Lipid-Based Nanoformulations for Peptide Delivery

Home , Solid lipid nanoparticle

International Journal of Pharmaceutics 502 (2016) 80–97

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

journa l homepage: www.elsevier.com/locate/ijpharm

Review

Lipid-based nanoformulations for peptide delivery

a, b a a b

Nada Matougui *, Lukas Boge , Anne-Claire Groo , Anita Umerska , Lovisa Ringstad ,

b a,c

Helena Bysell , Patrick Saulnier

Inserm U1066 (Micro et Nanomédecines biomimétiques), Angers, France

SP Technical Research Institute of Sweden, Stockholm, Sweden

Centre Hospitalier Universitaire (CHU), Angers, France

A R T I C L E I N F O A B S T R A C T

Article history: Nanoformulations have attracted a lot of attention because of their size-dependent properties. Among

Received 29 October 2015

the array of nanoformulations, lipid nanoformulations (LNFs) have evoked increasing interest because of

Received in revised form 28 January 2016

the advantages of their high degree of biocompatibility and versatility. The performance of lipid

Accepted 13 February 2016

nanoformulations is greatly inﬂuenced by their composition and structure. Therapeutic peptides

Available online 17 February 2016

represent a growing share of the pharmaceutical market. However, the main challenge for their

development into commercial products is their inherent physicochemical and biological instability.

Keywords:

Important peptides such as insulin, calcitonin and cyclosporin A have been incorporated into LNFs. The

Nanoformulations

association or encapsulation of peptides within lipid-based carriers has shown to protect the labile

Lipids

Peptides molecules against enzymatic degradation. This review describes strategies used for the formulation of

ﬁ

Drug delivery peptides and some methods used for the assessment of association ef ciency. The advantages and

drawbacks of such carriers are also described.

Contents

1. Introduction ...... 80

2. Lipid-based carriers ...... 82

2.1. Nanoemulsions and micellar systems ...... 82

2.1.1. Nanoemulsions (NEs) ...... 82

2.1.2. Micellar systems: phospholipid micelles ...... 83

2.2. Liposomes ...... 84

2.3. Liquid crystalline nanoparticles (LCNPs) ...... 86

2.4. Solid lipid nanoparticles (SLNs) ...... 88

2.5. Nanostructured lipid carriers (NLCs) ...... 90

2.6. Lipid nanocapsules (LNCs) ...... 92

3. Conclusion ...... 93

Acknowledgements ...... 93

References ...... 93

1. Introduction submicronic size of the particles is another parameter that favors

cell internalization (Yuan et al., 2008). They have also been

Lipidic systems have the potential to enhance the internaliza- explored as potential vehicles for speciﬁc-site drug delivery to

tion of drugs into the cell due to the afﬁnity of the lipid materials to various organs/tissues/systems such as the lymphatic system,

the membrane. In the case of lipid nanoformulations, the brain, lung and the skin (Khan et al., 2013; Liu et al., 2007b; Müller

and Keck, 2004; Pandey and Khuller, 2005). In the ﬁeld of

nanomedicine, they offer interesting alternatives to other colloidal

ﬁ

* Corresponding author at: INSERM UMRS-U1066 MINT “Micro et nanoméde- systems, enhancing drug ef cacy, and providing controlled and

”

cines biomimétiques Bâtiment IBS-IRIS 4, rue Larrey, 49933 Angers cedex 9, France. convenient drug release.

E-mail address: [email protected] (N. Matougui).

http://dx.doi.org/10.1016/j.ijpharm.2016.02.019

N. Matougui et al. / International Journal of Pharmaceutics 502 (2016) 80–97 81

The lipid components of lipid-based nanoformulations (LNFs) preferred to conventional surfactants. The latter are known to

are generally phospholipids, cholesterol and triglycerides (Copland insert themselves into the membrane bilayers and induce

et al., 2005; Rawat et al., 2008), but also bile salts and free fatty hemolysis when incubated with erythrocytes. Lipid surfactants

acids (Liu et al., 2007a). These excipients are relatively innocuous, act via the formation of steric barriers to prevent colloid

biocompatible and biodegradable in vivo. They are extracted from destabilization (coalescence, aggregation, . . . ) (Sarker, 2005).

natural sources or can be the derivatives of natural substances. A Lipid nanoparticulate delivery systems can be adjusted so as to

number of these lipids, such as phosphatidylcholine, stearic acid, have drugs adsorbed or linked to the particle surface, incorporated

cholesterol and glycerol monooleate have been used in FDA- into the polymer/lipid shell, or encapsulated within the particle

approved pharmaceutical applications and have well-established core. As a consequence, the pharmacokinetic and pharmacody-

safety proﬁles with appropriate toxicological data (Rowe et al., namic parameters of the drug can be improved and release can be

2009; Working and Dayan, 1996); this is their major advantage controlled. Furthermore, drugs can be protected from a harsh

compared to other carriers such as polymeric particles (e.g., environment, and undesired side effects can be avoided due to

dendrimers), carbon nanotubes, quantum dots and metal nano- targeted delivery.

particules (e.g., gold and iron nanoparticles) (Goodman et al., 2004; Signiﬁcant advances in biotechnology and molecular biology

Hu et al., 2011; Jain et al., 2010; Kostarelos, 2008; Soenen and De over recent years have resulted in the emergence of novel

Cuyper, 2010). Although lipid nanoparticles can be considered as molecules with the potential to offer signiﬁcant improvement in

relatively safe, and the type of lipid is an important factor affecting the treatment and prevention of diseases. The new biotherapeutics

toxicity. Cationic lipids promote non-speciﬁc binding to circulating include novel peptide and protein drugs. Therapeutic peptides are

blood cells such as erythrocytes, lymphocytes and endothelial cells typically molecules made of 2–100 amino-acids, presenting

(Pedroso de Lima et al., 2001). Cationic liposomes also demonstrate interesting biological functions and, being derived from natural

a greater activation of the human complement system compared to components, are well tolerated following administration. Peptides

neutral liposomes (Semple et al.,1998). Conversely, the presence of can be used to treat a broad range of diseases including cancer,

negatively charged lipids (e.g., phosphatidylglycerol, phosphati- cardiovascular diseases, infection, metabolic diseases and central

dylserine) on nanoparticle surfaces decreases their ability to nervous system disorders (Parmar, 2004).

penetrate the negatively-charged cell membranes (Fischer et al., Since Lypressin, a vasopressin analogue, was launched by

2003; Larson et al., 2007). They can also constitute binding sites for Novartis (Pichereau and Allary, 2005); efforts have not ceased to

plasma opsonins, which favor nanoparticle uptake by macro- multiply in an attempt to exploit the therapeutic potential of many

phages (Nichols, 1993). Coating the nanocarrier with hydrophilic peptides. Peptide-based therapeutics now constitute one of the

polyethylene glycol (PEG) is a prevalent strategy used to decrease fastest-growing classes of new drugs. In fact, almost half of the

the immunogenicity of charged particles (Nichols, 1993; Samad molecules in the pipelines of pharmaceutical companies are

et al., 2007). Currently-approved liposomal products are composed peptides (Dimond, 2010). However, the therapeutic potential of

of neutral lipids with or without PEGylated phospholipids (Samad peptides is hampered by a number of physico-chemical and

et al., 2007; Sarker, 2005). biological instabilities that impede their development and

When formulating nanoemulsions and nanosuspensions, translation to the clinic. These inherent limitations include: Low

PEGylated lipids, glycerophospholipids and derivatives are stability (proteolytic degradation); low oral bioavailability

Fig. 1. A hypothetical, pseudo-ternary phase diagram of an oil/surfactant/water system with emphasis on nanoemulsions, the emulsion phase, and the existence ﬁelds of

conventional micelles and reverse micelles.

