Research Article
Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Synergistic Enhancement of Cellular Uptake With CD44-Expressing Malignant Pleural Mesothelioma by Combining Cationic Liposome and Hyaluronic Acid–Lipid Conjugate

https://doi.org/10.1016/j.xphs.2019.06.012Get rights and content

Abstract

Malignant pleural mesothelioma (MPM) is a highly aggressive form of cancer, with a median survival of less than 1 year. It is well known that the hyaluronan (HA) receptor CD44 is highly expressed by MPM cells and is reported to be correlated with a poor prognosis. We herein report on the development of a new type if drug delivery system against CD44 that involves the use of lipid nanoparticles (LNPs) equipped with a new type of HA derivative. In this study, we evaluated HA-lipid conjugation (HAL) via the end of the HA molecule through reductive amination, a process that allowed the carboxylate group to remain intact. As a result, the HAL-modified LNP appears to be a potent nanoparticle for dealing with MPM. Surprisingly, the use of a combination of a cationic lipid and HAL had a synergistic effect on cellular uptake in MPM and consequently permitted an anti-cancer drug such as cis-diamminedichloro-platinum(II) (CDDP). Intrapleural injection of CDDP-loaded HAL-LNP (1.5 mg/kg as CDDP) per week significantly suppressed the progression of this type of cancer in an MPM orthotopic model. These results suggest that HAL-modified LNP represents a potent delivery system for MPM cells that express high levels of CD44.

Introduction

Malignant pleural mesothelioma (MPM) is a rare, but aggressive form of cancer, which originates in mesothelial cells of the pleura. MPM is mainly caused by a long exposure (typically 30-40 years) to asbestos.1 Although the disease is predicted to peak in approximately 2025 in the EU, Australia, and Japan,2, 3 asbestos is still consumed in developing Asian countries.4 In addition, the median survival is 9 to 12 months from the first diagnosis.5 Given these facts, developing an innovative therapy for MPM has become an urgent issue.

Several therapies, including chometherapy, surgery, and radiation therapy, have been explored for MPM patients. Historically, extrapleural pneumonectomy or extended pleurectomy decortication was frequently performed on MPM patients. However, in the American Society of Clinical Oncology, only such surgical resections of solid tumor are not recommended because of the high risk and difficulty associated with operation, plus the fact that these procedures are largely insufficient.6 Instead, they recommend multimodal therapy, for example, surgery with prechemotherapy or postchemotherapy (neoadjuvant or adjuvant). The latest studies revealed that a combination therapy involving an anti–vascular endothelial cell growth factor–A antibody (bevacizumab) and an immune checkpoint inhibitory antibody against programmed cell death–1 (nivolumab) and programmed cell death–L1 (ipilimumab) in addition to surgery and neoadjuvant therapy can significantly improve clinical outcomes for MPM patients.7, 8, 9, 10

Although the chemotherapy plays an important role in MPM treatment, the response ratio of even the currently most popular regimen for MPM (cis-diamminedichloro-platinum(II) (CDDP)-pemetrexed combined chemotherapy) is only 41.3%.11 Despite a large extent of progress in therapy for MPM, clinical outcome is significant, but limited and there is no second-line treatment.12, 13 Therefore, a more effective type of chemotherapy would be required for the treatment of MPM patients.

To efficiently deliver therapeutics, such as small molecules and nucleic acids, we recently developed a series of actively targeting-type lipid nanoparticles (LNPs), which are equipped with a ligand that specifically recognizes target cells using peptides and biomolecules.14, 15, 16, 17 Such ligand-equipped LNPs can specifically deliver them to cancer cells and are highly selective for specific target cells and consequently circumvent adverse effects derived from the unintended accumulation of anti-cancer therapeutics in off-target organs.18, 19 Thus, targeted-LNPs have shown prominent therapeutic outcomes in a mouse model.20 To target MPM, we focused on hyaluronan (HA) as a specific ligand. HA is a negatively charged polysaccharide that comprised N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) units.21 CD44 is a well-known receptor for HA.22, 23 Although a high expression level of CD44 is known to occur in a wide range of cancers,24 CD44 expression is significantly relevant to the diagnosis and prognosis of MPM.25, 26 It therefore appears that HA would be a potent ligand for targeting MPM.

A number of groups have reported on the use of HA-decorated LNPs for targeting cancer cells that express high levels of CD44. The modification of LNPs with HA follows 2 possible pathways: electrostatic interaction and covalent bond formation via the carboxylic acid groups in HA. To associate LNPs with negatively charged HA, cationic lipids, such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), are used. Typically, the prepared cationic LNPs are simply incubated in an HA solution to coat the LNPs with HA.27, 28 In the other route, prepared LNPs containing lipids with primary amine are incubated with HA in the presence of carbodiimide and N-hydroxysuccinimide to form an amide bond between the amine group and the carboxylate group in HA.29, 30, 31, 32 In both cases, the carboxylic acid group in HA is used to develop noncovalent or covalent binding. In a previous report, however, an X-ray crystal structure analysis of an HA-CD44 complex indicated that the oxygen atoms of the carboxylic acid groups in HA were associated with Ala102, Ala103, and Tyr83, which play a key role in the recognition process.33 To circumvent this interaction via carboxylate groups, we designed HA derivatives that are conjugated to the lipid through reductive amination at the reducing end of the HA molecule (HAL). We then optimized the resulting HAL-containing LNPs in terms of intracellular uptake and delivery efficiency to target MPM cells that express high levels of HA in an in vivo model mouse.

