1

Applications of self-assembling peptides

in controlled drug delivery

Sotirios Koutsopoulos, Ph.D.

Dru

g c

oncentr

ation in b

lood

Time

Ctoxic

Ceffective

Problems associated with drug administration

2

The importance of Control:

• Bio-compatible

• Non-toxic, non-immunogenic

• Bio-degradable

• Easy to handle

• Protect the cargo

• Deliver small, large, hydrophilic and hydrophobic molecules

• Programmed dose and time release for as long as it is required

• Flexibility & patient compliance

• Reduced risk of side effects from overdose

World drug delivery market: $17 - 29 billion for 2006$33 - 67 billion for 2009$65 - 97 billion for 2013

Most common systems

1. Electronic devices e.g., insulin pumps

2. Particle- and matrix-based systems e.g. polymers or inorganic materials

3. Liposomes

The importance of Control:

3

The importance of Control: 1. Devices

• First controlled drug delivery systems were built in the 1960s

• Insulin reservoir (like a regular syringe)

• Controller (computer chip)

• Glucose sensor

• Infusion device (pump) to deliver insulin to the body through a needle

The importance of Control: 2. Polymer micro- & nano-particles, matrices, and hydrogels

Poly-lactic-co-glycolic acid (PLGA)

Poly-ethylene glycol (PEG)

Poly-phosphoesters

Poly-vinyl alcohol (PVA)

Poly-ethylene oxide (PEO)

….

Clinical studies show:

Immune reaction to polymer surfaces

Degradation products may be toxic

Often toxic initiators are used

Not very efficient for protein delivery

Nanoparticles – microparticles

Composite polymer materials

Micelles

Colloids

Nanogels

Emulsions

Pluronic acid micelles (PEO/PPO)

Thin films

Polymer brushes

Hydrogels

4

Associated with cell toxicity effects

Immune response in some patients

Research started in late 1960s

Not many unique liposome-based drugs

(most contain phosphatidylcholine)

Doxil (1993, ovarian cancer, myeloma) poor industrial reproducibility

The importance of Control: 3. Liposomes

Lipid bilayerWater soluble

drug

Lipid soluble drug

AntibodyPEG protective layer

5-7 nm12-16 aa

1-cm

Self-assembling peptides

100-nm

2.5 nm5-7 aa

5

(RADA-RADA-RADA-RADA)

Hydrogel consisting of self-assembling peptides

At physiological conditions0.15 M NaCl & 3 < pH < 8

Liquid Gel

Tissue specific injectable drug release system

Hydrogel consisting of self-assembling peptides

Water solution

Drugs

6

Phenol Red

8-hydroxypyrene-1,3,6-trisulfonic

acid trisodium salt (3-PSA)

Phenol1,3,6,8-pyrenetetrasulfonic acid tetrasodium

salt (4-PSA)

Bromophenolblue

Release of model drugs through peptide hydrogels

1 cm

0.5 % w/v

Release of model drugs through peptide hydrogels

Measure diffusion for 1 week

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

220 320 420 520 620 720

1-15min

1-30min

1-45min

1-1.5hrs

1-3hrs

1-6hrs

1-9hrs

1-12hrs

1-24hrs

1-48hrs

1-96hrs

1-168hrs

Wavelength (nm)

Ab

so

rban

ce

5 mm

0.5 % w/v

7

Release of model drugs through peptide hydrogels

Slow release depending on charge and MW of the model drug

rh = 0.9 nm

rh = 1.0 nm

Release of model drugs through peptide hydrogels

Release depending on charge of the model drug

8

Release due to specific interactions of molecules with nanofibers

rh = 0.8 nm

rh = 1.1 nm

Release of model drugs through peptide hydrogels

Release of model drugs

9

Large and unstable molecules (structure held by weak forces)

Easily damaged at mild storage conditions

Rapidly eliminated by the body

Some are difficult to obtain in large quantities

Protein drug delivery

• FDA has approved >200 proteins for therapeutic uses

• In 2014, protein drugs returned a revenue of ca. $70 billion

Common administration routes are not suitable for proteins

Proteins are cleared from

circulation (frequent injections)

