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. 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 cm3/g) and surface area (700–1000 m2/g), tunable size (50–200 nm) and
shape, robustness and easy surface modification, have revolutionised the field of
controlled drug delivery”. The downside however, is their aggregation in
physiological conditions and non-specific binding in protein containing solutions.

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

was reported as a novel drug carrier. 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.

Collagen has been used with liposomes for drug delivery, to alter surface
properties of liposomes and as gel matrix to sequester liposomes. 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 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.

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% Lechitin
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. Equi-volume
collagen was added to study fibrillogenesis.

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: 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 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.
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 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

Fig4: Fibrillogenesis of collagen in different solvents

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. This suggests that the polarity of the solvent affects the
process of fibrillogenesis. Handbook of Nanomaterials for Industrial Applications.
(n.d.). Retrieved from https://www.sciencedirect.com/science/book/9780128133514
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
 ACS Nano2010484371-4379
Publication Date:July 12, 2010
https://doi.org/10.1021/nn901376h
Copyright © 2010 American Chemical Society 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 ibid