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Project 1: Elastin-like polypeptide stabilizes recombinant cyclophilin into a nanoparticle; Project 2: Encapsulation of Rhesus theta defensin-1 (RTD-1) in PEG-PLGA nanoparticles for aerosol deliv...

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Project 1: Elastin-like polypeptide stabilizes recombinant cyclophilin into a nanoparticle; Project 2: Encapsulation of Rhesus theta defensin-1 (RTD-1) in PEG-PLGA nanoparticles for aerosol deliv...

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Content 1
Project 1: Elastin-like polypeptide stabilizes recombinant cyclophilin
into a nanoparticle.

Project 2: Encapsulation of Rhesus theta defensin-1 (RTD-1) in PEG-
PLGA nanoparticles for aerosol delivery in CF

Project 3: A Biomimetic Solution to the Generation of Hemoglobin-
based Oxygen Carriers

By
Hao Guo

A Thesis Presented to the FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree, MASTER OF SCIENCE (Pharmaceutical Sciences)
August 2016

2
Acknowledgement

To Dr. Andrew Mackay for guiding me through these scientific investigations.

To Dr. Paul Beringer for enlightening my interests in this research area.

To Dr. Sarah Hamm-Alvarez for supporting me in autoimmune disease-related
exploration.

Great help with these projects has been received from Tim Bensman, Santosh
Peddi, Jugal Dhandhukia, Mihir Shah, Zhe Li.

3
Table of Contents
ACKNOWLEDGEMENT .................................................................................................................. 2
PROJECT 1: ELASTIN-LIKE POLYPEPTIDE STABILIZES RECOMBINANT CYCLOPHILIN
INTO A NANOPARTICLE. ............................................................................................................... 4
ABSTRACT ............................................................................................................................................... 4
INTRODUCTION ....................................................................................................................................... 4
RESULTS .................................................................................................................................................. 6
DISCUSSION ........................................................................................................................................... 11
EXPERIMENTAL PROCEDURES ............................................................................................................. 11
Recombinant plasmid construction .................................................................................................. 11
Drug encapsulation .......................................................................................................................... 12
HPLC method of CsA ....................................................................................................................... 13
Cell uptake study ............................................................................................................................... 13
FIGURES AND TABLES ........................................................................................................................... 14
REFERENCE ........................................................................................................................................... 22
PROJECT 2: ENCAPSULATION OF RHESUS THETA DEFENSIN-1 (RTD-1) IN PEG-PLGA
NANOPARTICLES FOR AEROSOL DELIVERY IN CF .............................................................. 24
ABSTRACT ............................................................................................................................................. 24
INTRODUCTION ..................................................................................................................................... 24
RESULTS ................................................................................................................................................ 29
DISCUSSION ........................................................................................................................................... 30
EXPERIMENTAL PROCEDURES ............................................................................................................. 31
Preparation of RTD-1 encapsulated microparticles ........................................................................ 31
Encapsulation efficiency dertermination ......................................................................................... 32
FIGURES ................................................................................................................................................ 33
REFERENCE ........................................................................................................................................... 36
PROJECT 3: A BIOMIMETIC SOLUTION TO THE GENERATION OF HEMOGLOBIN-
BASED OXYGEN CARRIERS ........................................................................................................ 39
ABSTRACT ............................................................................................................................................. 39
INTRODUCTION ..................................................................................................................................... 39
Significance of blood substitute ....................................................................................................... 39
Information about hemoglobin ........................................................................................................ 40
Challenges ......................................................................................................................................... 41
Hemogobin-ELP-based blood substitute .......................................................................................... 42
RESULTS ................................................................................................................................................ 44
DISCUSSION ........................................................................................................................................... 46
EXPERIMENTAL PROCEDURES ............................................................................................................. 46
Biosynthesis and purification of A192 ............................................................................................. 46
Chemical Conjugation method ......................................................................................................... 47
FIGURES ................................................................................................................................................ 48
REFERENCE ........................................................................................................................................... 53

4
Project 1: Elastin-like polypeptide stabilizes recombinant
cyclophilin into a nanoparticle.

Abstract
As a potent macrolide immunosuppressant, cyclosporin A (CsA) is used to treat
multiple autoimmune diseases, including autoimmune-mediated dry eye disease,
rheumatoid arthritis and psoriasis. Despite its potency, CsA has poor solubility,
poor bioavailability, and induces serious adverse drug reactions, including
nephrotoxicity and neurotoxicity. To overcome these limitations, CsA has been
formulated for systemic and topical delivery using advanced drug carriers
including emulsions and liposomes. Here we explore a new strategy to carry CsA
that utilizes its human target, a protein called cyclophilin, to which CsA binds with
a Kd of 36.8 nM. Due to its low MW (1.2 kD), cyclophilin is below the renal
filtration cutoff (<40 kD) and would be rapidly filtered from the blood by the
kidneys. To overcome this limitation, we used recombinant protein-engineering to
increase the molecular weight of cyclophilin through recombinant fusion with a 73
kD elastin-like polypeptide (ELP). Surprisingly, this fusion protein (CA192)
promoted assembly of nanoparticles with stability over 2 days. Most importantly,
these fusion proteins efficiently solubilize CsA, which they released with a half-
life of 128.3 h and 172.2 h under dialysis and in incubation with an excess
concentration of albumin. Our findings suggest that ELP fusions have the unique
property of stabilizing fusion proteins, which may have utility as drug carriers.

Introduction
Cyclosporin A (CsA) is a well-known lipophilic cyclic immunosuppressant peptide
containing 11 amino acids and works by blocking T-cell proliferation and
inhibiting the release of inflammatory cytokines, such as IL-2 and IFN-γ
5
(Stevenson et al., 2012). It has been widely used in prevention of rejection after
organ transplantation and in modulation of inflammatory responses in several
autoimmune disorders, such as rheumatoid arthritis and psoriasis (Colombo and
Ammirati, 2011). The immunosuppressant effect of cyclosporin is to inhibit the
activity of T cells by preventing the dephosphorylation of NFAT, which is nuclear
factor of activated T cells, by binding to cyclophilin. This complex can bind and
inhibit the enzymatic region of calcineurin, a calcium and calmodulin dependent
phosphatase, which, under normal circumstances, is responsible for activating the
transcription of IL-2. Under normal circumstances, once the antigen is presented to
the T cell surface, a signaling cascade that activates calcineurin can be initiated.
Activated calcineurin can dephosphorylate NFAT. Dephosphorylated NFTA is
then translocated into the nucleus. The gene expression of IL-2 can be then
activated.

