UNIVERSITÀ DEGLI STUDI DI PARMA CENTRO INTERDIPARTIMENTALE MISURE

Corso di perfezionamento in TECNICHE DI MICROSCOPIA A FORZA ATOMICA

L'utilizzo dell'AFM nello Sviluppo di Prodotti Farmaceutici

Dott.ssa Francesca Buttini, Dipartimento Farmaceutico,Università degli Studi di Parma

(francesca.buttini@unipr.it)

Parma, 29 maggio 2009

The Atomic Force Microscope (AFM) The atomic force microscope (AFM) is a very high-resolution type of scanning probe microscope, with demonstrated resolution of fractions of an Angstrom, more than 1000 times better than the optical diffraction limit. The AFM was invented in 1986, and is one of the foremost tools for imaging, measuring and manipulating matter at the nanoscale.

AFM is one type of scanning probe microscope, which is used to image surface structures on a nm or even sub-nm level and to measure surface forces.

The AFM consists of a microscale cantilever with a sharp tip (probe) at its end that is used to scan the specimen surface. The cantilever is typically silicon or silicon nitride with a tip radius of curvature on the order of nanometers (~10-50 nm).

Atomic Force Microscopy:

Modes of Operation In contact mode, the probe is essentially dragged across the sample surface. During scanning, a constant bend in the cantilever is maintained. As the topography of the sample changes, the z-scanner must move the relative position of the tip with respect to the sample to maintain this constant deflection. (deformation and damage of soft samples (e.g., biological and polymeric materials)

In non-contact mode, both the tip-sample separation and the oscillation amplitude are on the order of 1 nm to 10 nm, such that the tip oscillates just above the surface contamination layer, essentially imaging the surface of, for example, the adsorbed surface moisture. Instead of scanning the sample at constant deflection, the surface is scanned at constant reduction of the oscillation amplitude. As a result the tip is not in mechanical contact with the surface during the scan.

Tapping mode tends to be more applicable to general imaging in air, particularly for soft samples, as the resolution is similar to contact mode while the forces applied to the sample are lower and less damaging.

The only real disadvantages of tapping mode relative to contact mode are that the scan speeds are slightly slower and the AFM operation is a bit more complex, but these disadvantages tend to be outweighed by the advantages.

In tapping mode, the cantilever oscillates close to its first bending mode resonance frequency, as in non-contact mode. However, the oscillation amplitude of the probe tip is much larger than for non-contact mode, often in the range of 20 nm to 200 nm, and the tip makes contact with the sample for a short duration in each oscillation cycle.

Surface Roughness

The root-mean squared surface roughness (rms) of each sample was calculated from the AFM height data over a 10 µm x 10 µm area using equation:

where n is the number of points in the topography profile and yi is the distance of asperities (i) from the centre line. €

Rrms =1n

yi2

i=1

n

∑

AFM in Pulmonary Drug Product Development

1.  Nebuliser: aqueous solution or suspension aerosolized by air-jet, ultrasound, pumps

2.  Dry Powder Inhaler (DPI): drug products designed to dispense powders for inhalation. Driven by patient inhalation

3.  Metered Dose Inhaler (MDI): drug dissolved or suspended with propellant in pressurized dispenser and aerosolized using a metering valve and actuator

Deposi'onprocessthatcausestheinspiredpar'clestobecapturedin

respiratorytractthroughcontactwithwetspace

MECHANISMS impaction sedimentation interception diffusion

FACTORS (patient variability)

particle velocity drug particle aerodynamic diameter

> 5 µm

2 - 5 µm

< 2 µm

Powder for Inhalation: The Pharmaceutical-Technology Paradox

Particles of inhalation powder have to be small for aerosolization and deposition, but large enough to

allow metering during manufacturing of dosage form.

“Formulation Strategies” to overcome the problem

1.  Ordered Mixtures with a Carrier 80-120 µm

Separation Energy Measurements A way to get information on the surface forces which are acting in a system and to illustrate the functioning of an AFM, is to use the “Force-Mode”.

This is done by moving the tip towards and away from the surface. Recording photodetector signal as a function of z-piezo elongation yields a curve which can be interpreted as a force-vs.-distance-curve. In this work this technique has been employed to measure the drug-carrier adhesion force modifying the original cantilever

Force of Adhesion

5 µm

Area: Wad (µJ)

Apex: Fmax (nN)

Preparation of Drug Probes Colloid probes are prepared by mounting an individual drug particle (approximate diameter 3-5 µm) onto a V-shaped tipless silicon nitride cantilever (spring constant k = 0.58 N/m, Veeco Instruments Ltd., Cambridge, UK) using a quick-setting epoxy resin together with the custom-built microscope.

transitionmicroscope

glassslide

securingboltPTFEslideholder

z

x

y

x,y,ztransitionstagewithcantileverholderandcantilever(red)

NP-O10 Veeco Tipless probe

The Probe Spring Constant CANTILEVER: is a small micro-precision- machined rectangular or triangular piece of silicon or silicon nitride with a shiny surface. The spring constant of a cantilever can be estimated from its geometry and the properties of the material it is made from: thicker and shorter ones tend to be stiffer and have higher resonant frequencies. Cantilevers with spring constants between 0,005 N/m and 40 N/m are the most common used.

The extension of springs can be described by HOOKE’S LAW:

F= -k*s The force F you need to extend the spring depend on the range s by which you extend it. You can allocate a spring constant k to any spring.) The effective spring constant can be determine measuring the resonant frequency of the different cantilever and k is calculated using Cleveland Eq.:

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k = (2π )2 M1

(1/v12) − (1/v2

2) V1: unloaded res frequency V2: resonant frequency with a mass M1

Force of Adhesion using AFM

100 µm

A B

20 µm

5 µm

Force separation measurements are collected using layered imaging mode to produce multiple force distance curves (n = 250) between each drug probe at three surface sites over a 10 µm x 10 µm area. As the variability in probe contact radius geometry was unknown each study was performed at least in triplicate (n = 3-4 probes for each measurement).

SEM Microparticles Morphology

Bud-PVA88 Bud-PVA80

Bud-PVA-PVP Bud-0,01PVA Bud-0,01PVP

Budesonide r.m.

5 µm 5 µm

5 µm 5 µm 5 µm

3 µm

SEM Carrier Morphological Analysis

Smoothed Lactose Lactochem® Lactopress®

100 µm 100 µm 100 µm 20 µm 20 µm 20 µm

2.0µm

2.0µm

2.0µm

Roughness: 235.5 ± 11.0 nm Roughness: 196.2 ± 31.4 nm Roughness: 770.7± 47.5 nm

Carrier AFM Topography Analysis

Smoothed Lactose Lactochem® Lactopress®

Lactose Surface Topography

AerodynamicResults(NGI@30L/min)

(n=3,mean±StDev.)

Smooth Lactose Lactopress Lactochem

FPF%

(<5µm)

MMAD

(µm) GSD

FPF%

(<5µm)

MMAD

(µm) GSD

FPF%

(<5µm)

MMAD

(µm) GSD

Budesonide 29.1±0.7 1.68±0.0 2.10±0.05 14.6±0.7 3.71±0.2 2.11±0.21 17.7±0.9 3.16±0.4 2.32±0.12

Bud-PVA88 23.9±2.0 4.86±0.1 2.16±0.04 30.8±1.8 4.61±0.2 2.07±0.05

Bud-PVA80 22.3±1.8 5.47±0.3 2.09±0.06 31.1±1.3 5.26±0.1 2.21±0.04 20.1±0.7 5.75±0.1 2.05±0.06

Bud-PVA-PVP 31.3±1.7 5.41±0.4 2.28±0.09

Bud-0.01PVA 52.8±1.0 2.29±0.1 2.18±0.04 34.5±0.8 2.71±0.4 2.10±0.02 13.5±2.6 3.31±0.1 2.21±0.04

