Orally administered anti-cancer nanocarriers loaded with therapeutic proteins

257 Pages·2016·30.3 MB·English

Orally administered anti-cancer nanocarriers
loaded with therapeutic proteins
by
Mahidhara.V.N.L.Ganesh
(M.Sc Biotechnology)
Submitted in fulfillment of the requirements for the degree of
Doctor of Philosophy
Deakin University
May, 2012

Abstract:
Cancer is an abnormal chronic inflammation and still remains a significant cause of
morbidity and mortality with more than 10 million new cases per year. Dietary
neutraceuticals along with recent innovations in genetic engineering could be used as an
alternative therapeutic strategy to overcome adverse side effects imposed by
chemotherapeutics. 100% iron saturated multi-functional, anti-cancer bovine lactoferrin (Fe-
bLf) and dominant negative survivin (Sur R9-C84A) are such proteins which are recently
proved to be anti-cancerous. Nevertheless, short plasma half lives of bio-molecules like these
demand painful infusions over a short time periods, an issue was tried to address by using
nanotechnology. Moreover, oral delivery of these therapeutic proteins could be made possible
by using bio-degradable nanoformulations utilizing the advantages of bio ceramic/polymeric
combinations. Also, nano structures could have better uptake in tumour vasculature based on
the enhanced permeability and retention effect. Thus, the hypothesis examining the ability of
these nanoformulations not only to conserve the native configuration of these bio-active
proteins but to also entail their tumour specific delivery can therefore be studied.
This study demonstrated the formulation of a novel alginate enclosed, chitosan coated
ceramic anti-cancer nanocarriers (ACSC NCs) designed to aid as an additional protective
system towards gastrointestinal sustainability of these fragile biomacromolecules. These NCs
were loaded with Fe-bLf along with Sur R9-C84A in order to develop a novel platform to
carry anti-cancer proteins and/or peptides for oral therapy. Thus, ACSC NCs carrying Fe-
bLf, Sur R9-C84A or a combination of both were prepared by using nanoprecipitation and
ionic gelation methods. Size, morphology, internalization, gastrointestinal sustainability
profiles and in vitro protein release of the NCs under varying pH – were determined for all
three combinations of NCs. Cytotoxicity of the Fe-bLf/Sur R9-C84A loaded NCs was studied
in colon and breast cancer cell lines. Cellular uptake of these NCs in vitro in colon/breast
cancer lines was analyzed, by measuring the endocytosis and transcytosis.
Additionally, the study examined the enhanced anti-cancer efficacy, bio-activity and tissue
distribution of orally fed nanoformulated Fe-bLf in vivo as an extension of a previous study
performed in our lab. A diet containing Fe-bLf loaded NCs was given to the experimental
nude (C57BL 6 nu/nu) mice and the mice were subsequently challenged with xenogenic
(human colon and breast cancer cells) tumours. To be able to investigate the possible route
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for the internalization of the NCs and their apoptotic efficacy, real time analysis of the genes
involved in the internalization pathways and pro/anti-apoptotic mRNA were analyzed.
Electron microscopy and dynamic light scattering (DLS) results showed sphericity of the
NCs with a size of 205 ± 15 nm for the CSC inner core and increased to 322 ± 27.2 nm after
enclosing with alginate in order to form a final ACSC NCs. SDS PAGE followed by Western
blotting, using specific antibodies against bLf confirmed the structural integrity of the protein
after the nanoformulation; X-ray diffraction studies indicated amorphous properties of the
NCs. Confocal microscopy and Flow cytometry determined the qualitative and quantitative
internalization of rhodamine labeled NCs, upon treating the two cancer cell lines. Entrapped
protein in the NCs enhanced their cellular uptake by around 3 fold compared to control/void
NCs without protein. For example, void NCs have showed 25.1 ± 3.7% internalization in 1 h
where as Sur R9-C84A loaded [55.4 ± 1.6% (P<0.05)]; Fe-bLf alone loaded [58.6 ± 6.0%
(P<0.05)] and a combination of Fe-bLf and Sur R9-C84A [64.3 ± 4.6% (P<0.05)] have
shown increased uptake. In vitro cytotoxic and cell proliferation assays have established an
improved anti-cancer efficacy of the proteins upon nano encapsulation. Transcytosis studies
have shown that the NCs cross the membrane with no/minimal damage to the Caco-2 cell
monolayer as a confirmation of endocytic and transcytotic mechanisms.
