Research paper
Liposomal vasoactive intestinal peptide for lung application: Protection from proteolytic degradation

https://doi.org/10.1016/j.ejpb.2008.04.015Get rights and content

Abstract

Inhalative administration of vasoactive intestinal peptide (VIP) is a promising approach for the treatment of severe lung diseases. However, the clinical use of VIP is limited by the fact that the peptide is prone to rapid degradation mechanisms and proteolytic digestion. Accordingly, VIP exhibits a very short period of activity in the lung. To overcome this problem, we have designed a liposomal drug delivery system for VIP and characterized it in terms of its potential to protect VIP from enzymatic cleavage. The proteolytic conditions of the lung, the target site of aerosolic administered VIP, were mimicked by bronchoalveolar lavage fluid (BALF), a lung surfactant solution, obtained by fiberoptic bronchoscopy. Thus, the stability of VIP was assessed by its resistance to enzymatic degradation in BALF, using a combination of high pressure liquid chromatography with mass spectrometry. We found that free VIP was rapidly digested, whereas liposomal-associated VIP remained intact. By fluorescence spectroscopic techniques using fluorescent-labelled VIP we got strong indications that the tight association of VIP with the lipid membrane is only minimally affected upon incubation with BALF. Loading capacity and stability of EtCy3-VIP loaded liposomes were measured by fluorescence fluctuation spectroscopy. Finally, the protective properties of the liposomes were also expressed in the maintained biological activity of the peptide incubated with BALF.

Introduction

Vasoactive intestinal peptide (VIP) is a cationic 28 amino acid peptide and a member of the secretin/glucagon family [1]. It is widely distributed within the body, including lung, gastrointestinal tract, heart, kidney and brain [2]. VIP exerts a variety of biological functions, including pulmonary and bronchodilation [3], vasodilation [4], smooth muscle relaxation [1], anti-inflammatory and immunomodulatory effects [4], [5], [6]. Moreover, it functions as a neurotransmitter and neuromodulator [7]. VIP performs its biological effects through specific, G-protein-coupled membrane receptors, namely VPAC1, VPAC2 and PAC1 [8], [9].

As several diseases are characterized by a deficiency of VIP in the tissue, a treatment with exogenous VIP was suggested to result in a therapeutic benefit [5]. Among these disorders are cystic fibrosis [10], ulcerative colitis [11] and primary pulmonary hypertension (PPH) [2], [6]. The latter is a lung disease of unknown origin, which is characterized by persistent elevation of the pulmonary artery pressure [12]. According to this, the small blood vessels in the lung undergo changes, resulting in resistance to blood flow through the lung circulation [13]. Petkov et al. [6] demonstrated that PPH patients display, beside a lack of VIP, an increased VIP-receptor expression and binding affinity. Moreover, this group showed the suitability of pulmonary application of exogenous VIP for the treatment of PPH although the half-life time of VIP in the lung was extremely short.

In other lung diseases, such as asthma or chronic obstructive pulmonary diseases, however, the expression of VIP-degrading proteases is significantly increased [14], [15], a fact, which again hampers the clinical use of VIP as a therapeutic agent.

Generally, the pulmonary administration route of drugs, which act in the respiratory tract, exhibits several advantages over alternative routes, including a rapid drug effect at the site of action, a reduced dose, a reduction in systemic side-effects, the evasion of first pass hepatic metabolism [16] and an enhanced bioavailability of the delivered drug [17].

However, the biological effects of inhaled VIP are short-lived, due to several degradation mechanisms including spontaneous hydrolysis [2], enzymatic cleavage by, for instance, neutral endopeptidases (NEP) [4], [18], mast cell chymase and mast cell tryptase [19], or antibody-catalyzed degradation [7]. The most prevalent cleaving sites of VIP by NEP are at the amino acid positions 7–8 (Thr-Asp) and 25–26 (Ser-Ile), which leads to the major cleavage products, VIP1–25 and VIP26–28 and to a less extent to VIP1–7 and VIP 8–28 [4]. To this end, all these fragments are no longer physiologically active [20].

Accordingly, to circumvent these degradation problems, stable analogues of VIP were synthesized by modifying the primary structure of the peptide [1], [21]. Unfortunately, all these approaches were accompanied by a decrease or loss of the biological activity of the peptide; thus, none of these analogues have ever reached clinical trials or were ever therapeutically applied [2].

Hence, many attempts have been made to design a proper delivery system for VIP in order to stabilize the peptide by encapsulation in, or association with, e.g. liposomes [1], [22], [23]. Liposomes are typically made from natural, biodegradable and non-toxic phospholipid molecules and are able to encapsulate a variety of drug molecules into their aqueous interior or to bind them into or onto their lipid membranes. Thus, all these properties make them attractive candidates for the use as drug delivery vehicles (for reviews, see [24], [25], [26], [27]).

The pharmacokinetics of drugs can be modified by varying the liposomal composition [28]. Above that, a careful choice of the lipids is also necessary to influence the fate of the liposomes within the body. Grafting the liposomal surface with hydrophilic polymers, such as polyethylene glycol (PEG), leads to the formation of sterically stabilized liposomes, which display a longer life-time in the circulation, compared to conventional liposomes, due to a reduced recognition rate by macrophages [27], [29], [30], [31].

