This document discusses various characterization techniques for nanostructured lipid carriers (NLCs). It describes techniques such as photon correlation spectroscopy, scanning electron microscopy, transmission electron microscopy, and zeta potential analysis that can be used to analyze the morphology and size of NLCs. It also discusses differential scanning calorimetry and X-ray diffraction for analyzing the crystallinity and structure of lipids in NLCs. Additional techniques covered include rheology for viscosity, NMR for mobility within NLC cores, and in vitro and in vivo studies for evaluating drug release and absorption.

1. EVALUATION OF NANOSTRUCTURED LIPID
CARRIERS
Presented by:
Mr. Pankaj Kumar
PhD Scholar
DPSRU New Delhi

2.  Morphology
 Photon correlation spectroscopy (PCS)
 Scanning electron microscopy (SEM)
 Transmission electron microscopy (TEM)
 Zeta potential analysis
 Zetasizer
 Degree of crystallinity and lipid modification
 Differential scanning calorimetry (DSC)
 X-ray diffraction (XRD)
NLCs can be characterized by following methods

3.  Determination of viscosity
 Rheometer
 Drug content and Encapsulation efficiency
 Mobility of materials in inner core of NLC
 NMR
 Conformational order of hydrocarbon chains
 Raman spectroscopy

4.  In vitro characterization
 In vitro release
 Dissolution studies
 Ex vivo studies
 Gut permeation studies for intestinal absorption
 In vivo studies
 Pharmacokinetics
 Pharmacodynamics

5. Photon correlation spectroscopy (PCS)
 PCS is nondestructive technique, which give
information about size based on the equivalent
hydrodynamic diameter in liquid dispersions.
 PCS measure the polydispersity index (PI) provided
by the particle size distribution of the sample.
 Low PI (0.1–0.25) indicates narrow size distribution
whereas PI above 0.5 indicates a broad size
distribution
 PCS shows NLC being non-spherical particles differ
from a nanoemulsion with respect to Brownian motion
Morphology
A. Khosa, S. Reddi, and R. N. Saha, “Nanostructured lipid carriers for site-specific drug delivery,” Biomed.
Pharmacother., vol. 103, no. April, pp. 598–613, 2018, doi: 10.1016/j.biopha.2018.04.055
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6. Scanning electron microscopy (SEM)
 SEM gives information about the surface
characteristics and three dimensional morphology of
the particle.
 For SEM Nano dispersion is usually converted into a
powder, mostly by freeze drying
 The resolving power of SEM is ∼3–4 nm which is quite
low as compared to TEM
 SEM does not provide any information about the
internal structure of the particles
 Cryo field emission scanning electron microscope
(CryoFESEM) has been successfully used to evaluate
NLC.
Morphology
M. A. Iqbal, S. Md, J. K. Sahni, S. Baboota, S. Dang, and J. Ali, “Nanostructured lipid carriers system:
Recent advances in drug delivery,” J. Drug Target., vol. 20, no. 10, pp. 813–830, 2012, doi:
10.3109/1061186X.2012.716845.
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7. Transmission electron microscopy (TEM)
 TEM usually provides a two-dimensional image
of the internal structure of the nanoparticles.
 The resolving power of TEM is around 0.4 nm
 For SEM sample needs optimum thickness
which is about a few hundred nanometers
 Cryo TEM has been successfully used to
evaluate NLC
Morphology
A. Khosa, S. Reddi, and R. N. Saha, “Nanostructured lipid carriers for site-specific drug delivery,” Biomed.
Pharmacother., vol. 103, no. April, pp. 598–613, 2018, doi: 10.1016/j.biopha.2018.04.055
7

8. Zeta potential analysis
 Zetasizer used to measure particle and molecular size
from less than a nanometer to several microns and
charge measurements
 ZP dependent on the particles, pH, ionic strength and the
types of ions present.
 ZP gives important information on the long-term stability
of nanoparticles and their tendency to agglomerate
 ZP of ±60 mV indicates excellent stability
 ZP can easily be measured using analytical instruments
based on the principle of electrophoretic/electroacoustic
mobility
Zeta potential analysis
A. Khosa, S. Reddi, and R. N. Saha, “Nanostructured lipid carriers for site-specific drug delivery,” Biomed.
Pharmacother., vol. 103, no. April, pp. 598–613, 2018, doi: 10.1016/j.biopha.2018.04.055
8

