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High Throughput Crystallization of Peptides Using the iCrystal System and Metal-Assisted and Microwave-Accelerated Evaporative Crystallization (MA-MAEC) Technique

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ABSTRACT
Title of Thesis:
HIGH THROUGHPUT CRYSTALLIZATION OF GLUTATHIONE USING THE ICRYSTAL SYSTEM AND METALASSISTED AND MICROWAVE-ACCELERATED EVAPORATIVE CRYSTALLIZATION (MA-MAEC) TECHNIQUE.
Anish Bhandari, Master of Science
Thesis Advisor:
Kadir Aslan, Ph.D.
Department of Chemistry
Metal-Assisted and Microwave-Accelerated Evaporative Crystallization
(MA-MAEC) is a novel technique in the field of evaporative crystallization led by
the Aslan Research Group. The MA-MAEC technique is based on combined use
of microwave heating (speeds up the process) and metal nanoparticle structure
such as silver, gold, copper, Indium tin oxide (ITO), nickel (provides selective nucleation sites) to yield quality crystals of individual amino acids, small molecules,
and peptides in a very short period of time.
This Master of Science thesis focuses on the crystallization of glutathione
(GSH), a tripeptide on 95-well iCrystal plates using a mono-mode microwave
cavity (the iCrystal system) via MA-MAEC technique to yield high quality crystals.
The previous studies of GSH crystallization using the MA-MAEC technique with
Silver Nano films (SNFs)-deposited 21-well iCrystal platform proved most effective based on the crystallization time and the overall quality of GSH crystals at
500 mg/mL concentration. Sodium acetate was chosen to be the best solvent for
crystallization. In this study, we put together the underlying mechanism studied in
the prior research of GSH crystallization to successfully perform the crystallization on an ITO plated 95-well iCrystal plate at 500 mg/mL concentration at 5, 10,
30, 60 and 120 minutes time intervals hereby to enhance the crystallization process. Time of crystallization, crystal size, Fourier transform infrared spectroscopy
(FTIR) and X-ray diffraction (XRD) were used to characterize crystals.
The results demonstrated that the MA-MAEC technique affords for the
rapid crystallization of GSH in a high-throughput fashion using the iCrystal monomode microwave system. The crystallization time was reduced by up to 3-fold
compared to control conditions (room temperature). Crystal morphology of the
product crystals was similar to those reported in the literature.
HIGH THROUGHPUT CRYSTALLIZATION OF PEPTIDES USING THE ICRYSTAL SYSTEM AND METAL-ASSISTED AND MICROWAVE-ACCELERATED
EVAPORATIVE CRYSTALLIZATION (MA-MAEC) TECHNIQUE
by
Anish Bhandari
A Thesis Submitted in Partial Fulfillment
of the Requirements for the Degree
Master of Science
MORGAN STATE UNIVERSITY
December 2017
ProQuest Number: 10617293
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HIGH THROUGHPUT CRYSTALLIZATION OF PEPTIDES USING THE ICRYSTAL SYSTEM AND METAL-ASSISTED AND MICROWAVE-ACCELERATED
EVAORATIVE CRYSTALLIZATION (MA-MAEC) TECHNIQUE
by
Anish Bhandari
has been approved
December 2017
THESIS COMMITTEE APPROVAL:
_________________, Committee Chair
Kadir Aslan, Ph.D.
________________
Dereje Seifu, Ph.D.
________________
Birol Ozturk, Ph.D.
________________
Gabrielle L. McLemore, Ph.D.
ii
DEDICATION
This work is dedicated to Mr. Bharat Bhandari and Mrs. Sabita Bhandari.
iii
ACKNOWLEDGEMENT
First and foremost, I would like to offer my sincerest gratitude to my advisor, Dr. Kadir Aslan, who has supported me throughout my thesis with his patience, knowledge and encouragement. I could not wish for a better advisor/mentor. In my daily work, I have been blessed with cheerful and helpful group
of students, the one and only the Aslan Research Group members.
I would also like to thank the rest of my thesis committee for their encouragement, and insightful comments: Prof. Birol Ozturk, Prof. Dereje Seifu and
Prof. Gabrielle McLemore. I would like to thank my family for continuous support
and encouragement throughout my study period and during hard times. I would
also like to thank Ms. Brittney for introducing me to the iCrystal system and helping me run the experiments. Thank you Enock, Cynthia, Carisse, Zeinab, Bridgit,
Shay for your help.
