The document summarizes research aimed at developing a targeted co-delivery system for methotrexate (MTX) and a p53-derived peptide (p53i) using human serum albumin (HSA). The researchers synthesized fatty acid-conjugated versions of MTX and p53i to allow incorporation into HSA. They also generated recombinant HSA fusion proteins containing p53i or a control peptide. In vitro studies showed the fusion proteins increased p53 expression and promoted apoptosis. Co-delivery of fatty acid-conjugated MTX with the fusion proteins may enhance cytotoxicity compared to single agents and provide a new targeted drug delivery platform.

1. Wuyuan Lu et al. JBC, 398, 200-213 (2010)
The E3 ubiquitin ligase, MDM2, negatively regulates the activity of p53 via two main
mechanisms: (1) reduces transcriptional activity by sequestering the N-terminal
transactivation domain of p53 and (2) ubiquitylates lysines in the C-terminal domain of p53,
thus promoting p53 degradation by the proteosome. The structural basis for the first
mechanism has been well characterized and consists of a minimum binding sequence within
19-26p53 that forms an amphiphilc α-helix containing three critical residues; Phe19, Trp23,
and Leu26. These residues are located within a hydrophobic cavity of MDM2. Previous in
vitro studies by Bernal and colleagues have demonstrated reactivation of the p53 tumor
suppressor cascade following treatment with a small peptide bearing the key interacting
residues. We plan to use a solid phase peptide synthesis method to generate a 14 amino
acid length peptide (p53i) containing the minimum binding sequence necessary for MDM2
interaction.
In recent years, the advent of small peptide therapeutic agents has resulted in the ability
to enhance target specificity and blunt toxicity compared to small molecule drugs. Despite
this, serum instability and rapid renal clearance have plagued their widespread usage.
Lipidation and enhanced plasma protein binding are two strategies capable of extending the
half life of prodrugs. Our plan is to combine these methods by synthesizing p53i conjugated
to FA (FA-p53i); creating a species capable of being incorporated and transported by HSA.
The application of the synthesis described above is to develop an efficient drug carrier
system utilizing HSA for the targeted co-delivery of methotrexate (MTX) and FA-p53i. Such a
system offers the unique ability for one drug formulation to simultaneously deliver two anti-
cancer agents to a single cell and thus, promote a supra-additive effect on apoptosis. MTX is
a well described and widely used chemotherapeutic agent whose mechanism of action relies
on the inhibition of key enzymatic activities required for DNA synthesis. It will thus serve as a
model drug for the development of this platform technology.
Introduction
Reactivating the p53 Pathway Using Human Serum Albumin to
Co-Deliver Methotrexate and a p53-Derived Peptide
Michelle R. Joshi1, Nianhuan Yao1, Zhiyu Li1
1Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of the Sciences, Philadelphia, PA 19104
Caspase-3 Acl I p53i Spe I Nhe I
DEVDG ETF SDLWKLLPETAA TS AS Amino acid sequence
DNA sequence
Caspase-3 PMI ScaI SacII Nhe I
DEVDG TSFAETWALLSP PR AS Amino acid sequence
DNA sequence
Cargo: FA-Methotrexate and FA-p53i
1) Methotrexate
 MTX has been conjugated to:
• Albumin Clin. Cancer Res. 9, 1917-26 (2003).
• Gelatin Pharm. Res. 17, 1309-15 (2000).
• Fibrinogen Cancer Lett. 148, 189-95 (2000).
• Polyethylene glycol (PEG) Bioconjug Chem. 13, 773-85 (2002).
• Tumor-targeting Ab Nat. Biotechnol. 23, 1137-46 (2005).
 Conjugation has been shown to increase plasma retention and
enhance accumulation in tumor tissue
2) p53-Derived Peptide
 Designed to inhibit native p53-MDM2
interaction
3) Design and construct a recombinant HSA C-terminal fusion protein
2
HSA
p53iMTX
alternative strategy
wt p53
high
affinity
peptide
PMI
Methods
Results
Figure 2
1 2 3 4 5 6 7 8
HSA:FA-FITC-p53i (1:4)
unlabeled FA-FITC (pmol) 120 240 480360 960720- -
FA-bound HSA
Free FA
Figure 2: FA-FITC-p53i forms
a stable complex with human
serum albumin that is not
displaced by the presence of
unlabeled FA. HSA/FA-FITC-
p53i complexes were allowed to
form at a 1:4 molar ratio
(HSA:FA-FITC-p53i; 120
pmol:480 pmol) as described in
Methods. Next, samples were
incubated with increasing
concentrations of unlabeled FA. The absence of a lower band indicates FA-FITC-p53i was
not displaced by the unlabeled FA even at the highest concentration. The upper band
corresponds to the less mobile HSA/FA complex, while the lower band contains unbound
free FA-FITC or free FA-FITC-p53i.
