Applications of multifunctional poly(glycidyl methacrylate) (PGMA) nanoparticles in enzyme stabilization and drug delivery Tristan DeVere Clemons, BSc (Hons) This thesis is presented for the degree of Doctor of Philosophy of the University of Western Australia School of Chemistry and Biochemistry School of Animal Biology 2013
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Applications of multifunctional poly(glycidyl methacrylate) (PGMA) nanoparticles in enzyme stabilization and drug delivery Tristan DeVere Clemons, BSc (Hons)
This thesis is presented for the degree of Doctor of Philosophy of the University of Western Australia
School of Chemistry and Biochemistry
School of Animal Biology
2013
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Abstract Nanotechnology, although a science in its infancy, has the potential to revolutionize the
medical profession by improving on traditional drug delivery methods and transforming
how disease and injury are currently diagnosed, monitored and treated. The effective
delivery of small molecule drugs, peptides and proteins to a diseased or injury site has
faced considerable barriers in the past including premature clearance from the body, off
site toxicity and poor bioavailability or pharmacokinetics. Nanoparticles can be used to
help improve these characteristics by aiding delivery of therapeutics which otherwise
show little efficacy without assisted delivery. In this work, poly(glycidyl methacrylate)
(PGMA) nanoparticles have been synthesized as delivery vehicles incorporating a range
of surface functionalities and imaging probes to allow successful tracking of these
nanoparticles throughout testing. These delivery vehicles have been used in a range of
applications suggesting the broad applicability and suitability of functionalized PGMA
nanoparticles in medicine. The nanoparticles were shown to aid in the delivery of a
therapeutic peptide to modulate activity of the L-type calcium channel of cardiac tissue
as well as thermally stabilize industrially relevant enzymes through nanosurface
interactions. Finally, the potential of the nanoparticle’s as DNA delivery vectors for
gene silencing in cancer models was investigated. Further to these three delivery
applications of the functionalized PGMA nanoparticles, work will be presented herein
on the development of a novel spectrophotometric assay suitable for the detection of the
activity of the therapeutic enzyme chondroitinase ABC (chABC). This newly presented
assay was superior when compared to the traditional methods used for detecting the
activity of chABC. This assay was used to investigate a range of formulations including
functionalized PGMA nanoparticles, in an attempt to stabilize the therapeutically
relevant chABC at 37 °C, to prolong its activity and in turn improve its effectiveness as
a therapeutic in the treatment of central nervous system injuries.
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Table of Contents
Abstract ................................................................................................................................... iii Table of Contents ................................................................................................................. iv Acknowledgements ............................................................................................................. ix Abbreviations ....................................................................................................................... xi Published Works ................................................................................................................. xv Conference presentations .............................................................................................. xvii Statement of candidate contribution .......................................................................... xix Thesis Prelude .................................................................................................................... xxi Chapter 1 ................................................................................................................................. 1 Introduction and literature review ................................................................................ 1
1.1 Nanoparticles in modern medicine ................................................................... 1 1.1.1 Nanoparticles for drug delivery ............................................................................................ 2 1.1.2 Micelles, liposomes and dendrimers for drug delivery applications .................... 5
1.2 Polymeric nanoparticles and nanocapsules as drug delivery vehicles .......... 8 1.2.1 Methods for the preparation of polymeric nanoparticles .......................................... 9
1.3 Nanoparticle and cell interactions .................................................................. 13 1.3.1 Nanoparticle endocytosis ....................................................................................................... 16 1.3.2 Strategies to enhance cellular internalization ............................................................... 18
1.4 Multifunctional nanoparticles ........................................................................ 19 1.4.1 Targeted nanoparticles and ‘stealth’ coatings ............................................................... 21 1.4.2 Imaging agents and multifunctional nanoparticles .................................................... 24 1.4.3 Magnetic resonance imaging ................................................................................................ 29 1.4.4 Magnetic resonance contrast agents ................................................................................. 31 1.4.5 Fluorescent probes for biological imaging ..................................................................... 33 1.4.6 Theranostic nanoparticles and the combination of imaging and treatment together ......................................................................................................................................................... 34
1.5 Assessing nanoparticle toxicity ....................................................................... 35 1.6 Summary of the literature and thesis rationale ............................................. 37 1.7 Introduction to series of chapters ................................................................... 39
2.1 An introduction to polymeric nanoparticles for drug delivery .................... 43 2.2 Multimodal PGMA nanoparticles with a PEI functionalized surface ........ 45 2.3 Multimodal PGMA nanoparticles with a PEGylated surface ...................... 51 2.4 In vitro toxicity and cellular internalization studies ...................................... 54 2.5 Conclusion ......................................................................................................... 55 2.6 Detailed methods of nanoparticle synthesis and characterization .............. 55 2.6.1 Nanoparticle synthesis ............................................................................................................ 55 2.6.2 Nanoparticle characterization ............................................................................................. 57 2.6.3 In vitro testing of nanoparticles ........................................................................................... 57
Chapter 3 ............................................................................................................................... 59 Multifunctional polymeric nanoparticles for the delivery of the therapeutic AID peptide in cardiac ischemia-‐reperfusion injuries .......................................... 59
3.1 Current treatment of cardiac ischemia-reperfusion injuries ....................... 59 3.2 Loading of the therapeutic AID peptide to the nanoparticles ...................... 62 3.3 A comparison of cellular uptake and biodistribution ................................... 64 3.4 Nanoparticle and TAT-mediated delivery of the AID peptide reduces
