Formulation Screening of Perfluorocarbon Emulsions for Use in Acoustic Droplet Vaporization

Ian Sebastian, Mario Fabiilli, J. Brian Fowlkes, and Paul Carson

Department of Biomedical Engineering & Department of Radiology, University of Michigan, Ann Arbor, Michigan

Background: Perfluorocarbon Emulsions

• Micrometer sized emulsions with a perfluorocarbon (PFC) dispersed phase can be made using an emulsifier like bovine serum albumin (BSA) or polar lipids.

• Gaseous PFCs are common in many commercially available ultrasound (US) contrast agents.

Shell (albumin or lipid)

Superheated core(e.g. C5F12 - bp 29oC)

PFP

Saline with Albumin

Vial is Shaken with a High Speed Vial Shaker

Saline containing albumin-coated PFP droplets

Advantages of PFP

• PFP has a normal boiling point of 29°C. In the human body (37°C) PFP should boil. However the PFP droplets can be systemically administered and remain in a superheated liquid state, which destabilizes the emulsion slightly.

• PFP is also biocompatible; it has properties similar to other higher boiling PFCs that were researched for use as artificial blood substitutes.

• It is biologically inert and can dissolve high concentrations of O2.

• PFP is also hydrophobic and lipophobic.Molecular Structure of PFP

Acoustic Droplet Vaporization

• This PFP emulsion is destabilized by ultrasound pulses causing the liquid PFP to vaporize. This is known as acoustic droplet vaporization (ADV).

Superheated Droplets Gas bubbles

Low amplitude US

High amplitude US

Droplets Bubbles

Dialysis Tubing

B-mode US image from - Kripfgans et al. Ultrasound Med Biol (2000). 26:1177-1189.

Applications: Embolotherapy

• The expansion of the PFP gas can cause the occlusion of blood flow in capillary beds.

• Decreases in blood flow to canine kidney tissue is seen when ADV is performed in the renal artery

x

Focus

Increased echogenicity from PFP gas in renal cortexRenal Artery

Transverse Plane US images of externalized canine kidney

Applications: Embolotherapy

ADV Performed on Canine Kidney

Application: Embolotherapy

-100

-80

-60

-40

-20

0

20

40

R caudal anterior

cortex

R middle anterior

cortex

R cranial anterior

cortex

R caudal posterior

cortex

R middle posterior

cortex

R cranial posterior

cortex

L caudal anterior

cortex

L middle anterior

cortex

L cranial anterior

cortex

L caudal posterior

cortex

L middle posterior

cortex

L cranial posterior

cortex

% reduction of regional perfusion

Before ADV Post ADV

Before treatment of left kidney

After treatment of left kidneyBlood flow changes before and after

ADV in the right kidney (control).

Changes in Regional Perfusion of a Canine Kidney before and after ADV Treatment

Application: Drug Delivery

• It is possible to create a two phase emulsion; one phase containing PFP, the other containing an oil with a dissolved chemotherapy drug.

• Cancer specific ligands can be incorporated into the external surfactant allowing the droplets to bind to the vasculature of a tumor via the recognition of a specific receptor, such as vascular endothelial growth factor receptor(VEGF-R2).

• ADV would then release the chemotherapy drug at the tumor, and create selective embolization of the tumor. Alternatively, the hypoxic conditions created by the embolization could be used to activate a chemotherapy pro-drug.

Application: Drug Delivery

PFC

Drug-containing phase

Water-soluble surfactant

Oil-soluble surfactant

PFC-soluble surfactant

Two-Phase Emulsion

Vasculature

Motivation

The size of the PFP droplets in the formulation is crucial to the success of in vivo applications.

Droplet size Pro Con

Small •More efficient drug delivery

•Do not lodge in capillaries

•Less gas produced requiring more droplets for embolization

•More difficult to vaporize

Large •Large amount of gas upon vaporization

•Become lodged in lung capillaries

Lung Filtration of the Emulsion

0

2

4

6

8

10

12

14

16

0 5 10 15 20 25 30

AlbuminAlbumin (Lung Filt)LipidLipid (Lung Filt)

Vol

ume

Per

cent

(%

)

Droplet diameter (micron)

Larger droplets are filtered when they get lodged in lung capillaries. Thus, it is good to avoid larger droplets. These plots show droplet volume and number distributions as a function of diameter before and after passing through a theoretical first-pass filtration based on lung capillary diameter distributions (J.C. Hogg, Phys. Rev. 1987: 67(4): 1249-1295). Notice that a significant volume of albumin droplets are filtered on the first pass.