When administrated topically, CsA has been broadly used to treat dry eye
syndrome (DES), a multifactorial disease of the ocular surface caused by decreased
tear production which affects an estimated 5-30% of the population(Cornec et al.,
2015; Janine, 2007), by suppressing ocular surface inflammation. Because of its
hydrophobic property, the only commercially available topical administration form
of CsA is as an oil-in-water emulsion eye drop, which leads to poor ocular
tolerance, low bioavailability, and instability (Gupta and Chauhan, 2011). In
addition, when administrated through intravenous injection, CsA can potentially
lead to a number of serious adverse drug reactions (ADRs) because of its narrow
therapeutic window (Mahalati et al., 2001). Below the therapeutic window, CsA
cannot effectively inhibit T cell proliferation and the release of related cytokines,
its major therapeutic actions, while above the therapeutic window, it is known to
6
elicit severe side effects including nephrotoxicity and neurotoxicity (Survase et al.,
2011).

Derived from human tropoelastin, Elastin-like polypeptides (ELPs) consist of
pentameric repeats of (Val-Pro-Gly-Xaa-Gly)n where Xaa is the guest residue and
n is the length of the repetitive units. ELPs have a unique inverse transition
behavior. Below their transition temperature (Tt), they are highly water soluble but
once the temperature rises above their Tt, ELPs undergo a phase separation process
and self-assemble into different kinds of coascervates including different size
particles (Dhandhukia et al., 2013). This phase separation is a fully reversible
process and can be used to effectively purify ELP-conjugated materials (Shah et
al., 2013). Phase behavior can be precisely controlled by adjusting the
hydrophobicity of guess residue “Xaa” and number of pentapeptide repeats “n”
(Urry, 1997). Since ELPs are biodegradable (Shah et al., 2012) and non-
immunogenic, they have the potential to be excellent drug carriers(Shah et al.,
2013; Shi et al., 2013)

Results
Through molecular cloning, we successfully fused the cytosolic sequence of the
human receptor of CsA, cyclophilin A (CypA), to a particular ELP, A192, which
has the amino acid sequence of G(VPGAG)192Y (Figure 1A, Table 1). The CypA-
A192 (CA192) fusion protein is designed to help in solubilizing the poorly soluble
CsA and to function as a drug carrier to improve the CsA safety profile when
administered systemically. Unlike free CsA, as the molecular weight of CA192
well exceeds the renal filtration cutoff, fusion-bound drug should have
significantly reduced renal clearance. The results reported herein describe the
7
physical properties of the resulting carrier as well as the release characteristics of
CsA bound to CA192.

The molecular weight of purified fusion protein was verified by SDS-PAGE
stained with copper chloride (CuCl
2
). The parent ELP, A192, served as a control.
CypA has a molecular weight of 18kDa. Combined with the 73.6kDa molecular
weight of A192, the molecular weight of CA192 should be around 91.6kDa. This
shift for the CA192 is seen on SDS-PAGE (Figure 1B).

The thermal responsiveness property of CA192, along with the parent A192, was
characterized using UV-Vis by measuring their optical density of these constructs
at 350nm, where neither fusion protein nor plain A192 contributes significant
absorption. Both ELPs at different concentrations (5 µM to 100 µM) were subject
to a precisely controlled temperature increase from 25 to 75
°
C at a rate of 1
°
C/min.
The optical density profile representing the ELP phase separation behavior is
shown in Figure 2A. The Tt of ELPs is defined as the temperature at which the first
derivative of the optical density with respect to the temperature reaches a
maximum. At 25µM concentration, the Tt of CA192 was found to be 45.4
°
C.
Consistent with our previous finding (Wang et al., 2015), the Tt of CA192 was also
found to be a function of concentration: Tt = b–mLog
10
[C
ELP
], where the intercept
“b” is equal to 47.1, the slope “m” equals to 1.3 and [C
ELP
] represents the fusion
protein concentration (Figure 2B, Table 1). Finally, CA192 exhibited a two-phase
transition, distinct from A192. These findings suggest that the CypA moiety was
interacting to modulate the properties of A192 to change the properties of this
nanoparticle.

8
CsA was encapsulated in the fusion protein based on a previously reported two-
phase solvent evaporation method (Shi et al., 2013). To determine the
encapsulation efficiency and characterize the release profile, we established a
reversed-phase high-performance liquid chromatography (RP-HPLC) analysis
method to measure the CsA concentration in CA192. The CsA retention time was
found to be 2.5 min and the CsA encapsulation efficiency was then determined to
be 48.6±4.0% (p<0.05).

To evaluate the characteristics of CsA released from CA192 in vitro, its release
profile from the fusion protein was characterized by performing dialysis against
PBS at 4°C. Samples were collected from the dialysis cassette at different time
points between 0 and 192 h and analyzed by RP-HPLC. The release profile fits a
one-phase decay model with a half-life of 128.3 h (Figure 3). To further validate
the release profile, as a control group, with the same encapsulation method, human
albumin was explored to load CsA. The entrapment efficiency was determined to
be 4.8%, significantly lower than CA192 (Figure 4A). In order to mimic the
physiological situation where fusion-loaded drug could be displaced by albumin,
we dissolved human albumin into a PBS solution of CsA-loaded CA192 and
adjusted the albumin concentration to 1 mM, the physiological concentration of
albumin. The mixture was incubated at 37°C and samples were collected at
different time points up to 48 h. Another round of ITC was performed to purify
CA192 from the mixture, followed by RP-HPLC analysis. No significant drug loss
from fusion carrier to albumin was observed within 2 days (Figure 4B). Albumin
cannot deplete CsA from CA192 carrier effectively within a 48 h period,
suggesting the high binding affinity between CsA and CA192 is likely to be
maintained when administrated systemically.

9
The hydrodynamic radius (R
h
) of both the loaded and unloaded fusion protein,
along with plain A192, was measured via dynamic light scattering (DLS) using a
DynaPro Plate Reader II from Wyatt Technology. Before the DLS measurement,
solutions were filtered through 0.2 µM pore size filters. The concentration of each
solution was then adjusted to 20 µM. 60 µL from each sample was pipetted into
three different wells on the plate reader and covered by 15 µL mineral oil each to
avoid solvent evaporation. Centrifugation was then performed to remove air
bubbles. DLS intensity revealed that unloaded CA192 has a mean R
h
of around
64.8±0.7 nm (p<0.05), significantly outsizing that of the plain A192 of 7.3±0.6 nm
(p<0.05) (Figure 5A). Based on this DLS method, we performed a stability assay
to explore the stability of our fusion protein by measuring the mean R
h
shift during
incubation at physiological temperature. Briefly, the fusion solution was incubated
at 37°C for different periods of time up to 48 h and the mean R
h
of CA192 at these
different time points was measured with DLS. This assay demonstrated that the
mean R
h
of CA192 is well maintained throughout 48 h, suggesting good stability
and stable aggregation status at physiological temperature (Figure 5B). There
might be some slight aggregation occurring in the CA192 protein solution.
Through contributing most of the intensity, this aggregated form could lead to the
significantly bigger mean size of CA192 than plain A192. To determine if this was
the case, further separation was conducted with size exclusion chromatography
(SEC). This process was performed at 4°C using a Hiload 26/600 column (26×600
mm, particle size 24-44 µM). Elution was achieved with an isocratic flow rate of
2.6 mL/min and the detection wavelength of 214 nm. Fragments with absorption
above 10 mAu were collected and concentrated with a Spin-X concentrator (pore
size 10 kDa). Two fractions were observed after SEC separation (Figure 5C). Both
species were reanalyzed through DLS, demonstrating that 99.2% by mass of
fraction 2 exhibited a R
h
of 6.5±0.1 nm (p<0.05) and seemed fairly monodisperse.
10
Fraction 1, on the other hand, exhibited significantly higher heterogeneity. (Figure
5D) Through measuring the CA192 concentration of both fractions, we determined
that 20.8% by mass in solution is represented by the multimerized particles. Multi-
Angle static Light Scattering (MALS) was then applied to evaluate the aggregation
and oligomeric state of CA192. The molecular weight of fraction 2 was determined
to be 181.0±5.6 kDa (p<0.05), suggesting that a dimerization process was
occurring in the solution. Thus, we concluded that the CA192 fusion protein is
mostly maintained as dimerized nanoparticles with a 6.5 nm hydrodynamic radius.