damage following ischemia-reperfusion injury .............................................. 68 3.5 Conclusions and future work .......................................................................... 72 3.6 Detailed methods .............................................................................................. 73 3.6.1 Nanoparticle synthesis and characterization ................................................................ 73 3.6.2 Synthesis of the AID peptide ................................................................................................. 73 3.6.3 AID peptide attachment to PGMA nanoparticles ......................................................... 73 3.6.4 Isolation of guinea-‐pig ventricular myocytes ................................................................ 73 3.6.5 Uptake studies with cardiac myocytes ............................................................................. 74 3.6.6 in vitro fluorescence assays ................................................................................................... 74 3.6.7 Ischemia-‐reperfusion model ................................................................................................. 75 3.6.8 CK and LDH assays .................................................................................................................... 75 3.6.9 Cardiac biodistribution studies ........................................................................................... 76 3.6.10 Statistical analysis ................................................................................................................... 77
Chapter 5 ............................................................................................................................... 95 The development of a spectrophotometric assay suitable for quantitative and kinetic analysis of chondroitinase ABC (chABC) activity ...................................... 95
5.1 chABC as a potential therapeutic intervention for central nervous system
injuries ................................................................................................................ 95 5.2 Current assay methods for chABC activity can be improved ..................... 97 5.3 Comparison of the novel WST-1 assay to traditional assays for chABC
activity ................................................................................................................ 98 5.4 Conclusion and potential applications of the WST-1 assay ....................... 105 5.5 Materials and detailed methods .................................................................... 106 5.5.1 Materials ..................................................................................................................................... 106 5.5.2 WST-‐1 chABC assay ............................................................................................................... 106 5.5.3 DMMB chABC assay ............................................................................................................... 106 5.5.4 Absorbance at 232 nm chABC assay ............................................................................... 106 5.5.5 Substrate inhibition experiments .................................................................................... 107 5.5.6 Kinetic analysis with the WST-‐1 assay .......................................................................... 107 5.5.7 Activity of chABC versus trehalose stabilised chABC .............................................. 107
Chapter 6 ............................................................................................................................ 109 Attempts at stabilizing the therapeutically relevant enzyme chondroitinase ABC (chABC) ...................................................................................................................... 109
6.1 chABC for the treatment of central nervous system injury ....................... 109 6.2 PGMA nanoparticles do not impart thermal stability to chABC .............. 110 6.3 Investigation of PEG as a thermal stabilizing agent and cryoprotectant . 112 6.4 Assessment of chABC activity at 37 °C ........................................................ 115
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6.5 Recent advancements for the removal of chondroitin sulfate proteoglycans
and the use of chABC as a therapeutic intervention .................................... 117 6.6 Materials and detailed methods .................................................................... 120 6.6.1 Materials ..................................................................................................................................... 120 6.6.2 WST-‐1 chABC assay ............................................................................................................... 120 6.6.3 Time course experiment comparing PGMA nanoparticles and trehalose ..... 121 6.6.4 Evaluating chABC activity in the presence of free PEI ............................................ 121 6.6.5 Evaluating chABC activity in basic conditions ........................................................... 121 6.6.6 Investigation of PEG as a thermal stabilization agent ............................................ 121 6.6.7 Investigation of PEG as a cryoprotectant ..................................................................... 122 6.6.8 Time course analysis of chABC activity ......................................................................... 122
Chapter 7 ............................................................................................................................. 125 Multifunctional polymeric nanoparticles for gene delivery and RNAi in breast and colon cancer models. .............................................................................................. 125
7.1 c-Myc an appropriate target gene ................................................................ 126 7.2 RNAi technology and the problem of delivery ............................................ 126 7.3 Design of the nanoparticle non-viral vectors ............................................... 129 7.4 Summary of key findings ............................................................................... 132
Chapter 8 ............................................................................................................................. 133 Conclusions and future work ....................................................................................... 133
8.1 PGMA multifunctional nanoparticles .......................................................... 134 8.2 PGMA nanoparticles and peptide delivery to cardiac ischemia-reperfusion
injury ................................................................................................................ 136 8.3 PGMA nanoparticles for the stabilization of chABC ................................. 138 8.4 PGMA nanoparticles for RNAi technology ................................................. 139 8.5 Final remarks ................................................................................................. 140
Appendix A – Elemental analysis calculation of PEI attachment to nanoparticle surface by mass ........................................................................................................ 143 Appendix B – 1H-NMR spectra of carboxylic end functionalized poly(ethylene glycol) and poly(ethylene glycol) bound to poly(glycidyl methacrylate). ........... 144
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Appendix C - Standard curve used for the calculation of WST-1 molar absorption coefficient. ............................................................................................. 145 Appendix D – Chapter 7 – in vitro and in vivo analysis ....................................... 146 Appendix E – Published works not included directly in the thesis .................... 167
1. T. Clemons, N. K. Tangudu, V. K. Verma, S. S. Beevi, G. Mahidhara, T. Hay, M.
Raja, R. A. Nair, L. E. Alexander, A. B. Patel, J. Jose, N. M. Smith, B. Zdyrko,
A. Bourdoncle, I. Luzinov, A. R. Clarke, L. D. Kumar & K. S. Iyer. Breast and
colon cancer tumour regression through the delivery of c-Myc shRNA
conjugated to multifunctional polymeric nanoparticles. 4th International
Nanomedicine Conference, 1st July 2013. Sydney, Australia.
2. (Invited) T. Clemons. How tiny science can have a big impact on students. 2013
Conference of the Science Teachers Association of Western Australia, 18th May
2013. Perth, Australia.
3. T. Clemons, H. Viola, I. Swaminathan, L. Hool. Polymeric nanoparticles for the
treatment of ischemia-reperfusion injury. Australian Nanotechnology Network
Early Career Research Symposium 2012, 15th December 2012. Melbourne,
Australia.
4. (Invited) T. Clemons. How tiny science can have a big impact on students –
Nanotechnology outreach. Australian Nanotechnology Network Early Career
Research Symposium 2012, 16th December 2012. Melbourne, Australia.
5. T. Clemons, H. Viola, I. Swaminathan, L. Hool. Nanoparticles as a delivery
vehicle for the alleviation of cardiac ischemia-reperfusion injury. University of
Western Australia, School of Chemistry and Biochemistry 2012 research forum,
2nd November 2012. Perth, Australia.
