BIOM9311 Laboratory Report

Abstract
This experimental procedure investigated the nature and rate of diffusion in different drug
delivery systems namely ones using a membrane structure or a monolithic matrix. With the
molecule being diffused kept constant, it provided a good basis upon which to compare the
diffusion occurring. The drug was simulated using a blue dye with a molecular weight of
792Da and the membrane and matrix being composed of sodium alginate. It was found that
the total diffusion over a 120 minute period showed that the membrane system showed
almost 3 times higher diffusion while the monolithic system showed a slower more linear
diffusion. In addition to this, the diffusion across a membrane was investigated by using
molecules of different sizes (the smaller BSA and larger lgG molecule) along with different
membranes. It was shown that the size of the molecule played a role in reducing diffusion
rate across both membranes but also interestingly revealed that selective permeability
played a role in the rate of diffusion where membrane 2 showed a higher rate for the larger
lgG molecule.

Introduction
When looking at mass transfer that occurs in drug delivery and transfer across membranes,
diffusion is the key mechanism by which this occurs, however convection can come into play
given certain conditions. Diffusion is driven by the concentration gradient between two
areas. In drug delivery two scenarios are analyzed, one where a bolus of drug is surrounded
by a semi permeable membrane which the drug will diffuse through and the other a
monolithic device where the drug is distributed through a polymer matrix from which is
diffuses out. (Graham 1984) The transfer across a membrane is determined not only by the
size of the molecule in question and the size of the holes of the membrane but also whether
the membrane is selectively permeable to a specific molecule. This experimental procedure
aims to compare differences in the rates and nature of diffusion between monolithic and
membrane drug delivery systems, with the hypothesis that the diffusion rate from the
monolithic system will be higher due to the increased thickness through which the drug will
need to diffuse. It also aims to determine how different membranes and atomic weights
relate to diffusion in practice and whether selective permeability can be observed with a
hypothesis that a higher atomic weight will exhibit a lower diffusion rate.

Experiment 1
Materials and Methods
In order to perform this experimental process, one will require 0.2g Sodium alginate
dissolved in 10mL of water with 1mL of blue food dye added, 0.5g of calcium chloride in
50mL of water and 4 beakers each filled with 50mL of water, 56 1.5mL Eppendorf tubes, a
magnetic stirrer, plastic pipettes, metal strainer, stopwatch and an absorbance measuring
apparatus.

Place the calcium chloride solution on the magnetic mixer working at 300rpm, draw
approximately 2mL of the blue Sodium alginate solution into a pipette and hold the end of
the pipette 5 cm above the surface of the calcium chloride. Quickly release 20-30 drops of
the alginate into the calcium chloride. If creating monolithic beads, let the forming beads
stir for 10 mins before removing from the stirring solution. If creating membrane beads,
only let them remain in the stirring solution for 20 seconds. (Tønnesen 2002) To remove the
beads from the solution, use a metal strainer and tissue paper to remove the last remnants
of the solution. Setup the four beakers of water to be stirred on the magnetic stirrer, and
place 5 beads in each beaker, 2 beakers with the membrane beads and the other 2 with
monolithic beads. At equal time intervals, take a sample of the fluid from each beaker into
an Eppendorf tube and measure its absorbance at 628nm.

Results
The absorbance readings from the various points in time for each of the 4 beakers used has
been depicted in the following graphs showing the absorbance vs time.

Monolithic 1 Monolithic 2
0.16 0.16

0.14 0.14

0.12 0.12
Absorbance
Absorbance

0.1 0.1

0.08 0.08

0.06 0.06
0.04 0.04
0.02 0.02
0 0
0 50 100 150 0 50 100 150
Time (min) Time (min)

Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3

Membrane 1 Membrane 2
0.45 0.45
0.4 0.4
0.35 0.35
0.3 0.3
Absorbance
Absorbance

0.25 0.25
0.2 0.2
0.15 0.15
0.1 0.1
0.05 0.05
0 0
0 50 100 150 0 50 100 150
Time (min) Time (min)

Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3

In order to compare the results of the two different beads, an average was taken of the
three absorbance readings for each of the four experiments, the two monolithic
experiments data were averaged, as well as the two membrane experiments. These two
sets of data were then plotted on the same absorbance vs time graph below.

Absorbance vs Time
0.45

0.4

0.35

0.3
Absorbance

0.25

0.2

0.15

0.1

0.05

0
0 20 40 60 80 100 120 140
Time (min)

Monolithic Beads Membrane Beads

The absorbance value gives a good indication of the quantity of drug (food dye) that has
made its way into the aqueous solution surrounding the bead. The membrane system
exhibited a value of almost 0.4 for absorbance while the monolithic system only reached
approximately 0.14 for absorbance. Assuming a linear relationship between the absorbance
and quantity of drug in the solution, the membrane system released almost 3 times more
drug into the solution.

Also notable, for both systems, it appears that the absorbance over time exposes an inverse
exponential relationship, however the membrane system exhibited a trend that could be
considered linear over the first hour of reading.
As can be seen from the photos above, the final beads in the membrane system appear to
be a lighter shade of blue than the monolithic beads, which is a clear indication that the
membrane system released more “drug”. However there is an additional factor involved,
the volume of left over solid material is lower for the membrane system as can be seen by
the deformed state of the capsule. This also scales down the left over quantity of drug in the
system. These two factors combined possibly reflect the fact that the quantity released by
the membrane system was 3 time higher.

