A Package of Established Analytical Tools to Investigate the Solid-State Alteration of Lipid-Based Excipients

Summary

This publication shows the application of x-ray diffraction and differential scanning calorimetry as gold standards for investigating the solid-state of lipid-based excipients (LBEs). Understanding the solid-state alteration in LBEs and its effect on the performance of pharmaceutical products thereof is the key factor for manufacturing robust lipid-based dosage forms.

Abstract

Lipid-based excipients (LBEs) are low-toxic, biocompatible, and natural-based, and their application supports the sustainability of pharmaceutical manufacturing. However, the major challenge is their unstable solid-state, affecting the stability of the pharmaceutical product. Critical physical properties of lipids for their processing-such as melt temperature and viscosity, rheology, etc.-are related to their molecular structure and their crystallinity. Additives, as well as thermal and mechanical stress involved in the manufacturing process, affect the solid-state of lipids and thus the performance of pharmaceutical products thereof. Therefore, understanding the alteration in the solid-state is crucial. In this work, the combination of powder x-ray diffraction and differential scanning calorimetry (DSC) is introduced as the gold standard for the characterization of lipids' solid state. X-ray diffraction is the most efficient method to screen polymorphism and crystal growth. The polymorphic arrangement and the lamella length are characterized in the wide- and small-angle regions of x-ray diffraction, respectively. The small-angle x-ray scattering (SAXS) region can be further used to investigate crystal growth. Phase transition and separation can be indicated. DSC is used to screen the thermal behavior of lipids, estimate the miscibility of additives and/or active pharmaceutical ingredients (API) in the lipid matrix, and provide phase diagrams. Four case studies are presented in which LBEs are either used as a coating material or as an encapsulation matrix to provide lipid-coated multiparticulate systems and lipid nanosuspensions, respectively. The lipid solid-state and its potential alteration during storage are investigated and correlated to the alteration in the API release. Qualitative microscopical methods such as polarized light microscopy and scanning electron microscopy are complementary tools to investigate micro-level crystallization. Further analytical methods should be added based on the selected manufacturing process. The structure-function-processability relationship should be understood carefully to design robust and stable lipid-based pharmaceutical products.

Introduction

Lipids are a class of materials that contain long-chain aliphatic hydrocarbons and their derivatives. They cover a broad range of chemical structures, including fatty acids, acylglycerols, sterols and sterol esters, waxes, phospholipids, and sphingolipids¹. The use of lipids as pharmaceutical excipients started in 1960 for embedding drugs in a wax matrix to provide sustained release formulations². Since then, lipid-based excipients (LBEs) have gained extensive attention for diverse applications, such as modified drug release, taste-masking, drug encapsulation, and enhanced drug bioavailability. LBEs can be applied in a broad range of pharmaceutical dosage forms via versatile manufacturing processes, namely, hot-melt coating, spray-drying, solid lipid extrusion, 3D printing, tableting, and high-pressure homogenization, among others. Dosage forms such as tablets, orally disintegrating films, multiparticulate systems, nano and microparticles, pellets, and 3D-printed forms are the result^2,3,4.

LBEs possess the "General Recognized as Safe" status, low toxicity, good biocompatibility, and improved patient tolerance. Their natural origin and broad availability allow them to empower green and sustainable pharmaceutical manufacturing. Nevertheless, the use of LBEs has been associated with unstable dosage forms. Alterations in the properties of lipid-based products after storage have been widely reported. The solid-state of LBEs and the existence of lipid polymorphism are considered the main reasons for the instability of lipid-based dosage forms^5,6,7,8.

The mechanical and physical properties of lipids are closely related to their crystallization properties and the structure of their crystal network, which shows distinct hierarchies of structural organization. When lipids are used in the manufacturing of pharmaceutical products, the crystal structure is affected by the process parameters applied, such as temperature, organic solvents, shear, and mechanical forces, which in turn affects the performance of the pharmaceutical product^{5,7,9,10,11,12}. To understand this structure-function relationship, it is important to know the fundaments of lipid crystallization and crystal structure and analytical methods to screen them.