6. T. Clemons, M. Fitzgerald, S. Dunlop, A. Harvey, B. Zydyrko, I. Luzinov, S.
Iyer, K. Stubbs. Nanoparticles for enzyme stabilization. XI International
conference on Nanostructured Materials (Nano2012), 28th August 2012. Rhodes,
Greece.
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7. T. Clemons, H. Viola, I. Swaminathan, L. Hool. Polymeric nanoparticles for
delivery to cardiac myocytes. Molecular Imaging Symposium, 1st May 2012.
Sydney, Australia.
8. T. Clemons, M. Fitzgerald, S. Dunlop, A. Harvey, B. Zydyrko, I. Luzinov, S.
Iyer, K. Stubbs. Thermal stabilization of industrial and medically relevant
enzymes in the presence of nanoadditives. International Conference on
Nanoscience and Nanotechnology, 6th February 2012. Perth, Australia.
9. (Invited) T. Clemons, C. Evans, D. Ho. Because the small things make a big
difference. Science Teachers Association of Western Australia - Future Science
Conference, 2nd December 2011. Perth, Australia.
10. T. Clemons, C. Evans, B. Zydyrko, I. Luzinov, M. Fitzgerald, S. Dunlop, A.
Harvey, S. Iyer, K. Stubbs. Nanoparticles for multimodal enzymatic therapy in
the central nervous system. Combined Biological Sciences Meeting, 26th August
2011. Perth, Australia.
11. T. Clemons, C. Evans, B. Zydyrko, I. Luzinov, M. Fitzgerald, S. Dunlop, A.
Harvey, S. Iyer, K. Stubbs. Stabilization of enzymes against thermal inactivation
with multifunctional polymeric nanoparticles. Fifth International Conference on
Advanced Materials and Nanotechnology, 10th February 2011. Wellington, New
Zealand.
12. T. Clemons, C. Evans, K. Stubbs, M. Fitzgerald, S. Dunlop, A. Harvey, I.
Luzinov, B. Zydyrko, I. Swaminathan. Multifunctional polymeric nanoparticles
for the stabilization of enzymes against thermal inactivation. OzBio 2010 The
molecules of Life – From Discovery to Biotechnology, 29th September 2010.
Melbourne, Australia.
13. T. Clemons, C. Evans, K. Stubbs, M. Fitzgerald, S. Dunlop, A. Harvey, I.
Luzinov, B. Zydyrko, I. Swaminathan. Multifunctional polymeric nanoparticles
for the stabilization of enzymes against thermal inactivation. Australian
Research Network for Advanced Materials/Australian Research Council
Nanotechnology Network joint conference, July 22nd 2010. Adelaide, Australia.
xix
Statement of candidate contribution This thesis contains the results of work carried out by the author within the School of
Chemistry and Biochemistry and the School of Animal Biology at the University of
Western Australia during the period of January 2010 to June 2013.
The work presented herein contains no materials which the author has submitted or
accepted for the award of another degree or diploma at any university and, to the best of
the author’s knowledge and belief, contains no material previously published or written
by another person, except where due reference is made in the text.
Tristan DeVere Clemons
2013
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Thesis Prelude Nanotechnology and its integration into biology and medicine has been a rapidly
developing field with significant breakthroughs constantly being made. The work
conducted during this PhD was aimed at furthering this relationship between
nanotechnology and biology by improving the delivery of therapeutics to treat a range
of diseases and injuries. Specifically, the aims of the research were to:
1. Synthesize and characterize a novel multimodal PGMA nanoparticle platform
with the potential for use as a drug delivery vehicle.
2. Assess the efficacy of this delivery vehicle for the delivery of biologically
relevant payloads such as peptides and plasmid DNA in in vitro, ex vivo and in
vivo models of cardiac ischemia-reperfusion injury and breast and colon cancer
respectively.
3. Assess the ability of the synthesized polymeric nanoparticles to impart thermal
stability to enzymes for the end goal of producing a delivery vehicle for the
enzyme chondroitinase ABC to central nervous system injuries.
For the aforementioned aims to be achieved, a comprehensive understanding and
review of the literature regarding nanoparticle design, synthesis and characterization
techniques, nanoparticle structures and the addition of imaging agents and surface
ligands was required. The review of the literature, presented in Chapter One, covers
a range of fields as is expected from a multidisciplinary project. The use of
nanoparticles in drug delivery was explored, providing a review of the different
types of nanoparticles currently being used for medical applications and their
inherent merits and pitfalls. The synthesis, use and advantages of polymeric based
nanoparticle systems were discussed in order to provide a clear rationale for the
choice of using polymeric nanoparticles in this work.
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Secondly, as the nanoparticles that have been developed are intended for medical
applications, an understanding of the interactions of these nanoparticles at a cellular
level as well as the clearance mechanisms by which nanoparticles are removed from
the body are also discussed. The incorporation of imaging modalities into
nanoparticles to allow them to act in both a therapeutic as well as a diagnostic role is
important for nanoparticle tracking in vivo, and hence is also explored in the review.
Finally, the review covers a range of clinically relevant imaging modalities
appropriate for integration into the polymeric nanoparticle system. The review in
Chapter One provides a broad background of nanoparticles in medicine while also
assessing the considerations to be made in synthesizing new nanoparticles to be
suitable as drug delivery vehicles. Each subsequent chapter in the thesis will begin
with a short introduction to the literature specific to that chapter to provide the
relevant information required to understand the research problem, injury or disease
and the role in which nanoparticles can play in addressing these issues directly.
Chapter 1
Introduction and literature review
1.1 Nanoparticles in modern medicine
Nanotechnology is characterized by the creation and use of engineered materials or
devices that have at least one dimension in the range of 1-100 nm in size.1
Nanotechnology exploits the physical and chemical properties of nanoparticles, which
as a result of their size, are remarkably different from both atomic species and bulk
materials.2 Since the properties depend on the dimensions of the nanostructure, reliable
and continual changes can be achieved by changing the size of single particles. The best
example of this is quantum dots where altering the size of the quantum dot particle can
change the optical emission properties of the material (Figure 1.1).
