Urry D.W. (Ed.) What Sustains Life? : Consilient Mechanisms for Protein-Based Machines and Materials

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9.4 Medical Applications

515

a period of nearly 3 months at the level of

2.2|Lim/day (compared with

1.9|im/day

for

Leu-enkephalin amide). As expected, delivery

shuts off abruptly in about 3 months.

By changing the oil-like nature of groups in

the polymer, different levels of release can be

obtained. By proper design of the protein-

based polymer, different levels of constant

release are obtained, and, for a constant surface

area of an adequately sized depot, the amount

released can be constant for periods of more

than 3 months (see Figure 9.39).

9.4.5.2 Negatively Charged

Pharmaceuticals Delivered by Positively

Charged Polymers: Consideration of

Steroid Phosphates and Oligonucleotides

9.4.5.2.1 Loading Negatively Charged

Dexamethasone-Phosphate and

Betamethasone-Phosphate into a Series of

Positively Charged Model Proteins

For this example the series of microbially

prepared Model Proteins x' through xiv' in

Table 5.5 is used. Changes in the value of Tt as

the drug is added to a solution of each of the

series of increasingly hydrophobic elastic

protein-based polymers provide the loading

profile and present visualization of the relative

affinities of drug with model protein. The data

are given in Figure

9.40.

The differences in the

loading profiles and dexamethasone-phosphate

(Figure 9.40A) and betamethasone-phosphate

(Figure 9.40B) are very minimal, although there

is the slightest suggestion that the affinity would

be slightly greater for the latter.^^

9.4.5.2.2 Release Profiles for Negatively

Charged Dexamethasone-phosphate and

Betamethasone-phosphate from a Series of

Positively Charged Model Proteins

The release profiles for negatively charged dex-

amethasone-phosphate and betamethasone-

phosphate from a series of positively charged

model proteins are given in Figure 9.41. The

results are not as extraordinary as with the pos-

itively charged drug/negatively charged

polymer

cases.

In the case of drug delivery com-

bining negatively charged drug with positively

charged polymer, the polymer does not dis-

0.5 1.0

mol

drug/2mol Lysine

0.5 1.0

mol

drug/2mol Lysine

FIGURE

9.40. Loading profiles for (A) dexametha-

sone-phosphate and (B) betamethasone-phosphate

using the series of Lys (K)/Phe (F)-containing

Model proteins x' through

xiv'

in Table

5.5.

(Repro-

duced with permission from Urry et al.^^)

perse as readily in phosphate buffered saline.

The difference arises from the effectiveness of

chloride ion in lowering the value of Tj for

model proteins containing lysine (Lys, K)

residues. Under conditions of physiological

saline,

0.15

NaCl, CI" replaces the phosphate

of the drug. Nonetheless there occurs a period

of about 100 days between 70 to 170 days when

the releases hold within a good band that could

be adjusted to fit the desired therapeutic range

for the drug. Preparation of a protein-drug

complex that would provide the desired 100

day release would involve the proper amount

of drug being titrated into solution to form a

drug-polymer complex having the equivalence

shown at 70 days in Figure 9.41. In general,

516 9. Advanced Materials for the Future

desired levels of release would be achieved by

the size and shape of the depot and the

hydrophobicity of the model protein.^^

9.4.5.2.3 Controlled Release of Antisense

Oligonucleotides from a Series of Positively

Charged Model Proteins

Antisense oligonucleotides are parts of the

genetic material that can be used to block the

expression of certain genes. For example, a

A 1.0

0 50 100 150 200 250

days

B 1.0-

100 150

days

200 250

catheter with a balloon on its end is inserted

into a blocked coronary artery, and the balloon

is inflated to open the artery for improved

blood flow. A common problem is that this inju-

rious process induces a repair response, and an

associated cellular proliferation leads to reclo-

sure,

as has happened with a number of well-

known people, for example. Vice President

Cheney.

If, however, a controlled release device could

also be deployed that released low levels of the

correct antisense oUgonucleotide, or sRNAi,

the expectation is that reclosure could be

prevented. Thus our initial task is to see if we

can design bioelastic polymers that could give

rise to controlled release of negatively charged

oligonucleotides. In this case the bioelastic

polymer is designed with positive charges and

with different amounts of more oil-like groups.