Injection,

subcutaneous

or intravenous

Protein delivery through the lungs is difficult (e.g., Exubera)

Inhalation

Low permeability of large moleculesSkin

Proteins/enzymes are degraded in the stomach or they are not absorbed through the intestines

Oral

10

Study protein delivery through peptide hydrogels using proteins that:

• are well characterized and widely used

• have a wide range of MW and pI

• have therapeutic interest

Controlled release of proteins

Trypsin inhibitorMW 20.1 kDa

pI 4.6

LysozymeMW 14.7 kDa

pI 11.0

Human IgGMW 145.0 kDa

pI 7.2

BSAMW 66.0 kDa

pI 5.3

• High sensitivity and resolution (time/spatial)

• Detect molecules inside the hydrogel & in the supernatant

• Measure diffusion coefficients and concentration inside and outside the gel

Fluorescence Correlation Spectroscopy (FCS)

Detection volume (1 μμμμm x 1 μμμμm x 2 μμμμm)

Volume of detection is 1.5 fL

0.4 µm

laser beam

2 µm

1 µm

11

PBS

Gel + Protein

Watersolution

Protein

PBS

Protein release experiment

0 10 20 30 40 50 60

0.0

0.2

0.4

0.6

0.8

Lysozyme

Trypsin inhibitor

BSA

IgG

Mt/M

Time (hours)

Protein release through (RADA)4 hydrogel

Slow release depending primarily on the MW of the protein

8

12

0 20 40 60 80 100

0.0

0.2

0.4

0.6

0.8

1.0M

t/M

Time (days)

Protein release through (RADA)4 hydrogel

1.0 %

1.5 %

0.5 %

8

Protein release through peptide hydrogels

0 20 40 60 80 100

0.0

0.2

0.4

0.6

0.8

1.0(RADA)

4 1.0 %

(KLDL)3 0.6 %

(RADA)4 1.0 % (core) + (KLDL)

3 0.6 % (shell)

Mt/M

Time (days)

8

(RADA)4 + (KLDL)3

13

QuestionQuestion:

What is the state of the proteins after being released from the What is the state of the proteins after being released from the

hydrogels?hydrogels?

Protein release through peptide hydrogels

Study the released proteins:

• Spectroscopy (CD, Fluorescence)

• Activity tests, bioassays

Protein release through peptide hydrogels: CD spectroscopy

200 220 240

-80

-40

0

40

200 220 240

200 220 240-60

-40

-20

0

20

Wavelength (nm)

[θ ] x

10

-4 (

deg c

m2 m

ol-1

)

200 220 240

Lysozyme

Trypsin Inhibitor BSA

IgG

14

320 340 360 380

1

2

3

4

320 340 360 380

320 340 360 3800

1

2

3

4

Wavelength (nm)

Flu

ore

sce

nce

In

ten

sity (

a.u

.)

320 340 360 380

Lysozyme

Trypsin Inhibitor BSA

IgG

Protein release through peptide hydrogels: Fluorescence

Protein release through peptide hydrogels: Biological activity

Measure the hydrolysis of the cell membrane of M. lysodeiktikus

0

20

40

60

80

100

120

Native Lsz Released

No

rma

lize

d a

ctiv

ity o

f L

yso

zym

e

Measure the suppression of trypsin activity

0

2000

4000

6000

8000

0 1 2 3 4

conc. of Trypsin Inhibitor (mg/ml)

No

rma

lize

d a

citi

vity

of T

ryp

sin

Native Trypsin Inhibitor

Released Trypsin Inhibitor

15

Protein release through peptide hydrogels: Biological activity

Measure binding efficiency of monoclonal

IgG against antigen

Protein release through peptide hydrogels: Biological activity

Native or released

antibodies

Immobilized antigen

0 100 200 300 4000

20

40

Hydrogel released IgG

-dF

(H

z)

Time (s)

