NLCs)

CHAPTER

ROLE OF EXCIPIENTS IN FORMULATION DEVELOPMENT AND BIOCOMPATIBILITY OF LIPID NANOPARTICLES (SLNs/NLCs)

30

Slavomira Doktorovova*, Ranjita Shegokar**, Eliana B. Souto* *University of Coimbra, Coimbra, Portugal **Freie Universität Berlin, Berlin, Germany

CHAPTER OUTLINE 1 Introduction....................................................................................................................................811 2 Role of Excipients in SLNs and NLCs................................................................................................813 2.1 Currently Used Excipients in SLNs and NLCs..............................................................814 2.2 Surface Modification Agent and Ligands.....................................................................825 2.3 New Comers in Excipient Market................................................................................834 2.4 Liquid Lipids............................................................................................................835 3 Conclusions....................................................................................................................................836 References............................................................................................................................................837

1 INTRODUCTION There is an increasing interest for lipid nanostructures in the development of new drug-delivery systems. Their versatility relies on the improved physicochemical properties of lipids and surfactants to develop systems for several administration routes with optimized drug loading and release properties, longer shelf life and lower cytotoxicity. The extent and duration of drug action depends on how much of the drug reaches the target site and how long it stays there. The concentration time relationship at the site of action is a function of the amount and rate of dose delivered to the absorption site and the drug’s pharmacokinetics. To achieve and keep the therapeutic levels, lipid nanostructures have been proposed as these can be tailored to deliver the drug at controlled rates for extend periods of time, which is pharmaceutically relevant for short acting drugs, with shorter plasma half-lives and narrower therapeutic ranges. Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) are in the solid state (either amorphous or crystalline), and can have a diameter between 50 nm and 1 µm, whereby the size depends mainly on the production method and on the composition of lipid matrix (i.e., lipid and surfactant). Nanostructures for Novel Therapy Copyright © 2017 Elsevier Inc. All rights reserved.

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CHAPTER 30  ROLE OF EXCIPIENTS

Generally, the mean particle size ranges between 150 and 300 nm (Keck et al., 2014), which influences the colloidal stability, the drug loading, the pharmacokinetics and pharmacodynamics. SLNs and NLCs are produced by variety of methods or combinations of them are as follows (Muller et al., 2011): 1. microemulsion and double emulsion technique method 2. high pressure homogenization (HPH) 3. solvent evaporation or diffusion method 4. solvent injection method 5. microfluidization 6. sonication or ultrasonication 7. membrane contactor technique 8. supercritical fluid technology Depending upon the method, the use and amount of excipient varies, for example, in case of microemulsion method, the amount of lipid excipient used in composition is less compared to the HPH method. Another, for example is the use of solvent in method 3 and 4, which is not used in other methods. Fig. 30.1 lists main components and steps of the methods used. For the production of stable dispersions, it is mandatory to have the adequate qualitative and quantitative composition of excipient for a particular production method. Although the chosen excipient may in certain cases be optimized, the production under excessive energy during particle disruption may result in an instable product. Researchers can take advantage of a design of experiment (DoE) approach and mathematical calculations to determine the right excipient concentration, drug–excipient ratio.

FIGURE 30.1  Schematic Representation of the Types of Commonly Used SLNs/NLCs Production Methods, Process Steps, and Excipient Type Similar processes are applicable for NLC production; the only difference is Phase 1 contains mixture of liquid lipid and solid lipid along with surfactant.

2 ROLE OF EXCIPIENTS IN SLNs AND NLCs

813

For reproducible in vivo results and for stable dispersion, monodispersed populations are required, which can be obtained by selecting the appropriate excipient concentration especially lipid, surfactant, and their ratio followed by parameter settings of production method. Briefly, SLNs typically consist of three essential components, that is, solid lipid, surfactant, and water. These lipid particles are composed only of fats/lipids, which are solid at body and room temperature, while in NLCs a certain percentage of solid lipid is replaced by a liquid lipid (i.e., oil) but keeping the matrix also solid. SLNs and NLCs are able to absorb and/or encapsulate hydrophilic/lipophilic drugs, thus protecting them from chemical and enzymatic degradation. Furthermore, the encapsulated drug may be prevented from crystallization, thus, forming a solid solution. As a foreign material, the drug incorporation in the crystal lattice of the nanoparticle carrier material can happen to a limited extent, mainly due to its limited solubility in the stable crystal form. For that reason, the loading capacity of SLNs and NLCs is lower than that of an equally concentrated nanoemulsion (Bunjes et al., 1996; Westesen et al., 1997). The main focus of this chapter is to discuss the role of major excipients, namely, lipids, oils, and surfactants in the formulation of stable lipid nanoparticles. The significance of other complimentary excipients is also addressed. Furthermore, an overview of commonly used and upcoming promising excipients is given. The biocompatibility and toxicity of lipid nanoparticles is reviewed at the end of the chapter.

2  ROLE OF EXCIPIENTS IN SLNs AND NLCs The biological performance of lipid nanoparticle is greatly influenced by their overall composition and nature of excipients used. Lipid nanoparticles, namely, SLNs and NLCs, employ several types of excipients, for example, solid lipids, liquid lipids (oils), surfactants, and surface modifying agents. Compatibility of these excipients with each other and with the drug is of high importance. The main steps (from commonly used methods) for the selection of an excipient in the production of SLNs and NLCs are listed later: 1. solid lipid selection 2. liquid lipid selection 3. solid-liquid lipid compatibilities 4. solvent selection (as per production method used) 5. surfactants (anionic, cationic, and nonionic) 6. surface modifiers/ligand addition 7. auxiliary excipients—antioxidants, preservatives, tonicity adjuster 8. excipients for down scaling—production of dry mass—either by lyophilization (cryoprotectants) or spray drying (adsorbents) Successful formulation of stable lipid nanoparticle depends on multiple factors. It starts from the selection of excipient, process optimization to selection of packaging. Selection of the most appropriate excipient further depends upon the several factors, starting with the nature of drug to be incorporated, its melting point, solubility determination of drug in oil/lipid, and drug recrystallization behavior are major factors. Based on the drug properties, excipients selection (mainly solid lipid) needs to be done. Route of drug administration, like, oral, dermal, and intravenous makes further differentiate excipient selection. For parenteral formulation, excipients used must be safe and have a FDA recognized GRAS (generally regarded as safe) status while dermal lipid nanoformulations can employ excipients from cosmetic market.

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CHAPTER 30  ROLE OF EXCIPIENTS

Lipid nanoparticles are more physiological and biocompatible compared to polymeric carriers. This makes lipid selection more critical as it comprises major part of excipients used. Many properties of lipid like polarity, mucoadhesiveness, melting point, degradation behavior by gut enzymes, recrystallization (precipitation) behavior, bulk properties, length of composite lipids, compatibility with other solid lipids (in case of combination used), degree lipophilicity, temperature sensitivity, contact angle with water/buffer are critical factors in production of stable dispersion. GRAS approved lipid from different categories is used in production of lipid nanoparticles. These include fatty acids, glycerides, and waxes. There is no generalized rule on use of specific lipid or oil with specific drug. As discussed previously, compatibility, polymorphism, and miscibility with drug are key parameters. Several cationic lipids are being employed for gene drug delivery due to enhanced transfection efficiencies. Branched cationic lipids are most common due to lower cytotoxicity potential than single branched (refer lipid section, 2.1.1). Chemical nature of lipid (modified chemical structure), miscibility of lipid with other used solid or liquid lipids offer further advantages. In general, polar oils due to the presence of hydroxyl groups further offer miscibility with other lipids and can improve miscibility thereby increasing thermodynamic stability. Miscibility can be an essential component for the formulation stability of SLN. Wendt et al. (2013) discussed the efforts by USP/NF in modernization of fixed oil excipient monograph. Understanding the complex nature of such excipient, USP/NF planned to make available reference material for such excipients. US pharmacopeia’s excipient expert committee is evaluating oil excipient monographs which are registered as “oil” (vegetable oil, petrochemical oil, and essential oils). Currently, 31 monographs are under evaluation. Fixed oils (24 out of 31) included in USP/NF are mainly refined oils composed mainly of triglycerides and their derivatives. The main aim of modernizing these monographs is to control adulteration or contaminations by other low purity oils (Wendt et al., 2013). This type of adulteration can affect hydrolysis, oxidation, and degradation behavior of oil, as well as from lipid formulation. NLCs, which are based on oil part, are affected by this type of adulteration and can results in unstable product, causing expulsion of encapsulated drug thereby resulting in agglomeration or settling of formulation. Surfactants play an important role in the prevention of agglomeration, and maintaining the dispersitivity. They can be selected based on compatibility and stability profile with particular lipid–oil matrix. In case of solvent emulsification methods, type and amount of solvent (type I, II, II) play an important role. Commonly used solvent is ethanol and other cosolvents like polyethylene glycol, glycerol, and propylene glycol are used in formulation of lipid nanoparticles. Additives, such as antioxidants are incorporated into SLN to reduce oxidation. The commonly used antioxidants are propyl gallate, α-tocopherol, and butylated hydroxy toluene. Based on the way of addition of antioxidants (during production or after production) can affect the stability of SLNs/NLCs. Besides that, if SLNs/NLCs dispersions are being incorporated into end dosage form like cream or gel a compatibility profile needs be checked. The role of excipients in final dosage form and its effect on SLNs/NLCs performance is out of the scope of this chapter. Toxicity potential based on the route of administration is equally determining factor in selection of lipid. In general, cationic surfactants have high toxicity than anionic and nonionic surfactants, while amphoteric surfactants have lowest toxicity.

2.1  CURRENTLY USED EXCIPIENTS IN SLNs AND NLCs By definition, SLNs are composed from a solid lipid and stabilizing surfactant layer. NLC is composed of a lipid matrix formed by a lipid blend—most frequently mixture of oil and a solid lipid or eventually two-lipid matrix forming lipids. Solid lipid is meant to be a lipid that is in solid state at room, as

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well as at body temperature (i.e., has melting point > 37°C). There is evidence of melting point (Tm) decrease during SLNs/NLCs production, well described by Thompson–Gibbs equation—the decrease in individual particle diameter leads to depression of melting point. The higher decrease in particle size, greater Tm drop occurs. The practical consequence of this phenomenon is that many solid lipids traditionally used for SLN/NLC formulation do not form solid matrix at the end of SLNs/NLCs production. One of the main challenge associated to SLNs and NLCs, and their physicochemical stability during shelf life, is the polymorphic transformation of the lipid matrix. It is the ability of the matrix to crystallize in more than one distinct crystalline species with different internal lattice structures. Lipid crystallization is an important point for the performance (both in vitro and in vivo) of the lipid nanoparticles. The internal structure of the lipid matrix changes in variety of ways, as lipids can crystallize in different threedimensional structures. Crystallization of bulk triglycerides from the melt after rapid cooling usually occurs in the less stable α modification, which transforms via the β′, into the more stable β modification upon heating or during storage time. The α-form (hexagonal) is the least stable with a lower melting point and latent heat, whereas the β-form (triclinic) is the most stable with higher melting point and higher latent heat. The transformation of α to β′ (orthorhombic) and β is irreversible and occurs toward a more hydrodynamic stable system. In SLNs and NLCs, these transformations are faster than in the bulk material, which lead to a change in the relative fraction of the polymorphic forms. Depending on the chemical nature of the lipid excipient and on the production process parameters, different fractions of α and β′ modifications may occur. This phenomenon can lead to a reduction in the melting point, or more precisely, to changes in the form and shift of the melting peak. These created polymorphic forms are not long-term stable, leading to a gradual transformation to more stable modifications, which means increasing content of β′/βi and finally β. This is not desired because the change in lipid structure is responsible for drug expulsion during storage and changes in the release profile of incorporated drug, as well as changes in particle size parameters. There are many possibilities for loading drugs in lipid matrices. In general, when a crystal is formed, foreign molecules can be incorporated by replacing host molecules in the lattice by a guest molecule or by being incorporated in between host molecules. Drugs may also be localized in between the lipid lamellae, resulting in increase of the lattice spacing “d” that can be analyzed by X-ray diffraction studies. The drug can also be present in form of amorphous clusters, mainly localized in the imperfections of the crystal. In this case, drug accommodation is improved when the lipid crystal has more imperfections. Thus, drug loading can be increased by using crude lipid mixtures or by controlled nanostructuring of the lipid matrix, by creating as many imperfections as possible. Lipids employed for the production of nanoparticles include triglycerides and their mixtures, fatty acids, and waxes. The selection of the lipids for the production of the solid matrix is carried out by a solubility screening test based on the solubility of drug to give a visually clear solution in lipid melt under light against naked eye. The drug and varying quantities of selected lipid in glass vials are heated above the melting point of lipid in controlled temperature water bath. After melting the lipid in vials the solubility of drug is visually checked in the melt. The solubility of the drug in the lipid melt is determinant for higher loading capacity and higher encapsulation efficiency, that is, higher lipid solubility of the drug will result in higher encapsulation parameters. Depending on the variety of factors, a fat may exist in one crystalline form or it may be a mixture of several different crystal polymorphs. Fats are polymorphic and transform systemically through a series of successive crystalline forms without change in chemical structure. The polymorphic transitions of lipid matrix can be influenced by the addition of one or more substances or other fats. The thermal

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characteristics, crystal habit, texture, and appearance of the lipid matrix are relevant when determining its suitability as drug-delivery system. Since the properties of suspended colloids or dispersed materials may differ significantly from those in the bulk, comprehensive structural investigations are mandatory. The incorporated drug participates in the microstructure of the system and may even influence it due to molecular interactions, especially if the drug possesses amphiphilic and/or mesogenic properties. It is observed that SLNs composed of pure triglycerides (e.g., Dynasan (R) 114) could eventually maintain liquid or liquid crystalline state for considerably long periods of time, and only triglycerides with chains of C16 and longer could recrystallize and form solid lipid crystals (Bunjes et al., 2000; Bunjes and Westesen, 2001). In fact, most of the reported SLN/NLC formulations do not show 100% recrystallization index, which means that certain part of lipid excipients is always present in its liquid state. In case the formulation does not reach solid state in the end of production procedure, a supercooled melt is obtained. This will tend to crystallize within short period of time, which may disrupt the surfactant layer and lead to uncontrolled coalescence of lipid droplets and formation of large crystals. In some special cases, a structure with characteristics of liquid crystals can be obtained and if properly stabilized, may show excellent physicochemical stability. In some cases, intentionally or unintentionally, a nanoemulsion may be obtained. By intentional selection of lipids with low Tm, for example, for the purpose of formulation of thermosensitive drugs or peptides, a solid matrix is not obtained and major part of lipid excipient is present in its melted form. Unintentionally, the same may happen when selecting a lipid with low Tm and high Tm depression. Additionally, a lipid blend may create a eutectic blend, where an additional Tm drop occurs. These observations confirm, how critical is type and nature of lipid selection to produce stable SLNs/NLCs. The other condition of selection of a lipid matrix-forming lipid is its safety status. The original SLN/NLC patented by Gasco and Müller relied only on excipients well known from other pharmaceutical oral solid forms, dermal semisolid dosage forms, or cosmetic products, and therefore have well known and favorable safety profile. Most of the excipient used for lipid nanoparticle formulation holds GRAS status, which gives SLN/NLC a great advantage over many others colloidal drug-delivery systems from the regulatory point of view. An issue to consider is the actual route of administration of the intended SLNs/NLCs, exactly because most of the excipients are accepted by dermal or oral route, but not other routes of administration, for example, intravenous route. In this section, a comprehensive list of currently used excipients for SLN/NLC formulation and their role in SLN/NLC functionalities is discussed as follows: 1. Solid lipids—lipid matrix forming lipids, that is, lipids intended to form the (solid) lipid core. 2. Oils, that is, the lipids or lipophilic excipients that are intended to integrate the (solid) lipid core and decrease its crystallinity. 3. Surfactants—the material, often polymeric, intended to stabilize the lipid crystal suspension, maintain uniform particle size distribution and prevent particle growth. 4. Surface modifiers—the ingredients which add special function and impact in vivo effects.

2.1.1  Solid lipids The most frequently used solid lipids for SLN/NLC formulation are from following category: 1. Triglycerides, that is, glycerol esters with three (monoacid) saturated, linear fatty acids. 2. Partial glycerides (mono- and diglycerides), that is, glycerol esters with 1–2 saturated, linear fatty acids, and 2 or 1 free hydroxyl groups, respectively.

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3. Waxes, that is, esters of fatty acids and fatty alcohols. 4. Fatty acids, that is, saturated linear carboxylic acids with chain length of C12 and more. 5. Fatty alcohols, that is, saturated linear primary alcohols with chain length of C12 and more. Often single solid lipids are employed in production of SLN/NLC. Based on the requirement of formulation or nature of API, combination or blend of lipids is used. Main limitations in using such blends are control over degradation behavior over the time, stability of chemical modification form, and concentration selection of each lipid in blend. The role of the solid lipid is to form the lipid nanoparticle core and form a matrix that can accommodate the active ingredient (one or eventually more than one). All of these materials are intended to solidify (after processing at temperature>Tm) or precipitate (when processed by aid of an organic solvent that is removed in the final step of production process) and form crystalline platelets or spheres with reduced dimensions. As mentioned earlier, even though the lipids used for SLNs/NLCs production are solid at room temperature, the final SLN/NLC particles are rarely 100% recrystallized. The amount of recrystallized mass can be controlled to certain amount by selection of excipients with lower or higher Tm, as needed, and by using a blend of lipids, which usually leads to decreased crystallinity (Doktorovova and Souto, 2009). Higher portion of solid lipid mass contribute to sustained release of the drug, provided that the drug is incorporated in particle core, and to drug protection from environmental factors that might deteriorate the drug. On the other hand, lipid nanoparticles with decreased crystallinity are believed to be more stable (i.e., maintain the average particle size and size distribution) and, more importantly, due to limited polymorphic phase transitions, are less prone to drug expulsion upon solid lipid crystalline structure changes. It is generally accepted that SLN/NLC form spherical or rounded particles, as appears in various scanning electron microscope (SEM) images. As sample preparation for SEM requires drying the sample, freeze-dried SLN are often visualized. The freeze-dried SLN often require presence of a cryoprotectant, and often form redispersible aggregates. Evidence provided by transmission electron microscope (TEM) images shows that SLN prepared from pure glyceryl trimyristate form platelets composed of individual crystal layers, evidenced by a stair-like structure observed on platelet edge (Bunjes et al., 2007). In order to obtain a drug-delivery system that provides all the advantages that it is capable to provide, the selection of lipid matrix forming lipid is essential. The most important factors to consider for matrix forming lipids are: 1. Drug solubility in the lipid in question, to obtain high encapsulation efficiency and loading capacity. 2. Tm of the lipid, with respect to physicochemical characteristics of the active to be incorporated (temperature of decomposition, temperature at which degradation occurs). 3. Tm of the lipid, considering that this Tm will most probably decrease after processing, especially when using preparation methods based on processing lipid melt. This decrease may be advantageous (decreased crystallinity, decreased rates of drug expulsion) but can also compromise some of the functionality features of SLN/NLC, namely the capacity to control drug release and protect the drugs from light, humidity, or acidic/basic environment. Typical pure lipids used for SLN/NLC formulations are indicated in Table 30.1, together with their most important physicochemical characteristics. Examples of SLNs/NLCs together with findings on physical state of solid lipid matrix and its influence on drug encapsulation capacity can be found in our previous review (Souto et al., 2011b). Briefly, SLN/NLCs composed of pure lipids are rarely the

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CHAPTER 30  ROLE OF EXCIPIENTS

Table 30.1  List of Commonly Used Lipids (in Pure Form) in the Formulation Development of Lipid Nanoparticles Type

Lipids

Fatty Acid Chain Length

Melting Range (°C)

Availability

1

Glyceryl tribehenate

—

81–85

Dynasan (R) 122 (Io)

1

Glyceryl tristearate

C18

70–73

Dynasan (R) 118 (Io)

1

Glyceryl tripalmitate

C16

61–65

Dynasan (R) 116 (Io)

1

Glyceryl trimirystate

C14

55–58

Dynasan (R) 114 (Io)

1

Glyceryl trilaurate

C12

43–46

Dynasan (R) 112 (Io)

2

Glyceryl monostearate

C18

66–77

Imwitor (R) 491 (Io)

3

Cetyl palmitate, syn. Hexadecyl decanoate

C16(OOH)C16

43–57

Crodamol (R) CP (Cro), Kollicream (R) CP 15 (Ba)

4

Stearic acid

C18

56–72

Kolliwax (R) S (Ba)

5

Cetyl Alcohol

C16

46–52

Crodacol (R) C90 EP (Cro), Ginol (R) 16/95—(Io), Kolliwax (R) CA (Ba)

5

Stearyl Alcohol

C18

57–60

Crodacol (R) S95 EP (Cro) Ginol (R) 18/95—(Io), Kolliwax (R) SA (Ba)

The combination of lipids can be used to create imperfections in lipid lattice, to enhance drug loading. The types of lipid: (1) triglycerides, (2) partial glycerides (mono- and diglycerides), (3) waxes, (4) fatty acids, (5) fatty alcohols. Details on structure are explained in text. Ba, BASF GmbH, Germany; Io, IoI Oleo GmbH, former Cremer Oleo GmbH and Sasol GmbH; Cro, Croda Healthcare GmbH, Germany.

best lipid matrix to accommodate drugs, due to extensive polymorphic phase transitions that occur during storage and eventually lead to drug expulsion. Triglycerides tend to recrystallize in metastable α polymorph with loose fatty acid chains packing, which transitions into the most stable β polymorph, either directly (α →β) or through temporary formation of metastable β′ polymorph (α → β′ → β). The most stable β polymorph is the one with most dense fatty acid chain packing. This structural reorganization leads often to premature drug expulsion from the solid lipid matrix. The rate of polymorphic phase transitions is mainly influenced by surfactants present in the system: both Span 65 (Aquilano and Sgualdino, 2001) and phosphatidylcholine (Bunjes and Koch, 2005) were shown to delay the α →β transition for several months. Fatty acids show different polymorphic behavior but also suffer from drug expulsion upon lipid crystal reorganization and formation of a more densely packed structure. Stearic acid, which is the most frequently used solid lipid for SLN/NLC production, shows four polymorphs (A triclinic, B monoclinic, C monoclinic, and E monoclinic) and, additionally, several protypes. C polymorph forms upon recrystallization from melt and is the most stable at temperatures >32°C. Below this temperature, the most stable polymorph is the most densely packed B polymorph. High drug loading capacity of stearic acid was reported in the earliest years of SLN development, the latest reports, however, usually indicate limited drug loading capacity, often resolved by addition of oleic acid into the lipid matrix. Oleic acid also shows three different polymorphs (α,β,γ), but the most stable β polymorph melts at 16°C.

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The importance of lipid matrix physical state is related to drug encapsulation efficiency. Three models of drug incorporation were proposed in the early years of SLN development: 1. the drug-enriched core model, consisting of drug concentrated mostly in nanoparticle core and covered by a lipid shell, 2. the amorphous model, where the drug is molecularly dispersed within the lipid matrix, and 3. drug-enriched shell model, where the drug forms a layers condensed on a lipid core. The first two models are desired to obtain a system with sustained release properties. Amorphous drug–lipid system formation is often reported, but the fact is that obtaining a system in which the drug is homogenously distributed within lipid matrix is not easy at all. The drug quantity used must be completely soluble in the lipid in question not only at production temperature but also at room temperature, and should not partition only into lipid phase during the production. Absence of drug peak in differential scanning calorimetry (DSC) thermograph or wide-angle X-ray diffractogram is often regarded as sufficient proof of homogenous drug distribution in SLN/NLC system, however, at the drug loading typically achieved in SLN/NLC (<10%) but frequently well below <2% related to solid lipid content), an eventual peak of solidified or even crystallized drug may be undetected. The drug-enriched shell model was proposed for special drug–lipid combinations were the lipid crystallized before the drug during cooling step of preparation. Recent evidence for a curcumin-loaded trimyristin SLN suggest that this model may actually occur much more often than it might seem. The (lipophilic) drug, incompletely dissolved in lipid matrix, or released prematurely during production or storage, accumulates at water–lipid interface, that is, at SLN surface (Noack et al., 2012). Interestingly, even lipid nanoparticle systems with drug “only” attached to the surface may provide controlled drug release. In short, lipid or lipid blends need to be optimized as per nature of drug, dose of drug, and production method used.

2.1.2  Commercially available mixtures of solid lipids and fats The trend in SLN/NLC formulation is to prefer glycerides mixtures rather than pure triglycerides. The material is again inspired by established dermal and solid oral dosage forms and the fats used for their production. Glyceryl dibehenate (Ph. Eur) is, according to corresponding monograph, a mixture of tri-, di- and monoesters of glycerols of behenic acid (C22) and to minor extent stearic or palmitic acids. In Europe, Compritol 888 ATO (Gattefossé, S.A., France) is very well established in pharmaceutical industry, not only as release modifying agent but also as emulsifier (the mixture shows overall hydrophilic– lipophilic balance, HLB, of approx. 2) and viscosity enhancer (Aburahma and Badr-Eldin, 2014). It is one of the most frequently used lipids for lipid nanoparticle formulation. Another product that complies with the same pharmacopoeial monograph is Kolliwax GDB from BASF, Germany (former product name Speziol GDB PHARMA). Glyceryl distearate (Ph. Eur) is also a mixture of di- and triglycerides of stearic and palmitic acid (C16-C18, therefore also referred to as glyceryl palmito-stearate), required to contain 40%–60% diglycerides fraction and having overall melting point slightly lower than that of glyceryl dibehenate. Precirol ATO 5, is a product that most closely fulfils the pharmacopoeia definition for type I glyceryl distearate. Glyceryl monostearate 40–55 (Ph Eur) also requires a mixture that contains 40%–55% of monoglyceride and 30%–45% of diglycerides. Three types are defined according to the stearic acid content, being type I: the one with least stearic acid content (40%–60%, the other fatty acid summing 90% together with stearic acid is palmitic acid), type II requires 60%–80% glycerol substitution by stearic acid. The products that comply with definition of glyceryl monostearate type I are Geleol mono- and diglycerides NF from Gattefosse, France, and Imwitor 900P from IoI Oleo

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(former Cremer Oleo and Sasol, GmbH) Oleo (former Sasol, Germany). Kolliwax GMS II from BASF, Germany (known before as Cutina GMS V PH) and Imwitor 900K conform to pharmacopoeia type II definition. Apart from mixtures with 40%–55% content, IoI Oleo GmbH, Germany also provides Imwitor 491 containing 90% glyceryl monostearate. Apart from well-defined lipid mixtures, chemical companies also provide a portfolio of fats with different characteristics, currently used in dermal and cosmetic formulations, that were adapted with success in SLNs/NLCs or simply lipid nanoparticle formulations. Gelucire series of lipids from Gattefossé) are PEG containing mixtures of acylglycerides: lauryl macrogol-32 glycerides (Gelucire 44/14) or stearoyl macrogol-32 glycerides (Gelucire 50/13). Gelucires are defined by their melting point/HLB value. As discussed previously, even though most Gelucires have Tm  > 37°C, (the most frequently used for SLN/NLC formulation up to now is Gelucire 50/13), their Tm after processing to colloidal dimensions is expected to be decreased, leading to only partially crystallized lipid matrix. This can be advantageous for certain drugs or peptides and in preparation of paediatric/geriatric rectal and vaginal suppositories. There are already quite a few lipid nanoparticle formulations reported that are based on Gelucire 50/13 (Hazzah et al., 2016; Jeon et al., 2013), Gelucire 43/01 (Gonçalves et al., 2015; Nnamani et al., 2014) or Gelucire 44/14 with partially solid matrix. All of these lipid nanoparticle formulations resulted in low crystalline lipid matrix and were tested mostly for skin delivery of the encapsulated actives, some researchers successfully formulated then as a mucoadhesive lipid nanoparticle (Gonçalves et al., 2015; Hazzah et al., 2016). Witepsol series from IoI Oleo GmbH present lipids with melting point ranging from 32 to 37°C (but there is also Witepsol S series offer solid lipids with melting range up to 70°C). Some of them were successfully adapted for lipid nanoparticle formulation with low crystallinity. Witepsol H15 (melting range 33–35°C) was used for formulation of a sensitive polyphenol compound and Witepsol E 85 (Tm of 42–44°C) for chemotherapeutic actives (Ferreira et al., 2015; Martins et al., 2012). Beeswax and carnauba waxes are also used in lipid nanoparticle formulation. A summary of lipid mixtures currently used in formulation of SLN/NLC, is presented in Table 30.2.

2.1.3 Oils In order to solve the issues related to lipid nanoparticles composed only from solid lipids and solid lipid mixtures, NLCs were developed as a second generation lipid carrier with improved functionality. NLCs were proposed and proven to provide better physicochemical stability (i.e., maintenance of acceptable size distribution during longer period of time) and improved drug encapsulation. Theoretical models predicted oil droplet would integrate solid lipid matrix and thus decrease its crystallinity and provide better drug encapsulation efficiency. In reality, as proven by two independent research groups, the oil tends to accumulate on solid lipid surface, which at the same time contributes positively to NLC stabilization (Jores et al., 2004; Schwarz et al., 2012). By far the most frequently employed oils are mixtures of medium chain (C6–C10) triglycerides and their mixtures. As pure substances, only oleic acid and squalene are being typically used. The use of oleic acid was adapted from conventional dermal formulations as it is a readily available excipient supplied by many companies. It is valued for its skin permeation enhancer properties. Under certain circumstances and preparation procedures, oleic acid can form vesicular structures (Aquilano and Sgualdino, 2001). Squalene and isopropyl myristate are two other liquid lipids frequently used in NLC formulation. Squalene is triterpene compound produced by skin cells (mainly as precursor for cholesterol). It is used as moisturizer in cosmetic and dermal products, but is also approved for other routes of administration. Owing to advantageous properties in dermal products, squalene is often selected

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Table 30.2  List of Frequently Used and Commercially Available Lipid Excipients Employed in Formulation Development of Lipid Nanoparticle Composition (Ph. Eur. Monograph)

Brand Name

Supplier

Melting Range (°C)

Regulatory Status

Glyceryl dibehenate

Compritol (R) 888 ATO

Ga

69–74

D, O

Kolliwax (R) GDB

Ba

65–77

Glyceryl distearate type I

Precirol (R) ATO 5

Ga

53–57

O

Glyceryl monostearate 40-55 type I

Geleol Mono and Diglycerides NF

Ga

55–58

D, O

Imwitor (R) 900P

Io

54–64

—

Imwitor (R) 900K

Io

54–64

—

Glyceryl monostearate 40-55 type II

Kolliwax (R) GMS II

Ba

54–64

—

Lauroyl macrogol-32 glycerides

Gelucire (R) 44/14

Ga

42–47

O

Stearoyl macrogol-32 glycerides

Gelucire (R) 50/13

Ga

46–51

O

Cetearyl Alcohol, syn. Cetostearyl alcohol (C16/C18)

Crodacol (R) CS90 EP

Cro

Kolliwax (R) CSA 50

Ba

49–56

— —

D

Kolliwax (R) CSA 70

Ba

49–56

(Hard fat, adeps solidus)

Witepsol (R) Series, for example, Witepsol E85

Io

42–44 (E85)

Hydrogenated Palm oil

Softisan (R) 154

Io

53–58

Based on the route of drug administration selection can be done. Ba, BASF GmbH, Germany; Io, IoI Oleo GmbH, former Cremer Oleo GmbH, former Sasol GmbH, Germany; Cro, Croda Healthcare GmbH, Germany; Ga, Gattefossé, SA, France; Ph. Eur., European Pharmacopoeia.

as liquid lipid in NLC formulations for dermal delivery. Isopropyl myristate is available from BASF (Kollicream IPM, former isopropyl myristate PH) and is recommended for use in dermal formulations. Medium chain triglycerides (triglycerides, medium chain as per Ph. Eur.) are defined as triglycerides of capric (C10) and caprylic (C8) acid, in total at least 95% of the fatty acids present in the mixture. The most frequently used commercial lipid of this type is Miglyol 812 (IoI Oleo GmbH), another brand products are Kollisolv MCT 70 from BASF (former Myritol 318 PH) or Labrafac Lipophile WL 1349 from Gattefossé, France. Gattefossé provides a great variety of medium chain glycerides (MCT) mixtures and ethoxylated medium chain glycerides mixtures, the most frequently used in SLN/NLC formulations being Labrasol, syn. caprylocaproyl macrogol-8 glycerides, which is in fact an oil–water (o/w) stabilizer (HLB 14), and other products from Labrafac line and Labrafil product lines. Several other oils like coconut oil, jojoba oil, and other super refined oils are being used in formation of SLN/ NLC and are under investigation.

2.1.4 Surfactants The use of surfactants is crucial to maintain the lipid nanoparticle stability. The success of SLN in drug delivery is partly due to improved stabilization of lipid nanoparticles composed of solid matrix for a period of time longer than few days. The concept of SLN in the very beginning of their development

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CHAPTER 30  ROLE OF EXCIPIENTS

was the replacement of liquid lipids forming nano- or microemulsion by lipids that are solid at room temperature. Therefore, stabilization of SLN and NLC adapts many concepts for stabilization of o/w emulsions, including the selection of the surfactant. Being in fact a dispersion of solid matter in outer liquid phase, principles of suspension stabilizations are also valid for lipid nanoparticles. The behavior of lipid nanoparticles has partial resemblance to the behavior of both emulsions and suspensions. Lipid nanoparticles are of typical size, which regard them as a colloidal system. Surfactants play an important role in drug formulation, determining various properties of the colloidal system, such as its viscosity, and capacity to solubilize water-insoluble material. Surfactants have amphiphilic structure that are used to reduce the surface tension and facilitate the particle partition, that is, their hydrophilic groups oriented toward the aqueous phase and the hydrophobic groups oriented to the lipid (Rosen, 2004). In aqueous media, the surfactant molecules are oriented with their polar heads predominantly toward the aqueous phase while keeping their hydrophobic groups away from it. In vesicles, an aqueous phase will also occur in the interior of the structure. The selection of the surfactant mainly depends on the chosen lipid, since they need to be physicochemically compatible (Severino et al., 2012). The HLB is directly related to the solubility, that is, it is the balance of the size and strength of the lipophilic and hydrophilic groups of the surfactant (Severino et al., 2011). The required HLB (rHLB) of a final dispersion is predominantly dependent on the HLB of the lipid and on the HLB of the surfactant (and cosurfactant, if applied), and is determined applying the following equation (Ferreira et al., 2012): rHLB = % Lipid xHLBLipid  = %Surfactant xHLBSurfactant  + %Cosurfactant xHLBCosurfactant 

The rHLB is of high importance, as the selection of the optimized surfactant or combination of them contributes to the enhanced stability of the SLN and NLC dispersions. The blend of surfactants should adjust to the tails of the lipid chains in the interface to allow the coexistence of the oil droplets in a continuous aqueous phase. The interactions between lipids and surfactants are mainly electrostatic. These are physically adsorbed onto the surface or loaded within the lipid matrix. The hydrophilic groups of the surfactant increase the repulsive forces, depending on the volume size and chemical nature of the hydrophilic moieties of surfactants (Rosen, 2004). The surfactants typically used in the production of SLN and NLC are selected depending on their HLB) and also on the nature of the hydrophilic group. In general, nonionic emulsifiers do not show ionic charge (e.g., monoglycerides of long-chain fatty acids), such as Tweens, Spans, Mirjs tyloxapol, poloxamers, sugar esters and esters of stearic, palmitic, oleic, and lauric acids. Anionic or cationic surfactants may be important to improve the surface electrical charge to avoid particle aggregation and/or sedimentation. The cationic surfactants [e.g., stearylamine (Kuo and Chen, 2009; Pedersen et al., 2006; Vighi et al., 2010)], quaternary ammonium salts (Doktorovova et al., 2011; Doktorovova et al., 2012b; Tabatt et al., 2004), N,N-di-(β-stearoylethyl)N,N-dimethyl-ammonium chloride (Vighi et al., 2007, 2010) have a molecule moiety with positive charge. Anionic surfactants (e.g., sodium cholate and sodium taurocholate) have a negative moiety in the molecule, which improve the absorption of particles in the gastrointestinal tract. Phospholipids derived from soy or egg phosphatidyl choline have a variable fatty chain composition, and may also be used in the production of SLN and NLC. Soybean phosphatidyl choline contains more saturated fatty acyl chains than egg phosphatidyl choline (Souto et al., 2011a), and it has been reported to improve the emulsions stability, decrease particle size due to the amphiphilic properties (Schubert and MüllerGoymann, 2005; Schubert et al., 2006), and increase the skin permeation of topically administered

2 ROLE OF EXCIPIENTS IN SLNs AND NLCs

823

formulations (Cui et al., 2006; Dreher et al., 1997). When cosurfactants are used, stabilized emulsions are usually obtained when both surfactants are of the same hydrocarbon chain length (Schmidts et al., 2009, 2010). Different alkyl polyglucosides (APGs) were used as surfactants in the production of cetyl palmitate SLN (Keck et al., 2014). Stabilization with APGs led to smaller SLN and contact angle analysis of the SLN dispersions thereby demonstrating good wettability, high surface activity, a high tendency to remain at the o/w interface, and the improved wetting of cetyl palmitate. The diffusion velocity and the ability to cover newly created surfaces in a shorter time, depends on the molecular weight of the surfactant. Lower molecular weight surfactants move faster therefore are expected to be more efficient than those of higher molecular. The authors also found that the crystallinity of SLN increased with an increase in the alkyl chain length of the surfactants. Changes in temperature, concentration of surfactant, additives in the liquid phase, and structural groups in the surfactant may all cause changes in the mean size, shape, and aggregation number of nanoparticles. Aqueous SLN and NLC dispersions of low viscosity have potentially the risk of transformation into a viscous gel, resulting in loss of their colloidal size and increase of particle size, and risk of aggregation (Mehnert and Mäder, 2001). Usually, these changes are unpredictable and occur very fast. To avoid gel formation, the physicochemical stability must be improved by the addition of cosurfactants with high mobility (Westesen and Siekmann, 1997), but also storage at temperatures below room temperature, under dark conditions and under nitrogen atmosphere may prevent particle growth (Freitas and Müller, 1998). The surfactants for lipid nanoparticle stabilization are mostly o/w emulsifiers/stabilizers, that is, hydrophilic surfactants, mostly polymeric, with HLB > 8. Only one surfactant may not be sufficient to provide sufficient stabilization, therefore combinations of surfactants are frequently used. The combinations often include an o/w together with a w/o surfactant. The selection of surfactant is purely empirical, although some systematic approaches were presented: (1) similarly to emulsion stabilization using the HLB principle. Severino and coworkers showed that a solid matrix with known required HLB value can predict its stabilization in an emulsion system, and may also require similar HLB when formulated as SLN. This method was used for SLN composed of stearic acid, material for which the required HLB value is well known and easily available from current literature (Severino et al., 2011). Keck et al. (2014) used the (2) contact angle approach: aqueous solution of intended surfactant was required to form droplets with low contact angle when deposited on solid lipid surface. Great majority of reported SLN/NLC formulation relies on three surfactants: Polysorbate 80, Poloxamer 188, and phosphatidylcholine (syn. lecithins) of different origin (soy, egg). Less frequently, other types of Poloxamer than the 188 are used, usually Poloxamer 407. Polyoxyethylene sorbitan monooleate, (syn. polysorbate 80) is an o/w surfactant with HLB of approximately 15, accepted by regulatory agencies around the world for pharmaceutical products, including intravenous products. It is a partial ester of sorbitan, containing polyethylene glycol 20 chains and an oleic acid chain. Other types of polysorbates (sorbitans substituted with different fatty acids) are far less frequently used for SLN/NLC stabilization. It is marketed as stabilizer and holds GRAS status. Commercially, it is made available by various companies, for example, Kolliphor PS 80 from BASF, or Tween 80 from Croda. Tween trademark is property of Uniquema, but is used frequently as synonym for polysorbate 80 irrespective of its origin. Polysorbate 80 critical micellar concentration is as low as 0.02%, despite this number is not the most correct for polysorbate in presence of other (lipid) material, it is expected that apart from accumulating on lipid surface, it might form micelles on its own or mixed

824

CHAPTER 30  ROLE OF EXCIPIENTS

micelles with other surfactants present in the system. These colloidal systems might also contribute to solubilization of the encapsulated drug. Solubilization of the active ingredient by polysorbate present in lipid nanoparticle formulation is not a defect, but it may have consequences on drug release, showing biphasic behavior. There are several reports in the literature of drug being actually more soluble in polysorbate 80 than in the solid lipids considered for SLN/NLC formulation. Again, contribution of polysorbate might not be detrimental but it is worth verifying if the use of a lipid matrix actually brings advantage over a solubilization only by polysorbate or surfactant mixture. Special attention must be given to the concentration range used of polysorbate 80 in the formation of SLN/NLC. Poloxamer 188 is a nonionic triblock copolymer consisting of polyoxyethylene (hydrophilic block) and polyoxypropylene (lipophilic) units, showing HLB of > 24 (BASF—Nutrition & Health). In many cases, Poloxamer 188 is sufficient on its own to stabilize SLN, especially those composed of Compritol 888 ATO. It should be notes that at higher concentrations (>10%) Poloxamers form hydrogels; the effect on the overall lipid nanoparticle formulation should therefore be carefully evaluated. Furthermore, Poloxamer 188 shows melting point of around 52°C, which can be confusing when evaluating SLN/NLC thermal behavior. If present in sufficient quantity, a small melting peak of Poloxamer may be observed in differential scanning thermographs, exactly in the temperature range where some triglycerol polymorphs melt. In aqueous environment, Poloxamer 188 also forms micelles. In addition, Poloxamer 188 shows thermoreversible gelling properties. Up to now scarcely used, the good stabilization and solubilization properties together with gelling properties provide an opportunity for formulation of thermosensitive systems by using a Poloxamer type that shows gel transition at body temperature. Apart from Poloxamer 188, Poloxamer of different molecular weights is less frequently used, for example, Poloxamer 407. BASF offers wide range of grades with acceptable pharmaceutical quality (Kolliphor P series, former brand name Lutrol F (BASF, 2013)). Another supplier of Poloxamers is Croda (Synperonic series). Phosphatidylcholine, syn. lecithin, is also readily available in most research laboratories that deal with formulation and therefore is also frequently used for SLN stabilization. Phosphatodylcholine forms bilayer structures under wide range of hydration and temperature. It is generally accepted that phosphatidylcholine accumulates at solid lipid surface and acts as a stabilizing agent, but many authors also regard it as lipid matrix forming lipid. In case of lipid emulsion containing both triglycerides and phospholipids (e.g., Lipofundin MCT), it is known that the various colloidal structures coexist in the dispersion, including nanoemulsion droplets and multilamellar vesicles (liposomes). The only proof of phospholipids being actually associated with SLN surface is from Heiati et al., 1996. Considering later known facts about solid lipids recrystallization behavior in nanoparticles, these reports, however, has some drawbacks: SLN used for the experiments were based on trilaurine, which is known to form supercooled melts rather than solid matrix unless they are frozen after production and return to room temperature. As there is no cooling step mentioned in the report of Heiati, it is possible that the experiments were performed with supercooled trilaurine matrices, that is, with liquid droplets of trilaurine, which obviously have different behavior than suspensions of solid (crystalline) triglycerides. As adsorption on solid surfaces is not the same as adsorption on liquid–liquid interface, this theory should not be automatically considered true for suspensions. Tocopheryl polyethylene glycol 1000 succinate (TPGS) is less frequently used in SLN/NLC formulation. Commercially it is available as Kolliphor TPGS from BASF (former Speziol TPGS Pharma) is solubilizing and stabilizing agent, and at the same time it is hydrophilic form of tocopherol, that is, vitamin E, providing thus some antioxidant properties, too. TPGS is sometimes regarded as solid lipid

2 ROLE OF EXCIPIENTS IN SLNs AND NLCs

825

due to its Tm of 38°C, but the compound shows HLB of 13 making it suitable for stabilization of o/w of solid lipid-in-water systems. Other less frequently used surfactants in SLN/NLC formulation include: • Polyoxyethylene (20) stearyl ether, syn. steareth-20 (Brij S20 from Croda, former names Brij78 or Volpo S20), is a nonionic, o/w emulsifier (HLB 15) and liquid crystal stabilizer from group of ethoxylated fatty alcohols. Its use in SLN/NLC is limited mostly to nanoparticles produced by microemulsion template method, frequently in combination with TPGS and/or emulsifying wax (Dong et al., 2009; Howard et al., 2011; Ma et al., 2009). It was also used successfully for encapsulation improvement of doxorubicin (Siddiqui et al., 2012). • Macrogol 15 hydroxy stearate, marketed as Kolliphor HS 15 (formerly known as Solutol HS 15) is a water soluble, nonionic solubilizer with HLB 14–16, making it suitable for o/w emulsion formulation and therefore obviously also interesting for stabilization of lipid/water colloidal suspensions. Its use is by far more prominent in lipid nanocapsules where it is one of crucial excipients (Huynh et al., 2009). Another important characteristic of this surfactant is the ability to inhibit P-glycoprotein (P-gp) function. Its use in SLN/NLC is limited, but has long tradition form the very first years of SLN/NLC development up to most recent examples (Baek and Cho, 2015; Soni et al., 2014). • Polyglyceryl-3 dioleate, Plurol Oleique CC 497 from Gattefosse, a water insoluble w/o surfactant with HLB of approx. 3. It is marketed as a surfactant and bioavailability enhancer. In SLN/NLC, it was used both as a stabilizing agent (Shegokar and Singh, 2012a) and as an oil in NLC (Zhang et al., 2014). Apart from the most frequently used surfactants, some research groups pointed their investigation in characterization of suitability of “new” surfactants like Solutol HS 15, Plurol oleic, and Plantacare series, that is, those that are not typically used in SLN/NLC formulations. These are listed in Table 30.3.

2.2  SURFACE MODIFICATION AGENT AND LIGANDS Methods used for surface modification of SLN are in general deeply inspired by the methods used for liposome functionlization. This is mainly true for postinsertion method, adapted by many groups for SLN use, and inclusion of special functionalized lipids in the formulation. Another approach is linking the coating agent to an excipient believed to be an integral part of SLN/NLC by a covalent bond using N-hydroxy succinimide/1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (NHS/EDC) chemistry, an approach well validated in polymeric particles (Yu et al., 2012). A summary of surface modifications and methodology used in provided in Table 30.4. Polyethylene glycol (PEG) coating is used to obtain lipid nanoparticles with hydrophilic surface, which would enable longer circulation time by avoiding clearance by reticuloendothelial systems (RES) (Howard et al., 2008). Longer presence in systemic circulation improves bioavailability and is especially interesting for delivery of chemotherapeutic drugs by systems that rely on enhanced permeation and retention (EPR) effect (Maeda, 2001). In a similar manner than in liposomes, SLN/NLC are modified by inclusion of PEG conjugated either to distearoyl phosphatidyl ethanolamine (DPSE-PEG) or stearic acid residue (Polyoxyethylene-stearate, SA-PEG) in the formulation. DPSE is regarded by many authors as surfactant; indeed it was shown that inclusion of DSPE-PEG could improve mean particle size and stability. SA-PEG is used for its structural similarity to simple stearic acid, a common

826

CHAPTER 30  ROLE OF EXCIPIENTS

Table 30.3  Less Frequently Used Surfactants and Their Types in Formulation Development of Lipid Nanoparticles Surfactant Type

Brand Names

HLB

References

C8-10 fatty alcohol glucoside

15–16

Keck et al. (2014)

Glucopon (R) 425 N/HH (Co)

Alkyl polyglucosides of natural C8-14 fatty alcohols

12–13

Glucopon (R) 600 CS UP (Co)

Alkyl polyglucosides of natural C10-16 fatty alcohols

11–12

Plantacare (R) 1200 UP (Co)

C12-16 fatty alcohol glucoside

16–17

Alkyl polyglucosides. Plantacare (R) 810 UP (Co)

Polyhydroxy surfactants

Composition/Important Characteristics

Plurol (R) Stearique WL1009 (Ga) Polyglycerol 6-distearate

9–10

Kovacevic et al. (2011)

Plantacare (R) 810 PL (Co)

Caprylyl/capryl glucoside

15–16

Alkyl esters of PABA

NA

decyl, dodecyl, and tetradecyl esthers of p-amino benzoic acid

—

Chandra et al. (2011)

Sucrose esters

Surfhope SE Pharma D1216 (Mi) Sucrose laurate HLB 16

16

Surajit et al. (2014a,b)

Surfhope SE Cosme C1416 (Mi),

Surcrose mirystate HLB 16,

C1616 (Mi)

Sucrose palmitate HLB 16

C1816 (Mi)

Sucrose stearate HLB 16

Quillaja saponins

Quillaja saponaria wood extract, composed mainly of predominantly of glycosides of quillaic acid. (An).

Quillaja saponaria wood extract

13.5

Salminen et al. (2014, 2016)

Chlorogenic acida

N.A. (Genex Life Science Pvt Ltd, New Delhi, India)

Chlorogenic acid

—

Khan et al. (2016)

Cationic arginine ester

CAE (Ai)

PCA Ethyl Cocoyl Arginate syn.

—

Patil-Gadhe and Pokharkar (2014)

DL-Pyrrolidonecarboxylic acid salt of L-cocyl arginine ethyl ester

Manufacturers: (Ai) Ajinomoto, Tokyo, Japan (An) Andean QDP Ultra Organic, represented by Desert King International, San Diego, CA, USA, (Co) Cognis, Germany, currently part of BASF, Ludwigshafen, Germany, (Ga) Gattefosse, Saint-Priest, France, (Mi) Mitsubishi-Kagaku Foods Corporation, Tokyo, Japan a Used in combination with Poloxamer 188, PABA = p-amino benzoic acid.

Table 30.4  List of Excipients Used as Surface Modifiers in Lipid Nanoparticle Formulation to Offer Unique Functionality or Targeting Potential Attachment Method

Outcomes

References

PEG 2000

SA-PEG2000 as surfactant

Higher conc. Of SA-PEG2000, more DOX transported into brain Significantly decreased DOX concentration in heart and liver

Zara et al. (2002)

PEG 2000

SA-PEG2000 as surfactant

Enhanced permeability through mucus, enhanced permeation through cell monolayer (Caco-2/HT29) Increased relative bioavailability versus simple SLN

Yuan, Fan et al. (2014)

PEG2000

SA-PEG2000 as surfactant

Enhanced biochanin A bioavailability after oral administration in Sprague-Dawley rats versus drug suspension

Wang et al. (2015)

PEG2000

DSPE-PEG as surfactant

Accelerated release of salbutamol sulphate from PEG-SLN compared to simple SLN

Hong et al. (2006)

PEG 1000, polysaccharide and serum albumin

Solutol as surfactant

Enhanced bioavailability of SLN when surface modified with polysaccharide > serum albumin > PEG. Superior safety profile over uncoated NP

Shegokar and Singh (2012)

PEG2000

DSPE-PEG as surfactant

Sustained release Increased t1/2 Good protective effect in rat ischemia-reperfusion injury model.

Gao et al. (2008)

PEG2000

DSPE-N-PEG2000 as surfactant

PEG 2000

PEG2000-peptides

Preferential biodistribution to tumor tissue, decreased distribution to lung, liver, and kidney (MMP cleavable PEG-peptide functionalized SLN)

Zheng et al. (2014)

Folate

Folate-PEG-Chol as surfactant

Improved survival of Balb/C mice bearing murine lung carcinoma (M109) tumor (Folate-targeted PTXL SLN)

Jain et al. (2008)

Folate

Folate-SA as part of formulation

Increased brain targeting versus uncoated formulation (DTX and ketoconazole). Higher brain permeation than marketed formulation of DTX (Taxotere)

Venishetty et al. (2013a)

Folate

FA-PEG-DSPE as part of formulation

Improved cisplatin anticancer effect in vitro (HeLa) and in vivo (BALB/c mice)

Zhang et al. (2016)

Biotin

SA + (SLN, amino group) + EDC. HCl (crosslinker) + NHS + Biotin (carboxyl group)

Enhanced bioavailability after oral administration to SpragueDawley rats

Zhou et al. (2015)

Lee et al. (2007)

2 ROLE OF EXCIPIENTS IN SLNs AND NLCs

Surface Material

827

(Continued)

Table 30.4  List of Excipients Used as Surface Modifiers in Lipid Nanoparticle Formulation to Offer Unique Functionality or Targeting Potential (cont.)

828

Surface Material

Attachment Method

Outcomes

References

Ferritin

DSPE+ EDC. HCl + Ferritin (carboxyl group)

Enhanced cellular uptake, increased anticancer efficacy of encapsulated chemotherapeutic drug (5-fluoro-uracyl)

Jain et al. (2008)

Transferrin

DSPE (SLN, amino group) + EDC. HCl (crosslinker) + Transferrin (carboxyl group)

Enhanced bran uptake of quinine hydrochloride from Tf-SLN (i.v., albino rats) compared to solution or simple SLN

Gupta et al. (2007)

Transferrin

Transferrin-PEG-PE adsorption (on cationic SLN)

Enhanced in vitro and in vivo transfection in hepatocellular carcinoma (HepG2) cell line and Kupfer Cells in Balb/c mice

Jing et al. (2012)

Transferrin

Transferrin-PEG-PE adsorption (on cationic SLN)

Enhanced in vivo transfection and DOX delivery in model lung (A549) tumor in C57BL/6 mice

Han et al. (2014)

Ovalbumin

Adsorption from 5% mannitol solution to cationic SLN

Enhanced IL-12 release from murine BMDDC ex vivo enhanced immune response to ovalbumin in vivo (mice)

Patel et al. (2007)

β-d-Galactosides

β-d-galactosides with long fatty chain (nC18H37) and 2 or 10 Polyoxyethylene units as aglycon moiety (G2, G10) as parts of formulation

Increased accumulation in mice liver; increased t1/2 in blood plasma

Lian et al. (2008)

CHAPTER 30  ROLE OF EXCIPIENTS

Mannose

Covalent bond; Schiff base formation between mannose aldehyde group and secondary amine on SLN surface (containing SA+)

Increased uptake of FITC-labeled mannosylated formulation in A549 and MCF-7 cells, compared to simple SLN and DOX solution Significantly increased bioavailability, t1/2 and MRT in Balb/c mice versus DOX solution, decreased cmax

Jain et al. (2010)

Mannose

Covalent bond; Schiff base formation between mannose aldehyde group and secondary amine on SLN surface (containing SA+ and DSPE)

Increased uptake of rhodamine-labeled mannosylated formulation in A549 Decreased hemolytic activity versus simple SLN and PTX solution

Sahu et al. (2015)

Mannose

Man-C6-Chol as part of LNP formulation

Targeting to alveolar macrophages Decreased Rifampicin encapsulation efficiency Similar pharmacokinetic parametrs versus simple LNP

Song et al. (2015)

Mannose

Monomannosyl-dioleoyl glycerol as part of SLN formulation

Mishra et al. (2010)

Attachment Method

Outcomes

References

Mannan

Mannan-PEG-PE adsorption (on cationic SLN)

Enhanced in vitro and in vivo transfection in hepatocellular carcinoma (HepG2) cell line and Kupfer Cells in Balb/c mice

Jing et al. (2012)

Galactose

N-hexadecyl lactobionamide as part of SLN formulation

Improved liver targeting in Wistar rats (i.v.)

Wang et al. (2010)

Galactose

Covalent bond; Schiff base formation between galactose aldehyde group and secondary amine on SLN surface (containing SA+)

More sustained DOX release in vitro Decreased hemolytic activity of galactosylated DOX-loaded formulation versus nongalactosylated Increased cytotoxicity toward A549 cells

Jain et al. (2015)

Concacavalin A

SA+(SLN, amino group) + EDC (crosslinker) + Concacavalin A (carboxyl group)

Increased mitoxanthrone accumulation in lung tumor (A549 in Balb/C mice)

Mahor et al. (2010)

Phenylalanine

DSPE(SLN, amino group) + EDC (crosslinker) + Phenylalanine + NHS (carboxyl group)

Enhanced bioavailability in rat brain

Vyas et al. (2015)

l-Arginine

Adsorption

Increased uptake by human brain-microvascular endothelial cells (HBMECs)

Kuo and Lin (2009)

Histidine tagged polyarginine (cell penetrating peptides)

Chelation to nickel complex (SLN component DOGS-NTA-Ni)

Enhanced drug (celecoxib, ketoprofen) permeation into skin

Desai et al. (2012), Shah et al. (2012)

Pentapeptide (Thr-Lys-Pro-Pro-Arg)

Pentapeptide-PEG2000-SA as part of SLN formulation

Increased uptake by Wistar rat macrophages in vitro and in vivo

Zhao et al. (2013)

Pentapeptide

Pentapeptide-PEG2000-SA as part of SLN formulation

–

Fan et al. (2014)

Oligo-chitosan (3–6 kDa)

Adsorption

Enhanced mucoadhesive properties compared to simple NLC

Luo et al. (2010)

Oligo-chitosan (8–42 kDa

Adsorption Secondary crosslinking by glutaraldehyde

Improved DOX loading capacity More sustained drug release (further improved by crosslinking)

Ying et al. (2011)

Chitosan (50 kDa)

Adsorption

Improved transcellular transport through cell monolayer (Caco-2) Enhanced absorption (of encapsulated insulin) from GIT versus simple SLN

Fonte et al. (2011)

829

(Continued)

2 ROLE OF EXCIPIENTS IN SLNs AND NLCs

Surface Material

Table 30.4  List of excipients used as surface modifiers in lipid nanoparticle formulation to offer unique functionality or targeting potential (cont.)

830

Surface Material

Attachment Method

Outcomes

References

Chitosan (50–190 kDa)

Inclusion in aqueous phase during SLN production

Improved SLN stability Enhanced mucoadhesive properties versus simple SLN Enhanced cellular uptake Evidence of thick chitosan layer formation around lipid core (FT-IR, TEM)

Luo et al. (2015)

Chitosan

Adsorption Secondary crosslinking by glutaraldehyd

—

Dharmala et al. (2008)

Alginatea

Inclusion in (outer) aqueous phase during SLN production

Improved stabilization (ZP) of SLN Enhanced mucoadhesive properties

Fangueiro et al. (2012)

Hyaluronic acid

Adsorption (on cationic SLN)

Increased cellular uptake of targeted SLN versus simple SLN (almost inexistent uptake). SK-OV-3 cells

Ghalaei et al. (2014)

Hyaluronic acid

Alendronate–hyaluronate graft polymer adsorption on (negatively charged) NLC surface

Enhanced irinotecan-NLC cytotoxicity toward multidrug resistant cell line (Colo-320) P-gp inhibition, tumor targeting in vivo (Balb/c mice) Decreased hemolytic activity versus uncoated NLC Decreased uptake by macrophages versus uncoated NLC

Negi et al. (2014a,b)

CHAPTER 30  ROLE OF EXCIPIENTS

Hyaluronic acid

Hyaluronic acid-protamine-siRNA complex adsorption on cationic SLN surface (containing DOTAP)

Successful gene silencing in vitro (HepG2, ARPE-19) CD44mediated endocytosis and cavelae-dependent cell internalization (with possible lysosome evasion)

Torrecilla et al. (2015), Apaolaza et al. (2014)

Wheat germ agglutinin

Covalent bond; PVA (surfactant) crosslinked with glutaraldehyd at acidic pH and linked to WGA amino group

Enhanced binding and uptake by cell monolayer (Caco-2) Enhanced adhesion to rat intestinal mucosa ex vivo Improved bioavailaility after oral administration (Sprague-Dawley rats)

Liu et al. (2010), Liu et al. (2011)

poly(Allylamine hydrochloride)+ poly(diallyldimethyammonium chloride)+ poly(acrylic acid)

Layer-by-layer deposition of polyelectrolytes—polyelectrolyte multilayers Adsorption (to SLN)

SLN (slightly negative ZP) bind best to multilayers with polycationic outmost layer PEGylated surfaces repelled SLN

Finke et al. (2013)

Beta-hydroxybutyric acid

Beta-hydroxybutyric acid-SA+ conjugate as part of formulation

Enhanced brain uptake of DTX versus unmodified DTX-SLN and Taxotere

Venishetty et al. (2013b)

BMDDC, Bone marrow derived dendritic cells; Conc., concentration, DOGS-NTA-Ni = 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) imidodiacetic acid] succinyl nickel salt, DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane chloride a Used in combination with Poloxamer 188, PABA = p-amino benzoic acid.

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excipient for SLNs/NLCs. SA-PEG is an amphiphilic molecule; it might also contribute to stabilization of SLN/NLC itself. Due to the possibility of formation of separate colloidal object by DSPE-PEG itself (see earlier), SA-PEG might be more suitable excipient for PEGylated SLN/NLC formulation. Despite encouraging data on colloidal carriers modified by PEG, recent reports show that PEGylation is not a straightforward solution for improvement of bioavailability. It was observed that repeated doses of PEG-coated liposomes (Ishida et al., 2005) or SLN (Zhao et al., 2012a) actually lead to increased clearance of the colloidal system. This is described as accelerated blood clearance (ABC) phenomenon. The first dose of PEGylated colloidal carrier induces IgM type antibody production, which reacts with PEGylated surfaces after second and subsequent doses, and marks such carrier for removal by RES (Zhao et al., 2012a,b). Another theory clarifies that crucial factor is PEG distribution on the surface of a colloidal carrier: while low density of PEG chains (typically < 9% PEG per mm2) on the nanoparticle surface promote “mushroom-like” configuration of PEG chains, higher concentrations induce “brush-like” configuration. The mushroom-like configuration is recognized by phagocytic cells, leads to complement activation and to rapid clearance of colloidal system in question (Amin et al., 2015). Brush-like configuration is believed to repel opsonins and thus assure longer circulation time. This issue was not addressed in SLN/NLC. While there are many reports of PEGylated SLN/ NLC showing improved t1/2 and bioavailability, indeed most of the currently available reports are based on single dose administration. Therefore, PEGylation might not be a universal solution for RES avoidance. Furthermore, Shegokar and coworkers showed that PEG adsorption on a nanosuspension (stabilized by surfactants frequently used in SLN/NLC formulation) influences the quantitative distribution of adsorbed plasma proteins and apolipoproteins, but does not change their qualitative pattern. As a consequence, the protein adsorption pattern reveals good possibilities for macrophage recognition, confirmed by biodistribution study in rats which affirmed increased distribution to liver and spleen (Shegokar et al., 2011a). As an alternative to PEG coating, different hydrophilic or amphiphilic polymers were tested. Polyvinyl pyrolidone (Ishihara et al., 2010) and polyglycerols (Abu Lila et al., 2013) we suggested as promising alternatives. Polyglycerol derivates can also serve as surfactant for SLN/NLC stabilization, as shown by Kovacevic et al. (2011). Interestingly, hydrophilic “coating” can also be achieved by using poloxamers, very frequently used in SLNs/NLCs. Many encouraging in vivo reports might owe their success exactly to poloxamer outer layer of the colloidal carrier. We have shown that Poloxamer 188 is effective in shielding the SLN (even with positive surface charge) from blood plasma protein adsorption. Only limited amount of bound plasma proteins were found by 2D-PAGE, none of them being known opsonin. Such protein corona promotes longer circulation times (Doktorovova et al., 2012a). Surface modification by alginate coating aims at improved mucoadhesive properties, especially for formulations intended for oral administration. Alginate is a polysaccharide consisting of β-d-mannuronic acid and α-l-gluronic acid and typically bears negative surface charge (at neutral pH). Adsorption on SLN/NLC surface was assured by inclusion of alginate in outer aqueous phase of SLN prepared by double emulsion method (w/o/w). The authors observed that alginate could actually improve stability of SLN/NLC, probably by contribution to both steric and electrostatic repulsion between individual particles. (Fangueiro et al., 2012). Shegokar et al., studied in vivo effect of SLN after surface modification with PEGs, polysaccharide and serum albumin. The surface modification significantly enhanced the cellular uptake and bioavailability of stavudine encapsulated SLN. The type of surface modification showed different degree of bioavailability upon intravenous administration (Shegokar and Singh, 2012b).

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In order to enhance mucoadhesive properties desirable for oral formulation or interaction with sialic acid on eye surface for ocular formulations, chitosan is frequently used in SLN/NLC formulations. Chitosan, syn. Poly-d-glucosamine or deacetylated chitin, has gained much attention in Pharm. Res. for its biocompatibility, biodegradability, mucoadhesive properties and capacity to form stable complexes with nucleic acids. Chitosan is a weak base (pKa ≈ 6) (Lavertu et al., 2006), therefore it is soluble at acidic pH and at that pH it is positively charged and binds readily to SLN surface, which is negative (unless modified by inclusion of cationic components). Furthermore, chitosan shows high affinity to lipid surfaces. Several chitosan types are commercially available, ranging from chitosan to various chitosan salts and including chitosan of different molecular weights. Oligochitosans and chitosan salts are soluble in water (at neutral pH). Simple incubation of SLN with chitosan solution was used by different research groups; the reported results indicate that adsorption of chitosan was successful. Luo et al. (2015) proved recently by Fourier-transform infrared spectroscopy (FT-IR) and transmission electron microscopy (TEM) that chitosan forms a thick layers around (solid) lipid core. Therefore, chitosan can also contribute to stabilization of SLNs/NLCs, by both steric and electrostatic mechanisms. Various authors also noted chitosan could improve drug-loading capacity. In order to further reinforce chitosan surface layers, Dharmala et al. (2008) and Ying et al. (2011) used glutaraldehyde to promote crosslinking of chitosan hydroxyl groups. This approach (referred to as ionic gelation) is frequently used in formulation of chitosan nanoparticles, although with different crosslinking agents (e.g., tripolyphosphate) and usually leads to nanoparticles with better physicochemical properties and drug loading (Katas and Alpar, 2006). Hyaluronic acid (HA), syn. d-glucuronic acid and d-N-acetyl glucosamine, typically with very high molecular weight, is an anionic polysaccharide frequently found in extracellular matrix of various tissues. Its biological activity includes participation in regulation of wound healing/tissue regeneration (including skin lesions), namely by promoting cell proliferation and migration. Rationale for its use may be improvement of skin disorders including dry skin conditions or skin repair (Sandri et al., 2013). Apart from its skin effects, HA might also regulate cancer progression and metastization as the major ligand to CD44 receptor. Indeed, many types of invasive carcinomas express CD44 receptor, controlling signaling pathways involved in tumor progression (Horta et al., 2015). Attachment of HA to SLN/ NLC surface was mostly achieved by ionic bond between negatively charged HA and cationic SLN, especially formulated for the purpose of HA adsorption. Some of these formulations served for the purpose of gene delivery (namely siRNA delivery, Apaolaza et al., 2014; Torrecilla et al., 2015) and therefore contain multiple cationic components to assure sufficient positive charge density. In another work, Cetyl pyridinium Chloride (CPC) a water-soluble antimicrobial quaternary ammonium compound (QAC), having one long-chain alkyl group and pyridine moiety is used in lipid nanoparticle production. The feasibility of positively charged NLC production is performed using CPC as a stabilizer cum active for topical application (Shegokar et al., 2009). Also for the purpose of targeted delivery to malignant cells, ligands of lectin receptors were explored. These are various carbohydrates, most frequently used as galactose or mannose residues coating on SLN/NLC surface, that should promote receptor mediated cellular internalization of the whole nanoparticle. Therefore, covalent link to a SLN component, most frequently a surfactant with available amino group (SA+ or DSPE) was used. Mannose was subject to ring opening reaction in acidic environment and subsequently reacted with the amine present on SLN/NLC surface, forming a Schiff base. Another approach used for mannose coating was prior synthesis of mannose derivates with high

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lipophilicity [e.g., 3(mannos-1-yl)cholesterol] that can be included in SLN/NLC formulation, where the hydrophilic mannose moiety should be oriented to SLN/NLC surface and the lipophilic part should prefer adsorption or mixing with the rest of excipient forming the lipid core. For the purpose of enhanced skin penetration (Patlolla et al., 2010; Shah et al., 2012) or overcoming the blood–brain barrier (Venishetty et al., 2013a), cell-penetrating peptides are considered a promising strategy. Histidine-tagged polyarginine of different chain length (i.e., arginine residues) were adsorbed on SLN surface using the chelate complex formation, an approach used for isolation of histidine tagged peptides (Lauer and Nolan, 2002). In the case of SLNs, this approach resulted in increased transdermal delivery of two nonsteroidal antiinflammatory drugs. Another approach for attaching peptides to SLN surface is linking the peptide covalently to PEG chain of SA-PEG which then can be included in SLN formulation. Another surface modification approach aims at a specific targeting to a selected cell population. Apart from functionlization with monoclonal antibodies and their fragments, transferrin and folate are frequently used for surface modifications. The rationale is in increased metabolic needs of malignant tissues, reflected in folate and transferrin (Tf) receptor over expression. Folate (folic acid, FA) attachment is typically facilitated by inclusion of FA-PEG or Tf-PEG functionalized lipids in SLN/NLC formulation. These functionalized lipids (SA-PEG-FA, DSPE-PEG-FA or Cholesterol-PEG-FA, Tf-PEG-SA) are commercially available.

2.2.1  Complementary excipients Freeze-drying is often required for long-term storage of SLN/NLC, especially those with positive surface charge and special formulations that for any reason cannot guarantee suitable storage time in dispersion. Despite reports that it is possible to freeze-dry cationic SLN without use of any cryoprotectant (Vighi et al., 2007), most frequently the cryoprotectant is needed to assure good dispersion quality after rehydration of the product. Most frequently used cryoprotectants are carbohydrates; these include: • monosaccharide: glucose or mannose • disaccharide: lactose, sucrose, fructose, or the most efficient trehalose • sugar alcohol: mannitol, sorbitol Additionally, polymers like polyvinyl pyrollidone were successfully tested as cryoprotectants (Singh and Shegokar, 2011). Selection of the most suitable cryoprotectant is most often based on empirical optimization, as there is no rule yet that would identify suitable excipient combinations. Apart from acceptable appearance of the product, freeze-dried SLN/NLC should also show ability to redisperse easily into dispersion with similar size distribution characteristics to those before freeze-drying. Furthermore, the product should assure that the redispersion capacity is maintained over time of storage, which can be an additional challenge for product development (Doktorovova et al., 2014a). Similar selection of carbohydrates, but mainly mannitol, lactose, and sucrose were tested for SLN spray drying. There is also a report on spraydried SLN powder with excellent properties for pulmonary deposition prepared without any additional excipient. The same report also indicates mannitol as an especially suitable excipient (Dolatabadi et al., 2015). Further reports on SLN spray drying include the use of ethanol (Blasi et al., 2013) or methanol (Freitas and Mullera, 1998). The type and concentration of cryoprotectant can further affect plasma stability of redispersed system.

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The selection of excipient is considered crucial in assuring overall biocompatibility and safety of SLN/NLC. The theory is that it is the surfactant that has major influence of SLN/NLC biocompatibility. Generally, most of the marketed surfactants are safe for topical or oral use and if used in moderate quantities, do not cause harm on cellular or tissue levels. As we have shown before, there is no traceable effect of the three most frequently used types of surfactants on cell viability (Doktorovova et al., 2014b). Caution is in place when using cationic surfactants or surface modifiers for their known induction of cell membrane damage, oxidative stress and interference with intracellular lipid trafficking. The potential toxicity, metal contamination, miscibility, solvent capacity, and disposability must be taken into consideration. Several reports confirm role of excipients in determination of bioavailability and toxicity potential, it should be very well noted that the route of administration plays critical role and affects the toxicity potential, for example, SLN injected intravenously will show different toxicity compared to the SLN applied dermally with same composition. Besides that scaling up nanoparticles and role of excipients is separate topic itself for discussions. The successful scale up of lipid nanoparticles using various capacity homogenizers using same excipient composition to lab scale production is reported. However, the production parameters at large scale significantly affects the final particle size distribution (Shegokar et al., 2011b).

2.3  NEW COMERS IN EXCIPIENT MARKET In March 2015, Evonik has launched RSPO-certified preservative-free palm kernel oil–based surfactant Tego Betain P 50 C. Chemically, it is cocamidopropyl betaine and contains 38% active betaine. A patented manufacturing process assures high quality (with low levels of amidoamine and chloro acetic acids) and microbial purity of surfactant. Tego Betain P 50 C, an amphoteric surfactants can be used in preparation of lipid nanoparticles and can offer viscosity to skin and hair care formulations. For pharmaceutical application, the concentration optimization will be needed to exert optimal effect. Innospec Speciality Chemicals launched Pureact SLMI-85, a sulphate free isethionate ester based surfactant to expand their portfolio of taurates and isethionates based surfactants. It can be used as primary or secondary surfactant and has comparable flash foam properties to that sodium laureth sulphate and has excellent stability over wide range of pH. Another leading Surfactant manufacturer Stepan has launched in collaboration with Elevance surfactant Steposol Met-10U derived from natural oils mainly targeted to displace solvents. This product has main applications household, adhesive cleaning, and in paint removal. Recently, Naturex launched Sapnov, a natural foaming agent and surfactant extracted from quillaia. Sapnov is a nonionic and water soluble surfactant. Other examples of surfactant introduction include Colonial chemical’s 100% natural coconut based surfactant, Colafax PME, a monoalkyl phosphate, which can be used as alternative to sodium lauryl sulfate and ammonium laureth sulfate. It can be employed as primary or secondary surfactant and Cognis’s, Plantacare, Plantaren, Plantapon LGC product line which can be employed in development of SLN-based products for skin care. In 2014, Symrise launched a PEG-free solubilizer called SymSol PF-3 to be in competition of PEG-free products and this surfactant can solubilize lipophilic substances. It can be used as an emulsifier or coemulsifier in o/w formulations. It showed superior solubilizing performance in comparison to PEG-containing products (i.e., PEG-40 Hydrogenated Castor Oil) and other PEG-free solubilizer, The Company has applied for patent protection for SymSol PF-3. In 2007, Advanced Materials Inc. has launched innovative emulsifier named Pemulen, a polymeric emulsifiers based on high molecular weight polyacrylic acid polymers as o/w stabilizer. Various grades of Pemulen are available based on

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their oil emulsifying capability, for example, Pemulen TR-1, Pemulen TR-2. Another class of surfactants called Gemini surfactant is in boom. It combines monomers of two surfactant linked chemically. These surfactants offer improved surface active, wetting properties at typically exhibit much lower critical micelle concentration strong dependence on spacer structure, special aggregate morphology, and strong hydrophobic microdomain. These types of surfactants are being widely explored mainly in skincare, gene delivery compared to other drug delivery routes (Sekhon, 2013). Pierre Potier prize winner sustainable green chemistry based Sepiclear G7 (heptyl glucoside) is launched by Seppic. It is a nonionic surfactant derived from sugar and castor seeds. It helps in solubilization of essential oils, perfumes and vitamin E, in aqueous media. Another example of PEG-free solubilizer includes TEGO Solve 61 a naturally derived solubilizer for lipophilic and natural oils by Evonik. It is polyglyceryl esters (polyglyceryl-6 caprylate; polyglyceryl-4 caprate; polyglyceryl-4 cocoate; polyglyceryl-6 ricinoleate) and is effective for the solubilization of very lipophilic ingredients like fatty or natural oils. In 2014, Longvida launched new product from Douglas laboratories for nutritional supplement application based on solid lipid particle (SLPTM) technology. Formulation composed of curcumin with neurophenol. The composition showed 65 times higher bioavailability and exhibited longer half-life compared to regular curcumin. Now, Capsugel’s Lipidex Technology Platform allows integration of lipid, liquid, and semi-solid fill technologies. This platform allows customer to find tailored solutions for the many issues like low solubility drugs, poor therapeutic performance, and life cycle management. NanoLipid Restore CLR, a semi-finished product developed by Chemisches Laboratorium Dr. Kurt Richter, Germany and distributed by Pharmacos India. It is a NLC dispersion containing black current seed oil and is used in the cosmetic product line IOPE from Amore Pacific, South Korea. Other NLC incorporated products are Nanorepair Q10, Nanovital Q10, Cutanova (Dr. Rimpler, Germany) and Surmer (Isabelle Lancray, France). CEA-Leti, France developed two DDS platform based on lipids, namely Lipidots (lipid nanodroplet for diagnostic/drug delivery, 20-180 nm) and LipiVAC (vaccine DDS for antigens) has been developed. Most recent (September 2015) acquisition between the Lubrizol Corporation, a Berkshire Hathaway technology driven company, with Particle Sciences, a leading contract manufacturing and drug delivery solutions organization headquartered in Bethlehem, PA. This acquisition will now together offer variety of drug delivery solutions to the market across a variety of dosage forms.

2.4  LIQUID LIPIDS 40 years market player Fuchs, Germany offers highly refined white oils based on mineral hydrocarbons, consisting of saturated aliphatic hydrocarbons. The white oils are produced to meet the European Pharmacopoeia standards for light and liquid paraffin, and also meet the requirements for the USP/NF mineral and light mineral oil USP. In 2014, Exxon Mobil Corporation has launched two white oils called Marcol and Primol as an ideal solution for cosmetic and pharmaceutical applications. These oils offer excellent hydration and emolliency. Another market player Medisca offers variety of pharmaceutical, nutraceutical essential and fixed oils, for example, coconut oil, cod liver oil, eucalyptus oil (natural), lavender oil (natural). Stability of drug is concern for pharmaceutical industry, some polar (monoglycerides, diglycerides), free fatty acids, plant sterols, coloring matter (chlorophyll, carotene), and oxidative impurities present in oil excipients can cause degradation of drug and ultimately endanger the stability of formulation.

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Keeping this in mind Croda chemicals has launched Super Refined Oils, processed by flash chromatographic process. Super Refined Oils has reduced impurities and are suitable in pharmaceutical and health care formulations. Croda currently market, Super Refined Castor, cotton seed, olive, peanut, sunflower, sesame, soyabean oil, etc. Super Refining by chromatography helps to purity excipients and control the quality by controlling the moisture; residual catalyst (peroxide and aldehyde) without altering original chemical structure. Super Refined PEGs and are polysorbate, propylene glycol are also made available by Croda. Another supplier, Reitzer Pharmaceuticals offer Tea Tree Oil extracted from Melaleuca alternifolia has been used in many cosmetic and pharmaceutical applications. This oil has an antifungal, antimicrobial, and antiinflammatory healing oil and is suitable to use in eczema, psoriasis, and acne. Thirty years of heritage makes Avanti polar lipids a leading supplier of lipids to pharma industry. Various grades of lipids are available starting from class antigenic lipids, biotinylated lipids, deuterium labeled lipids, diglycerols, diglyceride pyrophosphates (DGPP), fluorescent phospholipids, fluorescent sphingolipids, functionalized PEG-Lipids, glycerol based lipids, glycolipids, lanthanide chelating lipids, maleimido containing lipids, natural lipids, PEG-lipids, phosphatidylinositols, phosphatidylserine, phytosphingosine to several synthetic lipids. Corden pharma has a wide phospholipid portfolio to offer for drug delivery, pharmaceutical, and biotechnological applications. Several lipids are available from monoglycerol, diglycerol, phosphatidyl acids, phosphoglycerols, phosphoserine, phosphoethanolamines, phosphocholines, methoxy PEGylated phospholipids, phosphoglycerols with heterogeneous fatty acid chains, cholesterol, 2-lyso phospholipids, ether lipids, cationic lipids, and fatty acid ate the main categories. As stated earlier, the choice of excipient is highly depend on the route of administration. Currently, most of the excipients are approved for topical application and some for oral use. Researchers are kindly advised to check their GRAS status before their use in intravenous applications. Overall leading excipient (mainly surfactant and lipid) companies are expanding their portfolio with innovative excipients to offer multiple and optimum choice of excipient for their drug delivery and final dosage form.

3 CONCLUSIONS Lipid nanoparticles especially SLNs and NLCs are well trusted and long researched (almost 25 years) as a drug delivery systems in pharma for various biomedical applications. Literature has extensively discussed and recognized the role of excipient in formulation, stability, in vitro and in vivo effects of the lipid nanoparticles. Biocompatibility of lipid nanoparticles is highly dependent on nature and type of excipient used. The extensive use of wide ranged excipient in cosmetic and dermal application is reported, followed by average use in oral and pulmonary drug delivery. However, excipients administered via intravenous route are really limited and need to follow more strict guidelines on metal impurities, toxicity considerations beside their GRAS status. From the very first step of “drug–lipid” screening till the conversion of lipid nanoparticle dispersion to dry powder and scaling up, excipients play critical role and their performance varies on case to case basis based on nature of other excipients and production method. The excipient companies are well aware of customer needs and limitations during formation of “functional” lipid nanoparticles. They are trying to overcome these issues by offering innovative products like surfactant cum surface modifier, functionalized lipids, PEG free ingredients, or refined excipients. Industries are now also offering their knowledge on understanding of excipients and its functionality related critical attributes as a part of QbD (quality by design approach) along consistency

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information and elemental impurity profile. Overall, excipient is soul of formulation development and biocompatibility performance of lipid nanoparticles and must be selected intelligently to achieve optimum performance.

ACKNOWLEDGMENT Doktorovova S. is recipient of postdoctoral scholarships from Portuguese Science and Technology Foundation (Fundação para a Ciência e Tecnologia) under ref. SFRH/BPD/101650/2014.

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