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

Advanced Drug Delivery Reviews 59 (2007) 478 – 490 www.elsevier.com/locate/addr

Solid lipid nanoparticles as a drug delivery system for peptides and proteins ☆ António J. Almeida a,⁎, Eliana Souto b a

Unidade de Ciências e Tecnologia Farmacêuticas, Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, P-1649-003 Lisboa, Portugal b Department of Pharmaceutics, Biopharmaceutics and Biotechnology, Free University of Berlin, Kelchstr. 31, D-12169 Berlin, Germany Received 2 October 2006; accepted 24 April 2007 Available online 1 May 2007

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. © 2007 Elsevier B.V. All rights reserved. Keywords: Solid lipid nanoparticles; Solid lipid microparticles; Proteins; Peptides; Vaccines; Drug incorporation

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Current approaches to protein delivery . . . . . . . . . . . . . . . . . . . 2.1. Particulate carrier systems . . . . . . . . . . . . . . . . . . . . . . 3. SLN as carriers for peptide and protein drugs. . . . . . . . . . . . . . . . 3.1. Protein incorporation in SLN. . . . . . . . . . . . . . . . . . . . . 3.1.1. Microemulsion-based SLN . . . . . . . . . . . . . . . . . 3.1.2. High pressure homogenisation (HPH). . . . . . . . . . . . 3.1.3. Solvent emulsification–evaporation . . . . . . . . . . . . . 3.1.4. Solvent emulsification–diffusion . . . . . . . . . . . . . . 3.1.5. Lipid particles from supercritical fluid (SCF) technology . . 3.2. Loading onto preformed lipid nanoparticles by sorption procedures . 3.3. Protein release and stability . . . . . . . . . . . . . . . . . . . . . 4. Delivery of SLN to mucosal surfaces . . . . . . . . . . . . . . . . . . . . 5. SLN as vaccine carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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This review is part of the Advanced Drug Delivery Reviews theme issue on “Lipid Nanoparticles: Recent Advances”. ⁎ Corresponding author. Tel.: +351 21 7946409; fax: +351 21 7937703. E-mail address: [email protected] (A.J. Almeida).

0169-409X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2007.04.007

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1. 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/HIVand 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 [2–6]. 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

Fig. 1. Biotechnology medicines in development by therapeutic category (after [1] with permission).

479

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 [8,12–19]. 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.

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2. 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 human clinical trials, most of the New Drugs being developed require appropriate formulation and delivery systems. Pharmaceutical formulation of peptides and proteins comprises the preservation of their biological activity during an acceptable shelf-life and ideally the effective and safe transport and delivery to its site of action. Unfortunately, there is no single strategy to follow in formulating such a product and proteins have to be evaluated on a case-by-case basis [6]. As already discussed, the most common method for protein and peptidebased drug delivery is by injection but parenteral administration of some therapeutic proteins has not been effective because the drug is cleared too rapidly from the body [21]. Such disadvantages have provided the impetus for the development of site-specific delivery systems that enable delivering of lower doses, gaining access to specific body compartments and to concentrate a therapeutic dose at a specific site of action without resulting in undesirable side effects. Initial approaches to address the problems associated with peptide and protein drugs have included chemical modification, altering amino acid sequences to reduce degradation by enzymes and antigenic side effects, or fusion to immunoglobulins or albumin to increase half-life [22]. Due to complication of structural modifications, excipients with stabilizing properties are frequently used including sugars, surfactants, salts, polyols, poly (ethylene glycol) (PEGs), polymers, metal ions, and amino acids [6]. Depending on the protein, these traditional stabilizers may increase protein stability only to a limited level and alternative approaches have been explored. For example, several authors have reported that cyclodextrins may act as chemical chaperones, avoiding aggregation of several proteins, such as calcitonin, insulin, lysozyme and somatropin [3]. Chemical conjugation with PEG (PEGylation) is currently considered one of the most successful techniques to prolong the residence time of protein drugs in the bloodstream and in a few cases it was also demonstrated to confer certain targeting properties [23]. It lowers the plasma clearance rate by reducing the metabolic degradation and receptor-mediated uptake of the protein from the systemic circulation. PEGylation also reduces immunogenicity and improves the safety profile of the protein by shielding immunogenic epitopes [3]. It has also been reported that PEGylation may increase the oral absorption of proteins [24]. Several PEGylated proteins are currently on the market, including PEG-adenosine deamidase (Adagen®, Enzon), pefilgrastim (Neulasta®, Amgen), PEG-L-asparaginase (Oncaspar®, Enzon), pegvisomant (Somavert®, Pfizer), PEG-alpha-interferon-2b (PegIntron®, Schering-Plough) and PEG-alpha-interfer-

on-2a (Pegasys®, Roche). As an alternative to PEGylation proteins may be conjugated to naturally occurring hydrophilic polymers of N-acetylneuraminic acid (polysialic acids). Polysialylation increased catalase and asparaginase stability in the presence of proteolytic enzymes or blood plasma. The antigenicity of asparaginase was reduced upon conjugation with polysialic acids, leading to increased circulatory half-life even in pre-immunised mice and suggesting polysialylation as a promising approach in protein delivery [25,26]. Controlled release formulations show numerous advantages, including protecting the protein over an extended period from degradation or elimination, increased patient compliance and also the ability to deliver the protein at the site of action, thereby reducing systemic exposure. Some interesting reviews [8,27] focus on strategies that have been used to deliver efficiently therapeutic proteins by different administration routes (Table 1). 2.1. Particulate carrier systems An established strategy to protein delivery consists of attaching the drugs to suitable particulate carrier systems, whereby the in vivo fate of the drug molecule is determined by the properties of the carrier system rather than those of the protein. As a result, there is an increased therapeutic index by controlling the rate and site of drug release. With this purpose microencapsulation and nanoencapsulation techniques have been developed, involving coating and isolating bioactive substances in envelopes of protective