iv
TABLE OF CONTENTS
List of Tables………………………………………………………………………….vii
List of Figures………………………………………………………………………….ix
CHAPTER 1: INTRODUCTION……………………………………………………1-3
CHAPTER 2: LITERATURE REVIEW………………………………………………5
2.1 Molecular characteristics and modeling of GSH crystals……………………5
2.2 The Novel MA-MAEC procedure and performance………………………….7
2.3 Different phases of crystal growth and challenges…………………………...10
CHAPTER 3: EXPERIMENTAL DESIGN
3.1 Materials…………………………………………………………………………..11
3.2 Methods…………………………………………………………………………...11
3.2.1 Solvent Selection Study……………………………………………………….11
3.2.2 Preparation of Sodium Acetate buffer……………………………………….11
3.3 Preparation of GSH solution……………………………………………………12
3.4 Preparation of ITO (Indium tin Oxide) films…………………………………...12
3.5 Crystallization of GSH using MA-MAEC technique…………………………..13
3.6 Characterization of GSH crystals………………………………………………15
CHAPTER 4: RESULTS AND DISCUSSION
4.1 Solubility of Glutathione…………………………………………………………16
4.2 Summary of crystallization time and crystal size…………………………….16
4.3 Crystallization of 500 mg/mL GSH at 5 minutes interval…………………….17
v
4.4 Crystallization of 500 mg/mL GSH at 10 minutes interval…………………..22
4.5 Crystallization of 500 mg/mL GSH at 30 minutes interval…………………..27
4.6 Crystallization of 500mg/mL GSH at 60 minutes interval……………………32
4.7 Crystallization of 500mg/mL GSH at 120 minutes interval…………………..36
4.8 Individual crystal growth assessment …………………………………………40
4.8 Characterization of GSH crystals by Fourier Transform Infrared
Spectroscopy (FTIR)………………………………………………………..48
4.9 Characterization of GSH crystals using X-ray diffraction…………………...56
CHAPTER 5: CONCLUSION……………………………………………………….63
CHAPTER 6: REFERENCES………………………………………………………64
vi
LIST OF TABLES
Table 1. The powder pattern XRD data generated from Diamond Crystal Impact
Software showing d-spacing, reflection parameters d (h, k, l), 2-theta
and corresponding multiplicity factors…………………………………….7
Table 2. Summary of results for the crystallization of GSH from 500-mg/mL on
circular crystallization platforms (95 wells) at different time intervals
using MA-MAEC technique………………………………………………….17
Table 3 Summary of Good and Bad wells in different trials at 5 minutes
Interval………………………………………………………………………20
Table 4 Summary of Good and Bad wells in different trials at 10 minutes
Interval………………………………………………………………………25
Table 5. Summary of Good and Bad wells in different trials at 30 minutes
Interval………………………………………………………………………30
Table 6 Summary of Good and Bad wells in different trials at 60 minutes
Interval………………………………………………………………………34
Table 7. Summary of Good and Bad wells in different trials at 120 minutes
Interval………………………………………………………………………38
vii
Table 8. An individual GSH crystal size at 60 minutes under different
experimental conditions…………………………………………………...40
Table 9. Single crystal growth observed at different conditions and different
time interval heating at 500 mg/mL GSH concentration……………….41
Table 10. Comparison of FTIR results of 500 mg/mL GSH at different time
intervals to the literature…………………………………………………49
viii
LIST OF FIGURES
Figure 1. Structures of glutamic acid, glycine and cysteine acid………………..1
Figure 2. Structure of Glutathione…………………………………………………..2
Figure 3. An illustration of Orthorhombic crystal system GSH tri-peptide
molecule in its Unit cell obtained using Diamond Software………….6
Figure 4. Schematic depiction of crystal formation in growth with MA-MAEC…9
Figure 5. Schematic diagram of preparation of 95-well PMMA crystallization
plate………………………………………………………………………..13
Figure 6. The iCrystal microwave used in the MA-MAEC technique…………...14
Figure 7. Optical image of GSH crystals grown using iCrystal system at 70 W
in an ITO deposited 95 wells circular platform in 5 minutes time
interval…………………………………………………………………18
Figure 8. Average crystal size and number of 500 mg/mL GSH at 5 minutes
time interval………………………………………………………………19
Figure 9. A pictorial description of “GOOD” and “BAD” crystals obtained at
three different experimental trials for 5 minute time interval………..21
Figure 10. Optical image of GSH crystals grown using iCrystal system at 70 W
in an ITO deposited 95 wells circular platform in 10 minutes time
interval………………………………………………………………….23
Figure 11. Average crystal size and number of 500 mg/mL GSH at 10 minutes
time interval………………………………………………………………24
Figure 12.A pictorial description of “GOOD” and “BAD” crystals obtained at
three different experimental trials for 10 minute time interval……….26
Figure 13. Optical image of GSH crystals grown using iCrystal system at 70 W
in an ITO deposited 95 wells circular platform in 30 minutes time
interval…………………………………………………………………..28
ix
Figure 14. Average crystal size and number of 500 mg/mL GSH at 30 minutes
time interval………………………………………………………………29
Figure 15. A pictorial description of “GOOD” and “BAD” crystals obtained at
three different experimental trials for 30 minute time interval………31
Figure 16. Optical image of GSH crystals grown using iCrystal system at 70 W
in an ITO deposited 95 wells circular platform in 60 minutes time
interval……………………………………………………………………..33
Figure 17. Average crystal size and number of 500 mg/mL GSH at 60 minutes
time interval……………………………………………………………….34
Figure 18. A pictorial description of “GOOD” and “BAD” crystals obtained at
three different experimental trials for 60 minute time interval………35
Figure 19. Optical image of GSH crystals grown using iCrystal system at 70 W
in an ITO deposited 95 wells circular platform in 120 minutes time
interval…………………………………………………………………….37
Figure 20. Average crystal size and number of 500 mg/mL GSH at 120 minutes
time interval………………………………………………………………38
Figure 21. A pictorial description of “GOOD” and “BAD” crystals obtained at
three different experimental trials for 120 minute time interval………39
Figure 22. An individual crystal growth at room temperature (control)………....43
Figure 23. An individual crystal growth of GSH at 5 minute interval……………43
Figure 24. An individual crystal growth of GSH at 10 minute interval…………..44
Figure 25. An individual crystal growth of GSH at 30 minute interval…………..44
Figure 26. An individual crystal growth of GSH at 60 minute interval…………..45
Figure 27. An individual crystal growth of GSH at 120 minute interval…………45
Figure 28.(a&b) Linear function representation of a single crystal growth at
