4 physicochemical analysis of hyaluronic acid powder … · 89 4 physicochemical analysis of...

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4 Physicochemical Analysis of Hyaluronic Acid Powder for Cosmetic and Pharmaceutical Processing

Lubomír Lapčík, Eva Otyepková, Barbora Lapčíková, Michal Otyepka, Jakub Vlček and Ivana Kupská

4.1 Introduction

Granular materials are used in many different applications and industries, such as basic construction materials, agriculture, fillers for synthetic polymers, cosmetic and pharmaceutical processing and the food industry [1]. Many practical engineering applications involve handling, flow and storage of bulk solids (e.g., pelletising, particle size reduction, tableting, mixing, packaging) and so require knowledge and understanding of flow properties of the particular solids and surface energy distribution. Several conventional properties of powders are known to affect how they flow (such as particle size and shape, moisture content and surface chemistry) but these properties do not consistently correlate with experimental powder flow behaviour. This has led to the requirement for the measurement of more specific powder flow properties such as internal and wall friction, bulk density, cohesion and flow function.

Hyaluronic acid (HA), a high molecular weight (MW) biopolysaccharide, was discovered by Meyer and Palmer in 1934 in the vitreous humour of cattle eyes [2]. HA is a member of a group of similar polysaccharides that have been termed ‘connective tissue polysaccharides’, ‘mucopolysaccharides’, or ‘glycosaminoglycans’. These polysaccharides include chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and heparin. HA is a

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Hyaluronic Acid for Biomedical and Pharmaceutical Applications

linear, unbranched polymer. Meyer and co-workers found HA to be composed of a repeating disaccharide unit that consists of N-acetyl-d-glucosamine and d-glucuronic acid linked by a b (1-4) glycosidic bond [3]. The disaccharides are linked by b 1-3 bonds to form the HA chain. In addition to its presence in the vitreous body, HA occurs in many living substrates such as the extracellular matrix and synovial fluids [2-5]. In practice, HA is used in wound healing, because it supports tissue reconstruction. During the first few days of tissue repair, endogeneous HA is the predominant glycosaminoglycan present in wounds and forms the template necessary for reconstruction following injury [2]. A detailed knowledge of flow and surface properties of HA powder material is important for practical pharmaceutical applications, e.g., in drug formulation, construction of wound healing dressings, tablets, capsules, dry powder inhalation formulations.

That is why, in this chapter, we present the results of our current research focused on powder flow and surface properties analysis of HA powder material by means of inverse gas chromatography (iGC) and powder rheometer testing. These are both excellent tools for characterisation of the physicochemical properties of powder materials.

4.2 Methods for Hyaluronic Acid Powder Analysis

4.2.1 Theoretical Background

The surface free energy of a solid can be described as the sum of the dispersive and specific contributions. Dispersive (apolar) interactions, also known as Lifshitz-van der Waals interactions, consist of London interactions which originate from electron density changes but may include both Keesom and Debye interactions [6, 7]. Other forces influencing the magnitude of surface energy are Lewis acid-base interactions which are generated between an electron acceptor (acid) and an electron donor (base). Details of the widely accepted theoretical

Physicochemical Analysis of Hyaluronic Acid Powder for Cosmetic and Pharmaceutical Processing

91

treatment of the estimation of solid surface free energy by selective wetting measurements are described in detail in our review article [6].

The dispersive component of the surface energy SDc can be calculated

from the retention time obtained from iGC measurements of a series of n-alkane probes injected at infinite dilution (concentration within the Henry´s portion of the adsorption isotherm) [8]. Two approaches are used for the evaluation of these dependencies, the first one according to Schultz and co-workers [9] Equation 4.1 and the second one according to Dorris and Gray [10] Equation 4.2:

RT V a LD

N SD

C2In/ /

N A

1 2 1 2

c c= +` `j j (4.1)

Where:

T: Absolute temperature;

a: Cross sectional area of the probe molecule;

ln: Natural logarithm;

R: Universal gas constant;

NA: Avogadro´s number;DLc : Dispersive component of surface free energy of the liquid probe;

SDc : Dispersive component of the surface free energy of the solid;

VN: Retention volume; and

C: A constant.

