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Peptide-based emulgel microparticles for the sustained release of drugs in the treatment of dental diseases Research Proposal
Ronak Patel Word count: 3117 (excluding appendices)
BL4209
G20644031
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Contents 1.0 Abstract ............................................................................................................................................ 2
2.0 Introduction ...................................................................................................................................... 3
2.1 Aims and Objectives ......................................................................................................................... 5
3.0 Methodologies proposed for the experiments ............................................................................... 5
3.1 Formulation ...................................................................................................................................... 6
3.2 Characterization ............................................................................................................................... 6
3.2.1 Fourier-trasform infrared spectroscopy ....................................................................................... 6
3.2.2 Microscopy .................................................................................................................................... 7
3.2.2.1 Fluorescence Microscopy ........................................................................................................... 7
3.2.2.2 Scanning Electron Microscopy (SEM) ........................................................................................ 8
3.2.2.3 Transmission Electron Microscopy (TEM) ................................................................................. 8
3.2.2.4 Atomic Force Microscopy (AFM) ............................................................................................... 9
3.2.3 Rheology ........................................................................................................................................ 9
3.2.4 Dynamic Light Scattering (DSL) ..................................................................................................... 9
3.3 Stability ........................................................................................................................................... 10
3.4 Drug Loading and Entrapment Efficiency ...................................................................................... 10
3.5 Drug Release Studies ...................................................................................................................... 11
4.0 Methods of Analysis ....................................................................................................................... 11
5.0 Limitations ...................................................................................................................................... 11
6.0 Anticipated Outcomes and Scope .................................................................................................. 12
7.0 Annotated References ................................................................................................................... 13
8.0 References ...................................................................................................................................... 15
10.0 Appendix ....................................................................................................................................... 19
A.1 Suppliers and Cost of Chemicals .................................................................................................... 19
A.2 Gantt Chart ..................................................................................................................................... 20
A.3 Risk Assessment ............................................................................................................................. 21
A.4 Ethics .............................................................................................................................................. 23
A.5 COSHH form ................................................................................................................................... 28
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1.0 Abstract
Peptide based emulgel microparticles epitomise an auspicious alternative to current drug
delivery methods. Previously the FEFK peptide has demonstrated its effectiveness, as it can
self-assemble at the oil/water interface to form ordered emulgels of outstanding
mechanical rigidity. However, a high peptide concentration is required in combination with
an enzyme hence here it is proposed, to exploit the FGEFGK as an alternative in the
encapsulation of microbial oils (Melissa oil) and model hydrophobic drugs (Curcumin and
Nile red) to study their release profiles for possible applications in the field of dental disease
treatment. In this proposal the methods for: the formulation, characterisation, stability
testing, release studies and data analysis are explored, with justifications of the chosen
methods. Additionally, anticipated problems and limitations of the research are considered,
along with the scope of the proposed research. If the anticipated results are verified, the
possible applications of the peptide based emulgel microparticles are immeasurable in the
field of drug delivery and cosmetics.
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2.0 Introduction
Since the dawn of modern medicine peptides and proteins have gained the most interest
due to their involvement in biological processes. Recently however, peptide-based
hydrogels have monopolized the field due to their potential applications in the fields of;
tissue regeneration (Laverty, 2017), cell culture (Worthington et al., 2015), cell therapy
(Gomes et al., 2016), biosensors (Lian et al., 2016) and most importantly drug delivery
(Habibi et al., 2016). Of the several peptide systems available for hydrogel synthesis the
most beneficial are the self-assembly peptides, not only do they self-assemble, they are
highly tuneable, easily functionalised, biocompatible and biodegradable (Bai et al., 2014).
Molecular self-assembly is defined as the spontaneous organisation of molecules under
thermodynamic equilibrium conditions into structurally stable arrangements via
noncovalent interactions such as hydrogen bonding, hydrophobic interactions and π-
interactions (Zhang and Altman, 1999). Self-assembling peptides can be categorised into
dipeptides, cyclic peptides, amphiphilic peptides, α helix/ coiled-coil peptides and β-sheet
peptides (Fan et al., 2017), which form different types of nanostructures as shown in figure
1.
Self-assembly and gelation can be triggered by light, pH, time, temperature and enzymes
(figure 2). The peptide of interest in this project is the FEFK tetrapeptide (F, phenylalanine;
E, glutamic acid; K, lysine) discovered by Zhang and Altman (1999), however it requires a
thermolysin enzyme or a high concentration (>120 mg/mL) (Guilbaud et al., 2013) to trigger
gelation, hence to overcome this, the peptide will be functionalized to FGEFGK (FG,
phenlyglycine) (figure 3). These amphiphilic, β-sheet forming tetrapeptides form elongated
Figure 1- Illustrating peptide self-assembly into different nanostructures. Adapted from Fan et al., (2017).
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fibres and above a certain critical gelation concentration (CGC) the fibres entangle to form
multi-dimensional networks with the ability to trap water (Elsawy et al., 2016), hence form
hydrogels (figure 2).
Hydrogels can be categorised as chemical or physical gels based on their crosslinking
patterns (Kopeček and Yang, 2009). These crosslinking interactions can be disrupted by
temperature, pH, light and the presence of specific solutes, hence they can be triggered to
release their payload, which is beneficial in regards to drug delivery. Gels facilitate a faster
release of drugs compared to other semisolid preparations, as they have a higher aqueous
component, which allows a greater dissolution rate (Khullar et al., 2012). However, a
complication arises when hydrophobic drug delivery is in question. Hence to overcome this
limitation emulgels can be formulated (a combination of emulsions and gels). They can be
either oil-in-water (lipophilic drugs) or water-in-oil (hydrophilic drugs) (Naga et al., 2014).
To date, emulgels have not been adapted for the delivery of antimicrobial oils for
oral/dental health, which is the gap this research will fill. One of the antimicrobial oils of
interest is the Melissa oil from the Melissa officinalis (Lemon Balm) species of the Lamiaceae
family (Mimica-Dukic et al., 2004). Due to the high levels of phenolic acid (rosmarinic acid)
Figure 2- Exemplifying the self-assembling and gelation process of β-sheet forming tetrapeptides, adapted
from Boothroyd et al., (2013)
Phenylalanine
Phenylglycine
Figure 3- Demonstrating the chemical structure of the amino acids phenylalanine (left) and phenlyglycine
(right) adapted from, Sigma Aldrich (2017).