different conditions with their corresponding R squared values..46-47
x
Figure 29.Rate of crystal growth at different conditions………………………….47
Figure 30. FTIR of 500 mg/mL GSH at 5 minutes time interval at different
trials……………………………………………………………………….50
Figure 31 FTIR of 500 mg/mL GSH at 10 minutes time interval at different
trials……………………………………………………………………….51
Figure 32. FTIR of 500 mg/mL GSH at 30 minutes time interval at different
trials……………………………………………………………………….52
Figure 33. FTIR of 500 mg/mL GSH at 60 minutes time interval at different
trials……………………………………………………………………….53
Figure 34. FTIR of 500 mg/mL GSH at 120 minutes time interval at different
trials……………………………………………………………………….54
Figure 35. FTIR of 500 mg/mL GSH at room temperature (control)……………55
Figure 36. X-ray Diffraction analysis of GSH crystals obtained after
crystallization of 500 mg/ml GSH at 5 minutes interval……………..57
Figure 37. X-ray Diffraction analysis of GSH crystals obtained after
crystallization of 500 mg/ml GSH at 10 minutes interval…………….58
Figure 38. X-ray Diffraction analysis of GSH crystals obtained after
crystallization of 500 mg/ml GSH at 30 minutes interval……………59
Figure 39. X-ray Diffraction analysis of GSH crystals obtained after
crystallization of 500 mg/ml GSH at 60 minutes interval……………60
Figure 40. X-ray Diffraction analysis of GSH crystals obtained after
crystallization of 500 mg/ml GSH at 120 minutes interval………….61
Figure 41. X-ray Diffraction analysis of GSH crystals obtained after
crystallization of 500 mg/ml GSH (control)………………..………….62
xi
Figure 2. Structure of Glutathione
Glutathione is a major antioxidant involved in nutrient metabolism and
regulations including functions like DNA and protein synthesis, signal transduction, cell proliferation, apoptosis etc. 3. GSH depletion can cause various pulmonary diseases, myocardial ischemia, aging and Parkinson’s disease. Low levels
of intracellular GSH can lead to immunodeficiency and improper lymphocyte
function. Molecular crystals are among the most marketed pharmaceuticals.
Crystal form is very important to the performance in dosage for some compounds
that show challenges to drug delivery such as low solubility to aqueous environment, slow dissolution in gastrointestinal media and low permeability4. A thorough understanding of the relationships between physical structures and polymorph properties of pharmaceutical solids is important in selecting appropriate
ingredients for the advancement of the drug product5. A model mechanism of
studying rapid crystallization the MA-MAEC technique has proved efficient for the
crystal growth of smaller amino acids, such as, L-alanine6,7,L-alanine with LLeucine additive8, L-alanine with L-valine and L-tryptophan additive9, glycine10, L2
arginine acetate11,Acetaminophen12 and larger molecules like glutathione13 , lysozyme11,14, and proteins15.
Crystallization is an essential tool for studying the structure and properties
of a molecule. There are variety of ways one can perform crystallization. It requires time and patience to grow crystals that are of high quality. In the past, the
MA-MAEC technique of crystallization has not only proved to be effective with
rapid crystallization of compounds but also with delivery of high quality crystals.
Crystal morphology is also a key factor in the development of crystals for production in pharmaceutical, optoelectronics and explosives16. The average time of
crystallization of high quality GSH crystals using the MA-MAEC technique in an
iCrystal system was less compared to any other techniques or microwavesystems17.
The focus of this Master’s thesis includes production and processing of
high quality GSH crystals in a high-throughput fashion using the iCrystal system.
The MS thesis statement is “High Throughput Crystallization of Peptides using
the iCrystal System and Metal-Assisted and Microwave-Accelerated Evaporative
Crystallization (MA-MAEC) Technique.” This work comprises the results from
earlier studies of GSH crystallization using the MA-MAEC technique used as reference data. It is important to note that this research study introduces 95-well
PMMA platforms and ITO films into the crystallization process, which is different
from the studies conducted on GSH in the past. The study of L-alanine crystallization using MA-MAEC technique was however successfully performed in 95 and
204 wells18. The results obtained from this experimental work will be compared to
3
GSH crystallization data collected at room temperature.
4
CHAPTER II: Literature Review
GSH is an important biomarker and has a striking importance in the field
of therapeutics. The compounds that is known to decrease GSH levels and increase the chances of tumors have led GSH to become useful tool in the development of cancer treatment and therapy1. GSH as a solid drug defines its performance and directly affects the pharmaceutical efficacy as some compounds
undergo a variety of phase transformations during their formulation process,
which provides a challenge in GSH ‘s stability and bioavailability. A thorough understanding of the relationship between the solid state and the crystal structure of
the similar phases has been utilized for optimizing formulation strategies to design suitable protocols to avoid problems5. The MA-MAEC technique is built on
the basis of using of metal nanostructures embedded onto a surface and low
power microwave heating for selective and rapid crystallization of molecules 19.
This review emphasizes the following aspects: 1) molecular characteristics and
modeling of GSH crystals 2) the MA-MAEC technique and 3) different phases of
crystal growth and various challenges.
2.1 Molecular characteristics and modeling of GSH crystals
The molecular configuration of GSH crystals remains crucial to the understanding of its structure on a fundamental level. The tripeptide glutathione crystallizes in an orthorhombic system (azbzc;αzβzγ=90°) with space group P212121
and unit cell (simplest repeating unit in a crystal) parameters of a= 28.05 Å,
b=8.8 Å, and c=5.63 Å20. Figure 3 shows graphic ball and stick representation of
5
GSH tripeptide in a unit cell along different axes. Table 1 provides an overview of
the d-spacing, 2-theta angle, the Miller planes and its corresponding multiplicity
factors.