Table 1 Delivery routes and novel technologies for therapeutic peptides and proteins (modified from references [28,29]) Delivery routes

Formulation and device requirements

Invasive Direct injection: intravenous Liquid or reconstituted solid (i.v.), subcutaneous (s.c.), (syringe), i.v. injected liposomes. intramuscular (i.m.), intracerebral vein (i.c.v.) Depot system (s.c. or i.m.) Biodegradable polymers, liposomes, permeable polymers (not degradable) microspheres, implants. Non-invasive Pulmonary

Oral

Nasal

Transdermal

Buccal, rectal, vaginal, ocular

Liquid or powder formulations, nebulisers, metered dose inhalers, dry powder inhalers. Solids, emulsions, microparticulates, nanoparticulates, with or without absorption enhancers. Liquid, usually requires permeation enhancers, nanoparticulates. Iontophoresis, electroporation, chemical permeation enhancers, prodrugs, sonophoresis, transfersomes. Gels, suppositories, bioadhesives, microparticulates.

References

[6,30]

[31–34]

[6,18,35,36]

[36–38]

[12,35]

[17,39,40]

[13–16]

A.J. Almeida, E. Souto / Advanced Drug Delivery Reviews 59 (2007) 478–490

materials until such time as their activity is needed. Polymeric hydrogels, nanoparticles and microspheres, and lipid-based drug delivery systems such as fat emulsions, liposomes and solid lipid nanoparticles (SLN) are all examples of particulate carrier systems for protein delivery. Fat emulsions are a delivery system for lipophilic drugs, which can be incorporated easily into the oil droplets. This carrier system allows the reduction of side effects but is thermodynamically unstable. Therefore, emulsions often tend to agglomerate or even break and the drug is rapidly released once it reaches the blood stream. Moreover, incorporation of hydrophilic proteins and peptides in the internal phase of w/o emulsions is difficult to achieve. Emulsions have been used as vaccine adjuvants to enhance the immune response to many protein antigens, acting mainly by marked depot effect, proper antigen presentation and lymphatic targeting [41]. An o/w emulsion (MF59®, Chiron Corporation) has been the first adjuvant to be licensed for human use after alum. Liposomes are a well established and extensively investigated particulate carrier system that has been successfully employed for the controlled release and site specific drug delivery. Liposomes consist of one or more phospholipid bilayers separated by internal aqueous compartments [34]. Differing with reference to their dimensions, composition, surface charge and structure, liposomes show the ability to entrap enzymes and proteins. The attractiveness in the application of liposomes resides on the compatibility of the constituent components with the body system thereby presenting low inherent toxicity. They are simple to prepare, and their composition can be varied to obtain more efficient preparations. It is therefore surprising that there are still so few liposome formulations on the market, owing perhaps to some in vivo instability that may cause rapid uncontrolled release of the drug from the formulation [42]. Nanoparticles and microspheres represent a wide class of delivery systems mainly studied for parenteral administration, but their applications to the mucosal routes are also most interesting. They have been sought, not only as a means of protecting the peptides from degradation and prolonging their half-lives in vivo, but also because they show excellent controlled release properties and act as immunological adjuvants for protein antigens [43]. Depending on the biodegradable material used, particle size distribution and degradation or erosion kinetics, various delivery profiles and therapeutic applications may be achieved. Currently the most prominent materials are biodegradable polyesters [e.g. poly(lactide) (PLA), poly(lactideco-glycolide) (PLGA), poly-ε-caprolactone (PCL) and poly (ortho esters)]. The lactide/glycolide co-polymers have a long history of safe use in human medicine but the commercial use of PLGA microspheres containing peptide and protein drugs is limited to parenteral delivery of luteinising hormone-releasing hormone (LHRH) analogues (e.g. Lupron Depot®, Takeda Abbot; Decapeptyl Depot®, Debiopharm), human growth hormone (Nutropin Depot®, Genentech) and octreotide acetate (Sandostatin LAR®, Novartis). Also relevant in current research are polysaccharides (e.g. alginate and chitosan), polyanhydrides and solid lipids such as the physiologically cleavable medium and long chain diacylglycerols and triacylglycerols.