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content they display several therapeutic properties such as, antimicrobial, antifungal,
antioxidant, sedative, carminative and antispasmodic (Barros et al., 2013). Hence an emulgel
formulated for the sustained release of Melissa oil can be utilized in the treatment of dental
abscesses caused by microbial infections (Inagaki et al., 2017). The oil has further
applications in the treatment of heart conditions, anxiety, insomnia, migraines, depression
and immunological disorders, which can be explored if the formulation is successful
(Encalada et al., 2011). In 2015, Ramanauskienė et al., formulated Melissa oil emulgels using
carbomer 980, methylcellulose and propylene glycol however, self-assembling peptides
have not been utilized as of yet.
In addition, Bai et al., (2014) observed a similar set of self-assembling peptides formed fibrils
that adsorbed at the oil-water interface, stabilizing emulsions and forming microparticles as
shown in figure 4. If time permits, these microparticles may also be studied by loading them
with model drugs such as curcumin or Nile red, to study their release kinetics.
2.1 Aims and Objectives
1. Optimization of the formulation process of these Melissa oil loaded peptide-based
emulgels.
2. Studying the drug release kinetics from the emulgels
3. Producing peptide-based microparticles and loading them with model drugs
(Curcumin and Nile red) to study the different parameters that control their release
kinetics.
3.0 Methodologies proposed for the experiments
This section will outline the methods and techniques that will be employed in the project
and the rationale behind employing them over other techniques.
Figure 4: Right- demonstrating the self-assembly process of aromatic peptides at the oil/water interface
and Left- a oil-in-water microparticle stabilised by fibrous network. Adapted from Bai et al., (2014).
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3.1 Formulation
To achieve the first aim of the project different formulations utilizing the FGEFGK peptide will
be synthesized. A simple formulation process will be employed similar to that described by
Elsawy et al., (2016) and Bai et al., (2014). The peptides will be formed using a peptide
synthesiser and 5 g will be dissolved in HPLC grade water (200 µL) by sonication. The pH will
be adjusted to 5.5 to trigger gelation via titration with sodium hydroxide (NaOH) (3 x 15 µL).
Instead of using chloroform however, the Melissa oil (5 x 50 µL) will be added and hand
shaken or vortexed (30 seconds) to form oil in water emulsions, a further 35 µL of HPLC
grade water will be added to make the final volume up to 500 µL.
These formulations will differ in the peptide concentrations as well as the oil to peptide
ratio. To evaluate these formulations different characterization techniques will be
employed. To assess the optimal peptide concentration, different concentrations will be
tested (0 mg/µL, 10 mg/µL, 20 mg/µL, 30 mg/µL, 40 mg/µL and 50 mg/µL) each one
formulated 3 times to ensure statistical significance. Once the optimal concentration has
been determined, the optimal ratio of oil to water will be assessed in the following water:
oil ratios; 1:9, 3:7, 5:5, 7:3 and 9:1. During these manipulations all other variables will be
controlled and kept constant such as the speed of the vortex, amount of NaOH added,
temperature and Melissa oil sample.
To form emulgel microparticles the emulsion solvent evaporation method will be employed
similar to that exploited by Ischakov et al., (2013). Rather than using an oil, a more polar
solvent is required such as chloroform (5 x 50 µL) to dissolve the model drugs (curcumin and
Nile red), evidentially the final step would require solvent evaporation to leave drug loaded
microparticles.
3.2 Characterization
To assess the optimal formulation as described before, various characterization techniques
will be employed to assess the; self-assembly process, stability, rheology and visualize the
emulgel microparticles.
3.2.1 Fourier-trasform infrared spectroscopy
Spectroscopy is the study of the interaction between electromagnetic radiation and matter,
and the reaction measurements of radiation intensity and wavelength. The main type of
spectroscopy used will be fourier-trasform infrared spectroscopy (FTIR) using a Bruker optics
vertex spectrophotometer. The main purpose of FTIR will be to determine the H-bonding
that underpin the β-sheet formation of the peptides. Moreira et al., (2016) utilized a
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method in which the samples were prepared using deuterated oxide phosphate buffer and
placed between 2 calcium fluoride windows parted by a polytetrafluoroethylene spacer.
Over 25 scans at a revolution of 1 cm-1 were taken in the region between 1570 and 1710 cm-
1. Identical methods were used by Scott et al., (2015) and Bai et al., (2014). Amide I
absorption caused by the stretching vibrations of the C=O and C-N groups in the region of
1615 to 1640 cm-1 is indicative of a β-sheet formation of peptides. Amide II absorption
caused by N-H bending, C-N and C-C stretching in the region of 1670 to 1698 cm-1 is also
suggestive of a β-sheet formation. Using this method, it is possible to prove not only the
formation of β-sheets but also how ordered they are.
3.2.2 Microscopy
Multiple microscopic techniques will be employed to image the microparticles, resulting in
both qualitative data (physical appearance) and quantitative data (the average size of
microparticles and thickness of the peptide fibres).
3.2.2.1 Fluorescence Microscopy
The technique outlined by Bai et al., (2014) will be employed, involving the use of the dyes;
Fluorescein isothiocyanate (FITC) and Thioflavin T (ThT). The FITC labels the aqueous phase
of the emulsion allowing the visualization of oil-in-water emulsions, results are to be
expected similar to those shown in figures 5 and 6. The ThT is used as the gold standard for
selectively staining amyloid fibrils which are formed of β-sheets (Biancalana and Koide,
2010), hence in this case they can be used to confirm the self-assembled peptide structures
as β-sheets. The samples are usually prepared similar to that of reflective light microscopy,
the only difference being the use of fluorescent dyes. The slides are then mounted on an
epi-fluorescent upright microscope and imaged using appropriate software such as ImageJ.
The only disadvantage of this method, is the phenomenon known as photobleaching, which
means the samples would have to be processed straight after the addition of the dye
(Philpott et al., 2014).
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3.2.2.2 Scanning Electron Microscopy (SEM)
Disregarding the high cost of this technique it is a very beneficial method of visualising the
microparticles as 3 dimensional images. Additionally, they can achieve a resolution of 1 nm,
much greater than any light microscopy technique. Sample preparation is therefore
vigorous and problematic, however when done right it can produce unparalleled images (de
Boer et al., 2015). In this context SEM will be used to visualise the emulgel film and the
microcapsules left after solvent evaporation.
The samples will be prepared using 2 different techniques; freeze drying and air drying. The
freeze-drying process usually entails the use of freeze driers, liquid nitrogen and tert-Butyl
alcohol (cryoprotectant) which may take up to a few days to completely dry the sample.
Once the samples are dried, they are sputter coated under vacuum conditions using a
Polaron SEM coating system with gold/palladium to enhance the image by making the
surface of the particles electrically conductive (Goldstein et al., 2017). The samples are then
imaged using a scanning microscope at 10 kV (Moreira et al., 2016).
