Preparation and pharmacodynamics of niclosamide micelles

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Abstract

Niclosamide (NIC) has been found to have outstanding antitumour activity in recent years, but its clinical application has been limited by its poor water solubility. In this study, nanomicelles (PEG2K-Fmoc-Ibuprofen micelles, PEG2K-FIbu) were prepared as carriers to improve the solubility and bioavailability of NIC. Nuclear magnetic resonance (NMR) confirmed its successful preparation. The drug-carrying micelles (PEG2K-FIbu/NIC) were then prepared using a thin film dispersion method. PEG2K-FIbu and PEG2K-FIbu/NIC were analysed in detail in terms of particle size, polymer dispersion index, stability and encapsulation efficiency. Finally, the in vivo effects of PEG2K-FIbu/NIC were tested in a mouse liver cancer model. The particle size of PEG2K-FIbu was 11.11 nm. The encapsulation rate was 70.4% and the tumour inhibition rate was 55.98%. Tumours were significantly reduced and damage to the liver was greatly reduced. The histopathological sections and serum biochemical indices also showed that the symptoms were significantly improved after administration. In summary, PEG2K-FIbu/NIC is a potential drug delivery system of NIC to effectively treat liver cancer, providing an effective dosage form for NIC.

Introduction

Liver cancer is one of the most common malignancies and the third most deadly cancer in the world. Despite advances in treatment techniques, its morbidity and mortality continue to increase, and its mortality rate is second only to that of lung cancer and colon cancer [[1], [2], [3]]. It has also been reported that it is the fastest growing cause of cancer deaths in Australia [4].

Niclosamide, a salicylanilide derivative used for the treatment of tapeworm infections, is safe, well tolerated, inexpensive and readily available. However, in recent years niclosamide has been found to have an anti-tumour effect. NIC has been found to play a role in tumours such as breast, lung and ovarian cancers [6]. It inhibits tumour cell proliferation and induces apoptosis by interfering with Wnt/β-catenin, Stat3, Notch, NF–K and Hedgehog signalling pathways [[6], [7], [8], [9]]. Although novel mechanisms of niclosamide continue to be discovered, most of these studies are still in their infancy. The direct target of niclosamide is still unclear, and its anti-tumour mechanism of action needs to be further elucidated. Also, prospective controlled clinical studies are lacking. However, the clinical application of niclosamide is limited by its insolubility in water. How to improve the water solubility of niclosamide has become a problem. So we decided to greatly improve the aqueous solubility of clonidine and increase its clinical application through the preparation of clonidine dosage forms. Nanosolubilisation of insoluble drugs is an important way to improve bioavailability. Nanopreparations can improve the solubility of insoluble drugs, which can be more easily absorbed by the body and thus exert their efficacy. Also the small particle size of nano-formulations makes it easier to reach the lesion [10]. Therefore, we prepared polymeric micelles in which niclosamide was encapsulated for drug delivery purposes. Polymer micelles have been shown to improve drug solubility and reduce toxicities compared to conventional carriers [11]. Polymeric micelles are therefore used to increase their solubility and bioavailability. Polymer micelles consist of hydrophilic and hydrophobic chain segments that form core-shell nanomicelles with an inner lipophilic and outer hydrophilic structure when both are above the critical micelle concentration (CMC). A common hydrophilic polymer is polyethylene glycol (PEG), which is often used to encapsulate hydrophobic cores. In recent years, polymeric micelles have been widely used in preclinical studies [12]. Polymer micelles with a stable cross-linked structure have been prepared by cross-linking. Currently, the (non-)reversible crosslinking of specific parts of the micelles is mainly achieved by physical and chemical reactions, which not only improve stability but also protect the drug from damage. There are three types of crosslinking: nuclear crosslinking, shell crosslinking and core-shell interface crosslinking [13,14].

In this experiment, PEG2K-FIbu micelles were prepared, consisting of a hydrophilic chain segment of PEG, a Fmoc motif and the hydrophobic structural domain of ibuprofen. PEG is highly biocompatible and helps the micelles to circulate in the bloodstream for a long period of time. Fmoc is a protective group for amino acids and significantly improves drug-carrying capacity and stability. Ibuprofen is a non-steroidal anti-inflammatory drug that reduces the incidence of cancer with long-term or regular use. Ibuprofen on the skeleton relieves pain in cancer patients, improves the stability of micelles, as well as increasing the sensitivity of tumours to the drug. PEG2K-FIbu micelle has been used to encapsulate paclitaxel and has been shown to have an inhibitory effect on breast cancer [15].

In this study, PEG2K-FIbu polymeric micelles were synthesized for the first time. PEG2K-FIbu/NIC was prepared and characterised by a thin film dispersion method. Finally, a tumour mouse model was established and the antitumour activity of hepatocellular carcinoma cells was measured in vivo.

Section snippets

Chemicals and reagents

Anhydrous ethanol, anhydrous ethyl ether and dimethyl sulfoxide were purchased from Tianjin Lianlong Bohua Pharmaceutical Chemical Co., Ltd. Fmoc, DCC and DMAP were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. PEG was purchased from Shanghai Tisi Aicheng Industrial Development Co., Ltd.

Mice and cell culture

Healthy Balb/c male mice and Kunming male mice with a body weight of 18 ± 2 g were provided by the Animal Center of Shenyang Pharmaceutical University. The mice were exposed to light (12 h

H NMR spectroscopy

The hydrogen NMR spectra were shown in Fig. 2. According to the NMR spectra, we can find some characteristic peaks. The signals at 3.63 ppm were attributed to PEG methylene hydrogen (- CH2 -). The signals at 3.38 ppm were attributed to the chemical shift for PEG terminal methoxy (- OCH3). The signals from 1.25 to 1.74 ppm were the carbon chain single peak characteristic peak. The benzene ring in the Fmoc moiety were at 7.0–7.3 ppm. The benzyl hydrogen signals in ibuprofen were at 2.03 ppm.

Discussion

In this study, we found that PEG2K-FIbu micelles could help niclosamide to better inhibit the growth of hepatocellular carcinoma. Ibuprofen on PEG2K-FIbu could play a role in relieving cancer pain and improving the stability of the skeleton. Inhibition by ibuprofen also increases the sensitivity of niclosamide to tumours, thereby enhancing the anti-cancer activity of the drug-loaded micelles against tumours. In addition, PEG2K-FIbu micelles have good stability. In vivo, PEG2K-FIbu/NIC showed

Conclusion

PEG2K-FIbu is a novel drug delivery vehicle in which ibuprofen increases the stability of micelles, relieves cancer pain and increases the sensitivity of tumours to NIC. The PEG2K-FIbu micelles have good stability, and the hydrophobic drug NIC can be encapsulated in them by the film dispersion method, which can greatly increase the water solubility of NIC, forming PEG2K-FIbu/NIC and greatly exerting its clinical application. .

In vivo, PEG2K-FIbu/NIC inhibited the growth of tumours in mice with

Declaration of interest

The authors report no conflicts of interest.

Acknowledgements

This work was financially supported by National Natural Science Foundation of China(81973284), Liaoning Province Science and Technology Administration (2019-ZD-0456), and Liaoning Education Department project (LJKZ0944).

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1

Jiarong Hang and Yu Chen contributed equally to this work and should be considered co‐first authors.

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