Characterization of nanoparticles & its regulatory aspects

1. Characterization of
Nanoparticles and its
Regulatory aspects
Name : Vivek Vyas
M.Pharm (Pharmaceutics)
K.B.I.P.E.R

2. 1. Particle size
2. Zeta potential
3. Structure and crystallinity
4. Nanoparticle yield
5. Drug entrapment efficiency
6. In-vitro release
7. FDA Considerations For Regulation Of Nanomaterial Containing
Products
8. References
Evaluation of Nanoparticles :

3. SIZE & SHAPE ANALYSIS :
I. MICROSCOPIC TECHNIQUES :1
a. Optical microscopy : useful in evaluating particle > 1µm.
Limitation : Tedious and less resolution.
b. Electron microscopy: Particle shape and morphology is
determined.
• Have greater resolution(10A - 1µm).
• Aqueous samples do not survive high vacuum, hence
special techniques of sample preparation are necessary
prior to electron microscopy to prevent microstructure
changes.

4. i. Scanning electron microscopy (SEM) :
• Principle : SEM images the sample surface by scanning
with a high energy beam of electrons, under high
vacuum.
• Electrons interact with the atoms that make up the
sample producing signals that contain information about
surface topography, composition, electrical conductivity
etc.
• Procedure : Samples are coated with electrically
conductive materials like gold, platinum, osmium,
graphite etc.
• Coating prevents static electric charge accumulation on
specimen during electron irradiation.

5.  Alternative to coating is to increase the bulk conductivity
by impregnation with osmium.
• Size range : 1-5nm in size.
• Disadvantages :
 Sample must be dry.
 Coating agent may change the morphology and size of the
particle.
 Provides only 2D projection.

6. SEM image of MICROSPHERE

7. SEM image of PELLETS

8. SEM image of GOLD NANOPARTICLES

9. ii. Transmission electron microscopy (TEM) :
• Principle : Beam of electrons is transmitted through an
ultra thin specimen, interacting with specimen as it passes
through it.
• An image formed from electrons transmitted through it is
magnified and focused by objective lens.
 Graphene, a carbon nanomaterial, one atom thick, is used
as platform.
It is transparent to electrons.
• TEM is of two types:
 Negative stain TEM
 Cryo – TEM

10. •Negative stain TEM : In this method, liposomes are embedded in
thin film of electron dense heavy metal stain.
• Negative stains used are ammonium molybdate, phosphotungstic
acid in case of liposomes composed of neutral or negatively
charged phospholipids.
• Uranyl acetate is used for positively charged lipids.
•
•Cryo -TEM : Sample can be viewed directly in TEM (at temp of -
196oC)
• Sufficient contrast is given to frozen sample by osmium
tetroxide.
• At such temperature, vapour pressure is low; hence, preservation
of microstructure is possible despite high vacuum.
• Disadvantages :
• Due to fluid property of dispersion, prior to freezing, thickness of
sample various from center (thin) to outside (thick film).
• Hence, actual size distribution cannot be known.

11. TEM image of MESOPOROUS SILICA NANOPARTICLES

13.  Coulter counter :
• In this technique , fine particles to be characterized are
placed in an electrolyte and a stream of suspension is
passed through an orifice between two electrodes.
• Size of fine particles is deduced from measured resistance
change between
electrodes.
• The resistance causes a voltage pulse, directly proportional
to particle volume of individual particle.
• When concentration is low, each voltage pulse corresponds
to an individual particle. Hence, size distribution can be
established.

14. Limitations :
 Only particle size > 400 nm are detected.
Particles must be spherical for accurate
volume measurements.
Particles that are non-spherical or porous
will give volumes larger than their actual
volumes.

15. II. DIFFRACTION AND SCATTERING TECHNIQUES :
 Laser light scattering technique :
• It is quick method for determination of size.
• Applied for particles < 1 µm.
• Rayleigh’s theory holds good for particles < 200nm, which
considers scattering intensity is proportional to sixth potency
of particle diameter.
•Diffraction technique:Laser diffraction can be applied for
particles >1µm
• Fraunhofer theory refers to proportionality between intensity
of diffraction and square of particle diameter.
• Scattering intensity depends on scattering angle, absorption ,
size of particles as well as refractive indices of both particles
and dispersion medium.

16. SURFACE CHARGE :1,3
• It is determined by zeta potential.
•Definition : zeta potential is the potential between tightly
bound surface liquid layer of particle and electroneutral layer.
•It provides the measure of net surface charge on the particle and
potential distribution at the interface.
•It is calculated using Helmholtz- Smoluchowski equation,
4ƞПµ
ζ = X 103
ɛE
•where ζ = zeta potential
ƞ = viscosity of dispersion medium.
µ = migration velocity.
ɛ = dielectric constant.
E = potential gradient between electrodes.

19. Surface Hydrophobicity :
Important influence on intraction of nanoparticles with
biological environment.
Several methods have been used,
1. Hydrophobic interaction chromatography.
2. Two phase partition.
3. contact angle measurement.