Furthermore, superior anti-tumour efficiency and nano toxicity was measured in vivo in nude
mice bearing xenogeneic tumours. Mice pre-treated with a diet containing the NCs were
observed to reject the tumours induced with colon and breast cancer cell lines (Caco-2 and
MDA-MB-231) derived from human origin. Histopathological and immunohistochemical
studies demonstrated the desired internalization of nanoparticles in tumour tissues without
any nanotoxicity. Real time PCR analysis indicated an over expression of transferrin
receptors (Tfr) and low density lipoprotein binding receptors (LRP) specific to lactoferrin
along with transferrin receptors, as an indication of a possible route of internalization. Anti-
apoptotic genes such as survivin and B-cell lymphoma (Bcl) family of genes were found to
be down regulated with treatment of the NCs, whereas up regulation of pro apoptotic Bax,
FAS and the executors such as caspase 3 indicated promotion of apoptosis in the tumours by
the NCs.
Taken together, the data from this study suggest for the first time that the ACSC NCs system
is efficient in encapsulation of the bio-molecular drugs Fe-bLf/SurR9-C84A by maintaining
their bio-activity and significantly enhancing their anti-cancer efficacy. Nano encapsulation
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of proteins further improved the cellular internalization of the NCs. Fe-bLf delivered as an
oral nanoformulation offers enhanced anti-tumorigenicity. No signs of nanotoxicity or
biodistribution in RES organs confirmed the biodegradability of the NCs. Herein, as a proof
of concept, the NCs were successfully tested in colon and breast cancer neoplasms, this
principle could be extended for other solid tumours. Nevertheless, the NCs could be used to
extend for the cure of difficult to treat malignancies. Future experiments related to insights of
up/down regulation of angiogenic mediators and inhibitors could suggest a clear mechanistic
view of the anti-cancerous nature of these novel nanocarriers.
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Acknowledgements
I am heartily thankful to my principle supervisor, Associate Professor Jagat R. Kanwar,
whose encouragement, guidance and support from the day one to the final level with his
advice, dedication and unsurpassed knowledge enabled me to develop an understanding of
the subject. His impact on me as a supervisor and mentor is immense.
The high-quality advice, support and friendship of my co-supervisor, Dr. Rupinder K.
Kanwar, has been invaluable on an academic, philosophical and personal level, for which I
am extremely grateful.
I shall concede the support of Deakin University for International Postgraduate Research
Scholarshipand AISRF grant for the financial support to carry research with the sophisticated
technology provided.
I thank Bio Deakin, ITRI Directors & staff, especially to Mrs. Elizabeth Laidlaw, Dr. Andrew
Sullivan, Dr. Pavel Cizek, Mr. Rob Pow, Dr. Ben Allardyce, Ms. Kim Durrant Ms. Sandy
Benness, Mr. John Robin, Dr. Chris Hurren and Ms. Leanne Costa for their help. I also want
to thank animal house staff, Dr Nick Branson, Mr. Adrian Cooper, Ms. Tania Thorpe, Mr.
Bruce Newell and Ms. Mary McGlynn for their support with the animal handling and
training.
I thank my fellow mates of the LIMBR group, Sara, Hannah, Rasika, Sishir, Jessica, Rangam,
Kislay, Sneha, Neha, Yogesh, Bhasker, Amy, Fawzi, Jayanth, Ajay, Nester, Ronak, Alicia,
Olja and Sam for their constant help, motivation and combined learning.
To my family and friends for their love, encouragement and support, which motivated me to
work muchharder to achieve and set new goals for the life.
Last but not least I thank the almighty goddess Gayatri for all those days with new beginnings
and successful endings throughout the past 160 weeks of my PhD studentship.
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Table of Contents Page No.