Section snippets

Materials

HA (MW 50,000) was generously provided by the Kewpie Corporation. Distearoyl-sn-glycero phosphoethanolamine, 1-stearoyl-2-oleoyl-sn-glycero phosphoethanolamine, dioleoyl-sn-glycerol phosphoethanolamine, and poly(ethylene glycol) (PEG)2000-distearoyl-sn-glycero phosphoethanolamine were purchased from NOF Corporation (Tokyo, Japan). DOTAP, 1,2-distearoyl-3-trimethylammoniumpropane, and dimethyldioctadecylammonium were purchased from Avanti Polar Lipids (Alabaster, AL). The WST-8 reagent was

Optimization of HAL-Modified LNPs With CD44-Positive MPM Cells

In initial experiments, CD44 expression in 3 cell lines of MPM cells was evaluated by flow cytometry. The results indicated that the HMM3 and H226 cells expressed high levels of CD44 and the HMM1 expressed moderate levels (Fig. 2). The HMM3 and H226 cells were subsequently used as CD44-positive cell lines, whereas HMM1 cells were used as a CD44-negative cell line. We then investigated the effect of HA and HAL on the cellular uptake of LNP. In previous reports, LNP was modified with HA by

Conclusion

We report herein on the design of a HA-conjugated lipid derivative (HAL) and show that the use of a combination of HAL and a cationic LNP resulted in a selective, synergistic cellular uptake by CD44-positive MPM cells. An LNP containing 10 mol% of a cationic lipid modified with 2.0 mol% HAL was found to have the highest ability to target MPM cells. The optimized HAL-LNP encapsulating CDDP achieved successful therapy for MPM-bearing mice.

Acknowledgments

The authors wish to thank Dr. Milton S. Feather for appropriately modifying the manuscript. This study was supported, in part, by Ministry of Health, Labour and Welfare, Japan, by Ministry of Education, Culture, Sports, Science and Technology, Japan, by Japan Society for the Promotion of Science, Japan KAKENHI (grant no. 18K18351) and by The Mochida Memorial Foundation for Medical and Pharmaceutical Research, Japan.

References (49)

  • Y. Matsumura et al.

    Significance of CD44 gene products for cancer diagnosis and disease evaluation

    Lancet

    (1992)
  • S. Chono et al.

    An efficient and low immunostimulatory nanoparticle formulation for systemic siRNA delivery to the tumor

    J Control Release

    (2008)
  • R. Ran et al.

    Enhanced gene delivery efficiency of cationic liposomes coated with PEGylated hyaluronic acid for anti P-glycoprotein siRNA: a potential candidate for overcoming multi-drug resistance

    Int J Pharm

    (2014)
  • D. Landesman-Milo et al.

    Hyaluronan grafted lipid-based nanoparticles as RNAi carriers for cancer cells

    Cancer Lett

    (2013)
  • S. Arpicco et al.

    Hyaluronic acid-coated liposomes for active targeting of gemcitabine

    Eur J Pharm Biopharm

    (2013)
  • H. Goudarzi et al.

    Enhancement of in vitro cell motility and invasiveness of human malignant pleural mesothelioma cells through the HIF-1α-MUC1 pathway

    Cancer Lett

    (2013)
  • L. Mo et al.

    PEGylated hyaluronic acid-coated liposome for enhanced in vivo efficacy of sorafenib via active tumor cell targeting and prolonged systemic exposure

    Nanomedicine

    (2018)
  • K. Takara et al.

    Design of a dual-ligand system using a specific ligand and cell penetrating peptide, resulting in a synergistic effect on selectivity and cellular uptake

    Int J Pharm

    (2010)
  • Y. Xiang et al.

    Chloride channel-mediated brain glioma targeting of chlorotoxin-modified doxorubicine-loaded liposomes

    J Control Release

    (2011)
  • H. Shmeeda et al.

    Delivery of zoledronic acid encapsulated in folate-targeted liposome results in potent in vitro cytotoxic activity on tumor cells

    J Control Release

    (2010)
  • A.A. Mujoomdar et al.

    Hyperthermic chemoperfusion for the treatment of malignant pleural mesothelioma

    Semin Thorac Cardiovasc Surg

    (2008)
  • H. Ando et al.

    Advanced therapeutic approach for the treatment of malignant pleural mesothelioma via the intrapleural administration of liposomal pemetrexed

    J Control Release

    (2015)
  • J.C. McDonald et al.

    The epidemiology of mesothelioma in historical context

    Eur Respir J

    (1996)
  • L.R. Chirieac et al.

    Clinicopathologic characteristics of malignant mesotheliomas arising in patients with a history of radiation for Hodgkin and non-Hodgkin lymphoma

    J Clin Oncol

    (2013)
  • Cited by (0)

    Conflicts of interest: The authors declared that there is no conflict of interest.

    This article contains supplementary material available from the authors by request or via the Internet at https://doi.org/10.1016/j.xphs.2019.06.012.

    View full text