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ON
BY
Sindhu M 2017B1A30712P
AT
June-2019
A REPORT
ON
BY
Sindhu M 2017B1A30712P
AT
June-2019
ACKNOWLEDGEMENTS
I would like to thank the Practice School Division, BITS Pilani and CSIR-CLRI
for giving me the opportunity of working in a research lab. I am very thankful
to Dr. Manoj Kannan, my PS-1 instructor, who diligently coordinated with the
host institute. He helped us to choose the right project and ensured that our PS-I
course was congenial in all aspects and educative. I am grateful to Dr. G.C.
Jayakumar, my mentor at CLRI, who gave me the project, guided me and
reviewed my work with constructive feedback. I sincerely thank Ms. Yasothai,
Ms. Kurinji Malar, Ms. Saranya and Mr. Peter, the PHd students at CHORD lab,
who helped me with my wet lab work. My work could not have been completed
without the cooperation of the SEM lab and the CSIF facility. I would also like
to thank Joel Joseph, Rahul, Deepa and Hamira, the other interns working in my
lab, for helping me with my work keeping me motivated.
BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCE
PILANI (RAJASTHAN)
Practice School Division
Abstract: The report aims to summarise the outcomes of the Practice School(PS)-1 course . It gives
an overview of the PS Station and then moves on to explain the project undertaken- Mesoporous
phospholipids for drug delivery. Mesoporous particles containing ordered pore structure, and high
surface area can be used for many practical applications. Mesoporous phospholipids, containing
collagen to modify its surface properties were prepared, to be used in drug delivery.
Date: Date:
INTRODUCTION:
Nano-porous materials containing pores between 2nm and 50nm in size are classified
structure and high specific surface area render them desirable as adsorbents, for
catalysis, and as hosts for different kinds of molecules 1. Biological applications include
biological matrices among others. Mesoporous Silica Nano-particles ( MSNs) have been
extensively studied as drug delivery agents, since 2001. “Their features such as well-
ordered internal mesopores (typically ca. 2–6 nm) with large pore volume (0.6–1
3 2
cm /g) and surface area (700–1000 m /g), tunable size (50–200 nm) and shape,
was reported as a novel drug carrier 4. Unique and desired properties of both liposomes
from the properties of MSNs mentioned above, MPPs are also expected to exhibit the
Collagen has been used with liposomes for drug delivery, to alter surface properties of
liposomes and as gel matrix to sequester liposomes 6. The present work aims to include
collagen in MPPs. The properties of collagen in non polar environment and in the
presence of lipids were studied, to better understand the physical and chemical
properties of collagen containing MPPs. Then MPPs containing collagen were prepared
COLLAGEN EXTRACTION:
Collagen was extracted from rat tail preserved at -4 degree Celsius. The tail was cut at
the corner and teased, exposing tendons, which were extracted and kept in 0.5N NaCl
3 ACS Nano2010484371-4379
Publication Date:July 12, 2010
https://doi.org/10.1021/nn901376h
Copyright © 2010 American Chemical Society
5 ibid
6
solution. To obtain collagen in it’s monomeric form, it was incubated in 0.5N Sodium
Acetate solution. It was then completely solubilised in 0.5M Acetic Acid by overnight
SDS-PAGE:
SDS-PAGE of collagen was performed to verify that the extract contained type-I
The behaviour of collagen in Acetic acid, non polar solvents and in the presence of
lipids were studied and compared. The non polar solvents used were t-butyl alcohol
and hexane in 2:1, 1:1 and 1:2 ratios in solvents 1,2, and 3 respectively.
6% Lechitin was added to each of the three solvents prepared above. True solution
Different volumes of water were added to each of the three prepared 6% Lecithin
solutions, and were incubated in a water bath at 50 degree Celsius. The maximum
volume of water that was soluble in the solutions were taken to be their water holding
capacities. The above determined concentrations were used to study the behaviour of
The behaviour of collagen in Acetic acid, non polar solvents, and in the presence of the
FIBRILLOGENESIS OF COLLAGEN:
7
UV Spectroscopy:
The UV Spectrum of collagen in Acetic acid, in the three non polar solvents, and in the
presence of lipids were recorded, between 200 and 800nm. Water was used as the
blank for collagen in Acetic acid, while the non polar solvents were used as blank for
collagen in non polar solvents and in the presence of lipids. Fibrillogenesis of collagen
Collagen was dissolved in each of the above mentioned solvents in accordance with
the determined water holding capacities by heating to 50 degree Celsius. They were
cooled at -80 Degree Celsius for one hour. The lower layer containing collagen and
lipid was retained while the upper layer containing the solvent was discarded. This was
FTIR-ATR.
An alpha region consisting of two bands, a beta region consisting of two bands and a gamma region
consisting of a single band were obtained, conforming the
presence and type of collagen ( Collagen I) in the sample ( Fig
1).
were recorded and it was observed that there was a vertical shift in the spectra and
variation in the
(B
A C
more the upward shift, the less polar the solvent was. The polar nature of collagen
suggests that this maybe due to lesser solubility of collagen as the polarity of solvent
decreases. The absorbance peak was found to be around 210nm in the presence of
lipids also ( Fig 3). However, in the presence of lipids the absorbance values rose from
200 to Absorbance maxima, unlike in the spectra recorded sans lipids. This suggests
that
Fig3: UV-Vis spectrum of collagen in the presence of lipids. (A) is the spectrum of
The sigmoidal curve, obtained for collagen dissolved in acetic acid was also observed
for collagen dissolved in solvent 1. In solvents 2 and 3, which are less polar, a
significantly different. Curve was obtained. The absorbance values decreased to a
constant value. Whereas, in the presence of lipid, none of the curves were sigmoidal.
(B)
(A)
(C)
This suggests that the polarity of the solvent affects the process of fibrillogenesis. ( Fig
4)
fibrillogenesis curve of collagen dissolved in 0.5M Acetic acid. (B) shows the
fibrillogenesis curve of the three solvents in the presence of lipid. ( C )shows the
solvent used. The viscosities of collagen in solvents 1,2, and 3 were found to be 1.44,
viscometer.
MPPs containing collagen were prepared and their properties were studied. Composed
of Lecitin and Collagen, it is anticipated to be safe for human internal use. Hence, the
prepared mesoporous particles can be further studied for their use as controlled drug
delivery agents. MSNs are known to be useful in drug and bio molecular delivery, bio-
imaging, bio sensing and bio catalysis. MPPs, which are expected to be more versatile