BE351 Lab 5

Drug Delivery

Rose Hulman Institute of Technology

Terre Haute, Indiana

Team
Alex Obatake
Kally Morozin
Maddie Mosscrop
Kevin Wuest

January 17, 2018

In fulfillment of the requirements of the course:

BE351-01
BE Lab
Dr. Buckley and Dr. Dosmar
Abstract
The release characteristics of an alginate hydrogel bead drug delivery system were evaluated
using tartrazine dye release and light spectroscopy. Dye release samples from the alginate beads
were collected at 5 minute intervals for 60 minutes and measured for absorbance. A standard
curve, generated by measuring the absorbance of 0.5 g/L tartrazine stock at varying
concentrations from 0% to 100%, was used to convert the absorbance values of the dye release
samples to concentrations. With the assumed 100% encapsulation efficiency, the initial mass of
tartrazine dye encapsulated in the 12 beads was calculated to be 0.0653 mg. The initial burst,
taking place over the first five minutes, was found to be 0.0302 mg. The total amount of
tartrazine dye released was observed to be 0.123 mg corresponding to a total of 188% drug
release. The average drug release per minute was found to be 0.00168 mg/min. Total drug
release time could not be determined with the collected data since trial did not last long enough
for the alginate beads to stop releasing tartrazine dye.

Methods
A spectrophotometer and known stock solutions were used to perform drug release tests on beads
made of a dyed alginate solution, composed of 0.2 g alginate for every 10 mL of stock with
0.5g/L tartrazine stock, being dropped into calcium chloride (6% wt CaCl).

Bead Production​. Alginate beads were produced by slowly dripping tartrazine alginate solution
into a well plate of 3 mL calcium chloride solution. After waiting 1 minute for crosslinking, the
calcium chloride solution was removed via pipetting and divided into three spectrophotometry
well plates. The beads were then rinsed with DI water and this solution was placed into another
three spectrophotometry wells after removal. Encapsulation efficiency was assumed to be 100%.

Dye Release Trials​. At five minute intervals, 1 mL of solution around the beads was removed
from the well plate and replaced with an equal amount of new DI water. The removed solution
placed into the same spectrophotometry plate. Dye concentrations were quantified using a
spectrophotometer at a wavelength of 475 nm with an endpoint assay and an automix time of 5
seconds. Release profiles were conducted over the course of 60 minutes. All release profiles
were performed in triplicate.

Standard Curve Construction​. A standard curve of 0.5 g/L tartrazine stock, at concentrations
found in Table 1, was developed to assess the concentration of drug release profiles. The
concentrations of tartrazine were placed in well plates to be tested in triplicate using a
spectrophotometer at a wavelength of 475 nm with an endpoint assay and an automix time of 5
seconds. A graph of the standard curve is shown in Figure 1 with a best fit curve.

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Table 1. ​Volumes of water and tartrazine used to make varying concentrations of solution to
produce the standard curve.

Concentration (%) Volume H​2​O (μL) Volume tartrazine (μL) Total (μL)
100 0 300 300
75 75 225 300
50 150 150 300
30 210 90 300
25 225 75 300
10 270 30 300
5 285 15 300
0 300 0 300

Figure 1.​ Standard curve produced through finding the absorbance of a wavelength at 475 nm at
each concentration of tartrazine solution with an R2 value of 0.9818.

Drug Release Calculations. ​The absorbance of the drug release profiles were compared to the
standard curve of tartrazine, using the equation found in Figure 1, to find the concentrations of
tartrazine in the drug release profiles. The cumulative release profile for concentration was found
by summing the concentration release profile at each time point. The percent drug release profile
was found by comparing the cumulative release profile to the initial encapsulated tartrazine dye
amount. This was used to determine total drug release, total percent drug release, initial burst,
and average minutely release of the tartrazine from the alginate beads.

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Results
The absorbance of the amount of tartrazine dye inside each well plate was determined using the
spectrophotometer and can be seen in Table 2.

Table 2. ​Mean absorbance, concentration, cumulative concentration and cumulative percent

release of drug release profiles for each 5 minute time interval measured with standard
deviations.
Time Mean Absorbance Mean Cumulative Cumulative
(minutes) (unitless) Concentration Concentration Percent released
(mg/mL) (mg/mL) (%)

5 0.255 ± 0.014 0.0303 ± 0.014 0.0303 ± 0.014 46.3 ± 21.4

10 0.154 ± 0.029 0.0183 ± 0.029 0.0485 ± 0.029 74.3 ± 44.4

15 0.108 ± 0.005 0.0128 ± 0.005 0.0613 ± 0.005 93.9 ± 7.7

20 0.101 ± 0.004 0.0120 ± 0.004 0.0733 ± 0.004 112 ± 6

25 0.088 ± 0.004 0.0104 ± 0.004 0.0838 ± 0.004 128 ± 6

30 0.065 ± 0.003 0.00771 ± 0.003 0.0915 ± 0.003 140 ± 5

35 0.060 ± 0.001 0.00712 ± 0.001 0.0986 ± 0.001 151 ± 2

40 0.050 ± 0.010 0.00593 ± 0.010 0.105 ± 0.010 160 ± 15

45 0.045 ± 0.003 0.00451 ± 0.003 0.110 ± 0.003 168 ± 5

50 0.038 ± 0.003 0.00451 ± 0.003 0.114 ± 0.003 175 ± 5

55 0.036 ± 0.001 0.00427 ± 0.001 0.119 ± 0.001 182 ± 2

60 0.034 ± 0.001 0.00403 ± 0.001 0.123 ± 0.001 188 ± 2

These absorbance values were plugged into the equation produced by the standard curve,

Absorbance
C oncentration = 8.4286
(1)

in​ ​order to find the concentrations, see Figure 2, and the cumulative concentrations, see Figure 3.