3.2.2.3 Transmission Electron Microscopy (TEM)
In contrast to SEM, TEM has much greater resolution and it can be used to confirm the
ability of the peptides to form extended β-sheet fibrillar structures. The average width of
the fibres can also be calculated using this technique. In TEM the samples are first diluted by
double distilled water and placed in a 300-400 mesh carbon-coated copper grid (Tian et al.,
2011). Subsequently, they are negatively stained using ammonium molybdate 2% for water
samples, 1% aqueous methylamine vanadate for water/chloroform samples (Moreira et al.,
2016). On the contrary Ischakov et al., (2013) used 1% uranyl acetate for negative staining.
Figures 5 and 6: illustrating expected results from fluorescence microscopy, Left- showing oil-in-water
emulsions with FITC labelling the aqueous phase and Right- demonstrating the β-sheets formation at the
oil/water interface. Adapted from Bait et al., (2014).
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After a brief time, the excess negative staining is removed and the samples are dry, they are
viewed on a TEM operating at 80-200 kV depending on the microscope (Hilal et al., 2017).
3.2.2.4 Atomic Force Microscopy (AFM)
AFM is a scanning probe microscopic technique; hence it can be used to measure local
properties such as height, friction and magnetism. In the context of emulgels and
microparticles, AFM can be used to confirm the results found from TEM and SEM in regards
to the morphology of the fibres. Sample preparation involves; depositing the samples onto a
freshly cleaved mica surface, after a brief time to allow the sample to air dry, the sample is
scanned in air under ambient conditions using an AFM (intermittent contact mode) with an
appropriate spring constant, drive amplitude and cantilever oscillation. To save time it is
suggested to collect data using random spot surface sampling (Moreira et al., 2016). The
data is then processed using Nanoscope Analysis software (Elsawy et al., 2016).
3.2.3 Rheology
Rheology is the study of the flow of matter and can be used to determine the viscoelasticity
of a media. To assess the mechanical properties of the emulgels and to assess how they will
behave in-vivo, these investigations are essential. Additionally, there is a direct link between
the β-sheet structure and viscosity, loss of which results in the increase of elasticity as
shown by Elsawy et al., (2016). The assessments are carried out on a stress-controlled
rheometer. Strain sweeps and radial frequency sweeps should be carried out to obtain G’
and G’’.
3.2.4 Dynamic Light Scattering (DSL)
To determine microparticle size diameter, polydispersity and zeta potential, a Zetasizer (for
particles in the range of 0.3 nm to 10 µm) or a Mastersizer (0.1 µm to 1 mm) can be
employed. It is important to determine these aspects as they predict the behaviour of the
particles in-vivo and have an effect on drug release. Additionally, these values can be used
to compare with other studies to test the validity and reliability of the results. Sample
preparation is fairly straight forward; the sample is diluted or dissolved (if dry) until the
solution is clear and placed into the DLS for analysis (Delmar and Bianco-Peled, 2016).
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3.3 Stability
The ability for the peptides to stabilize an emulsion at the interface will be compared to
traditional surfactant such as sodium dodecyl sulphate (SDS), polysorbate (Tween) 20 and
80. The method used by Bai et al., (2014) will be adopted, however rather than only
comparing long-term stability for 2 weeks it will be tested for 10 weeks (room temperature).
Additionally, the thermo-stability (temperature of up to 60 °C for 3 hours) and salt stability
(100 mM of phosphate, chloride and thiocyanate) will also be tested. The concentrations
employed for each surfactant (peptide and traditional) will be kept constant along with the
volumes of the formulation. To assess each experiment, pictures will be taken straight after
formulation and then after each experiment (weekly for long-term stability to assess change
over time). This method is relatively; swift, easy and doesn’t require additional equipment
to test stability.
3.4 Drug Loading (DL) and Entrapment Efficiency (EE)
The method described by Venkataharsha et al., (2015) will be employed in which, the
Melissa oil and curcumin will be quantified using UV-VIS spectrophotometry at an
absorbance of 264 nm (de Ciriano et al., 2010) and 422 nm (Delmar and Bianco-Peled, 2016)
respectively and Nile red using fluorescence spectroscopy at an excitation wavelength of
546 nm and an emission wavelength of 628 nm (Delmar and Bianco-Peled, 2016). 100 mg of
sample (emulgel or microparticles) will be dissolved in a solvent (10 mL methanol and 50 mL
dichloromethane) and sonicated for 30 minutes and then left for 24 hours, then tested. A
calibration curve (0-50 µg/mL) will be established by dissolving known amounts of the oil,
curcumin and Nile red in the same amount of solvent. Blanks will also be tested by
measuring the absorbance of unloaded microparticles to adjust for error. The drug loading
and entrapment efficiencies will be calculated using the following formulas. The reason for
using this method is due to its simplicity.
𝐷𝑟𝑢𝑔 𝐿𝑜𝑎𝑑𝑖𝑛𝑔 % = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑙𝑜𝑎𝑑𝑒𝑑
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑝𝑒𝑝𝑡𝑖𝑑𝑒 × 100
𝐸𝑛𝑡𝑟𝑎𝑝𝑚𝑒𝑛𝑡 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 % = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑙𝑜𝑎𝑑𝑒𝑑
𝑇𝑜𝑡𝑎𝑙 𝑑𝑟𝑢𝑔 𝑎𝑑𝑑𝑒𝑑 × 100
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3.5 Drug Release Studies
To assess the second and third aims, the method by Delmar and Bianco-Peled (2016) will be
adopted as its validation process is short compared to HPLC methods. 1 g of the emulgel or
100 mg of the microparticles will be placed in a vial containing 30 ml of simulated saliva fluid
(Duffó and Castillo, 2004) or phosphate buffer saline (PBS) (pH = 7.4, 75 mM) solution. The
vials will then be placed in a water bath at 37 °C and shaken at a rate of 100 rpm (to
simulated the conditions of the mouth). Periodically (every hour), 200 µL of the medium will
be withdrawn and tested using spectrophotometry or fluorescence spectroscopy (see
section 3.4). The same volume of fresh media will be added back to keep the volume
constant. The control (blank) will involve the use of unloaded emulgel and microparticles.
Only the best formulation (using the results from sections 3.1-3.4) of the emulgel and the
microparticles will be tested in triplets and shown in a release graph of % Mt/ M∞
(accumulative mass of drug released until time t, Mt, normalized by the release at long times
M∞) over time (hours) for up to 48 hours.
4.0 Methods of Analysis
As all experiments will be done in triplets (n=3), the quantitative data will be expressed as
mean ± standard error of the mean. Statistical analysis will be performed on SPSS by ANOVA
tests for multiple comparisons (for DLS, DL/EE and drug release studies) and the p < 0.05 will
be statistically significant. Additionally, data from microscopy will be shown as images.
