

Histology studies

The histopathological results of erlotinib liposomes and PEGylation liposomes were observed at the end of pharmacokinetic studies. Animals were anesthetized, and their hearts, livers, spleens, lungs, and kidneys were dissected and washed with cold saline. The organs were pressed between filter pads, weighed, and then fixed in 10% neutral formalin using standard techniques and stained with hematoxylin and eosin for histopathological examination. All tissue samples were examined and graded under light microscopy with 5,000× magnification.

Statistical analysis

Statistically significant differences between values obtained under different experimental conditions were determined using two-tailed unpaired Student’s t-test.

Result and discussion

Table 1 shows the addition of PEG that did not produce significant influence of erlotinib liposomes in terms of particle size and PDI. The average particle size of erlotinib liposomes and PEGylation erlotinib liposomes was 100.1±2.6 and 102.4±3.1 nm, respectively. The PDI of all samples was <0.8. The EE of erlotinib in liposomes and PEGylation liposomes were (84.7%±2.9%) and (85.3%±1.8%), respectively. The high EE in the formulation might be related to the strong hydrophobicity of erlotinib. Table 1 also gives the stability data of particle size of erlotinib liposomes stored at 4°C. After 3 months of storage, no dramatic change such as visible aggregation, drug content changes, and precipitation in the appearance of liposomes occurred. TEM images (Figure 1) showed that the liposomes dispersed well with a uniform shape and no changes during storage.

Table 1

Characterization of PEGylation erlotinib liposomes before and after storage at 4°C (n=3)

Formulations	Time	Particle size (nm)	EE (%)	PDI
PEGylation erlotinib liposomes	0 day	102.4±3.1	85.3±1.8	<0.45
Erlotinib liposomes	0 day	100.1±2.6	84.7±2.9	<0.43
PEGylation erlotinib liposomes	1 month	101.5±2.3	84.8±2.2	<0.47
Erlotinib liposomes	1 month	101.7±1.4	84.3±1.6	<0.48
PEGylation erlotinib liposomes	2 months	101.9±2.6	84.6±2.3	<0.56
Erlotinib liposomes	2 months	102.3±2.4	84.1±2.1	<0.58
PEGylation erlotinib liposomes	3 months	102.3±2.3	83.1±1.3	<0.63
Erlotinib liposomes	3 months	102.9±3.1	83.9±2.8	<0.61

Abbreviations: EE, entrapment efficiency; PDI, polydispersity index.

Figure 1

TEM images of PEGylation erlotinib liposomes and erlotinib liposomes before and after storage at 4°C (n=3).

Notes: (A) 0 day; (B) 3 months; (C) 0 day; (D) 3 months. Magnification ×10,000.

Abbreviation: TEM, transmission electron microscopy.

In vitro drug release

The in vitro release of erlotinib from the liposomes was investigated. Figure 2 shows the release profiles of the three groups. Compared with the group of rapid release of free drug, the other two liposome groups exhibited similar and sustained release, and no initial burst release was observed. Slow release occurred due to the fact that with the dissolution and diffusion mechanisms on the lipid matrices, the solubilized or dispersed drug could only be released slowly. As illustrated in Table 2, the in vitro drug-release kinetic model of erlotinib release from the PEGylation liposomes in PBS fits well with the Higuchi equation: Q =8.928t1/2−1.237 (r=0.993). Thus, it was speculated that the sustained-release property of erlotinib liposomes could enhance cycle in vivo. The in vitro release results indicated that the release of erlotinib from PEGylation liposomes was similar to that from ordinary liposomes, demonstrating that the PEG modification did not affect erlotinib release. The addition of modified materials did not lead to significantly reduced release speed. A possible reason could be that the quantity of added material did not greatly increase the steric hindrance of liposomes.

Figure 2

The in vitro drug-release profiles of erlotinib-loaded liposomes of two types and of free drug.

Note: Data are expressed as mean ± SD (n=6).

Abbreviation: SD, standard deviation.

Table 2

Release kinetic of erlotinib-loaded liposomes (n=6)

Parameters	Erlotinib liposomes		PEGylation erlotinib liposomes
	Equation	Correlation coefficient (r)	Equation	Correlation coefficient (r)
Zero-order	Q =2.372t −1.209	0.892	Q =3.281t −1.112	0.929
First-order	Ln(1−Q) =2.093t +0.192	0.933	Ln(1−Q) =2.371t +0.293	0.934
Weibull	lnln(1/(1−Q)) =1.298 lnt +0.381	0.938	lnln(1/(1−Q)) =3.271 lnt +0.234	0.926
Higuchi	Q =6.867t1/2−0.878	0.989	Q =8.928t1/2−1.237	0.993

