Unlocking the potential of microfluidic assisted formulation of exenatide-loaded solid lipid nanoparticles

Abstract

Exenatide, a first-in-class GLP-1 receptor agonist, is used to control glycaemic levels in type 2 diabetes. There are two approved injectable formulations: one solution for immediate action and one dispersion for prolonged action. Oral exenatide has low bioavailability due to poor gastrointestinal stability and absorption.

To address these obstacles, we designed Solid Lipid Nanoparticles (SLN) including DOTAP in the formulation to yield high exenatide encapsulation by hydrophobic ion pairing and DSPE-PEG_2kDa to convey colloidal stability and mucus diffusivity. The microfluidic production of SLN yielded 9.7 % exenatide encapsulation and 94.2 % loading efficiency. SLN exhibited solid cored-spherical morphology with sizes of about 120 nm and zeta potential of + 53 mV. The SLN surface charge was modulated by DSPE-PEG_2kDa coating; 10 and 30 w/w% DSPE-PEG_2kDa /lipid ratios yielded slightly positive and neutral zeta potentials, respectively. All SLN formulations provided exenatide protection from proteolytic enzymes. The non-PEGylated SLN resulted in a twofold increase of exenatide delivery across Caco-2 cell monolayers compared to the peptide solution. The 10 w/w% SLN PEGylation reduced the exenatide delivery compared to non-PEGylated SLN through Caco-2 cell monolayers. However, the exenatide delivery with 10 w/w% PEGylated SLN across mucus-producing Caco-2/HT29-MTX coculture layer was 2-fold higher compared to the unformulated peptide, and 1.5 higher than non-PEGylated SLN. The 30 w/w% SLN PEGylation did not improve the peptide transport neither through Caco-2 cell monolayers nor through Caco-2/HT29-MTX coculture layer.

Highlights

Exenatide loading in Solid Lipid Nanoparticles (SLN) by microfluidic process.

Hydrophobic Ion pairing enhances Exenatide encapsulation in SLN.

SLN protects Exenatide from gastrointestinal enzymatic digestion.

PEG and cationic LNP surface balance enhances mucosal permeation.

Introduction

Therapeutic peptides represent a distinctive class of biopharmaceutical compounds renowned for their remarkable selectivity and efficacy in treating a diverse range of diseases. These include cancer , diabetes, ailments affecting cardiovascular and gastrointestinal systems, as well as those diseases deriving from cytokine deficiencies (Wang et al., 2022). Oral administration of peptides is regarded as a convenient choice in virtue of high patient compliance while posing relevant formulation challenges. Yet, this route of administration for therapeutic peptides is often limited by low stability in the gastrointestinal tract and poor absorption. Indeed, the stomach and intestine present chemical, biochemical and physical barriers, including low pH, enzymes, mucus, and biological membranes, that affect the bioavailability of peptides (Brown et al., 2020). Nevertheless, the approval of oral dosage forms of peptides, namely cyclosporin A (Sandimmune Neoral®), desmopressin (DDAVP®), and semaglutide (Rybelsus®), have boosted strong enthusiasm proving that the development of rationally conceived delivery technologies, including microemulsion, peptide cyclization, and use of absorption enhancers, can be successful to overcome the challenges of this administration route for peptides (Buckley et al., 2018, Keown and Niese et al., 1998, Zizzari et al., 2021). However, the bioavailability of marketed products mentioned above has remained low, reaching as little as 0.8 % in some instances (Overgaard et al., 2021), namely semaglutide, and even for a smaller highly hydrophobic cyclic peptide, cyclosporine, bioavailability hovers around 60 % (Wiącek et al., 2020). These findings underscore the compelling opportunities for enhancing formulation and delivery approaches to improve treatment outcomes.

Lipid-based nanocarriers are biocompatible formulations with ability to cross the intestinal barrier (Niu et al., 2016) that can be successfully exploited for oral delivery of drugs suffering of poor bioavailability. Nevertheless, the low drug loading represents a main limit for the delivery of therapeutic peptides. Indeed, therapeutic peptide encapsulation into lipid based nanocarriers suffers from the low affinity between the hydrophobic carrier matrix and the hydrophilicity of peptides. Self-emulsifying drug delivery systems (SEDDs) and self-micro emulsifying drug delivery systems (SMEDDS) have been developed to overcome this limitation, SEDD technology is based on hydrophobic ion pairing (HIP), which quenches the hydrophilic character of the peptides by ion interaction with oppositely charged hydrophobic molecules(Ristroph and Prud’homme, 2019). Few studies have demonstrated that the HIP significantly improves the loading efficiency into SEDDS and SMEDDS (Griesser et al., 2017, Niu et al., 2016) of a wide range of polypeptides and small proteins including insulin(Claus et al., 2023, Liu et al., 2019), lysozyme (Šahinović et al., 2023), leuprorelin(Hintzen et al., 2014), exenatide (Phan et al., 2020), calcitonin(Wibel et al., 2023) and other peptides(Jörgensen et al., 2023). HIP has also been explored for production of nanostructured lipid carriers (NLC) and solid lipid nanoparticles for oligopeptides like leuprolide and desmopressin (Dumont et al., 2019a, Dumont et al., 2019b).