/SD

N a

RT In V V

4 A CH CH

N C H N C H

2 2

n n n n

2 2

1 2 4 2 2cc

=+ + +^ ^ ^hh h (4.2)

Where:

aCH2 : Surface area of a CH2 unit (~0.6 nm2);

n: Number of carbon atoms in alkane probes; and

γCH2 : Free energy (approximately 35.6 mJ/m2).

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The shear stress (t) generated along a defined plane depends on the normal stress (s) exerted on this plane. If a material is subjected to a shearing action, a characteristic relation is obtained between normal and shear stresses for each material. This relationship is graphically shown in s-t coordinates (Mohr diagrams) and the straight line obtained finally is the yield locus for a bulk material [11].

All failure stress states for a given consolidation stress are represented by the Mohr stress circle, which is both tangential to this yield locus and passes through the origin, representing the unconfined yield stress state. The major principal stress associated with this circle is the unconfined yield strength, fc, of the material. There is one yield locus for each critical consolidation stress and one unique value of unconfined yield strength for each major principal critical consolidation stress. Direct shear testers measure the bulk strength of materials by first generating the yield locus and then constructing the unconfined Mohr circle stress state from the data. While under a certain consolidation load, the specimen inside the cell is pre-sheared to a condition of continual deformation without volume change (critical state) [11]. The major principal stress in the steady state flow is called the major consolidation stress (s1). It is determined by drawing the steady-state Mohr circle passing through the point (sc, tc) which represents the consolidation conditions in shear tests. The circle is tangential to the yield locus and the intersection of the circle with the normal stress axis gives the s1 value. Unconfined yield stress (sc) is the maximum normal stress value when a solid having a free and stressless surface flows or deforms. While the yield locus of a solid is known, sc is found by drawing an unconfined yield stress Mohr circle at a tangent to the yield locus and passing through the origin (s = t = 0) [12]. There is a corresponding value of sc for each consolidation stress (s1). The flow function of the material is obtained by plotting sc against s1 values. The flow index (ffc) is defined as the inverse slope of the flow function. Based on the magnitude of the flow index, the powder materials are classified as: hardened ffc<1; very cohesive ffc<2; cohesive ffc<4; easy flowing ffc<10; free flowing ffc>10 [12].


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4.2.2 Experimental

iGC was conducted using a surface energy analyser (SEA). Samples were placed in 4 mm (internal diameter) columns, to give a total surface area of approximately 0.5 m2. The following eluent vapours were passed through the column: nonane, octane, hexane and heptane. All reagents were of analytical grade. The injection of vapours was controlled to pass a set volume of eluent through the column to give pre-determined fractional coverage of the sample in the column. The retention time of the vapours by the particles gives an indication of the surface properties of the material, including the surface energy. By gradually increasing the amount of vapour injected, it is possible to build up a surface heterogeneity plot.

Specific surface area measurements were made using a Micromeritics TriStar 3000 surface area and porosity analyser using the nitrogen Brunauer–Emmett–Teller (BET) technique.

Thermogravimetric analysis (TGA) and differential thermal analysis (DTA) experiments were performed in a simultaneous DTA-TGA apparatus (Shimadzu DTG 60). Throughout the experiment, the sample temperature and weight-heat flow changes were continuously monitored. The conditions of measurement were: heat flow 10 °C/min and dynamic atmosphere of nitrogen (50 ml/min). The range of temperature measurement was from 40 to 500 °C.

Scanning electron microscopy (SEM) images were captured on a Hitachi 6600 FEG microscope operating in the secondary electron mode and using an accelerating voltage of 1 kV.

Powder rheology measurements were performed on a FT4 powder rheometer. All experiments were performed at laboratory ambient temperature (24 °C) and at a relative humidity of 43%.

