Thermo-responsive Hydrogels for Intravitreal Injection and Biomolecule Release

Thermo-responsive Hydrogels for Intravitreal

Injection and Biomolecule Release

Ph.D. Thesis

Department of Chemical and Biological Engineering

Pawel W. Drapala

Advisor: Victor H. Pérez-Luna

1. Background and Significance

Age-Related Macular Degeneration (AMD)

Specific Aims

poly(ethylene glycol) (PEG) hydrogels

2. Thermo-Responsive Hydrogels

poly(N-isopropylacrylamide) (PNIPAAm),

Transition Temperature & Swelling

Volume Phase Transition Temperature (VPTT)

3. Copolymer Synthesis, Characterization and Degradation

Degradable Cross-links

Chain Transfer Agents (CTAs)

Selection of Hydrogel Formulations

Presentation Outline

Background and Significance

Thermo-Responsive Hydrogels

Copolymer Synthesis, Characterization and

Degradation

Release of Therapeutic Proteins

Biocompatibility of Drug Delivery System

Conclusions

4. Release of Therapeutic Proteins

Release of Proteins from Nondegradable Hydrogels

Higuchi Analysis of Diffusive-Controlled Systems

Effect of CTAs on Protein Release

PEGylated & Tethered IgG Release

5. Biocompatibility of Drug Delivery System

Bioactivity & Potential Cytotoxicity of Drug Delivery System

Cytotoxicity of Release Samples

Bioactivity of Release Samples

6. Contributions & Conclusion

Age-Related Macular Degeneration (AMD)

Normal Vision Age-related macular

degeneration

Incidence Rate: ~ 1 in 1,359 (~ 200,000 people in the United States)[1]

Elevated levels of Vascular Endothelial Growth Factor (VEGF)

“Wet” AMD:

• angiogenesis

• vascular leakage

• damage to photoreceptors

• vision loss

Angiogenesis Inhibitors:

• Avastin® & Lucentis®

• Injected into the vitreous every 4 to 6 weeks (half-life: 4.32 days)[2]

• Halt progression of wet AMD

• May lead to complications

[1] Facts About Age-Related Macular Degeneration. National Eye Institute. 2010.

[2] S. J. Bakri, M. R. Snyder, J. M. Reid, J. S. Pulido, and R. J. Singh. Pharmacokinetics of Intravitreal Bevacizumab. Ophthalmology,114(5):855-859, 2007.




Degradation



Conclusions

Aim 1. Determine the optimal hydrogel composition for localized drug delivery.

Hydrophobic/Hydrophilic Balance

Kinetics of Phase Change

Aim 2. Increase the duration (extend the therapeutic effect) of protein release from

thermo-responsive hydrogels.

Degradation kinetics of hydrogel crosslinks

Covalent Attachment of Proteins to the Hydrogel

Aim 3. Evaluate potential toxicity of degradation products and bioactivity of the released

angiogenesis inhibitor proteins.

Cytotoxicity of the drug delivery system

Activity of released anti-VEGF agents from the hydrogels

Specific Aims

Central Hypothesis: better treatment of wet AMD can be achieved by

localized and prolonged release of active angiogenesis inhibitor

proteins using thermo-responsive hydrogels by tailoring of hydrogel

structure, degradability, and controlling protein-polymer interactions.




Degradation



Conclusions

poly(ethylene glycol) (PEG) hydrogels

APS

PEG-DA PEG Hydrogel




Degradation



Conclusions

Hydrogels are hydrophilic 3-D networks of polymer chains High water content preserves protein bioactivity - ideal for protein drug delivery applications

Hydrogels are prevented from dissolving due to chemical or physical cross-links Protects the encapsulated proteins from immune recognition and clearance.

PEG hydrogels: nontoxic, non-immunogenic, anti-fouling

Can be polymerized under mild conditions via free radical polymerization:

poly(N-isopropylacrylamide)

(PNIPAAm)

NIPAAm

PEG-DA

PNIPAAm-co-PEG Hydrogel




Degradation



Conclusions

∆ Time ∆ Temp.