0

20

40

60

IgG injection

IgG injection Native IgG

4.0 µg/ml

2.0 µg/ml

1.0 µg/ml

0.5 µg/ml

4.0 µg/ml

2.0 µg/ml

1.0 µg/ml

0.5 µg/ml

-d

F (

Hz)

16

Smart hydrogel material for controlled release

KA6-NH2 (KAAAAAA-CONH2)

2.4 nm

2.6 nm 2.4 nm

DA6-COOH (DAAAAAA-COOH)

Lipid-like self assembling peptides

ac-A6K-NH2 (acetyl-AAAAAAK-CONH2)

2.6 nm

ac-A6D-OH (acetyl-AAAAAAD-COOH)

17

100 150 2000

5

10

15

20

Co

un

t n

um

ber

Nanovesicle size (nm)

100 150 200 2500

5

10

15

20

Co

un

t n

um

ber

Nanovesicle size (nm)

ac-A6K-CONH2

ac-A6K-CONH2

KA6-CONH2

Lipid-like self assembling peptide nanovesicles: AFM

20 40 60 800

10

20

30

Nanovesicle size (nm)

Cou

nt

nu

mb

er

20 40 600

10

20

30

Nanovesicle size (nm)

Co

un

t n

um

ber

ac-A6D-COOH DA6-COOH

Lipid-like self assembling peptide nanovesicles: AFM

18

Lipid-like self assembling peptide nanovesicles:Carboxyfluorescein encapsulation and release

0 1 2 3 4 5 6 70

20

40

60

80

100

ac-A6K-CONH

2

KA6-CONH

2

ac-A6D-COOH

DA6-COOH

% C

um

ula

tive

CF

Rele

ase

Time (hours)

Lipid-like self assembling peptide nanovesicles:Nile Red integration in the peptide bilayer

0 1 2 3 4 5 6 70

20

40

60

80

100

% C

um

ula

tive

Nile

re

d r

ele

ase

Time (hours)

ac-A6K-CONH

2

KA6-CONH

2

ac-A6D-COOH

DA6-COOH

inte

nsity

Flu

ore

scen

ce

560 600 640 680

inte

nsity

Wavelength (nm)

19

3 h 24 h 48 h0

10000

20000

30000Control

0.2 mg/ml ac-A6K-CONH

2

1.0 mg/ml ac-A6K-CONH

2

0.2 mg/ml KA6-CONH

2

1.0 mg/ml KA6-CONH

2

0.2 mg/ml ac-A6D-COOH

1.0 mg/ml ac-A6D-COOH

0.2 mg/ml DA6-COOH

1.0 mg/ml DA6-COOH

Cel

ls/w

ell

Lipid-like self assembling peptides: Epithelial cell proliferation

Lipid-like self assembling peptides: Drug absorption

Caco-2 cell monolayer model and rat intestine (everted sacs) are used to predict drug absorption (oral delivery)

0 50 100 150 200 2500

200

400

600

800

1000

1200

1400 Control

ac-A6K-CONH

2

KA6-CONH

2

ac-A6D-COOH

DA6-COOH

Time (min)Cum

ula

tiv

e F

ITC

-dex

tran

tra

nsp

ort

(p

mo

l/cm

2)

0

2

4

6

8

10

120 min30 min

FD-4 FD-4FD-4 + A6D FD-4 + A

6D

FD

-4 p

erm

eab

ilit

y (

nm

ol/cm

2)

50% increase

Caco-2 cell monolayer Rat intestine

20

Injectable peptide hydrogel

• Release small molecules & proteins of broad MW (e.g., Lsz, IgG)

• Hydrogel contains up to 99.5% water, active compound loading depends on the solubility of the drug

• Release kinetics depend on hydrogel density

• Functionality assays show that released proteins are active

Lipid-like peptide nanovesicles

• Release of hydrophilic and hydrophobic drugs

• Release kinetics depend on amino acid sequence and drug properties

• Peptides are not cytotoxic

• Increase transport through the epithelial layer

Conclusions

Self-assembling peptides are biocompatible, biodegradable, non-toxic, non-immunogenic, transparent which are ideal for biomedical applications