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3. 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 systems, SLN production techniques do not need to employ potentially toxic organic solvents, which may also have deleterious effect on protein drugs. Furthermore, under optimised conditions they can be produced to incorporate lipophilic or hydrophilic drugs and seem to fulfil the requirements for an optimum particulate carrier system [44,47]. Their colloidal dimensions and the controlled release behaviour enable drug protection and administration by parenteral and non-parenteral routes thus emphasising the versatility of this nanoparticulate carrier. Publications have described the use of lipid nanoparticles by parenteral routes including biodistribution and pharmacokinetic studies upon i.v. administration [48–52]. Higher amounts of drug were also found in the brain after i.v. injection, suggesting the potential use of SLN as a brain delivery of drugs such as doxorubicin, tobramycin, not capable of crossing the blood brain barrier [48–51]. Topical application either for therapeutic or cosmetic purposes is a research area in which SLN have already reached the market (NanoRepair Q10®, Dr. Rimpler) [53–55]. At the same time, the characterisation of the mechanisms of particle uptake and translocation at mucosal sites, now widely accepted, has opened an enormous field of investigation on the therapeutic applications of SLN using mucosal routes [50–52,56–60] (cf. Section 4 below). 3.1. Protein incorporation in SLN The SLN production is based on solidified emulsion (dispersed phase) technologies. Therefore, due to their hydrophilic nature most proteins are expected to be poorly microencapsulated into the hydrophobic matrix of SLN, tending to partition in the water phase during the preparation process, which is further enhanced by the use of surfactants as emulsion stabilizers. In addition, SLN can present an insufficient loading capacity due to drug expulsion after polymorphic transition during storage, particularly if the lipid matrix consists of similar molecules. However, lipids are versatile molecules that may form differently structured solid matrices, such as the nanostructured lipid carriers (NLC) and the lipid drug conjugate nanoparticles (LDC), that have been created to improve drug loading capacity (reviewed by Wissing and Müller [47]). Since the mid 1990's, authors have regularly published promising results concerning the incorporation of several peptides and proteins in solid lipid particulate carriers (Table 2). Therapeutically relevant peptides (e.g. calcitonin, cyclosporine A, insulin, LHRH, somatostatin), protein antigens (e.g. hepatitis B and malaria antigens) and model protein drugs (e.g. bovine serum albumin and lysozyme) have been investigated for drug release

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Table 2 Peptide and protein molecules incorporated in lipid micro- and nanoparticles Peptide/ Protein

Particulate system

Method of preparation

Incorporation efficiency

Cumulative release

Protein stability/ biological activity

References

Antide

LM

N85%

≤ 60%/24 h

100% BA

[61]

Antide

LM

N85%

≤ 40%/24 h

100% BA

[61]

BSA BSA BSA BSA-FITC Calcitonin CyA

LM LM SLN LM SLN SLN

93% intact 100% intact n.a. n.a. BA proved in vivo n.a.

[62] [63] [64] [65] [66,67] [68,69]

SLN SLN SLN SLN SLN Lipospheres SLN LM

58.1–69.5% 13–62% protein content n.a. n.a. N90% 95.4–97.8% 78.5–93.9% 96.6–97.8% n.a. 96.1% 13% protein content 88.4% n.a. 50.4–69.4% 58.2–67.9%

≈ 30%/24 h ≈ 80%/24 h n.a. n.a. 4%/6 h n.a.

CyA CyA CyA CyA CyA CyA Gonadorelin HBsAg

Milling of drug-lipid solid solutions (co-melting) Milling of drug-lipid solid solutions (solvent stripping) Solvent evaporation (w/o/w) Coating with lipid using SCF Adsorption onto SLN Adsorption onto LM Solvent evaporation (w/o/w) HPH hot dispersion HPH cold dispersion HPH hot dispersion HPH hot dispersion HPH hot dispersion Warm microemulsion (o/w) HPH hot dispersion Self emulsification Solvent displacement Solvent evaporation (w/o/w)

n.a n.a. n.a. b5%/2 h n.a. n.a. 80%/14 days n.a.

[70] [71,72] [73] [74] [75] [76] [77] [62]

HSA

SLN

Adsorption onto SLN

n.a. n.a. BA proved in vivo n.a. BA proved in vivo BA proved in vivo 100% intact N98% intact; BA proved in vivo n.a.

Insulin Insulin Insulin Insulin Insulin Insulin Insulin Insulin [D-Trp-6] LHRH JEAg

LM LM SLN LM SLN SLN SLN SLN SLN

Solvent evaporation (o/w) Melt-dispersion (o/w or w/o/w) Solvent evaporation (w/o/w) Solvent diffusion (w/o/w) Warm microemulsion (w/o/w) Solvent evaporation (w/o/w) Solvent displacement Supercritical CO2 (PGSS) Warm microemulsion (w/o/w)

12.4–32.4% (stealth) 7–43.5% n.a. (non-stealth) 45–94% b60%/3 days Some aggregation ≈ 50% or N80% b60% /3 days Some aggregation n.a. 45%/1 h n.a. 78–84% ≈ 30%/24 h Intact protein 37.8% n.a. n.a. 67.9% n.a. BA proved in vivo 26.8% n.a. n.a. 75% 100%/4 days BA proved in vivo 90% ≈ 10%/8 h n.a.

LM

Solvent evaporation (w/o/w)

73.8%

Lysozyme MAg R32NS1 Ovalbumin Ovalbumin Somatostatin Somatostatin

SLN Lipospheres

HPH cold dispersion Melt-dispersion (o/w)

43.2–59.2% N80%

SLN SLN LM LM

Melt-dispersion (o/w) Adsorption onto SLN Solvent evaporation (o/w or w/o/w) Melt-dispersion (o/w)

N80% 70–97% b75% 65–97%

Thymocartin LM

Solvent evaporation (o/w or w/o/w)

b10% or b50%

Thymocartin LM

Melt-dispersion (o/w or w/o/w)

b90% or 90–100%

Thymopentin SLN

Warm microemulsion (o/w with ionic pair or w/o/w)

5.2% or 1.7%

≈ 20%/ 10 days n.a. n.a. n.a. 53%/24 h n.a. 70–80%/ 14 days 65–90%/ 5 days 65–90%/ 5 days 10%/6 h

[78] [79] [79] [80] [81] [82] [82] [82] [83] [84]

