Pardeep K. Gupta
Philadelphia College of Pharmacy
University of the Sciences
Philadelphia, PA 19104
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Recombinant DNA technology opened the doors for the use of proteinbased therapeutics
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Limited success in the development of protein-based drugs is mainly due
to
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Their low plasma circulation times and instability in the GI tract
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Poor Patient compliance due to the need for frequent parenteral administration
Hence a need for developing sustained release drug delivery systems for
protein drugs
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Attractive option for delivering proteins because:
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High surface-to-volume ratio: enables high drug loading
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Possible to deliver drugs inside cells or even the nucleus
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Increased plasma circulation times
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Modulated release possible
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Active targeting is possible
Disadvantages: Made of Polymers; represent hydrophobic surfaces for
Proteins

Protein Adsorption

Spontaneous phenomenon;
occurs when proteins encounter
relatively hydrophobic surfaces

Protein Unfolding occurs Loss
of Protein Structure and Activity

Inert hydrophilic surfaces (e.g.
PEG Coated) prevent Protein
Adsorption
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Modified Surfaces: Reversible
Protein Adsorption
http://what-when-how.com/nanoscience-and-nanotechnology/biosurfaces-water-structure-at-interfaces-part-1-nanotechnology/
Expression System
E.Coli
Physiological Significance
Tissue differentiation , cell proliferation and
protein synthesis in bones, cartilage, liver, immune
system.
Used in treatment for
Short stature (pediatrics), Metabolic deficiencies,
chronic renal failure, sepsis, burns
Reasons for development of drug delivery
systems:
Daily dose injections, especially in pediatrics
Molecular Weight:
22,000 Daltons, non-glycosylated
Primary Structure:
191 amino acid peptide
Secondary Structure:
4 alpha helices; 2 cysteine bonds
Receptors:
GHR and GHBPs; One hGH molecule binds to 2
membrane bound receptors to activate
intracellular signals and biological activity
Known Marketed Products:
Humatrope, Genotropin, Norditropin
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Globular protein; single polypeptide chain of 191 amino acids
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Molecular weight is 22kDa; pI is 5.3;

Two disulfide bonds- C53-C165 and C182-C189
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Almost 55 +/- 5% of the polypeptide backbone exists in the right
handed α-helical conformation

4 α-helical bundles form the architectural core of the protein and
are surrounded by extended side chains and loop structures
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Absorption maxima is at 277nm

Fluorescence properties are mainly due to Indole moiety of
tryptophan and to a small extent due to tyrosine residues

Literature studies show that substrate affinity is a very
important factor in hGH adsorption
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Previous experiments in the lab have shown similar results:

On hydrophobic surfaces hGH adsorption is considerably high, irreversible
and associated with substantial structural change

On hydrophilic surfaces hGH adsorption is relatively lower, maybe
reversible to some extent & is associated with less structural changes

Conditions of the micro-environment are significant
Polystyrene
Nanoparticles
OUR
PROJECT
Recombinant
Human Growth
Hormone
Can a surface be developed which interacts reversibly with r-hGH?
Can a protein-coated surface serve this function
successfully?
Carboxyl-& Amino-functionalized Nanoparticles
5 Covalent Linkers: different
end groups & Length
Albumin-coated
nanoparticles
Lysozyme-coated
Nanoparticles
pH, Ionic Strength
Studying Interactions of r-hGH: With the
protein-coated Nanoparticles
Casein-coated
Nanoparticles
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A protein-coated colloidal surface was prepared & characterized to study its interactions
with rhGH
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Determination of Surface bound proteins was attempted using traditional and modified
BCA colorimetric assay
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Reversible r-hGH adsorption occurred due to the effect of following forces:

Hydrophobic Surfaces

Electrostatic forces
Which of these forces would be the dominant driving force for rhGH adsorption/desorption
was found to be influenced by the following characteristics of the new surface:

Isoelectric point and net charge of surface bound protein
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pH and Ionic Strength of desorption
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Bio-conjugation linker used to tether model protein to surface
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Presence of 1% BSA in the desorption

