Summary of Completed Work Fall and Spring 2014

Goals

The overall goal of this project would be to find a pathway to make drug delivery via pulmonary administration more potent (without raising toxicity) even when the drug passes through a mucous layer. Within the past few years, pulmonary administration has become more popular than oral administration. A few advantages for pulmonary administration is that it avoids the first-pass metabolism because it is directly transferred into the lungs thus enabling a quick response from the drug. This process also allows the drug to bypass the gastrointestinal tract which prevents the drug from being digested. This method ends up being a simpler way to administer the song while allowing for maximal absorption since it allows a compromise between pH solubility and pH permeability (Meng-Lund, 2014).

The transport of the mucosa is calculated through the use of a modification of Fick’s Law ( Error:Reference source not found) which assumes a steady-state equilibrium condition. Given the amount of time allowed for diffusion, a steady-state assumption can be maintained instead of a pulse assumption

(which would require a smaller surface area of exposure than what the diffusion chamber would provide). The apparent diffusion coefficient is lower because mucous is sticky and can clog filters. Therefore, it is not possible to hold mucous in membrane for experimental purposes since the membrane would be clogged. A more appropriate equation for the diffusion cell is given in Equation 2. This shows that the concentrations should have a logarithmic relationship with time and has

permeability a factor involved in the slope. The other variables are S, for surface area, and Vd, the volume of the donor cell.

The amount of diffusion that has occurred can be measured via FRAP (fluorescent recovery after photobleaching) or through ultraviolet light (Flanagan & Donovan, 2001).

When measuring the diffusion of a drug or particle, through a mucous slab or a known volume of the mucous, the mucous must be able to be accurately sudivided into sections at the end fo the experiment (Flanagan & Donovan, 2001). This could possibly require a higher concentration of PGM (pig gastric

Equation 1 Fick's First Law

Equation 2 Diffusion Cell Calculation

mucin) which would deviate from the normal range for human mucosa. The usual concentration for the mucous is 2% PGM. Typical rheologic data for the PGM mimetic is shown in Figure 1. Rheology is determined through values for G’, the bulk shear storage modulus (elastic), and G”, the bulk shear loss modulus (viscous). This displays whether the mimetic reacts appropriately to fluid flow.

Another issue that could occur during these experiments is the possibility for particles to crosslink to the mucous. This could skew the apparent diffusion coefficient for some particles which would result in poor data. To make the error from this as minimal as possible, a coating of low molecular weight polyethylene glycol will be applied to the particles to reduce association of particles with mucous and prevents protein adsorption (Lai, Wang, & Hanes, 2000).

Making drug delivery more effective through the mucous layer is also beneficial for patients who suffer from cystic fibrosis. Patients with cystic fibrosis have a mucous composition different from a healthier individual. The best place to start for finding a better way to get drug delivery through a mucous layer of different composition would be to find the optimum size for a nanoparticle in a normal mucous layer. This would give a good starting point to find the optimal size for nanoparticles within a patient with cystic fibrosis. This is also beneficial for pulmonary drug delivery as whole. Finding the ideal range for nanoparticle penetration for heatlhy people and people with cystic fibrosis would increase the efficacy of the drug and, therefore, allow pulmonary drugs to be approved at a faster rate since a higher percentage of people would be reached with the drug. Certain trends known about mucous infected with cystic fibrosis is the elastic modulus (G’) being dramatically greater than the viscous modulus (G”). This gives the mucous a less viscous nature and is the reason why mucal blockage have a higher chance of occuring in pulmonary and trachealbronchial tissue. Sample rheologic data of typical cystic fibrosis mucosa is in Figure 2. A possible strategy to deal with the different viscoelastic properties is adding a mucolytic agent to improve penetration of the drug, such as Dornase alfa or N-acetyl L-cysteine (Lai, Wang, & Hanes, 2000).

