Download Drug Delivery by Hydrogels Objectives

Drug Delivery by Hydrogels
Objectives—
1. To understand the fundamentals of solute transport in hydrogels and
apply that to measure the diffusivity of a drug in hydroxyethyl
methacrylate hydrogel.
2. To prepare a macroporous pHEMA hydrogel and under the
differences between a transparent and a macroporous pHEMA gel.
Introduction
In the last few decades, a number of novel approaches have been developed for
controlled drug delivery of ophthalmic drugs. However, most treatments of ocular
disease are still based on topical application of eye drops to the surface of the eye. Due
to the short residence time, only about 1–5% of the applied drug penetrates the cornea
and reaches the intraocular tissues. To maintain therapeutic levels of drug
concentration, frequent instillation of drops with large drug loadings are required, which
is inconvenient for patients. Moreover, the drug that gets absorbed in conjunctiva or in
the nasal cavity eventually reaches other organs through the blood circulation leading to
side effects. To overcome the drawbacks of eye drops, several ophthalmic drug delivery
systems have been proposed such as suspension of nanoparticles, nanocapsules,
liposomes, ocular inserts like collagen shields and Ocusert, and therapeutic contact
lenses. Among these, contact lenses have been widely studied due to the high degree
of comfort and biocompatibility. On instillation of medicated contact lens in the eye, drug
diffuses through the lens matrix into the thin tear film named post-lens tear film (POLTF)
trapped between the lens and the cornea, and the drug has a residence time about 30
min in the eye. An increase in the residence time leads to a significant increase in the
bioavailability. Both mathematical models and clinical data suggest that the
bioavailability for ophthalmic drug delivery can be as large as 50%, which is an order of
magnitude larger than that for drops. In the previous experiment you prepared a pHEMA
hydrogel, which is used as a contact lens. In this experiment, you will measure the
diffusivity of a glaucoma drug timolol in the gel for the purpose of designing a contact
lens for delivery of timolol to the eyes.
Theory
You have already learnt the basics of solute transport in hydrogels. You will use
the same approach of loading and release to measure the diffusivity of timolol in the
hydrogels. In this experiment, after loading a drug (timolol) in the hydrogel, the gel is
soaked in pure PBS (buffer) for drug to be released. Under the assumptions that drug
transport is diffusion controlled and that perfect sink conditions exist (that the
concentration of solutes in the release medium is approximately equals to zero
regardless of time), we can write the following equations for 1D Fickian diffusion:
C
 2C
D
t
y 2
(1)
Ct, y  h  0
(2)
C
t , y  0  0
y
(3)
C  y , t  0   Ci
(4)
Where C is drug concentration in gel, D is diffusivity, h = half thickness of gel and C i is
the initial concentration of drug in gel. The above set of equations can be solved to:
(5)
Where RD (%) is the percentage of drug released. At short time interval, the equation
can be simplified to:
(6)
From the slope of a plot of RD% vs sqrt (t), we can estimate diffusivity.
In the previous experiment you measured conductivity to obtain the salt
concentration in the solution. The drug timolol does not change the conductivity to the
same extent as salt so an alternate approach must be used to measure its
concentration. Here we will use UV-Vis spectroscopy which is a commonly used
method for measuring concentrations. This technique relies of the interaction of the
molecules with UV and visible spectrum of the light, typically from 190 nm to 1100 nm.
The interaction of molecules with light in this range of the wavelengths typically leads to
electronic transitions, which is manifested in the absorption spectra, which is a
characteristic of the molecule. The absorption spectrum is a plot of the absorbance as
a function of the wavelength. The absorbance (A) is measured by the UV-Vis
spectrophotometer by measuring the intensity of the transmitted light I and then using
the relationship
𝐼 = 𝐼0 10−𝐴
where I0 is the intensity of the incident light. The absorbance is linearly related to the
concentration, A = εcl, where ε is the absorptivity of the molecule, c is the concentration,
and l is the path length.
To use the UV-Vis spectroscopy as an analytical method we must first prepare a
calibration curve which related the absorbance to the concentration. The calibration
curve can be prepared at any wavelength but typically the wavelength of the maximum
absorbance is used to allow measurement of dilute solutions. By measuring the
absorbance for a range of solutions one can obtain the calibration curve and then use
that in future to convert the measured absorbance to the concentration.
Procedure:
A) To be done by TA 1 day before the experiment
A1) Prepare 50 ml of 0.1 mg/ml timolol solution by mixing 0.005 g of timolol in 50 ml of PBS (buffer) and
stirring it overnight.
A2) Drug loading
1. Take 3 clean glass vials with caps and label them 1L, 2L and 3L.
2. After the gels have been extracted and dried, take 3 of the gel pieces and weigh them and note the
weights.
3. Put 1 gel piece in each glass vial.
4. Add the drug solution. The volume of drug solution in ml = 100 x weight of dry gel in g. Note the volume
of solution added.
5. Close the vials. Store them away from direct sunlight.
B) Drug release: Experiment done by students
B1) Setting the baseline for measuring drug concentration
1. Take a disposable cuvette and put approx. 1.5 – 2 ml of PBS (buffer) using a transfer
pipette. Put this inside the cuvette slot in the UV-vis spectrophotometer. Set this as
the baseline at a wavelength of 291 nm (absorption peak for timolol).
