Microfluidic preparation and optimization of Kollicoat ® IR-b-PCL polymersome for co-delivery of Nisin-Curcumin in breast cancer application

This work aimed to develop amphiphilic nanocarriers such as polymersome based diblock copolymer of Kollicoat ® IR −block-poly(ε-caprolactone) (Kollicoat ® IR-b-PCL) for potential co-delivery of Nisin (Ni) and Curcumin (CUR) for treatment of breast cancer. To generate multi-layered nanocarriers of uniform size and morphology, microfluidics was used as a new technology. In order to characterise and optimize polymersome, design of experiments (Design-Expert) software with three levels full factorial design (3-FFD) method was used. Finally, the optimized polymersome was produced with a spherical morphology, small particle size (dH < 200 nm), uniform size distribution (PDI < 0.2), and high drug loading efficiency (Ni 78 % and CUR 93 %). Furthermore, the maximum release of Ni and CUR was found to be roughly 60 % and 80 % in PBS, respectively. Cytotoxicity assays showed a slight cytotoxicity of Ni and CUR −loaded polymersome (N- Ni /CUR) towards normal cells while demonstrating inhibitory activity against cancer cells compared to the free drugs. Also, the apoptosis assays and cellular uptake confirmed the obtained results from cytotoxic analysis. In general, this study demonstrated a microfluidic approach for preparation and optimization of polymersome for co-delivery of two drugs into cancer cells.

Introduction

Breast cancer ranks as the second most common cause of cancer-related deaths among women. There are different risk factors, which can increase the probability of developing breast cancer such as age, sex, family history, inherited genes, radiation exposure, alcohol intake and obesity (Sharma et al., 2010). In general, there are different treatment options for treatment of breast cancer such as surgery to remove the tumor, radiotherapy, hormone therapy and chemotherapy (Smith et al., 2004, Waks and Winer, 2019). Among these methods, chemotherapy has been generally employed for the treatment of breast cancer. Depressingly, most chemotherapeutic drugs have poor water solubility, non-specific toxicity on cancer cells, low bioavailability in blood and serious side effects such as nausea, diarrhoea, hair loss, anaemia and infections (Azim et al., 2011, Kamińska et al., 2015). Therefore, it is necessary to develop new drugs with the aim of identifying cancer cells and reducing drug resistance.

Antimicrobial peptides (AMPs) also called host defense peptides (HDPs) are a class of small peptides that found in most living creatures such as bacteria, fungi, plants, insects, birds, fish, amphibians, and mammals (Bahar and Ren, 2013). Yet, over 5000 AMPs have been identified or synthesized in a wide variety of organisms (Bahar and Ren, 2013, Ramazi et al., 2022). AMPs have potential antimicrobial or anticancer properties. Anticancer peptides (ACPs) are a series of short cationic peptides composed of 10 to 60 amino acids such as arginine and lysine. They can further differentiate between cancerous and normal cells by several mechanisms such as electrostatic interactions between the ACPs and the cancer cell membrane, the membrane fluidity of cancer cells, and the lower cholesterol content in cancer cell membranes compared to normal cells (Gaspar et al., 2013, Swithenbank and Morgan, 2017). Due to their lower drug resistance, selective toxicity against cancer cells, and lower side effects compared to chemotherapy and traditional therapies, ACPs are promising candidates for breast cancer treatment (Aghamiri et al., 2021, Swithenbank and Morgan, 2017).

Amongst AMPs, nisin (Ni) is one of the smallest cationic antimicrobial peptides, produced through the fermentation of the gram-positive bacterium Lactococcus lactis (Liu and Hansen, 1990). Due to non-toxicity of Ni for animals and its safety for human consumption, it was accepted as a food preservative by the World Health Organization (WHO) in 1969 and by the Food and Drug Administration (FDA) in 1988 (Kamarajan et al., 2015). Ni has been considered as an effective antitumor agent because of its excellent toxicity leading to apoptosis of cancer cells. Due to the cationic segments of the amino acids, Ni integrates into the cell membrane and it acts as a cell membrane disruptor and apoptotic pathway activator (Goudarzi et al., 2018, Zainodini et al., 2018). In recent years, Ni has been considered as a novel anti-cancer drug in breast cancer treatment. However, low serum bioavailability of Ni and poor uptake and bio-distribution in body have limited its clinical applications (Biswaro et al., 2018, Monfared et al., 2023).

Curcumin (CUR), is an active component in turmeric powder (Curcuma longa) that attracted the attention of researchers due to its beneficial pharmacological activities such as anti-oxidant, anti-inflammatory, wound healing, anti-coagulant and anti-cancer (Naksuriya et al., 2014). Several studies have demonstrated that CUR is an effective anticancer natural drug that induces apoptosis in cancer cells and cell cycle arrest in the G0/G1 or G2/M stages (Obaidi et al., 2022, Sa and Das, 2008). Despite many pharmacological effects of CUR, its retention time in the body is restricted because of its low stability and water solubility, hepatic elimination, and poor absorption at tumor site (He et al., 2016, Moghaddam et al., 2020).