Abstract……………………………………………………….......... i
Acknowledgements…………………………………........................ iv
List of publications………………………………………………..... xi
1-List of Figures…………………………………………………......... xiii
List of tables……………………………………………………….... xvi
List of Abbreviations ……………………………………………….. xvii
Chapter 1 : Introduction 1
1.1. Foundationsand background of the proposed study 1
1.2. Significance of this study - hypothesis and research question 3
1.3. Aims and objectives 5
Chapter 2 : Literature review 9
2.1 Preface 9
2.2 Is the cancerous state a chronic inflammation? 11
2.3 Apoptosis –a well planned suicide 13
2.4 Angiogenesis and tumour progression 15
2.5 Modulators of Angiogenesis 18
2.5.1Angiogenic factors 18
2.5.1.1. Integrins 18
2.5.1.2. Vascular endothelial growth factor 18
2.5.1.3. Angiopoietins 18
2.5.1.4. Fibroblast growth factors 19
2.5.1.5. Transforming growth factor(cid:69)(cid:3)(TGF(cid:69)(cid:12) 19
2.5.1.6. CXC-Chemokines 19
2.5.2 Angiostatic factors 19
2.5.1.1.Angiostatin 20
2.5.1.2.Thrombospondin-1 20
2.5.1.3.Endostatin 20
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2.6 Need for alternative therapy in cancer cure 22
2.7Uses of micro RNAs in cancer therapy 23
2.8Survivin –a hot anti-cancer target 26
2.9Natural anti-cancer molecules in alternativetherapy 29
2.9.1 Lactoferrin 31
2.10 Iron homeostasis and its sequestration by lactoferrin 32
2.10.1Does excessive iron causes toxicity? 33
2.10.2Sequestration of iron by iron-binding proteins 34
2.11Advantages of delivering anti-angiogenic/ anti-cancer molecules withinnanocarriers
37
2.12Uses of nanoparticles in cancer treatment 41
2.13 Use of inorganic biomaterials in medicine 43
2.14 Bio ceramics used in medical applications 43
2.15 Uses of bioceramics as nanoparticles 46
2.16 Oral delivery of anti cancer therapeutics 49
2.17 Scope for using multi layered polymeric/ceramic nanocarriers 52
Chapter 3 : Preparation and characterization of ACSC NCs with naturalLf 54
3.1Introduction 54
3.2Results 56
3.2.1 Standardization of formulation conditions for ACSC Lf NC 56
3.2.2 Physical characterization of ACSC Lf NCs 59
3.2.3 Analysis of the natural bLf configurationafter the formulation 62
3.2.4 Cell viability studies 64
3.2.5 In vitrointernalization studies of CSC Lf NCs in Caco-2 cells 66
3.2.6 Internalization of NCs in Caco-2 cells – Confocal microscopic and
immunocytochemical studies 68
3.2.7 In vitrotransport of CSC NCs via Caco-2 cell monolayer 70
3.3Discussion 72
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Chapter 4 : Validation of bio-availability of the Sur R9-C84A after encapsulation inthe ACSC
NCs 77
4.1Introduction 77
4.2Results 79
4.2.1Sur R9-C84A loaded NCs show spherical morphology with 330nm size 79
4.2.2ACSC NCs show controlled release profiles of the encapsulated protein 81
4.2.3Cell apoptotic studies shows maximum cell death with NCs treatment 83
4.2.4 Cellular uptake of CSC Lf NCs via Caco-2 cell monolayer(quantitative study) 86
4.2.5 Cellular uptake of CSC LfNC via Caco-2 cell monolayer (qualitative study) 88
4.2.6 Down regulation ofendogenous survivin expression after treatment with NC 90
4.3Discussion 92
Chapter 5 : Dual protein loaded nanocarriers for in vitro evaluation of anti-cancer efficacy 96
5.1Introduction 96
5.2Results 98
5.2.1 Characterization of NCs 98
5.2.2 Protein loaded NCs shows controlled release profiles 100
5.2.3 Chemical bonding and the thermal stability assessment of NCs 102
5.2.4 Cell apoptosis assessment after treatment with nanocarriers 104
5.2.5 In vitrointernalization profiles of the NCs 107
5.2.6 Quantitative determination of NCs internalization in cancer cells 109
5.2.7 Determination of mitochondrial membrane potential ((cid:39)(cid:92)m) after the treatment
111
5.2.8 Endocytosis of CS CFS NCs 113
5.3Discussion 115
Chapter 6 : In vivoanti-tumour efficacy of ACSC NC encapsulating Fe-bLf 119
6.1Introduction 119
6.2Results
6.2.1 Physicochemical characterization of Fe-bLf loaded ACSC NCs 122
6.2.2 Characterization of crystal structure for the ACSC NCs 124
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