Graphs and tables will be employed where ever possible. The results from the research will
be disseminated via a presentation and a thesis.
5.0 Limitations
The greatest limitation of the study is the proposed time, it may not be possible to carry out
all the experiments proposed in this document. Additionally, as the peptide-based emulgel
microparticles have not been formulated using the FGEFGK peptide, they may not even form
stable microparticles, which is why the emulgels will be formulated and drug release tested
first, which are stable as shown by Caplan et al., (2000; 2002a; 2002b). Although the
literature suggests that the peptide emulgels/microparticles will release their payload in a
sustained manner, this may not be true, if so the formulations can still be tested for
immediate release of microbial oils or drugs that are hydrophobic and poorly soluble in
water such as the model drugs employed (curcumin and Nile red), essentially improving
their solubility as they would enter solution as micelle structures (Ischakov et al., 2013).
Furthermore, access to all stated instruments may not be possible (i.e. TEM and SEM),
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hence those features may not be tested. Finally, the experiments carried out will be in-vitro,
and for the product to be marketed it would require in-vivo testing followed by clinical trials.
6.0 Anticipated Outcomes and Scope
This research will build on the work of; Bai et al., (2014), Elsawy et al., (2016),
Ramanauskienė et al., (2015) and Ischakov et al., (2013), to demonstrate the ability of
peptides to form emulgels and microparticles for the sustained release of microbial oils and
drugs for dental treatments. Additionally, to assess the optimal formulation characteristics
for stability and longest sustained release (up to 48 hours). If successful this will be the first
research of its kind to lay the ground work for dental medications with long lasting effects
reducing the need to apply gels every few hours to once daily, a beneficial trait in teething
infants. If peptide microparticles are successfully formulated, they will have possible
applications in multiple oral administrations ranging from the treatment of heart disease to
the treatment of cancers.
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7.0 Annotated References
Bai, S., Pappas, C., Debnath, S., Frederix, P., Leckie, J., Fleming, S. and Ulijn, R. (2014).
Stable Emulsions Formed by Self-Assembly of Interfacial Networks of Dipeptide
Derivatives. ACS Nano, 8(7), pp.7005-7013.
The authors concluded successful stabilization of water-in-oil and oil-in-water emulsions by the self-assembly of nanofibrous networks at the interface. In addition, they also found that peptides were far superior at stabilizing emulsions than traditional surfactants such as SDS. However, they classed long term stability as 2 weeks and no statistical tests were carried out. On the other hand, they made substantial contributions to the field and their methods employed such as stability testing, fluorescence microscopy and SEM will be adopted in this research. The study is relevant as they employed peptides to form microparticles the only difference is that in this research a different peptide sequence will be utilized. The fluorescent dyes exploited in this study are also selected in the proposal.
Delmar, K. and Bianco-Peled, H. (2016). Composite chitosan hydrogels for extended release
of hydrophobic drugs. Carbohydrate Polymers, 136, pp.570-580.
In this paper the authors proved that microemulsions trapped within a genipin crosslinked hydrogel significantly improved the solubility of hydrophobic drugs (curcumin and Nile red) compared to the free drugs and just the loaded microemulsions. Although this study doesn’t employ peptide based microparticles, its methodology used to study the drug release is comprehensive and will be used in this research as the release medium is only 30mL, sufficient to represent the volume in the mouth. Additionally, the hydrophobic model drugs will also be employed as this study provides sufficient information on how to measure their release (i.e. curcumin; spectrophotometry at 422 nm and Nile red; fluorescence spectroscopy 546 nm and 628 nm). The only disadvantage of the study is that peptide based system may behave differently.
Elsawy, M., Smith, A., Hodson, N., Squires, A., Miller, A. and Saiani, A. (2016).
Modification of β-Sheet Forming Peptide Hydrophobic Face: Effect on Self-Assembly and
Gelation. Langmuir, 32(19), pp.4917-4923.
The authors successfully functionalized a F9 peptide, resulting in a loss of β sheet conformation; a decreased association and bundling of peptide fibres; and an increase in elasticity. The paper is useful as the methodologies used such as FTIR, TEM, rheology and AFM to assess the self-assembly, morphology and topology are provided in enough detail to replicate and will be applied in this research. However, the study did have inadequate statistical information and limited applications. Furthermore, the peptide they’ve used as
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the control is similar to the peptide derived in this proposal, hence some of the results are expected to be similar such as those from FTIR. Ischakov, R., Adler-Abramovich, L., Buzhansky, L., Shekhter, T. and Gazit, E. (2013).
Peptide-based hydrogel nanoparticles as effective drug delivery agents. Bioorganic &
Medicinal Chemistry, 21(12), pp.3517-3522.
The results from this study indicated that the Fmoc-FF peptide based hydrogels nanoparticles have potential applications in as delivery systems by encapsulating a variety of drugs (doxorubicin and 5-flurouracil). Although they utilized a different peptide, the methodology used in characterising (DLS) the nanoparticles is effective, provided in sufficient detail to replicate and will be exploited in this research. Additionally, they provide an alternative method to produce nanoparticles which could be explored if the method in this proposal fails. The paper can also be used to compare microparticle/nanoparticle characteristics and release profiles.
Moreira, I., Sasselli, I., Cannon, D., Hughes, M., Lamprou, D., Tuttle, T. and Ulijn, R.
(2016). Enzymatically activated emulsions stabilised by interfacial nanofibre networks. Soft
Matter, 12(9), pp.2623-2631.
Moreira et al., successfully demonstrated that the Fmoc-YL peptide enzymatically activated
nanofiber formation at the interface stabilizing chloroform-in-water emulsions. The study is
useful as the analytical tools used to assess the interfacial network are effective and will be
imitated in this research. The negative staining reagent used in TEM (ammonium molybdate
2%) has been proposed for this research. It should be noted however that due to their
system requiring an enzyme to trigger gelation some of the methods may have to be altered
slightly. Additionally, they didn’t produce any statistical data, hence it’s difficult to say if
their results are reproducible. The stains used for fluorescence microscopy are also
suggested in the proposal (fluorescein isothiocyanate and thioflavin T).
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8.0 References
Bai, S., Pappas, C., Debnath, S., Frederix, P., Leckie, J., Fleming, S. and Ulijn, R. (2014).
Stable Emulsions Formed by Self-Assembly of Interfacial Networks of Dipeptide
Derivatives. ACS Nano, 8(7), pp.7005-7013.
Barros, L., Dueñas, M., Dias, M., Sousa, M., Santos-Buelga, C. and Ferreira, I. (2013).