Methods

Membrane Diffusion Study Protocol

1. Set previously made buffer to mix for 30 minutes then obtain 50 mL sample for experiment.2. Setting up the stock solution

a. Particles: Obtain stock particle solution and sonicate for 10 minutes followed by vortexing the solution 3 times for 30 seconds (repeat this step 3 times)

b. Other dyes: Weigh and mix required amount of dye into the buffer to achieve desired concentration

Figure 2 Rheologic data for CF mucosa (Pavan G. Bhat, 1996)

3. Dilute stock solution to desired concentration4. Vortex 3 times for 30 seconds followed by sonicating for 10 minutes (only for particles)5. Set up diffusion chamber

a. Wash all part of diffusion cellb. Place 50 mL buffer sample in 37 ° C water bathc. Cut out membrane at an appropriate size for doughnut section of chamber to hold (have

shiny side pointing upd. Plug all receivers but leave one space open for collecting samplee. Add 2 mL of buffer to each chamber with a stir bar in each chamber followed by 200 μL

of particle solution. Start timer once particle solution is added.6. Collect 200 μL sample of each chamber in following manner:

Time (minutes) Chamber A Chamber B Chamber CT1T2T3T4T5

*Remember to replenish 200 μL with sputum buffer after taking sample7. Prepare Standard Plot (refer to Standard Plot protocol) of known concentrations by setting up

Plate 1 accordingly:

1 2 3 4 5 6 7 8 9 10 11 12AB 1 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256C 2 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256D 3 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256EFGH

8. Set up Plate 2 accordingly:

1 2 3 4 5 6 7 8 9 10 11 12A (T1 minutes) (T2 minutes) (T3 minutes)B (A) X X X (A) X X X (A) X X XC (B) X X X (B) X X X (B) X X XD (C) X X X (C) X X X (C) X X XE (T4 minutes) (T5 minutes)F (A) X X X (A) X X XG (B) X X X (B) X X XH (C) X X X (C) X X X

*Use 50 μL of sample in each well

How to make a PTT cross-linked mucus mimetic:

Day 1:

1. Prepare 100 ml of sputum buffer (fresh)

2. Using the 35 mL amber glass vials, create 29 mL of a 2% PGM III solution in sputum buffer.

3. Place the tube(s) on the tube rotator (inside the walk-in refrigerator) and allow them to mix for 24 hours.

Day 2:

4. After 24 hours of mixing on the tube rotator, take the amber vials of your mucin solution back to the lab

5. Prepare a 15% solution (in water) of potassium tetrathionate. Mix this vigorously to ensure it has all dissolved.

6. Add 1 ml of this PTT solution to the 29 mL of mucin solution. Mix by hand for 5 minutes.

7. Place the vials in a 37C water bath. Make sure to lightly mix them every day to keep the mucin from settling. Also make sure that the water in the bath doesn’t evaporate (Fill in more if you need to)

Day 8:

8. Your mucin solution has now cross-linked for 6 days (required for best results). Now run rheology. Ask me or Edwin about how to do that when the time comes.

Results

A B C

10 L1 M1 N1

20 L2+0.1L1 M2+0.1M1 N2+0.1N1

30 L3+0.1*(L2+0.1L1) M3+0.1*(M2+0.1M1) N3+0.1*(N2+0.1N1)

Using time points at 30 seconds, 3 minutes, 6 minutes, 9 minutes, and 12 minutes, the results showed that while the dye’s concentration reached equilibrium on both sides within three minutes, the receiving cell showed an increase in the dye from 30 seconds to 3 minutes.

0 2 4 6 8 10 12 140

200

400

600

800

1000

1200

1400

Nuclear Fast Red 1-12-2015

ABC

Time (min)

Abso

rban

ce

Nuclear fast red had a fairly consistent absorbance spectrum which made it apparent that the time interval for measurements were too large. After these trials, it was necessary to use smaller time intervals.

0 1 2 3 4 5 6 7 8 9 100

0.020.040.060.08

0.10.120.14

f(x) = 0.0298145251619544 ln(x) + 0.0715622044316601R² = 0.917515148194065

f(x) = 0.027951434540564 ln(x) + 0.0419312899750891R² = 0.950540747160485f(x) = 0.0222970513930229 ln(x) + 0.0253772331148432R² = 0.944229662490104

Trypan Blue Trial in PBS 2-10-15

A Logarithmic (A) BLogarithmic (B) C Logarithmic (C)

Time (minutes)

mg

in R

ecei

ver

Comparison of the three trials for diffusion study 2-10-15 for trypan blue. Even though the amount of mass transfer that occurs is different for each trial, the trend is overall the same. Possible reasoning for why the mass transfer is different could be attributed to some differences between each membrane or the donor sample not being well mixed when added to the membrane.