Construction of recombinant HSA fusion proteins
• Amplified rHSA-p53i DNA by PCR
• Ligated rHSA-p53i into pPICZα A vector (Invitrogen)
• Transformed competent E.Coli cells using plasmid DNA & select for
zeocin resistance
• Isolated single colonies from overnight culture & grow up in selection
media
• Purified plasmid DNA & confirmed DNA sequence by restriction digest
• Linearized plasmid DNA & transformed Pichia yeast cells
• Expressed protein in Pichia strain, isolated and purified recombinant
protein
Negative control
742 bp
4608 bp
1763 bp
3587 bp
23K bp
9.4K bp
6.6K bp
4.4K bp
2.3K bp
2K bp
KasI/XhoI digest SpeI/XbaI digest
Caspase-3 Acl I p53i Spe I Nhe I
DEVDG ETF SDLWKLLPETAA TS AS Amino acid sequence
DNA sequenceKasI
Background
Co-Delivery
Combination Therapy
Vs.
Both anti-cancer agents reach target
Can use two species with complimentary mechanisms
to promote a robust apoptotic response
Ease of formulation & administration
One agent → multiple effects
Enhanced therapeutic effect at lower doses
Targeted co-delivery reduces side effects due to
toxicity in normal tissues
Benefits of Co-Delivery
Co-Delivery Approach
• Most abundant plasma protein
• Molecular weight = 66.5 kDa
• Long half-life
• Solublizing agent for long chain FA’s
• Binds a number of drugs
• Proven lack of toxicity and immunogenicity1
• Accumulates in malignant and inflamed tissue2
Nat Struct Biol. 5(9), 827-35 (1998).
Carrier: Human Serum Albumin1
Background
N-protected C-terminal amino acid residue was anchored via its
COOH group to the hydroxyl group of Wang resin. Side chain
functional groups of amino acids were masked with permanent
protection groups. The N-terminal amino group was protected by
a temporary moiety that can be removed for coupling to the next
residue. Deprotection/coupling process was repeated until
desired sequence was complete. Peptide was then released
from resin and side chain protecting groups were removed.
Resulting peptide was detected by ESI/MS and purified by HPLC.
Mol. Biotechnology. 33, 242-254 (2006).
Solid phase peptide synthesis
Methods
Synthesis of FA-Modified FITC, FA-Modified p53i,
and FA-Modified MTX
Fmoc-Lys(Alloc) was first coupled to Wang resin. After Fmoc deprotection, palmitic acid was
coupled to the a- amino group of Lys. After Alloc deprotection, Fmoc-SS-linker and NHS-
Fluorescein were coupled sequentially. The final product was cleaved using TFA.
Albumin/Fatty Acid Mobility Shift Assay
For experiments designed to detect albumin/FA-p53i complex formation, 120 pmol albumin
(dissolved in 1X PBS) was incubated +/- FITC-labeled FA-p53i at desired molar ratios.
Experiments aimed at detecting displacement of FA-FITC-p53i by unlabeled FA included an
initial incubation carried out as described above, but at a fixed albumin:FA-FITC-p53i molar
ratio. Following this incubation, unlabeled FA was added at increasing molar ratios up to 1:8
(albumin:unlabeled FA). Reactions were conducted in 20 µl of PBS under room temperature
for 30 minutes. The products were separated using Tris-Boric polyacrylamide gel 12 mA for
20 minutes. The gel was visualized under 305 nm UV.
Cells were plated in standard growth media at approximately 40% confluence and allowed to
attach overnight. On day 2, MTX, FA-MTX, HSA/MTX, HSA/FA-MTX complexes or HSA
fusion proteins were added at the indicated concentrations and allowed to incubate at
37ºC/5%CO2 for the indicated time period. Cell proliferation was then measured by CyQuant
assay (Invitrogen). Data represented three replicas at indicated concentrations.
