Nanomaterial Drug Products: Current Experience and...

PQRI Workshop on Nanotechnology

Nanomaterial Drug Products: Current Experience and Management of Potential Risks

January 14-15, 2014 USP Meeting Center

1:40 pm

Analytical Considerations for the Characterization of Nanomaterial Drug Products

Christie Sayes, Ph.D.

RTI International

[email protected]

Simplified Process Flow for Nanomaterial Drug Products R&D

2

Synthesis & Formulation

Physical & Chemical

Characterization

Safety & Efficacy

Evaluation

© Christie M. Sayes 2013

Detailed Process Flow

3

Chemical Synthesis

Dose-Response Size range and distribution

Amount (weight or number

per unit volume)

Impurities

Structure (organic,

polymeric)

In vitro Sciences

In vivo Sciences

Knockdown, transgenic

Surface characteristics

Synthesis & Formulation

Physical & Chemical

Characterization

Safety & Efficacy

Evaluation

Time Course

Pre-clinical, clinical

Epidemiology

Physicochemical

Properties

Liquid v. Aerosol Phase

Emulsification

Stability and Expiration

Dating

Formulations

Dose Metrics

Morphology (crystalline)


Synthesis Approaches

1 mm 0.1 nm 1 µm

4

Chemical methods: •Organic synthesis •Chemical reduction •Aerosol seeded-growth

Bottom-up strategy

Silver salt

NP

NP

NP

NP

Top-down strategy

Physical methods: •Photolithography •Laser-beam processing •Mechanical techniques


Nanomaterial Drug Products in the Real World

• Precursor

• Reducing agent

• Capping agent

• Molar ratio of precursor to reducing agent

• Molar ratio of precursor to capping agent

• Reaction temperature

• Reaction time

• Cleanliness of glassware

Nanopartilce

Size

Stable

Purity

Shape

5 © Christie M. Sayes 2013

6

Chemical Properties on the Nanometer Size Scale Lipid/Protein

Oxidation

H2O2

OH·

Fe2+

Fenton Reaction

Protein Adhesion

Inorganic Molecule Adhesion

O2

O2.-

Spontaneous ROS

Generation

e-

O2

O2.-

Photo-oxidation

TiO2 e-

UV

CeIV CeIII

Redox Cycling

e-

Dissolution and Release of Ions

Cd2+

Fe2+ Ag+

Zn2+ Al+


7

Physical Properties on the Nanometer Size Scale

Chemical Composition

Atomic Emission Spectroscopy (AES)

Energy Dispersive X-ray Spectroscopy

(EDS)

Surface

Specific Surface Area

Zeta potential as a measure of surface

charge

Reactivity via chemical reaction

Water solubility

Iso-Electric Point (IEP)

Size and Shape

Diffraction Patterns

Nanoparticle Tracking Analysis

Electron Microscopy (EM)

Dynamic Light Scattering (DLS)


Dynamic Light Scattering as a measure of aggregation

8

individual particles

particle agglomeration

200 600 1000 1400 Size (nm)

• Dynamic light scattering is also

known as photon correlation

spectroscopy or quasi-elastic light

scattering

• The detector measured fluctuations

in the scattered laser light

• These fluctuations are due to

Brownian motion

• Typical spectra of nanoparticle

suspensions in aqueous solution


Electron Microscopy as a measure of particle size

9

As with any other microscopy method, electron micrographs are a two dimensional representation of a three dimensional object

Size of the Analysis Region

Size regime

Type of analyses

Instrument

> 100 µm to 0.1 mm

bulk analysis

> 100 nm to 0.1 µm

SEM “micro analysis”

X-ray emission spectrometry (XES) Electron backscatter patterns (EBSP) Auger electron spectrometry (AES) X-ray photoelectron spectrometry (XPS)

< 100 nm to 0.1 µm

TEM “nano analysis”

X-ray emission spectrometry (XES) Transmission electron diffraction (SAD, CBED) Electron energy loss spectrometry (EELS) Atom probe

INFORMATION OBTAINED: • Size • Morphology • Aggregation • Elemental composition

microscopy.tamu.edu © Christie M. Sayes 2013

Nanoparticle Tracking Analysis as a measure of aggregation

• Nanoparticle Tracking Analysis (NTA) visualizes nanoparticles in liquid suspension

