1. i
UNIVERSITY OF PUERTO RICO
FACULTY OF NATURAL SCIENCES
BIOLOGY INTERCAMPUS DOCTORAL PROGRAM
RIO PIEDRAS CAMPUS & MEDICAL SCIENCES CAMPUS
Nano-sized delivery systems for
potential cancer therapies:
Modification of protein formulations to
improve pharmacological efficacy
DOCTORAL DISSERTATION
Submitted for Partial Fulfillment of
The Requirements for the Degree of
Doctor in Philosophy
In the subject of
Biology
By
Yamixa Delgado Reyes
San Juan, Puerto Rico
June 1, 2015
2. ii
DEDICATION
To God, my parents Luz & Dario, my husband Ismael, and Rosie:
I successfully accomplished this journey thanks to all of you.
In memory of Eliseo Rivas
“Cancer may have won the battle against your body,
but now your soul is free and your legacy remains alive.”
3. iii
TABLE OF CONTENTS
TABLE CAPTIONS.......................................................................................................... vi
FIGURE CAPTIONS........................................................................................................ vii
ABBREVIATIONS .......................................................................................................... xii
PAGE OF APPROVAL................................................................................................... xiii
AUTHOR’S BIOGRAPHY............................................................................................. xiv
PEER REVIEWED PUBLICATIONS..............................................................................xv
AKCNOWLEDGMENTS ............................................................................................... xvi
ABSTRACT.................................................................................................................... xvii
Chapter 1
INTRODUCTION ...............................................................................................................1
1.1 Proteins in biotechnology.......................................................................................1
1.2 Protein instability problem .....................................................................................2
1.3 Chemical modification of protein pharmaceuticals................................................5
1.3.1 PEGylation.............................................................................................9
1.3.2 Glycosylation........................................................................................11
1.4 Passive targeted nanoparticles for cancer therapy ..............................................13
1.4.1 Lipid-based formulations......................................................................17
1.4.1.1 Fatty acids ................................................................................18
1.4.1.1.1 Oleic acid ...........................................................................20
1.4.2 Protein-based formulations...................................................................21
1.4.2.1 α-Lactalbumin..........................................................................22
1.4.2.2 Cytochrome c...........................................................................24
REFERENCES ..................................................................................................................27
Chapter 2
CHEMICAL GLYCOSYLATION OF CYT C TO IMPROVES PHYSICAL AND
CHEMICAL PROTEIN STABILITY ...............................................................................33
3.1 BACKGROUND..................................................................................................33
3.2 RESULTS AND DISCUSSION...........................................................................36
3.2.1 Synthesis and characterization of the Cyt c neo-glycoconjugates ........36
3.2.2 Protein structural changes of Cyt c glycoconjugates.............................38
3.2.3 Stability of Cyt c glycoconjugates under stress conditions...................42
3.3 CONCLUSION ....................................................................................................51
REFERENCES ..................................................................................................................51
4. iv
Chapter 3
DEVELOPMENT OF BAMLET LIPID-PROTEIN COMPLEXES NANOPARTICLES
AND THE ELUCIDATION OF ITS CYTOTOXIC COMPONENT...............................55
4.1 BACKGROUND..................................................................................................55
4.2 RESULTS AND DISCUSSION...........................................................................57
4.2.1 Synthesis and characterization of BAMLET complexes ......................57
4.2.2 Identification of the cytotoxic component in BAMLET .......................63
4.2.3 BAMLET cytotoxicity in cancer and normal cell lines ........................70
4.2.4 BAMLET cell death pathway................................................................72
4.2.5 Long-term storage stability of BAMLET..............................................74
4.3 CONCLUSION. ...................................................................................................76
REFERENCES ..................................................................................................................78
Chapter 4
DEVELOPMENT OF HAMLET-LIKE CYTOCHROME C-OLEIC ACID
NANOPARTICLES FOR CANCER THERAPY .............................................................82
5.1 BACKGROUND..................................................................................................82
5.2 RESULT AND DISCUSSION.............................................................................85
5.2.1 Characterization of HAMLET-like complexes .....................................85
5.2.2 HAMLET cytotoxicity against cancer and normal cell lines................89
5.2.3 Cell death pathway, cell uptake and nucleus co-localization................90
5.3 CONCLUSIONS..................................................................................................95
REFERENCES ..................................................................................................................96
Chapter 5
CONCLUDING REMARKS.............................................................................................99
Appendix I
EXPERIMENTAL PROCEDURES................................................................................101
I.1 MATERIALS AND METHODS - CHAPTER 2 ...............................................101
I.1.1 Materials...............................................................................................101
I.1.2 Methods................................................................................................101
I.1.2.1 Amine-reactive functionalization of Dextran 10 kD ..............101
I.1.2.2 Amine-reactive functionalization of Dex-COOH...................102
I.1.2.3 Glycosylation of Cyt c............................................................102
I.1.2.4 Circular dichroism spectroscopy ............................................103
I.1.2.5 Peroxidase pseudo-activity assay ...........................................103
I.1.2.6 Moisture- and temperature-induced structural instability ......103
I.1.2.7 Oxidative stress assay.............................................................104
6. vi
TABLES
CHAPTER 1
Table 1.1 Some proteins actually used or under research as potential
pharmaceuticals…………………………………………………....…....................4
Table 1.2 FDA-approved PEGylated drugs.…………………………………….……….......8
Table 1.3 Most recent glycoprotein-based drugs in the market…………………………….10
Table 1.4 Clinically approved nanoparticle systems………………………………….........15
CHAPTER 2
Table 2.1 Functionality determination for Cyt c glycoconjugates.…………………………38
Table 2.2 Results of the capability of Cyt c and Dex3(1 kD)-Cyt c to activate apoptosis after
their exposure to water-organic solvent (o/w) interface conditions...……………48
CHAPTER 3
Table 3.1 Characterization of the synthesized BAMLET complexes.……………………..58
Table 3.2 Characterization of FA after ultra-sonication by DLS.…………………………..71
Table 3.3 Activation of caspase-3 and caspase-9 by OA and BAMLET………………….76
CHAPTER 4
Table 4.1 Characterization of HAMLET-like complexes...………………………………...87
Table 4.2 Capability of various preparations to activate caspase-3 and -9 in cell-free
assay…………………………………………………………………………….92
7. vii
FIGURES
CHAPTER 1
Figure 1.1 Representation of PEGylated particles shielded against
opsonisation…….....................................................................................................7
Figure 1.2 Benign cells vs malignant cells: Hallmarks of cancer and tumor
microenvironment …………………………………….…………………………16
Figure 1.3 Scheme of the passive targeting by the enhanced permeability and retention
(EPR) effect……………………………………………………………………...19
Figure 1.4 Cartoon structural representation of the horse heart cytochrome c (1HRC PDB)
and bovine α-lactalbumin (1F6S PDB)…………………………..........................22
CHAPTER 2
Figure 2.1 Representation of the chemical glycosylation of Cyt c using monofunctionally
activated glycans………………………………………………………………....40
Figure 2.2 Far UV (A), near UV (B), and heme (C) region CD spectra of 0.6 mg/ml Cyt c
and Cyt c glycoconjugates in 100 mM phosphate buffer at pH 7.4 and 20°C…...41
Figure 2.3 Effect of β-mercaptoethanol exposure of Dex3(1 kD)-Cyt c on the far-UV CD,
near-UV CD, and heme region CD spectra..……………………………………..45
Figure 2.4 Crystal structure (1HRC.pdb) of horse heart Cyt c (A). Cyt c has 19 solvent-
exposed Lys residues (5, 7, 8, 13, 22, 25, 27, 39, 53, 55, 60, 72, 73, 79, 86, 87, 88,
99, 100). The figure was generated using PyMol. Horse heart Cyt c sequence (B)
from Uniprot P00004.……………………………………………………………46
Figure 2.5 Degradation of Cyt c and Cyt c glycoconjugates by 4 mg/ml trypsin at 37°C. Each
experiment was performed in triplicate, the values averaged, and the error bars are
the calculated SD………………………………………………………………....46
Figure 2.6 Degradation of Cyt c and Cyt c glycoconjugates by 5 mg/ml α-chymotrypsin at
37°C. Each experiment was performed in triplicate, the values averaged, and the
error bars are the calculated SD………………………………………………….49
Figure 2.7 The effect of 75% relative humidity on the structure of Cyt c and its
glycoconjugates. After incubation, the glycoconjugates were lyophilized and
dissolved in 10 mM phosphate buffered saline at pH 7.3. Each experiment was
performed in triplicate, the values averaged, and the error bars are the calculated
SD...………………………………………………………………………………50
8. viii
Figure 2.8 The effect of incubation at 50°C on the structure of Cyt c. After incubation, the
glycoconjugates were lyophilized and dissolved in 10 mM phosphate buffered
saline at pH 7.3. Each experiment was performed in triplicate, the values
averaged, and the error bars are the calculated
SD………………………………………………………………………………...51
Figure 2.9 Scanning electron microscopy (SEM) images of lyophilized Dex3(1 kD)-Cyt c
after the precipitation via solvent displacement with (A) acetonitrile and (B)
acetone as desolvating agent……………………………………………………..51
CHAPTER 3
Figure 3.1 A schematic representation of the syntheses conditions for the coupling of OA
moieties to bovine α-LA to obtain BAMLET. For details on the properties of the
complexes prepared by the three methods please see Table
3.1………………………………………………………………………………...58
Figure 3.2 Fluorescence emission spectra ( exc = 295 nm) of the three BAMLET complexes
synthesized in this work and of native -LA……….……………………………59
Figure 3.3 SEM images of the three BAMLET complexes synthesized. In agreement with
DLS measurements (Table 3.1) the images display spherical
nanoparticles……………………………………………………………………..62
Figure 3.4 Modeling of OA binding to α-LA in the BAMLET complex. The blue colored
residues were found to be responsible for OA interactions with the α-LA
surface…………………………………......……………………………………..63
Figure 3.5 Fluorescence emission spectra (λexc= 295 nm) of the BAMLET complexes [0.5
mg/ml] after incubation with 3 mg of trypsin for 72 h.…………………………65
Figure 3.6 HeLa cell viability after 6 h of incubation with the BAMLET complexes and the
-LA component of the complex. A. Cytotoxicity of BAMLET complexes
synthetized using different conditions. The BAMLET complexes were adjusted to
a concentration of 120 µM of OA and LinOA. B. Experiments with -LA were
adjusted to 117 mM, the same as the BAMLET complexes in Fig 3.6A. C.
