Research paper
Solid lipid particles for oral delivery of peptide and protein drugs I – Elucidating the release mechanism of lysozyme during lipolysis

https://doi.org/10.1016/j.ejpb.2013.07.017Get rights and content

Abstract

The mechanism of protein release from solid lipid particles was investigated by a new lipolysis model in a biorelevant medium containing both bile salts and phospholipids. Lysozyme, a model protein, was formulated into solid lipid particles using four different types of lipids, two triglycerides with different chain-length of fatty acyl groups i.e. trimyristin (TG14) and tristearin (TG18), and two lipid blends dominated by diglycerides and monoglycerides, respectively. The release of lysozyme from the solid lipid particles and the lipid hydrolysis process were assessed in the lipolysis model, while the change in particle surface during the lipolysis process was evaluated using scanning electron microscopy. The lysozyme release profiles from TG14 and TG18 as well as diglyceride particles correlated well with the release of free fatty acids from the lipid particles during the lipolysis and therefore exhibited a lipase-mediated degradation-based release mechanism. The release of lysozyme from monoglyceride particles was independent on lipase degradation due to the instability of the lipid matrix in the lipolysis medium. In conclusion, the established lipolysis model is successfully used to elucidate the drug release mechanism from solid lipid particles and can potentially be used in rational selection of lipid excipients for oral delivery of peptide/protein drugs.

Introduction

Oral delivery of peptide and protein drugs is generally limited due to their degradation in the gastrointestinal tract (GIT) and low permeability across the intestinal epithelium [1]. Solid lipid particles have been proposed as promising drug carriers for oral delivery of peptides and proteins [2], [3] and have shown enhanced bioavailability for example calcitonin, growth hormone, and insulin upon oral administration to rats [4], [5], [6], [7]. The increased bioavailability could be due to the protection of proteins from degradation in the GIT by lipid particles [5], [8], [9] or permeation enhancing effects of lipids [10], [11]. Because of the increasing interests in lipid-based particles for oral delivery of proteins, a suitable lipolysis model is highly desirable for the elucidation of drug release from such delivery systems.

The release of small molecule weight drugs from lipid-based formulations for oral administration is often tested using a lipolysis model like the dynamic in vitro lipolysis model [12], [13]. To the best of our knowledge, the release of a protein drug from a lipid-based drug delivery system has never been reported in this kind of model probably due to the challenges related to: (1) degradation of proteins in the lipolysis medium, specifically the degradation by proteases from pancreatic extract commonly used in lipolysis studies, and (2) low drug load, which demands very sensitive and specific analysis tools to analyze the protein content in the lipolysis medium. Furthermore, most work with lipolysis models has been done on lipid emulsions [14] although some solid lipid formulations have been subjected to lipase to study the rate of lipid hydrolysis of solid lipid nanoparticles [15], [16], [17], [18] and of lipid depot systems [19], [20].

The presence of bile salts and phospholipids has a strong impact on the lipid degradation process and therefore needs to be included in a biosimilar in vitro lipolysis medium [21], [22]. The porcine pancreatic extract usually employed in lipolysis experiments consists of a mixture of enzymes excreted from the pancreas [23] and may be considered inappropriate to study the mechanism of protein release due to the presence of proteases, which will cause protein degradation. Even though pure pancreatic lipase could be used for lipolysis, it is very costly, especially in cases where in vivo relevant activities are required. A microbial lipase from Thermomyces lanuginosus may be considered as a suitable surrogate for pancreatic lipase, because it resembles the 1,3-specificity of pancreatic lipase [24], [25], [26] and results in a similar fatty acid (FA) composition following hydrolysis of soybean oil compared to pancreatic lipase [27]. Microbial lipases are active without the presence of co-lipase [28]; the T. lanuginosus lipase shows reduced activity at high levels of bile salt, but still retains its activity at a bile salt concentration of 8 mM [29]. Additionally, this lipase is available as a stable and highly active solution, making it possible for the in vitro lipolysis to attain similar enzyme activities as the pancreatic lipase in the human intestine in the fasted state (550 U/mL) [30].

The aim of the present study was to establish an in vitro lipolysis model to elucidate the release mechanism of protein drugs from solid lipid particles. In this study, lysozyme was chosen as the model protein, and solid lipid microparticles (SLM) with different type of lipid excipients were prepared in order to investigate the effect of lipid excipients on the release of protein drugs. Scanning electron microscopy (SEM) was used to visualize the particles and the changes in their surface during the lipolysis process in order to gain more insight into the release mechanism.

Section snippets

Materials

The lipids Dynasan 114 (glyceryltrimyristate, TG14) and Dynasan 118 (glyceryltristearate, TG18) were kindly provided by Cremer Oleo (Hamburg, Germany), and the lipids Precirol ATO 5 (Glyceryl distearate type 1, GDS) and Geleol mono- and diglycerides NF (Glycerol monostearate 40–55 type 1, GMS) were from Gattefossé (Lyon, France). Thermomyces lanuginosus lipase A solution (100,000 U/g) was a gift from Novozymes (Bagsværd, Denmark). Phosphatidylcholine (Lipoid S PC, >98%) was purchased from Lipoid

Physicochemical characterization of SLM

The size of the TG14 and GMS particles was significantly larger than that of the TG18 and GDS particles for all size parameters (Table 3). All particles were observed to be spherical by SEM (Fig. 1). The TG and GDS particles had smooth surfaces although small dents were visible on some of the particles, while the particles prepared from GMS showed major imperfections with big holes on the particle surface. Similar morphology of TG particles has been reported by Reithmeier et al., who observed

Conclusion

The release mechanism of proteins from SLM depends on the physicochemical properties of the lipid excipients. The TG and GDS particles exhibit a lipase-mediated degradation-based release mechanism for lysozyme. This gives the possibility of retarding the release of lysozyme with TG containing longer chain FA due to a slower digestion rate. The GMS particles are not suitable for controlling the release of lysozyme as they show excessive degradation of the lipid matrix in the lipolysis medium,

Acknowledgements

We would like to thank Dorthe Ørbæk (University of Copenhagen, Denmark) for her help with SEM and Harald Hansen (University of Copenhagen, Denmark) for providing access to the Typhoon TM FLA 7000 biomolecular imager.

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