Thermo-responsive Hydrogels for Sustained Anti-VEGF Delivery

Thermo-responsive Hydrogels for Intravitreal
Injection and Biomolecule Release

Pawel W. Drapala

Ph.D. Thesis
Department of Chemical and Biological Engineering

Advisor: Victor H. Pérez-Luna

Presentation Outline

1. Background and Significance 4. Release of Therapeutic Proteins
 Age-Related Macular Degeneration  Release of Proteins from
(AMD) Nondegradable Hydrogels
 Specific Aims  Higuchi Analysis of Diffusive-
 poly(ethylene glycol) (PEG) hydrogels Controlled Systems
2. Thermo-Responsive Hydrogels  Effect of CTAs on Protein
Release
 poly(N-isopropylacrylamide)
(PNIPAAm),  PEGylated & Tethered IgG
Release
 Transition Temperature & Swelling
5. Biocompatibility of Drug
 Volume Phase Transition Temperature
(VPTT) Delivery System
 Bioactivity & Potential
3. Copolymer Synthesis, Cytotoxicity of Drug Delivery
Characterization and Degradation System
 Degradable Cross-links  Cytotoxicity of Release Samples
 Chain Transfer Agents (CTAs)  Bioactivity of Release Samples
 Selection of Hydrogel Formulations 6. Contributions & Conclusion

Copolymer Synthesis,
Background and Thermo-Responsive Release of Biocompatibility of
Characterization and Conclusions
Significance Hydrogels Therapeutic Proteins Drug Delivery System
Degradation

Age-Related Macular Degeneration (AMD)

 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 Normal Vision Age-related macular
• damage to photoreceptors degeneration

• 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

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.

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

Degradation

poly(ethylene glycol) (PEG) hydrogels
 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:

APS

PEG-DA PEG Hydrogel

Degradation

poly(N-isopropylacrylamide)
(PNIPAAm)
PNIPAAm-co-PEG Hydrogel
NIPAAm

PEG-DA

∆ Temp. ∆ Time
Intravitreal
Injection

Degradation

Transition Temperature & Swelling

Swollen Collapsed
Hydrophilic Hydrophobic State
State (7°C) (37°C)
N o rm a liz e d A b s o rb a n c e

70
�� − ��

S w e llin g R a t io
1.0
60 �� =
��
0.8
50 8 m M P E G -D A
1 2 m M P E G -D A
0.6 1 6 m M P E G -D A
40
0.4 30
C ross-linker:
0 mM
0.2 4 mM 20
8 mM
0.0 12 m M 10
16 m M
0
30 32 34 36 38 40 20 22 24 26 28 30 32 34 36 38 40
T em perature ( o C ) T e m p e ra tu re ( o C )
Degradation

Volume Phase Transition Temperature
(VPTT)
[PEG-DA]* VPTT • The VPTT can be readily manipulated
0 mM 32.4 °C (± 0.3) by Hydrophobic/Hydrophilic
monomer ratios[3]
4 mM 33.4 °C (± 0.1)  PEG elevates VPTT
8 mM 34.7 °C (± 0.4)  Poly(L-lactic acid) (PLLA) decreases the
VPTT
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)
* PEG MW = 575 Da
2 mM 33.2 °C (± 0.6) 32.0 °C (± 0.6) ♯ PEG MW = 3400 Da

3 mM 33.9 °C (± 0.9) 32.9 °C (± 0.7)

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

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

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

PLLA-b-PEG rate of ester Acry-PLLA-b-PEG-b-PLLA-Acry
hydrolysis in-vivo[4,5]:
 hydrophobicity
 steric effects
 cross-linking density
 length of the PLLA oligomer
 autocatalysis
 size/charge of the encapsulated
biomolecules
Lactic Acid PEG
[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

Degradation Profiles
• 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 ��

Room Tem perature (24 o C) Physiological T em perature (37 o C )
120 30
PNIPAAm -co-PEG-b-PLLA PN IPAAm -co-PEG -b-PLLA (degradable)
(degradable) PN IPAAm -co-PEG (nondegradable)
100 25
PNIPAAm -co-PEG

S w e llin g R a tio
S w elling R atio

(nondegradable)
20
80
15
60
10

40
5

20 0
0 5 10 15 20 0 5 10 15 20
Tim e (days) Tim e (days)
Degradation

Chain Transfer Agents (CTAs)