Cellular uptake of α-LA-FoA..…………………………………………………...66
Figure 3.7 HeLa cell viability after 6 h of incubation with 120 µM of monounsaturated (OA),
polyunsaturated (LinOA), and saturated (SA) C18 FA. OA-PDPH is OA with a
blocked carboxy group…………………………………………………………..67
Figure 3.8 FTIR spectra of OA, PDPH and OA-PDPH. The characteristic stretching
vibrational mode of primary amines is observed in the PDPH spectrum at 3305
and 3260 cm-1
. These peaks are not present in the OA-PDPH spectrum instead
there is the N-H stretch at 3215 cm-1
. This value is distinctive of a secondary
9. ix
amine suggesting the reaction between OA and PDPH occurred. OA does not
present any amine peak…………………………………………………………68
Figure 3.9 MTS viability assay of different OA preparation methods in HeLa cells. Samples
labeled with the * were prepared using sonication prior to cell incubation.
Conditions: 120 µM FA, sonication for 2 min at an energy setting of 130
watts...……………………………………………………………………………69
Figure 3.10 SEM images of the different FA studied after sonication. All unsaturated FA
formed micelles of < 200 nm in diameter………………………………………71
Figure 3.11 HeLa cell viability assay to determine the LC50 after incubation with BAMLET
(LA-OA Tris-HCl 60) and OA at different concentrations. OA alone killed HeLa
cells with the same efficiency at equimolar concentrations as its formulation as
BAMLET.………………………………………………………………………..72
Figure 3.12 Non-selective cytotoxicity of BAMLET (α-LA-OA Tris-HCl 60) and OA towards
normal cells (Cho-K1 and NIH/3T3) and cancer cells (HeLa and A-549) after 6 h
of incubation. The values are the mean of quadruplicate measurements and the
error values are the calculated S.D…………………………………………........73
Figure 3.13 Confocal microscopy of HeLa cells after 6 h of incubation; (A-D) were incubated
with DAPI (blue) and PI (red); (A) control; (B) cells after incubation with 117
mM of denatured α-LA; (C) cells treated with 80 µM of BAMLET (α-LA-OA
Tris-HCl 60); (D) cells treated with 80 µM of OA alone……………………….75
Figure 3.14 Growth inhibition of HeLa cells after 24 h of incubation with 120 µM of
BAMLET (α-LA-OA), BAMLET-like complex (α-LA-LinOA) prepared by Tris-
HCl 60 method, and their components (α-LA, OA and LinOA)……………….77
Figure 3.15 Cytotoxicity of BAMLET (α-LA-OA Tris-HCl 60) and OA towards HeLa cells
after incubation at 50ºC and 75% of humidity for 14 days…………………….78
CHAPTER 4
Figure 4.1 (A.)The synthesis conditions for the coupling reactions of BSA and Cyt c to OA
to obtain HAMLET-like complexes. (B.) Representation of the HAMLET-like
lipid/protein NP, possible intracellular delivery, and activation routes…...……85
Figure 4.2 Fluorescence emission spectra ( exc = 280 nm) of the HAMLET-like complexes
synthesized in this work. ……………………………………………………….87
Figure 4.3 SEM images of the HAMLET-like complexes synthesized. In agreement with
DLS measurements (Table 5.1) the images display spherical
nanoparticles……………..……………………………………………………..88
10. x
Figure 4.4 LC50 determination after incubation of HeLa cells with Cyt c-OA and OA at
different concentrations. The ** indicate statistically significant differences
compared with OA alone as a control;
p<0.01.………………………………………………………………………….89
Figure 4.5 Non-specific cytotoxicity of 120 μM of HAMLET-like complexes and OA
towards normal cells (Cho-K1 and NIH/3T3) and cancer cells (HeLa and A-549)
after 6 h of incubation. The asterisks indicate significant differences: *: p<0.05;
**: p<0.01; and ***: p<0.001.………………………………………………….91
Figure 4.6 Apoptosis determination after 6 h of incubation with HAMLET-like complexes.
(A-B) were incubated with DAPI (blue) and PI (red); (A) 80 µM BSA-OA
complex; (B) 80 µM Cyt-OA complex.………………………………………...93
Figure 4.7 Cellular-uptake and nucleus co-localization. (A-B) were incubated with FITC-
HAMLET-like complex (green), DAPI (blue), and FM4-64 (red); (A) 80 µM
BSA-OA complex; (B) 80 µM Cyt-OA complex.……………………….94
11. xi
ABBREVIATIONS
ABTS 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
aa Amino acids
Apaf-1 Apoptotic protease activating factor-1
ATCC American type culture collection
CD Circular dichroism spectroscopy
BAMLET Bovine α-lactalbumin made lethal to tumor cells
BSA Bovine serum albumin
Cyt c Cytochrome c
CMC Critical micellar concentration
Dex-COOH Dextran hexanoic acid
DLS Dynamic light scattering
EDC Ethyl-3-[3-dimethylamino-propyl]carboiimide HCl
EPR Enhance permeability and retention
FA Fatty acid
FoA Folic acid
FBS Fetal bovine serum
FDA Food and drug administration
FITC Fluorescein isothiocyanate
FTIR Fourier transform infrared spectroscopy
HAMLET Human α-lactalbumin made lethal to tumor cells
HeLa Human cervical cancer cells
HEPES Hydroxyethyl)-1-piperazineethanesulfonic acid
12. xii
α-LA α-Lactalbumin
LinOA Linoleic acid
MEM Minimum essential medium
MTS Tetrazolium salts
MW Molecular weight
NHS N-hydroxysuccinimide
NHS-Lac Mono-(lactosylamido)-mono-(succinimidyl) suberate
NHS-Dex Mono-(dextranamido)-mono-(succinimidyl)suberate
NMR Nuclear magnetic resonance
NP Nanoparticles
OA Oleic acid
ω Omega
PDPH 3-[2-pyridyldithio] propionyl hydrazide
PEG Polyethylene glycol
PMS Phenazine methosulfate
RES Reticuloendothelial system
SA Stearic acid
SEM Scanning electron microscopy
TNBSA Trinitrobenzene sulfonic acid
13. xiii
ACCEPTED BY THE FACULTY OF NATURAL SCIENCES OF THE BIOLOGY
INTERCAMPUS DOCTORAL PROGRAM OF THE UNIVERSITY OF PUERTO RICO RIO
PIEDRAS CAMPUS AND MEDICAL SCIENCE CAMPUS IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR IN PHILOSOPHY
IN THE SUBJECT OF
BIOLOGY
CONCENTRATION
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
_______________________________________________
Kai Griebenow, PhD
THESIS DIRECTOR
DISSERTATION COMMITTEE:
_______________________________________________
Carlos González, PhD
_______________________________________________
Gabriel Barletta, PhD
_______________________________________________
Carlos Cabrera, PhD
_______________________________________________
Tugrul Giray, PhD
_____________________________________________
Dr. Tugrul Giray, PhD
CHAIRMAN
DEPARTMENT OF BIOLOGY
San Juan, Puerto Rico
June 1, 2015
14. xiv
AUTHOR’S BIOGRAPHY
Yamixa Delgado Reyes was born on October 6, 1984 in Humacao, Puerto Rico. Her
parents are Darío Delgado Díaz and Luz D. Reyes Nieves. She is the youngest of five siblings:
Jenny, Dario, Ebony, Gilbert and Sherly. All her childhood, she lived on the mountains of
Naguabo.
Yamixa graduated from Ana Roqué de Duprey High School in Humacao in 2002. The
same year, she was admitted to the University of Puerto Rico- Humacao to continue studies in
science. From 2004 to 2007, she had the great opportunity to work with Dr. Gabriel Barletta in
his bioorganic research laboratory in collaboration with Dr. Marc Legaut from the University of
Puerto Rico-Bayamón and Dr. Vidha Bansal from the University of Puerto Rico-Cayey. In 2007,
she received her bachelor degree in science with major in industrial chemistry. After her
graduation, she continued working with Dr. Barletta for eight months as an assistant researcher
until she entered graduate school. In 2008, she was admitted to the graduate program of the
Department of Chemistry at the University of Puerto Rico Rio Piedras Campus. After the
approbation of the qualifying exams, she transferred to the intercampus biology graduate
program with a major in biochemistry at University of Puerto Rico Rio Piedras Campus. She has
been working on her doctoral research project within the applied biochemistry and biotechnology
laboratory under the guidance of Dr. Kai Griebenow. As a graduate student, Yamixa has been
investigating the modification of proteins by glycans and fatty acids to develop potential drug
delivery systems for cancer therapy. Her graduate research projects have been presented at many
local and international scientific meetings. Furthermore, she will be finishing her PhD with three
first-authored scientific articles and five co-authored articles published in diverse peer-reviewed
journals. During her doctoral research, she earned different fellowships from NASA Puerto Rico
Space Grant Consortium (NASA-PRSGC), National Institute of Health (NIH), and National
Science Foundation (NSF).
Yamixa will complete the requirements for the Doctor of Philosophy degree in Biology
with concentration in biochemistry and biotechnology in June 2015.
15. xv
PEER REVIEWED PUBLICATIONS
Publications leading to the development of this doctoral thesis
Delgado Y, Morales-Cruz M, Hernández-Román J, Hernández G & Griebenow K (2015)
Development of HAMLET-like cytochrome c-oleic acid nanoparticles for cancer therapy.
Journal of Nanomedicine Nanotechnology (In press).
Delgado Y, Morales-Cruz M, Figueroa CM, Hernández-Román J, Hernández G & Griebenow K
(2015) The cytotoxicity BAMLET complex is regulated by the oleic acid and independent of α-
lactalbumin component. FEBS Open Bio 5, 397-404.
Morales-Cruz M, Delgado Y, Figueroa CM, Molina A & Griebenow K, Passive and active
targeting: the novel approaches for cancer therapy (Under Revision).
Delgado Y, Morales-Cruz M, Hernández-Román J, Martínez Y & Griebenow K (2014)
Chemical glycosylation of cytochrome c improves physical and chemical protein stability. BMC
Biochemistry 15, 16.
Additional publications
Morales-Cruz M, Figueroa CM, González-Robles T, Delgado Y, Molina A, Méndez J, Morales
M & Griebenow K (2014) Activation of caspase-dependent apoptosis by intracellular delivery of
cytochrome c-based nanoparticles. J Nanobiotechnol 12, 33.
Méndez J, Morales-Cruz M, Delgado Y, Orellano EA, Morales M, Figueroa CM, Monteagudo A
& Griebenow K (2014) Intracellular delivery of glycosylated cytochrome c Immobilized in
mesoporous silica nanoparticles Induces apoptosis in HeLa cancer cells. Mol Pharm 11, 102-
111.
Bansal V, Delgado Y, Legault M & Barletta G (2012) Low operational stability of enzymes in
dry organic solvents: changes in the active site might affect catalysis. Molecules 17, 1870-1882.
Castillo B, Delgado Y, Barletta G & Griebenow K (2010) Enantioselective transesterification
catalysis by nanosized serine protease subtilisin Carlsberg particles in tetrahydrofuran.
Tetrahedron 66, 2175-2180.
Bansal V, Delgado Y, Fasoli E, Griebenow K & Barletta G (2010) Effect of prolonged exposure
to organic solvents on the active site environment of subtilisin Carlsberg. J Mol Catal B: Enzym
64, 38-44.
16. xvi
ACKNOWLEDGMENTS
This doctoral dissertation was performed between January 2011 and May 2015 in the
Department of Biology, University of Puerto Rico. I feel really thankful to a lot of people and I
would like to express my gratitude to all the UPR professors, colleagues, and co-authors.
God, thanks for all the blessings in my life and the capability you put on me. I wish to
thank mami y papi for their support, love, and for giving me the tools to pursue my dreams. My
family: all of them fill a special place in my heart and I appreciate that they were there to
encourage me. My partner of life, Mael, thanks for been by my side giving me strength and
support when I thought to give up.
I really need to acknowledge my thesis mentor, Dr. Kai Griebenow for his unconditional
advice, for believing in my scientist skills, allowing me to conduct my research, and providing
any assistance and materials requested. I also dedicate this work and give special thanks to Dr.
Gabriel Barletta, the first professor who believed in me as a scientist, gave me the opportunity to
improve my knowledge, and to develop a lot of research techniques in his laboratory during my
BSc. I would also like to recognize the assistance of Dr. González, Dr. Cabrera, and Dr. Giray
who were more than generous for agreeing to serve on my committee, for their
recommendations, and all patience throughout the entire process.
I feel really thankful with the lab girls of “la covacha”: Moraima, Cindy, Anna, Zally,
and Freisa. I can’t forget your help with scientific issues and obviously, the good free times.
Undoubtedly, I could not finish without say thank you to Dr. Betzaida Castillo. She has been my
friend and a role model during my PhD.
Finally, I want to thank the financial support provided by grants from National Institutes
of Health (NIH SC1 GM086240), NASA Puerto Rico Space Grant Consortium Fellowship
(NASA-PRSGC NNX10AM80H), NIH Research Initiative for Scientific Enhancement Program
(RISE R25GM061151), Alfred P. Sloan Foundation and National Science Foundation Alliance
for Minority Participation Bridge to the Doctorate Program (NSF AMP-BDP HRD-0832961).
17. xvii
ABSTRACT
Proteins play a key role in the regulation of cell processes and recognition. The discovery
of proteins with pharmaceutical application has increased their importance in drug design. Thus,
a lot of new pharmaceuticals employ proteins (~ 300) to design new treatments that target
specific cellular processes. There are several challenges that most protein formulations face
during their use in biomedical pharmacology. The problem relies on the structural and chemical
protein instability and the general lack of knowledge about this matter. Proteins can be easily
denaturated by the stress environment during formulation (e.g., organic solvent exposure),
storage (i.e., high moisture and temperature), and parenteral delivery (i.e., acidic pH,
inmmunological and proteolytic response).
