Regular ArticlepH – Responsive colloidal carriers assembled from β-lactoglobulin and Epsilon poly-L-lysine for oral drug delivery
Graphical abstract
Introduction
Oral delivery is one of the most widely used routes of administration for small drug molecules as it is associated with high patient acceptance, pain free self-administration and cost-effective manufacturing. However, oral administration requires overcoming low solubility, poor bioavailability, poor stability and indigent membrane permeability of many drugs leading to low therapeutic levels via the gastrointestinal tract (GIT) [1]. To overcome these issues, many advances have been made in formulation science including formation of solid dispersions [2], smart coatings to achieve pH and enzyme mediated drug delivery [3], [4], as well as micro and nanoparticles [5]. Amongst these, nanoparticles have attained prominence as carriers for hydrophobic and hydrophilic drugs [6].
Nanotechnology has delivered radical changes in the pharmaceutical field including drug delivery [7], diagnostics [8], nutraceuticals [9] and preparation of biomaterials [10]. Traditional drug delivery systems have drawbacks such as emergence of drug resistance, non-specific targeting, poor drug stability and burst drug release leading to unwanted side effects [11]. To address these limitations, many innovative nanomaterials such as liposomes, dendrimers, polymers and inorganic nanoparticles have been developed as “smart” drug-delivery systems that have capacity to provide site-specific delivery [12], [13], [14]. These nanomaterials have the potential to increase biodistribution of drugs by protecting them from degradation and carrying drug directly to the targeted site [15], [16]. However, most of these nanoparticles suffer from various disadvantages such as low encapsulation efficiency (polymers), low acid stability (liposomes), high toxicity (quantum dots, dendrimers), and synthesis limitations (peptide dendrimers) [17]. Furthermore, traditional nanoparticle formation involves use of toxic solvents [18], multiple synthesis and loading steps leading to poor loading capacity, and burst release of drugs.
The ideal carrier for the development of nanoparticle based oral delivery systems for hydrophobic drugs should possess high encapsulation efficiency, biocompatibility, biodegradability and low toxicity. β-lactoglobulin (BLG) has emerged as a nutraceutical for preparation of a variety of dosage forms, such as polymer hybrid [19], hydrogels [20] and micro-nanoparticles [21], which have shown enhancement in delivery of hydrophobic nutraceuticals like curcumin [22], quercetin [23] and resveratrol [24]. BLG is a natural by-product obtained after separating components from milk. Because of its gelling and emulsifying properties, it is mostly used as an ingredient in processed foods [25]. The binding interactions between BLG and hydrophobic nutraceuticals have not been definitively characterised, but they have been reported to involve hydrogen bonding [26], hydrophobic interactions or electrostatic interactions [27].
Although, BLG-based delivery systems have many advantages including pepsin resistance [28], the drug is usually released immediately in the gastric environment thereby exposing the drug to acid-catalyzed decomposition [28]. Thus, these systems cannot protect the drug from harsh gastric conditions nor deliver the drug to target sites in the intestine. Succinylation is a mild chemical process in which succinic acid is grafted onto the ε-amino group of lysine residues via an amide bond, resulting in conversion of the basic (pKa ~10) lysine sidechain into an acidic (pKa ~4) side-chain. This technique has been used in food industry for many decades to increase food protein’s emulsifying properties. Succinylation has also been used to improve protein’s solubility at an intestinal pH by increasing protein charge density above pH 4.5. Consequently, after succinylation proteins have been able to retard the encapsulated drug release in acidic pH and promote the drug release at an intestinal pH [13], [29], [30], [31]. This is particularly important in designing colon targeted and pH responsive drug delivery systems. Caillard et al. were first to formulate delayed release tablets using succinylated β-lactoglobulin [29]. They found that due to succinylation, solubility of BLG was decreased at acidic pH and increased at pH > 5.2. These tablets have excellent properties as an enteric delivery system. They delay the release of drug in simulated gastric fluid and accelerate it in simulated intestinal fluid [29]. This work emphasises the possible applications of Succ. BLG as a functional nanocarrier for pH responsive oral formulations with controlled release. Recently Teng et al. attached ethylenediamine onto the aspartic and glutamic acid side chains of BLG by amidation to form cationic BLG with a desirable solubility profile and marked improvement in mucoadhesion [32]. However, such systems would not provide much needed pH responsiveness combined with improved permeation.
Apart from poor solubility and stability in the GIT, many drugs also suffer from low permeability across the intestinal membrane. Cationic polymers such as chitosan, have been extensively used in order to improve drug residence time in the gut and improve permeability across epithelium [33]. However, polymers such as chitosan are insoluble in neutral pH, which may have adverse impact on drug release and permeability. Epsilon poly-l-lysine (E-PLL) is a homopolymer of 25 to 35 l-lysine residues linked together by amide bonds between the carboxyl group of one residue and ε-amino group of the adjacent residue [34]. E-PLL is poly-cationic in nature and has potential applications in the field of medicine for drug delivery due to its biodegradable, non-toxicity for humans and water soluble properties [35]. We hypothesised that cationic E-PLL can interact with anionic succ. BLG through electrostatic interactions to form nanostructures (BCP). Furthermore, succ. BLG and E-PLL nanostructures can be stabilised by covalent cross-linking through amide bond formation (BCEP). Importantly, E-PLL is a food grade preservative and GRAS (Generally Recognised As Safe) approved by FDA as delivery carrier [36]. Hence, we hypothesised that combining cationic polymer with succ. BLG would make the formulation pH- responsive while retaining positive charge to achieve improved permeation and desired cellular uptake.