20. Nanoparticle yield :
% yield = Actual weight of product *100
Total weight of excipient & Drug

21. ENTRAPMENT EFFICIENCY 2
Centrifugation :
•Nanoparticle dispersion is to be centrifuged at 20,000 rpm
for 1hr to collect the supernatant liquid
•The collected liquid was filtered to measure the free
drug concentration after suitable dilution with a fresh
buffer and can be analyzed using HPLC or UV
Spectrophotometric.
•Entrapment efficiency = Wt. of drug incorporated/Wt.
of drug initially taken × 100

22. Protamine aggregation method :
•It is used for negatively charged or neutral liposomes.
•Dispersion is precipitated with protamine solution and
subsequently centrifuged at 2000 rpm.
•By analysing material in supernatant and liposomal pellet,
encapsulation efficiency can be determined.

23. CRYSTAL STRUCTURE :-1) Differential
scanning calorimetry :1,3
•It quantifies the enthalpic changes during endothermic and
exothermic phase transitions.
•Two aluminium plates are compared, one empty and the other
containing sample.
•Heat input of sample is adjusted so that its temperature
matches those of the reference pan.
•At the phase transition point, extra heat is required to
maintain the rise in temperature of the sample pan equal to that
of reference and is recorded directly.
Differential thermal analysis :
•It measures the temperature differences between reference and
sample.

25. 2) X-ray diffraction : 1,3
•When a monochromatic x-ray beam is focused on a crystal,
atoms scatter the x-ray beam, in specific pattern.
Bragg’s equation : nλ = 2d sinθ
λ - wavelength of x-rays
θ - angle of incidence
d - interatomic distance
•A typical interference pattern arises due to specific repeat
distances of the associated interlayer spacing, d.
•Larger terms for d in the region of long range order are
registered by the small angle x-ray diffraction technique.
•For short range order, registered by wide angle x-ray
diffraction technique.
•Interferences are detected in two ways, film detection and
registration of x-ray counts with scintillation counters.

26. 3) FTIR Spectroscopic Analysis:-
•The infrared spectra are recorded on Fourier Transform
Spectrometer in the mid–infrared region (MIR) within the
range (400-4500 cm-1).
•Due to the complex interaction of atoms within the molecule,
IR absorption of the functional groups may vary over a wide
range.
•However, it has been found that many functional groups give
characteristic IR absorption at specific narrow frequency
range. Multiple functional groups may absorb at one particular
frequency range but a functional group often gives rise to
several characteristic absorptions.
•Stretching & bending vibrations are varied after formulation
can be observed. Thus, the spectral interpretations should not
be confined to one or two bands only actually the whole
spectrum should be examined.

27. INVITRO RELEASE5:
▪ The key objectives of in vitro release testing are one or more of
the following:
(a) assessing the effect of formulation factors and manufacturing
methods on the drug product,
(b) routine assessment of quality control to support batch release,
(c) substantiating product label claims,
(d) establishing an in vitro in vivo correlation/relationship
(IVIVC/R),
(e) assuring product sameness under the SUPAC guidelines,
(f) as a compendial requirement

28. ▪ In vitro Dissolution:-
(a) USP I (basket): 900 mL buffer at 100 rpm;
(b) USP II (paddle): 900 mL buffer at 100 rpm;
(c) USP IV (flow through cell): 900 mL buffer at a flow
rate of 1.6 mL/min (peristaltic pump, closed loop)
through a cell (internal diameter = 25 mm) and 0.2 𝜇m
membrane disc filter;
(d) dialysis bag (MWCO 12 kDa, inner volume = 7 mL)
placed into a USP II (paddle) in vitro release tester
(outer volume = 900 mL, paddle rpm = 100).

29. •Invitro release profile can be determined using standard dialysis,
diffusion cell or ultrafiltration technique.
Dialysis Method:
• 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. As with the other methods, several
adaptations of the DM have been reported in literature with key
differences in set-up, container size, and molecular weight cut-off
(MWCO) .
With the regular dialysis technique, 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.

30. In general, the volume enclosed in a dialysis bag (inner media) is
significantly smaller than the outer media. For instance, inner
media volumes reported in literature range from 1 to 10 mL,
whereas the outer media volume is much greater, typically around
40– 90 mL.
Thus, container size will depend on the total volume of release
media required for the in vitro release study. 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

31. Diffusion cell : evaluated in phosphate buffer utilizing
double chamber diffusion cells on a shaker stand.
•A millipore hydrophillic low-protein binding membrane
is placed between two chambers.
Ultrafiltration : Nanoparticle suspension is added
directly into a stirred ultrafiltration cell containing buffer.
• Samples are collected through ultrafiltration membrane
using less than 2 bar positive nitrogen pressure and
assayed for the released drug using standard procedure.