At the molecular level, the smallest unit of a lipid crystal is termed a "unit cell." A regular three-dimensional repetition of unit cells builds the crystal lattices, with stronger molecular interactions alongside their lateral directions than the longitudinal ones, explaining the layered construction of lipid crystals. The repeated cross-sectional packing of hydrocarbon chains is known as sub-cell^1,12,13 (Figure 1). Lamellae are the lateral packing of lipid molecules. In the crystal package, the interfaces between different lamellae are made of methyl end groups, whereas the polar glycerol groups are placed at the interior parts of the lamella¹⁴. To differentiate each fatty-acid chain in the lamella, the term leaflet is employed, which represents a sublayer composed of single fatty-acid chains. Acylglycerols can be arranged in double (2L) or triple (3L) leaflet chain lengths¹⁴. The surface energy of the lamellae drives them to epitaxially stack to each other, to provide nano-crystallites. Different processing factors such as cooling temperature and rate affect the number of stacked lamellae and thereby, the crystallite thickness (~10-100 nm). Aggregation of crystallites leads to the formation of spherulites in micro-scale, and the aggregation of spherulites provides the crystal network of LBEs with defined macroscopic behavior¹³.

Solid-state transitions start at the molecular level. The geometrical transition from one sub-cell to another is called polymorphism. Three major polymorphs of α-, β'-, and β-form are usually found in acylglycerols, ordered according to increased stability. Tilting of the lamella with respect to end-groups occurs during polymorphic transitions^1,13. Storage and melt-mediated polymorphic transitions are experienced by LBEs. Storage transitions occur when the metastable form is stored below its melting temperature, whereas melt-mediated transitions happen as the temperature rises above the melting point of a metastable form provoking melting and successive crystallization of the more stable form.

Furthermore, phase separation and crystal growth can also occur. Phase separation is driven by initial multiphasic crystallization and growth of one phase or more. Particle-particle interactions, including sintering, molecular interactions, microstructural features, and foreign components, can also trigger crystal growth^1,5.

Monitoring the solid-state transitions of LBEs and their impact on the performance of dosage forms is of significant importance. Among others, differential scanning calorimetry (DSC) and x-ray diffraction, specifically simultaneous small and wide-angle X-ray scattering (SWAXS), are two gold standards for assessing lipid solid-state.

DSC is commonly used to measure the enthalpy changes of the material of interest associated with the heat flow as a function of time and temperature. The method is widely used for the screening of thermal behavior of lipids, such as possible pathways of melting and crystallization, corresponding temperature and enthalpy of different polymorphic forms, as well as minor and main fractions of lipid compositions. These data can be used to depict the heterogeneity, multiple phases, and lipid polymorphism^5,7,13.

X-ray diffraction techniques are the most powerful methods for structure determination in the solid state. Possessing ordered nanostructure with repeated lamellae, the reflection of x-ray beam from lipid crystals can be investigated using Bragg's law:

d = λ/2sinθ     (Equation 1)

where λ is the x-ray wavelength of 1.542 Å, θ is the diffraction angle of the scattered beam, and d is the interplanar spacing of repeated layers, defined as lamella length in lipids. The small-angle region of the x-ray can be perfectly used to detect the long-spacing pattern and calculate the lamella length (d). The larger the repeated distance d, the smaller the scattering angle (1-15°, small-angle region) since d is inversely proportional to sin θ. The sub-cell arrangement of lipids can be characterized as the short-spacing pattern in the wide-angle region of the x-ray diffraction. Both the long- and short-spacing patterns of lipids (lamella length and sub-cell arrangement) can be used to indicate the monotropic polymorphic transformation. For example, the α-form (hexagonal) can be altered to β (triclinic) due to a change in the angle of tilt of the chains, with alterations in the lamella length (long-spacing pattern, in the small-angle region, 1-15°) and in the cross-sectional packing mode (short-spacing pattern, in the wide-angle region, 16-25°) (Figure 2).

The information obtained from the SAXS region can further be used to investigate the crystal growth by measuring its thickness (D) via the Scherrer equation¹⁵:

D = Kλ/FWHMcosθ     (Equation 2)

Where, FWHM is the width in radians of the diffraction maximum measured at a half-way height between the background and the peak, generally known as full-width at half-maximum (FWHM); θ is the diffraction angle; λ is the X-ray wavelength (1.542 Å) and K (Scherrer constant) is a dimensionless number that provides information about the shape of the crystal (in case of the absence of detailed shape information K = 0.9 is a good approximation). Please note that the Scherrer equation can be used to estimate mean crystal sizes of up to about 100 nm since the peak broadening is inversely proportional to the crystallite size. Therefore, its application is useful for determining the thickness of nanoplatelets and, indirectly, the number of aggregated lamellae. Examples of using this well-known approach for screening the crystal properties of lipids in the pharmaceutical formulation development and the corresponding instability in product performance can be found in^{5,12,16,17,18}.

