MARK BEST

353 Pages·2014·5.92 MB·English

ORAL MUCOSA-NANOPARTICLE
INTERACTIONS AND UPTAKE
PATHWAYS IN FORMULATION
EXCIPIENT PROFILING
MARK BEST

A Thesis Submitted in partial fulfilment for the
degree of Doctor of Philosophy

March 2014

The School of Pharmacy & Biomolecular
Sciences
The University of Brighton
ABSTRACT
Nanomaterials are generally defined as chemical substances or materials that contain particles
with one or more dimensions less than 100 nanometres in size. They may be either engineered
or naturally occurring, but have unique properties due to a vastly increased surface area to
volume ratio when compared to non-nano (bulk) materials. This provides the potential for the
development of a wide range of enhanced formulations with superior efficacy including
applications in oral healthcare. As the properties of a material change at the nano-scale, there
are concerns that the toxicological profile of these materials may also change. Size is only one
factor; changes in shape, surface chemistry, chemical composition, porosity and solubility all
contribute to the overall biological toxicity profile of a nano-scale ingredient. Established links
between the specific properties of a nanomaterial and toxicity are not well understood, leaving
an important data gap in the literature. The purpose of this work was to utilise in vitro oral
epithelial models for the assessment of safety profiles of nanomaterials for applications in next
generation oral care products.
Four commercially sourced nanomaterials were analysed, alongside respective bulk counterparts
already found within oral care product formulations. These nanomaterials comprised of two
nano-zinc oxides (ZnO), silicon dioxide (SiO2), titanium dioxide (TiO2) and hydroxyapatite
(Ca5(OH)(PO4)3). Comprehensive characterisation of each material was carried out using a
range of analytical techniques to identify any structure-function relationships in vitro. Initial
toxicity screening experiments were conducted using a non-keratinised oral epithelial cell
monolayer (H376 cell line) with both cell viability and lysis analysed using MTT and LDH
assays respectively. Materials were investigated further using two 3-dimensional tissue models
representative of the main tissue types constituting the human oral mucosa: non-keratinised
buccal (RHO) and keratinised gingival (GIN-100) models. Nanomaterial uptake in the models
was investigated using confocal microscopy with a styrl dye (FM 1-43). This led to the
development of a novel, high throughput fluorescent assay as a potential method for screening
nanoparticle-uptake.
Results highlighted the complexities involved with nano-characterisation in biological media
using current techniques. A wide variety of particle shapes and sizes were recorded between
different nanomaterials, with results being dependent upon the sample preparation steps and
specific methods of analyses used. These disparities represent the current challenges
experienced by both researchers and regulators of nanotechnology at the present time. ZnO
was observed to be the most cytotoxic material during monolayer screening, at concentrations
exceeding 0.3125% w/v when delivered in protein-free media. Differences between bulk and
nanomaterial properties were recorded for all the materials, except for TiO2, but these did not
necessarily transfer to effects seen in the more representative 3-D models. Cytotoxicity results
from both RHO and GIN-100 models exemplified the disparity between sensitivity of
monolayer and the natural stratified tissue structure of human oral mucosa. Keratinised gingival
tissue models showed significantly greater durability over the less robust buccal model, in both
cytotoxicity assays and IL-1α cytokine response. Of all materials examined, cellular uptake was
only observed for nano-SiO2. This was the only material detected trafficking inside the cell using
the FM 1-43 styryl dye assay, with confocal data serving to verify the analysis of nanoparticle
internalisation using fluorescence.
In conclusion, nanomaterials pose considerable difficulties during formulation and analysis in
healthcare products. The risk of potential uptake and bioaccumulation or translocation to
particularly sensitive areas of the body also requires further investigation. Nanomaterials have to
be assessed on a case by case basis, and robust/consistent regulatory strategies developed to
enable industry to produce and market novel but safe nanoparticle containing formulations.
Risks to human health may be less of a hazard when applied to fully functioning healthy human
tissue, especially in comparison to existing bulk material effects and current, accepted irritant
ingredients (e.g. Sodium lauryl sulphate).