As shown in Figures 9.38 and 9.39, polymers

have been designed to form devices that result

in different constant levels of release for a con-

stant surface area. As shown in Figure 9.41,

a significant controlled release was achieved

using a small negatively charged drug with a

series of positively charged polymers. Figure

9.42, using more than 10-fold larger negatively

charged pharmaceutical, an oUgonucleotide,

with the same series of positively charged

polymers demonstrated a remarkable release

profile. Oligonucleotides, being very potent

pharmaceuticals, require but a small level of

release, such that the drug delivery devices in

this in vitro assay can release therapeutically

competent levels for months, as shown in the

lower curve in Figure 9.42. This demonstration

of controlled release of oligonucleotide by prin-

cipals associated with BRL resulted in the 1999

CRS-Prographarm Outstanding Pharmaceuti-

cal Paper that was awarded at the 27th

International Symposium on Controlled

Release of Bioactive Materials in July 10,2000,

in Paris.

FIGURE 9.41. Release pfofiles for (A) dexametha-

sone-phosphate and (B) betamethasone-phosphate

using the series of Lys (K)/Phe (F)-containing

Model Proteins x' through xiv' (K/5F) in Table 5.5.

Note the constant release for the most hydrophobic

Model Proteins xiv' (K/5F) in (A) and xiii (K/4F) in

(B).

(Reproduced with permission from Urry et al.^^)

9.4.5,3 Loading of Protein into

and Release from Transductional

Protein-based Polymers

When considering designed Tt-type transduc-

tional protein-based polymers for controlled

9.4 Medical Applications

517

• Polymer

• Polymer II

Polymer

A Polymer III

Decreased

release due to decrease of

surface area

in the

conical portion of

tube.

50 60 70

Days

FIGURE

9.42.

Release of negatively charged antisense hydrophobicity and pKa shifts, Model Proteins x'(I)

oligonucleotide from Lys-containing, positively through xiv'(V) in Table 5.5. (Reproduced with per-

charged polymers with systematically increased mission from Woods.^^)

release of an identified pharmaceutical, initial

insight into their effectiveness derives from the

curves.

This is apparent on examination

of the loading curves for dexamethasone-

phosphate and betamethasone-phosphate

shown in Figure 9.40 and on comparison to the

release curves of Figure 9.41. For these nega-

tively charged drugs, positively charged poly-

mers were considered, that

is,

the K/nF series of

Lys (K)/Phe (F)-containing Model Proteins x'

through xiv' in Table 5.5. The affinity between

charged drug and oppositely charged polymer

increases as the polymer becomes more

hydrophobic, because the AGap-derived driving

force for ion pairing increases with hydropho-

bicity. This is evidenced by the steepness of

the drop in the values of Tt for the onset of

the inverse temperature transition as the drug

is titrated into the solution of the polymer. In

general, the tighter interaction, as evidenced by

the greater decrease in Tt, results in a lower

and more constant release profile. Thus, when

considering the potential for controlled release

of a particular pharmaceutical such as a natural

protein, we look at the loading curves of

chosen transductional elastic protein-based

polymers.

9.4.5.3.1 Loading and Release Curves for

Soybean Trypsin Inhibitor

The net charge on the protein becomes the first

issue in the choice of charge on the polymer. In

general the polymer is chosen with the oppo-

site charge from that of the net charge on the

protein. This allows for the hydrophobically,

AGap-driven ion pairing that has the associated

effect of lowering the temperature for the onset

of the inverse temperature transition. The

protein of interest here is 20,000 Da protein,

soybean trypsin inhibitor (STI). It has a pi of

4.5,

that

is,

at pH 4.5 it contains an even number

of positive and negative charges. This means at

the physiological pH of

7.5

that STI carries a net

negative charge. Accordingly, the obvious

choice of protein-based polymer comes from

the K/nF series of Lys (K)/Phe (F)-containing

Model Proteins x' through xiv' in Table 5.5,

as used above for the dexa- and betamethasone

phosphates and oligonucleotides. The loading

curve is given in Figure 9.43 A, and Figure 9.43B

shows the release data in the more common

plot of percentage released as a function of time

for the original elastic protein-based polymer

from the mammalian elastic fiber, (GVGVP)n

518 9. Advanced Materials for the Future

Loading curve for Soybean Trypsin Inhibitor

60-

20 H

lOH

K/3F: (GVGVP GVGVP GKGVP-

GVGVP GVGFP GFGFP)39(GVGVP)