In this work, we assess the ability of succ. BLG and E-PLL to form uniform nanocomplexes and its impact on improving solubility, pH responsive release, permeability and anti-inflammatory activity of curcumin, a model hydrophobic drug. Curcumin, a main component of turmeric, is a polyphenolic nutraceutical obtained from the plant Curcuma longa. Curcumin have been used for the prospective treatment of cancer and inflammation [37], [38], [39]. However, it has also been categorized as a BCS class IV drug with poor solubility as well as poor intestinal permeability [40]. This poor water solubility and permeability of curcumin limits its efficacy as an anti-inflammatory drug. To overcome these limitations, curcumin has been complexed with proteins to improve its stability as well as solubility [41]. In this study, we explored the potential of BLG, succ. BLG and E-PLL nanocomplexed with curcumin to enhance the biological activity of curcumin in-vitro and in mouse derived 3D intestinal organoid cultures in the presence of inflammatory stimuli to model the effects on intestinal inflammation.
Section snippets
Materials
β-lactoglobulin (BLG) (>98%) from bovine milk, succinic anhydride, curcumin (CUR), pepsin, pancreatin, trypsin, acetonitrile (HPLC grade, purity > 99.9%) were purchased from Sigma Aldrich. Epsilon poly-l-lysine (>95% E-PLL) was purchased from Carbosynth Limited, UK. Ethanol (90%) was purchased from Merck, Germany, Acetic acid (>99% analytical grade was acquired from Chem-Supply – UQ store, and Snakeskin® dialysis tubing (MWCO 10 kDa) was purchased from Thermo scientific, USA.
Succinylation of β-lactoglobulin
BLG (200 mg) was dispersed in phosphate buffer (20 mL) at pH 7.4. Succinic anhydride (50 mg) was added to the BLG solution in 10 mg aliquots and stirred at room temperature (25 °C) for 1 h. The pH of the solution was maintained between 7.5 and 8.5 using 2 M NaOH. After the pH of the solution was stabilized at 8.0, the solution was stirred for further 20 min. The solution was dialysed in nanopure (Type I) water for 24 h at 4 °C with changing of water 5 times at an interval of 4 h and the volume
Particle size distribution and zeta potential
The particle size distribution and zeta potential of nanoparticles were characterised by a dynamic light scattering (DLS) instrument (Malvern Nano-ZS) as per manufacturer’s instruction. Please note that cumulants analysis was used to measure Z-Average and PDI. The intensity and number mean were obtained by fitting a multiple exponential to the correlation function to obtain the distribution of particle sizes (such as Non-negative least squares (NNLS) or CONTIN.
Free curcumin, BLG, succ. BLG,
Characterisation of nanoparticles
The particle size distribution and zeta potential of nanoparticles were determined according to the method shown in characterisation section. The average size of free curcumin was ~1.7 µm but when it was complexed within BLG, succ. BLG, BCP and BCEP particles, the particle size of complex decreased to < 300 nm (Table 1). The nanocomplex of curcumin with BLG and succ. BLG showed homogenous particle size distribution (274 ± 38 and 149 ± 5 nm), PDI (0.47 ± 0.02 and 0.10 ± 0.01) and highly negative
Conclusion
So far, the studies on the use of nutraceutical-based proteins to generate colloidal protein-drug nanocomplexes only improved the dispersibility and solubility of poorly soluble drugs such as Curcumin, Resveratrol, Thymol etc. However, majority of these formulations suffers from premature drug release and only some improvement on drug’s permeability.[31], [42], [62], [65], [66], [67], [68] To the best of our knowledge this is the first study to demonstrate the use of succinylated BLG complexed
CRediT authorship contribution statement
Naisarg Pujara: Investigation, Visualization, Methodology, Formal analysis. Rabina Giri: Visualization, Writing - review & editing. Kuan Yau Wong: Visualization, Writing - review & editing. Zhi Qu: Formal analysis, Writing - review & editing. Prarthana Rewatkar: Visualization, Writing - review & editing. Md. Moniruzzaman: Formal analysis, Writing - review & editing. Jakob Begun: Writing - review & editing. Benjamin P. Ross: Supervision, Writing - review & editing. Michael McGuckin: Supervision,
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
Authors are thankful for research maintenance funds provided by School of Pharmacy, The University of Queensland and funding provided by Mater Medical Research institute. Dr Popat is thankful to National Health and Medical Research Council of Australia for Career Development (GNT1146627), Early Career Fellowship (GNT1146627) and project grant. Authors are also thankful to Centre of Microscopy and Microanalysis (CMM) at UQ and TRI for providing facilities.
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