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CONTENTS
ABSTRACT ...................................................................................................................................i
LIST OF TABLES ...................................................................................................................... v
LIST OF FIGURES .................................................................................................................. vi
ACKNOWLEDGEMENTS ................................................................................................. xiv
AUTHORS DECLARATION ............................................................................................... xv
ABBREVIATIONS AND DEFINITIONS ....................................................................... xvi
1 INTRODUCTION ............................................................................................................ 1
1.1 Nanotechnology and nanomaterials ........................................................................ 1
1.1.1 Nano-scale properties ........................................................................................ 2
1.2 Nanotoxicology ........................................................................................................... 4
1.2.1 Exposure routes ................................................................................................. 6
1.2.2 Nanomaterial capacity for systemic toxicity ................................................... 9
1.2.3 Dose metrics ..................................................................................................... 10
1.2.4 Nanomaterial toxicity testing strategy ........................................................... 13
1.2.5 Nanomaterial cytotoxicity mechanisms ........................................................ 16
1.2.6 Uptake pathways .............................................................................................. 24
1.2.7 Endocytosis ....................................................................................................... 27
1.2.8 Carrier mediated transport .............................................................................. 31
1.2.9 Paracellular transport routes ........................................................................... 32
1.3 The human oral mucosa .......................................................................................... 36
1.3.1 Keratinisation .................................................................................................... 38
1.3.2 Physical barriers in the human oral mucosa ................................................. 46
1.4 Nanomaterials in industry ....................................................................................... 47
1.4.1 Nanomaterials in Oral Healthcare ................................................................. 49
1.5 Aims and objectives of the thesis ........................................................................... 53
2 GENERAL METHODS ................................................................................................ 57
2.1 Materials ..................................................................................................................... 57
2.2 Methods ..................................................................................................................... 61
2.2.1 Characterisation of materials .......................................................................... 61
2.2.2 Cell culture ........................................................................................................ 76
2.2.3 Biochemical assays ........................................................................................... 82
2.2.4 Cell imagaing ..................................................................................................... 92
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2.2.5 Statistical analysis .............................................................................................. 96
3 CHARACTERISATION OF MATERIALS ............................................................... 97
3.1 Results ........................................................................................................................ 99
3.1.1 SEM analysis of particle size and morphology ............................................ 99
3.1.2 TEM analysis of particle size and morphology .......................................... 106
3.1.3 EDS analysis of chemical composition ....................................................... 112
3.1.4 DLS nanoparticle hydrodynamic diameter measurements and material
polydispersity ................................................................................................................... 117
3.1.5 NanoSight nanoparticle tracking analysis (NTA) of nanomaterial size
distribution ....................................................................................................................... 134
3.1.6 Nanomaterial zeta potential measurements ............................................... 143
3.2 Discussion ................................................................................................................ 149
3.2.1 Size determination .......................................................................................... 150
3.2.2 Colloidal stability ............................................................................................ 163
3.2.3 Temperature effects on nanomaterial characteristics ................................ 167
3.2.4 Evaluation of nanomaterial characterisation .............................................. 167
4 IN VITRO MONOLAYER SCREENING OF MATERIALS ............................. 170
4.1 Cytotoxicity screening results ............................................................................... 172
4.1.1 LDH assay results........................................................................................... 172
4.1.2 MTT assay results ........................................................................................... 177