Bud-0.01PVP 52.3±1.5 2.21±0.1 2.23±0.10 20.7±3.3 3.39±0.2 2.30±0.03

Drug Aerodynamic Distribution using different Lactose carrier

0

10

20

30

40

50

60

70

Troath Pre-Separator

Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 Stage 6 Stage 7 Filter

Dru

g D

ep

osi

tio

n (

%)

Lactochem

Lactopress

Smooth Lactose

Drug‐CarrierAdhesionForce:AFMResults

Smooth Lactose Lactopress Lactochem

Fmax (nN) Wad (µJ) Fmax (nN) Wad (µJ) Fmax (nN) Wad (µJ)

Budesonide 410 ± 182 44 ± 27 377 ± 213 37 ± 30 274 ± 122 18 ± 15

Bud-PVA88 125 ± 74 10 ± 7 - - - -

Bud-PVA80 177 ± 36 14 ± 6 - - - -

Bud-PVA-PVP - - 248 ± 97 16 ± 12 - -

Bud-0.01PVA 241 ± 82 22 ± 13 365 ± 270 30 ± 30 303 ± 174 20 ± 18

Bud-0.01PVP 139 ± 81 9 ± 5 244 ± 97 27 ± 11 - -

?

F. Buttini. J Pharm Sci. 2008 Mar; 97(3):1257-67.

SEM:Analysisofdrug‐carrierblends

Bud RM/Smooth Lactose Bud PVA88/Smooth Lactose

50 µm 50 µm

Smooth Lactose Topography Imaging and Rugosity Analysis

Roughness Surface Value: 227.1 ± 45.2 nm (mean value ± standard deviation, n=6)

Drug-Carrier Interaction for a novel antiasthmatic drug

Problems: low dose (1 µg) drug particle aggregation

The interaction force is too big!

Rappresentazione della forza di adesione mostrata da lotti di polvere micronizzata contro il lattosio levigato, (n=2250 ± dev.standard). Il CHF materia prima ha mostrato due popolazioni di dati.

Valori di Forza massima di adesione (Fmax) e lavoro di adesione (Wad) tra farmaco e carrier. (n=2250±dev.standard).

Smooth Lactose Batch

Fmax (nN) Wad (µ J)

11.03 ± 1.27 413.15 ± 131.85 CHF 4226 pure

42.68 ± 4.63 2115.69 ± 344.31

CHF SD 22.46 ± 3.17 957.28 ± 290.41

CHF:Lat 50:50 12.89 ± 3.34 711.87 ± 221.31

Lactose SD 27.71 ± 5.65 1991.62 ± 369.30

Budesonide pure 57.40 ± 6.56 4266.79 ± 430.78

CHF:Bud SD 1:100 22.25 ± 3.13 1430.36 ± 328.37

Analisi SEM delle microparticelle

10 µm 10 µm

CHF materia prima verso lattosio levigato

The Drug Probe: A sphere-like shape decreases the data variability

Forza di Coesione farmaco-farmaco

•  La forza di coesione (Fco) tra la singola particella di Farmaco materia prima incollata all’estremità del cantilever e l’agglomerato di farmaco materia prima è stata misurata utilizzando la seguente equazione:

•  dove deflmax (nA) rappresenta il valore di massima deflessione del cantilever, s la pendenza (nA/nm) della curva di allontanamento del cantilever dall’agglomerato e k il valore di spring constant (nN/m) del cantilever.

•  A causa della bassa durezza ed irregolarità del substrato (agglomerato), le curve sono state ottenute manualmente. Sono state condotte 50 misurazioni da cui sono stati ricavati i valori di defl e s per la misura della forza di coesione.

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Fco =defl maxs

⋅ k

Tentativo eseguito per la determinazione della forza di coesione Farmaco-Farmaco

Fad: 62.90 nN ± 11.75 (mean ± SD, n=50)

Cantilever con particella di ma te r i a p r ima ve rso agglomerato di farmaco (superficie soffice!)