Figure 3. An illustration of orthorhombic crystal system GSH tri-peptide
molecule in its unit cell obtained using Diamond Software.
6
2Theta [°]
d-spacing [Å]
Intensity (105)
h
k
l
Multiplicity
22.363
3.9723
328
0
2
3
4
21.036
4.2199
132
1
1
3
8
31.938
2.7999
651
0
0
1
2
25.937
3.4325
313
1
2
1
8
43.91
8.3773
413
0
1
1
4
Table 1. The XRD pattern data generated from Diamond Crystal Impact Software
showing d-spacing, reflection parameters d(h,k,l), 2-theta and corresponding
multiplicity factors.
2.2 The novel MA-MAEC technique and performance
To date, there are various methods of crystallization of organic molecules
including proteins and peptides. Time is of the essence when it comes to crystallization technique and conditions. Crystallization of amino acids and smaller molecules using traditional evaporative crystallization technique may require up to
several hours to complete. Diao and his colleagues studied the role of surface
chemistry developed a basis for aiming to provide appropriate surface to control
nucleation during crystallization based on the nucleation induction time statistics
performed on Aspirin 21.
The Aslan Research Group has defined a new method called “the MAMAEC” technique for crystallization of amino acids and peptides within matter of
seconds. It is based on the combined use of microwave heating (speeds up the
7
process) and metal nanoparticle structure such as silver, gold, copper, ITO, nickel (provides selective nucleation sites)6. In addition, the metal nanoparticles act
as a microwave transparent medium that creates temperature gradients between
the metal surface and the solvent, which enhances the crystal growth process by
driving the molecule towards the cooler surface. The depiction of the mechanism
for the MA-MAEC technique is shown in the Figure 4 when microwaves are applied to a solution on a nanoparticle embedded surface (iCrystal plate), thermal
gradient progresses between the warm bulk solution and the cooler nanoparticle
layer due to the difference in thermal conductivities between the molecule of interest and the film surface. The result is mass transfer of molecule from warmer
solution to cooler nanoparticle area to bring the system to equilibrium. The continuous microwave heating brings more molecules to come in contact with already attached molecules in the nanoparticle layer19. Crystal growth continues
until all solution is evaporated. In the previous research by Lansiquot et. al, the
combined use of ITO dots and mono-mode microwave heating decreased the
crystallization time by 172-fold with number of crystals ten times more than that
of room temperature22. The PMMA platform offers circular shape for homogeneous heating; smaller size than microwave wavelength for effectiveness and ITO
films for the microwave-induced temperature gradient23. The iCrystal system and
the iCrystal plates have also been employed to demonstrate microwave accelerated bioassay24 technique for rapid detection of GFAP(Glial fibrillary acidic protein) and STX1 (Shiga Toxin 1) in buffer based on colorimetric and fluorescence
results25.
8
Figure 4. Schematic depiction of crystal formation in growth using MA-MAEC
technique.
In the previous research studies performed by the Aslan Research Group
in rapid crystallization of GSH peptides, it was concluded that the iCrystal microwave system was most effective compared to the conventional or the industrial
microwave. The Aslan Research Group observed the crystallization time and
crystal quality using three different types of microwave (conventional, industrial
and iCrystal) system. The crystallization time was accelerated by almost 7-fold
and the best quality crystals were produced while using the iCrystal plates with
(Silver nano-films) SNFs in the iCrystal system as compared to the other two microwaves13.
9
Homogeneous heating of the samples afforded by the iCrystal plate is
very important for the repeatability of the crystallization of GSH within the same
iCrystal plate. It is also important to ensure that there is a continuous and uniform
heating pattern along the crystallization plate. The 95-well iCrystal plates with
small circular shaped wells are designed to provide a uniform temperature distribution. Alabanza et. al. used COMSOL, computer simulation software that provided a predicted temperature variation and electric field distribution after 3 seconds of microwave heating in a conventional and found out that the temperature
variation between all wells of the PMMA differed by <1.0°C (predicted)6,26.
2.3 Different phases of crystal growth and challenges
There are three steps to crystallization (supersaturation> nucleation> crystal growth). Each step is very crucial and depends on the prior stages to a successful crystallization. Supersaturation is a dynamic force for crystal nucleation
and growth. Nucleation is the birth of crystal nuclei and can form spontaneously
or from the pre-existing crystals. Supersaturation and super cooling conditions
alone are not sufficient to cause crystallization in a system. The nuclei, which act
as the centers of crystallization, must exist before crystals can develop27. The
large local gradients in both concentration and temperature that are induced by
the generation of vapor at the heating surfaces and the vapor-liquid interface directly affects the mean size particles and particle size distribution (PSD) by increasing the nucleation rates28.
10
CHAPTER III: Experimental Design
3.1. Materials
Circular poly (methyl methylacrylate) or PMMA disks of 10 cm diameter
that were used in these experiments were purchased from McMaster-Carr (IL,
USA). Sodium acetate salt and L-glutathione used were purchased from SigmaAldrich (St. Louis, Missouri). Hydrochloric acid, pH (1-3) and ethyl alcohol solution were purchased from Pharmco-Aaper (Brookfield, CT), USA. The silicon isolator with 95 wells was designed by the Aslan Research Group and manufactured by Grace Bio Labs (Bend, OR, USA).
3.2 Methods
3.2.1 Solvent Selection Study
In the past, the Aslan Research Group identified the solvent used for the
crystallization of GSH. Solubility of GSH was studies using various solvents and
at varying temperatures. Of the solvents used, sodium acetate pH=4.6 at 50°C
proved to be most effective (MS thesis by Zaakan, A., 2016). All experiments in
this project were performed using sodium acetate buffer (pH=4.6).