82 N. Matougui et al. / International Journal of Pharmaceutics 502 (2016) 80–97

(injection required); risk of immunogenic effects; as well as a Partearroyo and Ostolaza, 1990). Therefore, biocompatible amphi-

challenging and costly synthesis process. philic lipid surfactants are usually preferred to conventional

Moreover, peptides can be prone to chemical and/or physical synthetic ionic and non-ionic ones. Lipid surfactants have low,

instability, mainly related to manufacturing and formulation critical micellar concentrations (CMCs). This property may

processes such as solvents, pH, temperature, ionic strength, high decrease the risks of erythrocyte lysis (which also concerns many

pressure, detergents, agitation and shearing (Hillery, 2001). other cells) in contrast with conventional synthetic surface active

Several peptide modiﬁcations have been explored to improve agents (Ashok et al., 2004; Torchilin, 2007). Three methods may be

peptide in vivo half-lives: The addition of carbohydrate chains; applied for the preparation of NEs: (i) high pressure homogeniza-

synthetic amino acids; polyethylene glycol molecules; as well as tion (an appropriate choice of surfactant and co-surfactant is

lipidation or cyclization strategies (Gentilucci et al., 2010). necessary), (ii) the low energy emulsiﬁcation method at constant

However, altering the chemical structure of the peptide drug temperature and (iii) the phase inversion temperature method

can potentially impair its therapeutic effect (Gante, 1994; Hruby (Tadros et al., 2004).

and Balse, 2000). Recent advances in drug delivery technology Due to their small droplet size, NEs are expected to provide an

offer a novel alternative to the drawbacks of using peptides. efﬁcient delivery of therapeutic agents in a targeted way. Such

Indeed, nanocarriers such as, polymeric nanoparticles, liposomes particles are more likely to adhere to membranes and to transport

and micelles seem to be a promising innovation to increase peptide bioactive molecules in a more controlled fashion. They can be

pharmacodistribution. administered orally, intra-nasally or topically onto the skin. Studies

Among drug delivery systems, lipid carriers offer a number of of transdermal transport using NEs has been investigated for

advantages making them interesting delivery vehicles for peptide various drugs including, tocopherol (Kuo et al., 2008), nimesulide

administration. In general, a nanocarrier needs to be composed of (Alves et al., 2007) and gene delivery (Wu et al., 2001). The

inert and biodegradable material and to be able to efﬁciently intranasal administration of NEs has been studied in order to

encapsulate and protect peptides against degradation, while at the achieve the targeting of curcumin (Sood et al., 2014) and

same time maintaining proper drug activity. Lipid-based nano- risperidone (Kumar et al., 2008) to the brain. Different generations

formulations can meet these requirements. The drug can be of emulsions and nanoemulsions used for parenteral drug delivery

adsorbed onto the particle surface or encapsulated within it. Due to (e.g., chemotherapy, anesthesia) have been considered in a review

their amphiphilic nature, some peptides and proteins are known to (Collins-Gold et al., 1990).

adsorb at solid-liquid interfaces in biological and non-biological NEs have also been tested for peptide delivery. Depending on

mechanisms such enzyme immobilization (Cao, 2005). The the hydrophilic lipophilic balance (HLB) of the peptide concerned,

adsorption efﬁciency depends on the nature of the peptide either O/W or W/O nanoemulsions can be considered as delivery

(charge, length, hydrophobicity) and its concentration in solution, systems. However, since the majority of therapeutic peptides are

by the properties of the adsorption matrix and by the solvent hydrophilic, most studies conducted on nanoemulsions as vehicles

(Haynes and Norde, 1994). This review describes a general state of for such molecules have explored water-in-oil nanoemulsions. The

the art dealing with the principal lipidic systems that can be used peptide is solubilized within the dispersed nanodroplets. The

as peptide carriers, their properties and their application in the mechanisms proposed for improving bioavailability using W/O

ﬁeld of peptide delivery. microemulsions were based on increased protection against in

vitro physical aggregation and in vivo enzymatic inactivation (Lim

2. Lipid-based carriers et al., 2012; Pattani et al., 2006) and/or the improvement of drug

absorption.

2.1. Nanoemulsions and micellar systems After cyclosporin A, which is an unusually lipophilic peptide,

the most studied peptide is insulin which is delivered parenterally.

2.1.1. Nanoemulsions (NEs) However, the parenteral route is the least acceptable route to the

Microemulsions can be assimilated to swollen micelles (ﬁlled patient. Thus, the oral administration of insulin still remains a

with water and/or oil) in thermodynamic equilibrium. These challenge for scientists. Its susceptibility to the strong acidic

systems are excluded from this review that focuses more on environment and the proteolytic activity in the gastrointestinal

nanoparticulate systems. Nanoemulsions (NEs) are kinetically- tract, limits its oral bioavailability. In this context, a number of

stable liquid isotropic dispersions composed of water, oil and studies have investigated NE-based systems for insulin delivery.

surfactants. At deﬁned stoichiometric ratios of the ingredients, the Sharma et al. (2010) reported on the incorporation of insulin. The in

formation of translucent nanoemulsions is spontaneous. NEs can vivo bioavailability of the NE-insulin was 10-fold higher, compared

be considered as being conventional emulsions that contains very to an insulin solution administered orally to healthy rats (Sharma

small droplets (Mason et al., 2006; Tadros et al., 2004) with et al., 2010). The improved bioavailability of the W/O NE systems

globular size in the range of 50–200 nm (Kong and Park, 2011). The was also observed for a lecithin-based NE of rh-insulin (Çilek et al.,

very small droplet size results in a signiﬁcant reduction of 2005). More recently, Li et al. (2013) have developed double

gravitational forces. Brownian motion may be sufﬁcient to emulsions (W/O/W) coated with alginate and chitosan. These NEs

overcome gravity, conferring long-term physical stability to NEs have been found to be highly efﬁcient for oral insulin delivery. The

(with no apparent ﬂocculation or coalescence). Ostwald ripening is in vitro insulin release from the system has been investigated. It

usually considered to be the major destabilization mechanism of was found that the peptide was better retained within the coated-

nanoemulsions. The solubility of the dispersed phase increases NEs compared to uncoated ones. At low pH (2.5), the conformation

with decreasing droplet radii. Hence the smaller droplets dissolve of the coating can prevent the premature release of insulin from

more rapidly, diffuse to, and deposit onto the larger drops. the inner phase in simulated gastric media. The in vivo oral

However, this phenomenon can be retarded by ensuring a administration of the coated-NEs was also investigated. These

monodispersity of the nanoemulsion (Liu et al., 2006). latter induced a signiﬁcant hypoglycemic effect compared to

NEs may be in the form of oil in water (O/W) or water in oil (W/ various formulations (insulin solution, uncoated nanoemulsions or

O) depending on whether the oil is dispersed as droplets in water, multiple emulsions), in either normal or diabetic rats. The

or vice versa (Fig.1). As stated previously, the insertion of surfactant improved effect could partly be attributed to the alginate/chitosan

monomers into cell membranes leads to membrane disruption and coating, which increases drug stability, improve permeability and

cellular lysis (Jumaa and Müller, 2000; Ohnishi and Sagitani, 1993; bioadhesion. Moreover, a more sustained and prolonged effect was

N. Matougui et al. / International Journal of Pharmaceutics 502 (2016) 80–97 83

reported compared to subcutaneous insulin administered as a Small-molecule drugs can be dispersed in the hydrophobic core

reference (Li et al., 2013). of the micelles (Cesur et al., 2009; Koo et al., 2011; Önyüksel et al.,

NEs have been investigated for the delivery of many other 2009), whereas peptides and water soluble drugs (e.g., doxorubicin

peptides. For example, polymyxinB (Pattani et al., 2006) and as displayed in Fig. 2) (Kuzmis et al., 2011; Lim et al., 2011; Wang

salmon calcitonin (sCT). The incorporation of the latter in the inner et al., 2010), are assumed to be present in the PEG micellar shell.

aqueous phase of a W/O/W emulsion appears to protect the Phospholipid micelles enable peptides to overcome the physico-

peptide against enzymatic degradation. sCT was further protected chemical and biological instability issues and can be used to

by the concomitant incorporation of a protease inhibitor, aprotinin, improve safety, efﬁcacy and biodistribution (Guo et al., 2005).

in the outer aqueous phase (Dogru et al., 2000). Prego et al. (2006) Polymyxin B (amphiphatic peptide) has been efﬁciently

designed a chitosan-PEG nanoemulsion for the oral delivery of solubilized in DSPE-PEG2000 micelles (Brandenburg et al., 2012).

peptides. The coated nanocapsules were reported to be more stable The phospholipid-peptide association can change the secondary

in gastrointestinal ﬂuids. Moreover, the pegylation of chitosan structure of the peptide, which provides better stability against

reduced the in vitro cytotoxicity of the nanoemulsions on the enzymatic degradation. Random coil conformation peptides

caco2 cell line. Finally, in vivo studies have shown the role of (Vasoactive Intestinal Peptide (VIP), Pituitary Adenylyl Cyclase-

