Characterization of Nanoparticles.

Faculty of Pharmacy, BBDNIIT
Lucknow
Characterization of
Nanoparticles
Presented by-
Manish Kumar Singh
M.Pharm, I Year
(Pharmaceutics)
Presented to-
Ms. Neelam Datt
Sr. Assistant Professor

Content
 Introduction
 Characterization of nanoparticles
 References
2

Introduction
Nanoparticles :
 The prefix “nano” comes from the ancient Greek vavoc through the
Latin nanus meaning very small.
 Nanoparticles are sub-nanosized colloidal structures composed of
synthetic or semi synthetic polymers.
 The drug is dissolved, entrapped, encapsulated or attached to a
nanoparticle matrix.
 The term nanoparticle is a combined name for both nanospheres and
nanocapsules.
3

Characterization of Nanoparticles
1. Size and surface Morphology
2. Specific Surface Area
3. Surface Charge and Electrophoretic Mobility
4. Surface Hydrophobicity
5. Density
6. Molecular weight Measurements of Nanoparticles
7. Drug Entrapment efficiency
8. Kinetic Study
9. Stability of Nanoparticles
10. Drug-Excipient compatibility studies
11. In-vitro Release Studies
12. Lamellarity
13. Phase Behaviour
14. Chemical Characterization (Liposomes)
15. Biological Characterization (Liposomes) 4

1- Size and Surface Morphology
Particle Size and size distribution:
The particle size is one of the most important parameters of nanoparticles.
The particle size and size distribution of nanoparticles can be determined
using numerous commercially available instruments.
A. Dynamic Light Scattering (DLS)-
 DLS measures brownian motion and relates this to the size of the
particles (Hydrodynamic diameter).
 Bias toward larger particles.
 We can determine polydispersity index (PDI), zeta potential and
aggregation of particles.
 Instrumentation - Zetasizer (Malvern panalytical tnstrument, UK),
Laser source, Photon detector, Polystyrene cuvettes/Quartz or optical-
quality glass cuvettes with caps.
 Dispersant – Water or whatever the dispersant used is.
5

Zetasizer
B. Nano Sight (NTA)-
 Nano sight helps in visualization and measuring nanoparticle size and
concentration with precision and accuracy.
 Nano sight instrument uses Nanoparticle Tracking Analysis (NTA) to
characterize nanoparticles from 10 nm – 2000 nm in solution.
6

 Characterization of aggregation state.
 Count of each and individual particle.
 Concentration 106 -109 particle per ml.
 Fluorescence detection.
 Low sample requirement.
Nano Sight
7

C. Scanning Electron Microscopy (SEM)-
 SEM is used to visualize the surface morphology of organisms, cells and
materials.
 Resolution is 1-2 nm.
 Can determine the elemental composition.
 Determine the size, shape, surface morphology.
Scanning Electron Microscopy
8

9
D. Transmission Electron Microscopy (TEM)-
 Resolution is 0.1 – 0.2 nm.
 Determine the internal structure or arrangements of the particles.
 Measure the size, size distribution, and morphology.
 Samples are prepared for imaging by drying nanoparticles on a grid that
is coated with a thin layer of carbon/formvar.

10
E. Atomic Force Microscopy (AFM)-
 AFM is an advanced nanoscopic technique that has been applied for the
characterization of PLA nanospheres and solid lipid nanoparticles (SLN).
 The AFM image can be obtained in aqueous medium.
F. Mercury Porositometry-
 It is equally suitable technique for sizing of nano-particulates.
 The freeze-dried nanoparticles are filled in a dilatometer under vacuum
and then measured with the help of a mercury pressure porositometer.

11
Particle Shape :
A. Scanning Electron Microscopy (SEM)-
 Resolution is 1-2 nm.
 SEM is used to visualize the surface morphology of organisms, cells and
materials.
 Determine the size, shape, surface morphology.
Gold nanoparticles Silver nanoparticles

12
B. Transmission Electron Microscopy (TEM)-
 Resolution is 0.1 – 0.2 nm.
 Determine the shape of the particles.
 Determine the internal structure or arrangements of the particles.
 Measure the size, size distribution, and morphology.
Gold nanoparticles

13
Crystallinity :
A. X-Ray Diffraction (XRD)-
 X-ray diffraction (XRD) is a powerful method for the study of
nanomaterials (materials with structural features of at least one
dimension in the range of 1-100 nm).
 XRD is also used to determine the thickness of thin films, as well as the
atomic arrangements in amorphous materials such as polymers.
 It is a powerful and rapid technique for identification of an unknown
material

14
Differential Scanning Calorimeter (DSC)-
 DSC is one of the most frequently used technique in the field of thermal
characterization of solid and liquid.
 DSC measurement information-
i. Polymorphism
ii. Degree of crystallinity
iii. Purity determination
iv. Decomposition behaviour
v. Melting/Crystallization behaviour
DSC

15
2- Specific Surface Area
Brunauer Emmett Teller (BET)-
 Gas adsorption or Nitrogen adsorption.
 Measure the specific surface area of nanoparticles including pore size
distribution.
 Determine porosity.