Several pharmaceutical approaches are available to overcome these problems to allow
high-dose protein delivery to the target tissue. Therapeutic formulations composed of surface-
modified proteins or lipid-proteins complexes can be synthetized to achieve specific
accumulation in the pathological area while improving the therapeutic index. In the case of
cancer therapy, some modified protein-based and micellar nanoparticles are actually FDA-
approved and show excellent pharmacological and clinical results.
Covalent modification, i.e., glycosylation (which is a post-translational modification that
consist in the covalent attachment of glycans to the protein), is a widely employed and fast
growing technology with the potential to improve the properties required for protein-based
biotherapeutics.
In the last decades, nanopartiulate drug delivery systems have opened new possibilities in
nanomedicine, especially in the field of protein formulations. Drug delivery systems have been
extensively utilized to enhance the efficacy of anti-cancer agents and to minimize systemic
18. xviii
toxicity. Numerous nano-sized platforms are being clinically approved in the drug delivery field
in order to obtain more effective therapeutics.
Fatty acids (FA) are one of the agents that are being used to develop new types of
intracellular delivery systems. Similarly, some FA such as monounsaturated oleic acid (OA) and
polyunsaturated linoleic acid (LinOA) exhibit in vivo cytotoxicity and antineoplasticity. This
novel property could support enhancement of drug potency.
For these reasons, in order to successfully utilize proteins as therapeutic agents, it is
necessary to prevent protein chemical and physical instabilities during the modification process.
Consequently, the goal of this project was to develop biologically stable protein formulations
for cancer therapy, such as bovine α-lactalbumin (α-LA), horse heart cytochrome c (Cyt c),
and bovine serum albumin (BSA) by covalent and non-covalent modification. The covalent
modification method studied was the attachment of the glycans lactose and dextran to Cyt c
discussed in the chapter 3. The non-covalent modification studied was the coupling of the FA
OA and LinOA to α-LA discussed in the chapter 4, and the coupling of OA to Cyt c and BSA
discussed in the chapter 5. All the materials and methods employed for the accomplishment of
this dissertation were described in the chapter 2.
This doctoral project represents an innovative application of therapeutically-relevant
proteins by applying surface-modifying strategies for the development of pharmaceutically-
stable protein formulations for cancer therapy.
19. xix
Polymeric particles in a wide variety of different shapes and sizes
Credit: Image from “How to make polymeric micro- and
nanoparticles” (2007) University of California College of
Engineering at Santa Barbara
Nano-sized delivery systems for
potential cancer therapies:
Modification of protein formulations
to improve pharmacological efficacy
20. Delgado (2015) Doctoral Dissertation
1
Chapter 1
Proteins are essential macromolecules to the organism in both cellular and molecular
functions, regulating most of the biological processes. Some of their biological functions include
enzyme catalysis, DNA processing, structural tissue components, transporting molecules, cell
signaling, and motility [1]. Proteins are encoded in genes and translated to amino acids (aa) that
are fold into unique 3-dimensional native conformation. The native-state protein structure is
stimulated by an effective attractive self-interaction arising from hydrophobicity [2]. The aa
composition and rearrangement convert the protein into an amphiphilic molecule that could be
easily chemically modified through the functional groups. Due to these unique functionalities
proteins are attractive alternatives for biomedical and nanotechnology applications [3]. However,
the problem of working with proteins is the chemical and physical instability that makes their
employment in nanomedicine difficult [4]. Conditions such as changes in pHs, temperatures, or
certain chemicals (organic solvents, salts, proteases) can cause tertiary structure perturbations
and thus, proteins may loose their bioactivity. In response, different approaches have emerged to
optimize the protein stability during formulation, after processing, and storage.
1.1 Proteins in biotechnology
Biotechnology was defined by The American Chemical Society as “the application of
biological organisms, biological molecules, systems, or processes by various industries to
learning about the science of life and the improvement of the value of materials and organisms
such as pharmaceuticals, crops, and livestock.” A biopharmaceutical is a biological product
made to develop pharmaceutical devices to treat or diagnose medical conditions and due to this,
today there are over 5000 biopharmaceuticals available [5].
21. Delgado (2015) Doctoral Dissertation
2
Proteins with therapeutic application are considered one of the most important
biopharmaceuticals. Since the 90s, by the advances in recombinant DNA technology, bacterial
expression, site directed mutagenesis, biocatalysts, metabolic engineering, and gene therapy, the
application of proteins and peptides in biotechnology rapidly increased [6,7], as shown in Table
1. In US, the Food and Drug Administration (FDA) granted 8 new protein formulations (i.e. 44%
of the total biopharmaceutical products) approvals in 2012 [8,9]. In contrast to small drug
therapeutics, most of the proteins are biocompatible and biodegradable because most of them are
inherently biological and our body has mechanisms for their degradation [10]. Another
advantage of proteins is the high physiological target specificity and low systemic toxicity
providing potent possible treatments for a lot of illness [11]. The specific biochemical
mechanisms of proteins promote the low administration doses helping to increase the patient
compliance [12]. In addition, high molecular weight (MW) proteins (>40 kDa) exhibit low renal
excretion promoting longer half-life circulation due to their hydrodynamic radius, while low
MW molecules are filtered through the glomerulus in short time [13,14]. From a
pharmacological view, most of the proteins show high solubility and an amphiphilic property
allowing the interactions with hydrophobic and hydrophilic moieties. This amphiphilic property
makes them an excellent material for NP synthesis [15]. Furthermore, proteins could be easily
chemically modified through the functional groups of the aa on the surface, conferring additional
target selectivity [16]. This promotes the specific action in the pathological place. But the fact
that proteins have a sophisticated structural conformation (primary, secondary, tertiary and
quaternary structure) causes the low physicochemical stability resulting in problems during
pharmacological process, systemic administration, and storage [17].
1.2 Protein instability problem
22. Delgado (2015) Doctoral Dissertation
3
Biopharmaceutical formulations need good structural stability in order to provide
acceptable shelf-life storage preserving the therapeutic efficacy. Researchers have been working
on the improvement of proteins as pharmaceuticals but it is still a persistent challenge due to
their biophysical properties inducing limited pharmacodynamic and pharmacokinetic profiles
[18].
The main problem of working with proteins to produce pharmaceuticals is their
inherendly limited chemical and physical stability to the environmental stress changes (e.g.,
extreme pHs, non-aqueous solvent exposure, high temperatures and moisture) during
formulation, administration and storage [19]. The intra- and inter-molecular interactions of the
protein primary structure can be perturbed during formulation and storage. These possible
adverse effects on the aa are redox (e.g., oxidation of disulfide bonds), deamidation, and the
destruction of labile Trp and Met side-groups [20]. Also proteins have the tendency to
experience secondary and tertiary structural damages during storage due to the fact that there is
only a minimal difference in thermostability between their folded and unfolded conformation
[21]. The three dimensional conformation of proteins is even more susceptible to environmental
stress because of the secondary and tertiary structure are due to energetically weak non-covalent
interactions i.e., hydrophobic, electrostatic, H-bonds, and London forces.These structural
changes raise protein unfolding and/or aggregation, and diminish its bioactivity [25]. After
administration proteins typically show short-term half-life and a tendency to undergo
denaturation. This is due to proteins are prone to in vivo degradation by endogenous proteolysis,
and the immunogenic response. There are proteases in the bloodstream and in the stomach that
can deactivate parenterally administered protein formulation.
23. Delgado (2015) Doctoral Dissertation
4
Table 1.1 Some proteins currently used or under research in pharmaceutical applications [22-24]
Biopharmaceutical Disease MW (kD) Indication
Insulin Diabetes 5.8 Inhibit the production of glucose and
remove the excess from blood.
Serum albumin Cancer 66.5 Tumor-selective accumulative carrier
Lysozyme Infection
Cancer
14.3 Antibacterial,
Antiviral,
Modulate tumor necrosis factor
generation.
Carboxypeptidase Cancer methotrexate toxicity treatment
Hepatitis B surface
antigen
Hepatitis B 24 Serologic marker
Interferon-alpha Hepatitis C
Cancer
19.4 Intiviral
Hyaluronidase Hydration
deficiency
54 Increase the absorption of other injected
drugs.