PEGylation degree of chitosan coating to enhance and prolong the Activating Polypetide (PACAP1-38), Glucagon-Like Peptide 1 (GLP1),

absorption of sCT (Prego et al., 2006). Secretin) adopt a helical secondary structures (active conforma-

tion required for resistance interaction) when associated with the

2.1.2. Micellar systems: phospholipid micelles phospholipid micelles (Dagar et al., 2003; Krishnadas et al., 2003;

Micellar systems consist of a mixture of water or oil and Lim et al., 2011; Önyüksel et al., 2006). Amino acid residues that are

surfactants (Fig. 1). When formulated in oil, they correspond to susceptible to proteolytic cleavage are also efﬁciently buried in the

well-known reverse micelles. Micelles form spontaneously and PEGylated shell. However, peptides with disulﬁde bonds such as

exist in dynamic equilibrium with a constant exchange of galanin are less liable to changes in their secondary structure after

monomers between the external medium and the micelles. The their incorporation or association with the micelles. Moreover,

micelles only form when the concentration of surfactant reaches Dagar et al. (2003) reported on the limited interaction of galanin, a

the CMC. As mentioned previously, the use of phospholipids neuropeptide hormone, with phospholipids because of the

instead of other polymeric surfactants, enables a reduction in the presence of disulﬁde bounds and low hydrophobicity (Dagar

concentration of the surfactant and hence improves the safety of et al., 2003). Thus peptides must have a ﬂexible structure and an

the system. appropriate hydrophobicity in order to promote favorable inter-

Recently, sterically stabilized micelles (SSMs), an example of actions with phospholipids.

lipid micelles, received much attention as safe drug delivery In this type of delivery system, peptides are not chemically-

systems. SSMs are phospholipid micelles produced from PEGylated linked to the micelles and hence can be released from the micelles

lipids like DSPE-PEG2000 (distearoyl-phosphatidylethanolamine and interact with their targets in their active conformation

chemically conjugated to PEG2000). They can be considered as safe (Banerjee and Onyuksel, 2012). Peptides associated with phos-

and biocompatible nanocarriers for the delivery of small mole- pholipid micelles showed enhanced activity compared to peptide

cules, hydrophobic drugs and even amphiphilic peptides to their solutions due to the increased availability of intact peptides at the

site of action (Koo et al., 2005; Lim et al., 2011). SSMs form target site. GLP1 associated-SSM signiﬁcantly decreased the

spontaneously in the aqueous environment when diacyl lipid-PEG lipopolysaccharide (LPS)-induced inﬂammatory reaction in mice

phospholipids are present at a concentration above their CMC. The model of acute lung injury (Lim et al., 2011). The bioactivity of VIP-

conjugation of PEG2000 to the phospholipids confers conical shape SSM for rheumatoid arthritis was also investigated; again this

to each monomer facilitating their self-assembly into micelles association demonstrated a signiﬁcant improvement of safety and

(Ashok et al., 2004; Lim et al., 2008). Furthermore, PEG prevents efﬁcacy of VIP administered to a collagen-induced arthritis rodent

micelle recognition by opsonins and thus will not be managed by model. The circulation half-life was prolonged and a 13-fold higher

the reticulo-endothelial system. The conjugates have very low CMC accumulation of VIP-SSM in joints was observed compared to free

levels (ap. 1 mM) when compared to other surfactants. Hence, they VIP. Meanwhile, VIP-SSM reduced peptide-associated side effects

are relatively stable after dilution in vivo (Kuo et al., 2008). DSPE- (e.g., hypotension, dysregulation of the gastrointestinal system)

PEG2000 micelles are easy to prepare and appropriated for a scaled- (Sethi et al., 2013).

up process. They can be sterilized and lyophilized without the use However, phospholipid micelles are not adapted for the

of cryo/lyoprotectant (Lim et al., 2008). delivery of all types of peptide. Indeed, micelle association is

Fig. 2. Schematic illustrations, of the self-assembly of water-soluble drugs and micelles. (A) Neuropeptide Y and DSPE-PEG2000 micelles. (B) Doxorubicin and PEG-

Phosphatidylethanolamine micelles (Kuzmis et al., 2011; Wang et al., 2010).

84 N. Matougui et al. / International Journal of Pharmaceutics 502 (2016) 80–97

most likely governed, by electrostatic and hydrophobic forces evolved; long-circulating liposomes, surface-modiﬁed liposomes

between the respective charges and the hydrophobic amino-acid and stealth liposomes have been developed.

moieties. Hence, the stability of the associated peptides in the body Liposomes are formed spontaneously after the hydration of

will depend on the strength of the interaction between the peptide phospholipids. Additional steps are often necessary to modify their

and the PEG palisade which, in turn, depends on the secondary size and structure. Several preparation methods are described.

structure and the hydrophobicity of the peptide. Peptides with an Each process provides liposomes with given characteristics: Size

intrinsic a-helical structure are less likely to associate efﬁciently, and the number of bilayers. The process can also be selected

than peptides that are random coiled, due to the hydrophobic according to criteria such as encapsulation efﬁciency and the

interactions between the peptide and the PEG layer. Another stability of the drug to be encapsulated. Bengham’s method (i.e.,

limitation concerns peptide length. Very small peptides are not the dry ﬁlm hydration method) consists of lipids dissolution in a

able to adopt a stable conformation or may be over-masked in the suitable organic solvent. The solvent is then evaporated under

PEG palisade so that recognition and interaction with its receptors reduced pressure, until the formation of a thin ﬁlm. Finally the ﬁlm

may be limited. In contrast, very large peptide molecules may be is resuspended in an aqueous medium, above the phase-transition

less strongly attached and more exposed to chemical and/or temperature, resulting in the formation of MLV liposomes. This

enzymatic degradation. Önyüksel et al. (2009) have produced method is simple and widely used; however, its disadvantage is the

promising peptide nanomedicines with 17–36 amino acid residues low encapsulation ability (Bangham et al., 1965; Wagner and

(Kuzmis et al., 2011; Önyüksel et al., 2009, 2006). Vorauer-Uhl, 2011). Other methods, based on the replacement of

an organic solvent by an aqueous medium, are also used. At ﬁrst,

2.2. Liposomes lipids are dissolved in a solvent or a mixture of organic solvents.

The aqueous phase is injected into the organic phase (reverse-

Liposomes are vesicles composed of one or more aqueous phase method), or the organic phase is injected into the aqueous

compartments delimited by phospholipid bilayers. The supramo- phase (organic solvent injection method). The phospholipids stand

lecular organization of the phospholipids in bilayers is a at the interface between the two immiscible phases. After

consequence of the repulsive forces between the hydrophobic sonication, a W/O emulsion is obtained in which the phospholipids

tails of the phospholipids and water. As a result the polar head are organized in reverse micelles surrounding the aqueous

groups are oriented to the aqueous phases (inner and outer) while compartment. Evaporation of the organic solvent leads to the

the hydrocarbon tails are forced to face each other to form one or formation of the bilayers of the liposomes.

several bilayers (Jesorka and Orwar, 2008). These methods allow the formation of LUVs with high

Due to the presence of both lipid and aqueous phases in the encapsulation efﬁciency of both hydrophilic and lipophilic

liposome structure, they can be used as a carrier for water-soluble, substances. Generally, the incorporation of lipophilic drugs is

lipid-soluble, and amphiphilic materials (Khosravi-Darani et al., performed through their co-dissolution with the lipids (Wagner

2007; Mozafari et al., 2008). Depending on their lipophilicity, the and Vorauer-Uhl, 2011). Hydrophilic drugs are dissolved in the

cargo can be accommodated in the lipid bilayer, in the entrapped aqueous medium, whereas amphiphilic drugs can be dissolved in

aqueous compartment or at the water/lipid interface. both media. The processes of liposome preparation can produce

Liposomes are made of naturally-derived phospholipids. large vesicles with a heterogeneous size distribution. Therefore, it

Glycerophospholipids, such as phosphatidylethanolamine, phos- is important to adjust the formulation using a vesicle size

phatidylserine and phosphatidylcholine, are commonly used in reduction method such as sonication, high-pressure homogeniza-

liposome formulations. However, phosphatidylcholines remain tion, or extrusion through polycarbonate membrane.

the most used due to their stability in liposome formulations As a major requirement, liposomes should maintain their

(Handa et al., 1990). Liposomes can be composed of a single or integrity and prevent the premature release of the encapsulated

multiple phospholipid bilayers (uni- and multi-lamellar). Their peptide and release it, preferably, in an active form at the target

size varies from 25 to 100 nm for the small unilamellar vesicles site. Different strategies have been investigated to increase vesicle

(SUV), larger than 100 nm for large unilamellar vesicles (LUV) and a stability and control drug release (Table 1). One of them involves

few micrometers for the multilamellar vesicles (MLV) (Lasic, 1998; the modiﬁcation of their external surface by the optimization of

Vemuri and Rhodes, 1995). In regards to the composition and the the lipid composition of the membrane. Indeed, lipids with a high

mechanisms of drug delivery, conventional liposomes have transition temperature, for example dipalmitoyl

Table 1

Examples of coated liposomal formulations.