4.1.3 SEM visual analysis of material particle-H376 cell interactions .............. 182
4.2 Discussion ................................................................................................................ 190
4.2.1 Evaluation of nanoparticle cytotoxicity by colorimetric assays ............... 191
4.2.2 Cytotoxicity assessed through cell imaging ................................................ 197
5 NANOMATERIAL CYTOTOXICTY TESTING USING 3-DIMENSIONAL
IN VITRO MODELS ............................................................................................................ 203
5.1 Results ...................................................................................................................... 205
5.1.1 Cytotoxicity testing using the RHO model of non-keratinised oral
mucosal tissue .................................................................................................................. 205
™
5.1.2 Cytotoxicity testing using the EpiGingiva keratinised oral mucosal tissue
model ........................................................................................................................... 210
5.1.3 ICP-OES measure zinc ion concentration ................................................. 216
5.2 Discussion ................................................................................................................ 220
5.2.1 Assessment of in vitro toxicity testing ........................................................ 220
5.2.2 Comparisons of 3-D keratinisation models ............................................... 223
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5.2.3 Further investigation of potential cytotoxic mechanisms ........................ 226
5.2.4 Inflammatory response .................................................................................. 229
5.2.5 Risk implications of nanomaterial exposure on the oral mucosa ............ 233
6 UPTAKE POTENTIAL OF NANOMATERIALS IN VITRO .......................... 235
6.1 Results ...................................................................................................................... 237
™
6.1.1 SynaptoGreen uptake assay results ........................................................... 237
6.1.2 Confocal laser scanning microscopy imaging of particle uptake............. 242
6.1.3 TEM-EDX analysis of nanoparticle uptake in 3-D tissue models ......... 252
6.2 Discussion ................................................................................................................ 258
6.2.1 Nanomaterial uptake routes .......................................................................... 260
6.2.2 Endocytosis as a transcellular uptake pathway for nanomaterials putative
of interest in future oral healthcare applications ........................................................ 264
6.2.3 Paracellular transport of nanoparticles ........................................................ 269
6.2.4 Nanomaterial uptake potential summary .................................................... 273
7 GENERAL DISCUSSIONS AND CONCLUSIONS ............................................ 275
7.1 Study conclusions ................................................................................................... 275
7.2 Experimental limitations of the study ................................................................. 277
7.2.1 Difficulties associated with accurate nanoparticle characterisation ........ 277
7.2.2 In vitro models and the current limitations of toxicity testing ................ 279
7.3 Future study ............................................................................................................. 284
7.4 Original contribution to research ......................................................................... 286
8 REFERENCES............................................................................................................... 287
9 APPENDICIES .............................................................................................................. 333
9.1 H376 cell loss of cell adhesion in response to SDS cytotoxicity ..................... 333
9.2 SiO -ACROS-Bulk material characterisation ..................................................... 334
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LIST OF TABLES
Table 1.1. A table to demonstrate the details of key keratinised proteins found in oral
epithelial keratinocytes. Compiled from data within (Dale et al., 1990).
Table 2.1. Parameters used for automatic optimised particle size analysis using Malvern
Zetasizer ZS90 (Zetasizer v6.1). Information obtained from experimental data and
(Malvern, 1996).
Table 3.1. Nanomaterial particle diameter results analysed at 22°C and 37°C using the
DLS based particle sizing instrument (ZetaSizer Nano ZS90).
Table 3.2. Nanomaterial particle diameter results analysed at ambient temperature
using the NTA based particle sizing instrument (NanoSight LM10). All measurements
are the average of 6 runs (n=6) with standard deviation reported.
Table 3.3. Zeta potential of nanoparticles measured in three different solvent
dispersions at both 22°C and 37°C. The Smoluchowski's theory was used for
calculations of electrophoretic mobility of the particles based on laser doppling and M3-
a
PALS in the Malvern ZetaSizer NS90. All results stated are the average of 6 runs (n=6)
averaged from at least 4 measurements per run.
Table 3.4. pH measurement of nanomaterials diluted to a 0.001% w/v concentration in
either dH2O or PRF medium prior to Zeta Potential analysis. All measurements carried
out at room temperature, with results an average of 3 different readings carried out on
different days (n=3) with standard deviation reported.
Table 3.5. A summary of the particle size analysis for all current materials tested in all
methodologies reported within this chapter*. All results included are taken from
analysis carried out at 22°C.
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LIST OF FIGURES
Figure 1.1. Schematic of human body with pathways of exposure to nanoparticles,
affected organs, and associated diseases from epidemiological, in vivo and in vitro studies.