III III III

ill III III III

-o

w/w 0.1 0.2 0.3

Weight fraction of STI/30 mer

In vitro release profile for Soybean Trypsin Inhibitor

100

Time (hrs)

FIGURE

9.43. Soybean trypsin inhibitor (STI), a

protein model for loading and release studies using

positively charged elastic protein-based polymers of

varied hydrophobicity. (A) Loading curve for the

protein-based polymer K/3F: (GVGVP GVGVP

GKGVP GVGVP GVGFP GFGFP)39(GVGVP).

(B) Percent of STI released as a function of time

from K/3F and from (GVGVP)25i. (A. Pattanaik,

L.C. Hayes, and D.W. Urry, unpublished results.)

with n = 251, and K/3F: (GVGVP GVGVP

GKGVP GVGVP GVGFP GFGFP)39

(GVGVP). The release from the initial

uncharged base polymer represents a burst type

of release, whereas that from the designed K/3F,

Model Protein v, after the first several hours

establishes a low-level, steady release rate.

9.4.5.3.2 Loading Curves for Ovalbumin

Ovalbumin, a 45,000 Da protein, has a pi of 4.7.

Accordingly at pH 7.5, physiological pH, oval-

bumin contains a net negative charge, which

again suggests use of the K/nF series. In partic-

ular, the loading curves for K/OF: (GVGVP

9.4 Medical Applications

519

GVGVP GKGVP GVGVP GVGVP GVG

VP)22(GVGVP) and K/2F: (GVGVP GVGFP

GKGFP GVGVP GVGVP GVGVP)22

(GVGVP) are shown in Figure 9.44A. Polymer

K/OF does not sufficiently lower the value of Tt

to function at 37°C. The K/2F-ovalbumin

complex is used below to determine if it suffi-

ciently slows the release to function in vivo for

the elastic protein-based polymer to serve as an

adjuvant.

Ovalbumin Loading Curves

50-1

K/2F

K/OF

O O O

'OO

0 0.5 1 1.5

Weight fraction of Ovalbumin/30 mer

$ O

£-5

^=3

5 F

«j)5

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

DayO Day 14

Days post immunization

FIGURE

9.44. (A) Ovalbumin loading into K/OF:

(GVGVP GVGVP GKGVP GVGVP GVGVP

GVGVP)22(GVGVP) and K/2F: (GVGVP GVGFP

GKGFP GVGVP GVGVP GVGVP)22(GVGVP)

shows K/2F to be a possible delivery device. (A.

Pattanaik, L.C. Hayes, and D.W. Urry unpublished

results.) (B) The K/2F-ovalbumin complex elicits

ovalbumin-specific antibody formation, whereas

ovalbumin and K/2F alone exhibit no significant

antibody formation. (Z. Moldoveanu,

Mestecky, A.

Pattanaik, and D.W. Urry, unpubhshed results.)

9.4.5.3.3 Potentiation of Protein Antibody

Development When Conjugated with

Positively Charged Protein-based Polymer

The capacity for slow release of a protein (the

antigen) from a depot suggests the use of the

protein-based polymer as a controlled release

vehicle that may function as an adjuvant

for the development of a superior immune

response. As shown in Figure 9.44B, neither

ovalbumin alone nor the protein-based

polymer alone results in the significant forma-

tion of ovalbumin specific antibody in the sera.

When the ovalbumin-loaded protein-based

polymer is given, significant amounts of oval-

bumin specific antibody are detected in sera.

9.4.5.4 Nanoparticles Composed of a

Hydrophobic Series of Charged Elastic-

contractile Protein-based Polymers

Vaccines can carry either a net positive charge

or a net negative charge. Accordingly, their

release can be controlled much as shown in

Figures 9.38, 9.39, 9.41, and 9.42. The different

release levels would function as primary and

secondary immunizations and even for very

slow release as third immunizations.