Monitoring the solid-state of LBEs within each developmental stage through well-established analytical techniques provide an effective strategy for designing high-performance manufacturing processes and stable lipid-based pharmaceutical products.

This publication presents the critical application of a comprehensive solid-state analysis of LBEs for monitoring the changes in solid-state and its correlation to the alteration in the release profile of active pharmaceutical ingredient (API) from the pharmaceutical dosage form. Multiparticulate systems based on lipid-coated API crystals via hot-melt coating, and nano-lipid suspensions produced via high-pressure homogenization are taken as case studies. The focus of this publication is the application of powder x-ray diffraction and DSC as analytical tools. The first two examples show the effect of polymorphic transformation and crystal growth, respectively, on the alteration in API release from coated samples. The last example reveals the correlation between the stable solid-state of lipid and the stable performance of the pharmaceutical product in lipid-coated multiparticulate systems and in nano-lipid suspensions.

Protocol

1. Differential scanning calorimetry (DSC)

1. Instrument preparation
   1. Use a differential scanning calorimeter equipped with an intracooler, an autosampler, and the software for instrument control and data analysis.
   2. Switch on the nitrogen gas supply and set the pressure between 0.2–0.5 bar and power up the DSC instrument and the automatic sample changer.
   3. Open the software and activate the standby mode by clicking on the Yes button. Allow equilibration of the device for at least one hour
   4. Purge the furnace with nitrogen, click on the New Method icon and go to Method Definition. Activate the Temperature Modulation option in the overview window.  Go to header tab and select the method by clicking on “sample”.
   5. Go to the tab Temperature Program, select Purge 2 MFC and on Protective MFC, both at 50 mL/min.
   6. Insert the following measurement method: Standby at 20 °C, heating cycle at 5 K/min from 20 °C to above the melting temperature of lipid, isothermal holding at this temperature for 5 min, cooling cycle to 0 °C at 5 K/min to -20 °C, final emergency reset temperature at a temperature 10 degrees °C above the highest temperature of the program, and final standby temperature at 20 °C.
   7. Go to the calibration tab and select the appropriate temperature and sensitivity file. Save the method
2. Sample preparation and measurement
   1. Weigh 3–4 mg of each sample into aluminum crucibles. Record the exact weight loaded into each crucible and seal the aluminum crucible with a pierced lid.
   2. Place the crucibles in the autosampler tray and activate the autosampler mode in the software and load related method for each sample.
   3. Fill out the sample position, sample name, and weight of each sample, and the position of reference crucible in the Sample Tray View window and start the measurements.
3. Data analysis
   1. Open the raw data using the software for data analysis and plot the temperature versus heat flow, by clicking on the button “X-time / X-temperature”
   2. On the popped out window, click on “Hide isothermal segments”. On the left side of the screen, select only the curves that are to be analyzed (e.g. unclick the “Additional” data).
   3. Check the thermal behavior of lipids as endothermic and exothermic events of absorbed or released energy in the form of heat, respectively, as a function of temperature.
   4. Click on the curve, followed by Evaluation and Area, to calculate the enthalpy of fusion as the area under the curve of endotherms.
   5. Select the integration boundaries by moving the vertical lines around 2 to 3 degrees Celsius before and after the onset and endpoint of the peak.
   6. Select a linear baseline for the peak integration. The area between the curve and the baseline is proportional to the change in enthalpy. Click on Apply to finish the calculation. Similarly, calculate the enthalpy of crystallization as the area under the curve of exotherms
   7. Determine the onset of melting temperature (To) by clicking on the curve to be analyzed and then on Evaluation and Onset.
   8. Select the quantification boundaries by moving the vertical lines to the most straight section of the curve. This is usually around 5-10°C before and after the peak. Then, determine the melting temperature by clicking on the curve to be analyzed, followed by Evaluation and Peak. The obtained value is the peak maximum.