2. After the baseline is set, dispose the PBS and the cuvette. In another cuvette, add
the original timolol solution and measure the absorbance. Note the absorbance of the
loading solution (absloading =
)
B2) Drug release
1. Take 3 clean glass vials with caps and label them 1R, 2R and 3R.
2. From the vials already containing the drug soaked gels, take vial labeled 1L and with
a pair of tweezers, remove the gel. Wipe the gel surface very lightly with Kimwipes.
Put this gel in vial 1R. Using the pipettes, add the given amount of PBS (buffer). This
volume of buffer (ml) corresponds to 100 x wt. of gel (g). Start the stop watch. After
removing the gel from vial 1L, close the vial and store it.
3. Do this for the remaining 2 gels sets. But have a gap of 5 minutes between the 3 gel
releases. So the 2nd solution is added at t = 5 min and 3rd solution is added at t = 10
min.
4. At t = 15 min, remove approx. 1.5 ml of the solution from the 1 st vial 1R using a
transfer pipette and add it to a cuvette. Measure the absorbance and using the
transfer pipette, put it back as much as possible, again using the transfer pipette.
5. Repeat the point 4 for vial 2R at t = 20 min and for vial 3R at t = 25 min.
6. Repeat 4 and 5 at t = 30 min, 45 min, 60 min, 75 min and 90 min.
7. Now, measure the absorbance of the drug in the loading solution vials (1L, 2L and
3L)
Record the data and filled in the blanks below.
Time (Min)
15
30
45
60
Sample 1
Sample 2
Sample 3
75
90
Loading solution abs
Put these values in the excel sheet. Follow the instructions given in the excel sheet to
find the diffusivity value.
Preparation of a macroporous pHEMA gel
While transparency is a necessary requirement for gels designed to be contact
lenses, other biomedical devices are not limited by this requirement. The pHEMA gel
that you prepared is transparent because the pores in the gels are nanosized and so
cannot scatter visible light. To avoid formation of pores in the gel it is critical to ensure
that the initial formulation contains less water than what can be accommodated in the
gel without pore formation. In several applications it is preferable for have a highly
porous gel such as in tissue engineering where the pores provide the space for the cells
to grow and migrate. If the polymerization solution contains excess water, i.e., more
than the swelling capacity of the gel, the excess water will form pores in the gel leading
to formation of the macroporous gel. These gels are opaque because the pores are
bigger than the wavelength of light so they scatter the visible light. We will now learn
how to prepare macroporous gels and measure some of the key properties.
Procedure
1. Mix monomer HEMA (4 ml) to DI water (5 ml) and then add 5 ml of 1N NaCl.
2. Add 400 µl of crosslinker ethylene glycol dimethacrylate (EGDMA) and 10 mg of
3.
4.
5.
6.
7.
the initiator TPO to the solution prepared and mixed by stirring for 20 minutes.
Bubble Nitrogen for 15 minutes to remove the dissolved oxygen.
Poured the solution in suitable beakers and photo polymerized by placing on
UVB transilluminator for 40 min.
Take the gel out and break a small piece.
Weight the piece to determine the fully hydrated weight.
Squeeze water out of the gel and then weight again to determine the mass of
water that can be easily squeezed out. This water represents the water that was
present in the connected pores in the gel.
8. Dry the gel piece by blowing air for 15 min or put the gel into the oven and
measure the weight again. The weight loss during drying represents the weight
of the water trapped in the isolated pores.
9. Determine the ratio of the water in the connected pores and that in the trapped
pores. This ratio is a measure of the connectivity of the pores in the
macroporous gel.
10. Determine the ratio of the total water and the dry gel weight. This is a measure
of total swelling capacity of the gel.
Do steps 1-9 with three samples. Use the composition given above for the first sample,
and choose the other compositions on your own. You can also replace salt with sugar
in one of the samples. Comment on the effect of composition on the properties of the
gel.
𝑊0 −𝑊2
× 100%
𝑊0
After finishing this lab you should have learnt the basics of macroporous hydrogel
preparation by free radical polymerization. Macroporous hydrogels find applications in a
number of areas particularly related to tissue engineering. If you found this lab
particularly interesting you should research applications of macroporous hydrogels and
also various common hydrogels utilized in industry.
Glaucoma is a major ophthalmic disease and the second largest cause of
blindness. Research this disease to find out symptoms, effects and common
treatments.
Post lab exercise
1. Why macroporous gels opaque but the nanoporous pHEMA gels are
transparent?
2. List some commercial drug delivery patches available in the market.
3. How does timolol treat glaucoma? How is timolol typically delivered to the
glaucoma patients?
4. List other common drugs used for glaucoma therapy
5. What are the benefits of drug eluting devices compared to oral drug delivery for
drugs with a high metabolism in the liver?
Pre lab questions:
1. Write the differential equation that describes mass transport of molecules in
hydrogels?
2. What is a perfect sink condition?
3. What are the units of diffusivity?
4. Why types of transitions contribute to absorbance spectrum in the UV range?
5. What determines whether a given gel is transparent or opaque?
6. What is a macroporous hydrogel and what are some of the common applications?