Not only is nanotechnology interesting from a synthetic approach but this scale also
mirrors that of many biological targets and systems. Many proteins, viruses and
important biological molecules are in the size range of 1-10 nm and as a result,
structures that can be accurately designed on the nanometer scale have the ability to
interact on the cellular, sub-cellular and molecular levels with unique specificity.1, 3 This
specificity can result in explicit interactions within cells and tissues without causing
undesirable side effects.3 A major field of nanotechnology research is the synthesis of
nanoparticles for medical applications including disease diagnosis, imaging and most
importantly treatment through the delivery of therapeutics. It is envisaged that the
global market for nanotechnology related applications in the medical field could
increase to between $70-160 billion US by the year 2015.4, 5
2
Figure 1.1. A) Quantum dots possess unique photo-physical properties making them
ideal for applications in biological imaging due to the ability to tune the emission colour
by altering the quantum dot size (particle size increasing from left to right). B) Narrow
emission spectra along with efficient light absorption throughout a wide spectrum of
wavelengths make quantum dots suitable for a range of applications, especially in
biological imaging. Figure and caption modified from Zrazhevskiy et al. 2009.6
Nanotechnology and nanoparticle drug delivery vehicles provide an exciting prospect
for the delivery of therapeutics in the treatment of a range of diseases and injuries in
comparison to current clinical methods.7, 8 Nanoparticles in particular possess a range of
advantages as drug delivery vehicles including drug protection from clearance and
degradation, high levels of drug loading, the potential for multiple therapeutics to be
delivered from the same entity, preferential drug release at target tissues, modifiable
drug release kinetics and finally ease of nanoparticle modification for the incorporation
of imaging probes, targeting moieties and surface structure functionalities.9 This review
provides insight into some significant breakthroughs and also highlights some of the
challenges still facing this field as a prelude to the work conducted in this thesis.
1.1.1 Nanoparticles for drug delivery
In drug discovery it is easy to find a long list of drug candidates that, although
possessing high potency, are unsuitable for clinical application due to poor solubility or
3
poor circulation within the body. Often these candidates have been overlooked in
preference for drugs possessing lower potency but better solubility and half-lives.4
Nanotechnology has the potential to change this by rewriting the rules of drug discovery
and improving drug characteristics, which were previously seen as limiting or
significant enough to warrant a drug’s rejection.4 Nanoparticle based drug delivery
systems have been developed to ultimately improve the efficiency of delivery and to
reduce systemic toxicity of a wide range of therapeutics. The application of
nanoparticles and nanocapsules for drug encapsulation has looked to build on this
concept down to the nanoscale. The first generation of nanoparticles developed for drug
delivery often only provided one function: drug coating and protection to either enhance
drug solubility or circulation time. These nanoparticles are now currently being tested in
clinical trials with some gaining recent approval for clinical applications (Table 1.1).10
A wide variety of nanoparticle formulations have been used for drug delivery
applications including liposomes, dendrimers, microemulsions, micelles, solid lipid and
polymer nanoparticles, and soluble polymers that have a therapeutic attached via
biodegradable linkages (Figure 1.2). Particles already approved for clinical use include
those based on liposomes, biodegradable polymeric nanoparticles and polyethylene
glycol (PEG) or protein based nanoparticle drug conjugates.10, 11
4
Table 1.1. Nontargeted nanoparticles that have been approved for clinical use or undergoing clinical trials.10 PLA, poly(l-lactide); pAsp, poly(l-aspartic acid); PEG, poly(ethylene glycol) Pglu, polyglutamate; PAA, poly(l-aspartate); HPMA, N-(2-hydroxypropyl)-methacrylamide-copolymer
Genexol-PM Paclitaxel-loaded PEG-PLA micelle Breast cancer, lung cancer Approved NK911 Doxorubicin-loaded PEG-pAsp micelle Various cancers Phase 2 NK012 SN-38-loaded PEG-Pglu (SN-38) micelle Breast cancer Phase 2
NC-6004 Cisplatin-loaded PEG-Pglu micelle Various cancers Phase 1 SP1049C Doxorubicin-loaded pluronic micelle Gastric cancer Phase 3 NK105 Paclitaxel-loaded PEG-PAA micelle Breast cancer Phase 3
Polymer-drug conjugates-based nanoparticle
OPAXIO (Cell Therapeutics)
Paclitaxel combined with a polyglutamate polymer Ovarian cancer Phase 3
IT-101 Camptothecin conjugated to cyclodextrin-based polymer Various cancers Phase 1/2 HPMA-DOX (PK1) Doxurubicin bound to HPMA Lung cancer, breast cancer Phase 2
HPMA-DOX-galactosamine (PK2)
Doxorubicin linked to HPMA bearing galactosamine Hepatocellular carcinoma Phase 1/2
CT-2106 Camptothecin poly-l-glutamate conjugate Various cancers Phase 1/2
Albumin-based nanoparticle
Abraxane Albumin-bound paclitaxel nanoparticles Metastatic breast cancer Approved
5
Figure 1.2. Schematic structure of a range of nanoparticle formulations currently being
prepared for drug delivery applications. Nanospheres and nanocapsules are basically
small vesicles used to transport materials. Nanocapsules are a shell with an inner space
loaded with the drug of interest. Both systems are useful for controlling the release of a
drug and/or protecting it from the surrounding environment. A micelle is a spherical
conglomeration of amphiphilic molecules, such as cholesterol. In aqueous environments,
the molecules form a tight ball with the hydrophobic groups on the inside and the
hydrophilic groups on the outside. The reverse occurs in a non-aqueous environment.
Micelles are useful for encapsulating non-water soluble drugs to be administered
intravenously. Dendrimers are highly branched polymers with a controlled three-
dimensional structure around a central core. Dendrimers are easily functionalized and
can accommodate more than 100 terminal groups. Liposomes are spherical vesicles that
comprise one or more lipid bilayer structures enclosing an aqueous core. Liposomes can
also be functionalized to improve cell targeting and solubility. Figure from Sanna et al.