3.2.2 Preparation of Sodium Acetate Buffer
Sodium acetate (0.2 M) was prepared by dissolving 8.2 grams in 500 mL
deionized water. The pH of solution was adjusted to 4.6 by adding hydrochloric
acid or sodium hydroxide as needed.
11
3.3 Preparation of GSH solution
GSH solution of 500 mg/mL was prepared by adding required amount of
tripeptide in 1.0 mL of 0.2 M sodium acetate buffer (pH=4.6). The solution was
heated on a water-bath on a Corning magnetic stirrer/heater until all the powder
dissolved. Average temperature of the water-bath when all the powder dissolved
was (54-56) ºC. The solution was observed under the microscope (Swift Digital
M10 monocular microscope) for the absence of any undissolved particles or crystals.
3.4 Preparation of ITO (Indium Tin Oxide) films
ITO film (0.1755 mm thick x 300 mm width x 1 m length) was purchased
from MTI Corporation (Richmond, CA, USA). Circular ITO films (2 mm) were
made using the hole-puncher. The ITO films were then attached to the sticky part
of the 95 well silicon isolator. Circular PMMA disks were rinsed with ethyl alcohol
followed by de-ionized water and wiped dry using Kimwipes. Finally, the silicon
isolator with ITO was attached surface is attached to the circular PMMA disks
and pressed to avoid air bubbles underneath.
12
3.6 Characterization of crystals
Length of GSH crystals was determined by Swiftcam Imaging II software
and Motic images 2.0 software. Digital images were taken by 12 MP IPhone 7
camera. The infrared spectrum analysis was performed using FTIR spectrophotometer (Agilent Cary 630 FTIR technologies) in attenuated total reflectance
(ATR) mode within the spectra range of 4000 to 500 cm -1. X-ray diffraction data
was collected using Rigaku Miniflex spectrophotometer equipped with Cu-K (alpha) radiation. The XRD experiment was set to collect the diffraction peaks between 20° and 55° (2-theta angle).
15
CHAPTER IV: Results and Discussion
4.1 Solubility of GSH
In an earlier study performed by Aslan Research Group, sodium acetate
(pH 4.6) proved to be the best solvent for crystallization of GSH17. The solvents
used in this experiment were observed in room temperature and at 50°C. An optical microscope was used to observe the solubility and the solvent chosen was
based on its ability to dissolve the most solute at its given temperature.
4.2 Summary of crystallization time and crystal size
Table 2 displays the summary of data of GSH crystal growth at different
time intervals at fixed microwave power of 70W on a 95 well ITO-coated iCrystal
plate. For each time interval, each experiment was performed with different trials.
The images were taken at 0 minutes for all the trials and at their respective intervals. The crystallization time (final minutes) is the time the crystallization process
was stopped due to excessive clustering of crystals. The trial with the best results
was taken to derive Table 2. In Table 2, we can see that the 10-minute time interval heating yielded the most number of crystals (n=49) per well. The longest
crystal of 379.6 Pm was observed at the 120-minute time interval trial but it yielded the lowest number of crystals (n=18) of all the experimental interval trials. In
all the experiments, the length and number of crystals increased as the time increased at a point where the nucleation caused excessive aggregation and clustering of crystals in the well. The number of “Good” (wells with clear and countable crystals) and “Bad” (evaporated and uncountable) wells was counted, which
16
reflects the effectiveness of the experiment. In some experimental trials, several
tiny crystals were observed in the beginning of the experiments (t=0 minutes).
This is due to the lag in the experiment at times. It is very infrequent and the time
lag is negligible. We chose the best trial out of three based on crystals images.
Table 2. Summary of results for the crystallization of GSH (500 mg/mL) on circular crystallization platforms (95 wells) at different time intervals using the MAMAEC technique.
Microwave
Level
70 W
70 W
70 W
70 W
70 W
Control
Time In- Crystallization Time
Number of Crystals Average Length of Crysterval
(Final)
in Each Well (n)
tals in Each Well (Pm)
(Minutes)
(Minutes) *
5
10
30
60
120
-
40
40
60
60
120
40
35
49
46
35
18
26
185 ± 43.2
153 ± 37.3
161 ± 28.7
337 ± 61.9
380 ± 113
106 ± 35.3
.
4.3 Crystallization of 500 mg/mL GSH crystals at 5 minutes interval
Figure 7 shows the images were captured at 0 minutes and every 5
minutes after the initial time. The microwave heating was stopped after there
were multiple layers or overlapping crystals as it created difficulty in crystal
counts and measurements. Trials 1, 2 and 3 were stopped at 80, 60 and 40
minutes, respectively. Trial 2 was chosen to generate crystal number and size
data as the crystals images were clear and crystals were easy to count. The average size and number of crystals every 5 minutes were calculated. The data
was only restricted to the first 40 minutes as crystals started to cluster and overlap. The results from three trials show that there are more crystals formed in trial
17
Figure 8 gives a graphical representation of the average number and size
of crystals in each well, which was 35 and 184.8 Pm at the end of 40 minutes,
respectively. Here we can see the number of crystals and their size increases
with increase in time. We can see a reduction in crystal number between 35-40
minutes. This result may be due to excess nucleation and difficulty in counting
crystals (Note: only visible crystals that could be measured were taken into consideration while generating these data).
Average GSH (500 mg/mL)Crystal Size and Number at 5 minute intervals
Crystal # and Average Size
250
size average
no. of crystals
200
150
100
50
0
0
10
20
30
40
Time( minutes)
Figure 8. Average size and number of GSH (500 mg/mL) crystals at 5-minute
interval at 70W on an ITO-deposited iCrystal plate using the MA-MAEC technique.