BA proved in vivo

[85]

≈ 100% BA BA proved in vivo

[86] [87]

Intact protein Intact protein n.a. Intact protein

[88] [89] [90] [90]

Intact peptide

[79]

Intact peptide

[79]

Intact peptide

[91]

BA — biological activity; BSA — bovine serum albumin; CyA — cyclosporine A; FITC — flourescein-5-isothiocyanate; HBsAg — hepatitis B surface antigen; HSA — human serum albumin; HPH — high pressure homogenisation; JEAg— Japanese encephalitis antigen; LHRH — luteinising hormone-releasing hormone; LM — lipid microparticles; MAg R32NS1 — malaria antigen R32NS1; n.a. — non-available; PGSS — particles from gas saturated solution technique; SEDDS — self-emulsified drug delivery system; SFC — supercritical fluid technology.

kinetics, protein stability and in vivo performance (Table 2). Several methods have been used to incorporate proteins in solid lipid particles. 3.1.1. Microemulsion-based SLN The first attempts to encapsulate peptide drugs in SLN were those of Morel et al. [84,91], who used the warm w/o/

w microemulsion-based technique to incorporate [D-Trp-6] LHRH and thymopentin. Encapsulation efficiencies were generally low even when peptide lipophilicity was increased by forming an ionic pair with a counter ion. Similar results were obtained with cyclosporine A (CyA), a hydrophobic peptide that was also incorporated using a single (w/o) warm microemulsion [74].

A.J. Almeida, E. Souto / Advanced Drug Delivery Reviews 59 (2007) 478–490

3.1.2. High pressure homogenisation (HPH) In order to identify and characterise the physicochemical parameters governing protein incorporation in SLN, lysozyme was incorporated as a model drug using HPH by the hot and the cold dispersion techniques [86]. Lipid composition, surfactants and homogenisation conditions (temperature, pressure, number of homogenisation cycles) were found to be crucial for lysozyme encapsulation. Regardless of SLN composition, lysozyme tends to partition to the aqueous phase. Only up to 59% of encapsulation efficiency was obtained, resulting in a poor final protein content of 0.03% (w/w) in SLN. Nevertheless, lysozyme remained intact and active throughout the harsh encapsulation conditions, which reached 1000 bar/3 cycles at 50 °C. While studies performed with bovine serum albumin (BSA) have shown that the usual HPH conditions for SLN preparation strongly affect protein structure [92], lysozyme did not suffer any detectable damage as determined by electrophoresis. This is not surprising since lysozyme is a protein with a high structural stability with stronger internal coherence. Also human insulin and CyA remain stable throughout formulation using HPH procedures [73,93]. What was unanticipated was the increased catalytic activity of incorporated lysozyme, probably due to its immobilisation by adsorption at the SLN surface (Fig. 2). Interaction of lysozyme with the lipid matrix may induce conformational changes that correspond to a more active state. This phenomenon, called interfacial activation, has been reported for lipases and glucocerebrosidase [94–96], thus explaining our observations [86]. The HPH technique has been extensively explored for the encapsulation of CyA, with reproducible batch results and incorporation efficiencies above 90% [68–73]. Characterisation by X-ray analysis shows that the hydrophobic CyA molecule is dissolved in the lipid matrix [70]. An oral formulation of SLN (157 nm) containing 20% of CyA was compared in vivo with the commercial formulation (Sandimmun Neoral/Optoral®). The blood profiles observed after oral administration to young pigs revealed a fast absorption and similar mean plasma profiles between the SLN and the commercial formulation, although the

Fig. 2. Activity of lysozyme extracted from SLN formulations, prepared by the cold HPH, compared to the activity of the enzyme in the initial lipid mixture (mean ± SD; n = 3). Soft/CA — Softisan 142/cetyl alcohol; Wit/CA — Witepsol E85/cetyl alcohol; Sup — aqueous supernatant from SLN suspension (after [86] with permission).

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former did not produce an initial blood peak as observed with the reference formulation. The area under the curves (AUC) suggests that the SLN formulation is less prone to cause side effects by lacking blood concentrations higher than 1000 ng/ml [73]. Similar results were observed in Wistar rats treated orally with 316 nm CyA-loaded SLN, demonstrating the in vivo sustained release effect of these carriers [75]. Bekerman et al. [76] investigated the effect of composition and particle size of the CyA-loaded lipid nanoparticles on the oral bioavailability of this drug in human volunteers and found a correlation between the AUC and Cmax and the particle size of the formulations. Oral bioavailability decreased when the particle size preparations was reduced from 400 nm to 25 nm, which may be explained by differences in particle composition, mainly surfactants that can influence particle uptake at the GI tract. The importance of oral administration of CyA has led to the development of a clinical batch manufacturing unit for CyA-loaded SLN by the HPH — hot dispersion technique, producing 2 kg or 10 kg batches using a modified APV LAB 60 homogeniser (Lübeck, Germany) [97]. 3.1.3. Solvent emulsification–evaporation The solvent evaporation method is a widespread procedure for the preparation of polymeric microspheres and nanoparticles, being firstly used for SLN preparation by Sjöström and Bergenståhl [98]. The encapsulation efficiency of hydrophilic molecules is improved by double-emulsion (w/o/w) technique allowing the formulation of therapeutic proteins and antigens. Currently, much of the work carried out on protein antigen microencapsulation, including solid lipid particles, is based on this method, which avoids any thermal or pressure stress on the incorporated proteins [62,85]. The former research group reports the preparation of solid lipid microparticles composed of soy lecithin by a combination of the concepts involved in the preparation of liposomes and polymeric microparticles, claiming to have combined the advantages of both particulate carriers [62]. However, the use of organic solvents is related to the increase of toxicity of the final product [99]. Protein encapsulation in SLN prepared by this method was investigated with the aim to explore their potential as oral delivery systems [80]. Although these nanoparticles failed to control insulin release (45% burst effect, probably due to protein accumulation at particle surface during preparation), particle coating with Poloxamer 188 or PEG 2000stearate increased the stability of the nanoparticles in gastric and intestinal media, seeming to combine the advantages of nanoencapsulation and PEGylation. Under the same condition the uncoated lipid nanoparticles suffered aggregation and 80% degradation in 4 h, thus showing that coating enables oral administration. The same authors demonstrated the feasibility of the approach upon incorporation of calcitonin in SLN prepared with tripalmitin, or with a mixture of tripalmitin and Miglyol® 812 (thus forming a solid matrix containing oily nanodomains) [66,100]. Particles were coated with PEG 2000-stearate or chitosan. Encapsulation efficiency depended on the polymeric coating material, being N90% for the PEG-coated and 30.7% for chitosan-coated particles, which is caused by competition between drug and coating material for binding sites at the lipid surface.