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Figure 2. ​The concentration of the tartrazine released from the alginate beads containing 0.0653
mg of tartrazine found at 5 minute intervals for 60 minutes.

Figure 3. ​The cumulative amount of tartrazine released from the alginate beads containing
0.0653 mg of tartrazine over a 60 minute interval.

The initial amount of tartrazine dye encapsulated in the beads is necessary for the percent
cumulative release of tartrazine dye to be determined. The equation used to determine the total
volume of tartrazine inside of the beads was,

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 V alg = 43 πr3 n, (2)
Ex: V alg = 43 π(1.375mm)3 (12) = 131mm2 = 0.131mL,
where V alg is the total volume of the alginate solution used, r is the approximate radius of the
beads, and n is the number of alginate beads formed. This value was then multiplied by the
concentration of stock tartrazine solution, 0.5g/L, to calculate that the total amount of tartrazine
in the alginate beads was 0.0653 mg.

mtar = V alg C tar (3)

mg
Ex: mtar = (0.131mL)(0.5 mL ) = 0.0653mg ,
where mtar is the total mass of tartrazine encapsulated in the alginate and C tar is the
concentration of tartrazine in the stock tartrazine solution. Since 100% encapsulation efficiency
was assumed, 0.0653 mg was determined to be the total amount of tartrazine contained in the
alginate beads at time zero minutes.

Figure 4. ​The percentage of the total tartrazine dye released from the alginate beads containing
0.0653 mg of tartrazine over a 60 minute interval.

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Table 3. ​Drug release parameters obtained from a 60 minute time release of tartrazine from
alginate beads containing 0.0653 mg of tartrazine.
Encapsulation Total Drug Total Percent Initial Burst Average Minutely
Efficiency (%) Release Drug Release (%) from 5 mins Release (mg/mL)
(mg/mL) (mg/mL)
100 0.123 188 0.0302 0.00168

The encapsulation efficiency was assumed to be 100%. With this assumption, the total drug
released was 0.123 mg corresponding to a total of 188% drug release. The initial burst was
calculated to be 0.0302 mg which took place over the first 5 minutes. This was found through
Figure 2, where the initial burst corresponds to the initial peak of drug release before the steady
release. The average drug release per minute was calculated to be 0.00168 mg/min. This value
was calculated over the linear time period after the initial burst, which was minutes 10 to 60.
This was calculated using the difference between cumulative concentrations at the ends of the
linear portion divided by the length of the linear portion. The total time of release could not be
calculated since the cumulative graph does not plateau within the measured time.

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Appendix. ​Dye release profiles from spectrophotometer.
Well ID Name Well 475 Count Mean Std Dev CV (%)

SPL1 A1 0.248 3 0.255 0.014 5.666

B1 0.272

C1 0.246

SPL2 A2 0.134 3 0.154 0.029 18.887

B2 0.14

C2 0.187

SPL3 A3 0.105 3 0.108 0.005 4.553

B3 0.114

C3 0.106

SPL4 A4 0.104 3 0.101 0.004 3.57

B4 0.102

C4 0.097

SPL5 A5 0.091 3 0.088 0.004 4.953

B5 0.09

C5 0.083

SPL6 A6 0.064 3 0.065 0.003 4.92

B6 0.063

C6 0.069

SPL7 A7 0.061 3 0.06 0.001 1.935

B7 0.059

C7 0.059

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SPL8 A8 0.061 3 0.05 0.01 20.664

B8 0.047

C8 0.041

SPL9 A9 0.048 3 0.045 0.003 6.739

B9 0.046

C9 0.042

SPL10 A10 0.041 3 0.038 0.003 6.565

B10 0.038

C10 0.036

SPL11 A11 0.037 3 0.036 0.001 3.237

B11 0.035

C11 0.035

SPL12 A12 0.035 3 0.034 0.001 1.682

B12 0.034

C12 0.034

SPL13 F1 4.434 3 3.92 0.903 23.038

G1 4.448 4.441

H1 2.877

SPL14 F2 3.433 3 3.237 0.512 15.806

G2 3.621

H2 2.656

SPL15 F3 2.573 3 2.356 0.308 13.055

G3 2.491

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 H3 2.004

SPL16 F4 1.728 3 1.502 0.271 18.022

G4 1.576

H4 1.202

SPL17 F5 1.296 3 1.204 0.12 9.971

G5 1.247

H5 1.068

SPL18 F6 0.553 3 0.524 0.042 8.029

G6 0.544

H6 0.476

SPL19 F7 0.293 3 0.281 0.014 4.892

G7 0.284

H7 0.266

SPL20 F8 0.032 3 0.034 0.003 8.575

G8 0.037

H8 0.032

SPL21 F10 0.329 3 0.268 0.053 19.636

G10 0.234

H10 0.242

SPL22 F11 0.263 3 0.297 0.032 10.848

G11 0.327

H11 0.302