Cytotoxicity Assays
1 2 3 4 5 6 7 8 9 10
Figure 1: Recombinant
HSA fusion proteins
are able to form
complexes with FA-
FITC. Recombinant HSA
fusion proteins as well
as wild type HSA were
incubated at the
indicated molar ratios
Figure 1
with FA-FITC. The speed at which molecules move through the gel is dependent on size
and charge. The upper band corresponds to the less mobile HSA/FA complex, while the
lower band contains unbound free FA-FITC.
Results
p53
MDM2
GAPDH
untreated
Nutlin
rHSA-p53i
rHSA-PMI
FA-p53i
Figure 5 Figure 5: FA-p53i, rHSA-p53i, and rHSA-PMI
increase p53, but not MDM2 protein
expression. This is in contrast to the actions
of the cis-imidazoline analog, nutlin. SJSA-1
cells were plated in standard growth media and
allowed to attach overnight. On day 2, 10 µM
nutlin, rHSA-p53i, rHSA-PMI or FA-p53i were
added in RPMI media containing 1% FBS +
0.05% DMSO and allowed to incubate x 24 hrs.
Cell monolayers were then washed, lysed and
immunoblotted for p53 and MDM2 (Santa Cruz).
P53 protein expression increased by approximately 60% in rHSA-p53i-treated cells and
30% in both rHSA-PMI and FA-p53i treatments (as determined using Image J software),
while MDM2 expression did not change relative to untreated wells.
Figure 3
Figure 3: Cytotoxicity of
MTX, FA-MTX, HSA/MTX,
and HSA/FA-MTX
complexes. Cell
proliferation was measured
by CyQuant assay
(Invitrogen). Data
represented three replicas
at indicated concentrations.
Experiments were repeated
twice. MTX and FA-MTX showed comparable cytotoxicity in MDA-MB-435 and SKBR-3 cells.
In MCF-7 cell, cytotoxicity of FA-MTX was about 3 times lower than that of MTX. FA
modification on MTX has no obvious effects on in vitro cytotoxicity.
Figure 4
Figure 4: Effect of MTX and FA-MTX on H1299 cells
xenografts. Fifteen mice were randomly split into 3
groups for USP saline, MTX, and FA-MTX treatment.
Cancer cells were injected as described in Methods and
tumor size was measured 2X per week. MTX (25 mg/kg)
and FA-MTX (equal to MTX 4.15 mg/kg) were
administered i.p. once a week for 3 weeks. Based on the
relative tumor volume, FA-MTX with 1/6 dose of MTX
showed comparable or slightly better efficacy.
Results
Figure 6
Figure 6: Recombinant HSA
fusion proteins promote
apoptosis via caspase
activation. SJSA-1 cells were
plated in 96-well plates in
standard growth media and
allowed to attach overnight.
Recombinant HSA fusion
proteins, FA-p53i or nutlin were
added at the indicated
concentrations in RPMI media
containing 1% FBS + 0.05%
DMSO and allowed to incubate x
24 hrs. Cells were then washed
and caspase activation was
quantitated using a fluorimetric
Homogeneous Caspase Assay
(Roche). Phase micrographs of
10 µM-treated wells: (A)
untreated (B) nutlin (C) FA-p53i
(D) rHSA-p53i (E) rHSA-PMI.
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
control
nutlin
10
µM
FA-p53i10
µM
rH
SA-p53i5
µM
rH
SA-p53i10
µM
rH
SA-PM
I5
µM
rH
SA-PM
I10
µM
Treatment Conditions
Foldchangerelativetocontrol
A
ED
CB
Conclusion
FA-modification of MTX and p53i is a valid method to facilitate non-covalent
incorporation into HSA
Recombinant HSA fusion proteins already containing the integrated p53i and PMI
peptides are capable of delivering FA-MTX.
The increase in p53 protein expression by FA-p53i, rHSA-p53i and rHSA-PMI
confirms intracellular uptake and reactivation of p53.
rHSA fusion proteins already containing the integrated p53i and PMI peptides are
capable of promoting apoptosis via caspase activation.
Studies are currently underway to determine if rHSA fusion proteins co-delivered
with FA-MTX can enhance cytotoxicity compared to single agent administration.
Wuyuan Lu et al. JBC, 398: 200-213 (2010)
Recipient of the Joseph B. Schwartz Graduate Student Pharmaceutics Travel Fund Award
AM-12-03777