2. Small- and wide-angle x-ray scattering (SWAXS)

1. Instrument preparation
   1. Use an x-ray scattering system, composing a point-focusing camera fixed to a sealed-tube x-ray generator and equipped with a control unit and related software.
   2. Use cooper (λ = 1.54 Å) at 50 kV and 1 mA as an x-ray source and two linearly positioned sensitive detectors to cover both small- and wide-angle x-ray scattering regions.
   3. Ensure safety requirements to protect from x-ray exposure.
   4.  Switch on the cooling water system on the control unit, the vacuum pump, the gas valves, and the power and safety control system.
   5. Switch on the voltage control and purging valves for the detectors at a gas flow between 10–20 mL/min.
   6. Switch on the x-ray tube and the standby option and wait approximately 10 min. Switch off the standby mode and power up the x-ray tube to full power (>50kV) and wait at least 30 minutes.
   7. Start the control software and click on RESET TPF. Choose Tugsten filter and set position. Go to Position to fix the position of the Tungsten filter
2. Sample preparation and measurement
   1. Make sure that the samples are available as fine powder. If necessary, grind the samples gently at low temperatures to provide a fine powder.
   2. Fill the samples into special glass capillaries with an external diameter of approximately 2 mm, avoiding any air entrapment in the capillaries. Seal the glass capillary with wax and place it carefully into the capillary holder.
   3. Switch on the motor for sample rotation and close the vacuum valve until the pressure is beneath 5 mbar.
   4. In the software, fix the measurements setting by selecting a position resolution of 1024. Fix the exposure time to 1200 s.
   5. Setup the energy limits by clicking on the tap Tools, then click on energy and resolution, and click on restart. Setup the energy limits to a suitable range between 400-900.
   6. Open the safety shutter and start the measurements. Ensure that the measurement window shows a maximum of 80 counts per second. If this is not given, adapt the filter position.
3. Data analysis
   1. Export the data as p00 files. The data consists of the intensity of transmission and absorption versus the channel number and the diffraction angle.
   2. Transfer the data for evaluation to a statistical software and correct the data by normalizing the intensities using the scattering mass measured with the Tungsten filter.
   3. Create a plot of normalized intensity versus two times the diffraction angle [(2Θ) 2xtheta].
   4. Use the function of “screen reader” to find diffraction peaks in SAXS and WAXS regions.
   5. Apply Bragg's equation to compute the diffraction peaks with maximum intensity into short and long d-spacing for WAXS and SAXS regions, respectively.
   6. Calculate the ratios of the peak position of the SAXS region to find out the crystal symmetry of the lipids (e.g., lamellar, hexagonal, cubic).
   7. Use the main diffraction peak of the SAXS region to quantify the crystallite thickness (D). Fit the peak into a Gaussian function via classical least squares and obtain the FWHM by clicking on Analysis, Peaks and baselines, Peak analyzer, Open dialog.
   8. On the popped out window, select the option “Fit Peaks Pro”. Select a constant baseline with y = 0, and select the main diffraction peak of the SAXS region and click on “Fit control” to select the peak fit parameters.
   9. Choose the GaussAmp function. Set the parameters y_0, xc_1 and A_1 as fixed and obtain the FWHM from the fit. Use the Scherrer equation to compute the crystallite thickness.