9. Differential scanning calorimetry (DSC)
 DSC used techniques for structural characterization of
particles
 Two basic types of instruments are used for DSC
determinations heat flux DSC and power
compensation DSC
 DSC is used to determine the state and crystalline
nature of lipid dispersions in their native state such as
semisolid systems and freeze-dried powders
 In NLC, DSC helps in understanding the mixing
behavior of liquid lipids and provides information
related to polymorphic transitions, crystallization
order etc.
Degree of crystallinity and lipid modification
N. V. Shah, A. K. Seth, R. Balaraman, C. J. Aundhia, R. A. Maheshwari, and G. R. Parmar, “Nanostructured
lipid carriers for oral bioavailability enhancement of raloxifene: Design and in vivo study,” J. Adv. Res.,
vol. 7, no. 3, pp. 423–434, 2016, doi: 10.1016/j.jare.2016.03.002.
9

10. X-ray diffraction (XRD)
 XRD is a technique in which scattering of X-rays by atoms of a crystal creates an interference effect and the
resultant diffraction pattern helps in identifying as well as determining crystal structure and differentiation
of its various polymorphic forms.
 It detects electron density fluctuations on a length scale according to Bragg’s law: 2dSin θ=λ
 XRD used to determine the particle size, shape and
structure of the lipid nanoparticles.
 It also be used to identify various polymorphic forms
of lipids such as glycerides
Degree of crystallinity and lipid modification
N. V. Shah, A. K. Seth, R. Balaraman, C. J. Aundhia, R. A. Maheshwari, and G. R. Parmar, “Nanostructured
lipid carriers for oral bioavailability enhancement of raloxifene: Design and in vivo study,” J. Adv. Res.,
vol. 7, no. 3, pp. 423–434, 2016, doi: 10.1016/j.jare.2016.03.002.
10

11.  Rheometers are used to measure the viscoelastic properties of NLC.
 Rheological parameters are calculated from the measured values
torque, deflection angle, and speed using conversion factors
 Rheological experiments with NLC are generally performed in order
to evaluate the effects of temperature, aging during storage,
excipients and processing parameters.
 These studies also allow the quantification of the flow of a NLC for
quality control purposes.
Rheometer
Determination of viscosity
C. Puglia and F. Bonina, “Lipid nanoparticles as novel delivery systems for cosmetics and dermal
pharmaceuticals,” Expert Opin. Drug Deliv., vol. 9, no. 4, pp. 429–441, 2012, doi:
10.1517/17425247.2012.666967
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12.  Encapsulation efficiency (EE) is a critical factor which needs to be optimized during formulation
design as it has an impact on the drug release and also on the cost-effectiveness of the formulation
 It is the percentage amount of drug that gets entrapped in the nanoparticle and reflects the
efficiency of the NLC formulation
 For lipophilic drugs, the entrapment is high as the drug gets homogenously solubilized within the
lipid
 Lipids with imperfections in the crystal structure show higher EE. In NLC, the presence of liquid
lipids increases imperfections in the crystal structure, thereby further increasing the EE
Drug content and Encapsulation efficiency
N. V. Shah, A. K. Seth, R. Balaraman, C. J. Aundhia, R. A. Maheshwari, and G. R. Parmar, “Nanostructured
lipid carriers for oral bioavailability enhancement of raloxifene: Design and in vivo study,” J. Adv. Res.,
vol. 7, no. 3, pp. 423–434, 2016, doi: 10.1016/j.jare.2016.03.002
12

