A REPORT

ON

MESOPOROUS PHOSPHOLIPIDS FOR DRUG DELIVERY

BY

Sindhu M 2017B1A30712P

AT

CSIR- Central Leather Research Institute

A Practice School-I station of

BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCE-PILANI

June-2019
 A REPORT

ON

MESOPOROUS PHOSPHOLIPIDS FOR DRUG DELIVERY

BY

Sindhu M 2017B1A30712P

Prepared in partial fulfilment of the

Practice school-I Course No.

BITS F221/ BITS F231/ BITS F241

AT

CSIR-CLRI( Central Leather Research Institute), Chennai

A Practice School-I station of

BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCE-PILANI

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

Station: CSIR-CLRI( Central Leather Research Institute). Centre: Chennai

Duration: From: 21 May 2019 To: 13 July 2019

Date of Submission: 21/06/19

Title of the Project: Mesoporous phospholipids for dry delivery

ID No: 2017B1A30712P Name of student: Sindhu M Discipline: MSc Biology,BE EEE

Name of expert : Dr. G. C. Jayakumar Designation: Scientist

Name of the PS Faculty: Dr. Manoj Kannan

Key words: Mesoporous

Project Area(s): Drug delivery

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.

Signature of Student: Signature Of PS Faculty:

Date: Date:
INTRODUCTION:
Nano-porous materials containing pores between 2nm and 50nm in size are classified

as mesoporous particles according to IUPAC nomenclature. Their ordered pore

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

controlled drug delivery, enzyme encapsulation and phospholipid extraction from

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,

1Handbook of Nanomaterials for Industrial Applications. (n.d.). Retrieved from https://www.sciencedirect.com/science/book/9780128133514

robustness and easy surface modification, have revolutionised the field of controlled

drug delivery”2. The downside however, is their aggregation in physiological conditions

and non-specific binding in protein containing solutions 3.

Mesoporous phospholipid particles ( MPPs), composed solely of Phosphatidyl Choline

was reported as a novel drug carrier 4. Unique and desired properties of both liposomes

and mesoporous materials are expected to be exhibited by MPPs. Therefore, apart

from the properties of MSNs mentioned above, MPPs are also expected to exhibit the

properties of liposomes such as the accommodation of hydrophilic and hydrophobic

guest molecules, easy surface modification and high bio-compatibility 5.

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

and their properties were studied.

MATERIALS AND METHODS:

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

2 Mesoporous Silica Nanoparticles for Drug Delivery: Current Insights

María Vallet-Regí 1,2,*, Montserrat Colilla 1,2, Isabel Izquierdo-Barba 1,2 and Miguel Manzano 1,2

3 ACS Nano2010484371-4379
Publication Date:July 12, 2010
https://doi.org/10.1021/nn901376h
Copyright © 2010 American Chemical Society

4 Totally Phospholipidic Mesoporous Particles

Shaoling Zhang, Kohsaku Kawakami, Lok Kumar Shrestha, Gladstone Christopher Jayakumar, Jonathan P. Hill, and Katsuhiko Ariga
The Journal of Physical Chemistry C 2015 119 (13), 7255-7263
DOI: 10.1021/acs.jpcc.5b00159

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

incubation with stirring.

SDS-PAGE:

SDS-PAGE of collagen was performed to verify that the extract contained type-I

collagen. U.K. Laemmli’s protocol was followed 7.

COLLAGEN IN NON POLAR SOLVENTS AND IN THE PRESENCE OF LIPIDS:

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

was obtained by heating to 50 Degree Celsius.

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

collagen in the presence of lipids.

The behaviour of collagen in Acetic acid, non polar solvents, and in the presence of the

lipid solvents, prepared as mentioned above were studied.

FIBRILLOGENESIS OF COLLAGEN:

A solution of 10% NaOH in Phosphate buffer of pH 7.2 was prepared. Equivolume

collagen was added to study fibrillogenesis.

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

was recorded by measuring absorbance at 6 second time intervals at 313nm.

Preparation and study of mesoporous particles:

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

followed by lyophilisation to obtain mesoporous particles. The properties of the

prepared mesoporous particles were studied by Scanning electron microscopy and

FTIR-ATR.

RESULTS AND DISCUSSION:

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).

Fig 1: SDS-PAGE of Collagen

The spectra of collagen in the three non polar solvents and in the presence of lipids

were recorded and it was observed that there was a vertical shift in the spectra and

that there was no

variation in the

FIG2: UV-VIS SPECTRUM OF COLLAGEN

IN NON POLAR SOLVENTS.

(B
A C

absorbance peak at around 210nm ( Fig 2). The

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

collagen in solvent 1 in the presence 1% and 6% Lechitin. (b) is the spectrum of

collagen in solvent 2 and in the presence of 6% Lechitin. ( C ) is the spectrum of

collagen in the presence of 6% and 1% lipid.

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)

Fig4: Fibrillogenesis of collagen in different solvents. (A) shows the sigmoidal

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

fibrillogenesis curve of collagen in each of the three solvents.

Water Solvent 1 Solvent 2 Solvent 3

Time 47.62 109.8 116.67 151.20

( Seconds)
47.98 113.65 116.84 154.37

47.46 116.75 116.53 156.12

Mean 47.69 113.4 116.68 153.90

The physical properties of the collagen solutions were also found to differ with the

solvent used. The viscosities of collagen in solvents 1,2, and 3 were found to be 1.44,

1.274 and 1.77 mPa.s respectively ( Table 1).

Table1: Measurement of viscosity of collagen in non polar solvents using Ostwald

viscometer.

Conclusions and future directions:

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

and bio compatible can also be studied for such uses.