Particle Size And Surface Morphology

Published Date: 02 Nov 2017

1.8.1. Particle size and surface morphology4, 12

Electromagnetic radiation of shorter wavelengths must be used to observe the structures smaller than 1µm. Electron beams present this possibility. The development of electron microscopes has resulted in instruments that are able to routinely achieve magnifications of the order of 1 million and that can disclose details with a resolution of up to about 0.1 nm4.

The scanning electron microscope (SEM) is one of the most versatile instruments widely applied to surface microstructure imaging. SEM is a type of electron microscopy that images the sample surface of a solid specimen by using a focused beam of high-energy electrons.

Fundamental principles

The Scanning process and image formation in SEM depends on signals produced from elastic and inelastic interactions between the high energy electron beam and the specimen surface. When the primary electron beam bombards on sample surface, energetic electrons penetrate into the sample surface forming an excitation zone, known as the interaction volume before they collide with the specimen atom. The shape and size of the interaction volume depends on the accelerating voltage and atomic number.

Instrumentation

The major components of an SEM include the electron gun, electron lenses, sample stage, detectors, data output devices, and the vacuum system. Fig: 11 show a structure of a conventional SEM.

Figure: 11 Schematic diagram of a Scanning Electron Microscope4

Application

SEM is routinely used to analyze shapes and surface topography of samples. It is used to analyze spatial variation in chemical compositions by using elemental maps, and spot chemical analysis. It is also used to identify the microfabric and crystalline orientation of materials Though there are few limitations associated with SEM such as its applicability only for solid sample which are stable under vacuum, inability to detect very light elements (H, He,Li), and extensive sample preparation for nonconductive materials.

1.8.1(B) Transmission Electron Microscopy (TEM) 4, 5

Transmission Electron Microscope (TEM) is a type of microscopy technique which operates on the same basic principle as the light microscope except TEM uses a beam of electrons, instead of light. The image is formed by the interaction of the sample specimen when electron beams are transmitted through it. Due to the small de Broglie wavelength of electrons, it is possible to get significantly higher resolution down to 0.1 nm in TEM over light microscopy.

Fundamental principle

Image formation by TEM is based on the interaction of an electron beam with the object using various electron scattering mechanisms, illumination conditions, as well as the action of objective lens and arranged apertures. The TEM image contrast is due to elastic scattering of electrons. There are various contrast modes to improve the image quality. These modes include: bright and dark field; diffraction contrast; and phase contrast.

Figure: 12 Schematic diagram of the cross section of a Transmission Electron Microscope4

Instrumentation

The schematic diagram for a conventional TEM with its major components is depicted in Figure:12. In TEM, the emission source of electrons is a tungsten filament or lanthanum hexaboride source. They are also known as electron gun. Electromagnetic lenses are used to accelerate and focus the electrons into a very thin beam by varying the magnetic field of electromagnetic lenses. The interior of the microscope is evacuated to low pressure typically 10-4 Pa in order to minimize scattering of the electrons by air molecules and to increase the mean free path of the electron gas interaction. Depending on the density of the sample specimen used, some of the electrons will be scattered while some will be unscattered and hit at the bottom on to a fluorescent screen or on a layer of photographic film. The image can be detected by a sensor.

Preparation of sample

Preparation of samples for TEM analysis is specific to the material under study. For pharmaceutical and material sciences, the powder in the solid state is dissolved or dispersed in solvent and deposited onto a support mesh known as "grid". Usually a grid is 2.5 – 3 mm in diameter, with a 50-400 mesh and made up of copper, molybdenum, gold or platinum. Biological samples can be fixed onto the grid using a negative staining material such as uranyl acetate or by plastic embedding.

Application

Combined with good spatial resolution, ultra high magnification, TEM is widely used to obtain structural and compositional information of various materials. Recently, High resolution TEM (HRTEM) has been used to obtain a resolution of 0.2 nm.