• “Tracks” individual particles and gives information about size distribution

• Used in applications such as drug R&D, protein aggregation, and nanoparticle characterization

Microscope

Particles in liquid

Laser

Glass

Metal surface

Scattered particles


Diffraction Patterns as a measure of crystallinity

11

An electron diffraction pattern and

transmission electron micrograph of

crystalline C60 aggregates (ranging 20 to 200

nm in diameter)

30 40 50 60 70 80 2

q

An X-ray diffraction pattern of crystalline zinc

oxide nanoparticles (~30 nm in diameter)

• Diffraction patterns for nanocrystalline materials

• There are 2 common methods to acquire a diffraction pattern: • Electron diffraction (shown to the left

above) • X-ray diffraction (shown to the left below)

• Diffraction from a 3D periodic structure in a

crystal is called Bragg diffraction

• Bragg diffraction may be carried out using either light of very short wavelength like X-rays or matter waves like electrons


Specific Surface Area as a measure of porosity and area

12

• Specific Surface Area (SSA) is performed on a dry powder

• Most common method is the BET method (S. Brunauer, P. H. Emmett and E. Teller, J. Am. Chem. Soc., 1938, 60, 309.)

• BET theory aims to explain the physical adsorption of gas molecules on a solid surface and serves as the basis for an important analysis technique for the measurement of the specific surface area of a material

0 5 10 15 20 0

400

800

1200

1600

Surf

ace

Are

a (m

2/g

)

Radius (nm)

Particle surface area and diameter is an inverse squared relationship


Zeta Potential as a measure of charge

Zeta potential is the boundary of the

diffuse layer of ions within which the

particle acts as a single entity

13

NP + +

+ + + + + + + +

+ + + + + +

+ + + +

+

-

+

-

+

+

+

+

Electric Double

Layer

Slipping

Plane

Stern Layer

Zeta Potential

Surface

Charge

Distance from Nanoparticle Center

Surface < Stern Potential < Zeta Potential

What properties influence this measurement? • Surface functionalization

• Surfactant Ions

• pH of suspension medium

• Ionic strength of suspension medium

Zeta Potential Stability Interpretation for Biomedical Research

From 0 to ± 5 mV Rapid agglomeration Increased bioaccumulation Decreased efficacy

From 10 to ± 30 mV

Relatively unstable

From 30 to ± 40 mV

Moderately stable Greater biodistribution Heightened bioactivity

More than ± 40 mV

Good to excellent stability

NP

+

+ +

+ +

+ +

+ -


Redox Capacity as a measure of reactivity

14

NP

organic matter

e-

NP NP

+ +

+

+ +

+

+

+

- -

-

-

“Smart” Nanoparticles • Most chemical reactions involving nanoparticles

occur on the surface of the particle

• Stimuli responsive materials can be manipulated to enable reversible change in their physicochemical characteristics in response to changes in their environmental surroundings

• These “smart” stimuli responsive materials are sensitive to pH, temperature, signaling molecules, enzymes or drugs, and/or reactive species

• Redox chemistry on the nanoparticle surface is the most common chemical reaction

enzyme or other targeting moieties

pH

temperature

signaling molecules

reactive species

mutifunctional

nanoparticle


Water Solubility as a measure of fate and transformation

15

Nanoparticles are either hydrophobic (lipophilic)

or hydrophilic (lipophobic)

NP

nanoparticle core

micelle

PEGylation

changing pH + +

+

+ + +

+ +

+

altered surface charge

C60(OH)24

suspended in

water

C60

suspended in

toluene

OHOH

OH

OH

HO

HO

OH

OHHO

HO

HO

OH

OHHO

OHOH

C60 C60(OH)24

5000 mg/L in toluene 0.5 mg/L in toluene

10-8 mg/L in water 100,000 mg/L in water


Atomic Emission Spectroscopy as a measure of composition

16

Primarily used for metal-based nanoparticles, such as:

•Colloids

•Oxides

•Bimetallic semiconductors

It is one of the few quantitative methods in particle

physicochemical characterization.