In order to further validate the potential efficacy of CA192 working as a potent
drug carrier, we then performed in vitro cell uptake study. As shown in figure 6,
both CA192 and A192 shown obvious co-localization with lysosome, suggesting
efficient cell uptake when administrated.

In summary, using molecular cloning, we successfully constructed an ELP-based
CsA carrier, CA192. It maintains both the phase transition property characteristic
of ELPs and the CsA binding affinity of CypA. In comparison with its parent ELP,
A192, the constructed CA192 has a lower transition temperature that, however, is
still well above physiological temperature. CsA encapsulation efficiency of CA192
was determined to be 48.6±4.0%, significantly higher than that of albumin. The
half-life of CsA drug release from CA192 was determined to be 128.3 h. DLS-
based stability assay demonstrated that throughout 48 h at 37°C, no significant
hydrodynamic radius shift was occurring in the solution, implying good stability of
the construct. In addition, like A192, CA192 co-localized with lysosomes in HeLa
cells, which implies that both constructs are endocytosed.

11
Discussion
Depending on SEC result, we already know that there are two different aggregation
status of CA192 in solution. In terms of drug encapsulation capacity, these two
fractions were found to be very different. Our data suggested that only dimerized
CA192 in fraction 2 had the ability to bind CsA.

Since the Tt of CA192 is well above the physiological temperature, they will
remain soluble while circulating in human body after IV injection. But in order to
achieve most long-duration effect after SC administration, we need to investigate
other CypA-ELP that has a Tt below physiological temperature. Ideally, this fusion
will be able to form a depot in the body and provide long-duration effect.

Experimental Procedures
Recombinant plasmid construction
We designed the encoding sequence of CypA using Escherichia coli biased
codons. As indicated as follows, the custom encoding sequence flanked by
restriction recognition sites of NdeI and BamHI at the 5’ and 3’ ends was ordered
from Integrated DNA Technologies (IDT) as follows:

5’-
CATATGGTTAACCCGACCGTTTTCTTCGACATCGCTGTTGACGGTGAAC
CGCTGGGTCGTGTTTCTTTCGAACTGTTCGCTGACAAAGTTCCGAAAAC
CGCTGAAAACTTCCGTGCTCTGTCTACCGGTGAAAAAGGTTTCGGTTAC
AAAGGTTCTTGCTTCCACCGTATCATCCCGGGTTTCATGTGCCAGGGTG
GTGACTTCACCCGTCACAACGGTACCGGTGGTAAATCTATCTACGGTGA
AAAATTCGAAGACGAAAACTTCATCCTGAAACACACCGGTCCGGGTAT
12
CCTGTCTATGGCTAACGCTGGTCCGAACACCAACGGTTCTCAGTTCTTC
ATCTGCACCGCTAAAACCGAATGGCTGGACGGTAAACACGTTGTTTTCG
GTAAAGTTAAAGAAGGTATGAACATCGTTGAAGCTATGGAACGTTTCG
GTTCTCGTAACGGTAAAACCTCTAAAAAAATCACCATCGCTGACTGCG
GTCAGCTGGAAGGTTACTGATCTCCTCGGATCC-3’

The NdeI and BamHI restriction sites enabled the insertion of the sequence into the
pET-25b(+) vector. Besides these two sites, BseRI restriction site was placed right
ahead of the BamHI restriction site, enabling the ligation of the A192 encoding
sequence, which was synthesized by recursive directional ligation in a modified
pET-25b(+) vector (Janib et al., 2014b). After verifying the correct sequence
through DNA sequencing, the resulted plasmid with the fusion protein sequence
was first amplified in TOP10 competent cells and then transfected into BLR
competent cells for expression. After expression, centrifugation and lysis, the
CA192 fusion protein was further purified by inverse transition cycling (ITC) (Sun
et al., 2011) using the unique thermal responsiveness of ELPs.

Drug encapsulation
n aqueous phase phosphate-buffered saline (PBS) containing 300 µM CA192 was
mixed with an organic phase 90% hexane/10% EtOH containing 900 µM CsA.
Under a nitrogen environment and constant stirring, along with the evaporation of
organic solvent, CsA was gradually released into the aqueous phase and
encapsulated by the fusion protein. This was followed by high-speed centrifugation
and filtration to remove the excess insoluble drug.

13
HPLC method of CsA
Since the detection wavelength of CsA is 210 nm, a wavelength at which most
organic solvents contribute significant absorption, an isocratic elution mode was
utilized to diminish the impact of solvents on UV absorption. Briefly, CsA
separation and detection was achieved on a C4 reverse-phase HPLC column
(150×4.6 mm, particle size 5 µm) using an isocratic mobile phase composed of
95% MeOH and 5% H
2
O at a flow rate of 1 ml/min at room temperature.

Cell uptake study
In order to further validate the potential efficacy of CA192 working as a potent
drug carrier, we then performed in vitro cell uptake study. Briefly, we labeled
CA192 and A192 with rhodamine and HeLa cell lysosome with LysoTracker
Green (LTG). For the treatment group, HeLa cells were incubated with 30 µM of
rhodamine-labeled (rh)-CA192 and 70 nM of LTG for 120 min. For the control
group, HeLa cells were incubated with 30 µM of rhodamine-labeled (rh)-A192 and
70 nM of LTG for 120 min. Cells were then rinsed with warm HBSS three times,
maintained in the fresh culture medium, and imaged by confocal microscopy.
Images were acquired using a Zeiss laser scanning microscope 510 Meta NLO
confocal imaging system equipped with Argon, red HeNe, green HeNe laser, and a
Coherent Chameleon Ti-Sapphire laser (LSM) mounted on a vibration-free table
(Carl Zeiss, Thornwood, NY). All images were acquired using a Plan-Apochromat
63x Oil immersion lens with a working distance of 0.19 mm.