Also,

if the

delivery vehicle could be nanoparticles, absorp-

tion could be through a number of epithelial

linings available in the body that could replace

the often-uncomfortable injections. Figure 9.45

demonstrates formation of uniformly sized

nanoparticles made of a substantially oil-like

yet negatively charged protein-based polymer

that would be the candidate for slow release,^^

Thus,

the stage is set for vaccine delivery by

nanoparticles. Similarly, delivery of negatively

charged genes could be considered by properly

designed positively charged nanoparticles.

9.4.5.5 Elastic Protein-based Matrices as

Devices for Transdermal Delivery

9.4.5.5.1 Skin Permeation

Bioelastic patches or disks are shown in Figure

9.46 for different loading levels of dazmegrel.

The circular patches, cross-sectional area of 1.9

cm^, are 20 Mrad y-irradiation cross-linked

(GVGIP)26o, designated as X2°-(GVGIP)26o,

520 9. Advanced Materials for the Future

FIGURE

9.45. Electron microscopy of nanoparticles

comprised of Model Protein v in Table 5.5, namely,

(GVGVP GVGFP GEGFP GVGVP GVGFP

GFGFP)42(GVGVP). (Reproduced with permission

from Urry et al.^^)

that have been equilibrated in dipropylene

glycol/propylene glycol/water (2:1:1). Pro-

gressing from left to right, the dazmegrel levels

are 0, 0.1, 1, and lOmg/cm^. When placed on

dog skin or human skin, these disks release

dazmegrel into the skin as shown in Figure 9.47.

Penetration into greyhound dog skin was five

times greater than into human breast skin. For

in vitro greyhound skin permeation studies, the

intermediate dose of

1.9mg/disk

yielded a con-

stant release of dazmegrel from 4 to 48 hours,

as measured by recovery from the receptor and

dermis. The total recovery of dazmegrel was

88 ± 13%, and the retention by the bioelastic

membrane averaged

± 9.0%.Thus, it appears

that the intermediate level could be maintained

for a significantly longer period of time, as

demonstrated by the in vivo delivery study

immediately below.^^

9.4.5.5.2 In vivo Skin Permeation

In narrowing down the concentration range for

loading of the bioelastic membrane, an inter-

mediate low (0.5mg/cm^) and an intermediate

high (5.0mg/cm^) were used. In the greyhound

dog model as shown in Figure 9.48, by 24 hours

the level of dazmegrel in the epidermis and

dermis had reached a constant level with the

intermediate low level of loading, and that

therapeutically competent level remained

FIGURE

9.46. y-Irradiation cross-linked elastic disks

of X^°-(GVGIP)26o, equilibrated in dipropylene

glycol/propylene glycol/H20 (2:1:1) and loaded

with

0,0.1,1,

and lOmg/cm^ dazmegrel, a thrombox-

ane synthetase inhibitor for preventing capillary

constriction and the subsequent necrosis that leads

to a pressure ulcer. (Courtesy of Bioelastics research,

Ltd.)

9.4 Medical Applications

521

ri 0.6

WD 0.5

S 0.4

Exposure Period

I I 4 Hours

I 24 Hours

• 48 Hours

a, 6

Low (0.19 mg) Intermediate (1.9 mg)

Loading Dose

High (19 mg)

FIGURE

9.47.

vitro

release into the skin of the grey-

hound dog from y-irradiation cross-linked elastic

disks of X^°-(GVGIP)26o equilibrated in dipropylene

glycol/propylene glycol/HaO (2:1:1) and loaded with

low (0.19mg/disk), intermediate (1.9mg/disk), and

high (19mg/disk) levels of dazmegrel, the throm-

boxane synthetase inhibitor. (Reproduced with per-

mission from Kemppainen et al.^^)

essentially constant for 2 weeks, which was the

time for termination of the study. The thera-

peutic level was maintained for the 2-week

period, as demonstrated in Figure 9.49. At the

end of the 2-week period, 80% of the loaded

amount remained in the disk such that contin-

ued therapeutic release could be expected for

weeks to come.^^

Adhesive Tape

B-TSI-M ' ^ ^ A ^

& Occlusive Covering Staple

Adhesive Bandage

Occlusive Covering

B-TSI-M

Epidermis

Dennis

_-:: Subcutaneous

Tissue

FIGURE

9.48. Greyhound dog model for transdermal delivery: Placement of drug-laden patches on the back

of a dog to follow the release of the drug into the skin. Reproduced with permission from the phD disser-

tation of N.-Z. Wang, Auburn University, Auburn, AL, (1998).

522

9. Advanced Materials for the Future

2.5

go 1.5

a 0.5

-a

*a 0.0

1.0

0.00

Expo^prg Period

• IDay

B 7 Days

• 14 Days

Intermediate Low Intermediate High

Loading Dose

Intermediate Low Intermediate High

Loading Dose

FIGURE

9.49. Release at 1, 7, and 14 days from y-

irradiation cross-linked elastic disks of X^°-

(GVGIP)26o, equilibrated in dipropylene glycol/

propylene glycol/H20 (2:1:1) and loaded with inter-

mediate low (0.5mg/cm^) and intermediate high (5

mg/cm^) levels of dazmegrel, a thromboxane syn-

thetase inhibitor. For the intermediate low level of

loading, a constant pharmacologically effective dose

is released for 14 days with yet 80% of drug remain-

ing in the disk at 14 days. (A) Epidermis. (B) Dermis.

(Reproduced with permission from Kemppainen

et al.^^)

9,4,5,6 Bioelastic Patches for the

Prevention of Pressure Ulcer Formation

9.4.5.6.1 Prevention of Pressure Ulcers by

Providing a Drug-laden Cushioning Disc to

Cover a Bony Prominence

Pressure ulcers occur at bony prominences

where the skin is compressed either at an assis-

tive device interface or as might occur for the

bed-ridden or wheel-chair dependent. Due to

the elasticity of bioelastic materials, the bioe-

lastic membrane alone provides a helpful pro-

tective cushion. The bioelastic materials can

also be designed to have partitioning properties

similar to the skin itself for a drug useful in

blocking the biochemical changes that cause

the tissue necrosis leading to pressure ulcer

formation. In particular, dazmegrel blocks the

activity of an enzyme called thromboxane syn-

thetase, and it is the release of thromboxane

9.4 Medical Applications

523

that is thought to lead to vascular constriction

and poor circulation that results in tissue break-

down and pressure ulcer formation. Dazmegrel,

therefore, is identified as a thromboxane syn-

thetase inhibitor. ^^

9.4.5.6.2

Preliminary Studies Show

Drug-laden Bioelastic Disc to Prevent

the Tissue Necrosis Resulting in Pressure

Ulcer Formation

The bioelastic disks were then used in a dog

model for pressure ulcer formation to deter-

mine efficacy. The coaptation walking cast

model of Swaim et

al}^^'^^

was utilized in the

greyhound dog. In this model, severe pressure

ulcers develop at the medial malleolus (the

inner ankle bone), as evaluated in Table 9.8.

When a bioelastic disk without drug (B-M) is

fixed in place over the anklebone, the occur-

rence and severity of ulcers is significantly

decreased, as shown as the control (unloaded)

data in Table 9.9. In the limited studies to

date,

the presence of a loaded intact disk, the

bioelastic-thromboxane synthetase inhibitor-

membrane (B-TSI-M) prevented ulcer forma-

TABLE

9.8. Pressure ulcer study: Subjective observa-

tions of skin over the medial malleolus with no bioe-

lastic matrix patch.

Dog^

DP-SN"

DP-9N

DP-ION

DP-llN

Appearance of skin

Hyperemia

Not noted

Small

Large

Not noted

Epidermal

abrasion/

ulceration**

Present

Present (3)

Present (2)

Days in cast

^ DP-#N = No bioelastic patch.

^ These lesions were deeper into the dermis than the super-

ficial abrasions noted when bioelastic matrix patches were

present.

It was necessary to remove the casts from these dogs

before the scheduled 7 days. The casts had considerable

"strike through" on the surface overlying bony promi-

nences, indicating a significant underlying dermal lesion.

Source: Reproduced from Kemppainen et al.^^

tion, as shown as the treatment (loaded) data in

Table 9.9.