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The solvent evaporation method and a melt-dispersion technique without the use of organic solvents were compared for protein incorporation in lipid microparticles using insulin, thymocartin and somatostatin as model drugs [79,80]. The resulting microparticles were characterised with respect to particle size and morphology, biocompatibility, encapsulation efficiency and in vitro release behaviour. The encapsulation efficiency was high (Table 2) and release was influenced by the physicochemical properties of the proteins. Both methods were found to be suitable for the preparation of microparticles for s.c. and i.m injection. 3.1.4. Solvent emulsification–diffusion In another attempt to produce insulin-loaded solid lipid microparticles, Trotta et al. [81] used an o/w emulsion–diffusion method, obtaining an encapsulation efficiency of about 80%. The solvent displacement technique [101] was also successfully applied to the preparation of gonadorelin-containing SLN [77]. A comparison between the microemulsion, the solvent evaporation and the solvent displacement techniques, including the use of lectins in the formulation, was established using insulin as model protein [82]. It was found that particle size, zeta potential and encapsulation efficiency of insulin-loaded SLN depended on the preparation methods and on the presence of lectins. The solvent evaporation method produced the highest encapsulation efficiency (67.9%). Irrespective of the production method more than 50% of insulin accumulated at particles surface, reaching 86.9% in SLN produced by the solvent displacement technique [82]. 3.1.5. Lipid particles from supercritical fluid (SCF) technology More recently, very attractive new techniques based on SCF technology have been studied as useful alternatives for drying pharmaceutical protein formulations, and to produce solventfree particulate drug carriers. Carbon dioxide (CO2) has been used almost exclusively in SCF processing of pharmaceuticals because of its low toxicity, its relatively low critical temperature and moderate critical pressure, and its low cost [102]. The main advantages of such techniques include mild processing conditions, possible sterilising properties of supercritical CO2, ability of producing microparticles or nanoparticles in the form of dry powders and feasibility of scaling-up [103]. The SCF technology comprises several processes for micro/nanoparticle production such as rapid expansion of supercritical solution (RESS), particles from gas saturated solutions (PGSS), gas/supercritical antisolvent (GAS/SAS), aerosol solvent extraction system (ASES), solution enhanced dispersion by supercritical fluids (SEDS), which are selected according to the drug solubility in the SCF [103–105]. Several proteins have been processed by such SCF techniques, mainly by SAS and PGSS [103]. The extensive description of these methods being out of the scope of the present review, the reader is referred to recent reviews on particle formation using SCF [103,105]. With reference to solid lipids, the viability of using the PGSS process to obtain spherical hydrogenated palm oil-based solid lipid microparticles, for prolonged release of hydrophilic drugs such as theophylline was demonstrated by Rodrigues et al. [104,106]. In addition, its

application to protein molecules led to a modified SAS technique that combines the atomisation and the anti-solvent processes to prepare lysozyme spherical nanoparticles (100 to 400 nm) using water/ethanol solutions, while keeping enzyme integrity and stability throughout the process. Using a modified PGSS process to produce SLN, insulin protein was dissolved in dimethylsulfoxide (DMSO) and this solution was then incorporated into melted mixtures of tristearin, phosphatidylcoline and dioctyl sulfosuccinate [83]. The lipid mass was mixed with compressed CO2. Atomisation of this mixture resulted in SLN of a particle size b 500 nm presenting sustained release properties and preserving insulin biological activity. Unfortunately, the use of organic solvents even as mild as DMSO compromises the benign aspects of solvent-free SCF processing, especially when particles are intended for injection purposes. An original approach consists of coating protein crystals of a given particle size with solid lipids dissolved in supercritical CO2. As the drug is not dissolved and the process is carried out under mild conditions (e.g. 35 °C/200 bar or 45 °C/200 bar for 1 h) protein integrity is preserved. BSA crystals were coated using this process either with tripalmitin or Gelucire® 50-02, and the latter resulted in prolonged release lipid microcapsules (80% of intact BSA in 24 h) [63]. 3.2. Loading onto preformed lipid nanoparticles by sorption procedures The use of adsorption onto a preformed system as a loading technique is an approach widely described for polymeric nanoparticulate carriers. The smaller the particle, the more difficult it becomes to achieve high drug encapsulation efficiency and a good sustained release effect. To circumvent this problem, the drug can be adsorbed onto the particle surface rather than encapsulated within. Due to their amphipathic nature, proteins are known to adsorb, accumulating at solid–liquid interfaces in a wide range of biological and non-biological processes such as enzyme immobilisation [107]. The rate and extent of protein adsorption is influenced by the properties of the protein and its concentration in solution, by the characteristics of the adsorption matrix such as hydrophobicity and by the solvent properties [108]. This physicochemical behaviour, leading to adsorption, can negatively affect the stability of the medicines of which they are the active ingredients, particularly at low protein concentrations. Structural changes and loss of activity of protein molecules during adsorption and desorption from polymer surfaces is often mentioned as an example of the change in the structure of adsorbed proteins [109]. However, many proteins keep or, more rarely, increase their activity upon adsorption [94–96,110]. Scientists have used this tendency to adsorption as a means to load particulate carriers with proteins. Adsorption onto preformed lipid particles has been carried out with different purposes. BSA-FITC-labelled cationic lipid microparticles were used to measure in vitro particle uptake by macrophages [65]. Plasma proteins were adsorbed onto SLN with the aim of studying the interaction of blood components with i.v. injected SLN and improve circulation time [78,111,112].