Taken from (Buzea et al., 2007).
Figure 1.2. Flowchart outlining the current best strategy approach for the risk
assessment of nanomaterials. Adapted from ((SCENIHR), 2006).
Figure 1.3. Hypothetical cellular interactions of nanoparticles causing inflammatory
effects on lung epithelium. Adapted from (Oberdorster et al., 2005b).
Figure 1.4. The hierarchal oxidative stress model. Adapted from (Nel et al., 2006).
Figure 1.5. A model showing the simplified NF-kB signalling pathway that is activated
by oxidative stress in response to nanoparticle generated ROS in keratinocytes. Adapted
from (Huang et al., 2010b).
Figure 1.6. The main transport pathways associated with nanoparticle internalisation
through cell layers and membranes in the human body. Adapted from (Forth et al., 1987).
Figure 1.7. The main types of endocytosis. Taken from Mariana Ruiz Villarreal (free of
copyright), 2007.
Figure 1.8. Outline of the main events in receptor-mediated endocytosis. Figure taken
from (Grant & Donaldson, 2009).
Figure 1.9. A diagram showing the tight cell to cell adhesion in keratinised epithelium.
Adapted from (Cooper, 2000).
Figure 1.10. A schematic to show a to non-keratinised epithelial cell tight junction, at
the apical surface of stratified tissue structures. Taken from Mariana Ruiz, 2006.
Figure 1.11. A diagram showing the epithelial tissues of the oral mucosa, with its four
epidermal layers. Adapted from (Wertz & Squier, 1991).
Figure 2.1. A schematic representation of Zeta potential. Taken from (Kaszuba et al.,
2010).
Figure 2.2. The chemical structure of (a) SynaptoGreen™ and (b) FM®1-43X both
variants of N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium
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dibromide, styryl dyes. Taken from:
www.lifetechnologies.com/order/catalog/product/T3163
Figure 3.1. SEM images 1,000X magnification of particles (bulk and nano) in typical
agglomerates, as observed by imaging their distribution spread across the aluminium
specimen stub.
Figure 3.2. SEM images 100,000 X magnification of all particles (bulk and nano)
investigated for their interest within oral healthcare.
Figure 3.3. SEM images 300,000X magnification of all particles (bulk and nano)
investigated for their interest within oral healthcare.
Figure 3.4. Mean particle sizes of both nano and bulk materials measured from SEM
images taken between 1,000 and 100,000 times magnification.
Figure 3.5. TEM images 80,000X magnification of particles (bulk and nano) in typical
agglomerates, spread across the formvar coating on the copper grid.
Figure 3.6. TEM images at 75,000X magnification of bulk-sized particles included as
control materials for comparisons of nano-sepcific properties of nanoparticles of the
same chemicals.
Figure 3.7. TEM images at 350,000X magnification of nanoparticles investigated for
potential use within oral healthcare formulations.
Figure 3.8. Mean particle sizes of both nano and bulk materials measured from TEM
images taken between 3,000 and 180,000 times magnification.
Figure 3.9. EDS spectra of Hydroxyapatite (Ca (OH)(PO ) ) material (top) bulk,
5 4 3
(bottom) nanoparticle.
Figure 3.10. EDS spectra of Silicon dioxide (SiO ) material (top) bulk, (bottom)
2
nanoparticle.
Figure 3.11. EDS spectra of Titanium dioxide (TiO ) material (top) bulk, (bottom)
2
nanoparticle.
Figure 3.12. EDS spectra of Zinc oxide (ZnO) nanomaterial solutions (top) ZnO-
45009, (bottom) ZnO-45408.
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Figure 3.13. EDS spectra of Zinc oxide (ZnO) bulk powder .
Figure 3.14. DLS particle size distributions by intensity polystyrene standards dispersed
in dH O at 22°C (top) and 37°C (bottom).