14
Figures and tables

Figure 1 Construction of a cyclophilin-ELP fusion protein. A) Cartoon depicting
an ELP fusion protein, CA192 bound to CsA B) SDS-PAGE of CA192 stained
with copper chloride demonstrating the molecular weight shift after fusing CypA
to A192.

15

Figure 2 Phase transition property of CA192 and plain A192. A) The optical
density profile representing the phase separation behavior of both CA192 and plain
A192; B) The transition temperature of ELPs was found to be a function of
concentration: Tt = b–mLog10[CELP]. 95% confidence intervals were also shown
with dash lines. The values of slope and intercept are shown in table 1.

16

Figure 3 The release profile of CsA from CA192. The drug release follows a one-
phase decay model with a half-life of 128.3 h. The best fit release profile is shown
with a solid line. The 95% confidence interval of half-life ranges from 98.2 h to
176.9 h. 95% confidence intervals are shown in dashed lines.

17

Figure 4 CA192 shows much higher CsA binding affinity than albumin. A) In
comparison to CA192, human albumin demonstrates much lower encapsulation
efficiency of CsA. CA192 has a mean entrapment efficiency of 48.6±4.0%
(p<0.05). Albumin has a mean entrapment efficiency of 4.8±0.1% (p<0.05). Paired
t test, p<0.001. B) At physiological concentration, human albumin cannot deprive
CsA from the fusion effectively within 48h. The half-life was found to be 172.2 h,
comparable to the release half-life under dialysis.
18

Figure 5 DLS intensity data reveals that CA192 has a significantly higher
hydrodynamic radius than A192. CsA loading doesn’t change the mean R
h
of the
fusion. However, SEC-DLS revealed the majority by mass of CA192 has a R
h
of
6.5 nm. A) DLS intensity data reveals that unloaded CA192 has a mean R
h
of
64.8±0.7 nm (p<0.05), significantly outsizing plain A192 with a R
h
7.3±0.4 nm
(p<0.05). Loaded CA192 has a mean R
h
of 64.5±0.5 nm (p<0.05). B) The stability
assay revealed that the R
h
of CA192 is well maintained throughout 48h, suggesting
19
good stability of this constructed fusion protein and stable aggregation status at
physiological temperature. C) SEC was conducted to separate free CA192
nanoparticles depending on their particle sizes. Two fractions were observed. D)
99.2% by mass of fraction 2 was found to have a R
h
of 6.5±0.1 nm (p<0.05).

20

Figure 6 Both A192 and CA192 are co-localizing with lysosome in HeLa cells.
Lysosome was labeled by LysoTracker Green and ELP constructs were labeled
with rhodamine. Imaged by confocal microscopy.

21
Table 1. Protein-polymers evaluated in this study
Label Amino Acid Sequence
Exp. M.W.
[kDa]
a
Slope, m
[°C/Log 10(μM)]
b
Intercept, b
[°C]
c
Purity
[%]
A192 G(VPGAG) 192Y 73.6 8.3±0.28 72.3±0.41 98.1
CA192
MVNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENF
RALSTGEKGFGYKGSCFHRIIPGFMCQGGDFTRHN
GTGGKSIYGEKFEDENFILKHTGPGILSMANAGPNT
NGSQFFICTAKTEWLDGKHVVFGKVKEGMNIVEAM
ERFGSRNGKTSKKITIADCGQLEG(VPGAG) 192Y
91.6 1.3±0.27 47.1±0.39 98.8
a b
The ELP phase diagram as a function of temperature, T
t
, and concentration, C
ELP
, was fit to the following
relationship: T
t
= b – m Log
10
[C
ELP
], where b is the intercept at 1 µM and m are °C change for a 10-fold change in
concentration. mean ± 95% CI.

c
Polypeptide purity was assessed using SDS-PAGE and subsequent densitometry of copper chloride stained gel.

22
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24
Project 2: Encapsulation of Rhesus theta defensin-1 (RTD-1) in
PEG-PLGA nanoparticles for aerosol delivery in CF

Abstract
RTD-1 has been shown to have the promising therapeutic effect against cystic
fibrosis (CF) induced lung infection. Recent progress has proved its activity
against CF strains of Pseudomonas aeruginosa both in vitro and in vivo. But one
main challenge with aerosol delivery to the lungs is the relatively short retention
time due to rapid mucociliary and macrophage clearance mechanisms. Since recent
advances in drug delivery using nanoparticle technologies have indicated that
appropriate drug delivery vehicles can achieve sustained release to the airways,
here we are trying to encapsulate highly hydrophilic RTD-1 into PEG-PLGA-
based microparticles to achieve extended retention time in the lung, which might
imply better therapeutic effect against lung symptoms of CF.

Introduction
Cystic fibrosis (CF) is a genetic disorder that affects mostly the lungs, which is
characterized by a chronic cycle of airway obstruction, infection, and
inflammation, leading to remodeling, loss of pulmonary function, and eventual
respiratory failure, but also the pancreas, liver, kidney and intestine. In terms of
etiology, inherited in an autosomal recessive manner, CF is caused by the presence
of mutations in both copies of the gene for the cystic fibrosis transmembrane
conductance regulator (CFTR) protein. CFTR is involved in production of sweat,
digestive fluids, and mucus. When CFTR is not functional, secretions which are
usually thin can become thick instead.

25
Generally, the main signs and symptoms of cystic fibrosis are salty-tasting skin
(Quinton, 2007), poor growth, and poor weight gain despite normal food intake
(Hardin, 2004), accumulation of thick, sticky mucus (De Lisle, 2009), frequent
chest infections, and coughing or shortness of breath (O’Malley, 2009). Males can
be infertile due to congenital absence of the vas deferens (Makker et al., 2009).
Symptoms often appear in infancy and childhood, such as bowel obstruction due to
meconium ileus in newborn babies (Blackman et al., 2006). As the children grow,
they exercise to release mucus in the alveoli (Ratjen, 2009). Ciliated epithelial cells
in the person have a mutated protein that leads to abnormally viscous mucus
production (De Lisle, 2009). The causes of growth failure are multifactorial and
include chronic lung infection, poor absorption of nutrients through the
gastrointestinal tract, and increased metabolic demand due to chronic illness
(Hardin, 2004).