The one limitation at this medial malleolus

site of high shear pressure is an occasional (one

in four of the test cases, DP-7T) splitting or

tearing of the

disk,

with early signs for ulcer for-

TABLE

9.9. Pressure ulcer study: Subjective observations of unloaded and loaded bioelastic matrix patches

and skin over the medial malleolus (casts in place 7 days).

Dog/treatment^

Controls (unloaded)

DP-IC

DP-2C

DP-3C

Treatment (loaded)

DP-4T

DP-5T

DP-6T

DP-7T

Patch appearance

Intact, cloudy white

area

Intact, clear

Cloudy white area,

triangular tear distal

Intact, clear

Clear, triangular tear

cranial—distal

Appearance of skin under or immediately adjacent to patch

Hyperemia

Slight over entire

med. mal.

None

Present distal, not

traceable

None

Small cranial—distal

Small central and

proximal,^ slight,

not traceable

Slight distal, not

traceable

Superficial epidermal

abrasion/ulceration

2 small

None

Present distal

None

Small distal

Skin adjacent to patch

Nothing notable

Hyperemia and

epidermal

abrasion/ulcer—distal.

Ecchymosis—proximal

Nothing notable

" DP-#C = Unloaded bioelastic patch; DP-#T = loaded bioelastic patch; DP-#N = no bioelastic patch.

^ This proximal small area of hyperemia was noted before the cast was placed. It was covered by the patch and had not

gotten worse over the 7 day cast period.

Source: Reproduced from Kemppainen et al.^^

524

9. Advanced Materials for the Future

mation occurring at the location of the split.

Of note is that such high shear stress is not

expected in the usual appUcations, and even the

occasional splitting at this severe test site

appears to be solvable. A modified composition

has been prepared in which the force necessary

to initiate fracture has been doubled, as shown

in Figure 9.50. This should prevent the small

1400-

X20-(GVGIP)320

X20.(GVGIP)260

x20-(GVGVP)25i

300

% Extension

1400-

X20-(GVGIP)260

12 3 4 5

Ligament Length (nmi)

FIGURE

9.50. Notch-ligament length approach to the

determination of the essential work of fracture

shows doubled specific work of fracture for y-cross-

linked X^°-(GVGIP)32o as compared with previously

used X^°-(GVGIP)26o- Break stress data in Figure

9.20 suggest even more favorable break strengths are

yet possible with chemical cross-linking approaches.

(A) Stress-strain curves. (B) Essential work of frac-

ture (intercept). (Reproduced with permission from

Urry et al.^^

amount of splitting that did occur in the medial

malleolus site of high shear stress.

This success with the simple matrices of

X'°-(GVGIP)n and similarly of X2°-(GVGVP)n

for transdermal delivery does not yet bring to

bear the full power of the Tftransductional

protein-based polymers. Finer control yet will

occur as the forces arising out of the

apolar-polar repulsive free energy of hydra-

tion, AGap, are employed. Accordingly, there is

much to be optimistic about when considering

elastic protein-based polymers capable of

inverse temperature transitions for develop-

ment of transdermal delivery systems.

9.4.6 The Eye as a Special Site

for Applications

A great deal can and should be written about

the potential for applications in the eye of

elastic and plastic protein-based polymers with

the capacity for exhibiting inverse temperature

transitions. Some work has indeed been done in

this area, but the detaiUng of it will have to be

done in the future as time and space are limit-

ing in the present effort. Some of the applica-

tions are (1) drug delivery by means of eye

drops (perhaps with nanoparticles) or by

wafers placed under the eyelids; (2) drug deliv-

ery by means of an ocular plug inserted through

the sclera; (3) soft contact lenses with a greater

control of the refractive index and with less

fragile material that does not support forma-

tion of a protein coating; (4) Coatings of

intraocular lenses that would make them less

damaging to tissue when being implanted and

that would limit fouling of the surface; (5)

replacement of the vitreous humor following

surgical procedures; (6) as a biodegradable

elastic band following retinal reattachment

surgery that would disappear once its task has

been completed; (7) strabismus surgery for pre-

vention of compromising adhesions that follow

attempts to correct eye alignment by relocating

rectus muscles as discussed briefly above for

the prevention of adhesions section 9.4.4.3.2;

and (8) Drainage sleeve for reUeving pressure

of glaucoma.