3. Dissolution tests

1. API release from coated multiparticulate systems
   1. Use USP apparatus 2 (paddle) for dissolution studies.
   2. Fill the dissolution test vessels with phosphate buffer pH 6.8, and heat to 37 °C.
   3. Weigh triplicates of samples of coated particles equivalent to a single dose of API, and place the samples into dissolution test vessels.
   4. Start the paddle at a speed of 100 rpm.
   5. Set the auto-sampler to take samples of 1 mL at the following sampling points: 30 min, 60 min, 90 min, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 18 h, and 24 h.
   6. Analyze the samples via a suitable HPLC method^5,7,17.
   7. Analyze the data by plotting the cumulative API release versus time.
   8. Carry out the experiments for stored samples under long-term (25 °C, 60% relative humidity) and accelerated (40 °C and 70% relative humidity).
2. API release from solid lipid-nanoparticles (SLN)
   1. Prepare simulated lung fluid (SLF) by mixing 0.02% (w/w) of dipalmitoylphosphatidylcholine in Dulbecco's phosphate buffer saline (D-PBS), with the following composition: KCl (2.683 mM), KH₂PO₄ (1.47 mM), NaCl (136.893 mM), Na₂HPO_4·2H₂O (8.058 mM), CaCl_2·2H₂O (0.884 mM), and MgCl_2·6H₂O (0.492 mM). Pre-heat it at 37 °C.
   2. Use dialysis cassettes with a cellulose membrane bag of a controlled cut-off of 7,000 Da in triplicate for each sample.
   3. Assign one dialysis cassette for each sampling time: 0.5 h, 1.5 h, 3 h, 5 h, 7 h, and 24 h. Hydrate the dialysis cassettes for 2 min by immersing them in SLF. Then, carefully dry their surface with soft paper towels.
   4. Inject 3 mL of the sample (lipid-nanosuspension), equivalent to 600 µg of dexamethasone, into each cassette.
   5. Immerse each cassette in 200 mL of SLF at 37 °C (sink conditions) and agitate the system at 125 rpm.
   6. Take 200 mg samples from inside the cassette using a syringe at each determined sampling time.
   7. Determine the API content using the developed HPLC-MS method¹⁸.
   8. Calculate the API released from SLN by mass balance, according to¹⁸, briefly as the difference between the total amount of API in SLN and the remaining amount of API after sampling.
   9. Repeat the process for stored samples.

Results

Correlation between polymorphic transition of lipid and API release in lipid-coated API crystals:
API crystals coated with glycerol monostearate are measured via DSC and x-ray directly after coating and after 3 months of storage under accelerated conditions (40 °C, 75% relative humidity)⁷. Glyceryl monostearate is a multiphasic system containing 40%-55% monoglycerides, 30%-45% diglycerides, and 5%-15% glycerides, mainly tristearin¹⁹. The polymo...

Discussion

Powder x-ray diffraction and DSC were described in this manuscript as gold standards for the solid-state analysis of LBEs. Powder x-ray diffraction has the outstanding advantage of processing the measurements in situ, with minimum solid-state manipulation of samples during the measurements. Moreover, the same-filled capillaries can be stored under different conditions after initial measurements to investigate the solid-state alteration during storage. In this work, we focused on the wide- and small-angle regions...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CaCl2·2H2O	Sigma-Aldrich	223506
Cassettes with a cellulose membrane bag with a cut-off of 7000 Da, Thermo Scientific Slide-A-Lyzer 7K	Fisher Scientific Inco, USA
Control software of x-ray system	HECUS dedicated house equipment
Control unit of x-ray system	HECUS dedicated house equipment
Differential scanning calorimeter (DSC) aluminum crucibles and lids	Netzsch, Germany
Differential scanning calorimeter DSC 204 F1 Phoenix (NETZSCH, Germany).	Netzsch, Germany
Dipalmitoylphosphatidylcholine (DPPC)	Sigma-Aldrich	850355P
Dissolution paddle apparatus II, Erweka DT 828 LH	Erweka GmbH, Langen, Germany
Dynasan 116	IOI OLEO		Tripalmitin
Geleol	Gattefosse		Glyceryl monosterarate
KCl	Sigma-Aldrich	529552
KH2PO4	Sigma-Aldrich	P0662
Kolliphor P 188	BASF Chem Trade		Poloxamer 188
MgCl2·6H2O	Sigma-Aldrich	M2670
Na2HPO4·2H2O	Sigma-Aldrich	S9763
NaCl	Sigma-Aldrich	S9888
Netzsch DSC 204F1 Software Version 8.0.1	Netzsch, Germany	6.239.2-64.51.00
Origin Pro (OriginLab, Northampton, MA) (statistical software	OriginLab, Northampton, MA
Proteous Analysis Software	Netzsch, Germany
Tween 65			Polysorbate 65
Witepsol PMF 1683	IOI OLEO		Triglycerol ester of stearatic/palmitic acid (partially esterified)
Witepsol PMF 282	IOI OLEO		Diglycerol ester of stearic acid
X-ray HECUS system composed of a point-focusing camera and two linearly positioned sensitive detectors	HECUS dedicated house equipment