Most common atomization method is inductively

coupled plasma (ICP)

AES can gives information on:

•Total ion concentration

•Impurities

•REDOX state

•Presence of nanoparticle inside of a cell

Advantages of the technique

Determination of 68 metals

Ability to make ppb determinations on major components of a sample

Precision of measurements by flame are better

Analysis is subject to little interference

Most interference that occurs have been well studied and documented

Sample preparation is simple (often involving only dissolution in an acid)

Instrument easy to tune and operate

excitation

source

excited

atoms

selector

detector


UV-Visible-IR Spectroscopy as a measure of functionality

17

Peaks Description After incorporated

on nanoparticle

922 cm-1 Asymmetric wagging vibrations of the

terminal dihalovinlyl group Shifts to 911 cm-1

884 cm-1 Deformation vibrations of the

cyclopropane rings Shifts to 843 cm-1

1072 cm-1 (C=O)-O- stretching Shifts to 1077 cm-1

1250 cm-1 aryl-O stretch, out-of-phase C-O-C

stretching and ring vibrations

1607 cm-1 1401 cm-

1

Asymmetry flex vibration of C=O and symmetry flex vibration of C=O

Shifts to 1583 cm-1 and 1393 cm-1

Wedge 1 Wedge 2

Sooresh et al. ACS Journal of Applied Mat & Interfaces. 2011. 3: 3779-3789

INSTRUMENTATION: • Monochromator-based • High Energy Xenon Flash • Infrared spectrometer • Tuned “windows” to avoid

interferences

INFORMATION: • Surface plasmon properties which

can be interpreted as changes to particle’s surface (i.e. functionalization)

• Shift in bands will indicate interaction with other molecules in its vicinity


0

50

100

150

200

0 2500 5000 7500 10000

Energy eV

Energy Dispersive X-ray Spectroscopy as a measure of composition

18

• Energy dispersive spectroscopy as a micro-analytical tool can yield both quantitative and qualitative results

• These results are typically a spectra with signature peaks identifying atomic species preset in the sample

Fe

Fe

Cu

Cu

Si

Os P

Fe

Fe Cu

Cu

Os

Fe2O3

nanoparticles in a membrane

bound vesicle

A549 epithelial cell

incident X-ray

ejected electron detector

shells

or orbitals

spectra


pH and Iso-Electric Point as a measures of fate and transformation

19

- - - -

- - -

- - - - -

+ + + +

-

+ +

+ +

+

+ +

-

-

particle

double layer

zeta potential

influences

cellular viability

- - - -

- - -

- - - - -

+ + + +

-

+ +

+ +

+

+ +

-

- -

-

-

- -

+ - +

- +

- +

-

- + - +

+ -

+

- +

+

- -

-

+

- +

+ + + +

+

+ + +

- - - -

+

- -

- -

-

- -

+

+ +

+

+

+ +

pH=IEP

small agglomerate

small agglomerate

large agglomerate

• The model below is a schematic diagram of the effects of pH on a nanoparticle

• Surface charge is altered when the pH is

increased or decreased

0

500

1000

1500

2000

2500

-80

-60

-40

-20

0

20

40

60

80

1 3 5 7 9 11 13

Siz

e (n

m)

Zeta

po

ten

tia

l (m

V)

pH

• Titration of nanoparticles in ultrapure water (18.2 mΩ)

• In a model nanoparticle system,

the largest aggregate size would be observed at its isoelectric point (zeta potential=0 mV)


Identification of Risk Triggers

20

Goal: Develop methods

for the purpose of building a risk

management methodology for

nanomaterial drug products

Adapted from Tsuji J S et al.

Toxicol. Sci. 2006;89:42-50 © Christie M. Sayes 2013

A Life Cycle Approach

Pristine nanoparticles

Nano-enabled Products

Product

End-of-life

Well-characterized and understood

engineered nanomaterials

Wear & tear; Use vs. misuse

Pharmaceuticals Devices

Toxicity Assessment

Pristine

Pristine

End-of-life

End-of-life

Characterization

21

Packaging


200 nm

TiO2

Sooresh et al. Environmental Science & Technology, 2013, submitted.

22

Polymer + TiO2

500 nm

5 µm

A Life Cycle Approach

Other changes

• ROS generation decreases

• Signature peaks become less intense (XRD, EDS)

• FTIR bands shift


Nanomaterial Drug Products: Current Experience and...

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