The sample used was hyaluronate (sodium salt, of microbial origin) in the form of a white powder and with a MW of 0.7 to 0.9 MDa. It was kept in dry conditions in a desiccator (at the ambient temperature of 24 °C) for 4 weeks prior to the SEA and powder rheology experiments.

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4.3 Results and Discussion

The surface properties of HA powders were analysed by several experimental techniques. The specific surface area of HA powders was found to be 15 m2/g as observed by the BET technique, for a sample density of 1.00 g/cm3. To characterise the exact moisture content in the studied samples, DTA-TGA measurements were performed. These showed gradual sample mass loss with increasing temperature, having three distinct degradation regions (Figure 4.1). The first one, characteristic for water loss of 13.72 wt% up to 180 °C and the second and third regions, characteristic of a two-stage polysaccharide degradation starting at 180 and at 300 °C. The observed weight loss for the second region was 38.55 wt% and 12.26 wt% for the third region. Typical shapes of the particles of the HA powder studied are shown in Figure 4.2. These were characteristic spherical and cylindrical shapes of approximately 70 mm in diameter.

Temperature (°C)

100 200 300 400 500

TG

A (

wt%

)

40

60

80

100

Figure 4.1 TGA DTA degradation pattern of HA powder under study


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s_s_k_h-02 1.0kV 6.5mm x700 SE 50.0um

Figure 4.2 SEM image of studied HA powder

The surface energy profile and its components of the studied HA powder, based on iGC measurements data, are shown in Figure 4.3. The total surface energy and dispersive surface energy surface coverage dependencies showed exponentially decreasing curve patterns. The magnitudes of the determined absolute values of both quantities (total surface energy as well as of dispersive surface energy) were 34.0 mJ/m2 for 0% coverage and gradually decreased to 11.7 mJ/m2 for total surface energy and to 4.9 mJ/m2 for dispersive surface energy for 100% surface coverage. The highest energetic sites occupy approximately only 5% of the HA surface [13]. The significant difference in measured surface energy absolute values at low and high coverage indicates a high degree of inhomogeneity between the highest surface energy sites which have an approximately three-fold higher absolute value of the surface energy than the lowest energetic sites. The latter SEA-based absolute value of dispersive surface energy calculated by the Schultz method [9], correlates very well with the published data obtained from contact angle measurements of the apolar dispersive component of the surface tension, which was found

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to be 39.8 mJ/m2 [14]. As shown in Figure 4.3, the polar acid-base part of the surface energy coverage dependency is characteristic with minor linear increase with increasing coverage ranging from 0.7 mJ/m2 for 0% coverage up to 6.8 mJ/m2 for 100% coverage. The results obtained indicate dominance of the dispersive part of the surface energy. The surface energy distribution is characteristic with narrow distribution of the total surface energy ranging from 10-34 mJ/m2 with the maximum at 18.5 mJ/m2 (Figure 4.4). The total surface energy distribution is controlled by distribution of the dispersive part which ranges from 3.3-34.1 mJ/m2. It is evident that the polar surface active sites are of relatively low energy, again documenting the low polarity of sample surface. On the other hand, the dispersive part surface energy distribution is of a wider character reflecting the fact that a higher number of structural elements are responsible for this behaviour [13].

Coverage (n/nm)

0.00 0.05 0.10 0.15

Surf

ace

Ene

rgy

(mJ/

m2 )

0

10

20

30

Figure 4.3 Profile plot of total surface energy and its components for HA powder under study. Empty circle: total surface energy;

empty triangle: dispersive part of surface energy; and empty square: acid-base part of surface energy


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Surface Energy (mJ/m2)0 10 20 30

Are

a In

crem

ent

(%)