Coating Particle name Advantage Peptide Entrapment efﬁciency% Route of References

administration

Pectin Pectin-liposome Improved intestinal absorption eCT 49.70% Oral (Thirawong et al.,

nanocomplexes 2008)

Silice Silica-liposome Protection against lipolytic degradation and extended Insulin 70% Oral (Mohanraj et al.,

nanocapsules insulin release 2010)

PEG VIP-loaded liposomes Protection of the surface-attached VIP from enzymatic VIP n.a Lung delivery (Hajos et al.,

(VLL) degradation in the airway 2008)

Inhibition of particle aggregation

Mucin Mucin-Lip (cetyl- Improved stability Insulin 20–40% Oral (Iwanaga et al.,

mucin) 1997)

Chitosan Chitosan/trimethyl Mucoadhesive BSA 97% (before spray Nasal (K.H. Chen et al.,

chitosan drying) 60% (after) 2013)

Chitosan-aprotinin- Mucoadhesive Calcitonin 75% Oral (Werle and

MLV Takeuchi, 2009)

Carbopol Carbopol-MVL Mucoadhesive Insulin 58–62% Nasal and (Jain et al., 2007)

Sustained release ocular

N. Matougui et al. / International Journal of Pharmaceutics 502 (2016) 80–97 85

phosphatidylcholine (DPPC), have been found to increase liposome a-helical sturcture. This structural modiﬁcation protects the VIP

stability in the gastrointestinal ﬂuids and hence increase insulin from degradation and inactivation. Moreover, the a-helical form

retention (Kokkona et al., 2000). seems to be the preferred conformation for ligand–receptor

Silica liposome nanocapsules, developed by Mohanraj et al. interaction (Sethi et al., 2005). Insulin-loaded multivesicular

(2010) have also shown controlled insulin release and increased liposomes of 26–34 mm show a high protein loading of between

liposome stability. In the gastrointestinal environment, DPPC, 58% and 62% (Jain et al., 2007).

composed the liposomal surface is protonated, providing high Liposomes made of phospholipids containing the palmitoyl

silica coverage. In fact, at high silica:lipid ratios, coated liposomes group, such as palmitic acid, egg phosphatidylcholine (EPC), and

show increased protection and a controlled release of insulin. dipalmitoyl phosphatidylcholine (DPPC), enhance the pulmonary

Firstly, retention of insulin inside the particles in the simulated delivery of insulin in rats. Such phospoholipids, especially DPPC,

gastric ﬂuid, which is desirable because it reduces insulin gastric have been found to enhance the in vitro permeation through a

degradation and secondly, the release and accumulation of the human bronchial epithelial cell model. It is suggested that they

peptide in its active form in the intestine (Mohanraj et al., 2010). open tight junctions of pulmonary epithelial cells enhancing the

One can cite an incorporation of bioadhesive molecules (chitosan, paracellular transport of water-soluble drug. This in vitro

mucin, alginate) in the liposome membrane (K.H. Chen et al., 2013; observation correlated with the in vivo enhanced bioactivity.

Iwanaga et al., 1997; Thongborisute et al., 2006), or the attachment Moreover, no cytotoxic effect was identiﬁed which conﬁrms that

of synthetic polyethylene glycol derived phospholipids to their no epithelial cell abrasion occurred (Chono et al., 2009).

surface (sterically-stabilized liposomes). An increase in their Liposomes have been investigated for the delivery of antimi-

circulation half-life has been experimented due to their capacity crobial peptides. Omri et al., demonstrated that polymyxin B (PMB)

to escape into the immune system (Lasic et al., 1999). was efﬁciently incorporated into DPPC/Chol liposomes. The in vitro

Liposomes have been described as being administered via oral activity of encapsulated PMB was enhanced in several Gram-

(Das and Chaudhury, 2011), intravenous, and pulmonary routes negative bacteria. The intra-tracheal instillation of the liposomal

(Konduri et al., 2003; Vyas et al., 2004). Moreover, liposomes have formulation was assessed against Pseudomonas aeruginosa in a rat

been intensively studied to target drugs to the skin strata (Cevc, model of lung infection. The data showed that, compared to free

2004) and the lymph (Oussoren and Storm, 2001). Several studies PMB, lung retention of the drug was 5-fold higher. Moreover, the

have reported the use of liposomes as drug carriers in the liposomal PMB signiﬁcantly reduced the pulmonary bacterial

treatment of cancer (Ahmad et al., 1993; Mayhew et al., 1987), count (Omri et al., 2002). More recently, a study conducted by He

leishmaniasis (Alving et al., 1978), metabolic disorders (Gregor- et al. (2013) examined the pharmacokinetics and efﬁcacy of

iadis et al.,1982), and fungal diseases (Lopez-Berestein et al.,1985). intravenous liposomal PMB in a pneumonia model against two P.

Liposomes have also been investigated for gene delivery and aeruginosa isolates. Liposomes were found to be able to reach the

vaccine therapy (Gregoriadis, 1998). Liposomal drug development lung thanks to macrophage uptake. Hence, the drug can achieve

has led to the commercialization of several products, Doxil and effective concentrations both in systemic and lung compartments

1 1

Myocet ; two liposome-based anticancer drugs and AmBisome , (He et al., 2013).

a systemic liposomal formulation of amphotericin B, an anti-fungal Thirawong et al. (2008) prepared self-assembled pectin-

molecule (Torchilin, 2005). The most recent liposomal drug to liposome nanocomplexes in order to improve the intestinal

receive FDA approval, Marqibo , is a liposomal formulation of absorption of calcitonin (eCT). This enhancing effect is related to

vincristine that was approved in August 2012 to treat acute the mucoadhesive properties of the pectin and the extent of the

lymphoblastic leukemia at second or greater relapse. liposomal retention by the intestinal mucosa. Jain et al. (2007)

An extensive number of studies have explored the suitability of described novel mucoadhesive and multivesicular liposomes

liposomes for the delivery of peptides and proteins. Table 2 (chitosan-MLV, Alginate-MLV) as sustained release carriers for

summarizes some examples of liposomes encapsulating peptides insulin when administered via nasal and ocular routes (Jain et al.,

and proteins. The degradation of peptides can be prevented after 2007).

their incorporation inside the liposomal bilayers. Various param- The major disadvantage of liposomes is their instability during

eters are to be considered when using liposomes for peptide storage and in biological media (Ruysschaert et al., 2004;

delivery such as the composition of the liposomes, drug loading, Sulkowski et al., 2005), which is related to their phospholipid

and release from lipid vesicles as well as the size and the surface bilayer. In fact, the occurrence of oxidation and chemical hydrolysis

charge. Unilamellar, nano-sized Vasoactive Intestinal Peptide have been described for phospholipids in aqueous liposomal

(VIP)-loaded liposomes (VLL) have been designed and suggested dispersions. For this purpose, monitoring phospholipid integrity is

as a promising treatment against various lung diseases, e.g., asthma important to characterize the liposomal dispersions after prepa-

and pulmonary hypertension (Hajos et al., 2008; Stark et al., 2007). ration. In an aqueous liposomal dispersion, the ester groups of the

The association of the VIP with phospholipids results in a phospholipids can be hydrolyzed to free fatty acids and lysophos-

conformational change of the peptide from a random coil to pholipids. The high concentration of the lysophospholipids often

Table 2

Examples of peptide/protein loaded liposomes.

Protein/peptide Method of preparation Entrapment efﬁciency (%) References

Adanantyltripeptides Dry lipid ﬁlm – (Frkanec et al., 2003)

BSA Reverse evaporation 25–71 (Dai et al., 2006, 2005)

Double emulsiﬁcation 43–71 (Murakami et al., 2006)

Freeze thawing 20–45

Calcitonin Dry lipid ﬁlm 20 (Takeuchi et al., 2005, 2003)

Enkephalin Double emulsiﬁcation 50–80 (Ye et al., 2000)

Insulin Reverse evaporation 30–32 (Zhang et al., 2005)

Freeze thawing – (Goto et al., 2006)

Leishmania Antigen Freeze extrusion – (Badiee et al., 2007)

Human gamma globulin Dehydration-rehydration 30–31 (García-Santana et al., 2006)

86 N. Matougui et al. / International Journal of Pharmaceutics 502 (2016) 80–97

leads to an increase in permeability of the lipid bilayer and a various applications. A schematic representation of the different

destabilization of the system (Zuidam et al., 1995). Phospholipids LCPs and their corresponding dispersions are presented in Fig. 3

with unsaturated fatty acids are mainly targets for modiﬁcation below.

under oxidative conditions. Lipid oxidation can change the The most extensively studied lipid for the formation of LCPs and

integrity of the bilayer. This modiﬁcation can possibly cause drug LCNPs is glyceol monooleate (GMO), also known as monoolein.

leakage and a resulting fusion and/or aggregation. GMO is a naturally-occurring lipid that is well tolerated and can be

Recently, Pantze et al. (2014) developed a new gelatin dosage used in food and pharmaceutical formulations (Kulkarni et al.,

form of liposomes. The incorporation of liposomes in a solid matrix 2011). Chemical stability might be an issue when using GMO and

bypasses the problems of stability related to the liquid drug form. naturally occurring phospholipids. These lipids can be easily

Moreover, matrix liposomes show a higher stability in the GI-tract hydrolyzed by acids, degraded by enzyme-catalyzed reactions, and

and allow more sustained release. The gelatin is present both in the are subject to oxidative degradation. Glycerol dioleate (GDO),

inner core and the outer matrix, which slows the leakage of small diglycerol monooleate (DGMO) and soy phosphatidylcholine (SPC)

hydrophilic molecules and hence confers higher protection against are other lipids investigated to produce LCPs and LCNPs (Barauskas

degradation (Pantze et al., 2014). et al., 2010; Johnsson et al., 2005; Wadsäter et al., 2015, 2014).

Phytantriol has also been widely studied in the literature for the

2.3. Liquid crystalline nanoparticles (LCNPs) formation of cubosomes (Barauskas and Landh, 2003; Fraser et al.,

2013; Rizwan et al., 2011). This lipid is more chemically stable

Liquid crystalline phases (LCP) are formed spontaneously upon compared to glyceride-based lipids. This is due to the lack of ester

hydration of certain amphiphilic molecules, e.g., polar lipids. This bonds cleavable by lipase enzymes in the gastrointestinal tract

self-assembly of molecules into structures with a long range order (Nguyen et al., 2011, 2010). The phase diagrams for phytantriol and

is driven by the phenomenon often referred to as the “hydrophobic GMO are displayed in Fig. 4 and shows the rich phase behavior of

effect” (Milak and Zimmer, 2015; Chen et al., 2014). There are three these lipids.

main types of LCP: Lamellar, hexagonal and cubic. The formed LCNPs are formed by the dispersion of the lipids or a pre-formed

mesophase can be predicted by the geometrical shape of the polar cubic or hexagonal phase in the presence of a stabilizing

head group and the hydrophobic tail of the amphiphile, using the compound. The most frequently used stabilizer is the triblock

critical packing parameter (CPP) concept. The CPP is deﬁned by the copolymer Pluronic F127 (Barauskas et al., 2010; Chong et al., 2015,

ratio of the molecular chain volume to the head group area and the 2012; Larsson and Tiberg, 2005; Muller et al., 2010a; Zhai et al.,

critical chain length. Liquid crystalline nanoparticles (LNCPs) are 2011). Other stabilizers have also been studied, such as polyethyl-

dispersions of LCPs and have been investigated extensively over ene oxide (Myrj), pegylated phytanyl-copolymers, Polysorbate 80,

the last decades, due to their functional properties and potential in colloidal clay platelets (Laptonite) and b-caseine among others

Fig. 3. Different types of liquid crystalline phases; lamellar, hexagonal and cubic and their corresponding liquid crystalline nanoparticles, (reprinted from (Mulet et al., 2013)

copyright (2012), with permission from Elsevier). There are three types of inverse, bicontinuous cubic structures: primitive, gyroid and double diamond with the

corresponding space groups Im3m, Ia3d and Pn3m, respectively. The discontinuous (discrete cubic) phase consisting of close-packed micelles is represented by the face-

centered space group Fd3m. The scale bar equals 100 nm in cryogenic electron microscopy images.

N. Matougui et al. / International Journal of Pharmaceutics 502 (2016) 80–97 87

Fig. 4. The phase diagrams of phytantriol (top) and GMO (bottom) (Reprinted from (Mulet et al., 2013) copyright (2012), with permission from Elsevier). The co-existence of

liquid crystalline gel with water is a pre-requisite in order to make dispersions of LCNPs.

(Chong et al., 2015, 2012; Larsson and Tiberg, 2005; Muller et al., loading. Mulet et al. (2010) developed a high throughput screening

2010a, 2010b; Zhai et al., 2011). Two general approaches have been protocol for the preparation and characterization of LCNPs in

established to make LCNPs: A bottom-up and a top-down microliter plates based on the bottom up approach. This opens up

approach. The latter is the most frequently used protocol and numerous possibilities for future advances with respect to drug

involves the agitation of the lipids or preformed cubic phase by delivery development.

high-shear energy dispersing techniques. Such approaches involve The incorporation and release of drugs from LCNPs have been

ultra-sonication, high shear mixing (e.g., Ultra-turrax) and high investigated for a range of compounds. These include irinotecan,

pressure homogenization. A high temperature treatment cycle has omapatrilat, indomethacin, amphotericin, cinnarizine and vitamin

been shown to narrow particle size and the distribution of K (Boyd et al., 2006b; Esposito et al., 2005; Lopes et al., 2007, 2006;

cubosome dispersions of GMO (Barauskas et al., 2005). However, Nguyen et al., 2011; Tamayo-Esquivel et al., 2006; Yang et al., 2012;

this protocol might not be feasible to use when incorporating Z. Yang et al., 2014). LCNPs can be classiﬁed as a burst release

sensitive active compounds, such as protein and peptides. system for lipophilic drugs (Boyd, 2003; Boyd et al., 2006a). In

The bottom-up approach involves the initial mixing of the lipid terms of protein and peptide delivery, LCNPs have been

with a hydrotrope, e.g., ethanol, followed by dilution with an investigated as carriers for the delivery of insulin (Chung et al.,

aqueous solution containing the stabilizer (Spicer, 2005; Spicer 2002), protein vaccines (Gordon et al., 2012; Rattanapak et al.,

et al., 2001; Swarnakar et al., 2007 Swarnakar et al., 2007). Using 2012; Rizwan et al., 2011), somatostatin (Cervin et al., 2009) and

this protocol, there is no need for high energy mixing, but cyclosporin A (Lai et al., 2010; Lopes et al., 2006). A summary is

controlling particle size and particle size and distribution can be presented in Table 3. The main rationale for encapsulating peptides

more challenging. A schematic diagram of the different prepara- into liquid crystalline nanoparticles is to protect the peptides from

tion methods is displayed in Fig. 5. Different strategies for proteolytic degradation, but it is also a means to control and trigger

incorporating active substances, such as using poorly-soluble drug release.

drugs and peptides, have been investigated. These include loading The oral administration of protein and peptide drugs is a

during the manufacturing process as well as post-development challenging area since they are prone to enzymatic degradation.

Fig. 5. The different steps in top-down (left) and bottom-up (right) methods for making LCNPs. The bottom-up preparation protocol does not involve any high shear mixing,

but the efﬁcient incorporation of an active substance can be more challenging, as is the control of particle size.

88 N. Matougui et al. / International Journal of Pharmaceutics 502 (2016) 80–97

Table 3

Studies of peptide and protein incorporation into LCNPs.

Peptide/protein Molecular Liquid crystalline nanoparticles Entrapment efﬁciency References

weight (%)

Insulin 58 kDa “Nanocubicles” of MO and Pluronic-F127 100 (Chung et al., 2002)

Ovalbumin 45 kDa Cubosomes 33–73 (Rizwan et al., 2011)

Type 1: Phytantriol, pluronic F127, propylene glycol, PEG200.

Type 2: GMO, pluronic F127, propylene glycol, PEG200.) 47–53

Ovalbumin 45 kDa Cubosomes (phytantriol, propylene glycol, P407) in chitosan 85 (Gordon et al., 2012)

hydrogels

Ovalbumin 45 kDa Cubosomes: Phytantriol, poloxamer 407, propylene glycol 19–60 (Rattanapak et al., 2012)

Somatostatin 1.6 kDa Cubosomes (phosphatidylcholine, GDO, polysorbate 80) 10–20 (Cervin et al., 2009)

Cyclosporin A 1.2 kDa Cubosomes: GMO-Pluronic F127 86–92

Hexosomes: GMO, Oleic acid, Pluronic F127 No data (Lai et al., 2010; Lopes et al., 2006)

Moreover, the absorption of large compounds is limited. Interest- To conclude, LNCPs allow the incorporation of hydrophilic and

ingly, Chung et al. (2002) incorporated insulin into GMO-based hydrophobic compounds. The nanostructured internal morpholo-

cubosome-type particles (nanocubicles). Encouraging values of gy can provide controlled and sustained release properties of the

blood glucose concentration over time in vivo were reported, active compounds. Moreover, the solubility of poorly-soluble

compared to intravenously-administrated insulin. Lai et al. (2010) compounds can be increased, and sensitive actives can be

reported on the incorporation of cyclosporine A in GMO-based protected against degradation in LCNP-systems (Rizwan et al.,

cubosomes. They showed that the relative oral bioavailability of 2010; Shah et al., 2001). It has been shown that LCNPs undergo

cyclosporine A was about 178% when formulated in cubosomes phase transitions if exposed to lipase enzymes (Wadsäter et al.,

compared to the commercially available Neoral self-micro- 2014). Such transitions can destabilize the particles and limit

emulsifying concentrate. LCNPs have also been investigated for administration possibilities. Also, in-vitro cytotoxicity and hemo-

the topical administration of peptides. Lopes et al. (2006) have lytic activity has been reported for LCNP-systems (Barauskas et al.,

investigated the in vivo and in vitro skin penetration of cyclosporine 2010; Hinton et al., 2014; Murgia et al., 2010). Due to these ﬁndings,

A. They reported a 2-fold increase in skin penetration from in vitro the intravenous administration of LCNPs seems to be challenging.

studies. The skin concentration of the peptide was 1.5–2.8 higher in These factors need to be taken into account in the process of

vivo compared to peptides formulated in an oil formulation. No formulating new drug delivery systems based on LCNP.

skin irritation of the LCNPs was observed in this study.

The release of ovalbumin from cubosomes was investigated by 2.4. Solid lipid nanoparticles (SLNs)

(Rizwan et al., 2011). It was demonstrated that the release was

more sustained from formulations produced via the bottom-up Solid Lipid Nanoparticles (SLNs) are submicron colloidal

method, compared to cubosomes prepared by the top-down carriers (50–1000 nm) composed of lipids (0.1–30% w/w), which

approach. A more recent study concerning the in vivo immuno- are in a solid state at both room and body temperatures (Lucks and

logical response of cubosomes concluded that these particles have Muller, 1993). They are classically dispersed in aqueous media.

a great potential as sustained release vaccine delivery systems Depending on the type and the concentration of the lipid,

(Rizwan et al., 2013). In addition, a comparative study on the surfactant (0.5–5%), e.g., poloxamer 188, lecithin, polysorbate 80,

incorporation of ovalbumin into liposomes and cubosomes, phosphatidylcholine can be added in order to stabilize the lipidic

showed an almost 3-fold higher entrapment efﬁciency in core. The use of solid lipids instead of oils is an interesting

cubosomes compared to liposomes (Gordon et al., 2012). approach to achieve controlled drug release, because drug

Rattanapak et al. (2012) showed that topical delivery of ovalbumin diffusion in a solid lipid should be considerably lower compared

was increased from cubosomes compared to liposomes. to an oily phase. The lipids used may be triglycerides (trimeristyl,

In the majority of studies, peptides and proteins have been tristearin), mono-glycerides (glyceryl monostearate), fatty acids

incorporated during the preparation of the LNCPs. It is also possible (stearic acid, palmitic acid), steroids (cholesterol) or waxes (cetyl

to add peptides after the formation of the LCNPs and let adsorb to palmitate) (Mehnert, 2001).

the particles. Somatostatin was shown to spontaneously adsorb Different methods can be used to prepare SLNs (Table 4). High

onto LCNPs by Cervin et al. (2009). In vivo studies have shown that pressure homogenization (HPH) is commonly regarded as a

the half-live time of somatostatin increased when formulated reliable and powerful technique for the preparation of SLNs

together with LCNPs. (Mehnert, 2001). Nevertheless, some drugs may be fragile to high

Table 4

Different methods of SLNs preparation: Application to peptides.

Method Protein/peptide References

High shear homogenization Cyclosporin A (Almeida et al., 1997; Kim et al., 2009)

Hot homogenization Lysozyme

Cold homogenization

Solvent emulsiﬁcation/evaporation Thymocartin Insulin (Reithmeier et al., 2001)

SLN preparation using supercritical ﬂuids Insulin (Salmaso et al., 2009a)

Recombinant human growth hormone (rh-GH)

Microemulsion-based SLNs preparation Cyclosporin A (Gasco, 1993; Ugazio et al., 2002)

Double emulsion method Insulin (Gallarate et al., 2009; Garcia-Fuentes et al., 2003)

Reverse micelle double emulsion Insulin calcitonin (C. Chen et al., 2013; Liu et al., 2007a)

Coacervation technique Insulin leuprolide (Gallarate et al., 2011)

Solvent emulsiﬁcation-diffusion method Gonadorelin (Hu et al., 2004)

N. Matougui et al. / International Journal of Pharmaceutics 502 (2016) 80–97 89

shear stress and can be affected during the formulation process The drug-enriched core model occurs when the recrystalliza-

(Pérez et al., 2002). Microemulsion is another process to produce tion mechanism is the opposite to that described for the drug-

SLNs. However, the use of high temperatures (60–70 C) can also enriched shell model. The drug is solubilized in the lipid melt close

lead to the degradation of labile drugs. In addition, it is necessary to to its saturation solubility. The cooling decreases the solubility of

use high concentrations of surfactants and co-surfactants in the the active ingredient in the melt and leads to drug crystallization

related formulations. This can be undesirable with respect to prior to the lipid. Complete cooling leads to lipid crystallization

regulatory guidelines and applications. The double emulsiﬁcation that forms a shell around the drug-enriched core. This model is

(W/O/W) method represents a relatively simple and efﬁcient way associated with membrane-controlled release governed by diffu-

to prepare SLNs loaded with labile and hydrophilic drugs (Gallarate sion (Fick’s law) (Müller et al., 2002a).

et al., 2009). In this case, the drug and stabilizer are encapsulated in Many factors can affect the drug loading efﬁciency of SLNs: The

the inner aqueous phase of the W/O/W double emulsion. A drug solubility in the lipid melt, the miscibility of the melted drug

stabilizer is necessary to prevent drug partitioning to the outer with the lipidic environment, the chemical and physical structure

aqueous phase during solvent evaporation. This type of formula- of the solid matrix lipid, and the polymorphic state of the lipidic

tion is usually called “liposphere” due to its relatively larger materials. Obviously, water-soluble drugs are characterized by

particle size compared to SLNs. For example, Liu et al. (2007a), lower entrapment efﬁciency compared to more lipophilic drugs.

(2007b) used the reverse micelle-double emulsion strategy to However some research conﬁrms that under optimized conditions

enhance the drug loading efﬁciency of insulin as well to control its SLNs can be produced to incorporate hydrophilic peptides

sustained release while avoiding a signiﬁcant “burst effect”. They (Almeida and Souto, 2007).

obtained particles with a small size (around 110 nm) composed of Since the mid 1990s, interesting results concerning the

Sodium Cholate (SC) and Soybean Phosphatidylcholine (SPC) incorporation of several peptides and proteins in SLNs have been

mixed micelles, with high entrapment efﬁciency (98%), good published (Table 5). Peptides (e.g., calcitonin, cyclosporin A,

physical stability, and good sustained drug release (Liu et al., insulin, and thymopentin), protein antigens (e.g., hepatitis B,

2007a). malaria antigens) and model protein drugs (e.g., bovine serum

Muller, Mäder and Mehnert (Mäder and Mehnert, 2001; Müller albumin and lysozyme) have been investigated to enhance drug

et al., 2000) describe three different models for the location of a release kinetics and protein stability.

drug within SLNs: (i) the homogeneous matrix model, (ii) the drug- The method of SLN production plays a pivotal role to achieve

enriched shell model, and (iii) the drug-enriched core model successful drug loading. Emulsion techniques are more suitable for

(Fig. 6). The differences among these models are mainly due to the peptides (Hu et al., 2004). However, one limitation to these

formulation composition, i.e., the chemical nature of the active techniques is the use of toxic organic solvents. As a result, the use

ingredient, lipid and surfactant, as well as the process parameters of super critical ﬂuids has been proposed as an alternative, but

(Souto et al., 2005). small amounts of solvent are sometimes still necessary to dissolve

In the case of the matrix model, the drug is homogeneously the peptide (Salmaso et al., 2009a).

dispersed within the lipid matrix. It occurs for particles produced Garcia-Fuentes et al. (2003) were among the ﬁrst to study SLNs

by the cold homogenization technique without the use of a drug- for the delivery of hydrophilic peptides. Insulin stability and its

solubilizing-surfactant, or when lipophilic drugs are incorporated release from PEGylated SLNs made of tripalmitin and lecithin have

into SLNs produced by the hot HPH technique. Such models are been investigated. A burst release of insulin within the ﬁrst few

suitable for the incorporation of drugs that can show sustained hours indicated the localization of the peptide in the surface layers.

release. Nevertheless, drug stability appears to be compromised (Garcia-

For the drug-enriched shell model, a lipid core is surrounded by Fuentes et al., 2003). Another study conducted by Gallarate et al.

a drug corona. This model can be explained by a lipid-precipitation (2011) investigated the challenging incorporation of insulin and

mechanism that occurs during the cooling stage. A solid lipid core another model peptide, leuprolide, into SLNs. In fact, one limitation

forms when the recrystallization temperature of the lipid is of peptide incorporation is drug solubility in the lipid matrix.

reached with a concomitant increase in drug concentration in the Hydrophobic ion pairs of peptides are found to enhance peptide

liquid outer shell of the SLN. Finally, complete cooling leads to the lipophilicity. A drug-surfactant complex of peptides is formed

crystallization of the compound-enriched shell (Muchow et al., thanks to electrostatic interactions of anionic amphiphilic

2008). This model is best used for drugs that release very fast from surfactants and cationic peptides. After formation, neither peptide

SLNs and is highly desirable for dermatological application (Souto integrity nor the secondary structure were affected. Moreover, in

et al., 2004). vitro release studies demonstrated a more sustained release of

Fig. 6. Models of drug incorporation into SLNs.