19
Table 3. Summary of “Good” and “Bad” wells for three different trials at the
5-minute time interval.
Time Interval (minutes)
5
Trials
T1
T2
T3
“Good” Wells
53
86
46
“Bad” Wells
42
9
49
Table 3 summarizes the number of “Good” and “Bad” wells in three different trials. Trial 2 is highlighted in red as to signify that the data provided on average size and number of crystals was obtained from the trial. Figure 9 provides a
detail description of the “Good” and “Bad” wells along with the image of the 95well iCrystal plates that were used for the crystallization. Tend denotes the time
when crystallization was stopped. Trial 1, 2 and 3 were stopped at 80, 60 and 40
minutes, respectively. The term “Good” and “Bad” refers to the visibility of crystals in the respective wells. The “Bad” wells do have crystals but not of the same
visible quality as the Good wells.
20
4.4 Crystallization of 500 mg/mL GSH at 10 minutes interval
Figure 10 shows the images were captured at 0 minutes and every 10
minutes after the initial time. Microwave heating was stopped after there were
multiple layers or overlapping of crystals both of which create difficulty in counting and measuring crystals. Trials 1, 2 and 3 were stopped at 100, 90 and 80
minutes, respectively. Trial 1 was chosen to generate number and size data as
the crystals images were clear and crystals were easy to count. The average
number and size of crystals in each well was 49.0 and 153.3 Pm at the end of 40
minutes, respectively. The data was only restricted to first 40 minutes as crystals
started to cluster and overlap. Table 4 shows the total number of “Good” and
“Bad” wells. At the 10 minutes interval, the number of “Good” wells in all three
trials is highest compared to any other experimental interval. We saw overlapping
and clustering of crystals, as time passed.
22
Figure 11 is a graphical representation of the average number and size of
crystals in each well, which were 49.0 and 153.3 Pm at the end of 40 minutes,
respectively. Here, we can see the number of crystals and their size increases
with an increase in time.
Crystal # and Average Size(um)
Average Crystal Size and Number of GSH Crystals at 500 mg/mL at 10 minute time Intervals
250
average size
no. of crystals
200
150
100
50
0
0
10
20
30
40
Time ( Minutes)
Figure 11. Average size and number of GSH (500 mg/mL) crystal solution at 10
minutes time intervals at 70W on an ITO-deposited iCrystal plate using the MAMAEC technique.
24
Table 4. Summary of “Good” and “Bad” wells in different trials at 10-minute time
intervals.
Time Interval (minutes)
10
Trials
T1
T2
T3
Good Wells
76
83
89
Bad Wells
19
12
6
Table 4 gives us number of “Good” and “Bad” wells in three different trials.
Trial 2 is highlighted red to signify that the data provided on average size and
numbers of crystals were obtained from that trial. Figure 12 provides a detail description of the “Good” and “Bad” wells along with the image of the 95- well iCrystal plates that was used for crystallization. Tend denotes the time when crystallization was stopped. Trial 1, 2 and 3 were stopped at 100, 90 and 80 minutes, respectively.
25
4.5 Crystallization of 500 mg/mL GSH at 30 minutes interval
Figure 14 shows the images were captured at 0 minutes and every 30
minutes after the initial time for all three trials. Microwave heating was stopped
after there were multiple layers or overlapping of crystals both of which create
difficulty in counting and measuring crystals. Trials 1, 2 and 3 were stopped at
210, 150 and 120 minutes, respectively. Trial 3 was chosen to generate number
and size data as the crystal images were clear and crystals were easy to count.
The average number and size of crystals in each well was 46.0 and 160.5 Pm at
the end of 60 minutes, respectively. The data was only restricted to first 60
minutes as crystals started to cluster and overlap.
27
Figure 14 gives a graphical description of average crystal number and
size at 30 minutes time interval. The number and size of crystal increased with
increasing time. At the end of 60 minutes, the average number of crystals in each
well was 46 and their length was 160.5 Pm. Table 5 provides a summary of
“Good” and “Bad” crystals in three different trials. Trial 3 was chosen to generate
data for average number and size of crystals.
Crystal # and Avg. Size(um)
Average Crystal Size and Number of GSH (500 mg/mL) crystals at 30 minute time intervals
300
average size
no. of crystals
250
200
150
100
50
0
0
30
60
90
120
Time( Minutes)
Figure 14. Average size and number of GSH (500 mg/mL) solution at 30-minute
time intervals at 70W on an ITO-deposited iCrystal plate using the MA-MAEC
technique.
29
Table 5. Summary of “Good” and “Bad” wells at 30-minute time intervals.
Time Interval(Minutes)
30
Trials
T1
T2
T3
Good Wells
58
72
86
Bad Wells
37
23
9
Table 5 gives us number of “Good” and “Bad” wells in three different trials.
Trial 3 is highlighted in red to signify that the data provided on average size and
numbers of crystals were obtained from that trial. Figure 15 provides a detail description of the “Good” and “Bad” wells along with the image of the 95 -well iCrystal plates that were used for crystallization. Tend denotes the time when crystallization was stopped. Trial 1, 2 and 3 were stopped at 210, 150 and 120 minutes,
respectively.
