Development of a stable and high loaded liposomal formulation of lapatinib with enhanced therapeutic effects for breast cancer in combination with Caelyx®: In vitro and in vivo evaluations

https://doi.org/10.1016/j.colsurfb.2021.112012Get rights and content

Highlights

  • Lapatinib is a hydrophobic molecule.

  • Therapeutic effects of lapatinib can be enhanced using drug delivery systems.

  • Encapsulating lapatinib in a pegylated liposomal formulation can enhance therapeutic outcomes.

  • Liposomal lapatinib shows promising effects on inhibiting cell proliferation and inducing autophagy and apoptosis.

  • Co-administration of liposomal lapatinib with liposomal doxorubicin improves the survival time.

Abstract

Lapatinib, a dual tyrosine kinase inhibitor, has poor water solubility, which results in poor and incomplete absorption from the gastrointestinal tract. To overcome this obstacle, we designed a stable and high-loaded liposomal formulation encapsulating lapatinib and examined its therapeutic efficacy in vitro and in vivo on TUBO and 4T1 cell lines. We also assessed the impact of liposomal lapatinib on the extent of the tumor and spleen-infiltrating lymphocytes and the autophagy and apoptosis gene expression within the tumor site. Our results showed that liposomal lapatinib inhibits cell proliferation and significantly induces autophagy and apoptosis compared to control groups. Moreover, when it used in combination with liposomal doxorubicin, it extended the time to end from 22.4 ± 3.5 in the control group to 40 days in the TUBO cell line and from 29.2 ± 1.7 to 38.6 ± 2.2 days in 4T1 triple-negative breast cancer cell line, which reveals its promising effects on the survival of tumor-bearing mice.

Our results indicated the need for further evaluations to understand liposomal lapatinib’s potential effects on autophagy, apoptosis, and particularly on immune system cells.

Introduction

Breast cancer (BC) is one of the most prevalent cancer types in women and is ranked the second cause of death among all other cancer types [1]. BC is divided into different subtypes, and among them; the epidermal growth factor receptor-2 (HER2) positive breast cancers tend to have a poor prognosis, and the activated signaling pathway by this receptor can lead to cell proliferation, angiogenesis, and tumor growth [2]

A wide range of medications, such as tyrosine kinase inhibitors, has been used to treat patients with Her2/neu-expressing breast cancer [3]. Lapatinib is a tyrosine kinase inhibitor that inhibits both EGFR and HER2 receptors and has been approved by the FDA to deal with Her2/neu-expressing breast cancer [4]. Growing evidence indicates the effect of lapatinib on inducing apoptosis and autophagy and extending the survival time [5]. Several clinical trials have tested lapatinib in metastatic and aggressive kinds of Her2 overexpressing breast cancers. It has been approved for clinical use in combination with capecitabine [6] or letrozole [7]. Lapatinib is also being tested in various studies for the treatment of aggressive types of cancers. For instance, a recent study has shown promising effects on metastatic colorectal cancer [8] and likewise metastatic bladder cancer [9].

The only available lapatinib dosage form is 250 mg oral tablets, under the trade name of Tykerb® [10]. Lapatinib is approximately insoluble in water (log P = 5.4) and therefore has a poor, variable and incomplete absorption from the gastrointestinal tract [11]. Furthermore, it binds 99 % to plasma albumin [12]. These limitations cause extensive doses administration (1000−2500 mg/day) and consequently severe side effects like diarrhea and vomiting [13] that limit the clinical application of this molecule.

Incorporating small molecules into drug delivery systems can help reduce the side effects and potentiate their efficacy [14]. Liposomes are drug delivery systems composed of a lipid bilayer that can carry various soluble and insoluble molecules [15]. Their structure can be designed according to the desired function to expand their applications. Furthermore, liposomes are not only versatile, biocompatible, biodegradable structures but also they can protect the active ingredients from degradation [16]. Liposomes are widely used to deliver anticancer agents with the ability to target the tumor site. By passive or active targeting using these vehicles, while the therapeutic efficacy is potentiated, the side effects can be diminished [17].

This study aimed to develop a novel and stable liposomal formulation of lapatinib and assess its in vitro and in vivo efficacy. This process was carried out using Her2/neu-expressing or triple-negative cell lines to have enhanced speculation of the drug effects in vivo in mice models. Another aim of this study was to explore the lapatinib effect on the extent of autophagy and apoptosis in the tumor tissues and its impact on tumor- and spleen-infiltrating lymphocytes.

Section snippets

Materials

Lapatinib powder was purchased from Mesochem Technology CO., Ltd (Beijing, China). Hydrogenated soy L-α-phosphatidylcholine (HSPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (mPEG2000-DSPE) were purchased from Avanti Polar Lipid (Alabaster, USA). Cholesterol was purchased from Sigma-Aldrich (Steinheim, Germany). PMA/ ionomycin cocktail and antibodies (PE-Cy5-Mouse Anti CD4, PE-Cy5-Mouse Anti

Physicochemical characterization of liposomal lapatinib

Twenty different formulations (with various molar ratios of lipids) were prepared and characterized to reach the proper drug to lipid ratio and final specifications (Supplementary 1). Physicochemical characteristics of the optimum liposomal lapatinib formulation in terms of z-average (nm), PDI, surface charge (mV), entrapment efficiency percentage (EE %) were determined and are listed in Table 1. The optimum hydrated with succinate buffer (10 mM, pH 5.5) lapatinib liposome formulation consisted

Discussion

Lapatinib is a tyrosine kinase inhibitor [4] and despite its effectiveness in inhibiting the progression of malignant cells, it is almost insoluble in water with a high protein binding which hinders the bioavailability [8,17]. Moreover, several adverse effects have been reported in patients taking lapatinib, affecting its clinical use [25].

Various drug delivery systems have been utilized to overcome the drug delivery barriers. By incorporating drugs into the delivery systems with suitable

Conclusion

In the current study, we attempted to overcome the drawbacks of lapatinib by entrapping it into liposomes and improving the stability and drug-loading efficacy. Moreover, the combination of liposomal lapatinib with Caelyx® also increases the antitumor activity and improves the survival time. Furthermore, we found the promising effect of liposomal lapatinib on inhibiting cell proliferation and inducing significant autophagy and apoptosis, which needs further investigations to understand the

CRediT authorship contribution statement

Sara Shokooh Saremi: Conceptualization, Methodology, Investigation, Software, Validation, Writing.

Amin Reza Nikpoor: Methodology, Software.

Kayvan Sadri: Supervision (labeling of liposomes).

Amin Mehrabian: Writing- Reviewing and Editing.

Maryam Karimi: Visualization, Investigation.

Atena Mansouri: Visualization, Investigation.

Mahmoud Reza Jafari: Supervision.

Ali Badiee: Supervision.

Declaration of Competing Interest

All authors declare that there is no conflict of interest.

Mashhad University of Medical Sciences has funded this project (project number: 950953(.

Acknowledgments

This study was a part of Sara Shokooh Saremi’s Ph.D. thesis (Grant number: 950953) supported by Nanotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; support is gratefully acknowledged.

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