0.0

0.2

0.4

Figure 4.4 Distribution plot of total surface energy and its components for HA powder under study. Circle: dispersive part of surface energy; triangle: acid-base part of surface energy; and

square: total surface energy

The specific acid-base free energy distributions for applied selected probes reflect the ratio of structural components of the donor-acceptor character present at the HA solid/gas interface (Figure 4.5). The broadest distribution was found for acetonitrile ranging from -11.8 kJ/mol up to 15.0 kJ/mol specific (acid-base) free energy, and the narrowest one was for dichloromethane ranging from -10.8 to 2.3 kJ/mol. The majority of the polar character surface constituents in the energy range from -10.6 to 12.8 kJ/mol. Figure 4.5 shows the results of free energy profiles for selected probes with different polarity. It can be seen from the obtained free energy coverage dependencies that the highest reactivity non-polar sites at 0% coverage are of approximately seven-fold higher energy content (15 kJ/mol) in comparison to dichloromethane (2 kJ/mol).

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Specific (acid-base) free energy (kJ/mol)–10 –5 0 5 10 15

Are

a in

crem

ent

(%)

0.0

0.1

0.2

0.3

0.4

0.5

Figure 4.5 Specific acid-base free energy distributions of studied HA powder as observed for selected wetting probes. Square:

acetone; diamond: acetonitrile; triangle: ethanol

The results of the powder flow measurements are summarised in Figure 4.6, which shows the results of yield loci and Mohr´s circles at defined consolidation stresses for the studied HA sample. The relevant consolidation stress s1 is equal to the major principal stress of the Mohr stress circle which is tangential to the yield locus and intersects at the point of steady flow. It was found to be 16.4 kPa. The latter stress circle represents the stresses in the sample at the end of the consolidation procedure (stress at steady-state flow). It corresponds to the stress circle at the end of consolidation at the uniaxial compression test. The unconfined yield strength sc was found to be 5.08 kPa which results from the stress circle which is tangential to the yield locus and which runs through the origin (minor principal stress s2 = 0). Because the largest Mohr stress circle indicates a state of steady-state flow, the internal friction angle (31.9°) can be regarded as a measure of the internal friction at steady-state flow. For the samples studied, the effective angle of internal friction was observed to be 40.75°. It can be stated that materials with higher friction angles seem to have easy flow properties.


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Applied Normal Stress (Incipient) (kPa)0 4 8 12 16

Shea

r St

ress

(In

cipi

ent)

(kP

a)

0

2

4

6

8

10

Figure 4.6 Yield locus and Mohr’s circles of studied sodium hyaluronate powder (measured at 24 °C). Determined parameters:

cohesion = 1.41 kPa; unconfined yield strength sc = 5.08 kPa; major principal stress s1 = 16.4 kPa; friction coefficient ffc = 3.24; angle of internal friction j = 31.9 °; and effective angle of internal

friction je = 40.75°

4.4 Conclusions

For the sodium hyaluronate powder studied, the surface coverage dependence of the surface energy was found to be dominated by the dispersive energy part, thus indicating the low polarity character of the material. For 0% coverage, 30 mJ/m2 total surface energy was found. The surface structure of the studied polymer powder was found to be relatively inhomogeneous as reflected in the three-fold higher energy content for the highest energy sites (at 0% surface coverage) in comparison to the lowest energy sites as observed for 100% coverage (11.7 mJ/m2). The total surface energy distribution ranged from 10 to 34 mJ/m2 with a maximum at 18.5 mJ/m2. The major component of the total surface energy with respect to the

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magnitude was the dispersive one. With respect to the characterisation of macroscopic powder flow behaviour, the yield locus and flow function dependencies at different stress levels for the studied samples were determined. It was found that the character of the flow was cohesive (ffc = 3.24). The effective angle of internal friction was found to be 40.75° and the angle of internal friction was 31.9°.

Acknowledgments

Financial support from the Operational Program Research and Development for Innovations - European Regional Development Fund (grants CZ.1.05/3.1.00/14.0302 and CZ.1.05/2.1.00/03.0058) is gratefully acknowledged.

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7. J.F. Gamble, M. Leane, D. Olusanmi, M. Tobyn, E. Šupuk, J. Khoo and M. Naderi, International Journal of Pharmaceutics, 2012, 422, 238.

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