Intravitreal

Injection

Tem perature (oC )

30 32 34 36 38 40

No

rm

aliz

ed

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

0 m M

4 m M

8 m M

12 m M

16 m M

C ross-linker:

T e m p e ra tu re (o C )

2 0 2 2 2 4 2 6 2 8 3 0 3 2 3 4 3 6 3 8 4 0

Sw

ell

ing

Ra

tio

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 m M P E G -D A

1 2 m M P E G -D A

1 6 m M P E G -D A

Transition Temperature & Swelling

𝑄𝑆𝑤𝑒𝑙𝑙𝑖𝑛𝑔 =𝑊𝑆𝑤𝑜𝑙𝑙𝑒𝑛 −𝑊𝐷𝑟𝑦

𝑊𝐷𝑟𝑦




Degradation



Conclusions

Swollen

Hydrophilic

State (7°C)

Collapsed

Hydrophobic State

(37°C)

Volume Phase Transition Temperature

(VPTT)

• The VPTT can be readily manipulated by Hydrophobic/Hydrophilic monomer ratios[3]

PEG elevates VPTT

Poly(L-lactic acid) (PLLA) decreases the VPTT

[3] H. G. Schild. Poly (N-Isopropylacrylamide) - Experiment, Theory and Application. Progress in Polymer Science, 17(2):163-249, 1992.

[PEG-DA]* VPTT

0 mM 32.4 °C (± 0.3)

4 mM 33.4 °C (± 0.1)

8 mM 34.7 °C (± 0.4)

12 mM 35.3 °C (± 0.3)

16 mM 35.8 °C (± 0.1)

[cross-linker] PEG♯ PEG-b-PLLA

0.5 mM 32.1 °C (± 0.7) 29.8 °C (± 0.7)

1 mM 32.5 °C (± 0.9) 31.6 °C (± 0.7)

2 mM 33.2 °C (± 0.6) 32.0 °C (± 0.6)

3 mM 33.9 °C (± 0.9) 32.9 °C (± 0.7)

* PEG MW = 575 Da ♯ PEG MW = 3400 Da




Degradation



Conclusions

Degradable Cross-links: poly(L-lactic acid)

Acry-PLLA-b-PEG-b-PLLA-Acry

Lactic Acid PEG

PLLA-b-PEG rate of ester hydrolysis in-vivo[4,5]:

hydrophobicity

steric effects

cross-linking density

length of the PLLA oligomer

autocatalysis

size/charge of the encapsulated biomolecules

[4] Darrell Irvine. Molecular Principles of Biomaterials. MIT OpenCourseWare, 2006.

[5] J. L. West and J. A. Hubbell. Photopolymerized hydrogel materials for drug delivery applications. Reactive Polymers, 25(2-3):139-147, 1995.




Degradation



Conclusions

∆ Temp. ∆ Time

“Biodegradable”: material initially in solid or gel-phase, subsequently reduced to soluble fragments that are metabolized or excreted under physiological conditions (i.e. saline environment, pH = 7.4, 37 °C)

Degradation Profiles

Time (days)

0 5 10 15 20

Sw

ellin

g R

ati

o

20

40

60

80

100

120

PNIPAAm-co-PEG-b-PLLA (degradable)

PNIPAAm-co-PEG (nondegradable)

Room Temperature (24 oC)

Tim e (days)

0 5 10 15 20

Sw

ellin

g R

ati

o

0

5

10

15

20

25

30PNIPAAm -co -PEG-b-PLLA (degradable)

PNIPAAm -co -PEG (nondegradable)

Physiological Tem perature (37 oC )

• Swelling Ratios (below and above the VPTT) as function of incubation time for:

Nondegradable hydrogels cross-linked with Acry-PEG-Acry (PEG-DA)

Degradable hydrogels cross-linked with Acry-PLLA-b-PEG-b-PLLA-Acry

Molar concentrations: Cross-linker = 1 mM

PNIPAAm = 350 mM




Degradation



Conclusions

𝑄𝑆𝑤𝑒𝑙𝑙𝑖𝑛𝑔 =𝑊𝑆𝑤𝑜𝑙𝑙𝑒𝑛 −𝑊𝐷𝑟𝑦

𝑊𝐷𝑟𝑦

Chain Transfer Agents (CTAs)

PNIPAAm cannot exceed 32 kDa and PEG can not exceed 50 kDa for clearance by the renal system[6,7]

Lower the MW of growing PNIPAAm polymer chains using Glutathione CTA:

CTA-Initiated Growing Polymer Radical

Terminated Polymer Growing PNIPAAm Radical

[4] N. Bertrand, J. G. Fleischer, K. M. Wasan, and J. C. Leroux. Pharmacokinetics and biodistribution of N-isopropylacrylamide copolymers for the design of pH-

sensitive liposomes. Biomaterials, 30(13):2598-2605, 2009.