2
Figure 3.15. Demonstration of the difficulty in accurate measurement of DLS particle
size distributions by intensity of back scattered light by hydroxyapatite nanomaterial
dispersed in three different solvents at 22°C (a) dispersed in dH O, (b) PRF media, (c)
2
ethanol and (d) n = 6 mean values.
Figure 3.16. DLS particle size distributions by intensity of SiO nanomaterial dispersed
2
in three different solvents at both 22°C (top) and 37°C (bottom).
Figure 3.17. DLS particle size distributions by intensity of TiO nanomaterial dispersed
2
in three different solvents at both 22°C (top) and 37°C (bottom).
Figure 3.18. DLS particle size distributions by intensity of ZnO-45009 nanomaterial
dispersed in three different solvents at both 22°C (top) and 37°C (bottom).
Figure 3.19. DLS particle size distributions by intensity of ZnO-45408 nanomaterial
dispersed in three different solvents at both 22°C (top) and 37°C (bottom).
Figure 3.20. DLS ZetaSizer data showing the polydispersity index (y axis) versus the
averaged actual polydispersity width of particle diameters recorded during size
measurements (x axis), for all nanomaterials dispersed in each of three different solvents
at 22°C.
Figure 3.21. DLS ZetaSizer data showing the polydispersity index (y axis) versus the
averaged actual polydispersity width of particle diameters recorded during size
measurements (x axis), for all nanomaterials dispersed in each of three different solvents
at 37°C.
Figure 3.22. NTA size distribution expressed as number of particles of hydroxyapatite
nanomaterial dispersed in: dH O (blue), PRF media (red) or ethanol (green). Analysed at
2
room temperature (n = 6).
Figure 3.23. NTA size distribution expressed as number of particles of SiO
2
nanomaterial dispersed in: dH O (blue), PRF media (red) or ethanol (green). Analysed at
2
room temperature (n = 6).
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Figure 3.24. NTA size distribution expressed as number of particles of TiO
2
nanomaterial dispersed in: dH O (blue), PRF media (red) or ethanol (green). Analysed at
2
room temperature (n = 6).
Figure 3.25. NTA size distribution expressed as number of particles of ZnO-45009
nanomaterial dispersed in: dH O (blue), PRF media (red) or ethanol (green). Analysed at
2
room temperature (n = 6).
Figure 3.26. NTA size distribution expressed as number of particles of ZnO-45408
nanomaterial dispersed in: dH O (blue), PRF media (red) or ethanol (green). Analysed at
2
room temperature (n = 6).
Figure 3.27. The general classification of colloid stability in solution based on zeta
potential (ζ) measurements, negating charge on particle. Adapted from (Vallar et al., 1999).
Figure 3.28. Zeta potential measurements of nanoparticle surface charge, taken from
the data in Table 3.4.
Figure 4.1. A graph comparing the cytotoxic effects of bulk versus nano material
exposure to H376 monolayers at concentrations ranging from 0 to 0.25% w/v in serum
free growth media, for 5 minutes at 37°C/5% CO .
2
Figure 4.2. A graph comparing the cytotoxic effects of nanomaterial exposure to H376
monolayers at concentrations ranging from 0 to 0.25% w/v when delivered in different
media, incubated at 37°C/5% CO for 5 minutes.
2
Figure 4.3. A graph comparing the cytotoxic effects of bulk particle sized material
exposure to H376 monolayers at concentrations ranging from 0 to 0.25% w/v when
delivered in different media, incubated at 37°C/5% CO for 5 minutes.
2
Figure 4.4. A graph comparing the cytotoxic effects of additional control materials
exposed to H376 monolayers at concentrations ranging from 0 to 0.25% w/v when
delivered in different media, incubated at 37°C/5% CO for 5 minutes.
2
Figure 4.5. A graph comparing the cytotoxic effects of bulk versus nanomaterial
exposure, to H376 monolayers, in terms of cell viability calculated from MTT
metabolism at concentrations ranging from 0 to 0.25% w/v in serum free growth media
for 5 minutes at 37°C/5% CO .
2
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