Staphylokinase Myocardial
infarction
15.5 Profibrinolytic agent
Cytochrome c (Cyt
c)
Cancer 12.4 Apoptotic inductor
Uricase Gout
Hyperuricemia
Degrade uric acid
α-Lactalbumin
(α-LA)
Cancer 14.2 Fatty acid drug carrier
Aprotinin Surgery 6.5 Fibrin sealant patch
Immune globulin Wiskott-Aldrich
syndrome
162 Primary immune deficiency
disorders
Arginine deaminase Cancer 45 Metabolize arginine
24. Delgado (2015) Doctoral Dissertation
5
Furthermore the opsonins of the immunological system can detect and target the foreign material
in the body for the elimination of the monocytes and macrophages (Figure 1.1) [26].
Protein-based therapies are not readily taken up by cells as are small-drugs. Many
proteins exhibit their target extracellularly and nevertheless, other ones have their bioactivity in
the cell cytoplasm or organelles. In the last case, this is an additional difficulty for the protein-
based formulations development due to a lot of them being membrane impermeable by their
charge and high MW. This hinders the protein absorption across the cell bilayer inducing poor
bioavailability, bioaccessibility, and rapid renal kidney filtration [27,28]. For these reasons, the
widespread distribution of the protein formulation into different organs requires the drug
administration in larger doses, which exceed the therapeutic index inducing systemic toxicity.
For these drawbacks, researchers are actively looking for rational formulation strategies
that could overcome the overall protein instability on pharmaceutical applications. To overcome
these limitations, in the last decades researchers have been working on chemical modification of
proteins.
1.3 Chemical modification of protein pharmaceuticals
Formulating a protein drug for delivery though systemic administration, requires
multitude of strategies. The dosage form needs to stabilize the drug making it resistant to
environmental conditions. The innate limitations of proteins could be overcome integrating
chemical modification technologies during the development of the formulation. Established
mechanisms include: aa mutations, surfactants, proteins crosslinking and bioconjugates using
PEG, glycans and FA [29]. However some of these procedures significantly alter and destabilize
protein properties. Normally aa substitutions on protein structure result in the disruption of its
function [30,31]. The use of oil–aqueous interface and organic solvents irreversibly denatures
25. Delgado (2015) Doctoral Dissertation
6
most proteins [32,33]. Even though, the protein formulation promotes solubility at physiological
pH values, it must overcome various physiological barriers for its successful delivery.
For the aim of this thesis, we are focusing on covalent and non-covalent chemical
modification by glycans and FA of pharmaceutically-relevant proteins to improve their
unfavorable biophysical properties. Protein modification using PEG and glycans can be
performed by direct modification of exposed side-chain aa groups such as Lys and Cys [34].
Moreover, we hypothesize that chemical modification can protect from physiological and
environmental stress factors. Also this method could protect from the immune system, and the
excretion by the kidney [35,36]. These new approaches also have been developed to diminish
injection frequency and increase patient compliance [37,38].
26. Delgado (2015) Doctoral Dissertation
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Figure 1.1 Representation of PEGylated particles shielded against opsonisation. Adapted from [12].
28. Delgado (2015) Doctoral Dissertation
9
1.3.1 PEGylation
Long-term stable proteins are required in order to maintain their therapeutic efficiency
while resisting metabolic processes [39].
PEGs work on protein’s surface as decorative molecules that build a layer against aggressive
conditions, i.e., increase half-life circulation, protect from the RES, and proteolytic degradation
after administration (Figure 1.1). Also, these PEG molecules protect the protein during storage
environment (e.g. against high humidity and temperatures) [40]. Some explanations about how
these moieties influence protein stability include decreasing the solvent accessible area and
reducing hydrogen bonds between protein and solvent [21,41]. The decorator’s size,
modification degree, and the modification sites may result in a different level of stabilization,
and resistance against proteases [42]. PEG shielding also improved the tumor accumulation and
ability to penetrate solid tumors in vivo [43]. PEGylation of pharmaceuticals is the most utilized
technique to protect and stabilize proteins and even small drugs in the market (Table 1.2).
Limitations of PEGylated proteins were proposed when the results from in vitro
experiments differ from in vivo results. Different groups [44,45] discovered that high MW
(>60kD) PEGylated proteins can accumulate in the liver, exceeding the kidney excretion limit.
However, PEGylated proteins can also accumulate in the liver by the hydrodynamic volume of
PEGs. The most important pitfall of PEGylation arises from the prolonged circulation and
accumulation of PEG moieties in different organs during treatment, giving rise to the
macromolecular syndrome [39,46]. In the same way as PEGylation, glycosylation provides a
shield able to protect proteins from external stressing conditions but with the biological
degradability advantage.
29. Delgado (2015) Doctoral Dissertation
10
Table 1.3 Most recent glycoprotein-based drugs in the market [34].
Product
Trade name
(Company)
Disease
Number
of glycans
Production
system
Agalsidase alfa Replagal®
(Shire) Fabry 6 HF cells
Agalsidase beta Fabrazyme®
(Genzyme)
Fabry 6 CHO cells
Alglucosidase alfa Myozyme®
(Shire) Pompe 7 CHO cells
Alpha 1-antitrypsin
(α1AT)
Prolastin®
(Talecris
Biotherapeutics)
Congenital α1AT
deficiency with
emphysema
3 Tissue
fractionation
Antithrombin III Atryn®
(Ovation
Pharmaceutics)
Berinert®
(CSL)
Thromboelitic events 3–4 Milk
fractionation
C1-esterase-inhibitor Cinryze®
(CSL) Hereditary
angioedema
6
7
Plasma
fractionation
Choriogonadotropin
alfa
Ovidrel®
(EMD
Serono)
Female infertility 8 CHO cells
Darbopoetin alfa Arasnep®
(Amgen) Anemia associated
with chronic renal
failure
5
1
CHO cells
Dornase alfa Pulmozyme®
(Genzyme)
Cystic fibrosis 2 CHO cells
Drotrecogin alfa Xigris®
(Eli Lilly) Sepsis 4 HEK cells
Galsulfase Naglazyme®
(Genzyme)
Maroteaux-Lamy
syndrome
6 CHO cells
Nesiritide Natrecor®
(Scios) Dyspnea 2 E. coli using
recombinant
DNA technology
Glucocerebrosidase Cerezyme®
(Genzyme)
Type I Gaucher 4 CHO cells
30. Delgado (2015) Doctoral Dissertation
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1.3.2 Glycosylation
Another technique currently being explored to overcome disadvantages of PEGylation in
pharmaceuticals is glycosylation. Glycans are crucial biological components which vary from a
single monosaccharide to branched polysaccharides in a wide range of MW. The biological
function of glycans has been debated for years. Most mammalian plasma proteins, cell receptors,
and membrane-bound proteins are glycosylated. A variety of glycan biological roles have been
proposed, but the most recognized function is that glycans are tags. This helps the organism to
recognize intrinsic and extrinsic glycoconjugates to promote the organism’s survival [47]. In the
cell, glycans can be bound to proteins and lipids to form glycoproteins and glycolipids. Natural
glycosylation of proteins are not expressed during the translation, due to the fact that glycans is
not directly encoded in the DNA genome [48]. In the lumen of endoplasmic reticulum, glycans
are attached to the N atom of the Asn side chain or O atom of the Ser side chain of the nascent
protein polypeptide by the oligosaccharyltransferase [49]. This process is a co-translational
modification that occurs while the protein is been folded.
In the past decades, different natural glycoproteins were FDA-approved as
pharmaceuticals. The biological efficiency of glycoprotein drugs in the market increased by
expressing their intrinsic glycans (Table 1.3). Different groups are working on the development
of chemical glycosylation techniques as an alternative to conjugation with PEG. We have
introduced chemical modification to stabilize several pharmaceutically relevant proteins. We
have investigated the protein structure, function, dynamics, proteolytic stability, and
thermostability in our newly synthetized glyco-bioconjugates [18,21,34,50-55]. In our studies,
we used FDA-approved sugars as lactose and dextran. We found that glycosylation provides the
advantages typically associated with PEGylation (e.g. long term circulation, biocompatibility,
31. Delgado (2015) Doctoral Dissertation
12
and proteolytic protection [56-59]), and that it also overcomes the undegradability problem.