2013 and caption has been modified from Orive et al. 2009.12, 13
1.1.2 Micelles, liposomes and dendrimers for drug delivery applications
Micellar nanoparticles consist of a hydrophobic core which is surrounded by
amphiphilic block copolymers that have assembled around this hydrophobic core to
produce a core/shell architecture in aqueous media.13, 14 The hydrophobic core region of
6
the micelle acts as a reservoir for hydrophobic drugs; the hydrophilic exterior of the
micelle allows for nanoparticle stability in aqueous media.14 Micelles have the ability to
encapsulate a range of therapeutic cargoes including hydrophobic drugs,
oligonucleotides, proteins and imaging agents with considerably high loading levels (up
to 30% w/w).13, 15 Micelle nanoparticles have shown great promise as delivery systems
with a number currently in phase 3 clinical trials (Table 1.1). Micelles can also be
produced from stimuli responsive block copolymers to allow disassembly in the
presence of triggers such as pH, temperature, light or ultrasound.16 This allows for
targeted release of the therapeutic payload held within the micelle structure.
A recent study by Lee et al. encapsulated the photosensitive Protoporphyrin IX (PpIX)
within a pH responsive micelle based on the block copolymer of PEG-poly(β-amino
ester) (Figure 1.3).16 The pH of the microenvironment surrounding tumour tissue is
lower (pH 6.4-6.8) than that of normal tissue (pH 7.4).17, 18 This reduction in pH allows
for protonation of the tertiary amines present in the amino ester, resulting in an increase
in the hydrophilicity of the polymer.17 This change results in rapid demicellization in
the regions surrounding the tumour tissue and leads to the release of the encapsulated
photosensitizer PpIX. PpIX produces a strong fluorescent signal allowing the location
of PpIX surrounding the tumour microenvironment to be identified (Figure 1.3B).
Furthermore, when irradiated with the appropriate wavelength of light, PpIX produces
cytotoxic singlet oxygen (photodynamic therapy), which in turn destroys nearby tumour
cells (Figure 1.3D).
7
Figure 1.3. Polymeric micelles for optical imaging and photodynamic therapy. A)
Schematic illustration of PpIX-encapsulated pH-responsive polymeric micelles for
tumor diagnosis and photodynamic therapy. B) Fluorescence images after injection of
PpIX-encapsulated pH-responsive polymeric micelles. C) Ex vivo images of organs and
tumors. D) Tumour growth after injection and laser irradiation. Figure and caption from
Lee et al. 2012.16
Similar to micelles, liposomes are closed colloidal structures consisting of an aqueous
core surrounded by a phospholipid bilayer with their main application in the delivery of
aqueous biomolecules and hydrophilic drugs.13 Liposomes have the potential to entrap
relatively large amounts of hydrophilic drugs within their aqueous core or between the
lipid bilayer shell structure if the therapeutic is lipophilic.15 A major advantage of
liposomes is that they form spontaneously in solution and they essentially possess no
inherent toxicity due to the presence of the components of liposomes throughout the
body in all cell membranes.13 Liposomes have had great success in the delivery of
anthracycline based chemotherapeutics including doxorubicin, and daunorubicin for the
treatment of metastatic breast cancer,19, 20 ovarian cancer,19 and for the treatment of
AIDs related Kaposi’s sarcoma.21 An interesting application of liposomes for drug
8
delivery is the utilization of liposomes for the encapsulation and aerosol delivery of
vasoactive intestinal peptide (VIP) for the treatment of various lung diseases such as
asthma and pulmonary hypertension. A recent study by Hajos et al. found that
encapsulation of VIP within liposomes was successful in allowing VIP to avoid
enzymatic degradation once inhaled and deposited within the bronchi.22 This study
found that loading of the VIP within the liposomes for inhalation therapy improved the
pharmacological and biological activity of the VIP treatment in comparison to the
delivery of free VIP.22
Dendrimers are not nanoparticles per se but more strictly defined as a polymeric
macromolecule of nanometer dimensions composed of highly branched monomers that
emerge radially from a central core.14 Dendrimers can be biodegradable or non-
biodegradable structures. Natural polymers such as glycogen, and some proteoglycans
consist of a dendrimer like structure. However, for drug delivery, the synthetic polymer
poly(amidoamine) (PAMAM) is the most extensively studied.14, 23 PAMAM has been
shown to be effective for the binding and subsequent delivery of cisplatin both in vitro
and in vivo where it shows improved efficacy in comparison to cisplatin delivered
without the dendrimer.24 Properties that make dendrimers attractive for drug delivery
applications include monodispersed size distributions, modifiable surface chemistry,
multivalency, water solubility and an internal cavity available for drug loading.23 Due to
the ease with which dendrimer surface chemistry can be modified, the addition of
contrast agents, imaging probes and targeting ligands can be coupled with a therapeutic
for delivery, resulting in the production of dendrimer based multifunctional drug
delivery systems.23 Dendrimers can be produced with low cytotoxicity and surface
decoration of the dendritic structure with PEG can prolong its circulation half-life.
Although there is significant interest in dendrimers as drug delivery vehicles, few have
translated into clinical trials with Vivagel® the most promising candidate, currently in
phase 2 clinical trials.25 Vivagel® is a L-lysine dendrimer that contains a polyanionic
outer surface which exhibits antiviral activity against the sexually transmitted herpes
simplex virus (HSV) and the human immunodeficiency virus (HIV).25
1.2 Polymeric nanoparticles and nanocapsules as drug delivery vehicles
Polymeric nanoparticles and nanocapsules are solid formulations ranging in size from
10-1000 nm in diameter and can be synthesized from natural or artificial polymers.