The A549 lung cancer cell as a model cell was applied to assay the cellular cytotoxicity of erlotinib liposomes. MTT assay method was used to evaluate the viability of cells. The cellular viability of free drug, blank liposome, and erlotinib liposomes during the different incubation times is shown in Figure 3. Even after the cells were incubated for 48 hours with 0.3 mg/mL blank liposomes, the cellular viability was still >80%. This result indicates that blank liposomes had lower cellular cytotoxicity. By contrast, with the same concentration (free drug), the cellular cytotoxicity of erlotinib liposomes increased significantly under the same incubation condition, which should contribute to the increase in intracellular drug concentration by the transportation of liposomes. In addition, the two liposomes of erlotinib (with and without PEGylation) exhibited a similar trend of cellular cytotoxicity at the same concentration. With the extension of incubation time, liposomes of erlotinib (with PEGylation) showed a larger inhibitory effect on cells. PEG-modified liposomes exhibited high efficiency in the present study, which is expected to be introduced into the development of other drugs. Its value is reflected not only in the changes of erlotinib but also in the possibility of helping establish a platform to transfer the toxicity active substance to become an ideal candidate drug.22

Figure 3

The cellular viability of A549 cells cultured with blank liposomes and erlotinib-loaded liposomes during different incubation time in comparison with that of erlotinib liposomes (without PEGylation) at the same dose (n=6).

Note: Data are expressed as mean ± SD.

Abbreviation: SD, standard deviation.

The pharmacokinetic profile of erlotinib following administration in the three formulations was evaluated initially in nontumor-bearing rats, with results presented in Figure 4. A dose of 10 mg/kg free drug was chosen and employed throughout all studies based on previous studies performed by our group (data not shown). As expected, both the clearance and the volume of distribution of the drug were significantly lower when administered in liposomes as free drug (p<0.05). Compared to ordinary liposomes, those composed of PEGylation showed relatively long terminal half-lives (96.8±11.2 hours versus 92.7±10.1 hours), lower clearance rates (0.07±0.03 mL/kg versus 0.09±0.05 mL/kg), and higher area under the curve 0–∞ values (898.5±82.4 mmol/L*h versus 625.5±72.7 mmol/L*h). They were also significantly different from free drug (Table 3). The results also showed that erlotinib-loaded PEGylation liposomes could significantly change the pharmacokinetic behavior of drugs and improve drug bioavailability nearly 2 times compared to ordinary liposomes, which was due to the prolonged in vivo circulation of drug. Furthermore, when the drug is prepared into PEGylation liposome formulation, its pharmacokinetics is mainly determined by the characteristics of PEGylation liposome rather than the physical and chemical properties of the drug molecule.

Figure 4

Concentration–time curve of erlotinib in different formulations.

Notes: Free drug (blue line), liposomes (green line), and PEGylation liposomes (red line) (n=6). Data are expressed as mean ± SD.

Abbreviation: SD, standard deviation.

Table 3

Pharmacokinetic parameters of erlotinib after intravenous administration of free drug, liposomes, and PEGylation liposomes to rats (n=6)

Parameter	Intravenous administration
	Free drug	Liposomes	PEGylation liposomes
t1/2 (min)	28.9±8.3	92.7±10.1*	96.8±11.2*
AUC0–t (μg⋅min/mL)	168.8±18.3	574.3±65.3*	792.5±78.6*
AUC0–∞ (μg⋅min/mL)	186.7±19.6	625.5±72.7*	898.5±82.4*
MRT (min)	29.2±8.7	89.5±9.6*	92.7±10.1*
CL (L/kg/min)	0.21±0.11	0.09±0.05*	0.07±0.03*

Notes:

p<0.05 versus free drug.

Abbreviations: AUC0–t, area under the curve calculated to the last measured concentration; AUC0–∞, area under the curve from time 0 extrapolated to infinite time; CL, clearance; MRT, mean residence time; t1/2, half-life.

The histopathological examinations of the heart, liver, spleen, lung, and kidney were carried out to identify any damage done to the tissue. The microphotographs of the heart, liver, spleen, lung, and kidney were taken following their incubation with erlotinib formulations (Figure 5). No sign of damage, such as the appearance of epithelial necrosis or sloughing of epithelial cells, was detected.

Figure 5

Histopathological studies of the heart, liver, spleen, lung, and kidney.

Notes: (A) Free drug; (B) erlotinib liposomes; (C) PEGylation erlotinib liposomes. Magnification ×5,000.

Conclusion

In this study, PEGylation liposomes formulations of erlotinib were developed with an average particle size of 102.4±3.1 nm. TEM images showed that the liposomes dispersed well with a uniform of shapes and with no changes during the storage. The in vitro drug-release kinetic model of erlotinib release from the PEGylation liposomes in PBS fits well with the Higuchi equation. In vitro anticancer activity assay showed that the blank liposomes had lower cellular cytotoxicity and that the cellular cytotoxicity of erlotinib liposomes increased significantly at the same incubation condition, which should contribute to the increased intracellular drug concentration by the transportation of liposomes. The two liposomes of erlotinib (with or without PEGylation) exhibited a similar cellular cytotoxicity at the same concentration with no significant difference. Pharmacokinetic results indicated that erlotinib-loaded PEGylation liposomes can significantly change the pharmacokinetic behavior of drugs and improve the drug bioavailability nearly 2 times compared to the ordinary liposomes.