Another glycosylation benefit is the stabilization of the protein by forming glycan-protein
hydrogen bonds. Another group introduced a chemical glycosylation method using maltose and
dextrose sugars. However, those are not completely biodegradable [60]. Most recently, a
research group employed a technology developed in 2004 to form a glyco-dendrimer-protein
complex to construct a nanosized mannose-dendrimer- protein that inhibits the Ebola virus [61-
63]. A good example of important glycans for cancer therapy could be the attachment of sialic
acid and hyaluronic acid to anticancer drugs to recognize the overexpressed lectins or CD44,
respectively, on cancer cell surfaces [64-67]. This means that the conjugation of carbohydrates to
therapeutic proteins extends their pharmacological effectiveness [68].
A synthetic glycosylation drawback is that most of the available techniques produce a
mixture of glycoforms with different properties (e.g. pharmacodynamics) that must be adjusted
for the therapeutic application [69]. Site-directed mutagenesis to generate glycosidic linkages on
recombinant proteins is an approach to avoid the heterogeneous glycoconjugates [70].
In this dissertation, we will discuss in the 1.4.2.2 Cytochrome c and in the chapter 2, the
effects (i.e., stability, functionality, and structural changes) of chemical glycosylation of a
pharmaceutically-relevant protein: Cyt c.
1.4 Passive targeted nanoparticles for cancer therapy
Cancer is a group of diseases in which cells lose the ability to halt division due to the
dependence on different mutated oncogenes. Some of the well-determined oncogenes are RAS
(cell proliferation, differentiation and survival) [71], WNT (cell division and migration) [72],
MYC (cell proliferation and apoptosis) [73], ERK (cell proliferation) [74], TRK (survival and
differentiation) [75], and p53 (suppress tumors by regulating gene expression) [76]. Most of
32. Delgado (2015) Doctoral Dissertation
13
these genes are responsible for the development of the cancer hallmarks (Figure 1.2). The top
five chemotherapeutic drugs commonly used cancer treatments in 2013 by THE 2013 DRUG
TREND REPORT were methotrexate, Gleevec®
, Xeloda®
, Revlimid®
and Lupron Depot®
. These
anticancer drugs lead problems related to systemic toxicity and poor life quality of the patient.
Therefore, nanotechnology brings many potential benefits to pharmaceutical nanomedicine
including the passive and active targeting for treatment and detection of cancer (Table 1.4).
Nanomaterials are used to encapsulate drugs to minimize the drug degradation and inactivation
after administration in patients. The major advantages of nanomaterials include the
functionalization with steering molecules (e.g. proteins, PEG, antibodies, vitamins, lipids,
glycans, growth factors, and ligands) to overcome drug’s solubility and stability problems, to
increase the blood circulation half-life, to increases concentrations of the anticancer drug within
the tumor tissue, and to selectively deliver the drug to specific tissues or organs [77-80]. Many of
the developed delivery systems under investigation combine multifunctional targeting
capabilities in order to fight the most difficult cancer hallmarks, such as drug resistance and
metastasis [15].
Passive drug delivery exploits the structural differences between normal and tumor tissue.
It is possible to develop particles with an appropriate size to penetrate the tumor capillary
fenestrations and accumulate inside the tumor interstitium by the enhanced permeability and
retention (EPR) effect [81,82]. This EPR effect is characterized by impaired lymphatic drainage,
leaky tumor vasculature between the endothelial cells, and high interstitial pressure (10-50 mm
Hg) [83]. The particle size is an extremely important aspect to enter into the irregular tumor
vasculature that exhibits the EPR effect (Figure 1.3). The physical features of nanoparticles are
the size, shape, and charge to control specific delivery into pathological areas. Particle of >20 nm
33. Delgado (2015) Doctoral Dissertation
14
is necessary to avoid renal filtration [84,85]. The optimum size for nanoparticles will depend on
the biomedical application. The size of fenestrae is from 100 to 800 nm [81,86,87], unlike the
tight endothelium of normal vessels (i.e., 5-10 nm) [88]. Many nanoparticle formulations have
shown high stability in embedding both hydrophobic drugs and hydrophilic drugs.
Even more important than the diameter of the NP is the homogeniety of the particle. The
preparation methods for nanoparticles have to be reproducible and promote a monodisperse
sample. Polydispersity can cause low tumor accumulation, less efficiency and as a consequence,
lead to a higher requiered drug dose [89]. Another feature that the drug needs is a high MW
(>40 kD) because it has been demonstrated that it could accumulate spontaneously in tumors
[90]. The in vivo kinetics adsorption of macromolecules (e.g. proteins) depends on particle size
(i.e. around 200 nm) and surface hydrophobicity [91]. For intravenous administration of
anticancer drugs, generally the diameter size of the NP is < 500 nm [92].
34. Delgado (2015) Doctoral Dissertation
15
Table 1.4 Clinically approved nanoparticle systems [83,93,94]
Trade name Nanoplatform Disease Status (year) Company
Abelcet®
/
Ambisome®
Amphotericin B lipid
complex
Fungal infections Approved (1995) Enzon
DaunoXome® Liposome-daunorubicin
conjugate
Kaposi’s sarcoma Approved (1996) Galen Ltd.
Feridex®
Dextran-iron oxide Liver/spleen lesion
imaging
Approved (1996) Berlex Laboratories
Rapamune®
Antibiotic Colorectal cancer Approved (1999) Wyeth
Resovist®
Dextran-iron oxide Liver/spleen lesion
imaging
Approved (2001)
Europe
Bayer Schering
Pharma AG
Estrasorb®
Estradiol micelle emulsion Menopausal hot
flashes
Approved (2003) Novavax
OncoTCS®
Liposome-vincristine
conjugate
Non-Hodgkin’s
lynphoma
Approved (2004) Inex & Enzon
Myocet®
Liposome-doxorubicin
conjugate
Metastatic breast
cancer
Approved (2005)
Europe, Canada
Cephalon (Europe)
Sopherion (Canada)
Doxil/Caelyx®
PEGylated Liposome-
doxorubicin conjugate
Ovarian and breast
cancer
Approved (2005) Ortho
Biotech/Schering-
Plough
Abraxane®
Albumin-paclitaxel
conjugate
Metastatic breast
cancer
Approved (2005) Celgene
Megace®
Megestrol acetate Breast and
endometrial cancer
Approved (2005) Bristol-Myers
Squibb
TriCor®
Fenofibrate hypolidemic
agents
Reduction of
cholesterol
Approved (2005) AbbVie
Nanoxel®
Docetaxel-loaded PEG-
micelle
Breast cancer Approved (2007) Fresenius Kabi
DepoCyt(e)®
Sustained-release
cytarabine liposome
Lymphomatous
meningitis
Approved (2007) SkyePharma
Emend®
NK I receptor agonist Suppression of
chemotherapy-
induced nausea
Approved (2008) Merck
Genexol®
Polymeric micelle- Lung Cancer Phase II US, Samyang
35. Delgado (2015) Doctoral Dissertation
16
paclitaxel Approved Korea
Endorem®
Dextran-iron oxide Liver/spleen lesion
imaging
Approved Europe Guerbet
ThermoDox®
Heat-activated
doxorubicin liposomal
Breast and liver
cancer
Phase I/II US Celsion
Rexin-G®
Tumor-targeted
retrovector cyclinG1 gene
Sarcoma,
pancreatic cancer
Phase III US
Approved Philippines
Epeius
Biotechnologies
Figure 1.2 Benign cells vs malignant cells: Hallmarks of cancer and tumor microenvironment
[95, National Cancer Institute.com]
36. Delgado (2015) Doctoral Dissertation
17
1.4.1 Lipid-based formulations
Lipids play key roles as membrane components and in biological processes, such as
metabolic fuels working on storage, and transport of energy in the body. Also, lipids are poorly
water soluble with the tendency of self-association forming micelles when reaching the critical
micellar concentration (CMC) [96]. Several nano-sized micellar drug formulations have entered
in clinical trials and others are clinically approved e.g., Genexol®
, Nanoxel®
, Estrasorb®
(Table
1.4).