To overcome the limitations of combinatorial Ni and CUR for treatment of breast cancer, nanocarriers have attracted significant attention as they improve the bioavailability, biodistribution, solubility, and stability of antitumor drugs, and diminish drugs resistance (Haider et al., 2022, Kumar et al., 2021). In recent decades, a diverse range of nanocarriers composed of materials such as lipids, polymers and inorganic materials have been developed for cancer treatment (Sabit et al., 2022). Polymeric nanocarriers are colloidal and are defined as core–shell nanocarriers such as micelles or nanospheres, nanocapsules, liposomes, and polymersomes that are prepared from hydrophilic/hydrophobic polymers (Letchford and Burt, 2007). Among all varieties of polymeric nanocarriers, polymersomes are hollow spheres with a hydrophilic core and a polymer shell made of self-assembled amphiphilic block copolymers (LoPresti et al., 2009). Due to their ability to encapsulate both hydrophilic and hydrophobic drugs, high colloidal stability, passive and active targeting capabilities, significant biocompatibility, and long blood circulation times, polymersomes are promising nanocarriers for drug delivery to tumor cells in treatment of cancer (Hasannia et al., 2022, Köthe et al., 2020, Sharma et al., 2020).

For preparation of amphiphilic copolymers, kollicoat ® IR and polycaprolactone (PCL) polymers show a great potential in the field of cancer therapy. Kollicoat ® IR is a polyvinyl alcohol (PVA)-polyethylene glycol (PEG) graft copolymer (PEG: PVA, 1: 3) that is a novel polymer in treatment of cancer thanks to its water solubility, more accumulation in tumor environment, superior biocompatibility, and longer blood circulation time (Yamamoto et al., 2013). PCL is a biocompatible and biodegradable polymer that is used as a hydrophobic chain in synthesis of amphiphilic copolymers (Bagheri and Mahmoodzadeh, 2020).

To date, numerous strategies have been reported to produce polymersomes, including thin-film rehydration, sonication, direct injection, and microfluidics technology for fabrication of self-assembled vesicles (Anajafi and Mallik, 2015, Balasubramanian et al., 2016, Martin et al., 2023). Due to the lack of risk for sample contaminations, high efficiency of drug loading, ability to control the size, and possibility of forming multi-layered nanocarriers, microfluidics technology has been introduced as a promising method to produce polymersomes (Känkänen et al., 2023, Liu et al., 2020).

Different parameters influence the formulation of polymersomes in microfluidics device. In microfluidic technique, the ratio of aqueous phase to the organic phase or flow rate ratio (FRR) and total flow rate (TFR) are key factors in determining the obtained particle size and polydispersity of the polymersomes (Rebollo et al., 2022, Sedighi et al., 2019).

The aim of this study was to produce self-assembled polymersomes from kollicoat ® IR −b-PCL by novel microfluidic mixing using a toroidal mixer (TrM) for co-delivery of Ni and CUR which is beneficial in treatment of breast cancer. For this purpose, we optimized polymersomes with regards to their size and polydispersity index (PDI) with design of experiments (Design-Expert) software and three levels full factorial design (3-FFD) method. The physicochemical and morphological characteristics of the optimized polymersomes were assessed using dynamic light scattering (DLS) and transmission electron microscopy (TEM). The in-vitro cytotoxicity, cell growth inhibition, and apoptosis induction in MCF-7 cells were assessed using various formulations of Ni and CUR (free and loaded in polymersomes).

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Materials

Polyvinyl alcohol- polyethylene glycol graft-copolymer (Kollicoat ® IR) (Mw = 45,000 Da), ε −caprolactone (ε −CL), 2,2-Bis(hydroxymethyl) propionic acid (bis-MPA), Stannous octoate (Sn(Oct)2), Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP) were purchased from Sigma Co. Curcumin (CUR) was purchased from Bio Basic Inc. Nisin (Ni) from Lactococcus lactis and Dimethyl sulfoxide (DMSO) were purchased from Merck Chemical Co. Disodium hydrogen phosphate (Na2HPO4).

Sahar Salehi, Soheil Boddohi, Mohammad Adel Ghiass, Mehrdad Behmanesh, Microfluidic preparation and optimization of (Kollicoat ® IR-b-PCL) polymersome for co-delivery of Nisin-Curcumin in breast cancer application, International Journal of Pharmaceutics, Volume 660, 2024, 124371, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2024.124371.

Read also our introduction article on “Binder“ here: