Solid Lipid Nanocarrier Development Toolbox for Increasing Oral Bioavailability of API

Abstract

This article provides an overview of the processing techniques and the methods of characterization for evaluating Solid Lipid Nanocarriers (SLN) designed to improve the bioavailability of active pharmaceutical ingredients (API). It presents the commonly used formulation techniques for developing lipid-based solid nanocarriers and details the analytical approaches to assess these systems for size, morphology and zeta potential. Also discussed are the measures for encapsulation efficiency, drug release profile, intestinal permeability of the encapsulated API across cell models, and assessment of particle internalization.

Introduction

The bioavailability of orally administered Active Pharmaceutical Ingredients (API) depends primarily on their solubility properties in the gastro-intestinal milieu and their subsequent absorption across the intestinal epithelial cells. Indeed, drugs need to be dissolved in the gastro-intestinal fluids to be properly absorbed. When the dissolution rate is slower than the absorption rate of the API, the dissolution step becomes the limiting factor for achieving adequate bioavailability.¹ This is the case for poorly-water soluble drugs, belonging to the class II of the Biopharmaceutics Classification System (BCS). On the other hand, BCS class III drugs, exhibit good solubility but low permeability across the intestinal border. This is generally the case of large hydrophilic molecules, such as peptides and proteins, the absorption of which across the epithelium is hindered by the hydrophobicity of enterocytes phospholipidic bilayer, the small paracellular pathway and the tight junctions.

Advanced drug delivery systems are often selected as an efficient strategy to enhance the bioavailability of poorly bioavailable drugs. Among them, Solid Lipid Nanoparticles (SLN) are controlled drug delivery systems which small size favors the transport across the intestinal border and increases encapsulated drug distribution at the epithelial interface. Developed in the 90s, the SLN technology uses biodegradable and biocompatible lipid excipients which offer reduced cytotoxicity compared to other colloidal systems. Surfactants are typically added to the solid lipid carrier to increase the system stability. Nanostructured Lipid Carriers (NLC) were developed later to increase drug payload and overcome the problem of API expulsion during crystallization or phase transition of the lipids constituting SLN. Briefly, NLC are a derivative of SLN technology having a liquid lipid in addition to the solid lipids in the formulation.

A recent review has focused on the encapsulation of peptides in SLN.2 However, many of the processes described can be adapted to the encapsulation of the poorly soluble but highly permeable API belonging to the Biopharmaceutical Classification System (BCS) II category. In the case of the BCS II API, the active is simply dissolved or dispersed in the solid lipid carrier. For the highly soluble but poorly permeable (BCS III) category of API, however, it is necessary to first increase the API lipophilicity and thus its affinity for the solid lipid carrier. This may be achieved with either covalent lipidization, using Reversible Aqueous Lipidization technique,³ cyclization of the API backbone,⁴ or by non-covalent lipidization with the formation of Hydrophobic Ion Pairs⁵ or Hydrophobic Hbond Pairs.⁶

The discussions in this article apply to SLN and NLC to encapsulate both types of API.

SLN Preparation Techniques

SLN or NLC may be obtained by a variety of formulation techniques adapted to the properties of the API such as solubility, pKa, heatsensitivity, shear-sensitivity, and dose requirements.

Melt Dispersion Processes

A frequently used method for producing SLN is preparation and subsequent solidification of nanoemulsions. Typically, the API is dissolved or dispersed in the lipid phase, heated 5 to 10°C above the melting point of the solid lipid excipients. The aqueous phase, containing the emulsifier(s), is heated to the same temperature and poured into the lipid phase. High shear rates need to be applied to the system in order to break the resistance induced by the viscosities of both phases and to form new interfaces.

Only few devices like High Pressure Homogenizers (HPH) or ultrasonic equipments can provide sufficient shear rates to form nano-sized droplets. The preparation of a hot nanoemulsion may also be facilitated by a probe sonicator, a device that is useful for laboratory scale validation of formulation composition. It does however pose a potential risk of contamination by metallic residues during scale up. For this reason, the use of hot HPH is generally preferred over ultrasonic devices.

A coarse, hot emulsion generally formed by high shear homogenization is poured into the reservoir of the HPH system. The coarse emulsion is forced through an interaction chamber consisting of a narrow gap (few microns) of predefined shape (represented in Figure 1), under high pressure (100 to 2000 bars). The high shear rates resulting from the impact (collision) forces of the walls and the particles themselves enable significant size reduction.^7,8 Several passages in the interaction chamber are generally required to obtain a monodisperse hot nanoemulsion. A recent article describes the different formulation step to formulate NLC for lipidized peptides encapsulation.⁹

Whether by ultrasonication or hot HPH, most of the energy provided to the system is dissipated by Joule effect. Consequently, the formulation temperature rapidly increases which can potentially result in API degradation. For thermosensitive API, cold HPH process was developed. With this technique, the API is also dispersed or dissolved in the molten lipid phase which is then rapidly cooled by use of liquid nitrogen or dry ice. This material is then milled into microparticles and suspended in a cold emulsifier solution before injection into the HPH.

Although the processes described above are easy to set up, they may be deleterious to shear sensitive drugs such as DNA, albumin or erythropoietin.¹⁰ For these API, low energy melt dispersion processes such as membrane contactors can be used. With the latter, the molten lipid containing the API is continuously pushed through the membrane contactor of defined pore-size. The nanodroplets are formed at the surface of the membrane where an aqueous solution of emulsifier(s) circulates and sweeps away the particles.¹¹ Particle size will depend on the type of lipids, the process temperature, membrane pore size, and the flow velocity of the aqueous phase.

Microemulsion Templates

The method developed by Gasco et al. is based on a template, where a microemulsion is formed between the molten lipids containing the API and an aqueous emulsifier(s) solution. The nanoparticles are obtained by pouring the hot microemulsion into cold water. Depending on the formulation composition, particle sizes between 50 and 800 nm with a Polydispersity Index (PDI) range of 0.06 and 0.912 may be obtained.

Solvent-Based Processes

The use of solvents in pharmaceutical preparations is generally discouraged because of the toxicological issues resulting from potentially incomplete removal of the solvent. However, there are a number of solvent-based techniques to obtain solid lipid nanoparticles that are of interest for the encapsulation of very heat-sensitive API. Among these techniques, nanoprecipitation, represented in Figure 2 is one of the simplest and most used techniques to produce SLN and NLC. The technique is based on solvent affinity. A solution of lipid excipients and API dissolved in a water-miscible organic solvent is progressively added to water containing the emulsifier(s). This addition causes the spontaneous migration of the organic solvent to the aqueous phase leading to the instantaneous precipitation of the excipients as solid nanoparticles.¹³ The use of microfluidic devices can be a pathway to develop this technology to an industrial scale. Other techniques such as emulsion-diffusion or emulsion-evaporation can be considered. An interesting review from Knez and Weidner describes supercritical fluid extraction of emulsions (SFEE) techniques for the production of nanoparticles.¹⁴ SFEE can be used for the production of solid lipid nanoparticles to encapsulate API.¹⁵ One of the major advantages of this technique is the production of dry material.

In summary, most SLN and NLC are obtained as suspensions in water and require a drying step to stabilize the formulations and to process them in their final dosage form (capsule, tablet…). Freeze drying is generally preferred (after addition of a cryoprotectant) but spraydrying or fluid bed drying can also be considered.¹⁶ The advantages and the drawbacks of the commonly used processes are summarized in Table 1.

Nanocarrier Composition

Triglycerides, waxes, partial glycerides, and fatty acids of varying chain length or ester content constitute the “lipids” used in SLN and NLC. Generally, the higher the percentage of the formulation lipids, the larger is the size of the particles obtained. Moreover, an increase in the chain length of the lipids increases the viscosity of the lipid phase in melt processes. Consequently, higher shear rates are required with long lipid chains to reduce particle size.¹⁷ High levels of emulsifiers are needed to stabilize the SLN formation process where high shear rates create numerous interfaces among the particles. All types of emulsifiers may be used for this purpose which have been listed in a 2012 review by Mehnert et al.¹⁰

In the case of NLC, a liquid lipid fraction is added to the system to increase the mobility of the chains upon lipid crystallization. It can also be used to increase intestinal permeability, especially in the case of medium-chain triglycerides,¹⁸ or for their protective effect towards proteolytic degradation in the case of peptides.¹⁹ The liquid lipid should be sufficiently hydrophobic to remain in the nanoparticles, otherwise an exudation phenomenon may be observed.

Characterization of Lipid Nanoparticles

Particle Size

The functionality of nanoparticles is largely correlated with particle size mainly because the latter impacts drug release and absorption rate at the intestinal barrier that is one of the main obstacles to oral bioavailability. Particle size measurements also involve determination of the polydispersity index (PDI) which expresses the relative error between curve fit and experimental values, giving an overview of the quality of the dispersion and guaranteeing a homogeneous repartition of the API within the formulation and a good stability of the system.

Laser Diffraction (LD) and Dynamic Light Scattering (DLS) are commonly used techniques for measuring particle size. LD is based on Fraunhofer’s theory that relates the diffracted light intensity and the diff raction angle to the particle size (e.g. the smaller the particle the larger the angle of diffraction). A clear advantage of the LD technique is that it covers a wide range of sizes, from a few nanometers up to one millimeter. DLS, also called Photon Correlation Spectroscopy (PCS) on the other hand is based on Rayleigh diffusion, which states that light diff uses in all directions as long as the particles are small compared to the wavelength (generally 633 nm). The laser goes through all particles in Brownian motion which causes fluctuation in scattering intensities. Constructive and destructive interferences are generated, giving information about particle speed and consequently particle size (larger particles display slower movements). A less common technique to measure particle size in a more accurate manner is the combination of Field Flow Fractionation (FFF) with MultiAngle Light Scattering (MALS) which enables the separation and particle size analysis of several populations in polydispersed nanosuspensions.²⁰

All the techniques mentioned above provide a single value to characterize particle size. However, only perfect spheres can be described by a single number. Consequently, the values obtained for non-spherical objects correspond to the diameter of an equivalent sphere that has one property in common with the analyzed particles (equivalent volume, equivalent weight, minimum diameter, etc. Although all responses will be correct, the values will vary as a function of the selected property. It is then important to compare several techniques to obtain a more complete description of the system. The ideal solution is to complete the particle size analyses by observations of the morphology of the particles.

Morphology Assessment

Assessment of particle morphology is especially important in the case of solid lipid nanocarriers for which platelet structures are commonly observed upon lipid crystallization.¹⁰ The following are widely used microscopic tools for evaluating nanoparticle morphology.

Transmission Electron Microscopy (TEM) facilitates 2-D observations on interactions between the electrons transmitted to a thin sample and the material, providing a very high-resolution image. A prerequisite for this analytical technique is that the sample be electron transparent. Although this method is considered to be non-destructive, the electron beam can cause melting of the lipid nanoparticles hence affecting their structure and integrity. To preserve their morphology, it is therefore recommended that the analyses involve cryo-TEM whereby samples are frozen in liquid ethane prior to observations. Examples of SLN and NLC observations by cryo-TEM are provided in Figure 3. Scanning Electron Microscopy (SEM) provides information about the surface morphology and 3D structures of nanoparticles. Whereas TEM utilizes electron transmission through the sample, SEM measures electron transmission of the sample surface. A beam of high-energy electrons is emitted to generate different signals at the surface of the sample. Secondary electrons provide high resolution SEM images. Backscattered electrons (BSE) and diffracted backscattered electrons (EBSD) are used to determine crystal structures and orientations, as well as the photons, visible light and heat. An additional step for sample preparation may involve electrically insulating the samples by coating them with a thin layer of conducting material. For the same reasons as for TEM, it is recommended to cryogenically fix the samples before observation to maintain their structure under the electron beam.

Other less common methods can be used to evaluate particles morphology. In Cryo-Field Emission Scanning Electron Microscopy (cryo-FESEM), samples are frozen in liquid nitrogen and the measurements are conducted in the frozen state. Atomic Force Microscopy (AFM) technique provides high resolution (0.01 nm) 3-D topography. With this technique, samples can be directly analyzed; conductivity of the material is not required and the measurements can be realized in presence of solvents (no vacuum necessary).^7,21

Zeta-potential

Zeta-potential, or ζ-potential, represents the electric charge of the nanoparticles. It is measured by electrophoresis, a technique that identifies the movement of the charged nanoparticles in suspension under a magnetic field. Zeta-potential is a useful measure for predicting the stability of SLN in suspension as it relates to the tendency of the nanoparticles to repulse each other as particle electric charge increases. In oral delivery, Zeta-potential is also a tool to predict the intestinal absorption of the nanoparticles as neutral and anionic ones are more likely to cross the mucus barrier than positively charged nanoparticles.

Other characterization tools such Differential Scanning Calorimetry (DSC), X-ray diffraction or Proton-NMR can be used to study the inner structure of the particles and predict their stability and behavior.^10,22

Encapsulation Efficiency

The amount of API actually encapsulated in the nanoparticles is the most important measure of the process efficacy. It is calculated by subtracting the quantity of free drug in the supernatant from the overall amount of API in the nanosuspensions as follows:

Equation 1: Encapsulation efficiency calculation:

Drug loading is calculated after drying the nanoparticles and corresponds to the ratio of API within the nanoparticles:

Equation 2: Drug loading calculation

The total amount of API can be easily quantified after dissolution of the nanoparticles in an appropriate solvent. Measuring the amount of API in the supernatant is trickier as it requires a proper separation from the nanoparticles. A number of methods like ultrafiltration, field flow fractionation, and ultracentrifugation have been described in the literature. Lv et al. recently compared three commonly used techniques to separate nanoparticles from their dispersing environment. The study reported poor separation with centrifugation, especially with SLN, despite the high centrifugal forces applied. Gel permeation chromatography showed good separation of large macromolecules although the latter may adhere to the beads constituting the column, resulting in distortion of the results. Finally, the filtration/centrifugation method using membrane with pre-defined mesh size offered good separation of the nanoparticles, with only 0.32-0.93% drug leakage from the nanoparticles.²³

Drug Release

Evaluation of drug release from any dosage form is necessary to predict the fate of the drug after its administration to the patient. Contrary to macro-sized delivery systems, there is no “gold standard” established for evaluating drug release from nanoparticles. Nothnagel et al. have described different techniques of measuring drug release from nanoparticles in different media,24 dividing them into three categories: i)sample and separate ii) dialysis, and iii) in situ evaluation. All categories present advantages like simplicity, efficacy and also drawbacks like poor separation, retardation of release by dialysis bags, which should carefully be evaluated before selecting one method over another.

Evidently, release kinetics depend on the release conditions such as sink conditions, replacement of medium after aliquots withdrawal, type of medium, and agitation. Generally, the release studies are conducted in Simulated Gastric Fluid (SGF), Fasted State Simulated Intestinal Fluid (FaSSIF-V1 or V2) and more rarely in Fed State Simulated Intestinal Fluid (FeSSIF).

Intestinal Permeability

The intestinal barrier consists of a mucus layer and the intestinal epithelium. Mucus is a complex hydrogel, negatively charged and prevents the passage of large molecules. The epithelium is mainly composed of enterocytes (99%) with hydrophobic phospholipid bilayers that prevent the passage of hydrophilic molecules. These cells are covered with microvilli and are separated by tight junctions which limit the transcellular passage to small molecules. The other cells constituting the epithelium are endocrine cells (or L cells), Goblet cells responsible for the production of mucus and M cells known to initiate mucosal immunity responses. These latter cells are only present in specific regions known as Peyer’s patches. They are less covered in mucus and microvilli and are more conducive for the passage of large molecules. Once absorbed, the macromolecules are conducted to the lymphoid system. This absorption route presents the advantage of avoiding the first pass effect. Moreover, M cells are not restricted to small molecules so as enterocytes (limited to 50-100 nm).

In-vitro cell models

Caco-2 cells, Madin-Darby Canine kidney (MDCK) cells and Parallel Artificial Membrane Permeability Model (PAMPA) are the common in vitro models used in the evaluation of intestinal permeability of API and pharmaceutical preparations. In the case of PAMPA, an artificial lipid membrane is formed by a mixture of lecithin and an organic solvent. This technique is cost-effective and easy to set up which can be of great advantage in the early formulation development phase. However, its use is limited to the evaluation of passive transport.

MDCK cells use is less commonly reported than that of Caco-2 cells, although they are said to enable a more predictive intestinal apparent permeability and more inter-laboratory reproducibility than Caco-2 cells.

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Caco-2 cells were originally obtained from human epithelial colorectal adenocarcinoma and are widely reported in the literature for the evaluation of intestinal permeability. Indeed, under certain culture conditions these cells express many properties of enterocytes: tight junctions between adjacent cells, microvilli on apical face, and the expression of metabolizing enzymes. Intestinal permeability evaluation of API is evaluated on cell monolayers, following a 21-day incubation in culture medium (with medium changed every other day). The integrity of the monolayer is assessed by TransEpithelial Electrical Resistance (TEER) measurements which should be above 250 Ω.cm2 or by evaluation of the paracellular transport of known markers such as mannitol.

Caco-2 cells are interesting cell models to evaluate intestinal permeability. However, their high TEER value (generally around 500 Ω.cm2) and the absence of mucus make them less representative of the in vivo conditions. For these reasons, mucin-secreting models using HT29-MTX co-cultured with Caco-2 were developed. In the latter case the Caco-2 and HT29-MTX ratio is generally 3 to 1 but can vary if a specific region of the intestine needs to be modeled, as their number increases along the gut.^25,26,27 The presence of mucus can be evaluated by staining the cell monolayers with Alcian blue, as shown in Figure 4.

Follicle-associated epithelium model was developed to evaluate the effect of M cells on the intestinal permeability of the API. The monolayers are obtained after incubation of Caco-2 with Raji cells which help the conversion of Caco-2 cells into M-like cells.²⁸ This conversion can be monitored by a decrease in TEER values. Other complex in vitro cell models were developed to specifically study the impact of the API on inflamed intestinal disorder. These models were well described by Beloqui et al.²⁹

Cytotoxicity Evaluation

Before permeability evaluation of API, it is of utmost importance to measure the cytotoxicity of the formulation or the API itself. The most common cytotoxicity evaluation tests are MTT test, using a tetrazolium dye to quantify a number of living cells, the quantification of Lactate Dehydrogenase (LDH) released upon tissue damage, and the quantification of adenosine triphosphate (ATP) proportional to the number of living cells. MTT is a tetrazolium dye which is reduced by the enzymes present in viable cells. The incubation of the formulations with the cells followed by addition of MTT enable a colorimetric assay to quantify the viability of the cells. In contrast, quantification of LDH released enables an estimation of the number of lysed cells. Measurements of cytotoxicity provide indication on the range of formulation or API concentration that should be used for permeability evaluation without damaging cell monolayers which would distort the results.

Apparent Permeability Assay

Intestinal permeability evaluation is generally evaluated over 2 hours during which the formulation or the API is co-incubated with the cell monolayers grown on inserts. The integrity of the monolayers is evaluated before and after the incubation by TEER measurements. API quantification on the basolateral side enables the calculation of the apparent permeability (Papp, cm.s-1) as described in the following equation:

Where dQ/dt is the transport rate (μg/s), A is the surface area of the insert (cm²) and C0 is the initial concentration at the apical side (μg/mL).

These experiments can also be carried on freshly excised animal intestine (generally from rats) in Ussing chambers.

Interaction with Intestinal Cells

Evaluation of the interactions between the nanocarriers and the intestinal cells provides complementary information regarding their transport. In particular, it is possible to observe the internalization of the particles by the cells or, on the contrary, their retention at the apical side or by the mucus.

To observe the cellular uptake of the nanoparticles, confocal scanning laser microscopy (CSLM) images can be taken after fixation and staining of the monolayers or the tissue. The observations require the encapsulation of a fluorophore in the lipid-based nanocarrier and ensure the stability of the dye inside the carrier. With this objective in mind, it is recommended to label SLN or NLC with a dye from the indocarbocyanine family (DiD, DiO, Dil, …) which were proven to remain in the nanoparticles, unlike Nile Red or Coumarin 6.30 For example, Figure 5, shows the internalization of Precirol.ATO5 based SLN and NLC loaded with DiD by Caco-2 and co-cultured Caco-2/HT29-MTX monolayers. This observation can be confi rmed by flow cytometry measurements to quantitatively measure the uptake of the fluorophore-loaded nanoparticles by the cells.

The intestinal cell models and the techniques described above are important tools for evaluating the mechanism of transport of the API from nanoparticles – more specifically demonstrated for saquinavir loaded-NLC by Beloqui et al.^29,32,33

Conclusions and Perspectives

The critical considerations and options for developing, characterizing, and evaluating the efficacy of SLN and NLC for enhancing the oral bioavailability of BCS class II and III API have been reviewed. Clearly, the cited methods may be selected in accordance with the development objectives and more specifically in line with the API properties (environmental sensitivity, solubility, pKa), the intended dose, and the stage of the undertaking, be the early phase or the late, scale-up phase of the development. Also, it may be necessary to multiply the number of measurements in order to obtain the most objective description of the formulated system. Lastly, many of the tools described in this document can be adapted to other routes of administration such as topical or parenteral delivery.

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