Discussion
Diffusion relies on a difference in concentration in two regions of a chemical species. It
doesn’t require the movement of the fluid that it resides in, and for this reason the drug
should diffuse from either the monolithic or membrane devices in this experiment even
when completely still conditions are present (no movement of beads or the water). (Deen
1981) By stirring the water, the concentration of drug in the water can equalize across its
mass more quickly rather than relying on simply diffusion to do this, the net result of this is
that the water surrounding the bead will always have a minimum concentration of drug
possible, as this maximizes the concertation gradient between the water and the bead,
therefore increasing the rate of drug release. (Graham 1984) An additional possible effect of
stirring is that it introduces a convectional component, where water flow through the bead
occurs, increasing the release rate of the drug. (Geankoplis 2003)

The release rate of the drug would decrease as the molecule size increased, as the rate of
diffusion decreases. This is due to the fact that the larger molecules move slower and a
lower interaction time with the membrane. The stirring of the system, possibly increase the
speed of the larger molecules and thereby the interaction time. This would speed up the
release rate of larger molecule drugs.
Experiment 2
Materials and Methods
In order to carry out this experiment, one will require bovine serum albumin,
immunoglobulin, a side by side diffusion cell, membranes, pipettes, stirrers, Eppendorf
tubes and Eppendorf pipettes. To test a membrane, place a membrane in the cell, place a
solution of BSA, IgG and water in one side of the cell and water in the other side (3mL each).
Let the cells start mixing and at assigned times, take a sample from the water side of the cell
into an Eppendorf tube. Measure the absorbance of each molecule in each sample.

Results
The following plot shows each of the substances and its percentage diffusion with each of
the two membranes, providing 4 sets of data. Of the three absorbance readings calculated
for each time interval an average was taken and the average of each point is shown below.

Percentage Diffused vs Time

9

7
Percentage Diffused

0
0 10 20 30 40 50 60
Time (hr)

BSA-Membrane 1 BSA-Membrane 2
IgG-Membrane 1 IgG-Membrane 2

As can be seen from the plot at the time of final reading, the BSA exhibited the highest
percentage of diffusion for both of the membranes, with the highest rate being at almost
6% through membrane 1 while the BSA percentage for membrane 2 was lower at just under
5.5%. The IgG exhibited lower percentage diffusion for both membranes with a percentage
of just over 4.5% for membrane 2 and just under 2.5% for membrane 1, significantly lower
than all the other recorded values. Interestingly IgG diffusing through membrane two was
the only data to change ranking in percentage diffused for various times, up until 28
minutes it had the highest percentage, and at some point between 28 and 48 minutes it
dropped from 2nd to 3rd highest percentage.
It is clear from the graph that the both end values for the BSA were higher than the values
of IgG. It is also known that the IgG has more than twice the atomic mass of the BSA. Since
both membranes exhibit lower values for IgG and that the mass of IgG is lower, it suggests
that the lower the atomic mass the higher the rate of diffusion across the membrane.

The BSA diffused better through membrane 1 than membrane 2 at every sample taken,
where the percentage of the amount diffused was greater at each point. The data for each
membrane share similar profiles with what appears to be a constant offset between data
points.

The IgG has a significantly higher percentage diffusion across membrane 2 than membrane
1, the difference in percentage is much greater than the difference between the
membranes for the BSA substance.

Discussion
Diffusion can only take place when there exists a concentration gradient of a certain species
between two areas. Net diffusion will no longer occur at the point the concentrations reach
equilibrium, however as the gradient get smaller, the rate of diffusion tends towards zero.
(Geankoplis 2003)

As brought up in the results section, due to the diffusion rates of BSA being higher than the
ones of IgG for both membranes and that the BSA has a smaller atomic mass, it follows that
the lower the atomic mass, the higher the diffusion rate. As the atomic mass increases, the
speed at which is moves becomes slower relative to a smaller molecule, which reduces the
number of collisions with the membrane, resulting in a lower rate of crossing.

One of the surprising results of this experimentation is that membrane 1 exhibited a higher
amount of diffusion for BSA, membrane 2 showed a higher amount of diffusion for IgG.
Assuming that simply porosity was the only variable needed to describe how substances
diffused across a membrane, this result would make sense as a larger molecule should show
a lower diffusion in a less porous membrane which clearly isn’t the case here. (Deen 1981)
Clearly membrane 2 is selectively permeable to IgG to some degree as it is letting IgG pass
at a greater quantity that BSA which has a smaller atomic mass. Some biological tissues are
complex and have pathways that allows certain molecules to travel through either
continuously or at some other rate.

Conclusions
It was found that indeed the membrane drug delivery system had a higher rate of diffusion
by almost 3 times but the monolithic system exhibited a more linear release through the
middle of the recorded time interval. The transfer of species through membranes provided
results that matched the hypothesis made, where larger atomic masses decrease the
diffusion rate. However the lgG molecule showed a higher diffusion rate in membrane 2
despite being a larger molecule that the BSA molecule suggesting a selective permeability to
the lgG.
References
Deen, W., Bohrer, M. and Epstein, N. (1981). Effects of molecular size and configuration on
diffusion in microporous membranes. AIChE Journal, 27(6), pp.952-959.
Geankoplis, C. and Geankoplis, C. (2003). Transport processes and separation process
principles. Upper Saddle River, NJ: Prentice Hall Professional Technical Reference.
Graham, N. and McNeill, M. (1984). Hydrogels for controlled drug delivery. Biomaterials,
5(1), pp.27-36.
Gupta, P., Vermani, K. and Garg, S. (2002). Hydrogels: from controlled release to pH-
responsive drug delivery. Drug Discovery Today, 7(10), pp.569-579.
Tønnesen, H. and Karlsen, J. (2002). Alginate in Drug Delivery Systems. Drug Development
and Industrial Pharmacy, 28(6).