[5] T. Yamaoka, Y. Tabata, and Y. Ikada. Distribution and tissue uptake of poly(ethylene glycol) with different molecular-weights after intravenous administration

to mice. Journal of Pharmaceutical Sciences, 83(4):601-606, 1994.




Degradation



Conclusions

P ∙ +XR → R ∙ +XP

CTA Reaction

Glutathione

0 mg/mL 1 mg/mL 2 mg/mL 3 mg/mL 4 mg/mL1 mM2 mM3 mM4 mM5 mM6 mM7 mM

YesYesYes YesNo Yes YesNo No Yes YesNo No No YesNo No No No Yes

Glutathione Concentration (Chain Transfer Agent)


Molarity (Cross-linker)


Does the polymerization

produce a hydrogel?

Is the produced hydrogel injectable

via 30-gauge needle?


Yes No No No NoYes No No No NoYes Yes No No NoYes Yes Yes No NoYes Yes Yes Yes NoYes Yes Yes Yes NoYes Yes Yes Yes Yes







Degradation



Conclusions


Does the injectable hydrogel fully degrade within 30 days at 37 °C?

swollen hydrophilic state collapsed hydrophobic state partially degraded

collapsed state

∆ Temp. ∆ Time


NoNoNo Yes

No Yes YesNo Yes

NoNo







Degradation



Conclusions


Does the injectable hydrogel fully degrade within 30 days at 37 °C?

swollen hydrophilic state collapsed hydrophobic state partially degraded

collapsed state

∆ Temp. ∆ Time


NoNoNo Yes

No Yes YesNo Yes

NoNo







Degradation



Conclusions

Degradable Hydrogels

Time (days)0 2 4 6 8 10 12 14

Sw

elli

ng R

atio

0

20

40

60

Nondegradable Hydrogels (control)

Time (days)0 2 4 6 8 10 12 14

Sw

elli

ng R

atio

0

20

40

60

No Glutathione

0.5 mg/mL Glutathione


Degradation Profiles

Swelling Ratios (with and without Glutathione CTA) as function of incubation time:

Nondegradable hydrogels at 37 °C cross-linked with: Acry-PEG-Acry (PEG-DA)

Degradable hydrogels at 37 °C cross-linked with: Acry-PLLA-b-PEG-b-PLLA-Acry

CTA

[mg/mL] Qt=0

VPTT

[°C]

3 mM PEG cross-links

0 20.4 33.3

0.5 31.0 34.4

1.0 32.5 36.2

3 mM PLLA-b-PEG-b-

PLLA cross-links

0 23.9 32.9

0.5 34.7 34.1

1.0 37.4 35.0




Degradation



Conclusions

Release of Proteins from

Nondegradable Hydrogels

T im e (days)

0 10 20 30 40

IgG

Re

lea

se

d o

f E

nc

ap

su

late

d (

%)

0

20

40

60

80

100

IgG Release from PNIPAAm-co-PEG hydrogels

∆ Time




Degradation



Conclusions

Modes of Mass Transport:

Diffusion – due to concentration gradients

Convection – due to dehydration

Kinetics – due to hydrolytic degradation

24 °C 24 °C

24 °C 37 °C

∆ Time

∆ Temp.

Physiological

Temperature (37 °C)

Room Temperature (24 °C)

Higuchi Analysis of

Diffusive-Controlled Systems

𝜕𝐶

𝜕𝑡= 𝛻 ∙ 𝐷𝑔𝛻𝐶

𝑀𝑡𝑀∞= 1 −

8

2𝑛 + 1 2𝜋2∙ exp− 2𝑛 + 1 2𝜋2𝐷𝑔

𝛿2𝑡

∞

𝑛=0

𝑀𝑡𝑀∞≅ 4𝐷𝑔𝑡

𝜋𝛿2

12

T im e (days)

0 1 2 3 4 5

BS

A R

ele

as

ed

of

En

ca

ps

ula

ted

(%

)

0

20

40

60

80

100

Body Tem perature (37 oC)