 PNIPAAm cannot exceed 32 kDa and
PEG can not exceed 50 kDa for clearance CTA Reaction
by the renal system[6,7]
 Lower the MW of growing PNIPAAm P ∙ +XR → R ∙ +XP Glutathione
polymer chains using Glutathione CTA:

Growing PNIPAAm Radical Terminated Polymer

CTA-Initiated Growing Polymer 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

Selection of Hydrogel Formulations

Does the polymerization Glutathione Concentration (Chain Transfer Agent)
produce a hydrogel?
0 mg/mL 1 mg/mL 2 mg/mL 3 mg/mL 4 mg/mL
1 mM Yes No No No No
2 mM Yes No No No No
Acry-PLLA-b-PEG-b-PLLA-Acry 3 mM Yes Yes No No No
4 mM Yes Yes Yes No No
Molarity (Cross-linker) 5 mM Yes Yes Yes Yes No
6 mM Yes Yes Yes Yes No
7 mM Yes Yes Yes Yes Yes

Is the produced hydrogel injectable
via 30-gauge needle? Glutathione Concentration (Chain Transfer Agent)

1 mM Yes
2 mM Yes
Acry-PLLA-b-PEG-b-PLLA-Acry 3 mM Yes Yes
4 mM No Yes Yes
Molarity (Cross-linker) 5 mM No No Yes Yes
6 mM No No No Yes
7 mM No No No No Yes

Degradation

Selection of Hydrogel Formulations

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

Glutathione Concentration (Chain Transfer Agent)

1 mM No
2 mM No
Acry-PLLA-b-PEG-b-PLLA-Acry 3 mM No Yes
4 mM No Yes Yes
Molarity (Cross-linker) 5 mM No Yes
6 mM No
7 mM No

∆ Temp. ∆ Time

swollen hydrophilic state collapsed hydrophobic state partially degraded
collapsed state

Degradation

Degradation Profiles
Swelling Ratios (with and without Glutathione CTA) as CTA VPTT
function of incubation time: [mg/mL] Qt=0 [°C]
 Nondegradable hydrogels at 37 °C cross-linked with: 0 20.4 33.3
Acry-PEG-Acry (PEG-DA)
 Degradable hydrogels at 37 °C cross-linked with: 3 mM PEG cross-links 0.5 31.0 34.4
Acry-PLLA-b-PEG-b-PLLA-Acry 1.0 32.5 36.2
0 23.9 32.9
3 mM PLLA-b-PEG-b-
No Glutathione 0.5 34.7 34.1
PLLA cross-links
0.5 mg/mL Glutathione 1.0 37.4 35.0
1.0 mg/mL Glutathione

Nondegradable Hydrogels (control) Degradable Hydrogels
60 60
Swelling Ratio

Swelling Ratio
40 40

20 20

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

Release of Proteins from
Nondegradable Hydrogels
Ig G R e le a s e d o f E n c a p s u la te d (% )

IgG Release from PNIPAAm-co-PEG hydrogels
100
∆ Time
80 Room Temperature (24 °C)

60
24 °C 24 °C
40
Physiological
20 Temperature (37 °C)

0
0 10 20 30 40
T im e (d a ys) ∆ Time
∆ Temp.
 Modes of Mass Transport:
 Diffusion – due to concentration gradients 24 °C 37 °C
 Convection – due to dehydration
 Kinetics – due to hydrolytic degradation
Degradation

Higuchi Analysis of
Diffusive-Controlled Systems
B S A R e le a s e d o f E n c a p s u la te d (% )

BSA Release from PNIPAAm-co-PEG hydrogels 60

100 y = 27.868x + 4.8453
50 R² = 0.9851

80 40

30

Mt/M∞
60

20
40
10
T = 24°C
20
B ody T em perature (37 o C ) 0
0 0.5 1 1.5 2
0 R oom T em perature (23 o C ) tn
70
0 1 2 3 4 5

T im e (days) 60
y = 62.536x - 15.542
�� 50 R² = 0.8923
= �� ∙ ��
�� 40
∞
�� 8 − 2�� + 1 2 �� 2 ��

Mt/M∞
30
=1− ∙ exp ��
��∞ 2�� + 1 2 �� 2 �� 2 20
��=0
1 10
�� 2 T = 37°C
≅4 0
��∞ �� 2 0 0.5 1 1.5
tn

