1. Delivery of Therapeutic Proteins to The Injured Spinal Cord
Background and Motivation:
In primaryinsultcausesimmediatecelldeath,succeededbythedevelopment
of a toxic environmentthatpreventsregeneration.
SpinalCordInjury
Therapeuticproteinsareusedforenhancingtheregenerationof thespinalcordinjurysites:
(NT-3) is a neuroregenerative molecule, which is responsible for the
maintenance,proliferation,and differentiationofneurons[2].
(ChABC) is a bacterial enzyme that can degrade the Chondroitin Sulphate
Proteoglycans(CSPG)intheglialscar,thusclearsapathforaxongrowth[2].
Neurotrophin-3
Chondroitinase ABC
(a) (b) (c) (d)
(e)
Tail
Head GangliaHead Ganglia
Tail Ganglion Head Ganglia
b a, b a, b a, b, c
Kasra Tajdaran , Irja Elliott Donaghue , Malgosia Pakulska , Molly Shoichet
b c
Department of Chemical Engineering and Applied Chemistry; Institute of Biomaterials and Biomedical Engineering; Department of Chemistry, University of Toronto
The Institute of Biomaterials and Biomedical Engineering
UNIVERSITY OF TORONTO
The goal of this project is to investigate the sustained delivery of NT-3 from poly(lactic-co-
glycolic acid) (PLGA) nanoparticles and the affinity-based release of ChABC both in vitro and in
vivo from a hydrogel drug delivery system.
NT-3 release from PLGA nanoparticles :
Objectives:
ŸOptimise encapsulation efficiency of PLGA nanoparticles
ŸAnalyse NT-3 release in vitro
ŸAnalyse NT-3 release in vivo
ŸAnalyse ChABC release in vitro
ŸAdapt %5 MC gel for in vivo delivery
hydrophobic junctions
methyl cellulose
SH3-binding peptide
SH3-ChABCNT-3 loaded nanoparticleshydrophobic junctions
methyl cellulose hyaluronan
Primary
emulsion:
Sonicating PLGA in
dichloromethane
(DCM)
Secondary
emulsion:
Sonicating
emulsion + polyvinyl
alcohol (PVA) in H2O
primary
Solvent
Evaporation:
secondary emulsion
+ PVA in H2O
Double Emulsion Solvent Evaporation Technique
NanoparticlesPreparation
To study the influence of double emulsion solvent evaporation parameters variation on nanoparticle
encapsulation efficiency, Bovine Serum Albumin (BSA) was used as a model protein for NT-3. The
encapsulationefficiencywasdeterminedusingamicroBCAassay.
Table.1. Effects of double
emulsion technique
parameters variation on
encapsulationefficiency.
0
5
10
15
20
25
30
35
0 500 1000 1500 2000 2500 3000 3500
C(ng/mL)
Depth (µm)
45min
3h
6h
Figure.3. Release of NT-3 from the HAMC
combined with PLGA nanoparticles is analysed by
an Enzyme-Linked Immunosorbent Assay (ELISA).
Data shows that by increasing the sonication time
during nanoparticles preparation, a more
sustainedreleasecanbeobtained.
Optimizing nanoparticle encapsulation efficiency:
Analysing in vitro release of
NT-3 from PLGA nanoparticles:
Analysing release of NT-3
in spinal cord tissue:
Figure.4. NT-3 loaded HAMC is injected
intrathecally to the rat spinal cord. The spinal
cord tissues are analysed by performing ELISA,
in order to find NT-3 concentration in the spinal
cord.
=QQ
Gel weight over time
Original Gel Weight
UROP Summer
Research Award
Figure.5. A. Modified MC with SH3-binding peptide. B. Cumulative release profile for
MC and modified MC. Transient interactions between the binding pairs slows the
diffusionofSH3-ChABCfromthematrix,resultinginsustainedreleaseofSH3-ChABC.
Swelling Ratio (Q):
In order to prevent over-pressurizing spinal cord caused by swelling of the injectable
hydrogel, maximum swelling ratio (Qmax) needs to be 2.2. Based on previous studies
aQmaxof2.2isconsideredsafeinvivo[4].%5MCgelhasaQmaxequalto1.12,which
isinthesafezoneforinvivoinjection.