Test Diffusion Cell with Nuclear Fast Red with known mass of dye added to prove mass of dye is increasing in receiver:

0 1 2 3 4 5 6 7 8 9 100

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

f(x) = 0.0222970513930229 ln(x) + 0.0253772331148432R² = 0.944229662490104

A

Time (minutes)

mg

in R

ecei

ver

0 1 2 3 4 5 6 7 8 9 100

0.02

0.04

0.06

0.08

0.1

0.12

f(x) = 0.027951434540564 ln(x) + 0.0419312899750891R² = 0.950540747160485

B

Time (minutes)

mg

in R

ecei

ver

0 1 2 3 4 5 6 7 8 9 100

0.02

0.04

0.06

0.08

0.1

0.12

0.14f(x) = 0.0298145251619544 ln(x) + 0.0715622044316601R² = 0.917515148194065

C

Time (minutes)

mg

in R

ecei

ver

The results for these experiments show an expected trend for the trypan blue diffusion through the cells. The receiver increases on a logarithimic scale which agrees with calculations for the mass transfer. Unfortunately, the standard deviations were high for these trials. This was later corrected by switching the readings to absorbance.

Standard plots for trypan blue only yielded decent results when absorbance was measured. For fluorescence, the data was very unreliable and all over the place. After trying the standard plate and the quartz plate, it was determined that the fluorescence reading for trypan blue was unreliable and that absorbance was the better measure as shown by the trials.

0 0.02 0.04 0.06 0.08 0.1 0.120

0.050.1

0.150.2

0.250.3

0.35

f(x) = 2.98711794871795 xR² = 0.998290362547325

Trypan Blue Absorbance at 607 nm

A BC AVERAGELinear (AVERAGE)

Concentration (mg/mL)

Abso

rban

ce (O

D)

0 0.02 0.04 0.06 0.08 0.1 0.120

0.050.1

0.150.2

0.250.3

0.35

f(x) = 2.98523467643468 xR² = 0.998302823017831

Trypan Blue Absorbance at 607 nm

A BC AVERAGELinear (AVERAGE)

Concentration (mg/mL)

Abso

rban

ce (O

D)

Overall, I think the technique was alright for the standard plots, it was the fact I used a fluorescence measurement instead of absorbance measurement that was the issue.

Standard plot for Dextran with Rhodamine and preliminary testing of the diffusion cell with the dye showed that ideal time markers would be 5 min, 10 min, 20 min, 30 min, 60 min.

0 0.5 1 1.5 2 2.50

200400600800

100012001400

f(x) = 554.257407312457 xR² = 0.999254186780516

Standard Plot Fluorescence Ex. 540 nm Em. 625 nm

1 2 3Average Linear (Average)

Concentration (mg/mL)

Fluo

resc

ence

After time slots were determined, a diffusion study was done with three cells to observe the diffusion barrier during the time periods mentioned above.

0 10 20 30 40 50 60 700

0.05

0.1

0.15

0.2

0.25

0.3

f(x) = 0.0158511190238961 ln(x) + 0.19847630503188R² = 0.637688472854552

Diffusion Study Rhodamine with Dextran

Time (minutes)

Amou

nt in

rece

iver

(mg)

Again the logarithmic behavior is seen for this data. To fully capture the behavior, it could be possible to take smaller time points between 5 and 10 minutes to observe more of the logarithmic behavior.

0 0.2 0.4 0.6 0.8 1 1.20

100200300400500

600700

f(x) = 549.735314899591 xR² = 0.997944838407207

Standard Plot for Rhodamine Dextran 5-5-15

1 2 3Average Linear (Average)

Concentration (mg/mL)

Abso

rban

ce

0 2 4 6 8 10 12 140

0.02

0.04

0.06

0.08

0.1

0.12

0.14

f(x) = 0.0195701624835952 ln(x) + 0.0785964360582783R² = 0.987843899732322

Diffusion Study Rhodamine with Dextran 5-5-15

Time (minutes)

Rece

iver

Am

ount

(mg)

Using a smaller time scale, the logarithmic trend was observed for fluorescence with very little error as indicated by the small error bars. This shows that the logarithmic trend is the desired behavior observed for diffusion and should be expected for particles.

Rheological Properties of Mucin Mimetic which will be used in future transport studies:

Sample 1 shows modest differences between Day 2 and Day 14 after crosslinking should be completed. The range for the viscoelastic modulus decreases as the amount of time after crosslinking increases.