1.8.2. Surface charge of the nanoparticles

The Zeta potential of the nanoparticles was determined by laser Doppler anemometry using a Malvern Zetasizer also called Doppler Electrophoretic Light Scatter Analyzer11.The zeta potential, an important parameter when considering the stability of the nanoparticles in vitro12. The more negative or positive values of zeta potential are related to more stable particles; more repulsion between particles reduce the particle aggregation4. Mucoadhesion, on the other hand, can be promoted by a positive zeta potential value. The mucus layer itself is at a neutral pH value an anionic polyelectrolyte. Consequently, the presence of the positively charged groups on the particles could lead to electrical charge interactions between the mucus and the particles21.

The most widely-used theory for calculating zeta potential was developed by Smoluchowski in 1903. The theory is based on electrophoresis and can be expressed as4:

μ = ζε/η where,

(μ) is the electrophoretic mobility,

(ε) is the electric permittivity of the liquid,

(η) is the viscosity and

(ζ) is the zeta potential.

Table: 2 Zeta potential for colloids in water and their stability4

Zeta potential (mV)

Stability behavior of the colloid

0 to ±5

Rapid coagulation/flocculation

from±10 to ±20

Incipient instability

from±20 to ±40

Moderate stability

from±40 to ±60

Good stability

More than ±61

Excellent stability

Basic principle and Instrumentation4

Laser Doppler Electrophoresis (LDE) is based on the combination of electrophoresis and Laser Doppler Anemometry (LDA). It is used to measure velocities and thereby zeta potential of colloid particles. The technique is based on the measurement of light scattering to the determine particle size for diluted dispersions or suspensions when particles flow through a series of interference fringes. Most widely used instrument available is Zetasizer® with standard cell.

Figure: 13 Schematic of a Laser Doppler Electrophoresis instrument4

1.8.3. Fourier Transform Infrared Spectroscopy (FTIR) 4, 5

Advancements in computing techniques have enabled FTIR to become a popular tool to characterize various types of materials including polymers. FTIR is used for both qualitative and quantitative purposes. Molecular reaction mechanisms of biomolecules have been studied using time resolved FTIR. In Pharmaceutical research, FTIR is used to identify and analyze structure of drugs, excipients, polymorphism and dissolution. Drug polymer interaction studies can be performed using this technique in dosage forms containing nanoparticles. The FT-IR spectra of pure drug and nanoparticles loaded with drug were recorded to check drug polymer interaction and stability of drug22.

1.8.5. Entrapment and loading efficiency

Drug entrapment or encapsulation efficiency is a percentage value that describes the quantity of the drug material in the nanoparticles out of the total amount used in the process5. The drug content (or drug loading) percentage is the drug amount compared to the nanoparticle mass4. The entrapment into the nanoparticles is described by two important parameters: theoretical drug loading, which is the ratio between mass of drug used in synthesis and mass of polymer used in synthesis, and nanoparticle recovery, which is the ratio between mass of nanoparticles recovered and mass of polymer and drug used in synthesis. The drug content is calculated by the ratio of mass of drug in nanoparticles to mass of nanoparticles recovered24, and the drug entrapment by the ratio of mass of drug in nanoparticles to mass of drug used in synthesis. The quantitative determination of active component entrapped in nanoparticles is done by centrifugation method25.

The redispersed nanoparticles suspension was centrifuge to separate the free drug in the supernatant. Concentration of free drug in the supernatant was determined by UV-Vis spectrophotometyrically at desired wave length after suitable dilution if necessary23, 26.

The encapsulation efficiency was determined by using the following formula23:

Encapsulation efficiency (%)

= [1-(Drug in supernatant liquid / Total drug added)] ×100

The percentage drug loading capacity was determined using the following formula3:

% Drug loading

= [(Total amount of drug - Amount of free drug) /Nanoparticles weight]×100

1.8.6. Drug release study

Nanoparticles exhibit their special drug delivery effects in most cases by direct interaction with their environment, i.e., their biological environment. Drug release may occur by11.

Desorption of surface bound drug

Diffusion through the nanoparticle matrix

1.8.6(A) Methods of Measurement of Drug Release4, 11

For characterization purposes and for quality control reasons, the determination of the in vitro release of drug from nanoparticles is important. United States Pharmacopoeia (USP) methods are generally used to evaluate drug release profiles of conventional and novel drug delivery systems of macro size by using any of the USP-recommended dissolution test apparatus11. In case of micro and nanoparticulate systems, these apparatus are not usable due to the following reasons4:

Difficulty to achieve sink conditions with nanoparticles having a very high surface area in the existing USP methods.

