Solid lipid nanoparticles as a drug delivery system for peptides and proteins

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Abstract

Solid lipid particulate systems such as solid lipid nanoparticles (SLN), lipid microparticles (LM) and lipospheres have been sought as alternative carriers for therapeutic peptides, proteins and antigens. The research work developed in the area confirms that under optimised conditions they can be produced to incorporate hydrophobic or hydrophilic proteins and seem to fulfil the requirements for an optimum particulate carrier system. Proteins and antigens intended for therapeutic purposes may be incorporated or adsorbed onto SLN, and further administered by parenteral routes or by alternative routes such as oral, nasal and pulmonary. Formulation in SLN confers improved protein stability, avoids proteolytic degradation, as well as sustained release of the incorporated molecules. Important peptides such as cyclosporine A, insulin, calcitonin and somatostatin have been incorporated into solid lipid particles and are currently under investigation. Several local or systemic therapeutic applications may be foreseen, such as immunisation with protein antigens, infectious disease treatment, chronic diseases and cancer therapy.

Introduction

The increasing number of new molecules of biotechnological origin such as monoclonal antibodies, hormones and vaccines, as well as their therapeutic potential, makes protein delivery an important area of research. The 2006 PhRMA report “Biotechnology Medicines in Development” identifies 418 new biotechnology medicines for more than 100 diseases, including cancer, infectious diseases, autoimmune diseases, AIDS/HIV and related conditions (Fig. 1), which are in human clinical trials or under review by the Food and Drug Administration [1]. However, the therapeutic potential of peptide and protein drugs, as well as their clinical application, is often hampered by a number of obstacles to their successful delivery [2], [3], [4], [5], [6].

Protein stability is the balancing result between destabilizing and stabilizing forces. The formation and stability of the secondary, tertiary and quaternary structures of proteins are based on weak non-covalent interactions (e.g. electrostatic interactions, hydrogen bonding, van der Waals forces and hydrophobic interactions). Disruption of any of these interactions will shift this delicate balance and destabilize the proteins [4], [6]. Therefore, the chemical and physical stability of proteins can be compromised by environmental factors such as pH, ionic strength, temperature, high pressure, non-aqueous solvents, metal ions, detergents, adsorption, and agitation and shearing. Most of these factors are present in common manufacturing processes, including sterilisation and lyophilisation, which may damage the proteins, reducing their biological activity, inducing aggregation and render the proteins immunogenic, leading ultimately to precipitation [2], [3], [7].

Being highly vulnerable molecules, proteins usually present short in vivo half-lives, due to degradation by enzymes, either at the site of administration or in every anatomical location, on their way to the site of pharmacological action.

Diffusion transport of large molecules such as pharmaceutical proteins through epithelial barriers is generally slow resulting in poor absorption to the blood stream, unless specific transporters are available. Proteins' physicochemical properties make them unsuitable for absorption by the main routes and mechanisms. In the gastrointestinal (GI) tract the situation is even poorer due to degradation by acidic environment and proteases [8].

The most common mode of administration of pharmaceutical proteins is intravenous (i.v.) injections, which are usually not well tolerated by patients. Although clearance of i.v. injected proteins may range from a few minutes to several days, most proteins have short half-lives in the blood stream. After administration unwanted deposition may occur resulting in the need of frequent administration of high doses to obtain therapeutic efficacy [1], [5]. Both unwanted distribution and repeated high dose administration can lead to toxic side effects. The subcutaneous (s.c.) and intramuscular (i.m.) injection routes are also used for administration of biopharmaceuticals, being the former the most common one. Upon s.c. injection protein bioavailability may be as high as 100%, but also may be much lower, the fate depending on molecular weight, site of injection, muscular activity and pathological conditions [9]. While proteins over 16,000 Da can diffuse through the blood endothelial wall entering the blood capillaries at the site of injection, or enter the lymphatic system and reach the blood mainly via the thoracic duct, lower molecular weight proteins are predominantly absorbed in the blood circulation via the local blood capillaries [9]. Lymphatic transport is a slow process and the prolonged presence of the protein at the site of injection will expose it to enzymatic degradation [9], [10]. For these reasons, the effectiveness of potential peptide and protein drugs is dependent on a frequent administration regimen, which compromises the patient comfort and makes this route expensive.