Symptoms of CF can vary in different organs. We have been focusing on the lung
disease, which results from clogging of the airways due to mucus build-up,
decreased mucociliary clearance, and resulting inflammation.[17][18] Severe
inflammation and accompanied infection can cause structural changes to the lungs
and lead to other late-stage symptoms. Specifically, the early-stage symptoms are
characterized by incessant coughing, copious phlegm production and decreased
ability to exercise, which are usually caused by the thick mucus inhabited bacteria-
induced pneumonia. When it comes to later stages, changes in the architecture of
the lung can happen, including pathology in the major airways, and lead to
difficulties in breathing. Further development of CF can eventually exacerbate
other signs including hemoptysis, pulmonary hypertension, heart failure and
hypoxia. In terms of organisms causing lung infections in CF patients,
pseudomonas aeruginosa is the most common pathogen, which has been
26
demonstrated to be chronically present in the lungs of about 80% of adults with CF
(Farrell et al., 2008). The strong association between P. aeruginosa and the airway
infection has been also revealed by epidemiological studies (Bragonzi, 2010;
Paroni et al., 2013). Progressive loss of lung function and shortened survival can
be induced by this infection. P. aeruginosa in CF patients can develop resistance
and adaptations by forming biofilms within the CF lung environment, making the
treatment much harder. In addition, the emergence of P. aeruginosa strains that
have multiple drug resistance (MDR) is another barrier to treat CF. Previous study
has shown that approximately 25% of adults with CF are chronically infected with
MDR P. aeruginosa.

In addition to lung symptom, CF can also impair the function of sinuses, pancreas,
vas deferens and so on. In paranasal sinuses, the over retention of mucus may
cause blockage of the sinus passages and lead to infection, which may cause facial
pain, fever, nasal drainage and headaches. Chronic sinus infection-induced
inflammation can also help to develop overgrowth of the nasal tissue. Blocked
nasal passages can further increase breathing difficulties in addition to lung
impairment; The similar situation of thickened mucus in the lung has a counterpart
in secretion of pancreatic juice from pancreas. Blocking exocrine secretion could
result in irreversible damage to the pancreas, coming with painful inflammation
(pancreatitis). Once the pancreatic ducts are totally plugged, atrophy of the
exocrine glands and progressive fibrosis can be induced (Kumar et al., 2012).

Theta-defensins are a family of mamalian antimicrobial cyclic cationic peptides
(Conibear and Craik, 2014). They only present in leucocytes of “Old World”
primates, but no in human, gorilla, bonobo, and chimpanzee (Cole et al., 2004; Li
et al., 2014). The reason why theta-defensins are limited to “Old World” monkey is
27
that the terminal mutation in the theta-defensins happened before the evolutionary
appearance of hominids (Nguyen et al., 2003). Among theta-defensins family,
Rhesus Theta-defensin-1(RTD-1) is gaining more and more attention because of its
broad microbicidal activity. Studies have been showing that RTD-1 has potent
microbicidal activity against many bacteria, fungi, herpes simplex virus and HIV
type 1 (Tongaonkar et al., 2011; Tran et al., 2008). Depending on the potent
activity of RTD-1 against MRSA and P. aeruginosa (Tai et al., 2013) and the fact
that systemic administration of RTD-1 in vivo demonstrated significantly
improved survival rate in pre-clinical models of peritonitis and sepsis, Dr.
Beringer’s lab tested the hypothesis that RTD-1 has promising therapeutic effect in
CF lung infection and proved its both in vivo and in vitro activity against CF
strains of Pseudomonas aeruginosa (Beringer et al., 2016).

One main challenge with aerosol delivery to the lungs is the relatively short
retention time due to rapid mucociliary and macrophage clearance mechanisms.
Recent advances in drug delivery using nanoparticle technologies indicates that
appropriate drug delivery vehicles can achieve sustained release to the airways
(Tang et al., 2009; Vij et al., 2010). Therefore, we proposed to develop and
characterize a polyethylene glycol (PEG)-poly (lactic-co-glycolic acid) (PLGA)
nanoparticle formulation of RTD-1 for aerosol delivery in CF. A nanoparticle
formulation of RTD-1 could significantly improve its duration and efficacy
following pulmonary administration, which will promote its utility in CF patients.

Polyethylene glycol (PEG) is a polyether compound with many applications in
medicine as well as industrial manufacturing, which has a structure of
H−(O−CH
2
−CH
2
)
n
−OH. Depending on the difference of the length of the chain
and molecular weight, which leads to different physical properties and determines
28
the different applications of PEG, it can also be refered as PEO or POE. Typically,
PEG itself is used to refer to oligomers and polymers with a molecular weight
below 20,000 kDa, PEO to polymers with a molecular weight above 20,000 kDa
and POE to polymers with any molecular weight. Different initiators are used in
the polymerization process of PEG, which results to different forms of PEG.
Mothoxypoly (ethylene glycol), abbreviated mPEG, is the most common initiator.
Lately, various studies of PEG have significantly widened its medical application.
In addition to its conventional usage as an osmotic laxative, PEG has been found to
be able to attach to various protein medications and allow a slowed renal clearance.
As a result, PEG attachment can contribute to maximize the therapeutic effect of
therapeutic agents by extending the circulation period, reduced toxicity and longer
dosing interval.

Being biodegradable and biocompatible, poly (lactic-co-glycolic acid) (PLGA) has
been widely used in a host of Food and Drug Administration (FDA) approved
therapeutic devices. Ring-opening co-polymerization method is introduced to
synthesize PLGA from the cyclic dimers of glycolic acid and lactic acid. Different
forms of PLGA can be obtained by altering the molar ratio of lactide to glycolide
used for the polymerization. For example, PLGA 75:25 represents a copolymer
which is composed of 75% lactic acid and 25% glycolic acid. This molar ratio,
together with block structure, also has an impact on the crystallinity property of
PLGAs. Despite some exceptions, relationship between the degradation efficiency
and the monomer’s molar ratio has been found. Generally, higher proportion of
glycolide indicates higher hydrolysis degradation efficiency. In addition, PLGA
polymers that are end-capped with esters usually demonstrate slower degradation
(Samadi et al., 2013). In terms of biosafety profile, PLGA can be completely
degraded in the human body by hydrolysis of its ester linkage into original
29
monomers, lactic acid and glycolic acid. Both of them are normal metabolism by-
products and can be effectively processed by human body. Thus, when PLGA is
used for drug delivery or biomaterial application, toxicity should not be a concern.

Results
In order to encapsulate highly hydrophilic RTD-1 into PEG-PLGA microparticles,
the double emulsion-solvent evaporation method was established to fabricate these
particles. The microparticle morphology was characterized by scanning electron
microscopy (SEM) (Fig 1). Image J analysis revealed a mean particle size of
1.91µm. The entrapment efficiency was determined to be 2% by mass by high
performance liquid chromatography (HPLC) after extracting RTD-1 into straight
water from PEG-PLGA microparticles. Briefly, loaded microparticles undergo
lyophilization followed by dissolving back into dichloromethane. Then,
hydrophilic RTD-1 can be easily extracted into water using two-phase separation
method.

Then, the in vitro release study was investigated. Briefly, 105mg encapsulated
particles were suspended in 1ml acetate buffer. The mixture was then incubated
under 37
0
C and consistent shaking at 250rpm. At different time points, perform a
centrifugation and collect 100µL supernatant. The same amount of fresh acetate
buffer was then added back into the mixture to keep the volume constant.
Consistent with the drug release profiles of other PLGA-based drug delivery
platform (Patel et al., 2012), a burst release was observed within the first 30min,
which accounts for 53.8% of encapsulated drug. The following sustained release
was found to be able to last at least 48h (Fig 2).