Phenolic profiles of cultivated, in vitro cultured and commercial samples of Melissa
officinalis L. infusions. Food Chemistry, 136(1), pp.1-8.
Biancalana, M. and Koide, S. (2010). Molecular mechanism of Thioflavin-T binding to
amyloid fibrils. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1804(7),
pp.1405-1412.
Boothroyd, S., Miller, A. and Saiani, A. (2013). From fibres to networks using self-
assembling peptides. Faraday Discussions, 166, p.195.
Caplan, M., Moore, P., Zhang, S., Kamm, R. and Lauffenburger, D. (2000). Self-Assembly of
a β-Sheet Protein Governed by Relief of Electrostatic Repulsion Relative to van der Waals
Attraction. Biomacromolecules, 1(4), pp.627-631.
Caplan, M., Schwartzfarb, E., Zhang, S., Kamm, R. and Lauffenburger, D. (2002a). Effects
of systematic variation of amino acid sequence on the mechanical properties of a self-
assembling, oligopeptide biomaterial. Journal of Biomaterials Science, Polymer Edition,
13(3), pp.225-236.
Caplan, M., Schwartzfarb, E., Zhang, S., Kamm, R. and Lauffenburger, D. (2002b). Control
of self-assembling oligopeptide matrix formation through systematic variation of amino acid
sequence. Biomaterials, 23(1), pp.219-227.
de Boer, P., Hoogenboom, J. and Giepmans, B. (2015). Correlated light and electron
microscopy: ultrastructure lights up!. Nature Methods, 12(6), pp.503-513.
de Ciriano, M., Rehecho, S., Calvo, M., Cavero, R., Navarro, Í., Astiasarán, I. and Ansorena,
D. (2010). Effect of lyophilized water extracts of Melissa officinalis on the stability of algae
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and linseed oil-in-water emulsion to be used as a functional ingredient in meat products. Meat
Science, 85(2), pp.373-377.
Delmar, K. and Bianco-Peled, H. (2016). Composite chitosan hydrogels for extended release
of hydrophobic drugs. Carbohydrate Polymers, 136, pp.570-580.
Duffó, G. and Castillo, E. (2004). Development of an Artificial Saliva Solution for Studying
the Corrosion Behavior of Dental Alloys. CORROSION, 60(6), pp.594-602.
Elsawy, M., Smith, A., Hodson, N., Squires, A., Miller, A. and Saiani, A. (2016).
Modification of β-Sheet Forming Peptide Hydrophobic Face: Effect on Self-Assembly and
Gelation. Langmuir, 32(19), pp.4917-4923.
Encalada, M., Hoyos, K., Rehecho, S., Berasategi, I., de Ciriano, M., Ansorena, D.,
Astiasarán, I., Navarro-Blasco, Í., Cavero, R. and Calvo, M. (2011). Anti-proliferative Effect
of Melissa officinalis on Human Colon Cancer Cell Line. Plant Foods for Human Nutrition,
66(4), pp.328-334.
Fan, T., Yu, X., Shen, B. and Sun, L. (2017). Peptide Self-Assembled Nanostructures for
Drug Delivery Applications. Journal of Nanomaterials, 2017, pp.1-16.
Goldstein, J., Newbury, D., Michael, J., Ritchie, N., Scott, J. and Joy, D. (2017). Scanning
electron microscopy and x-ray microanalysis. 4th ed. New York: Springer, pp.116-150.
Gomes, E., Mendes, S., Leite-Almeida, H., Gimble, J., Tam, R., Shoichet, M., Sousa, N.,
Silva, N. and Salgado, A. (2016). Combination of a peptide-modified gellan gum hydrogel
with cell therapy in a lumbar spinal cord injury animal model. Biomaterials, 105, pp.38-51.
Guilbaud, J., Rochas, C., Miller, A. and Saiani, A. (2013). Effect of Enzyme Concentration of
the Morphology and Properties of Enzymatically Triggered Peptide Hydrogels.
Biomacromolecules, 14(5), pp.1403-1411.
Habibi, N., Kamaly, N., Memic, A. and Shafiee, H. (2016). Self-assembled peptide-based
nanostructures: Smart nanomaterials toward targeted drug delivery. Nano Today, 11(1),
pp.41-60.
17
Hilal, N., Ismail, A., Matsuura, T. and Oatley-Radcliffe, D. (2017). Membrane
characterization. 1st ed. Netherlands: Elsevier, pp.145-158.
Inagaki, Y., Abe, M., Inaki, R., Zong, L., Suenaga, H., Abe, T. and Hoshi, K. (2017). A Case
of Systemic Infection Caused by Streptococcus pyogenes Oral Infection in an Edentulous
Patient. Diseases, 5(3), p.17.
Ischakov, R., Adler-Abramovich, L., Buzhansky, L., Shekhter, T. and Gazit, E. (2013).
Peptide-based hydrogel nanoparticles as effective drug delivery agents. Bioorganic &
Medicinal Chemistry, 21(12), pp.3517-3522.
Khullar, R., Kumar, D., Seth, N. and Saini, S. (2012). Formulation and evaluation of
mefenamic acid emulgel for topical delivery. Saudi Pharmaceutical Journal, 20(1), pp.63-67.
Kopeček, J. and Yang, J. (2009). Peptide-directed self-assembly of hydrogels. Acta
Biomaterialia, 5(3), pp.805-816.
Laverty, G. (2017). Antimicrobial peptides as hydrogels for tissue regeneration and repair.
Peptides and Proteins as Biomaterials for Tissue Regeneration and Repair, pp.347--368.
Lian, M., Chen, X., Lu, Y. and Yang, W. (2016). Self-Assembled Peptide Hydrogel as a
Smart Biointerface for Enzyme-Based Electrochemical Biosensing and Cell Monitoring. ACS
Applied Materials & Interfaces, 8(38), pp.25036-25042.
Mimica-Dukic, N., Bozin, B., Sokovic, M. and Simin, N. (2004). Antimicrobial and
Antioxidant Activities ofMelissa officinalisL. (Lamiaceae) Essential Oil. Journal of
Agricultural and Food Chemistry, 52(9), pp.2485-2489.
Moreira, I., Sasselli, I., Cannon, D., Hughes, M., Lamprou, D., Tuttle, T. and Ulijn, R.
(2016). Enzymatically activated emulsions stabilised by interfacial nanofibre networks. Soft
Matter, 12(9), pp.2623-2631.
Naga Sravan Kumar Varma, V., Maheshwari, P., Navya, M., Reddy, S., Shivakumar, H. and
Gowda, D. (2014). Calcipotriol delivery into the skin as emulgel for effective permeation.