30
4.6 Crystallization of 500 mg/mL GSH at 60 minutes interval
The images were captured at 0 minutes and every 60 minutes after the
initial time. Microwave heating was stopped after there were multiple layers or
overlapping of crystals both of which create difficulty in counting and measuring
the crystals. Figure 16 shows the optical image of GSH crystals grown using
iCrystal system at 70 W. Trials 1, 2 and 3 were stopped at 120 minutes. Trial 3
was chosen to generate number and size data as the crystals images were clear
and crystals were easy to count. Figure 17 demonstrates the graphical representation of the average number and size of crystals in each well, which was 35.0
and 336.8 Pm at the end of 60 minutes, respectively. The data was only restricted to first 60 minutes as crystals started to cluster and overlap. The table below
shows the total number of “Good” and “Bad” wells. We do see the overlapping
and clustering of crystals as time passes.
32
Average Crystal size and number of GSH (500mg/mL) crystals at 60 minute time intervals
Crystal # and Avg. size(um)
500
average length
no. of crystals
400
300
200
100
0
0
20
40
60
80
Time ( Minutes)
Figure 17. Average size and number of GSH (500 mg/mL) crystals at 60-minute
time interval at 70W on an ITO-deposited iCrystal plate using the MA-MAEC
technique.
Table 6. Summary of “Good” and “Bad” wells in all trials at 60-minute interval.
Time Interval (Minutes)
60
Trials
T1
T2
T3
Good Wells
76
61
63
Bad Wells
19
34
32
Table 6 gives us number of “Good” and “Bad” wells in three different trials.
Trial 3 is highlighted in red to signify that the data provided on average size and
number of crystals was obtained from that trial. Figure 18 provides a detail description of the “Good” and “Bad” wells along with the image of the 95- well iCrystal plates that were used for the crystallization. Tend denotes the time when crys-
34
4.7 GSH (500mg/mL) crystallization at the 120-minute time interval
The images were captured at 0 minutes and 120 minutes after the initial
time. The microwave heating was stopped after there were multiple layers or
overlapping of crystals both of which create difficulty in counting and measuring
the crystals. Trials 1, 2 and 3 were stopped at 120 minutes. Trial 2 was chosen to
generate number and size data as the crystals images were clear and crystals
were easy to count. Figure 19 shows optical images of GSH crystals grown using
iCrystal system at 70 W in an ITO deposited 95-well platform. Figure 20 provides
a graphical representation of the average number and size of crystals in each
well that was 18.0 and 380 Pm at the end of 120 minutes, respectively. The data
was only restricted to the first 120 minutes as crystals started to cluster and overlap. The overlapping and clustering of crystals occurred as time passed. Table 6
provides the number of “Good” and “Bad” wells in all the trials at the 120-minute
interval GSH crystallization. Trial 2 was chosen to generate average size and
number data based on the visualization as it gave the best images. Figure 15 is
an optical image of “Good” and “Bad” wells in each trial. The arrowhead shows
their respective well number (Note: well numbers denote a select examples of
“Good” and “Bad “wells).
36
Crystal # and Average size(um)
Average Crystal Size and Number of GSH ( 500 mg/mL) crystals at 120 minute time intervals
600
size
no. of crystals
500
400
300
200
100
0
0
60
120
180
Time (minutes)
Figure 20. Average crystal size and number of GSH (500 mg/mL) at 120-minute
time intervals
Time Interval (Minutes)
120
Trials
T1
T2
T3
Good Wells
64
59
50
Bad Wells
31
36
45
Table 7. Summary of Good and Bad wells in all trials at 120 minutes interval
Table 7 shows the number of “Good” and “Bad” wells in three different trials. Trial
2 is highlighted in red to signify that the data provided on average size and numbers of crystals were obtained from that trial. Figure 21 provides a detail description of the “Good” and “Bad” wells.
38
4.8 Individual Crystal Growth Assessment
The growth of individual crystal was accessed by picking the best crystal in the
well and tracking its growth at various time intervals. Table 8 provides the length
of a single) was observed at 60 minutes interval of microwave heating the crystal
at 60 minutes at different microwave heating time intervals. The longest crystal
(429 µm) was observed at the 60-minute time interval of microwave heating. The
smallest crystal size of 256.4 µm was observed at room temperature (control). At
the 120-minutetime of microwave interval heating, size of the crystal was 552
µm. Table 9 provides the details of the growth of a representative crystal at various time intervals.
Table 8. Representative GSH crystal size at 60 minutes under different
experimental conditions
Microwave Time
Time (minutes)
Single crystal size (µm)
Control
60
256.4
5 minutes interval
60
347.9
10 minutes interval
60
309.0
30 minutes interval
60
187.9
60 minutes interval
60
429.0
120 minutes interval
60
289.5
40
squared values. Due to non-linear growth at control, 5 and 10 minutes interval,
the graph was broken down into two linear graphs each representing the growth
at their respective time. At control, the initial rate of growth of crystal (crystal
size/min) is 8.64 for the first 10 minutes and 2.83 from 20 through 40 minutes.
At 5 minutes time intervals of microwave heating, the initial rate of growth
of crystal until 15 minutes is 9.94 and as it progresses from (20 -40) minutes, the
rate is 4.67. At 10 minutes time intervals of microwave heating, the initial rate of
crystal growth is 9.43 until 20 minutes however, the rate drops to 2.39 from (2040) minutes. The graphical representation of 30, 60 and 120 minutes time interval heating are linear with crystal growth rate of 3.3, 6.26 and 4.82 respectively.
The data supports that the rate of crystal growth of glutathione can be controlled
by microwave heating. The heating and the cooling cycles of the microwave
heating has a positive effect on the crystal growth. Figure 29 provides the rate at
which the peptide glutathione (500 mg/mL) grows at different microwave heating
intervals.