Degradation

Effect of CTAs on Protein Release

Nondegradable Hydrogels No CTA Degradable Hydrogels
cross-linked with cross-linked with
0.5 mg/mL CTA
Acry-PEG-Acry Acry-PLLA-b-PEG-b-PLLA-Acry
1.0 mg/mL CTA
100 100
Released of Encapsulated (%)

80 80

60 60

40 40

20 20

0 0
0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14

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

Degradation

PEGylated & Tethered IgG Release

PEGylation PEGylated
IgG
Immunoglobulin G (IgG) Acry-PEG-SVA

Hydrogel Synthesis (with PEGylated IgG)
PEGylated IgG Release

∆ Temp. ∆ Time

Collapsed Hydrophobic Partially Degraded
Swollen Hydrophilic State
State Collapsed State
Degradation

SDS-PAGE Analysis of IgG PEGylation

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

Degradation

Release (at 37 °C) of IgG with varying
degree of PEGylation

No PEGylation
1:5 IgG to Acry-PEG-SVA Molar Ratio
1:15 IgG to Acry-PEG-SVA Molar Ratio

Nondegradable Hydrogels Degradable Hydrogels
cross-linked with cross-linked with
Acry-PEG-Acry Acry-PLLA-b-PEG-b-PLLA-Acry
100 100

80 80

60 60

40 40

20 20

0 0
0 2 4 6 8 10 0 2 4 6 8 10
Degradation

Bioactivity & Potential Cytotoxicity
of Drug Delivery System

Schematic Model of VEGF Pathway Inhibition
 Avastin® and Lucentis ® inhibit the binding of
VEGF to its receptor VEGFR-2
 Abrogate VEGF-induced neovascularization[7]
Receptor binding domain
Migration
VEGF Binding to VEGFR-2 Neovascularization
Proliferation

Sampling Schedule
PBS (control)
Extract Sample 1 Released Control
Bulk Avastin®
Extract Sample 2 Released Avastin®
Bulk Lucentis®
Encapsulation Release
Extract Sample 3 Released Lucentis®
Extract Sample 4 Released PEGylated Avastin®
PEGylation PEGylated Avastin®
Extract Sample 5 Released PEGylated Lucentis®
PEGylated Lucentis®

[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.
Degradation

Bioactivity & Potential Cytotoxicity
of Drug Delivery System
Avastin®/Lucentis® Schematic Model of VEGF Pathway Inhibition
 Avastin® and Lucentis ® inhibit the binding of
VEGF to its receptor VEGFR-2
 Abrogate VEGF-induced neovascularization[7]
Receptor binding domain
Migration
VEGF Binding to VEGFR-2 Neovascularization
Proliferation

Sampling Schedule
PBS (control)
Extract Sample 1 Released Control
Bulk Avastin®
Extract Sample 2 Released Avastin®
Bulk Lucentis®
Encapsulation Release
Extract Sample 3 Released Lucentis®
Extract Sample 4 Released PEGylated Avastin®
PEGylation PEGylated Avastin®
Extract Sample 5 Released PEGylated Lucentis®
PEGylated Lucentis®

[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.
Degradation

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

MTS Cytotoxicity of Buffer Extracts Protein Lost in Each Extraction Step
1.2 30
No Glutathione
Normalized Absorbance

1.0 25 0.5 mg/mL Glutathione

IgG Protein Loss (%)
1.0 mg/mL Glutathione
0.8 20

0.6 15

0.4 10

0.2 5

0.0 0
1st 2nd 3rd 4th 5th Control 1st 2nd 3rd 4th 5th Total
Buffer Extraction Step Buffer Extraction Step

[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.
Degradation

Cytotoxicity of Release Samples
0.5

0.45

0.4

0.35
Absorbance

0.3

0.25

0.2

0.15

0.1

0.05

0
PBS Avastin Lucentis Blank Avastin Lucentis Avastin Lucentis

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

Bioactivity of Release Samples
0.12

0.1
Absorbance

0.08

0.06

0.04

* * **
0.02 *
*
0
FBS PBS VEGF Avastin Lucentis Blank Avastin Lucentis Avastin Lucentis

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

Contributions & Conclusion

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.

Degradation

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

Thermo-responsive Hydrogels for Sustained Anti-VEGF Delivery

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