Drug Delivery Systems

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
Antnio J. Almeida a,, Eliana Souto b
a
Unidade de Cincias e Tecnologia Farmacuticas, Faculdade de Farmcia, 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 emulsificationevaporation . . . . . . . . . . . . .
3.1.4. Solvent emulsificationdiffusion . . . . . . . . . . . . . .
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: aalmeida@ff.ul.pt (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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A.J. Almeida, E. Souto / Advanced Drug Delivery Reviews 59 (2007) 478490
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 [26].
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 [8,1219].
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.
480
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]
[3134]
[6,18,35,36]
[3638]
[12,35]
[17,39,40]
[1316]
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--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.
481
3. SLN as carriers for peptide and protein drugs

Since their first description by Mller 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
Mller 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 [4852]. 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 [4851]. Topical application either for therapeutic
or cosmetic purposes is a research area in which SLN have
already reached the market (NanoRepair Q10, Dr. Rimpler)
[5355]. 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
[5052,5660] (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 Mller [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
482
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
58.169.5%
1362% protein content
n.a.
n.a.
N90%
95.497.8%
78.593.9%
96.697.8%
n.a.
96.1%
13% protein content
88.4%
n.a.
50.469.4%
58.267.9%
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
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
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 diffusion (w/o/w)
Warm microemulsion (w/o/w)
Solvent displacement
Supercritical CO2 (PGSS)
Warm microemulsion (w/o/w)
12.432.4% (stealth) 743.5% n.a.

(non-stealth)
4594%
b60%/3 days Some aggregation
50% or N80%
b60% /3 days Some aggregation
n.a.
45%/1 h
n.a.
7884%
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
73.8%
Lysozyme
MAg
R32NS1
Ovalbumin
Ovalbumin
Somatostatin
Somatostatin
SLN
Lipospheres
HPH cold dispersion

Melt-dispersion (o/w)
43.259.2%
N80%
SLN
SLN
LM
LM
Adsorption onto SLN
Solvent evaporation (o/w or w/o/w)
N80%
7097%
b75%
6597%
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 90100%
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.
7080%/
14 days
6590%/
5 days
6590%/
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].
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 [9496], 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% [6873]. 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).
483
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 (Lbeck, Germany) [97].
3.1.3. Solvent emulsificationevaporation
The solvent evaporation method is a widespread procedure
for the preparation of polymeric microspheres and nanoparticles,
being firstly used for SLN preparation by Sjstrm and
Bergensthl [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.
484
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 emulsificationdiffusion
In another attempt to produce insulin-loaded solid lipid
microparticles, Trotta et al. [81] used an o/w emulsiondiffusion
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
[103105]. 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 solidliquid 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 [9496,110].
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].
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
[113115]. 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 Mder [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
Mller 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
485
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.
486
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 ( 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 m 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 m [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].
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 m) being less effective than
smaller ones (10 m) [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 m) 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 m 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
487
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 Tcnico, Lisboa, Portugal, for the useful
scientific discussion.
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Advanced Drug Delivery Reviews 59 (2007) 478 490

Solid lipid nanoparticles as a drug delivery system for

A.J. Almeida, E. Souto / Advanced Drug Delivery Reviews 59 (2007) 478490

Fig. 1. Biotechnology medicines in development by therapeutic category

the gastrointestinal (GI) tract the situation is even poorer due to

A.J. Almeida, E. Souto / Advanced Drug Delivery Reviews 59 (2007) 478490

2. Current approaches to protein delivery

on-2a (Pegasys, Roche). As an alternative to PEGylation

Formulation and device

Buccal, rectal, vaginal,

Liquid or powder formulations,

A.J. Almeida, E. Souto / Advanced Drug Delivery Reviews 59 (2007) 478490

materials until such time as their activity is needed. Polymeric

3. SLN as carriers for peptide and protein drugs

A.J. Almeida, E. Souto / Advanced Drug Delivery Reviews 59 (2007) 478490

Milling of drug-lipid solid solutions

Adsorption onto SLN

Solvent evaporation (o/w)

12.432.4% (stealth) 743.5% n.a.

Solvent evaporation (w/o/w)

HPH cold dispersion

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

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

kinetics, protein stability and in vivo performance (Table 2).

w microemulsion-based technique to incorporate [D-Trp-6]

A.J. Almeida, E. Souto / Advanced Drug Delivery Reviews 59 (2007) 478490

3.1.2. High pressure homogenisation (HPH)

Fig. 2. Activity of lysozyme extracted from SLN formulations, prepared by the

A.J. Almeida, E. Souto / Advanced Drug Delivery Reviews 59 (2007) 478490

The solvent evaporation method and a melt-dispersion

application to protein molecules led to a modified SAS

A.J. Almeida, E. Souto / Advanced Drug Delivery Reviews 59 (2007) 478490

On the other hand, many papers describe the adsorption of

release period [63,66,77,79]. The most common explanation

A.J. Almeida, E. Souto / Advanced Drug Delivery Reviews 59 (2007) 478490

Fig. 3. Serum calcium levels after oral administration to conscious rats

Surface characteristics are a crucial parameter in the efficacy

The SLN physicochemical characteristics turn them an

A.J. Almeida, E. Souto / Advanced Drug Delivery Reviews 59 (2007) 478490

However, apart from the already mentioned experiments

reviewed are still at a basic level. For example, mucosal delivery

A.J. Almeida, E. Souto / Advanced Drug Delivery Reviews 59 (2007) 478490

A.J. Almeida, E. Souto / Advanced Drug Delivery Reviews 59 (2007) 478490

[83] P. Caliceti, A. Brossa, S. Salmaso, S. Bersani, N. Elvassore, A. Bertucco,

A.J. Almeida, E. Souto / Advanced Drug Delivery Reviews 59 (2007) 478490

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