42
Single crystal growth at room temperature (500 mg/mL GSH)
Single Crystal Size (um)
200
150
100
50
0
0
10
20
30
40
50
Time(minutes)
Figure 22. Single crystal growth at room temperature of 500 mg/mL GSH
Single crystal growth at 5 minute time intervals (500 mg/ml GSH)
Single crystal size (um)
250
200
150
100
50
0
0
10
20
30
40
50
Time (minutes)
Figure 23. Single crystal growth at 5 minute intervals of microwave heating of
500 mg/mL GSH
43
Single crystal growth at 10 minutes interval (500 mg/mL GSH)
Single crystal size (um)
250
200
150
100
50
0
0
10
20
30
40
50
Time (minutes)
Figure 24. Single crystal growth at 10 minute time intervals of
microwaveheating of 500 mg/mL GSH
Single crystal growth at 30 minute time intervals ( 500 mg/mL GSH)
Single crystal size (um)
500
400
300
200
100
0
0
20
40
60
80
100
120
140
Time (minutes)
Figure 25. Single crystal growth at 30 minute time intervals of microwave
heating of 500 mg/mL GSH
44
Single crystal growth at 60 minute time intervals (500 mg/ml GSH)
single crystal size (um)
800
600
400
200
0
0
20
40
60
80
100
120
140
Time (minutes)
Figure 26. Single crystal growth at 60 minute time intervals of microwave
heating of 500 mg/mL GSH.
Single crystal growth at 120 minute time interval ( 500 mg/mL GSH)
Single crystal growth (um)
1400
1200
1000
800
600
400
200
0
0
50
100
150
200
250
300
Time (minutes)
Figure 27. Single crystal growth at 120 minutes interval microwave heating of
500 mg/mL GSH.
45
Individual Crystal Growth of GSH (500 mg/mL) at different conditions
Figure 28a. Linear function representations of single crystal growth under
different conditions with corresponding R-squared values.
46
Figure 28b. Linear function representations of single crystal growth under
different conditions with corresponding R-squared values.
Rate of crystal growth at different conditions
Rate (crystal size per minute)
7
6
5
4
3
2
1
0
Control
5
10
30
60
120
Microwave heating time interval in minutes
Figure 29. Rate of crystal growth at different conditions.
47
4.9 GSH Crystal characterization using Fourier Transform Infrared
Spectroscopy (FTIR)
The infrared spectra of glutathione crystals grown at different time intervals were obtained and analyzed. Figures below show spectra for all the experimental trials for GSH (500 mg/mL) concentration at different time intervals of microwave heating using the iCrystal system. The spectra were compared to the
FTIR spectra of GSH from the literature. The table provides a comparison summary of FTIR results of GSH (500 mg/mL) at different time intervals of microwave
heating to those found in the literature. The table provides peak intensity at different wavenumbers particularly the peaks for thiol group(–SH) stretching at
2526 cm-1; νN-H stretching at 3346 cm-1 and 3250 cm-1 provides evidence of (–
NH2) group.; νC=O antisymmetric reported at 1713 cm-1 and 1280(δOH) indicates
the presence of a carboxyl (–COOH) group; asymmetric νCOO- at 1397 cm-1.
The band at 1075 cm-1 can be assigned as νC-N (stretching). In the other hand,
the FTIR analysis of GSH grown in the laboratory revealed wavenumbers that
were 8-10 units higher than those found in the literature. This difference is negligible can be attributed to the difference in sensors of a FTIR instrument. Similar
results were observed for the crystals that were grown at 5, 10, 30, 60 and 120minute time intervals. These results prove that the iCrystal system microwave
heating has no effect on the morphology of crystals during the MA-MAEC process.
48
4.10 X-ray Diffraction Analysis
The GSH (500 mg/mL) crystals grown at different time intervals (5, 10,
30, 60 and 120 minutes) were analyzed using powder X-ray diffraction (XRD).
The diffraction peaks look identical to the ones reported in the literature. XRD data further proved that MA-MAEC technique had no effect on GSH crystal structure29. Figures 36-40 provide a graphical representation of intensity versus 2theta degree (angle) of the diffraction patterns of GSH crystals. The three-digit
number enclosed in parenthesis on the top of the peaks indicates the plane at
which the x-ray bombards the crystalline material (GSH). The intensity is measured as counts per seconds that determine the number of times the GSH crystals
get hit by the projected photon from the X-ray. Figure 41 represents the powder
diffraction pattern under control conditions.
56
5. Conclusion and future work
In conclusion, we demonstrated that the iCrystal system is effective in
rapid crystallization of the GSH peptide. Sodium acetate at pH= 4.6 proved to be
the best solvent from our earlier studies from our laboratory. The use of 95-well
iCrystal platform for the MA-MAEC technique was effective in crystallization and
quality growth of GSH (500 mg/mL) crystals. Based on our experiment with various microwave heating time intervals, our data suggest that the 5-minute interval
of microwave heating produced more crystals of a larger size compared to the
other time intervals. The 95-well iCrystal platforms for the MA-MAEC technique
proved effective in the crystallization of GSH (500mg/mL). The functionality and
the morphology of GSH crystals analyzed via FTIR and XRD respectively were
not altered by our MA-MAEC technique.
In addition, the use of MA-MAEC technique significantly reduced the initial
crystallization time as well as the time needed for complete crystallization. The
use of microwave heating with the iCrystal platform yielded more crystals than
seen under control conditions (ITO at room temperature). The quality and size of
the crystals grown using the iCrystal were higher than those grown under control
conditions. Further studies can include optimization of the crystallization conditions, use larger peptides and proteins, and use a 204-well iCrystal platform to
grow crystals.
63
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