16
3- Surface Charge and Electrophoretic
Mobility
 The nature and intensity of the surface charge of nanoparticles is very
important as it determines their interaction with the biological
environment.
 The surface charge of colloidal particles in general and nanoparticles in
particular can be determined by measuring the particle velocity in an
electric field.
 Laser light Scattering technique, i.e. Laser Doppler Anemometry or
Velocimetry used for velocity determination.
 The colloidal stability is analyzed through zeta potential of
nanoparticles. This potential is an indirect measure of the surface
charge.
 The surface charge of the colloidal particles could also be measured as
electrophoretic mobility.

17
4- Surface Hydrophobicity
 The surface hydrophobicity of nanoparticles has an important influence
on the interaction of colloidal particles with the biological environment.
 Surface hydrophobicity can be determined by several techniques such as-
i. Hydrophobic interaction chromatography,
ii. Biphasic partitioning,
iii. Adsorption of probes,
iv. Contact angle measurements etc.
 X – ray photon correlation spectroscopy permits the identification of
specific chemical groups on the surface of nanoparticles.

18
5- Density
 Some polymeric nanoparticles specially polycyanoacrylate and poly
(methyl methacrylate) seems to have porous interior and they also
exhibit more irregular and rough surface.
 The density of nanoparticles is determined with helium or air using a gas
Pycnometer.

19
6- Molecular weight Measurements
of Nanoparticles
Molecular weight of the polymer and its distribution in the matrix can be
evaluated by gel permeation chromatography (GPC) using a refractive
index detector.
7- Drug Entrapment efficiency
 After centrifugation amount of drug present in supernatant (w)
determined by UV spectrophotometery.
 After that standard calibration curve plotted.
 Then amount of drug present in supernatant subtracted from the total
amount used in the preparation of nanoparticles (W).
 (W-w) is the amount of drug entrapped. % drug entrapment calculated
by formula-
% 𝑑𝑟𝑢𝑔 𝑒𝑛𝑡𝑟𝑎𝑝𝑚𝑒𝑛𝑡 = 𝑊 − 𝑤/𝑊 × 100

20
8- Kinetic Study
For estimation of the kinetic and mechanism of drug release, the result of
in vitro drug release study of nanoparticles were fitted with various kinetic
equation like-
i. zero order (cumulative % release vs. time)
ii. first order (log % drug remaining vs time)
iii. Higuchi’s model (cumulative % drug release vs. square root of time).
9- Stability of Nanoparticles
 Stability studies of prepared nanoparticles determined by storing optimized
formulation at 4°C ±1°C and 30°C ± 2°C in stability chamber for 90 days.
 The samples were analyzed after a time period like at 0, 1, 2, and 3 months for
their drug content, drug release rate as well as any changes in their physical
appearance (ICH Q1A (𝑅2).

21
10- Drug-Excipient compatibility
studies
 The drug- excipient compatibility studies was performed by using FT-IR
spectrophotometer.
 The FT-IR spectra of drug, polymer and formulations were analysed
separately and then correlated for incompatibility.
11- In-Vitro release studies
Release from nano-sized dosage forms can be assessed using one of the
following methods-
(a) USP I (basket type)
(b) USP II (paddle type)
(c) USP IV (flow through cell)
(d) Franz diffusion cell
(e) Dialysis method
(f) New Methods -
i. Electrochemical methods - Repetitive square-wave voltammetric
technique, differential pulse polarography.
ii. Non electrochemical methods - Calorimetry, Turbidimetry, and Laser
diffraction

22
A- USP I (basket type)-
 Rotation speed of 50, 100 rpm.
 Immersed in 900ml of media.
 Temperature maintained at 37±0.20°C.
 Required quantity of medium withdrawn
at specific time periods and same volume
of medium was replaced in the flask.
 The withdrawn samples were analyzed
using UV spectrophotometer.
B- USP II (paddle type)-
 Rotation speed of 50 rpm.
 Immersed in 900ml of media.
 Temperature maintained at 37±0.20°C.
 Required quantity of medium withdrawn
at specific time periods and same volume
of medium was replaced in the flask.
 The withdrawn samples were analyzed
using UV spectrophotometer.

23
C- USP IV (flow through cell)-
 Apparatus consist of a reservoir for the dissolution medium and a pump
that forces dissolution medium through the cell holding the test sample.
 900 mL buffer at a flow rate of 1.6mL/min (peristaltic pump, closed
loop) through a cell (internal diameter = 25 mm) and 0.2 𝜇m membrane
disc filter.