Saudi Pharmaceutical Journal, 22(6), pp.591-599.
18
Philpott, M., Rogers, C., Yapp, C., Wells, C., Lambert, J., Strain-Damerell, C., Burgess-
Brown, N., Gingras, A., Knapp, S. and Müller, S. (2014). Assessing cellular efficacy of
bromodomain inhibitors using fluorescence recovery after photobleaching. Epigenetics &
Chromatin, 7(1), p.14.
Ramanauskienė, K., Stelmakiene, A. and Majienė, D. (2015). Assessment of Lemon Balm
(Melissa officinalisL.) Hydrogels: Quality and Bioactivity in Skin Cells. Evidence-Based
Complementary and Alternative Medicine, 2015, pp.1-7.
Scott, G., McKnight, P., Tuttle, T. and Ulijn, R. (2015). Tripeptide Emulsifiers. Advanced
Materials, 28(7), pp.1381-1386.
Sigma Aldrich (2017). Phenylalanine and Phenylglycine. [image] Available at:
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Tian, Y., Devgun, J. and Collier, J. (2011). Fibrillized peptide microgels for cell
encapsulation and 3D cell culture. Soft Matter, 7(13), p.6005.
Venkataharsha, P., Maheshwara, E., Prasanna, Y., Reddy, V., Rayadu, B. and Karisetty, B.
(2015). Liposomal Aloe vera trans-emulgel drug delivery of naproxen and nimesulide: A
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engineering. Reactive and Functional Polymers, 41(1-3), pp.91-102.
19
10.0 Appendix
A.1 Suppliers and Cost of Chemicals
Appendix A.1 – A list of materials required for the proposed experiments including their; purity, supplier, pack
size, quantity required, unit cost and total cost. Along with the travel and equipment rental costs.
List of Materials Purity Supplier Pack Size Quantity Per Unit Total cost
Polysorbate 20 99% Sigma-Aldrich 100 g 1 £ 16.50 £ 16.50
Polysorbate 80 99% Sigma-Aldrich 100 g 1 £ 16.80 £ 16.80
Sodium dodecyl sulfate
99% ACROS Organics 25 g 1 £ 17.80 £ 17.80
Melissa oil 99% Aromatherapy Ltd 10 mL 1 £ 16.95 £ 16.95
Curcumin >95% Thermo Fisher 1 g 1 £ 33.48 £ 33.48
Nile Red 99% ACROS Organics 100 mg 1 £ 56.60 £ 56.60
Thioflavin T >95% Thermo Fisher 5 g 1 £ 20.80 £ 20.80
Fluorescein isothiocyanate
≥90% Sigma-Aldrich 100 mg 1 £ 21.10 £ 21.10
Sodium Hydroxide pellets
≥98% Sigma-Aldrich 1 Kg 1 £ 26.90 £ 26.90
HPLC grade water 100% Thermo Fisher 2.5 L 1 £ 36.12 £ 36.12
Chloroform ≥99.8% Thermo Fisher 500 mL 1 £ 23.65 £ 23.65
Potassium chloride >95% Thermo Fisher 500 g 1 £ 21.28 £ 21.28
Ammonium molybdate
≥99% Thermo Fisher 100 g 1 £ 18.30 £ 18.30
Calcium chloride dihydrate
≥99% Thermo Fisher 25 g 1 £ 16.70 £ 16.70
Sodium chloride >99.5% ACROS Organics 1 Kg 1 £ 12.90 £ 12.90
Potassium phosphate monobasic
≥99% ACROS Organics 25 g 1 £ 16.30 £ 16.30
Potassium phosphate dibasic
≥98 % ACROS Organics 500 g 1 £ 36.40 £ 36.40
Potassium bicarbonate
99% Honeywell Fluka 100 g 1 £ 11.90 £ 11.90
Potassium thiocyanate
≥99% ACROS Organics 250 g 1 £ 22.60 £ 22.60
Citric acid 99.50% ACROS Organics 250 g 1 £ 10.30 £ 10.30
Alpha-amylase 100% Thermo Fisher 5 g 1 £ 28.79 £ 28.79
Methanol ≥99.9% Honeywell Fluka 1 L 1 £ 17.62 £ 17.62
Dichloromethane ≥99% Thermo Fisher 500 mL 1 £ 25.72 £ 25.72
Equipment rental £ - £ -
Travel £ - £ -
Total £ 499.79
20
A.2 Gantt Chart
Appendix A.2- illustrating a Gantt chart for the planned activities, demonstrating the start and
duration of each. FTIR: Fourier-transform infrared spectroscopy, FM: fluorescence microscopy, AFM:
atomic force microscopy, TEM: transmission electron microscopy, SEM: scanning electron
microscopy, DLS: dynamic light scattering, DL: drug loading and EE: entrapment efficiency.
ACTIVITY PLAN START
PLAN DURATION WEEKS
1 2 3 4 5 6 7 8 9 10 11 12
Formulation 1 3
Stability 1 10
FTIR 3 2
FM 4 1
AFM 4 1
TEM 4 1
SEM 5 1
Rheology 5 1
DLS 5 1
DL & EE 6 2
Drug release 8 3
Data analysis 8 12
Report writing 10 12
0
A.3 Risk Assessment
RISK ASSESSMENT FORM
Risk Assessment For Assessment Undertaken
By
Assessment Reviewed
Service / Faculty / Dept:
School of Pharmacy and
Biomedical Sciences
Name: Ronak Patel Name:
Location of Activity:
Maudland building
Date: 08/01/2018 Date:
Activity:
Formulation and Drug
release testing of emulgel
microparticles
Signed by Head of Dept /
equivalent:
REF: Date:
List significant
hazards here:
List groups
of people
who are at
risk:
List existing
controls, or refer
to safety
procedures
etc:
For risks which
are not adequately
controlled, list the
action needed:
Remaining
level of
risk: high,
med or low
Chemical Hazards
Students/Staff Gloves, Safety
Glasses, proper
training on storage
and use
Low
Physical Hazards
Students/Staff Follow lab
procedure and
keep work area
tidy
Low
Spillage Students/Staff Follow lab
procedure, lab
coat
Low
Equipment
Students/Staff Follow protocols,
don’t allow waste
container to over
fill
Low
Fumes
Students/Staff Fume hood Low
1
Heat/Flames
Students/Staff Follow lab
procedure, proper
training
Fire safety
(extinguishers),
evacuation
Low
Electricity (wires)
Students/Staff Unplug all
appliances when
not used
Low
Poor lighting
Students/Staff Always working in
a well illuminated
room
Low
Name of Student Signature of Student Date
Ronak Patel
08/01/2018
Name of Supervisor Signature of Supervisor Date
Mohamed Elsawy
0
A.4 Ethics
UNIVERSITY OF CENTRAL LANCASHIRE
Ethics Checklist
All activities - undergraduate, postgraduate, research, commercial, knowledge transfer, evaluation, audit or teaching and learning - need ethical consideration.