90 N. Matougui et al. / International Journal of Pharmaceutics 502 (2016) 80–97

Table 5

Examples of peptide-loaded SLNs.

Peptide/Protein Lipophilicity Molecular weight Entrapment efﬁciency Route of administration References

BSA Lipophilic 66 kDa n.a Intravenous (Gualbert et al., 2003)

Salmon calcitonin Hydrophilic 3.2 kDa >90% Oral (Garcia-Fuentes et al., 2005a)

CyA Lipophilic 1.2 kDa 95.4–97.8% (hot dispersion) Topical (Penkler et al., 2003)

78.5–93.9% (cold dispersion)

96.6–97.8% Peroral (Olbrich, 2000)

90.8% Topical (Kim et al., 2009)

96.1% Oral (Müller et al., 2006)

88.4% Oral (Zhang et al., 2000)

Insulin Hydrophilic 58 kDa 37.8% Oral (Zhang et al., 2006)

67.9% Oral (Garcia-Fuentes et al., 2003)

26.8% Oral (Garcia-Fuentes et al., 2003)

75% (Salmaso et al., 2009b)

rhEGF Hydrophilic 6.4 kDa 73.9% Topical (Gainza et al., 2014)

BSA, bovine serum albumin; CyA, cyclosporin A; rhEGF, recombinant human epidermal growth factor; n.a, not available; PGSS, particles from gas saturated solution

technique.

leuprolide from SLNs, which indicated the efﬁcient entrapment of are based on a variety of lipid materials. Most of them are approved

peptide complexes within the lipid matrix (Gallarate et al., 2011). as GRAS and are physiologically well-tolerated (Blasi et al., 2013),

More recently, a study has explored the potential of the topical (ii) They have comparatively higher drug encapsulation efﬁciency,

administration of SLN-loaded recombinant human epidermal (iii) They can encapsulate both lipophilic and hydrophilic drugs (iv)

growth factor (rhEGF) to treat chronic wounds. The encapsulated They Improve drug stability in their lipid matrix and provide a

growth factor demonstrated enhanced in vitro and in vivo controlled and/or targeted drug release, (v) Their production can

bioactivity. SLN-rhEGF promotes cell proliferation on the con- be scaled up with good reproducibility, (vi) Their surface can be

cerned cell lines. The enhanced bioactivity is in part attributed to coated with hydrophilic polymers or surfactants, such as PEG to

sustained release, better cellular uptake, and higher activation of improve their bioavailability and/or increase their loading

cell proliferation compared to free rhEGF. The ability to release efﬁciency. However, some disadvantages have been noted such

active molecules in a controlled manner allowed better cell as a poor drug loading capacity due to the formation of a perfect

management of the growth factor as a result of the saturation of lipid crystal matrix (Kayser et al., 2004). Moreover, drug expulsion

the speciﬁc receptors. In vivo, SLNs were found to be well tolerated after polymeric transition during storage has been reported. One

with good occlusive properties that increased skin hydration. The can also note relatively high water contents in the related

intralesional administration of SLN-rhEGF in a full-thickness dispersions (70–99.9%). A major hindrance of SLNs is a low

wound model resulted in a signiﬁcant improvement of healing capacity to load hydrophilic drugs due to partition in the aqueous

in terms of wound closure compared to free rhEGF; this is phase during the preparation process (Cortesi et al., 2002). The

consistent with in vitro data (Gainza et al., 2014). drug loading of hydrophilic drugs can be enhanced by the

SLNs are often composed of physiological lipids. One can ﬁnd formation of an imperfect crystal (a combination of incompatible

different distribution and metabolism pathways available in the solid lipids) which limits drug expulsion or by forming a drug–

body, which can contribute signiﬁcantly to the in vivo fate of the surfactant complex (Gallarate et al., 2011).

carrier. The most important enzymes involved in SLNs degradation

are lipases present in various organs and tissues. An in vitro 2.5. Nanostructured lipid carriers (NLCs)

experiment indicated that solid lipid nanoparticles showed

different degradation velocities induced by the pancreatic lipase. Nanostructured lipid carriers (NLCs) are considered as an

The degradation was found to be dependent on their composition alternative to liposomes and nanoemulsions, due to various

(nature of the lipid matrix, stabilizing surfactant) (Müller et al., advantages, such as the ease of preparation, high drug loading

1996). capacity, and sustained drug release properties (Kayser et al.,

Depending on their application, solid lipid nanoparticles may 2004). NLCs represent a second generation of nanoparticles based

represent an alternative carrier system to other colloidal carriers on solid lipids and were developed to overcome potential issues

such as emulsions, liposomes and polymeric micro and nano- with SLNs (Mukherjee et al., 2009; Müller et al., 2002b). Compared

particles. One can emphasize the following advantages: (i) They to SLNs, NLCs are modiﬁed after the incorporation of liquid lipids

Fig. 7. Different types of NLC: I-the imperfect type; II-the amorphous type; III-multiple O/F/W type (Selvamuthukumar and Velmurugan, 2012).

N. Matougui et al. / International Journal of Pharmaceutics 502 (2016) 80–97 91

ﬁ

into a solid structure. This special structure enables better drug Recent studies have presented interesting ndings on the

ﬂ

accommodation, increasing the loading capacity for numerous in uence of oil-based carriers. One study shows that the amount

active compounds. Another advantage consists in lower water and the type of the carrier oil govern NLCs stability. As the oil

content in the ﬁnal particle suspension. Consequently, this concentration was increased, the crystallization and melting

structure minimizes possible drug release during storage (Müller temperatures decreased, the polymorphic transformation rate

et al., 2002b). Three types of NLC have been described (Fig. 7): increased, the particles became more spherical, and suspension

stability was improved. Moreover, less surfactant was necessary to

(i) The imperfect type consists of small amounts of chemically produce stable NLC suspensions than was required to stabilize SLN

different liquid lipids (oils) mixed with solid lipids. This suspensions without a carrier oil (Y. Yang et al., 2014; Z. Yang et al.,

incompatibility leads to imperfections in the crystal order 2014).

inside the lipid core allowing higher drug loading (Müller The methods used to produce NLCs for the delivery of peptides

et al., 2002b). and proteins are W/O/W double emulsion and the hot HPH

(ii) The amorphous type: The particles are solid but in an technique. Examples are calcitonin and cyclosporin A, that were

amorphous state. Crystallization upon cooling is avoided by associated to NLCs for oral delivery. NLCs-loaded cyclosporin A

the addition of lipids with a special structure (b forms) (e.g., have been produced via the hot HPH method (Müller et al., 2002a).

hydroxyl-octacosanyl-hydroxystearate and isopropyl-myris- NLCs intended for the delivery of calcitonin have been produced by

tate) (Jenning et al., 2000a,b; Kayser et al., 2004). Therefore, the W/O/W double emulsion technique, yielding an association

ﬁ

the consequent drug expulsion during storage is prevented ef ciency higher than 90% (Garcia-Fuentes et al., 2005a, 2005b).

ﬁ

(Radtke and Müller, 2001). This association is mainly attributed to the high af nity of the

(iii) The multiple types also referred to multiple Oil in Fat in Water cationic sCT to the negatively-charged lipids which make up the

(O/F/W) carriers. The solubility and dispersion of many core of the nanoparticles, and to the phospholipidic surfactant

lipophilic drugs in a liquid lipid is higher than in a solid lipid. (lecithin) present at the surface. In this way, the sCT is both

Therefore, an excess amount of oil is mixed with the solid lipid. adsorbed at the surface and incorporated in the lipidic core. In vitro

Above the solubility, a phase separation occurrs with the release studies showed a burst release, corresponding to the

formation of oily nano-vesicles within the solid lipid matrix surface adsorbed peptide followed by a continuous and slow

(Jenning et al., 2000a,b). Thus, the drug is dissolved in the oil release attributed to the peptide located in the inner part of the

and is protected by the surrounding solid lipids. nanoparticles. Nevertheless, the NLCs showed no enhanced

properties compared to SLNs (Garcia-Fuentes et al., 2005b). Gainza

Fig. 8. Preparation of LNCs by the Phase Inversion Temperature process.