9
Generally speaking, the major advantage of polymeric nanosystems over other nano-
delivery systems is their inherent stability and structural rigidity.13 These polymeric
nanoparticles often incorporate their therapeutic for delivery via drugs that are adsorbed,
dissolved, entrapped, encapsulated or covalently linked to the nanoparticle.26, 27 The
most commonly used synthetic materials for the synthesis of biodegradable polymeric
nanoparticles are poly(lactic acid) (PLA), poly(D-L-glycolide (PLG) or the copolymer
of these synthetic polymers being poly(lactic-co-glyoclic acid) (PLGA). due to their low
toxicity, biodegradability, FDA approval and tissue compatibility.27, 28 Biodegradable
nanoparticles based on these aforementioned polymers have been used for the delivery
of a range of therapeutics in vivo for the treatment of cancers,29 neurodegenerative
disorders,30 and for the controlled release of contraceptive steroids and fertility control
systems.31, 32
Nanoparticles synthesized from naturally occurring polymers such as chitosan, albumin
and heparin have been popular choices for the delivery of oligonucleotides, proteins and
small molecule drugs. Despite significant research in the use of polymers for
nanoparticle drug delivery systems only one, Abraxane, has been approved for clinical
applications to date.33 Abraxane is an albumin based nanoparticle system developed for
the delivery of paclitaxel, a proven chemotherapeutic agent, to metastatic breast
cancers.33 Furthermore, Abraxane is currently undergoing clinical trials for delivery to a
variety of other cancers including non-small-cell lung cancer (phase 2 trial)34 and
advanced nonhematologic malignancies (phase 1 and pharmacokinetics trials).35 Since
the use of polymeric nanoparticles forms the crux of this PhD thesis, the following
section will cover in detail the common methods used for their preparation.
1.2.1 Methods for the preparation of polymeric nanoparticles
A number of approaches have been developed for the synthesis of polymeric
nanoparticles most of which involve the use of block copolymers consisting of polymer
chains of differing solubilities. The more common techniques for polymeric
nanoparticle formulations include layer-by-layer (LbL) approaches, nanoprecipitation
(sometimes referred to as the solvent displacement method), emulsification, solvent
evaporation methods, and the salting out method. Further to these traditional methods,
techniques that make use of microfluidics, super critical technology and the premix
membrane emulsification method are increasingly favoured due to their potential for
10
producing highly monodispersed nanoparticles in high yields.28 Usually, the choice of
nanoparticle formulation method is dictated by the physicochemical properties of the
drug, the polymer intended for encapsulation and particle size requirements.9 The
common techniques for the preparation of polymeric nanoparticles from pre-formed
polymer are discussed in the following paragraphs paying special attention to the
associated merits and pitfalls of each method.
The LbL approach to producing polymeric nanoparticles is a highly versatile and
interesting nanoparticle engineering method. Typically, LbL particles are formed
through the consecutive deposition of polymers which interact with one another (e.g.
through electrostatic interactions or hydrogen bonding) onto a core particle template.36
This results in the formation of core-shell particles consisting of an ultrathin, highly
tunable multilayered polymer coating on particles of varying size (from 10 nm up to a
few microns), shape and composition.37, 38 Furthermore, if a sacrificial core is used in
this method the subsequent removal of this template allows the production of hollow
polymeric capsules.36 LbL particles have been successful in their efficient encapsulation
of cargoes, triggered release or degradation and targeted delivery through the
incorporation of antibodies.39, 40 An interesting recent study by Poon et al. developed
LbL nanoparticles with a pH responsive shell in order to improve cellular uptake of the
nanoparticles in an acidic tumour microenvironment.41 Carboxyl terminated quantum
dots were sequentially coated with iminobiotin-functionalized poly(L-lysine) (PLL),
neutravidin, and biotin-functionalized PEG. The nanoparticles produced consisted of a
fluorescent quantum dot core and alternate layers of PLL and PEG held together by the
strong physical interaction between biotin and avidin.41 The incorporation of PEG on
the nanoparticle surface increased circulation time until this layer was selectively
eroded by the acidity in the hypoxic tumour microenvironment.36, 41 This erosion of the
outer surface exposed the positively charged layers beneath the PEG coating resulting in
rapid cellular internalization and, in turn, tumour retention of the LbL nanoparticles.41
Despite the considerable promise surrounding LbL nanoparticles, these systems are still
in their infancy and thus the pharmacokinetics in disease models, the biocompatibility
and toxicity in vivo following delivery, are yet to be determined.36 This method can
also be time consuming with wash steps between layer depositions, and often the
requirement of a highly precise pairing of polymers to ensure a strong interaction
between layers and thus structural integrity once the sacrificial core is removed from the
polymeric shell.
11
The nanoprecipitation method involves an organic solvent that is miscible with an
aqueous phase and can also dissolve both the polymer and drug intended for
encapsulation.9 The organic phase (solvent), containing polymer and drug, is added
drop wise to the stirring aqueous phase (non-solvent) where, upon contact with the
water, the hydrophobic polymers and drug precipitate and spontaneously self assemble
into core shell like spherical structures in an attempt to reduce the system’s free energy.9
The nanoprecipitation method is simple and can be easily scaled up to industrial levels
as it only requires gentle stirring and no high stress shear. This method however is
limited to hydrophobic drugs, which are highly soluble in non-polar solvents but only
slightly soluble in water. The method also has challenges in determining an appropriate
polymer/drug/solvent/non-solvent system, which allows for nanoparticle formation with
high drug loading efficiency.28 Using a diblock copolymer of PLGA and PEG,
docetaxel loaded polymeric nanoparticles consisting of a PLGA core and a PEGylated
outer shell have been successfully produced with the nanoprecipitation method for in
vivo chemotherapeutic treatments.42 When targeted to and tested in an in vivo model of
prostate cancer, these targeted nanoparticles resulted in 100% viability (all animals
reached 109 days survival) and tumour reduction compared to docetaxel alone where
only 14% of mice reached the 109 day target.42
Emulsion techniques involving either a water in oil (W/O), an oil in water (O/W) or a
double emulsion (W/O/W) require the formation of an emulsion followed by an input of
high powered sonication or homogenization to produce nanoparticles from the emulsion.