Amount of rhGH added for adsorption
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GHR: 620 amino acid protein, 246 aa extracellular domain in circulating
form(GHBP); belongs to GH/Prolactin hematopoetic Class I cytokine
receptor family
GHBP-can act as agonist/antagonist depending on levels of GH, GHR
turnover, post-receptor regulation of GHR and plasma GHBP levels.
High affinity GHBP produced by proteolytic cleavage by TACE (receptor
ectodomain shedding); 5-20% circulating GH binds to low affinity GHBPits precise nature is unknown
GH at physiological conditions binds to GHBP in a 1:1 proportion; only at
supraphysiological conditions the stoichiometry changes to 1:2
Deficiency of GHBP causes Laron Dwarfism; increased GHBP levels are
seen in gonadal dysfunction; low levels are seen in Cirrhosis and
diabetes
Easy & Inexpensive
production &
characterization-e.g.
BCA assay works!
Eliminate tedious & expensive
expression & purification
procedures of proteins-Only the
important parts of the protein
chopped off & used
GHBP
peptides/fragments
Natural carriers for
GHBP; especially imp
for GH replacement
therapy where patients
have inherent GHBP
deficiency as well
Peptides are smaller &
hence more robust than
proteins-storage &
handling easier than
proteins
Biodegradable and porous
Polymeric Shell such as PLGA
to achieve sustained and/or
controlled release
G
H
GHBP
fragment
Subcutaneous/IV
injection; ocular,
nasal, transdermal
delivery
PLGA nanoparticle
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GHBP mutants were prepared using phGHbp and expressed in E.Coli
KS330
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The mutants were prepared using alanine-scanning mutagenesis.
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Concentrations were determined using UV absorbance and structure was
determined using CD spectrophotometer
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Binding constants were determined from competitive displacement
assays using I-125-labelled hGH as tracer
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Of residues 1-238 in hGHbp, removal of residues 7-28 does not affect
binding
11 residues contribute significantly to hGH binding affinity (Arg43,
Glu44, Ile103, Trp104,Ile105, Pro106, Asp126, Glu127, Asp164, Ile165
and Trp169).
Many of these play indirect structural roles by supporting and positioning
Trp104 and Trp169 for binding. On the other hand, side-chains in hGH
act in a more independent fashion.
The interface is composed mainly of side-chain interactions participating
in aliphatic-aromatic stacking interactions.
Binding surface on hGHbp is relatively “soft” and the binding
determinants are on loops; in case of hGH they are on the loops and the
surface is relatively “hard”.
The main functional epitope is hydrophobic framed by polar residues to
permit binding of only “correct” partners.
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Four segments in the cysteine-rich domain of the hGHbp that are
important to binding of hGH
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These residues include
Thr101 to Pro106>> Val125 to Asp132> Arg70 to Glu82 ~Glu42 to Glu44

There is electrostatic complementarity between an electropositive binding
epitope on hGH and an electronegative epitope on hGHbp
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Of the 10 charged-to-alanine substitutions in the hGHbp causing
reduction in binding affinity 7 are acidic residues.
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On the other hand on hGH 3 of the 5 disruptive binding sites are basic
residues and none are acidic residues.
Peptide
No.
Peptide Sequence
Part of GHBP Sequence
Naturally
Occurring
1
S-P-E-R-E-T-F-S
YES
NO
2
R-R-N-T-Q-E-W-T-Q-E-W-K-E
YES
NO
3
R-R-N-T-Q-E
YES
NO
4
W-T-Q-E-W-K-E
YES
NO
5
T-S-I-W-I-P
YES
NO
6
V-D-E-I-V-Q-P-D
YES
NO
7
T-S-I-W-I-P-Y-C-I-G-K-C-F-S-V-D-E-IV-Q-P-D*
NO
NO
8
T-S-I-W-I-P-Y-G-S-V-D-E-I-V-Q-P-D
NO
NO
9
K-V-D-K-E-Y-E
YES
NO
10
R-V-R-S-K-Q-R
YES
NO
*-Cysteine residues form a double bond
Graduate students who worked on this project

Snehal Atwe

Rajeshwar Motheram

Lisa Park

Preeti Desai

Ken Yin