Moreover, the association of VIP to negatively charged phospholipids induces a conformational change of the peptide from predominantly random-coiled to α-helical [5]. The latter is the preferred conformation for the ligand–receptor interaction [32], [33]. Whereas, the biological activity of liposomal VIP displays no differences, irrespective, whether non-PEGylated [34] or PEGylated [23] formulations are used.

In addition, liposomes feature a great suitability as drug carriers for pulmonary administration, as they exhibit sustained drug release and depot effects. Moreover, they show aqueous compatibility and are able to prevent local drug irritation reactions in the lung [35], [36]. Thereby, the PEGylation of the liposomes leads to a decreased degradation and absorption of the lipid vesicles in the lung [37]. Only recently, it was shown by Anabousi et al. [38] that PEGylation enhances the membrane integrity of the liposomes in lung surfactant and results in a lower fusion rate of the liposomes after nebulisation. Moreover, the stealth effect might reduce alveolar macrophage clearance.

Usually, the natural fate of inhaled liposomes is a transfer of their lipids to the intracellular phospholipid pool mediated by an interaction of the phospholipids with the pulmonary surfactant [17], [35]. This so-called epithelial lining fluid (ELF) covers the airways, particularly the alveoli [39], and it moderates the surface tension during breathing [40]. ELF can be obtained by bronchoalveolar lavage (BAL), which is performed during fiberoptic bronchoscopy [41]. The resulting surfactant solution, called bronchoalveolar lavage fluid (BALF), is a complex mixture of cells, including alveolar macrophages, lymphocytes, bronchial epithelial cells and mast cells [39]. In addition, it contains a wide variety of soluble components, such as phospholipids (78–90%), mainly phosphatidylcholine, proteins (5–10%), which are mostly released by epithelial cells [42] and neutral lipids (4–10%), mainly cholesterol [43]. Some of these surfactant proteins as well as the neutral lipids are responsible for the adsorption of phospholipids at the air–water interface in the lung [40], [44]. Besides a huge diversity of soluble proteins, BALF also contains several proteolytic enzymes, such as trypsin, cathepsin D and different carboxypeptidases [41], [45].

In this study, we have investigated the stability of a liposomal drug delivery system, intended for pulmonary administration of VIP, and we have tested the integrity of liposomal-associated VIP towards proteolytic degradation. Whereas the design and physico-chemical characterization of this system is already established in our laboratory [46].

In order to mimic most closely the metabolic conditions in lung environment, the degradation behaviour of VIP and possible changes in size and peptide load of the liposomes, were examined after incubation with the cell-free supernatant of BALF. In addition, stability experiments were conducted by incubation of the free peptide solution as well as of the liposomal-associated VIP with Krebs–Henseleit solution, a physiological buffer system, which is isoosmotic to 0.9% saline solution.

In this study, various methods were applied in order to evaluate a protective effect of liposomes towards proteolytic cleavage of VIP.

Section snippets

Materials and methods

Palmitoyl-oleoyl-phosphatidylcholine (POPC), lyso-stearyl-phosphatidylglycerol (lyso-PG) and polyethyleneglycol conjugated distearyl-phosphatidylethanolamine (DSPE-PEG2000) were purchased from Avanti Polar Lipids (Alabaster, AL).

VIP (amino-acid sequence HSDAVFTDNYTRLRKQMAVKKYLNSILN-NH2), N-terminally tryptophan (Trp) modified VIP (Trp-VIP) and EtCy3-VIP with the fluorescent marker Cy3 coupled to the N-terminal amino group of histidine were synthesized by piCHEM (Graz, Austria) using FMOC

Results and discussion

Recently, we have reported on a VIP loaded liposomal formulation, which is intended for the pulmonary application of VIP [46].

Here, we investigate the susceptibility of free and liposomal VIP towards enzymatic degradation, which rapidly occurs in the lung (for a review see [4]), and we address the question whether liposomal-associated VIP is thus protected from proteolytic digestion. To mimic the conditions after pulmonary administration, solutions of free VIP as well as empty and VIP loaded

Conclusion

In the present study, we have tested a liposomal drug delivery system, intended for the pulmonary administration of VIP, a cationic vasodilatory peptide that is highly sensitive to enzymatic degradation in the lung environment. We could demonstrate the stability of the unilamellar liposomes in respect of size and drug load efficiency under metabolic conditions, mimicked by BALF. Moreover, the protective role of liposomes on proteolytic cleavage of VIP was shown by fluorescence techniques.

Acknowledgements

The Austrian Nano-Initiative co-financed this work as part of the Nano-Health (Project No. 0200), the sub-projects NANO-LIPO, NANO-BREATH, NANO-FLU being financed by the Austrian FWF (Fonds zur Förderung der Wissenschaftlichen Forschung) (Project Nos. N202, N206, N209) and NANO-VIP by the FFF (Project No. 253-NAN).

We are grateful to Bernadette Zanner and Dorina Clay for technical assistance.

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