13. Mobility of materials in inner core of NLC
 Nuclear magnetic resonance (NMR) is important tools to evaluate the presence of other colloidal
structures, understand the dynamic phenomena and the influence of oily nano-compartments,
characteristic of NLC type III.
 This technique is noninvasive, analysis of the same sample repeatedly is possible
 NMR active nuclei of interest are 1H, 13C, 19F and 31P
 example, mobility of oil molecules has an impact on the width of the NMR signals arising due to
variation in proton relaxation time in liquid and solid/
 semi-solid state.
Nuclear magnetic resonance (NMR)
R. H. Müller, “Lipid nanoparticles: recent advances,” Adv. Drug Deliv. Rev., vol. 59, no. 6, pp. 375–376,
2007, doi: 10.1016/j.addr.2007.05.002.
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14.  The technique is used to map chemical and structural changes in molecules from their transition between
two vibrational states. Raman spectroscopy is a useful tool to study packing, conformation and any
changes in lipid chain arrangement after loading oils.
 The Raman data, thus, revealed that oil loading did not lead to changed lipid chain arrangement;
therefore, oil incorporation into the solid lipid matrix could not be confirmed.
 The Raman spectrum of the nanoemulsion is characteristic of random coiled chains, whereas NLC
formulations clearly showed the sharp bands vs (CH2), vs (C--C) and vs (C--C), thus indicating the highly
conformational order of the acyl chains
Raman spectroscopy
Conformational order of hydrocarbon chains
R. H. Müller, “Lipid nanoparticles: recent advances,” Adv. Drug Deliv. Rev., vol. 59, no. 6, pp. 375–376,
2007, doi: 10.1016/j.addr.2007.05.002.
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15.  In vitro cell cultures, studies showed a highly efficient drug transport through target cells.
 The drug release behavior of NLC is dependent on the proportion of oil, production temperature and emulsifier
concentration. The controlled Release of drug from the NLC can result in extended half-life and protection from
enzymatic degradation.
 Drug release from NLC can either be controlled by diffusion of the drug or erosion of the matrix depending upon
whether the drug is entrapped in the core of the NLC, in the shell or in the matrix.
 The drug release behavior of NLC is dependent on the type of solid lipid used, proportion of oil, production
temperature and emulsifier Concentration
 Particles of smaller size result in faster release compared to larger particles due to increase in the surface area
and shorter diffusion path
 Cold homogenization is a better technique as it results in a core enriched model of nanoparticle minimizing burst
release and providing prolonged release
In vitro release and dissolution studies
In vitro characterization
Continue…
A. Khosa, S. Reddi, and R. N. Saha, “Nanostructured lipid carriers for site-specific drug delivery,” Biomed.
Pharmacother., vol. 103, no. April, pp. 598–613, 2018, doi: 10.1016/j.biopha.2018.04.055
15

16. Continue…
 Release studies were performed to evaluate the in vitro drug release pattern using the dialysis bag technique
 The dialysis bag with a molecular weight cutoff of soaked in release medium for 12 hours before use
 Five milliliters of each test formulation was individually put in a dialysis bag that was firmly tied
 They were then immersed in 100 mL of phosphate buffered solution (pH 6.8) containing 20% (v/v)
polyethylene glycol 400 and maintained at 37°C±0.5°C with stirring at 100 rpm
 At each sampling time point, 1 mL was withdrawn and the volume was replenished with the same volume of
fresh medium.
 The samples were filtered through a 0.45-mm membrane and analyzed using HPLC
In vitro characterization
G. Yang, F. Wu, M. Chen, J. Jin, R. Wang, and Y. Yuan, “Formulation design, characterization, and in vitro
and in vivo evaluation of nanostructured lipid carriers containing a bile salt for oral delivery of
gypenosides,” Int. J. Nanomedicine, vol. 14, pp. 2267–2280, 2019, doi: 10.2147/IJN.S194934
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17. Gut permeation studies
 Ex vivo methods provide a theoretical means of estimating human intestine absorption.
 Adequate paracellular permeability is provided by the small intestinal epithelium, a mucus layer is
present in the model, and there is also expression of transport proteins and drug metabolism.
 Ex vivo sometimes inappropriately estimate the degree of oral absorption. Despite these shortfalls, ex
vivo methods are simple, and widely used in the design and testing of potential new drugs.
 In NLCs, this study is generally used for the oral and transdermal delivery to check the permeation by
taking gut or other organ
• Intestinal perfusion
• Everted gut sac experiment
• Ussing chamber
Ex vivo studies
A. Singh, Y. R. Neupane, B. Mangla, and K. Kohli, “Nanostructured Lipid Carriers for oral bioavailability
enhancement of Exemestane: Formulation design, in vitro, ex vivo and in vivo studies.,” J. Pharm. Sci.,
vol. 0, no. 0, Jun. 2019.
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18. The purpose of studying pharmacokinetics and pharmacodynamics is to understand the drug action,
therapy, design, development and evaluation
In Pharmacokinetic studies of NLCs, determine various parameters like AUC, Cmax, Tmax, Mean
Residence Time (MRT), Mean Absorption Time (MAT), Peak Occupancy Time (POT) etc.
This study is used to check the bioavailability of drug incorporated in NLCs.
Also check safe and tolerable levels of exposure
Pharmacodynamics study, determine the drug-response or concentration-response relationships
Also, determine the Mechanism of action of drug in body, Drug-receptor interactions and therapeutic
window
In vivo studies
G. Yang, F. Wu, M. Chen, J. Jin, R. Wang, and Y. Yuan, “Formulation design, characterization, and in vitro
and in vivo evaluation of nanostructured lipid carriers containing a bile salt for oral delivery of
gypenosides,” Int. J. Nanomedicine, vol. 14, pp. 2267–2280, 2019, doi: 10.2147/IJN.S194934
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Pharmacokinetics and Pharmacodynamics studies

19. Thank you