This checklist will identify whether a project requires an application for ethics approval, and to which committee it should be referred to. No field work, experimentation or work with participants can start until approval is granted. The questions should be completed by the Principal Investigator or supervisor of the proposed project. Where projects involve students, the Principal Investigator is always the supervisor/Director of Studies and never the student.
Principal Investigators, or supervisors/Director of Studies, are responsible for ensuring that all activities fall within the principles set down in the University Code of Conduct for Research and the University Ethical Principles for Teaching, Research, Knowledge Transfer, Consultancy and Related Activities. They are also responsible for exercising appropriate professional judgment in undertaking this review and evaluating the activity according to the criteria laid down in this checklist. If you are uncertain about any sections of this document, or need further information and guidance, please contact the relevant ethics committee.
If, on completion of the checklist:
• any question is answered ‘Yes’, then an application for ethical approval is required:- o For undergraduate and postgraduate taught projects, students should in the first instance
discuss the project and ethical issues with their supervisor. Unless the project is considered to be ethically complex or of a sensitive nature (e.g. involves vulnerable populations) submission for ethical approval should be sought through the relevant School Ethics Committee or process.
o For research, commercial and other projects, use the questions to help compile suitable evidence and submit an application to the relevant ethics committee.
• all questions are answered ‘No’ and you (the Principal Investigator) are not concerned with the ethical nature of the activity, then it is unnecessary to apply for ethical approval. However, it is still incumbent on you to observe the University’s Ethical Principles in the conduct of the activity and to record that:
o a review has taken place of the ethical aspects of the activity; and that o either no ethical issues have been identified or ethical issues have been identified
but that these have been addressed satisfactorily.
All research student registration proposals, irrespective of the outcome of the Ethics Checklist, need to be
submitted to the relevant ethics committee to be dealt with either by Chair’s Action or full review. See
specific guidance for research degree students at https://www.uclan.ac.uk/research/environment/assets/ethics_pack_for_research_degree_students_1718.pdf
1
Further details on the e-Ethics process, including an electronic version of this checklist, are available at
https://www.uclan.ac.uk/students/research/ethics.php
2
1 Project
1.1 Project Title
Peptide-based emulgel microparticles for the sustained release of drugs in the treatment of
dental diseases
1.2 Project type Staff
research
Research degree
(inc Prof Doc) PG taught X UG taught Commercial
1.3 Short
description
in layman's terms
[no acronyms or
jargon]
Formulation, Characterisation and drug release testing of peptide-based emulgel/microparticles containing Melissa oil or model drugs (curcumin or Nile Red)
1.4 Dates
Start: 01/05/2018 End: 10/07/2018
1.5 School of …..
Pharmacy and Biomedical Sciences
1.6 Project
supervisor /principal
investigator:
name, position and
original signature
Ronak Patel, Student
1.7 Co-workers:
names and positions
[e.g. student]
Supervisor: Dr. Mohamed Elsawy,
Lecturer in Pharmaceutics
Read any associated procedures and guidance or follow any associated checklist link, and delete, ‘Yes’ or
‘No’, for each characteristic.
If you respond ‘No’, then in your judgment you believe that the characteristic is irrelevant to the activity. You
may only tick ‘No’ to the main question (i.e. A, B, etc) where none of the statements in that section apply to
your activity.
If you are unsure whether to answer ‘Yes’ or ‘No’ to a question, you should answer ‘Yes’ and submit details
to relevant ethics committee for initial review.
3
A) Does the activity involve human participants, data or material e.g. as research participants
including the use of their data or using human tissue/fluid/DNA samples?
No
If Yes, and
Where the activity involves any external organisation for which separate and specific ethics
clearance is required (e.g. NHS; school; any criminal justice agencies including the Police, Crown
Prosecution Service, Prison Service, Probation Service or successor organisation) seek and gain
external ethics before submitting for UCLan ethical approval. Submission can be just the external
organisation ethics application paperwork – email details to [email protected] to check.
Where the activity involves the use of human tissue / DNA samples or body fluid seek and gain
relevant external ethics before submitting to relevant e-Ethics Committee. Submission can be just
the external organisation ethics application paperwork (e.g. Brain Tissue North West) – email
details to [email protected] to check.
Continued/…
For all other activities involving human participants, their data or materials*, complete and submit
UCLan Ethics Committee Application Form to relevant e-Ethics Committee – BAHSS; PSYSOC or
STEMH.
* such as :-
requiring participants to give informed consent;
potential imbalance of power and authority which might compromised the validity of participants’
consent;
researchers and/or participants in the potential disclosure of any information relating to illegal
activities; the observation of illegal activities; or the possession, viewing or storage of any material
(whether in hard copy or electronic format) which may be illegal
potential risk of physical, social, emotional or psychological harm, distress or discomfort to the
researchers or participants (Please note also the University’s Policy and procedures on Safeguarding
and Prevent);
deception of the participant be necessary during the activity;
aim to shock or offend (e.g. art)
invasion of privacy or access to confidential information about people without their permission
excavation and study of human remains
B) Does the activity involve isolation and culture of micro-organisms, or genetically modified
micro-organism?
No
4
If so, process via UCLan Biological Safety Committee before submitting to the relevant e-Ethics
Committee
C) Does the activity involve scientific procedures1 being applied to a vertebrate animal (other
than humans) or cephalopods?
No
If so, please email [email protected] requesting application form and submission deadlines for
AWERB
D) Does the activity involve collection of rare plants, endangered species or work in the
natural environment?
No
If so please submit this checklist together with outline details of the activity / UCLan’s role to
E) Does activity relate to military/defence/weapons or the Defence industry, including
excavation of battlefields, military installations, etc (i.e. site with unexploded bomb)?
No
If so please submit this checklist together with outline details of the activity / UCLan’s role to
F)
Are there any potential other ethical and political concerns?
e.g. Are you aware of any
• potential ethical concerns or political concerns that may arise from either the conduct or dissemination of this activity, e.g. unethical practices of companies funding this research; results of research being used for political gain by others; potential for liability to the University from your research?
• ethical concerns about collaborator company / organisation, e.g. its product has a harmful effect on humans, animals or the environment; it has a record of supporting repressive regimes; does it have ethical practices for its workers and for the safe disposal of products?