Exenatide (EXEN), is a synthetic 39-amino-acid peptide that has incretin properties similar to the human incretin hormone glucagon-like peptide 1 (GLP-1). EXEN, approved in 2005, was the first peptide drug used to treat type 2 diabetes. As a GLP-1 receptor agonist, exenatide marked a significant advancement in diabetes treatment by enhancing insulin secretion and controlling blood glucose levels (Malone et al., 2009). However its use is restricted to parenteral administration. Previous studies showed that HIP can be successfully exploited for EXEN loading into SEDDS, which resulted in oral bioavailability of approximately 28 % (Ismail et al., 2020). Nonetheless, the low stability of both the carrier system and the encapsulated drug(Maji et al., 2021) and the local toxicity and gastrointestinal discomfort (Chatterjee et al., 2016, Salawi, 2022) caused by the high surfactant content in the formulation (typically ranging from 30 % to 60 %) may limit the development of SEDDS for the oral delivery of peptides and proteins.

Additionally, when the drug diffuses with high mobility into the oil phase of lipid nanoemulsions, fast release of drugs is observed, which makes it difficult to have a sustained release. Thus, SLN represent an appealing option to nanoemulsions (i.e. SNEDDS) and SEDDs to tackle issues such as drug leakage and burst release (Mirchandani et al., 2021). Furthermore, they offer a variety of benefits, including stability upon administration, controlled drug release, and can be produced at a cost-effective reproducible large scale by microfluidic technology (Anderluzzi and Perrie, 2020). However, while the literature reports several studies focusing on the development of lipid particles encapsulating nucleic acids (Prakash et al., 2022), microfluidics is underrepresented for development of protein and peptide-loaded carriers. To the best of our knowledge, the incorporation of peptides into SLN through the HIP method via microfluidics has never been previously reported.

In this study, a widely used biodegradable cationic lipid, DOTAP, was exploited for HIP exenatide loading into SLN by microfluidics. The effect of critical production parameters and formulation composition on exenatide-loaded SLN physicochemical and biopharmaceutical properties were evaluated in order to set up an optimized production process. The EXEN-loaded SLN were decorated with polyethylene glycol (PEG) and the biopharmaceutical behaviour of the SLN was evaluated using in vitro epithelial models to explore the biocompatibility and the delivery efficacy.

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Materials

Exenatide was purchased from APExBIO (TX, USA). Hydrogenated soybean phosphatidyl choline (HSPC, Scheme S1) was a kind gift from Lipoid GmbH (Ludwigshafen, Germany). 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP, Scheme S1) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5.5) (Cy5.5-DSPE) were provided by Avanti Lipids (AL, USA). The wheat germ agglutinin-Alexa Fluor 488 conjugate (WGA-AF 488), (N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) (fluorescein-DHPE) and (2′-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazole trihydrochloride trihydrate) (Hoechst 33342) were purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA-USA). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG2kDa) was purchased from Laysan Bio (AL, USA). Mucin from the porcine stomach (type II), cholesterol, sterile Dulbecco’s phosphate-buffered saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM), Foetal bovine serum (FBS), penicillin–streptomycin solution (10.000 units penicillin, 10  mg streptomycin/mL), l-glutamine (200  mM), trypsin, and 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO-USA). The Caco-2 cell line (HTB-37) was provided by the American Type Culture Collection (ATCC, VA-USA) and the HT29-MTX cell line was kindly provided by Prof. Valentina Alessandria from DISAFA, University of Torino (Torino, Italy). All buffers, salts, solvents, and other reagents utilized were of analytical purity grade.

Büşra Arpaç Birro, Cristiano Pesce, Francesco Tognetti, Agnese Fragassi, Lisa Casagrande, Mariangela Garofalo, Stefano Salmaso, Paolo Caliceti, Unlocking the potential of microfluidic assisted formulation of exenatide-loaded solid lipid nanoparticles, International Journal of Pharmaceutics, 2025, 125686, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2025.125686.