Micelles are made of self-assembling amphiphilic molecules consisting of hydrophobic
core and hydrophilic shell. They are exploited due to their feasibility to incorporate even
hydrophobic or hydrophilic drugs [97]. This lipid arrangement provides a micellar corona for
protection from biodegradation by proteases and RES [98]. An additional benefit is that lipids
possess better stability than other surfactants due to their intrinsically low CMC [94]. Also, the
surface of the micelle can be chemically decorated to increase the stability of the delivery system
and to improve the therapeutic response [99,100]. When a protein is the active agent in the
formulation, lipid-based coating could confer protection for the susceptible protein structure and
induce cell internalization [101,102].
Liposomes are another type of lipid arrangement usefull for drug delivery systems.
Liposomes are spherical lipid bilayers separated by internal aqueous phases. Table 1.4
summarizes some of the NP systems that are composed of liposomal-drug conjugates (e.g.
Abelcet®
/Ambisome®
, DaunoXome®
, OncoTCS®
, Myocet®
, Doxil/Caelyx®
, DepoCyt(e)®
,
ThermoDox®
). Another advantage of liposomes systems is the ability to penetrate the blood
brain barrier, which permits drug delivery to treat brain malignancies [103]. This property was
discovered using a liposome-encapsulated horseradish peroxidase and a liposomal glucose
37. Delgado (2015) Doctoral Dissertation
18
oxidase showing after injection the presence of the enzymes in brain tissues [104,105]. The
following advantages could be realized with lipid-based formulations: targeted drug release,
improved drug stability, high drug content, use of lipo- and hydrophilic drugs, excellent
biocompatibility, inexpensive production, and easy sterilization [106].
1.4.1.1 Fatty acids (FA)
A FA is structurally long (e.g. C12-22) water-insoluble carboxylic acid with a
hydrocarbon chain usually derived from triglycerides and phospholipids. FA represent one of
the most important energy dietary source yielding large amount of ATP, i.e., grain, vegetable,
and fish oils, dairy and meat products. Some of the biological functions of FA are platelet
aggregation, lipid peroxidation, membrane flexibility, protein acylation, and gene regulation
[107]. Due to their insolubility, in the bloodstream free FA are transported by serum albumin. FA
could be divided in two groups: unsaturated and saturated FA. The saturated FA have no double
bonds and have a straight hydrocarbon tail. The most abundan saturated FA in animals is stearic
acid (SA,18:0). The unsaturated ones have one or more carbon-carbon double bonds in either cis
or trans configuration. These insaturations bend and restrict the hydrocarbon chain geometry of
the FA. For this reason membranes are composed of a mixture of different saturated and
unsaturated FA conferring specific cell flexibility. In the unsaturated FA, there are the omega
(ω)-3 (configuration: Cis-Δ9,12,15) (e.g. linolenic acid), ω-6 (configuration: Cis-Δ9,12) (e.g.
LinOA) and the ω-9 (configuration: Cis-Δ9). ω-3 and ω-6 are essential nutrients which must be
obtain from food and can be metabolized in the mammalian body by desaturases [108]. Essential
FA have to be ingested because humans lack the ability to add unsaturations to long FA. The
trans unsaturated FA, normally known as “trans fats” are not naturally synthetized and are
produced by the FA hydrogenation during the food processing.
38. Delgado (2015) Doctoral Dissertation
19
As regards to the therapeutic use, unsaturated FA proved to induce cell death (apoptosis and
necrosis), growth inhibition, and sensitize cells to anticancer drugs [109-112]. Also, unsaturated
FA could be internalized by peroxisome-activated receptor-mediated endocytosis or by diffusion
(e.g., flip flop) across the cell membranes [113,114]. Furthermore, even the saturated SA
demonstrated to be useful for the brain-targeting in an amphiphilic copolymer micellar
doxorubicin delivery system [115]. Non-covalent modification of proteins and peptides using FA
(e.g., SA or palmitic acid) was shown to enhance cellular uptake and targeting of the FA-protein
complex [116].
Figure 1.3 Scheme of the passive targeting by the enhanced permeability and retention (EPR) effect.
Tumor tissues usually lack effective lymphatic drainage and exhibit an irregular endothelial vasculature.
Nanoparticles (green) can extravasate into the tumor interstitium.
39. Delgado (2015) Doctoral Dissertation
20
1.4.1.1.1 Oleic acid (OA)
OA (18:1 Cis-Δ9; 282.5g/mol MW) is the most common monounsaturated FA in animals
and the major component of the cell membrane phospholipids (e.g., phosphatidylcholine). OA is
an abundant FA that can be found in olive, canola, coconut, soybean, almond, pecan, peanut,
macadamia, sunflower, grapeseed, sesame, poppyseed, and buckthorn oil [117,118]. In the body,
the biosynthesis of OA is in charge of stearoyl-CoA desaturase dehydrogenating SA. This FA
has been used in our cuisine since antiquity and modern studies show evidence of their
therapeutic benefits, i.e. decrease levels of low density lipoproteins [119] and reduce blood
pressure [120]. In 2006, Curi-Boaventura [114] demonstrated that OA cause in vitro cell death
by apoptosis, lipid peroxidation and reactive oxygen species.
Related to cancer, OA has demonstrated its application in the development of the delivery
systems (e.g. micelles and liposomes) and as an anticancer agent. An example of this is the
discovery of a FA-protein complex called Human Alpha lactalbumin Made LEthal to Tumor
cells (HAMLET) between the milk protein α-LA and OA responsible to cause cell death
exclusively in immature cells [121]. After a few years of studies, the arguments of cytotoxic
source of HAMLET/BAMLET (the bovine counterpart) have been debated presenting
contradictory results. Some arguments came from the development of HAMLET-like complexes
using α- LAs homologues i.e. lysozyme [122], β-lactoglobulin [123], parvalbumin [124], and
lactoferrin [125]. These complexes showed very similar HAMLET cytotoxic activities,
suggesting that the protein sequence does not influence the tumoricidal action. These results
proposed that OA component is the key of cytotoxicity of HAMLET.
40. Delgado (2015) Doctoral Dissertation
21
Another case is the sophorolipids which recently showed anti-inflammatory and
anticancer effects [126]. These are microbial glycolipids mixtures composed mostly of OA and
dimeric glucose forms. Human liver [127], pancreatic [128], and esophageal [129] cancer cell
lines showed susceptibility when were treated with sophorolipids. In addition, sophorolipids
have the capability to be easily surface-modifiable giving another advantage on the development
of delivery systems.
1.4.2 Protein-based formulations
Protein formulations have been broadly used for the development of cancer therapies.
Proteins are been used as the versatile natural equivalent polymer of the synthetic polymers for
the design of drug delivery systems. Over the years, proteins have shown the ability to be the
drug carriers for targeting in the enhancement of the delivery [130]. Also, they could improve the
pharmacokinetic outcomes of clinical therapeutic agents. The use of proteins in NP-based
formulations gained great interest due to their biological advantages over other polymers. Some
of the benefits were mentioned in section 1.1 Proteins in biotechnology. In addition, there are
some technical advantages for protein-based NP as the available methods to synthetize them and
the feasibility to characterize the proteinaceous component in the system, e.g. concentration and
size distribution. Proteins are biomolecules with special functionalities, such as their
amphiphilicity (as previously mentioned for the FA) that tolerate hydrophilic and lipophilic
interactions which confer the possibility to be utilized with many types of drugs [131]. However,
so far, even when proteins are actively used as therapeutic agents, there have been very few
studies applying the use of nanosized protein-based carriers for drug delivery. Some examples
are casein [132], β-lactoglobulin [133], human serum albumin [134], gelatin [135], transferrin
41. Delgado (2015) Doctoral Dissertation
22
[136], and zein [137]. Some of these are hydrophobic proteins—this left the door open for the
research of water soluble proteins as carriers.
Figure 1.4 Cartoon representation of the structures of A. horse heart cytochrome c (1HRC PDB)
and B. bovine α-lactalbumin (1F6S PDB).