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On the other hand, many papers describe the adsorption of antigens to polymeric microspheres and subsequent in vivo studies demonstrate their efficacy as vaccine delivery systems [113–115]. SLN of different lipid compositions were loaded by adsorption with ovalbumin, which kept its integrity and revealed a slow release profile (53%/24 h) with an initial 19% burst release, which is in accordance with previous publications [114]. Still in agreement with those findings, the langmuirian-type isotherm, was found to characterise best the adsorption processes of BSA [64] and calcitonin [66] onto SLN surface. Therefore, adsorption onto preformed is a SLN is perhaps the simplest loading procedure for proteins onto particulate carriers, avoiding the possible degradation of the protein molecules caused by the harsh conditions inherent to the majority of SLN production techniques. 3.3. Protein release and stability After a decade of publications concerning protein incorporation in solid lipid particle data on release mechanisms and full kinetic characterisation are still scarce (Table 2). In addition, no set criteria are available for the design of solid lipid particulate carrier for protein delivery, partly due to the variation in methodology and mode of evaluation. Nevertheless, data so far available allow concluding that prolonged in vitro release and subsequently sustained in vivo effects can be achieved for various pharmaceutical proteins [61,67,73,75,76]. The main factors influencing peptide and protein release from solid lipid particles are the physicochemical characteristics of the drug itself, particle size, lipid matrix composition, surfactants used, drug distribution throughout the matrix, method of preparation and production parameters [47,99,107]. For example, it was possible to modify the release profile of antide not only by changing the lipid matrix (Compritol® E ATO vs. Imwitor® 900), but also by a slight modification on preparation method [61]. Release from microparticles was faster whenever these were prepared with Imwitor® 900 (lower melting point matrix) or using the co-melting method (more wetted matrix). Interestingly, the use of organic solvents (benzyl alcohol and ethanol) in the solvent-stripping method did not affect protein stability. In general, the lipid matrices used in SLN or in lipid microparticles (reviewed by Mehnert and Mäder [99]) result in sustained protein release profiles, probably due to the nature of the lipid matrix itself and to the affinity of proteins to some formulation components [66]. Notice the remarkable peptide retention capacity of the microemulsion-based SLN produced by the research group of Gasco, which is independent on the drug and the lipid composition [74,84,91]. In many cases, however, burst release may be a problem since hydrophilic peptides and proteins tend to accumulate at the o/w interface during preparation remaining at particles surface, causing a burst effect. The core-shell incorporation model, with drug-enriched shell, of Müller et al. [116] has also been used to explain protein accumulation at SLN surface [82]. Although the burst release can be useful to deliver an initial dose [116], it is often considered as a failure of a putative controlled release formulation. In fact, burst release is common in protein-containing SLN [63,66,77,79,80,86], frequently followed by a well-defined slow

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release period [63,66,77,79]. The most common explanation suggested for this phenomenon is the adsorption of released protein molecules at the particles surface, which sometimes leads to incomplete release [66,79,80]. Protein formulation also depends on the ability of process to preserve protein integrity and activity. The characterisation of protein formulations should therefore include information concerning protein stability, which is the case of most authors herein reviewed (Table 2). Most of the aforementioned preparation techniques involve harsh conditions that somehow are prone to induce protein denaturation and degradation. On the one hand, the dispersion of protein molecules in emulsions containing organic solvents, high shear mechanical agitation, high pressure homogenisation and high temperatures may cause damage to protein structure or self-aggregation with consequent losses in physiological activity. On the other hand, research in solid lipid particles as protein delivery systems, particularly in protein stabilization, is benefiting from the know-how accumulated during the past decades in the field of polymeric microspheres and nanoparticles. It explains the success of most formulations of peptides and proteins in solid lipid particles. 4. 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) transcellular route: transport across intact epithelial membranes possibly by endocytosis; 3) paracellular route: transport through endothelial tight junctions. The data produced by several research groups in the last 25 years lead to the conclusion that both the extent of absorption and the mechanism of particle uptake at mucosal surfaces are dependent on particle diameter, surface hydrophobicity, surface charge, shape and elasticity, specific targeting ligands (e.g. lectins), physical and chemical stability and other factors such as vehicle properties and volume [118,119]. The opportunity thus created called the attention of research groups to the potential of SLN as delivery systems for the oral, nasal, pulmonary, ocular, and rectal, routes. Oral delivery of drugs incorporated in SLN is gaining interest. In vivo studies performed with orally administered lipid nanoparticles containing tobramycin [50,51], clozapine [52], rifampicin, isoniazid and pyrazinamide [120] have been reported. In addition, oral administration of SLN containing proteins has shown promising results. The uptake of PEG-coated and chitosan-coated lipid nanoparticles was demonstrated in CaCo-2 cells. However, rats treated orally with chitosan-coated nanoparticles containing calcitonin revealed a significant and prolonged hypocalcaemic effect as compared to the control calcitonin solution (Fig. 3) whereas PEG-coated particles did not show a significant effect [67], probably due to the chitosan ability to open tight junctions.

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Fig. 3. Serum calcium levels after oral administration to conscious rats of calcitonin in solution (□), or associated to PEG-coated nanoparticles (⋄) or CS-coated nanoparticles (▴) (mean ± SD, n = 6). ⁎Significant differences from the calcitonin solution (α b 0.01) (after [67] with permission).

Surface characteristics are a crucial parameter in the efficacy of oral lipid nanoparticulate formulations. The stabilizing effect of lectin-modified insulin-containing SLN was demonstrated in vitro by incubation with proteolytic enzymes [82]. After oral administration to rats, both insulin-loaded SLN and lectinmodified SLN reduced serum glucose levels to concentrations similar to those produced by a s.c. insulin injection, showing that SLN promoted oral absorption of insulin. Particulate uptake also occurs in the nasal mucosa, and particles up to approximately 1 μm have been shown to rapidly enter the bloodstream following intranasal administration [121]. The nasal-associated lymphoid tissue (NALT) is able of taking up and transporting particulate and soluble antigens [122]. However, it was demonstrated that the therapeutic outcome of nasally delivered antigen-containing colloidal carriers is highly dependent on the involvement of the lower respiratory tract [122,123]. The works of Saraf et al. [62] using lipid microparticles confirm these findings. Lipid microparticles containing protein antigens were able to reach the lungs and be taken up by lung macrophages, which contributed to an increased specific mucosal immune response (cf. Section 5). Growing attention has been given to the pulmonary route as an alternative non-invasive means for both local and systemic drug delivery using lipid particles, due to the fact that it provides a large absorptive mucosal. The lung offers a large surface area for drug absorption and the alveolar epithelium allows rapid drug absorption. The alveoli can be effectively targeted for drug absorption by delivering the drug as an aerosol, with mass median aerodynamic diameter less than 5 μm [18]. Although metabolic enzymes can be found in the lungs, the metabolic activities and pathways may be different from those observed in the GI tract, which makes pulmonary administration of many drugs very promising, particularly for peptides and proteins [19]. Despite the rapid onset of action and the success of current applications, the utility of the lung for drug delivery using particulate carriers is not fully appreciated. Most published data are limited to the identification and in vitro evaluation of physicochemical formulation parameters in order to achieve the desired therapeutic effect.

The SLN physicochemical characteristics turn them an appropriate pulmonary delivery system due to the correlation between diameter within the nanometer range, composition and ability for deep lung deposition. Both prolonged drug serum concentrations and lung drug retention are achievable by means of particulate delivery systems including SLN [124,125]. The in vivo fate of pulmonary administered radiolabelled SLN, as assessed using gamma-scintigraphy, after inhalation of aerosolised nanoparticles or by endotracheal delivery involves phagocytosis by alveolar macrophages [57,58]. Few minutes after lung deposition, inhaled nanoparticles translocate to regional lymph nodes suggesting that inhalation is an effective route to deliver drugcontaining SLN to the lymphatics. Accumulation in regional lymph nodes also suggests that the translocation mechanism of lipid nanoparticles may involve phagocytosis by macrophages followed by migration to the lymphatics, which indicates that inhalation could be an effective route for vaccine delivery. The ocular and rectal routes have also been successfully explored [59,60] but there are no reports on protein-containing SLN delivered by these routes. 5. 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 proteolytic degradation at mucosal sites, association with particulate carriers by microencapsulation or adsorption is an established strategy to improve vaccine efficacy. However, most of these polymers present problems associated with the costs and the potentially toxic organic solvents used for microsphere production. Among the biodegradable polymers used for antigen microencapsulation the PLA/PLGA are the most commonly used. PLA/PLGA microspheres are very useful antigen delivery systems that are ingested by macrophages and dendritic cells, providing lasting immunity thanks to sustained release at relatively predictable times of the microencapsulated or adsorbed material [126,127]. Concerning lipid-based systems, it has been established in several laboratories that liposomes can act as powerful immunological adjuvants inducing both cellular and humoral immunity for a variety of bacterial and viral antigens relevant to human disease. These seem to be effective when administered by different routes making an attractive vaccine delivery vehicle for the development of mucosal vaccines [128]. Protein antigens may be covalently linked to triacylglycerols using a controlled lipidation technique. Lipidated antigens have the ability to self assemble in water, mimicking viral particles that enhance both humoral and cellular immune responses at systemic and mucosal levels [129]. It was observed that lipid microparticles can trigger in vitro the internalisation of BSA by antigen-presenting cells [65].