24
D- Franz Diffusion Cell-
 Franz diffusion cell is a diffusion system that is used for characterizing
drug permeation through a skin model.
 The drug product is placed on the skin surface and the drug permeates
across the skin in to a receptor fluid compartment that may be sampled
at various times.

25
E- Dialysis Method : Dialysis bag (regular dialysis)-
 In this method, physical separation of the dosage forms is achieved by
usage of a dialysis membrane which allows for ease of sampling at
periodic intervals.
 The nanoparticles are introduced into a dialysis bag containing release
media (inner media/compartment) that is subsequently sealed and placed
in a larger vessel containing release media (outer media/compartment),
agitated to minimize unstirred water layer effects.
 In the regular dialysis technique, drug released from the nanoparticles
diffuses through the dialysis membrane to the outer compartment from
where it is sampled for analysis.

26
12- Lamellarity
 Lamellarity of vesicle i.e. numbers of bilayers present in liposomes is
determined using Freeze-Fracture electron microscopy and P-31
nuclear magnetic resonance analysis.
13- Phase Behaviour
 Liposomes and lipid bilayers exhibit various phase transition that are
studied for their role in drug release.
 Phase behaviour of liposomal membrane determines properties such as
permeability, fusion, aggregation and protein binding.
 Evaluated using Freeze-Fracture electron microscopy .
 They are more comprehensively verified by DSC.

27
14- Chemical Characterization
Parameters Methods/Instrumentation
Phospholipid concentration Lipid phosphorous content using Barlett assay/
Stewart assay, HPLC
Cholesterol concentration Cholesterol oxidase assay and HPLC
Drug concentration Appropriate method given in monograph for
individual drugs.
Phospholipid peroxidation UV absorbance, GLC
Phospholipid hydrolysis HPLC and TLC
Cholesterol auto-oxidation HPLC and TLC
Anti-oxidant detergent HPLC and TLC
pH pH meter
Osmolarity Osmometer

28
15- Biological Characterization
A- Sterility-
 Sterility test is performed to check the chances of contamination.
 Aerobic or Anaerobic culture used.
 Fluid thioglycollate medium is intended for anaerobic bacteria.
 Soya-bean casein digest medium is suitable for aerobic bacteria.
B- Pyrogenicity-
 Method- Rabbit fever response test or Limulus Amebocyte Lysate
(LAL) test.
 Performed to check the microbial metabolite (Pyrogens) which cause
fever or elevate body temperature.
 Rabbit fever response test- If a pyrogenic substance is injected to rabbit
vein, an elevation of temperature occur.
 LAL Test - It is an in-vitro method. Utilizing the gelling property of the
lysate of the amebocytes of Limulus polyphemus. In the presence of
pyrogens, a firm gel is formed within 60 minutes when incubated at
37°C.

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References
1. Vyas S.P., Khar R.K., “Targeted and Controlled Drug Delivery Novel
Carrier System”, 1st Edition, 2007, CBS Publishers and Distributors,
New Delhi, Page no-206,331,356-359.
2. Khar R.K., Vyas S.P., Ahmad F.J., Jain G.K., “The Theory and Practice
of Industrial Pharmacy:, 4th Edition, 2013, CBS Publishers and
Distributors, New Delhi, Page no- 887.
3. Tiruwa R. A review on nanoparticles – preparation and evaluation
parameters. Indian Journal of Pharmaceutical and Biological Research
(IJPBR) 2015; 4(2):27-31.
4. Pal S.L.; Jana U.; Manna P.K.; Mohanta G.P.; Manavalan R.
Nanoparticle: An overview of preparation and characterization. Journal
of Applied Pharmaceutical Science 01 (06); 2011: 228-234.
5. Kant S.; Kumar S.; Prashar B. A Complete Review On : Liposomes.
IRJP 2012,3(7).

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6. Kharia A.A.; Singhai A.K.; Verma R. Formulation and Evaluation of Polymeric
Nanoparticles of an Antiviral Drug For Gastroretention. IJPSN. Volume 4.
Issue 4. January-March-2012.
7. Sharma R.; Bisen D.P.; Shukla U.; Sharma B.G. X-ray diffraction: a powerful
method of characterizing nanomaterials. Recent Research in Science and
Technology 2012, 4(8): 77-79.
8. B. Akbari.; M. Pirhadi.; Tavandashti.; M. Zandrahimi. PARTICLE SIZE
CHARACTERIZATION OF NANOPARTICLES – A
PRACTICALAPPROACH. Iranian Journal of Materials Science &
Engineering Vol. 8, Number 2, Spring 2011.
9. D’Souza S. A Review of In-Vitro Drug Release Test Methods for Nano-Sized
Dosage Forms. Hindawi Publishing Corporation Advances in Pharmaceutics
Volume 2014, Article ID 304757, 12 pages.

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