30
In order to assess the anti-inflammation efficiency of RTD-1 encapsulated
microparticles in vitro, an in vitro study followed by IL-8 Elisa assay was then
conducted. Basically, confluent A549 cells, the adenocarcinomic human alveolar
basal epithelial cells, were treated with either plain PEG-PLGA microparticles or
RTD-1 encapsulated microparticles at the same mass for 24h. Cell culture
supernatants were collected respectively. IL-8 levels in both supernatant were
determined by IL-8 Elisa Assay Kit, which demonstrated a decreased IL-8 level of
about 10% (Fig 3).

Discussion
PEG-PLGA based microparticles or nanoparticles have been widely investigated
and used in drug delivery. But most of the time, they are used to encapsulate
hydrophobic drug. Although there are some successful attempts to encapsulate
hydrophilic drug of small molecular weight, our investigation of RTD-1
encapsulation into PEG-PLGA particles is still challenging in terms of the
encapsulation efficiency because of these following reasons:
1. There are too many factors that could impact this efficiency, which includes
the inner phase peptide concentration, outer phase surfactant concentration,
polymer specificity, organic solvent removal method, homogenization
condition and so on. All these conditions needs to be taken into
consideration when optimizing the encapsulation efficiency.
2. The highly acidic environment caused by the hydrolysis of PLGA can
deteriorate the peptide stability of RTD-1. experimental evidence on the
spatial and temporal distribution of pH in PLGA has been provided using
confocal microscopy, yielding minimal pH levels of 1.5 in the core area of
microspheres (Fu et al., 2000). Although the RTD-1 has been shown to be
stable at acidic environment (pH4.4), this highly acidic environment could
31
probably lead to insufficient peptide stability, which could lead to
incomplete release of the drug substance from the degrading PLGA, mostly
by non-specific adsorption of the compound to the matrix material,
formation of non-covalently bound aggregates or covalent aggregation
(Crotts and Park, 1998; van de Weert et al., 2000). To overcoming this
deleterious effect, co-encapsulation of basic additives or sugars will be used
to protect peptide in the following studies.
3. The amphiphilic property of PEG-PLGA copolymer leads to really low
solubility no matter in water or in common organic solvents, especially after
forming microparticles, which leads to incomplete drug extraction during the
two-phase separation method and makes it hard to determine the accurate
encapsulation efficiency.

Experimental Procedures
Preparation of RTD-1 encapsulated microparticles
As reported by previous study (Gupta et al., 2011), RTD-1 encapsulated
microparticles were prepared by water-in-oil-in-water double emulsion-solvent
evaporation method. Briefly, 60µL of internal water phase (W1) containing 0.3mg
RTD-1 (5mg/mL) was first emulsified in 500µL of dichloromethane (OP)
containing 10mg of PLGA and 2mg of PEG-PLGA copolymer. The resulting
emulsion was added into 2ml external water phase (W2) containing 1% polyvinyl
alcohol (PVA) and 3% sodium chloride (NaCl). This mixture then underwent
another sonication. In order to remove the rest dichloromethane and harden the
microparticles, the resulting double emulsion was stirred for 8h at room
temperature under fume hood. Microparticles were collected by centrifugation at
4000rpm for 15min. After being washed three times by ddH
2
O, these particles
were lyophilized for 48h to get free-flowing power.
32

Encapsulation efficiency dertermination
Aqueous two-phase extraction method was used to extract hydrophilic RTD-1
from polymeric microparticles. Encapsulated microparticles were first dissolved
into dichloromethane and added into a separatory funnel following by adding
certain amount of ddH
2
O into the funnel. The funnel was then closed and shaken
gently by inverting the funnel several times. Excess vapor needed to be released by
inverting the funnel and carefully opening the stopcock. When separation was
completed, both the top stopper and the stopcock were opened up and the lower
phase was collected by gravitation. When the bottom layer had been removed, the
stopcock was closed and the upper layer was poured out through the top into
another container. The RTD-1 concentration in collected water phase was then
tested by HPLC at 210mn.

33
Figures

Figure 1 The SEM image of RTD-1 encapsulated PEG-PLGA microparticles.
34

Figure 2 The release profile of RTD-1 from PEG-PLGA-based microparticles. A
sustained release followed by a burst release was observed.

0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0 10 20 30 40 50 60
Released Mass (mg)
Time (Hours)
35

Figure 3 The shift of IL-8 level in A549 cell culture media by treating with either
unloaded or loaded PEG-PLGA microparticles.

36
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T., Rahimian, S., Teunissen, E., Van Steenbergen, M., Amidi, M., and
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Zeitlin, P., Boyle, M.P., Fu, J., and Hanes, J. (2009). Biodegradable polymer
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nanobiotechnology 8, 1-18.

39
Project 3: A Biomimetic Solution to the Generation of Hemoglobin-
based Oxygen Carriers

Abstract
Emergency circumstances usually need large amounts of blood, where donated
blood may not be sufficient to fill the needs due to its limitation including storage
issue and type compatibility issue, as well as its limited resources. But free
hemoglobin released into the vasculature can rapidly scavenge nitric oxide and
thus lead to systemic vasoconstriction, decreased blood flow, increased release of
pro-inflammatory mediators and potent vasoconstrictors and a loss of platelet
inactivation. Renal toxicity can also be induced by rapid infiltration of free
hemoglobin from the kidney due to its low molecular weight. Here we proposed
that fusing hemoglobin to one particle elastin-like polypeptides can help to prevent
extravasation and reduce the nitric oxide scavenging-mediated toxicities. Chemical
conjugation has been investigated. Highly controllable molecular cloning will also
be studied.

Introduction
Significance of blood substitute
Over 4.5 million patients require blood transfusions throughout North America
each year. Blood transfusions are a life-saving intervention in a number of clinical
settings such as battlefield hemorrhaging, major surgical procedures, and anemia.
In events involving acute trauma, occurring in a serious car accident for example, a
victim may need almost 100 pints of transfused blood. Transfusion therapy has
therefore been an integral part of military medicine. As a vital component of blood,
red blood cells (RBCs) are the most transfused blood product in battlefield trauma
40
care; more than 54,000 units of RBCs are transfused every year in military
hospitals.

Information about hemoglobin
As the most common type of blood cell, red blood cells (RBCs) comprise
approximately 99% of the cells in blood, which are vertebrate organism’s principal
means to deliver oxygen to the body tissue and remove carbon dioxide from the
tissue via blood flow through the circulatory system. The reversible oxygenation
function of RBCs (i.e. a large volume of oxygen taken up in the lungs and
delivered to the tissues and the removal of carbon dioxide) is carried out by
hemoglobin (Hb), which is the iron-containing oxygen-transport metalloprotein in
the red blood cells of all vertebrates(Anthea et al., 1993) and accounts for 96% of
the dry weight of the RBCs in mammals. Hb has an oxygen-binding capacity of
1.34 mL O2 per gram(DE VILLOTA et al., 1981), which increases the total blood
oxygen capacity seventy-fold compared to dissolved oxygen in blood.

Similar to many other multi-subunit globular proteins, hemoglobin has a
quaternary structure coming from its four subunits in roughly a tetrahedral
arrangement. In most vertebrates, the hemoglobin molecule is an assembly of four
globular protein subunits. Each subunit is composed of a protein chain tightly
associated with an iron ion containing non-protein heme group. In adult humans,
the most common hemoglobin type is a tetramer (which contains four subunit
proteins) called hemoglobin A, consisting of two α and two β subunits non-
covalently bound, each made of 141 and 146 amino acid residues, respectively.
This is denoted as α2β2. The subunits are structurally similar and about the same
size. Each subunit has a molecular weight of about 16,000 daltons, for a total
molecular weight of the tetramer of about 64,000 daltons (64,458 g/mol).(Van
41
Beekvelt et al., 2001) Hemoglobin A is the most intensively studied of the
hemoglobin molecules. When oxygen binds to the first subunit of
deoxyhemoglobin, the first oxygen molecule increases the affinity of the remaining
subunits for additional oxygen molecules. As additional oxygen is bound to the
other hemoglobin subunits, oxygen binding is incrementally strengthened, so that
hemoglobin is fully oxygen-saturated at the oxygen tension of lung alveoli.
Likewise, oxygen is incrementally unloaded and the affinity of hemoglobin for
oxygen is reduced as oxyhemoglobin circulates to deoxygenated tissue. The
absorption spectra of oxyhemoglobin and deoxyhemoglobin differ. The
oxyhemoglobin has significantly lower absorption of the 660 nm wavelength than
deoxyhemoglobin, while at 940 nm its absorption is slightly higher.

Challenges
There are a lot of circumstances where large amounts of blood are required
immediately including civilian emergency, battlefield and so on. But donated
blood has its obvious limitations. First, red blood cells can only be stored for
limited period. The clinical practice has established a 21-day preservation limit at
2-6
0
C for red blood cell; Second, compatibility issue has to be taken into
consideration when it comes to donated blood cell. Third, the source of donated
blood is limited due to irregular donation.

Hemoglobin itself, although taking the responsibility of oxygen transportation,
cannot be injected into circulation system directly. A preclinical model mimicking
the injury during hemolytic states(Minneci et al., 2005), in which hemoglobin is
released into the circulation system, along with many other similar tests(Caron et
al., 1999; Gould et al., 2002; Vandegriff et al., 2003), has shown that, without
being contained by a red cell membrane, free hemoglobin released into the
42
vasculature can rapidly scavenge nitric oxide and thus lead to systemic
vasoconstriction, decreased blood flow, increased release of pro-inflammatory
mediators and potent vasoconstrictors and a loss of platelet inactivation(De
Caterina et al., 1995; Lin et al., 2001; Rother et al., 2005), which could eventually
create the conditions vascular thrombosis of the heart or other organs. Another
major challenge with free hemoglobin lies in its low molecular weight, which
permits rapid removal from the kidneys leading to renal toxicity.

In order to minimize such toxicities, a larger, more stable hemoglobin-based blood
substitute molecules are proposed to prevent extravasation and reduce the nitric
oxide scavenging-mediated toxicities. Many methods have been tried, including
polymerization and pegylation of hemoglobin.

Hemogobin-ELP-based blood substitute
Here in our lab, we have been trying to make the recombinant protein by fusing
elastin-like polypeptide (ELP), to hemoglobin though either chemical conjugation
or protein engineering.

Derived from human tropoelastin, ELPs consist of pentameric repeats of (Val-Pro-
Gly-Xaa-Gly)n and have a unique inverse transition behavior wherein below their
transition temperature (Tt), they are highly water soluble but once the temperature
rises above their Tt, ELPs undergo the phase separation process and self-assemble
into particles(Dhandhukia et al., 2013). This is a fully reversible process and can
be used to effectively purify ELP-conjugated materials(Shah et al., 2013). Phase
behavior can be precisely controlled through adjusting the hydrophobicity of guess
residue “Xaa” and number of pentapeptide repeats “n”(Urry, 1997). The ELP we
43
used in the preparation of recombinant hemoglobin is A192, with the amino acid
sequence of G(VPGAG)192Y and the molecular weight of 73.6kDa.

ELPs are attractive as hemoglobin delivery systems for at least five important
reasons: first, because ELPs can be genetically encoded, their synthesis from a
synthetic gene in a heterologous host (e.g., bacteria or eukaryotic cell) can provide
complete control over the amino acid sequence and molecular weight, two
variables that are not easy to precisely control in synthetic polymers. Second, ELPs
can be expressed from a plasmid-borne gene in E. coli to relatively high yields
(500 mg/L growth), which also makes them attractive for hemoglobin delivery
applications where large quantities of polymer are often required. Third, they can
be purified from E. coli and other cell lysates in batch process by exploiting their
inverse temperature phase transition without the need for chromatography, which
simplifies large scale purification of ELPs(Meyer and Chilkoti, 1999). Fourth,
ELPs can be engineered to approach the viscoelastic properties of native elastin
upon crosslinking. Fifth, they are biocompatible, biodegradable, and non-
immunogenic(Urry, 1997).

The versatility of ELPs have been underscored by the fact that ELPs can be
appended to other proteins since 1999 by Chilkoti et al (Meyer and Chilkoti, 1999).
In Dr. Mackay’s lab, ELP fusions are widely investigated to treat cancer and ocular
diseases (Janib et al., 2014a; Shah et al., 2013; Wang et al., 2014; Wang et al.,
2015). The feasibility and strategy of expressing hemoglobin through molecular
cloning have been investigated since 1985(Nagai et al., 1985), when Nagai et al.
described a method for the production and engineering of semisynthetic human Hb
in E. coli. Synthesized β-globin was found to be able to refold in vitro in the
presence of native a-globin and heme to gain a fully functional tetramer. Later in
44
1990(Hoffman et al., 1990), Stephen et al. successfully expressed fully functional
tetrameric human hemoglobin in E. coli through expressing both human α- and β-
globin from a single operon at the same time. Two different globin chains can fold
in vivo and incorporate endogenous heme. They also tried to express the α- and β-
globin separately and found that the presence of α- and β-globin in the same cell
stabilizes α-globin and aids the correct folding of β-globin.

In terms of chemical conjugation, carboxyl-reactive chemical groups in bio-
molecular probes are commonly used for labeling and crosslinking carboxylic
acids to primary amines. For aqueous crosslinking, the most readily available and
commonly used carbodiimides are the water-soluble 1-ethyl-3-(-3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC). EDC reacts with
carboxylic acid groups to form an active O-acylisourea intermediate that is easily
displaced by nucleophilic attack from primary amino groups in the reaction
mixture. The primary amine forms an amide bond with the original carboxyl
group, and an EDC by-product is released as a soluble urea derivative. The O-
acylisourea intermediate is unstable in aqueous solutions. Thus, N-
hydroxysuccinimide (NHS) or its water-soluble analog (Sulfo-NHS) is often
included in EDC coupling protocols to improve efficiency or create dry-stable
(amine-reactive) intermediates. EDC couples NHS to carboxyls, forming an NHS
ester that is considerably more stable than the O-acylisourea intermediate while
allowing for efficient conjugation to primary amines at physiologic pH (Fig 1).

Results
EDC-based chemical conjugation between hemoglobin subunits and A192 were
fabricated, which was proved by SDS-PAGE (Fig 2). Under reducing environment,
hemoglobin tetramer was disassembled, which is why the band representing plain
45
hemoglobin were located between 15-20 kDa. Interestingly, on lanes of fusion
samples, two bands above plain A192 can be found, which we think is A192
binding to either one subunit or two subunits of hemoglobin.

Then we used size exclusion chromatography (SEC) to separate fusion protein
from plain A192 depending on their difference of molecular weight. Two peaks
were observed (Fig 3). We believe the first peak represents fusion protein and
second peak stands for plain A192.

Those two peaks from SEC were collected separately: fraction 1 and fraction 2.
Using Dynamic Light Scattering (DLS), we can measure the hydrodynamic radius
of these two fractions. As shown in Fig 4, the hydrodynamic radium of fraction 1 is
11.4nm, significantly bigger than fraction 2, which has a radium of 7.4nm. 7.4nm
hydrodynamic radius of fraction 2 is consistent with our previous study of A192
(Janib et al., 2014a).

One important property of ELP is that it undergoes phase separation once the
temperature reaches its transition temperature. The thermal responsiveness
property of fusion protein, along with the parent A192, was characterized using
Ultraviolet–visible spectroscopy (UV-Vis) by measuring their optical density of
these constructs at 350nm, where neither fusion protein nor plain A192 contributes
significant absorption. As shown in Fig 5, after conjugation, the phase separation
property of ELPs is well maintained.

46
Discussion
Although this chemical conjugation method demonstrated the feasibility of fusing
hemoglobin to one of our ELPs, A192, there are too many uncontrollable factors
existing in this procedure. Considering EDC-based chemical crosslinking are
randomly happening in the solution, different protein combination can be achieved,
which leads to the big challenge of making consistent products. In order to solve
this problem, molecular cloning will be needed. We already designed the
hemoglobin-ELP expressing plasmid. The next step is to investigate the feasibility
of expressing fully functional hemoglobin-ELP fusion using Escherichia coli
expressing system.

Experimental procedures
Biosynthesis and purification of A192
As previously reported, the gene encoding A192 was synthesized by directional
ligation in a modified pET25b(+) vector (McDaniel et al., 2010). Then these
reconstructed plasmids were transfected into TOP10 cells for amplification. After
18h culture under constant shaking, a mini-preparation was contacted to obtain
purified plasmids. BLR cells transfected with these purified plasmids were then
introduced for A192 expression. After 24h incubation, expressing cells were
collected from the culture by centrifugation and underwent sonication to be lysed.
Following that, polyethyleneimine (PEI) was used to precipitate nucleic acids from
crude cell lysates. After another centrifugation, the supernatant was transferred to
50ml tubes. All the tubes were placed on 37 ºC heating block. 2M NaCl was then
added into each tube. When solution turns turbid (indicates ELP phase separation),
perform a Hot Spin (37˚C, 12 min, 4000 rpm). After Hot Spin, keep pellet. Place
those tubes containing the pellets on ice. Add 2 mL of cold PBS to re-suspend
47
pellet. Once re-suspended, mix all re-suspended liquid into 1 tube and then
perform a Cold Spin (4˚C, 12 mins, 12,000 rpm). After the Cold Spin, transfer all
the supernatant to one tube so that it’ll be one solution. This marks one cycle of the
first round of ITC (Inverse Transition Cycling). Repeat ITC at least two more
times.

The ELP concentration was determined by measuring A280 by Nanodrop 2000
spectrophotometer. The molar absorption coefficient of a peptide or protein is
related to its tryptophan (W), tyrosine (Y) and cysteine (C) amino acid composition
(Gill and Von Hippel, 1989; Pace et al., 1995). At 280nm, this value is
approximated by the weighted sum of the 280nm molar absorption coefficients of
these three constituent amino acids, as described in the following equation:
ε = (nW × 5500) + (nY ×1490) + (nC ×125)
where n is the number of each residue and the stated values are the amino acid
molar absorptivity at 280nm.

Chemical Conjugation method
Hemoglobin and A192 were first mixed at different ratios (1:1, 1:2, 1:4). Ten times
molar excess of EDC as the molarity of hemoglobin was then added into the
mixture. In order to achieve higher reaction efficiency, the same molarity of Sulfo-
NHS as that of EDC was also added into the reaction solution. After being rotated
overnight, ELP fusion, as well as plain ELP, was purified by another ITC from the
mixture.

48
Figures

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-
biology-resource-library/pierce-protein-methods/carbodiimide-crosslinker-chemistry.html
Figure 1 The scheme of chemical conjugation approach. The addition of Sulfo-
NHS can increase the reaction efficiency.

49

Figure 2 The SDS-PAGE demonstrating the shift of molecular weight after
chemical conjugation. In the first two lanes loaded by fusion protein, two bands
can be seen above the bands of plain A192, which we believe are A192 binding to
one or two subunit of hemoglobin.

50

Figure 3 The result of SEC purification. Two peaks can be observed, which
represents fusion and plain A192 respectively.

51

Figure 4 The DLS results revealing the different hydrodynamic radii of two
different SEC fractions. In fraction 1, which we think is fusion protein,
nanoparticles with a hydrodynamic radius of 11.4nm represent 99.7% of total
mass. In fraction 2, which represents plain A192, nanoparticles are monodispersed
and have a hydrodynamic radius of 7.4nm.

52

Figure 5 The result of UV-Vis temperature ramp showing that the phase separation
property of ELPs is well maintained after conjugation.

53
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Creator Guo, Hao (author)

Core Title Project 1: Elastin-like polypeptide stabilizes recombinant cyclophilin into a nanoparticle; Project 2: Encapsulation of Rhesus theta defensin-1 (RTD-1) in PEG-PLGA nanoparticles for aerosol deliv...

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 07/25/2017

Defense Date 05/20/2016

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag drug delivery,nanoparticle,OAI-PMH Harvest,protein engineering

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Advisor Mackay, J. Andrew (committee chair), Beringer, Paul (committee member), Hamm-Alvarez, Sarah (committee member)

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