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However, apart from the already mentioned experiments performed with model protein antigens [65,86,88,89] the incorporation of protein antigens into lipid particles for immunisation purposes is still under explored and only a few authors have described their use with real antigens. Experiments on the incorporation of recombinant malaria protein antigen R32NS1 into lipospheres, using a technique which involves the cooling of an emulsion prepared with melted lipids led to an increase in serum specific IgG response after i.m injection. Immune response lasted for at least 12 weeks after primary immunisation and it was found to depend on particle composition and particle size, with larger lipospheres (73 μm) being less effective than smaller ones (10 μm) [87]. In a comprehensive study Saraf et al. [62] produced positively and negatively charged lipid microparticles by the solvent evaporation technique (w/o/w) containing protein antigens (HBsAg) for intranasal immunisation against hepatitis B. The mean particle size obtained (1.6 μm) was appropriate for mucosal uptake, which was demonstrated by ex vivo with alveolar macrophages. When given intranasally to rats both anionic and cationic microparticles elicited higher specific mucosal antibody responses, when compared with the free and the alum-adsorbed antigen. This response was also higher than that obtained upon i. m. vaccination with the same formulations. The specific antiHBsAg sIgA levels induced intranasally by the cationic microparticle formulation were the highest in the nasal, pulmonary and salivary glands, as determined in nasal washes, in bronchoalveolar lavages and saliva, respectively. Only in serum, the intramuscularly administered alum-adsorbed antigen resulted in a superior IgG level. A recent report describes the oral immunisation of mice with solid lipid microparticles containing a Japanese encephalitis antigen. The microparticles of 1.6 μm in diameter were prepared by the double-emulsion solvent evaporation method and showed good in vitro uptake by the intestinal M-cells. The oral administration of the vaccine on weeks 0, 1 and 4 induced serum IgG titres higher than the level of protection, up to 14 weeks into the experiment [85]. Although still sparse, the existing information clearly indicates that, as for biodegradable microspheres, lipid microparticles act as effective vaccine carriers with immunoadjuvant properties by parenteral and mucosal routes. Immunity to antigens can be drastically improved through the administration of antigen-containing lipid particles The in vivo deposition observed in the respiratory tract upon intranasal administration [62] particles mechanisms of adjuvanticity is in agreement with the fact that the lung mucosa plays an important role in eliciting immune response to intranasally administered antigens [122,123]. 6. 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

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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 the use of organic solvents, it may be concluded that some proteins are able to endure the harsh formulation procedures, making possible protein formulation using SLN. Protein encapsulation efficiency depends not only on the lipid mixture employed or the technique used, but mostly on the protein molecule, confirming that every protein must be considered as a special case. The versatility of SLN as protein delivery systems has been demonstrated. Proteins and antigens may be attached to SLN for therapeutic purposes by parenteral routes or upon uptake at mucosal sites. The lipid matrix improves protein stability, avoiding proteolytic degradation after administration, and releasing the protein in a controlled manner. Finally, lipidbased formulations of pharmaceutical proteins, as important as cyclosporine A, insulin, calcitonin and somatostatin 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. Acknowledgements The authors are grateful to Prof. E. Gomes de Azevedo, Instituto Superior Técnico, Lisboa, Portugal, for the useful scientific discussion. References [1] B. Tauzin, Report: Biotechnology Medicines in Development, Pharmaceutical Research and Manufacturers Association, Washington DC, 2006. [2] A.M. Hillery, Drug delivery, the basic concepts, in: A.M. Hillery, A.W. Lloyd, J. Swarbrick (Eds.), Drug Delivery and Targeting for Pharmacists and Pharmaceutical Scientists, Taylor & Francis, London, 2001, pp. 1–48. [3] S. Frokjaer, D.E. Otzen, Protein drug stability: a formulation challenge, Nat. Rev. 4 (2005) 298–306. [4] A.K. Banga, Drug delivery today, Pharma Tech 2002, Business Briefing World Market Series, London, 2002, pp. 150–154. [5] D. Crommelin, E. van Winden, A. Mekking, Delivery of pharmaceutical proteins, in: M.E. Aulton (Ed.), Pharmaceutics: The Science of Dosage Forms Design, Churchill Livingstone, Edinburgh, 2001, pp. 544–553. [6] W. Wang, Instability, stabilization, and formulation of liquid protein pharmaceuticals, Int. J. Pharm. 185 (1999) 129–188. [7] W. Wang, Protein aggregation and its inhibition in biopharmaceutics, Int. J. Pharm. 289 (2005) 1–30. [8] D.K. Pettit, W.R. Gombotz, The development of site-specific drug-delivery systems for protein and peptide biopharmaceuticals, Trends Biotechnol. 16 (1998) 343–349. [9] D.J.A. Crommelin, G. Storm, R. Verrijk, L. de Leede, W. Jiskoot, W.E. Hennink, Shifting paradigms: biopharmaceuticals versus low molecular weight drugs, Int. J. Pharm. 266 (2003) 3–16. [10] M. Saltzman, Drug Delivery: Engineering Principles for Drug Therapy, Oxford University Press, New York, 2001. [11] N.M.K. Ghilzai, A. Desai, Facing the challenges of transmucosal absorption — buccal, nasal and rectal routes, Pharma Tech 2004, Business Briefing World Market Series, London, 2004, pp. 104–106. [12] M.I. Ugwoke, R.U. Agu, N. Verbeke, R. Kinget, Nasal mucoadhesive drug delivery: background, applications, trends and future perspectives, Adv. Drug Deliv. Rev. 57 (2005) 1640–1665.

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