1.4.2.1 α-Lactalbumin (LA)
α-LA is a mammalian milk protein composed of 123 aa (14 kD; Figure 1.4B) stabilized
by four S-S bridges and it can bind co-factors as Ca+2
and Zn+2
. This protein contains two
structural domains: a large alpha domain (four α-helices) and a small beta domain (a β-sheet and
loop) [145]. The fundamental function of α-LA is to produce lactose as part of the regulatory
subunit of the lactose synthase and the catalytic component of β-1,4-galactosyltransferase. α-LA
works enhancing the glucose affinity and inhibiting the polymerization of galactose. Also, the
structure of α-LA is highly composed of essential aa (i.e. 6 % of Trp, 11 % Lys and 6 % of Cys
molar content) [146,147]. At acidic pH, a conformational transition to α-LA occurs that is
considered a more flexible collapsed state well-known as the molten globule state [148]. In this
state the protein maintains most of its secondary structure but has a perturbed tertiary structure.
42. Delgado (2015) Doctoral Dissertation
23
This partially denatured α-LA has the capability to bind OA forming a cytotoxic complex
discovered in 1995 [121] by a research group that were studying the properties of breast milk.
This complex was named HAMLET/BAMLET.
In chapter 4, we are going to investigate the properties of this BAMLET complex and
the role of the OA and the bovine α-LA components due to its main cytotoxic constituent is still
under debate. We hypothesize that OA is the cytotoxic source and the α-LA protein just the OA
carrier. Thus, we synthetized three BAMLET complexes comprised of OA non-covalently
coupled to α-LA. These complexes were obtained using varying synthesis conditions to shed
light into the substantial debate on the role of the α-LA protein. Our data suggest that OA has to
reach critical micelle concentration to form active BAMLET particles, which have an
approximate hydrodiameter of 250 nm. Proteolysis experiments on BAMLET show that OA
protects the protein and likely is located on the surface consistent with a micelle-like
structure. Native or unfolded α-lactalbumin without OA lacked any tumoricidal activity. In
contrast, OA alone killed cancer cells with the same efficiency at equimolar concentrations as its
formulation as BAMLET. The contradictory literature results on the cytotoxicity of BAMLET
might be resolved by our finding that it was imperative to sonicate the samples to obtain toxic
OA. The cytotoxicity of the complex was modulated by the fatty acid: while polyunsaturated
LinOA was as cytotoxic as OA, the saturated stearic acid (SA) was not. Our data unequivocally
show that the cytotoxicity of the BAMLET complex is exclusively due to OA and that OA alone
when formulated as a micelle is as toxic as the BAMLET complex. Stability experiments show
that BAMLET retains more activity over time than the OA micelles alone, which suggest the
protein still has an important function probably in the delivery of the lipid payload in clinical
applications.
43. Delgado (2015) Doctoral Dissertation
24
1.4.2.2 Cytochrome c (Cyt c)
Cyt c is a highly water soluble hemeprotein (~12 kD MW; Figure 1.4A) associated with
few cellular functions, such as; peroxidase activity, in the oxidative phosphorylation in the
mitochondria, and as an inductor in intrinsic and extrinsic apoptotic process after its release to
the cytoplasm. In cellular respiration, Cyt c works as an electron transporter undergoing redox
reactions where the heme carries the electrons [37]. In the induction of apoptosis, Cyt c is
covalently bound to the cardiolipins on the inner mitochondrial membrane until the induction of
apoptosis by BAX oligomerization [38]. Thus, increase in mitochondrial calcium levels and
reactive oxygen species promote the oxidation of the Cyt c- cardiolipin complex. The free Cyt c
is released to the cytoplasm to interact with the apoptotic protease activating factor-1 (Apaf-1),
forming a multi-subunit complex known as the apoptosome [140].
As Cyt c is a very positively charged protein, mostly due to the 19 Lys residues. Some of
the interactions sites with Apaf-1 are Lys residues and residues near the solvent-exposed heme
group [141]. However, the nitration of the Cyt c Tyr 46, 48 and 74 demonstrated to impairs the
apoptosome formation and the activation of caspases demonstrating that other residues are
necessary for the interactions between Cyt c/Apaf-1 [142,143]. In presence of ATP, the
apoptosome complex triggers the cascade caspase by the activation of the executioners caspase-
9, caspase-3 and, caspase-7, which are responsible for completely killing the cell [144]. Due to
these roles, Cyt c has strong potential in the development of Cyt c-based formulations for cancer
therapy [138].
In chapter 2 and 4 we investigated the covalent and non-covalent modifications of Cyt c
by glycans and FA, due to, the use of proteins as therapeutic agents or as drug carriers in
formulations are still a challenge.
44. Delgado (2015) Doctoral Dissertation
25
Specifically in chapter 2 we explored chemical glycosylation of Cyt c as a method to
increase protein stability and thus enhance their long-lasting bioavailability. Thus, three different
MW glycans (lactose and two dextrans with 1 kD and 10 kD) were chemically coupled to surface
exposed Cyt c Lys residues using succinimidyl chemistry via amide bonds. Five neo-
glycoconjugates were synthesized, Lac4-Cyt-c, Lac9-Cyt-c, Dex5(10kD)-Cyt-c, Dex8(10kD)-Cyt-
c, and Dex3(1kD)-Cyt-c. Subsequently, we investigated glycoconjugate structure, activity, and
stability. Circular dichroism (CD) spectra demonstrated that Cyt c glycosylation did not cause
significant changes to the secondary structure, while high glycosylation levels caused some
minor tertiary structure perturbations. Functionality of the Cyt c glycoconjugates was determined
by performing cell-free caspase 3 and caspase 9 induction assays and by measuring the
peroxidase-like pseudo enzyme activity. The glycoconjugates showed ≥94% residual enzyme
activity and 86 ± 3 to 95 ± 1% relative caspase 3 activation compared to non-modified Cyt c.
Caspase 9 activation by the glycoconjugates was 92 ± 7% to 96 ± 4% (the same activation
observed for caspase 3 within the marging of error). There were no major changes in Cyt c
activity upon glycosylation. Incubation of Dex3(1 kD)-Cyt c with mercaptoethanol caused
significant loss in the tertiary structure and a drop in caspase 3 and 9 activation to only 24 ± 8%
and 26 ± 6%, respectively. This demonstrates that tertiary structure intactness of Cyt c was
essential for apoptosis induction. Furthermore, glycosylation protected Cyt c from detrimental
effects by some stresses (i.e., elevated temperature and humidity) and from proteolytic
degradation. In addition, nonmodified Cyt c was more susceptible to denaturation by a water-
organic solvent interface than its glycoconjugates, important for the protein formulation with
polymers. These results demonstrate that chemical glycosylation is a potentially valuable method
45. Delgado (2015) Doctoral Dissertation
26
to increase Cyt c stability during formulation and storage and potentially during its application
after administration.
In chapter 4, we investigated the development of HAMLET-like complexes composed of
OA coupled to Cyt c and as a non-toxic control, BSA. After we demonstrated that OA is the real
therapeutic agent, we hypothesized that by replacing α-lactalbumin with a protein with
bioactivity one should be able to synergistically increase the toxicity towards cancer cells. The
coupling of OA to Cyt c and BSA was performed at pH 8 and 45ºC and we obtained HAMLET-
like Cyt c-OA and BSA-OA complexes. The syntheses of HAMLET-like Cyt c-OA and BSA-
OA complexes micelles were performed at pH 8 and 45ºC and we loaded 10 and 53 molecules of
OA per molecule of Cyt c and BSA in the micelles, respectively. Our data indicate that OA
promotes protein structural changes characteristic of the protein-OA interactions in HAMLET.
We found that Cyt c-OA and BSA-OA complexes obtained had a circular shape and a diameter
of 123 and 169 nm. Cell viability tests revealed cytotoxicity of both complexes within 6 h
towards cancer (HeLa and A-549) and normal (Cho-K1 and NIH/3T3) cell lines. Some
selectivity towards killing cancer cells was displayed by Cyt c-OA. The cancer cell lines showed
less than 10% of viability when they were incubated with Cyt c-OA while normal cells showed
more than 20% of viability. BSA-OA killed both cell types with less efficiency than Cyt c-OA
and displayed no selectivity. Cyt c-OA complex revealed 50% of caspase-3 and caspase-9
activation in a cell-free assay while BSA-OA lacked any caspase activation. Confocal
micrographs showed the morphological changes characteristic of apoptosis induction by the
action of HAMLET-like complexes. This study demonstrates that using Cyt c increases the
potency of OA in HAMLET-like complexes.
46. Delgado (2015) Doctoral Dissertation
27
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