0.1 1 10 1000.01

0.1

1

10

100

1000SK-1-8-1 D14 Rheology - Frequency

Sweeps (n=3)

G' AverageG" Average

Frequency (rad/s)

Visc

oela

stic M

odul

us (P

a)

0.1 1 10 1000.001

0.01

0.1

1

10

100

1000

10000SK-1-8-1 D2 Rheology - Frequency

Sweeps (n=3)

G' Average

G" Average

Frequency (rad/s)

Visc

oela

stic M

odul

us (P

a)

Sample 2 shows more dramatic differences from Day 2 to Day 14 with the viscoelastic modulus showing a large decrease comparatively as the frequency increases. Even though the mimetic in a previous trial seemed good to use after 10 days, 14 days shows evidence of degradation of the crosslinkings. So for experimental purposes, it would be recommended to use the mimetic for diffusion studies within 7 days.

0.1 1 10 100

0.001

0.01

0.1

1

10

100

1000

10000 SK-1-8-1 D2 Rheology - Frequency Sweeps (n=3)

G' AverageG" Average

Frequency (rad/s)

Visc

oela

stic M

odul

us (P

a)

0.1 1 10 1000.01

0.1

1

10

100

1000

10000 SK-1-8-2 Rheology D2 - Frequency Sweeps (n=3)

G' AverageG" Average

Frequency (rad/s)

Visc

oela

stic M

odul

us (P

a)

0.1 1 10 1000.001

0.01

0.1

1

10

100

1000 SK-1-8-2 Rheology D14 - Frequency Sweeps (n=3)

G' Av-erage

Frequency (rad/s)

Visc

oela

stic M

odul

us (P

a)

0.1 1 10 1000.01

0.1

1

10

100

1000

10000 SK-1-8-2 Rheology D2 - Frequency Sweeps (n=3)

G' AverageG" Average

Frequency (rad/s)

Visc

oela

stic M

odul

us (P

a)

Measuring the rheology through the C60/4 rather than the C35/4 gave more reasonable results. This explains the odd results from the first tests. Further tests include waiting a few more days to see if crosslinking stabilizes or if mimetic continues to crosslink. This will give information on the immediacy for running diffusion studies with the mimetic.

0.1 1 10 1000.01

0.1

1

10

100

1000

SK-1-8-1 Rheology D10 - Frequency Sweeps (n=3)

G' AverageG" Average

Frequency (rad/s)

Visc

oela

stic M

odul

us (P

a)

Sample 2:

0.1 1 10 1000.001

0.01

0.1

1

10

100

1000 SK-1-8-2 Rheology D10 - Frequency Sweeps (n=3)

G' AverageG" Average

Frequency (rad/s)

Visc

oela

stic M

odul

us (P

a)

Some crosslinking has occurred within the past ten days, but not enough for the mimetic to be in the acceptable range for human mucous. Dan suggested a repeat of the experiment which will occur in the next couple of weeks. These results were possibly due to using old PTT, and was later found to be due to using the wrong mucin batch. More experiments will be run once reagents for the sputum buffer are verified because it unsure whether anhydous or monohydrate forms of a compount is needed.

Discussion/Conclusions/Future Work

The logarithmic trend for the diffusion of the various dyes were observed with reliable R2 values which means that this type of trend should be viewed in other transport studies as well. Conclusions from the mimetic formations were that the mimetic would hold for at least a week. This ensures a week’s worth of reliable testing for transport studies using the mimetic. Future work for this project include making the mucin mimetic from the correct batch and with the right reagents (once the anhydrous and monohydrate confusion is cleared), and doing transport studies with the dyes going through the mimetics. This will give a baseline estimate for what is expected when the particles will diffuse through the mucin mimetic as well. After finding the ideal particle size for the mimetic, the next plan would be to synthesize a mucin mimetic which has properties similar to CF mucosa. Then transport studies will be done to find an ideal particle size for that type of mimetic.

ReferencesFlanagan, D., & Donovan, M. (2001). Drug Transfer Through Mucus. Advanced Drug Delivery Reviews,

174-195.

Hamad, R., & Fiegel, J. (2013). Synthetic Tracheal Mucus with Native Rheological and Surface Tension Properties. Wiley Online Library, 1788-1798.

Lai, S., Wang, Y. Y., & Hanes, J. (2000). Mucus-Penetrating Nanoparticles for Drug and Gene Delivery to Mucosal Tissues. Advanced Drug Delivery Review, 158-171.

Meng-Lund, E. M. (2014). Ex Vivo Correlation of the Permeability of Metoprolol Across Human and Porcine Buccal Mucosa. J. Pharm. Sci., 103: 2053–2061.

Pavan G. Bhat, D. R. (1996). Drug Diffusion through Cystic Fibrotic Mucus: Steady-State Permeation. Journal of Pharmaceutical Sciences, 624-630.