32. FDA Considerations For Regulation Of
Nanomaterial Containing Products4
▪ General considerations for nanotechnology
products:-
■ Characterization
■ Safety
■ Environmental impact

33. Characterization Considerations
■ What are the forms in which particles are
presented to host, cells and organelles?
□ Soluble vs. insoluble particles
□ Organic vs. inorganic molecules
□ Nanoemulsions, nanocrystal colloid dispersions
□ Liposomes
□ Nanoparticles that are combination products (drug-
device, drug-biologic, drug-device-biologic)

34. ■ What are the standard tools used for characterization
of nanoparticle properties?
■ What are validated assays to detect and quantify
nanoparticles in drug product and in tissues?
■ How do we determine long and short-term stability
of nanomaterials (in various environments)?
■ What are the critical physical and chemical properties,
including residual solvents, processing variables,
impurities and excipients?
■ How do physical characteristics impact product
quality and performance?

35. ■ What are the critical steps in the scale-up and
manufacturing process for nanotechnology products?
■ How are characterization and manufacturing
procedures assessed for “personalized therapies”?
□ What is the level of characterization needed?
□ Preclinical: ADME, toxicology?
□ CMC: extent of physical characterization?

36. Safety Considerations
■ As particle size gets smaller, there may be size-
specific effects on activity, such as:
□ Will nanoparticles gain access to tissues and cells that
normally would be bypassed by larger particles?
□ Once nanoparticles enter tissues, how long do they
remain there?
□ How are they cleared from tissues and blood?
□ If nanoparticles enter cells, what effects do they have on
cellular and tissue functions (transient and/or permanent)?
□ Might there be different effects in different cells types?

37. ■ Route-specific issues:
□ Inhalation
■ Local respiratory toxicity
■ Distribution in respiratory tissues
■ Systemic bioavailability
□ Sub-cutaneous
■ Sensitization
□ Ocular
■ Intravitreal retention
□ Oral
■ Increased bioavailability
□ Dermal
■ Increased dermal and systemic bioavailability
■ Increased follicule retention
■ Distribution to local lymph nodes
■ Phototoxicity
□ IV
■ Hemocompatibility
■ Sterility
■ Different tissue distribution and half-life of API (with targeted
delivery and liposomes)

38. ■ ADME
□ What are the differences in the ADME profile, for
nanoparticles versus larger particles of the same drug?
□ Are current methods used for measuring drug levels in
blood and tissues adequate for assessing levels of
nanoparticles (appropriateness of method, limits of
detection)?
□ How accurate are mass balance studies, especially if
levels of drug administered are very low; i.e. can 100%
of the amount of drug administered be accounted for?
■ How is clearance of targeted nanoparticles
accurately assessed? If nanoparticles
concentrate in a particular tissue, how will
clearance be assessed accurately?
■ Can nanoparticles be successfully labeled
for ADME studies?

39. Environmental Considerations
■ Can nanoparticles be released into the environment
following human and animal use?
■ What methodologies would identify the nature, and
quantify the extent, of nanoparticle release in the
environment?
■ What might be the environmental impact on
other species (animals, fish, plants,
microorganisms)?

40. Current Preclinical Tests for
Safety Evaluation
■ Pharmacology
■ Safety pharmacology
■ Toxicology (including clinical pathology and histopathologic
analysis)
■ ADME
■ Genotoxicity
■ Developmental toxicity
■ Immunotoxicity
■ Carcinogenicity
■ Other

41. Adequacy of Current Preclinical
System?
■ Existing battery of preclinical tests is currently
believed to be adequate.
■ Why?
□ High dose multiples used
□ At least 2 animal species used
□ Extensive histopathology on most organs
□ Functional tests (cardiac, neurologic, respiratory,
reproductive, immune system, etc/…)
□ Extended treatment periods (up to 2 years for
carcinogenicity studies)

42. Future Testing Considerations
■ Types of preclinical screening tests that may be useful in
identifying potential risks (Screening IND?):
□ In vitro assays
□ In vivo assays
■ Role of new technologies to help identify potential toxicities:
□ Omics
□ Imaging (qualitative/quantitative)
■ What is the role of modeling:
□ In predicting exposure?
□ In predicting safety concerns?
□ In helping design of personalized therapies?

43. Are There Special Testing Requirements for
Nanotechnology Products?
■ Currently there are no testing requirements
that are specific to nanotechnology products.
■ CDER/FDA’s current requirements for
safety testing of products is very rigorous.
■ However if research identifies toxicological
risks that are unique to nanomaterials,
additional testing requirements may become
necessary.

44. •REFERENCES :
1. Vyas S.P. , Khar R.K. Targeted & Controlled Drug Delivery,
Novel Carrier Systems, CBS Publication ,2002 ,Page No.249-
277,331-387
2. Nanoparticles –A Review by VJ Mohanraj & Chen Y, Tropical
Journal of Pharmaceutical Research 2006; 5(1): 561-573
3. Jain N. K., Controlled and novel Drug Delivery, 1st edition
2001, CBS Publication; 292 - 301.
4. www.FDA.GOV/NANOTECHNOLOGY
5. X. Cao,W.W.Deng, M. Fu et al., “In vitro release and in vitroin
vivo correlation for silybin meglumine incorporated into
Hollow-type mesoporous silica nanoparticles,” International
Journal of Nanomedicine, vol. 7, pp. 753–762, 2012.
6. Google.com(images)