Gelation Time:
Figure.6. Effect of NaCl Concentration on %5
MC gelation time. Gelation time is adjusted
by changing the salt (NaCl) concentration of
the %5 MC gel. Similar gelation time as 1.4:3
HAMC [4] is obtained by 350mM NaCl
concentration. Gelation time is determined
usingTimeSweepRheology.
Adjusting %5 MC gel properties for in vivo release:
ŸNanoparticle encapsulation efficiency was increased to 95.6% by varying double
emulsion technique parameters. As the next step, NT-3 encapsulation efficiency will be
determinedbasedonthenewparameters.
ŸNT-3 was released over 21 day from PLGA nanoparticles in HAMC. Effect of sonication
periodonnanopaticlescharacteristicswillbedetermined.
ŸNT-3 can penetrate from HAMC into spinal cord tissue. NT-3 penetration from PLGA
nanoparticleswillbeanalysed.
ŸModification of MC with SH3-binding domains results in a more sustained release of
ChABC–Sh3.
Ÿ%5 MC gel with 350 mM NaCl concentration has desired in vivo functional properties.
ReleaseofChABCwillbeanalysedinvivo.
Conclusions and Future Work:
[1] Kang, Catherine E., Charles H. Tator, and Molly S. Shoichet. "Poly(ethylene glycol) modification
enhances penetration of fibroblast growth factor 2 to injured spinal cord tissue from an intrathecal
delivery system.." Journal of Control Release 114 (2010): 25-31. Print.
[2] R.C. Robinson, Radziejewski, C., Spragon, G., and Jones, E.Y., The structures of the
neurotrophin 4 homodimer and the brain-derived neurotrophic factor/neurotrophin 4 heterodimer
reveal a commonTrk-binding site, Protein Sci., 8 (1999) 2589-2597.
[3] Vulic, Katarina, and Molly S. Shoichet. "Tunable Growth Factor Delivery from
Injectable Hydrogels forTissue Engineering." JACS 134 (2011): 882-885. Print.
[4] Baumann, M. Douglas, Catherine E. Kang, Jason C. Stanwick, Yuanfei
Wang, and Molly S. Shoichet. "An injectable drug delivery platform for sustained
combination therapy." Journal of Controlled Release 138 (2009): 205-213. Print.
Figure.1.Significant degradation
occurs after compression injury.
Preventing this degradation and
regenerating the initial injury site
are key to effective treatment.
Image copyright(2005) by
MichaelCorrin.
Figure.2. An intrathecal drug delivery system. The
composite hydrogel is injected intrathecally at the
site of injury and remains localized between the
arachnoid and pia mater, releasing the drug load
into the spinal cord. Image copyright (2005) by
MichaelCorrin.
Figure.3. HAMC hydrogel combined with NT-3
loadedPLGAnanoparticles.
Batch
Number
Varied Parameters Encapsulation Efficiency ± SD (%)
1 73.7 ± 1.7
2 Reducing batch volume to ½ 70.0 ± 4.2
3 Reducing batch volume to ¼ 13.6 ± 2.2
4 Reducing batch volume to ¼ and decreasing
sonication time by 2 minutes
8.7 ± 0.8
5 Reducing batch volume to ¼ and doubling DCM
volume
95.6 ± 1.9
Standard Batch
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8
Cumulativepercentreleased(%)
Time (d)
Cumulative Release Profile for MC and Modified MC
MC
Modified MC with
SH3-Binding Peptide
[NaCL] (mM)
Effect of NaCl Concentraion on %5 MC Gelation Time
0
2
4
6
8
10
12
14
16
200 300 350 400
GelationTime(min)
%5 MC Gelation Time
1.4:3 HAMC Gelation Time
A B
Project Overview:
Affinity based ChABC release:
Drug Delivery System to Spinal Cord:
Current methods of drug delivery to the spinal cord, such as bolus intrathecal injection and
catheter minipumps are inefficient and prone to infection. The Shoichet lab has developed an
injectable drug delivery system using a blend of hyaluronan (HA) and methylcellulose (MC)
(HAMC) to achieve a localized, minimally invasive therapeutic release.
0
1
2
3
4
5
6
7
8
9
10
0 5 10 15 20 25 30
NT-3CumulativeMassReleased(ng/mg)
Time (d)
10min sonication
2 min sonication
*