Room Tem perature (23 oC)

BSA Release from PNIPAAm-co-PEG hydrogels y = 27.868x + 4.8453

R² = 0.9851

0

10

20

30

40

50

60

0 0.5 1 1.5 2

Mt/M

∞

tn

T = 24°C

y = 62.536x - 15.542 R² = 0.8923

0

10

20

30

40

50

60

70

0 0.5 1 1.5M

t/M

∞

tn

T = 37°C




Degradation



Conclusions

Effect of CTAs on Protein Release

Time (days)0 2 4 6 8 10 12 14

Re

lea

se

d o

f E

nca

psu

late

d (

%)

0

20

40

60

80

100

No CTA

0.5 mg/mL CTA

1.0 mg/mL CTA

Time (days)0 2 4 6 8 10 12 14

Re

lea

se

d o

f E

nca

psu

late

d (

%)

0

20

40

60

80

100


cross-linked with

Acry-PEG-Acry


cross-linked with





Degradation



Conclusions

Burst Release in initial deswelling phase: accounts for over 70% of total released protein.

PEGylated & Tethered IgG Release

∆ Temp. ∆ Time

Swollen Hydrophilic State Collapsed Hydrophobic

State

Partially Degraded

Collapsed State

PEGylation

Immunoglobulin G (IgG) Acry-PEG-SVA

PEGylated

IgG

Hydrogel Synthesis (with PEGylated IgG)

PEGylated IgG Release




Degradation



Conclusions

SDS-PAGE Analysis of IgG PEGylation




Degradation



Conclusions

Lane 1:

molecular weight marker

Lane 2:

No PEGylation control IgG

Lane 3:

IgG PEGylated at 1 to 5 molar ratio of IgG to Acry-PEG-SVA

Lane 4:

IgG PEGylated at 1 to 15 molar ratio of IgG to Acry-PEG-SVA

Release (at 37 °C) of IgG with varying

degree of PEGylation

Time (days)0 2 4 6 8 10

Rele

ased o

f E

ncapsula

ted (

%)

0

20

40

60

80

100

Time (days)0 2 4 6 8 10

Rele

ased o

f E

ncapsula

ted (

%)

0

20

40

60

80

100

No PEGylation

1:5 IgG to Acry-PEG-SVA Molar Ratio

1:15 IgG to Acry-PEG-SVA Molar Ratio


cross-linked with

Acry-PEG-Acry


cross-linked with





Degradation



Conclusions

VEGF

Receptor binding domain

Binding to VEGFR-2Migration

ProliferationNeovascularization

Bioactivity & Potential Cytotoxicity

of Drug Delivery System




Degradation



Conclusions

Avastin® and Lucentis ® inhibit the binding of VEGF to its receptor VEGFR-2

Abrogate VEGF-induced neovascularization[7]

Extract Sample 1

Extract Sample 2

Extract Sample 3

Extract Sample 4

Extract Sample 5

Bulk Avastin®

Bulk Lucentis®

PBS (control)

PEGylated Avastin®

PEGylated Lucentis®

Released Control

Released Avastin®

Released Lucentis®

Released PEGylated Avastin®

Released PEGylated Lucentis®PEGylation

Encapsulation Release

Sampling Schedule

Schematic Model of VEGF Pathway Inhibition

[7] A. Klettner and J. Roider. Comparison of bevacizumab, ranibizumab, and pegaptanib in vitro: Efficiency and possible additional pathways. Investigative

Ophthalmology & Visual Science, 49(10):4523-4527, 2008.

VEGF

Receptor binding domain

Avastin®/Lucentis®

Binding to VEGFR-2Migration

ProliferationNeovascularization

Bioactivity & Potential Cytotoxicity

of Drug Delivery System




Degradation



Conclusions

Avastin® and Lucentis ® inhibit the binding of VEGF to its receptor VEGFR-2

Abrogate VEGF-induced neovascularization[7]

Extract Sample 1

Extract Sample 2

Extract Sample 3

Extract Sample 4

Extract Sample 5

Bulk Avastin®

Bulk Lucentis®

PBS (control)

PEGylated Avastin®

PEGylated Lucentis®

Released Control

Released Avastin®

Released Lucentis®

Released PEGylated Avastin®

Released PEGylated Lucentis®PEGylation

Encapsulation Release

Sampling Schedule

Schematic Model of VEGF Pathway Inhibition

[7] A. Klettner and J. Roider. Comparison of bevacizumab, ranibizumab, and pegaptanib in vitro: Efficiency and possible additional pathways. Investigative

Ophthalmology & Visual Science, 49(10):4523-4527, 2008.