The single O/W emulsion technique is the most common and is used for the preparation
of hydrophobic polymeric nanoparticles containing hydrophobic drugs. The organic
components (drug and polymer) are dissolved in a water immiscible organic solvent (e.g.
dichloromethane), which is then emulsified under intense shear stress in an aqueous
phase containing an appropriate surfactant to aid in particle stabilization.9 The volatile
organic solvents are allowed to evaporate resulting in the self-assembly of nanoparticles
containing the encapsulated drug once again as a result of the system aiming to reduce
free energy.9 This method produces nanoparticles with high drug entrapment efficiency
although it is limited to drugs that are soluble in the same solvent as that used for the
polymer. Furthermore, particle monodispersity is difficult to achieve with emulsion
techniques.
12
Questions remain surrounding the scale up potential of this technology due to the high
energy requirements in homogenization and the use of toxic chlorinated solvents.28 The
single O/W emulsion technique is limited in its ability to only encapsulate hydrophobic
drugs and hence a double emulsion of W/O/W can be used for the incorporation of
hydrophilic drugs. The W/O/W method is still an emulsion technique, where a W/O
emulsion is first produced before emulsifying the mixture again to produce a final
nanoparticle in aqueous media.9 The salting out method is an extension of the emulsion
techniques described above where the mixing of the organic and the aqueous phases is
prevented by saturating the aqueous phase with electrolytes such as magnesium acetate,
magnesium chloride or calcium chloride.28 This method is advantageous in that it does
not require elevated temperatures and also avoids the use of toxic chlorinated solvents.
However, this method does introduce extensive nanoparticle washing steps to remove
the excess salts and also raises concerns with regards to waste and recyclability of the
large amounts of solvent and salts required.28
New approaches for polymeric nanoparticle preparations have looked to address some
of the limiting factors of the aforementioned technologies. Methods based on
microfluidic technology to produce rapid mixing techniques in microchannels such as
hydrodynamic flow focusing have been shown to produce polymeric nanoparticles
exhibiting narrow size distributions when compared to similar nanoparticles produced
by bulk synthesis techniques.43, 44 An interesting finding from a study by Karnik et al.
found that the polymeric nanoparticles produced from microfluidic methods were able
to achieve higher drug loading then those produced by bulk methods, a highly desirable
characteristic of nanoparticles intended for drug delivery.43 Super critical fluid (SCF)
methods based on the rapid expansion of a super critical solution containing the
polymer and drug to produce the nanoparticles have had great success recently. One
success in this area is that of the UK based spin off company, Critical Pharamceuticals,
which has commercialized a range of products through the use of supercritical methods
for advanced drug delivery of growth hormones. As solubility in SCFs can be up to a
million times higher than that under ideal gas conditions, the rapid expansion from
supercritical pressure to ambient pressures produces extremely high super saturated
solutions.28 These solutions when released from super critical conditions, rapidly result
in very homogenous nucleation conditions for the solute (i.e. polymer) producing
nanoparticles of narrow and reproducible size distributions.45 The premix emulsification
13
method combines the emulsification technique to produce a coarse ‘premix’ emulsion as
per techniques mentioned previously. This premix is then extruded through a Shirasu
porous glass membrane with high pressure to produce uniform nanodroplets.28 Wei et al.
prepared PLA nanoparticles by this method and found that several factors play a key
role in influencing the uniformity in nanoparticles produced including organic solvent
selection, the volume ratio of organic to the external aqueous phase, the pore size of the
microporous membrane and finally the transmembrane pressure used during
collection.46 This method has the advantage of high productivity, simplicity in operation
and the potential for industrial scale up by increasing the surface area of the membrane
or by connecting membranes in parallel.46
While polymeric nanoparticles have the potential to revolutionize drug delivery and
how diseases are treated, important issues still remain. Current synthetic methods,
although very successful at producing a large range of functionalized polymeric
nanoparticles have only been achieved on a small scale. Issues surrounding efficiency of
loading and recycling of by products must be addressed before these technologies can
be scaled up to industrial production. It is considered that this is a major limiting factor
preventing the successful integration of polymeric nanoparticles into the clinic and
market.28
1.3 Nanoparticle and cell interactions
In addition to accurate synthesis and drug loading, another integral consideration for
nanoparticles developed for drug delivery is how they interact with biological systems
once introduced into the body. For drugs with intracellular targets, often the cell
membrane can loom as a formidable barrier. The concept of nanoparticles, which can be
tailored to carry these drugs across the cell membrane and to relevant sub cellular
compartments, provides an attractive means to achieve improved drug trafficking. Proof
of concept studies in the 1970s have shown that sub-micron sized liposomes,47 as well
as synthetic polymer nanoparticles,48 were able to deliver and concentrate in cells,
therapeutics which previously were unable to do so on their own. The plasma
membrane is the barrier which protects the cell against unwanted intruders such as
pathogens, macromolecules and even nanoparticles from entering the cell from the extra
cellular space.49 It consists of a self-assembled bi-layer of lipids where the hydrophobic
interior of this layer is responsible for restricting the passage of water-soluble
14
substances into the cell. Although the passage of small molecules, amino acids and ions
occurs through specialized membrane protein pumps and selected ion channels on the
cell surface, the majority of nanoparticles must undergo some form of membrane
interaction before the process of endocytosis can occur.50 Endocytosis can occur
through a range of mechanisms (Figure 1.4) which can be broadly categorized into
either phagocytosis (cell ‘eating’ for solid particles) or non-phagocytic pathways (cell
‘drinking’ processes).5, 50 With reference to nanoparticles however, these classical
references of cell eating and drinking are not as relevant due to the ability of solid
nanoparticles to still be internalized through non-phagocytic pathways.51 It is important
to have an understanding of the relevant pathways of cell entry which could act on or
affect nanoparticle uptake as this will have direct effects on the drug physicochemical
characteristics as well as the intracellular fate of the nanoparticle carrier and in turn its
therapeutic cargo.51
Phagocytosis for the internalization of macromolecules and indeed most nanoparticles
occurs primarily in specialized cells known as phagocytes, which include macrophages,
monocytes, neutrophils, astrocytes and dendritic cells.52 Phagocytosis can be described
as a general three-step process. An important first step is recognition of the nanoparticle
by opsonin proteins in the bloodstream to tag the nanoparticle for phagocytosis.