10-1

100

101

102

4 8 12 16 20

AlbuminAlbumin (Lung Filt)LipidLipid (Lung Filt)

Num

ber

Per

cent

(%

)

Droplet diameter (micron)

Effect of Emulsion Composition on Droplet Diameter

2.0

2.1

2.2

2.3

2.4

2.5

2.6

2.7

4 5 6 7 8 9

Mea

n dr

ople

t di

amet

er (

mic

ron)

Number (n) of carbons (CnF

2n+2)

10-1

100

101

102

2 4 6 8 10 12 14

AlbuminLipid

Pe

rce

nt

(%)

larg

er

tha

n c

orr

esp

on

din

g d

iam

ete

r

Droplet diameter (micron)

These plots show how choice of emulsion composition can affect the size distribution. The left plot show that droplet diameter tends to increase as the number of carbons in the PFC increases (this is due to increased viscosity of the PFC). The right plot shows the difference between using lipids and albumin as the surfactant. Notice that lipid droplets are smaller overall.

General Materials and Methods

• For all experiments PFP was used as the dispersed PFC phase and albumin was used as the emulsifier.

• Droplet samples were prepared in 2 mL glass vials with rubber stoppers and emulsified using an amalgamator.

• A Coulter Counter was used to determine the size distribution and concentration of droplets in each sample.

Experiments: Long Term Emulsion Stability

• Goal: Asses shelf-life of the emulsion due to general emulsion stability issues.

• Many identical sample vials were created at once; then three vials were sampled and the size distribution of the emulsion was measured using a Coulter Counter. Three new samples were measured every week for 13 weeks.

Results: Long Term Emulsion Stability

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14

Diameter

% >10 micron

Dro

plet

dia

met

er (

mic

ron)

Percent (%

) of droplets greater than 10 micron in diam

eter

Time (weeks)

Notice that the increase in diameter over time is curved. The increase in volume is approximately linear. Since the average diameter of the emulsion begins to increase as soon as the emulsion is made, this suggests that the emulsion should not be stored for a long period of time before use after shaking, if small droplets are desired.

Conclusions: Long Term Emulsion Stability

• The linear increase in volume over time is predicted by models of Ostwald ripening. This suggests that Ostwald ripening is the primary driver of the emulsion changes, rather than coalescence.

• The increase in diameter with time may be a good way to generate larger droplets, but if small droplet are desired the vials should not be mixed until they are needed.

Experiments: Formulation Variations

• Goal: Optimization of formulation to maximize PFP emulsification and generate droplet parameters desirable for our current applications (smaller droplets that are very concentrated).

• The quantities of the three components in the solution were varied– Albumin, saline, and PFP

• Focus was placed on separating the effect of the vial and the solutions ability to mix effectively in it, and the effects of variations in the components

Results: Formulation Variations

0.0

2.0

4.0

6.0

8.0

10

0.0

5.0 108

1.0 109

1.5 109

2.0 109

2.5 109

3.0 109

3.5 109

4.0 109

0 5 10 15 20

Diameter

#/mL

Ave

rag

e d

rop

let d

iam

ete

r (m

icro

n)

Dro

ple

t con

cen

tratio

n (n

um

be

r of d

rop

lets p

er m

L em

ulsio

n)

Albumin (mg/mL)

The albumin concentration in the saline was varied from 1 mg/mL to 20 mg/mL with constant amounts of PFP and saline. Notice that the number of droplets per mL is highest and diameter is lowest when the albumin concentration is 4 mg/mL. This suggests that 4 mg/mL of albumin is the optimal concentration for the given amounts of saline and PFP.

Results: Formulation Variations

The plot shows the change in diameter as the amount of PFP was changed with a constant volume of 1 mL total (see the figure below). It seems evident, like in the previous experiment, that there is an intermediate value for the PFP to saline ratio that minimizes the diameter of the droplets in the emulsion. The two circled points occurred when the concentration of PFP was too high to be fully emulsified. The mixture remained in two phases at these points.

1.6

1.8

2.0

2.2

2.4

2.6

2.8

0 1 102 2 102 3 102 4 102 5 102 6 102 7 102

Diameter (um)

Dia

me

ter

(um

)

PFP with 1mL Volume (uL)

Results: Formulation Variations

109

1010

109

1010

1011

0 1 102

2 102

3 102

4 102

5 102

6 102

7 102

8 102

Number of Droplets per mL of Solution

Number of Droplets per mL of PFP in Solution

Num

ber

of D

ropl

ets

per

mL

of S

olut

ion

Num

ber of Droplets per m

L of PF

P in S

olution

Amount of PFP Used in Sample Prep (uL)

1.7

1.8

1.9

2.0

2.1

2.2

2.3

2.4

2.5

0 1 102

2 102

3 102

4 102

5 102

6 102

7 102

8 102

Diameter (um)

Dia

met

er (

um)

PFP Used in Sample Prep (uL)

Results: Formulation Variation

• Notice from the previous slide that when the amount of PFP is about 250 μL the number density of the droplets is maximized while maintaining a small diameter.