92 N. Matougui et al. / International Journal of Pharmaceutics 502 (2016) 80–97

et al. (2014) compared NLC-rhEGF and SLN-rhEGF. The data reduced to allow encapsulation of labile drugs, such as DNA lipidic

demonstrated no differences between the two formulations in complex or lipophilic peptide complex, with an increased amount

terms of in vitro and in vivo activity. However, the formulation of salt and the addition of polyglyceryl-6 dioleate (oleic Plurol )

parameters (preparation process and encapsulation efﬁciency) instead of Lipoid S75-3 (Morille et al., 2010; Vonarbourg et al.,

make NLC-rhEGF a better alternative (Gainza et al., 2014). 2009).

In order to improve NLCs efﬁciency, the surface modiﬁcation of Due to the fact that LNCs surface is composed of PEG-based

NLCs has been envisaged. For example, in the ﬁeld of ocular nonionic surfactants, these LNCs have stealth properties to avoid

delivery, thiolated NLCs (Cys-NLCs) of cyclosporine A were detection by the complement system, and uptake by the

successfully developed. The cysteine-polyethylene glycol stearate mononuclear phagocyte system (Vonarbourg et al., 2006). They

coating has shown to affect the afﬁnity and the residence time of can also be grafted with ligands to achieve active targeting (e.g.,

NLC in the ocular mucosa. In fact, the topical ocular administration tumor targeting) (Hirsjärvi et al., 2014). The attachment of

to rabbits, resulted in a better accumulation of the drug in the polysaccharides at the surface of the nanoparticles allows the

ocular tissues compared to non-thiolated nanoparticles due to the modiﬁcation of the physicochemical and biological properties of

mucoadhesive property of cysteine (Shen et al., 2010). the carrier. For example, one can improve the biodistribution

In brief, NLCs have been developed in order to overcome the proﬁle (Morille et al., 2010) and avoid non-speciﬁc interactions, or

limitations of SLNs. In most cases, the encapsulation efﬁciency was create interesting templates for further attachment of targeting

improved due to the increased solubility of the drug. Besides, the ligands (Béduneau et al., 2008). Recently, the surface of LNCs was

imperfect core formed after crystallization limits drug release from modiﬁed by the post-insertion of amphiphilic lipochitosan (LC) or

the carrier. Concerning in vitro and in vivo studies, the comparison lipodextran (LD) (Hirsjärvi et al., 2013).

between SLNs and NLCs showed, in many cases, no differences in Many lipophilic and amphiphilic drugs, have been encapsulated

drug efﬁcacy. Following these encouraging results, a large number into LNCs (e.g., ibuprofen) (Lamprecht et al., 2004), various

of studies are ongoing to explore, more extensively, the potential of hydrophobic anti-cancer agents (e.g., paclitaxel, etoposide) (Groo

NLCs as peptide carriers. et al., 2013; Saliou et al., 2013), and a camptothecin derivative, 7-

Ethyl-10-hydroxy-camptothecin (Sn38) (Roger et al., 2011). LNCs

2.6. Lipid nanocapsules (LNCs) have also been reported as efﬁcient gene carriers for cancer

therapy. DNA was associated with cationic lipids, i.e., DOTAP/DOPE

Lipid nanocapsules (LNCs), with sizes between 20 and 100 nm, to form lipoplexes, which has been encapsulated into LNCs

are biomimetic, synthetic nanocarriers used in drug delivery and (Vonarbourg et al., 2009). Unfortunately, their lipidic core is not

for imaging purposes (Huynh et al., 2009). The structure of LNCs is adequate as they stand to encapsulate hydrophilic molecules. To

a hybrid between polymeric nanoparticles and liposomes because improve this efﬁciency, some strategies have been described such

they contain an oily core, composed of medium-chain triglycer- as the incorporation of charged surfactants to the formulation of

ides, surrounded by a surfactant shell made of lecithin and LNCs and the post insertion of additional linkers. These strategies

PEGylated surfactants. The oily phase is composed of triglycerides promote the establishment of ionic interactions with charged

of capric and caprylic acids (Labrafac WL 1349). The hydrophilic drugs at the surface of the nanoparticles (Ramadan et al., 2011).

surfactant, Kolliphor HS 15, is a PEG derivative and is a mixture of The surface potential of the LNCs to be modiﬁed leads to a number

free PEG 660 and PEG 660 hydroxystearate. The aqueous phase of strategies to incorporate drugs onto the surface. The post-

consists of deionized water containing sodium chloride. Another insertion of charged polysaccharides allows the adsoption of

surfactant, Lipoid S75-3 composed of 70% of phosphatidylcholine charged drugs to the surface of the LNCs based on electrostatic and/

soya bean lecithin, is also added to signiﬁcantly increase LNCs or hydrophobic interactions. Chitosan-transacylated LNCs were

stability. The choice of the oily phase and the surfactant can be developed to adsorb negatively-charged siRNA (Messaoudi et al.,

modulated according to the properties of the encapsulated drug 2014). Other linkers have also been investigated, such as the

(Hureaux et al., 2009; Morille et al., 2010; Roger et al., 2009). All incorporation of DSPE-PEG2000-maleimide into the LNCs shell for

components are FDA approved for oral, topical and parenteral the conjugation of L1-peptide, a biomimetic peptide (Weyland

administration. et al., 2013).

The preparation of LNCs involves a phase inversion Another alternative, is to load the lipophilic core of the particles

temperature (PIT) process, which is a solvent free and low with reverse micelles that are more suitable for the encapsulation

energy procedure (Fig. 8) (Heurtault et al., 2002). Initially, the of hydrophilic drugs (Vrignaud et al., 2012, 2011). The hydrophilic

components are mixed together; the emulsion is then heated molecules are encapsulated in the oily core through their

and cooled several times between 60 C and 90 C to obtain solubilization in reverse micelles. After encapsulation, the system

reversible emulsion phase inversions. Higher temperatures lead remains stable and the encapsulation efﬁciency can reach up to

to W/0 emulsions following the dehydration of the polar 90% (Anton et al., 2010). This nanotechnology was successfully

surfactant heads while lower temperatures lead to classical O/ applied to the encapsulation of doxorubicin hydrochloride and

W emulsions. After several temperature cycles, rapid dilution erlotinib hydrochloride, two hydrophilic anti-cancer drugs

with cold water is usually performed at temperature corre- (Vrignaud et al., 2012, 2011).

sponding to the phase inversion zone, to elaborate and ﬁx the The development of Aqueous-Core Lipid Nanocapsules provides

ﬁnal LNC suspension. A simple rapid cooling (without dilution) an additional tool to encapsulate hydrophilic drugs such as

can also be successful. It has been demonstrated that these last peptides (Anton et al., 2009). The method of preparation is closely

formulation steps correspond to an instantaneous and irrevers- similar to the lipid core nanocapsules. The ﬁrst step consists of the

ible dispersion of the bicontinuous system which characterizes formulation of a W/O nanoemulsion. This system contains water

the Phase Inversion Zone. The obtained particles demonstrate a and an additional oil with a low HLB surfactant. It may be prepared

good level of stability (>18 months) under drastic in vitro by one of the conventional methods known in the art. However, the

conditions (change of pH, temperature, dilution and stirring) low-energy PIT method is preferred, since it may prevent the

(Heurtault et al., 2002). degradation of fragile molecules during encapsulation. The PIT

The size of the particles mostly depends on the non-ionic method, previously described, is modiﬁed in the ﬁnal step. The

surfactant concentration and on the amount of oil ﬁxed at the transitional bicontinuous system is instead diluted with a volatile

beginning of the process. Moreover, the temperature can be oil (e.g., pentane, isopentane), resulting in the formation of a W/O

N. Matougui et al. / International Journal of Pharmaceutics 502 (2016) 80–97 93

nanoemulsion. Finally, aqueous core nanocapsules are generated Acknowledgements

by adding an additional water phase (external) and simultaneously

removing the volatile oil through evaporation. We would like to thank the European Union’s Seventh

The incorporation of hydrophilic molecules can be performed in Framework Programme for research, technological development

different ways: and demonstration under grant agreement 604182.

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