Secondly, this signaling triggers the plasma membrane to form an invagination
preparing for the nanoparticle to be internalized and, finally the plasma membrane will
‘pinch off’ from the surrounding plasma membrane to engulf the nanoparticle producing
a discrete package bound by plasma membrane proteins within the cell (Figure 1.4).5, 53,
54 The internalized vesicle, known as a phagosome, is trafficked within the cytoplasm
until it becomes accessible to early endosomes. The phagosome then begins to acidify
and matures, fusing with late endosomes and finally lysosomes to form a
phagolysosome.52 The speed with which this process occurs is highly dependent on the
particle and its surface characteristics but typically the process can take from minutes to
hours.52
15
Figure 1.4. Pathways of entry into cells. Large particles can be internalized by
phagocytosis, whereas fluid uptake occurs by macropinocytosis. Both processes appear
to be triggered by and are dependent on actin-mediated remodeling of the plasma
membrane at a large scale. Compared with the other endocytotic pathways, the size of
the intracellular vesicles formed by phagocytosis and macropinocytosis are much larger.
Numerous cargoes can be endocytosed by mechanisms that are independent of the coat
protein clathrin and the fission GTPase, dynamin. Most internalized cargoes are
delivered to the early endosome via vesicular (clathrin- or caveolin-coated vesicles) or
tubular intermediates (known as clathrin- and dynamin- independent carriers (CLIC))
that are derived from the plasma membrane. Some pathways may first traffic to
intermediate compartments, such as the caveosome or glycosyl phosphatidylinositol-
anchored protein enriched early endosomal compartments (GEEC), en route to the early
endosome. Figure and caption from Mayor et al. 2007.54
Phagolysosomes become acidified due to the proton pump ATPase located in the
membrane of the phagolysosome; the recruitment of an enzyme cocktail to aid in the
degradation of the foreign body also occurs at this time.55 Although a minimum size of
0.5 µm is often considered the limit for phagocytosis, previous studies have shown
nanoparticles ranging from 250 nm to 3 µm in diameter can undergo in vitro
phagocytosis.51 Careful control of the nanoparticle surface coating and nanoparticle size
can play important roles in producing nanoparticles that can avoid phagocytic uptake.56
It is generally accepted however that the in vivo fate of nanoparticles is opsonization
followed by phagocytosis with little discrimination for nanoparticle composition, unless
16
the particles are very small in size (less then 100 nm), or more importantly possess a
specific hydrophilic coating (such as PEG) to aid in the avoidance of opsonin
recognition.51
Non-phagocytic pathways, normally referred to as pinocytosis, are not restricted to
specialized cells and contain processes that are used by almost all cells for the
internalization of fluids and solutes alike. Non-phagocytic uptake into cells can occur
through four main mechanisms: clathrin-mediated endocytosis, caveolae-mediated
endocytosis, macropinocytosis and other clathrin and caveolae independent processes
(Figure 1.4).51, 54 Clathrin-mediated endocytosis, the most common mechanism for
uptake, results in trafficking of cargoes into the lysosomal pathway for
biodegradation.53 Conversely, caveolae-mediated uptake has been shown to produce
caveolar vesicles which do not contain a degradative enzymatic cocktail and hence
caveolae-dependent uptake is seen as a mechanism which if targeted could avoid
trafficking of nanoparticles to the degradative lysosomal pathway.56 A third process
known as macropinocytosis is where actin derived protrusions from the cell membrane
can engulf cargoes, upon which the protrusion collapses to again fuse with the cell
membrane. The fate of cargoes which are internalized by macropinosomes can vary
however often they will fuse with lysosomes, which in turn acidify for the degradation
of the cargo.51 By having a better understanding of the variety of internalization
pathways by which nanoparticles can be internalized, a clearer understanding will be
gained as to what kind of environment nanoparticles may be exposed to once they are
internalized. This information is important, for example, when developing new
nanoparticles with site-specific drug release capabilities or biodegradation qualities, or
if the nanoparticle vehicle is engineered with specific escape mechanisms to avoid
degradation in endosomes.57, 58
1.3.1 Nanoparticle endocytosis
Nanoparticle size, shape and relative hardness can dictate which endocytosis pathway is
activated and utilized for nanoparticle uptake. A study by Rejman et al. investigated the
internalization of uniform spherical polystyrene nanoparticles of differing sizes in
murine melanoma cells (B16-F10).56 This study demonstrated that polystyrene spherical
nanoparticles with diameters of 50 and 100 nm were rapidly internalized in less than 30
minutes by a clathrin-mediated pathway.56 In comparison, larger nanoparticles (200 and
17
500 nm in diameter), also made from polystyrene, were internalized much more slowly
(2-3 h) and exhibit an 8-10 fold decrease in internalization when compared to the
smaller particles.56
The shape of nanoparticles has also been recently investigated to see the role it plays on
nanoparticle internalization. Gratton et al. investigated the internalization of a series of
nanoparticles in HeLa cells where the nanoparticles were fabricated to have differing
aspect ratios.59 High aspect ratio rod shaped nanoparticles were internalized in HeLa
cells at a greater rate than spherical nanoparticles of similar internal volume, a
phenomenon similar to that of the appreciable increase seen in the uptake of rod-shaped
bacteria in non-phagocytic cell lines.59 Even nanoparticle hardness can influence the
interactions of nanoparticles with the cell membrane and in turn can have a direct
influence over cell internalization. A recent study by Banquy et al. investigated the
internalization of similar particles of differing hardness, i.e. Young’s modulus.60 This
study found that 150 nm hydrogel nanoparticles with intermediate Young’s modulus
(35 and 136 kPa) were internalized by a range of different mechanisms in macrophages