Alternative non-injectable routes are currently assuming greater importance. Among these, mucosal absorption has been rather neglected in the advanced drug delivery market, perhaps because of the obstacles that still have to be overcome in order for these routes to become commercially viable alternatives for the delivery of a large number of biomolecules [8], [11]. The mucous surfaces of the body (mouth, eye, nose, rectum and vagina) offer less of a barrier than the skin or the GI tract to the systemic absorption of drugs and the advantage of bypassing the hepato-gastrointestinal first-pass elimination associated with the oral route. They are ideal for rapid absorption but practical difficulties include the fact that most mucosal sites are not suitable for dosage forms that must remain in place for a prolonged period [4]. Nevertheless, nasal, ophthalmic, buccal, rectal, vaginal, transdermal and pulmonary routes have been extensively studied for peptide and protein delivery [8], [12], [13], [14], [15], [16], [17], [18], [19].

Regardless the administration route many therapeutic proteins do not possess the required physicochemical properties to be absorbed, and reach or enter target cells, needing delivery and targeting systems that aim to overcome these limitations, and improve drug performance. In order to fulfil this requirement, particulate carriers such as liposomes, microspheres, micelles and nanoparticles, etc., are currently under development. This review outlines the research work in the field of solid lipid nanoparticles (SLN) and lipid microparticles as peptide and protein delivery systems and vaccine carriers, focusing on the encapsulation methods, release kinetics, protein stability throughout the formulation procedures and discussing the future trends of protein-containing SLN.

Section snippets

Current approaches to protein delivery

Over 125 biotechnology drugs are already available, and the US market for advanced drug delivery systems is currently estimated to be $75 billion, being expected to reach $121 billion by 2010 [1], [20]. In addition, generic drug companies view patent expirations as opportunities to launch generic copies of these drugs. The first biogeneric drug (Omnitrope®, Sandoz, a generic version of somatropin), has recently been approved by the European Commission. With so many new biotechnology drugs in

SLN as carriers for peptide and protein drugs

Since their first description by Müller et al. [44], SLN have attracted increasing attention as an efficient and non-toxic alternative lipophilic colloidal drug carrier prepared either with physiological lipids or lipid molecules used as common pharmaceutical excipients. Two main production techniques were then established: the high-pressure homogenisation described by Müller and Lucks [45] and the microemulsion-based technique by Gasco [46]. Unlike most polymeric microsphere and nanoparticle

Delivery of SLN to mucosal surfaces

In order to be absorbed at mucosal surfaces, therapeutic proteins may be attached to suitable particulate carriers that protect the drugs from degradation and improve permeability. The mucosal uptake and translocation of solid particles into the blood stream was for a long time a controversial issue. Different mechanisms of particle entry at several mucosal sites have been described, some less efficient than others, were proposed [117]: 1) transport via the M cells (antigen sampling); 2)

SLN as vaccine carriers

For a long time particulate carriers have been sought as vehicles for protein antigens. An extensive work has been developed in the area of vaccine formulation using various biodegradable polymeric nanoparticles and microparticles, which release their payload of antigen in a controlled manner and possess adjuvant properties by parenteral or mucosal administration routes [123]. Taking into consideration that most peptide or protein antigens are ineffective for mucosal immunisation due to

Conclusions

The importance of protein delivery lays emphasis on the potential of solid lipid micro- and nanoparticles as effective carriers for pharmaceutical peptides, proteins and vaccines. Although benefiting from the research carried out with biodegradable polymeric particles, many of the approaches herein reviewed are still at a basic level. For example, mucosal delivery and uptake of particulate systems is a subject, which is now starting to be understood. Although many incorporation methods avoid

Acknowledgements

The authors are grateful to Prof. E. Gomes de Azevedo, Instituto Superior Técnico, Lisboa, Portugal, for the useful scientific discussion.

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    This review is part of the Advanced Drug Delivery Reviews theme issue on “Lipid Nanoparticles: Recent Advances”.

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