Cytotoxicity of post-polymerization

buffer extracts

Unreacted acrylamide monomers[6] and TEMED[7] are toxic

Removed from hydrogels by extraction through gentle agitation in PBS

5 Extractions, 20 minutes each, buffer 20x hydrogel volume




Degradation



Conclusions

MTS Cytotoxicity of Buffer Extracts

Buffer Extraction Step

1st 2nd 3rd 4th 5th Control

No

rma

lize

d A

bso

rba

nce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Protein Lost in Each Extraction Step

Buffer Extraction Step

1st 2nd 3rd 4th 5th Total

IgG

Pro

tein

Lo

ss (

%)

0

5

10

15

20

25

30

No Glutathione



[6] A. S. Wadajkar, B. Koppolu, M. Rahimi, and K. T. Nguyen. Cytotoxic evaluation of N-isopropylacrylamide monomers and temperature sensitive poly(N-

isopropylacrylamide) nanoparticles. Journal of Nanoparticle Research, 11(6):1375-1382, 2009.

[7] C. G. Williams, A. N. Malik, T. K. Kim, P. N. Manson, and J. H. Elissee. Variable cytocompatibility of six cell lines with photoinitiators used for polymerizing

hydrogels and cell encapsulation. Biomaterials, 26(11):1211-1218, 2005.

Cytotoxicity of Release Samples

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

PBS Avastin Lucentis Blank Avastin Lucentis Avastin Lucentis

Absorb

ance

Stock Solution Released Released +

PEGylated

MTS cytotoxicity of hydrogel degradation products

Samples consisted of degraded PNIPAAm-co-PEG-b-PLLA hydrogels used for encapsulation and release of Avastin® or Lucentis®.

No statistical significance was detected compared to PBS control




Degradation



Conclusions

0

0.02

0.04

0.06

0.08

0.1

0.12

FBS PBS VEGF Avastin Lucentis Blank Avastin Lucentis Avastin Lucentis

Absorb

ance

Bioactivity of Release Samples

Stock Solution Released Released +

PEGylated

BrdU assay results of HUVEC proliferation.

FBS is the positive control and PBS is the negative control. All other samples were cultured in the presence of VEGF.

Thermo-responsive PNIPAAm-co-PEG-b-PLLA hydrogels were used for encapsulation and release of Avastin® or Lucentis®.

Standard deviation bars, *p < 0.001 vs. VEGF, **p < 0.05 vs. VEGF

* *

* *

**




Degradation



Conclusions

Contributions & Conclusion




Degradation



Conclusions

1. Optimized the precursor formulation so that the hydrogels are both injectable and hydrolytically degradable.

2. Established that the cross-linker molar concentration should fall in the range of 1 to 4 mM in order for the thermo-responsive hydrogels to exhibit a sharp coil-to-globule phase transition ca. 33 °C.

3. Reduced undesirable burst release in the initial swelling phase by tethering of biomolecules through PEGylation and subsequent attachment to the polymer chains.

4. Demonstrated that the hydrogel degradation products were nontoxic under in-vitro cell culture conditions.

5. Confirmed that angiogenesis inhibitors released from PNIPAAm-co-PEG-b-PLLA hydrogels were stable and bioactive.

Conclusion: localized and prolonged release (~2 weeks) of active

angiogenesis inhibitor proteins can be achieved using thermo-responsive

hydrogels by tailoring of hydrogel structure, degradability, and controlling

protein-polymer interactions.

Acknowledgements

Advisors

Victor H. Pérez-Luna

Eric M. Brey

Jennifer J. Kang-Mieler

Graduate Students

Yu-Chieh Chiu (cross-linker synthesis) and Bin Jiang (cell culture)

Undergrad Students

Diana Gutierrez and Alexa L. Beaver

Funding

The Lincy Foundation, The Macula Foundation, Veteran’s Administration




Degradation



Conclusions

Documents

Thermo-responsive Hydrogels for Intravitreal Injection and Biomolecule Release