Difficulty to separate dissolved drug from undissolved particulates while sampling.

Need for specific enzymes to release the drug from biodegradable polymeric particulates (colon-specific microparticulates).

Need for unconventional conditions of pH or temperature for specialized nanoparticulates (pH/temperature-sensitive nanoparticles).

. The following methods for the determination of the in vitro release have been used:

1. Side by side diffusion cells with artificial or biological membranes

2. Dialysis bag diffusion technique

3. Reverse dialysis sac technique

4. Ultracentrifugation

5. Ultra filtration (Centrifugal) technique

The dialysis technique is generally preferred10. Various researchers have proposed different methods with one common strategy of using synthetic membrane bag with specified porosity to hold the sample. The bag containing the sample is immersed in the recipient fluid, which is stirred at a specified rpm. The samples are withdrawn at regular intervals and are analyzed for the drug content4.

1.8.7. FACTORS AFFECTING DRUG RELEASE

Besides polymer erosion, a number of interrelated factors govern rate of release from particles. Mainly the physicochemical properties associated with particles such as size, shape, porosity, morphology etc11,27. These, in turn are influenced by a variety of factors, for example; method of preparation, formulation parameters to name a few10.

DRUG RELEASE KINETICS -MODEL FITTING OF THE RELEASE DATA

In order to analyze the drug release mechanism, in vitro release data were fitted into a zero-order, first order, Higuchi, Korsmeyer-peppas model28. Drug dissolution has been described by kinetic models in which the dissolved amount of drug (Q) is a function of the test time, t or Q=f(t). Some analytical definitions of the Q(t) function are commonly used, such as zero order, first order, Higuchi, Korsmeyer–Peppas models29.

1.9.1. Zero order kinetics4

The zero order rate Equation describes the systems where the drug release rate is independent of its concentration.

Q1 = Q0 +K0 t

Where;

Q1 the amount of drug dissolved in time t,

Q0 is the initial amount of drug in the solution (most times, Q0=0)

K0 is the zero order release constant.

ft = K0 t

Where;

ft = 1-(Wt/W0) and ft represents the fraction of drug dissolved in time t and

K0 the apparent dissolution rate constant or zero order release constant.

In this way, a graphic of the drug dissolved fraction versus time will be linear if the previously established conditions were fulfilled.

Use:

This relation can be used to describe the drug dissolution of several types of modified release pharmaceutical dosage forms, as in the case of some transdermal systems, as well as matrix tablets with low soluble drugs, coated forms, osmotic systems, etc.

1.9.2. First order kinetics4

The first order Equation describes the release from a system where the release rate is concentration dependent.

Kinetic equation for the first order release is as follows

Log Qt = log Q0 + K1 t/2.303

Where Qt is the amount of drug released in time t,

Q0 is the initial amount of drug in the solution and

K1 is the first order release constant.

In this way a graphic of the decimal logarithm of the released amount of drug versus time will be linear. The pharmaceutical dosage forms following this dissolution profile, such as those containing water soluble drugs in porous matrices, release the drug in a way that is proportional to the amount of drug remaining in its interior, in such way, that the amount of drug released by unit of time diminishes.

1.9.3. Higuchi model4

Higuchi describes drug release as a diffusion process based in the Fick’s law, square root time dependent.

Qt = KH t1/2

Where KH is the Higuchi dissolution constant treated sometimes in a different manner by different authors and theories. This relation can be used to describe the drug dissolution from several types of modified release pharmaceutical dosage forms, as in the case of some transdermal systems and matrix tablets with water-soluble drugs.

1.9.4. Korsmeyer–Peppas model4

To find out the drug release mechanism first 60% drug release data can be fitted in Korsmeyer–Peppas model which is often used to describe the drug release behavior from polymeric systems when the mechanism is not well-known or when more than one type of release phenomena is involved.

Log (Mt / M∞) = Log KKP + n Log t

Where,

Mt is the amount of drug release at time t.

M∞ is the amount of drug release after infinite time.

KKP is a release rate constant incorporating structural and geometrical characteristics

n is the release exponent indicative of the mechanism of drug release.