• When the volume of fluid in the vial becomes too large, the formation of the emulsion is impeded by the lack of vial headspace for agitation to occur effectively.

Results: Formulation Variation

The amount of PFP and total albumin was kept constant. Thus, the concentration of albumin was varied to maintain 3mg of albumin for every volume of saline. Notice that after 750μL the number of droplets drops off significantly. The most likely reason for this is after the total volume in the vial exceeds 1 mL, there is not enough headspace for the solution to mix and emulsify well. The large air space allows for a lot of very turbulent interactions to form the emulsion.

108

109

1010

109

1010

1011

0 2 102 4 102 6 102 8 102 1 103 1.2 10 31.4 10 31.6 10 3

Droplets per a mL of Solution

Droplets per a mL of PFP

Dro

ple

ts p

er

a m

L o

f S

olu

tion D

rop

lets p

er a

mL

of PF

P

Amount of Saline (uL)

Experiments: Centrifugation

The presence of large albumin (> 6μm) droplets prompted an investigation of centrifugation as a way to filter out larger particles. After centrifugation there were virtually no droplets larger than 6μm. The solution was too dilute after centrifugation so the supernatant was centrifuged. The resulting pellet was virtually the same distribution as the supernatant. These results suggested that a concentrated, small diameter solution could be achieved, but 95% of the original droplets were lost. This was unacceptable. Staged filtration was also tried, but the high number of droplets saturated the filters too quickly.

10-3

10-2

10-1

100

101

102

4 8 12 16 20

No CentrifugationSupernatantPellet of Supernatant

No

Cen

trifu

gatio

n

Diameter (um)

Experiment: ADV Threshold Measurements

• Goal: Determine whether the ADV threshold varies as a function of droplet diameter.

• Important to understand depending on the intended application; changes in ADV threshold may affect formulations differently in drug delivery versus embolotherapy.

• Simultaneously measure inertial cavitation (IC), which may be responsible for the ADV mechanism.

Change in ADV Threshold with Droplet Diameter

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

1 1.5 2 2.5 3 3.5 4 4.5 5

ADVIC

Thr

esho

ld (

MP

a)

Diameter (um)

*

*

Notice the ADV threshold decreases from 1-2.5 μm, and then it remains constant. At the same time the inertial cavitation threshold remains constant, and it occurs at a higher rarefactional pressure. Therefore, inertial cavitation is not necessary for ADV to occur. Additionally smaller droplets require higher rarefactional pressures for ADV to occur.

Experiment: Stability of the Emulsion to Temperature

• Goal: Confirm that the emulsion is stable at body temperature (37°C), and determine at what temperature the emulsion becomes unstable.

• The stability of the emulsion upon heating could influence the necessary storage conditions and choice of PFC for in vivo studies.

• It gives insight into the degree to which the PFP is stabilized by the emulsification process.

• The emulsion was diluted in saline and held at a constant temperature for one hour, and samples were taken at regular intervals.

Results: Stability of the Emulsion to Temperature

-10

-5

0

5

10

15

20

25

30

-6

-4

-2

0

2

4

6

20 30 40 50 60 70 80

Average Decrease in Number of Droplets

Average Decrease in Droplet Size (um)

Ave

rag

e D

ecr

ease

in N

um

be

r of

Dro

ple

ts

Avera

ge

De

crease

in D

rople

t Size (u

m)

Temperature (C)

Notice that the emulsion remains stable up to 50°C then at 60°C there is a dramatic drop in the number of droplets seen at the end of sampling. This suggests that the emulsion becomes unstable between 50°C and 60°C. Additionally, there is no significant change in the average diameter of the droplets, which suggests that the vaporization temperature does not change significantly with diameter of the droplet.

Final Conclusions

• There tends to be an intermediate range of concentrations for each component that is acceptable for proper emulsion formation.– The diameter and number of droplets changes some in this

range, but the largest changes in the size distribution of the emulsion is seen when emulsion components are changed.

• The ability of the mixture to mix properly has a very large impact on the quality of the emulsion.

• The increase in average size of the droplets seems to be driven largely by diffusion and Ostwald ripening rather than coalescence.

• Inertial cavitation does not seem to be necessary for ADV to occur.

• The emulsion is stable at temperatures below 50°C, and significant droplet loss due to vaporization occurs above 50°C.