No
If so please submit this checklist together with outline details of the activity / UCLan’s role to
1 Including interrupting an animal’s natural environment (e.g. tracking or observing wild deer)
0
A.5 COSHH form
THE CONTROL OF SUBSTANCES HAZARDOUS TO HEALTH REGULATIONS 2002 (as amended) C.O.S.H.H. RISK ASSESSMENT FORM.
1. School UCLAN 2. Assessors name Ronak Patel
3. Job title Student 4. Tel Ext 07946535986
5. Briefly describe the task/process:
Formulation, Characterisation and drug release testing of peptide-based emulgel microparticles containing Melissa oil or model drugs (curcumin
or Nile red)
6. Substances used or produced as by- products or waste
Approx. quantities
(Tick ✓) Exposure route Frequency and duration of exposure
Workplace exposure Limit.
(Tick ✓) Special requirements (including references to guidance, codes of practice etc).
inhale ingest inject absorb storage fire waste other
1. Melissa oil 250 µL ✓ ✓ 15 mins ✓ ✓
2. Peptide (FGEFGK) 5 g ✓ 15 mins ✓
3. Curcumin 4 mg/mL ✓ ✓ 48 hours ✓
4. Nile red 1 mg/mL ✓ ✓ 48 hours ✓
5. Chloroform 250 µL ✓ ✓ 15 mins STEL-29.7 mg/m3 ✓ ✓
6. Sodium dodecyl sulfate
5 g ✓ ✓ 15 mins 480 mins ✓
7. Citric Acid 30 mg/L ✓ ✓ 10 mins 480 mins ✓
8. Methanol 10 mL ✓ ✓ 5 mins STEL-333 mg/m3 ✓ ✓ ✓
9. Thioflavin T 3 mg ✓ ✓ 2 mins ✓
10. Fluorescein isothiocyanate
3 mg ✓ ✓ 2 mins ✓
11. Sodium hydroxide 15 µL ✓ ✓ 15 mins ✓
12. Alpha-amylase 2 g/L ✓ 10 mins ✓ ✓
1
13. Potassium chloride 720 mg/L ✓ ✓ 10 mins ✓
14. Ammonium molybdate
3 mg ✓ ✓ 2 mins STEL- 10 mg/m3 15 mins
✓
15. Calcium chloride 220 mg/L ✓ ✓ 10 mins ✓ ✓
16. Sodium chloride 600 mg/L ✓ ✓ 10 mins
17. Potassium phosphate
866 mg/L ✓ ✓ 10 mins ✓
18. Potassium bicarbonate
1.5 g/L ✓ ✓ 10 mins ✓
19. Polysorbate 20 5 g ✓ ✓ 15 mins ✓
20. Polysorbate 80 5 g ✓ ✓ 15 mins ✓ ✓
21. Dichloromethane 50 mL ✓ ✓ 5 mins STEL- 1060 mg/m3
✓ ✓ ✓
7. Are substances likely to be?
splashed 1-21 spilled 1-21 diluted 2-21 Decanted 1,3- 21
heated 1-7, 11-20
mixed 1-6,19,20; 7,11- 18; 8&21
sprayed
used in a poorly ventilated or confined space
8. Hazard classification: 9. Known health effects: 10. Results of relevant health surveillance
11. Results of exposure monitoring
Very Toxic 5,6,8 Corrosive 6,11,14,21 1-skin corrosion, oral toxicity, target
1- Acute toxicity LD50 mouse (561 mg/kg)
Toxic 1,5,6,8, 14
Irritant 3,4,5,6,7,11,12, 14,15,21
3-eye, respiratory and skin irritation
6- Acute toxicity LD50 rat (1,200 mg/kg)
Harmful 5,6,8, 14
Sensitising 12 4-eye irritation 5-harmful if swallowed;
8- Oral and dermal toxicity LD50 rat (2769 mg/kg)
Mutagenic 5 Toxic for Reproduction
toxic if inhaled; skin and eye irritant; may cause drowsiness; causes cancer; damage to unborn child; prolonged exposure caused organ damage
21- Oral toxicity rat LD50 (2000 mg/kg), inhalation toxicity LD50 rat (60.14 mg/L, 4h)
2
Carcinogenic 5,21 Biological Hazard
6-oral/inhaled toxicity, skin/eye irritation, organ toxicity
7-eye irritation
Approved Classification of Biological Hazard
8-skin/eye/respiratory irritation; oral and inhaled toxicity; causes damage to organs 11- skin burns and eye damage 12- may cause allergy or asthma symptoms 14- oral toxicity (cat 4); skin/eye corrosion and irritant; specific organ toxicity 15- eye irritation 21- skin/eye corrosion and irritation; carcinogenic, organ toxicity (single exposure), may cause drowsiness
12. Additional risks: for example circumstances where work will involve exposure to more than one substance hazardous to health, consider the risk presented by exposure to such substances in combination. Also, non-routine maintenance may present additional risk of exposure.
13. Persons likely to be exposed:
Staff ✓ Students ✓ Public Other (specify)
3
14. Risk (without precautions)
Unsure ➔ Consult your Line Manager and ask the Health, Safety & Environment Section to help, if required. ✓ High ➔ For high and medium risks, precautions must be taken to control the risks.
Medium
Low ➔ For low risks, consideration need only be given to easy, inexpensive precautions to reduce the risk further.
15. Hierarchy of preventative and control measures to reduce exposure (in order of priority)
Control Reference procedures, rules, records etc.
Effect of control measure
Change the task or process so that the hazardous substance is not required or generated.
Replace the substances with a safer alternative.
Totally isolate or enclose the process.
3 Partially enclose the process and use local exhaust ventilation. Use fume hood when working with toxic/harmful chemicals
Reduce chance of inhaling harmful fumes
Ensure good general ventilation.
Use a system of work that minimises the chance and degree of exposure.
1 Provide personal protective equipment (PPE). Lab coat, Gloves, Goggles, masks
Reduce chance of skin/eye contact with harmful substances
2 Train and inform staff in the safe system of work and risks. Training in proper use of equipment, mixing chemicals and lab etiquette
Reduce chance of accidents and improper procedures
4 Additional supervision. Supervision when working with hazardous chemicals
Reduce chance of accidents
Examination, testing and maintenance of engineering controls and/or PPE.
Monitoring of exposure.
Health Surveillance.
Other (specify).
4
16. Remaining risk
Unsure Consult your Line Manager and ask the Health, Safety & Environment Section to help.
High Date of assessment
Review date
Medium ✓ Low Consideration need only be given to easy, inexpensive precautions to reduce the risk further.
17. Emergency Arrangements:
Supervisor’s Name:
Mohamed Elsawy Signature: Date:
