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CHAPTER V
NANOTUBE DELIVERY FOR
EFFICIENT THERAPEUTICS
5.1 Therapeutic RNAi
Although initially RNAi was discovered accidentally it has evolved into
a powerful tool for probing gene function and in medicine. Being a negative
gene regulatory pathway, it helps in study of gene function by producing
knockdown/knockout phenotypes. This aids immensely in characterizing
gene function. In C. elegans, nearly all of the worm‘s predicted ~19,000
genes have been tested for the function by RNAi. Similar whole genome
knockdown strategies are being pursued in other organisms, including
plants. Apprehensions about deploying RNAi in mammalian systems were
abolished when the first hint that the technology might work was
demonstrated in early mouse embryos (Gil et al, 2000). But Tuschl and
colleagues emphasized the flexibility of these therapeutic siRNAs and its
profound applications to induce effective silencing in many mammalian cells.
(Elbashir et al, 2001). These small RNAs, which are chemically synthesized
mimics of Dicer products, are presumably incorporated into RISC and target
cognate substrates for degradation.
Two of the major advantages of siRNA over small molecule drugs are
that sequences can be rapidly designed for highly specific inhibition of the
target of interest. Second, the synthesis of siRNAs does not require a cellular
expression system, complex protein purification, or refolding schemes, and is
relatively uncomplicated. (Sah DWY et al, 2006) There are four major types of
anti-mRNA strategies:
Single-stranded antisense oligonucleotides (ODNs): A synthetic, small,
single-stranded ODN inhibits the translation of a specific gene by hybridizing
to the corresponding mRNA through Watson-Crick binding. (Stahel RA 2003)
Ribozymes: Ribozymes are catalytically active RNAs that cleave single-
stranded regions in RNA through trans-esterification or hydrolysis reactions.
(Emilsson GM 2003; Doudna JA 2002)
siRNAs: are 21-23nt in size complementary to the gene to be knocked down.
Sequence specific degradation of cognate mRNAs is the mechanism by which
it operates. Bertrand et al compared anti-sense ODNs with siRNA concluded
that siRNA technology is more efficient. Compared with antisense ODNs,
siRNAs inhibit the synthesis of target proteins with high specificity and at a
much lower dosage. (Bertrand JR 2002)
microRNAs: Endogenous, short, double-stranded, and noncoding RNA
molecules, that have been identified in a variety of organisms and certain
viruses. RNAi has been made stable and heritable by enforced expression of
the silencing trigger, usually as an inverted repeat sequence forming a
hairpin structure in vivo (Smith et al, 2000; Wang et al, 2000). Mature
microRNAs are typically 20–24 nucleotides in length and regulate target
mRNAs post-transcriptionally by interactions with partially mismatched
sequences in the 3′- untranslated regions of these messengers.
Merits and demerits
RNA interference has been declared ―Breakthrough of the year‖ by the
famous Science magazine in the early 21st century. It has an immense
impact on biomedical research and will lead to novel medical applications in
the future. RNAi could provide exciting new therapeutic strategies for
treating infections, cancer, neurodegenerative diseases, antiviral diseases
(e.g., viral hepatitis and human immunodeficiency virus 1, HIV-1),
Huntington's disease (Harper SQ, 2005; Rodriguez-Lebron E, 2005)
hematological diseases, (Gewirtz AM 2007) research and therapy, (Rohl T
2006; Christoph T, 2006) dominantly inherited genetic disorders, (Hickerson
RP 2006) and many other illnesses.
However, there are a few hurdles to overcome. Major ones include the
poor pharmacokinetic property of siRNA, off-target effects and interferon
response. (Dorsett Y 2004) siRNAs longer than 30 nucleotides lead to the
activation of the immune system in specialized highly sensitive cell lines and
at high concentrations, (Bridge AJ 2003) Other disadvantages like a low
transfection efficiency, poor tissue penetration, and nonspecific immune
stimulation by in vivo–administered siRNAs are major stumbling blocks in
therapeutic application. Nucleic acid drugs are highly charged and do not
cross cell membranes by free diffusion, which is another major issue to be
pondered by scientists in the application of siRNAs. Therefore, a targeting
technology that permits these small sized therapeutic RNAs to cross
biological membrane barriers have to be used for the delivery of RNAi. This
penetration of the cellular boundaries needs to be in stealth conditions so
that recognition by the immune signaling system is avoided. For the above-
mentioned reasons, there is a need for a suitable carrier that will provide a
stable complex. This complex should provide adequate protection to ensure
targeting and delivery of siRNA. It should more importantly be able to bridge
the gap between cell culture and animal models to allow for an efficient
siRNA delivery in vivo. Today the major limitation for the full therapeutic
potential of this approach is the lack of an efficient delivery system to target
and deliver the siRNA to the desired cells.
5.2 Delivery vehicles for RNAi applications
Any drug delivery strategy should achieve target specific delivery
combined with minimum off-target effects. It should also eliminate the
potential dosage errors and exhibit better maintenance of drug levels. The
need for fewer administrations, optimal use of the drug in question and
increased patient comfort are some of the clinical requirements. Many
groups are searching for an optimal delivery tool that can be systemically
administered, safely and repeatedly and will deliver the siRNA specifically
and efficiently to the target tissue. The importance of particle size for cellular
uptake has been demonstrated by many studies on targeted drug delivery. In
general, particles with an average diameter between 200 and 500nm could
be introduced into target cells via endocytosis and subsequently permeate
the nuclear membranes through the nuclear pores. Therefore, nanoscaled
systems in the range from 100 to approximately 500 nm are required. Fang
et al have investigated the influence of methoxy polyethylene glycol (MePEG)
on the molecular weight and particle size of stealth nano particles and on
their in vivo tumor targeting properties. The results of the study showed
PEG-PHDCA nanoparticles with higher MePEG molecular weight and smaller
particle size, which could achieve higher in vivo tumor targeting efficiency.
(Fang C 2006) Two major modes of delivery viral and non-viral are in use
presently. The use of viral vectors is a successful strategy as far as in vitro
research needs are concerned whereas non-viral methods are more popular
in vivo and could translate into effective therapeutic tools.
Non viral delivery of siRNA
Nonviral vectors seem to be promising tools for gene delivery, because
they are relatively safe and can be modified through the incorporation of
ligands for targeting specific cell types. However, the levels of gene
expression mediated by these vectors are low as compared with viral vectors.
Nanosized particles such as liposomes, polymeric micelles, lipoplexes and
polyplexes are often studied as drug carrier systems for nucleic acid delivery.
Cationic polymers are able to both condense large genes into smaller
structures and mask the negative DNA/siRNA charges, which are necessary
for transfecting most types of cells. (De Smedt SC 2000) Lipid-based gene
delivery systems, such as liposomes, are vesicles composed of a phospholipid
bilayer with an aqueous core, and for this reason hydrophilic as well as
hydrophobic materials could be packaged into liposomes. (Hirsch-Lerner D
2005; Zimmer A 1999; Leng Q 2007)
In vitro, chemically synthesized siRNA is most effectively introduced
into cells using electroporation, (Bartlett DW 2007; Fan Y 2006; Furset G
2007; Hagemann 2006; Michaela M 2007) nuclear or cytoplasmic micro-
injection, or commercially available lipid reagents such as Oligofectamine,
(Chen G 2006) and Lipofectamine (Zhang M,2008) Metafectene (Xu RX 2006)
and siPORT Amine (Li GY 2005). Because of the invasiveness of the
electricity and cytotoxicity of some lipid reagents, these transfection methods
are not commonly used for in vivo delivery.
A promising new nanoparticle technology for protein based gene
delivery, so-called proticles for the delivery of ODNs and siRNA was explored.
This technology uses an initial complex between human serum albumin and
protamine, whereas the nanoparticles are formed by selfassembly. As a
result of the positive surface charge of this complex, ODNs and siRNA
accumulate and ―ternary‖ nanoparticles result with a hydrodynamic
diameter in the range of 230–320 nm. In fact, the biodegradability of these
proticles' components shows almost no cytotoxicity. (Lochmann D 2005)
Viral delivery of siRNA
Cells of the immune system or primary cells cannot be efficiently
transfected by lipid delivery reagents. Thus, viral delivery of RNAi cassette-
containing vectors is a powerful alternative to lipid transfection. Viral
delivery is applicable for any mammalian cell type, including hard-to-
transfect, primary and nondividing cell types. Some advantages of viral
vectors are their ability to infect both dividing and nondividing cells (e.g.,
adenovirus), the stability of recombinant vectors, the large insert capacity,
and the potential to be produced at high titers. Certain viruses have the
ability to efficiently carry foreign genes and deliver them, thus correlating
with efficient gene expression. For example, viral vectors derived from
retroviruses, adenovirus, adeno-associated virus, herpesvirus, and poxvirus
are used in more than 60% of clinical gene therapy trials worldwide. (Walther
W 2000)
5.3 Nanotube based cellular delivery system
One of the ultimate goals of nanomedicine is to create medically useful
nanodevices that can function inside the body. It is envisioned that
nanodevices will be hybrids of biologic molecules and synthetic polymers
that can enter cells and the organelles to interact directly with DNA and
proteins. Additionally, nanomedicine will have an impact on the key
challenges in cancer therapy - localized drug delivery and specific targeting.
The newly developed nanodevices such as quantum dots, nanowires,
nanotubes, nanocantilevers, nanopores, nanoshells and nanoparticles are
the most promising applications for various cancer treatments.
Carbon Nanoshells - Nanoshells are nanoparticle beads that consist of a
silica core coated with a thin gold shell (Loo et al, 2005). They scatter specific
wavelengths of light across the visible and near-infrared (NIR) spectrum.
(Konig et al, 2000) Their ability to scatter light has potential for cancer
imaging.
Carbon Nanotubes - In the area of cancer therapeutics, carbon nanotubes
have primarily been used for transporting DNA cargoes into the cell and for
thermal ablation therapy. Kam et al have shown that single-walled carbon
nanotubes 1 to 2 nm in diameter and carrying a cargo of 15-mer DNA
adsorbed onto their surfaces can be internalized by cells and accumulate in
the cytoplasm without causing cytotoxicity and suggesting an energy-
dependent uptake mechanism. (Kam et al, 2005)
Dendrimers - Dendrimers are spherical polymers that are normally less than
5 nm in diameter. Their key useful feature is the polymer branches that
provide vast amounts of surface area to which therapeutic agents and
targeting molecules could be attached.
Quantum dots - Quantum dots are composed of 10–50 atoms, and they
confine electron-hole pairs to a discrete quantized energy level. When excited
with ultraviolet light, they fluoresce in different neon colors depending on
their size. Tumor cells labeled with quantum dots can be used to track
metastasis to specific tissues and organs. (Voura et al, 2004)
5.4 Results
Major focus in the development of nano structure for biomaterial delivery
relies on three important factors, chemical modification, biocompatibility and
minimal damage of the transported tissues/organs, etc. But each material
has its own limitation in biological system. The biggest obstacles in selection
of ideal nanomaterials however are the complex interaction between
nanomaterials and cellular environment, their progressive accumulation in
the live cells, inefficient degradation and other pharmacokinetic properties
including cell toxicity and immunogenicity.
Considerable efforts have been directed towards surface modifications
(Liang G, et al. 2007), multivalent attachment of small molecules (Weissleder
R, 2005) and coating for minimizing such effects. These measures also
favour in vivo transport through diversified biological organs and effective
tissue specific targeting. In recent years, fluorescence tagged nano-materials
are used for in vivo and in vitro tracking. One of the major disadvantages
was the rapid disassociation of fluorescent dye and loss of activity in a short
period of time. However, bleaching of fluorescent intensity was prevented
markedly by using nano-materials carrying intrinsic fluorescence rather
than relying on radiolabel or tagged dyes for indirect detection or
measurements.
T[2] K. Konig, Multiphoton microscopy in life sciences, J Microsc 200
(Pt 2) (2000), pp. 83–104. Full Text via CrossRef | View Record in Scopus |
Cited By in Scopus (370(Khe synthesis and characterization of supra-
molecular architectures based upon non-covalent interactions have attracted
significant research efforts over the last few years because of their biological
relevance and the ability to synthesize new materials. The key idea in such
studies is to design building elements, which allow controllable assembly to
give micro or nanomaterials, which exhibit fluorescence and offer
applications in biology and medicine. We have done the structural
characterization and fluorescent emission studies of two p- amino benzoic
acid based (PABA) nanotubes. Para-aminobenzoic acid (PABA) is frequently
used as structural compounds of commercial drugs (Kluczyk A, et al. (2002).
PABA is a naturally occurring non-protein amino acid. It is an effective
inhibitor of bacterial growth. Here, the linkers, lauric or stearic acid side
chain were coupled for making PABA into tube like nanostructures. The
stearic side chain emits intrinsic fluorescence in the green range while lauric
acid is embedded with Rhodamine B, on the tube wall. To trace PABA nano-
device through the complex biological system, in-depth fluorescence imaging
was performed using Confocal Laser Scanning Microscopy.
5.4.1 Structural characterization with advanced microscopy
The tubes after self assembly have been suitably prepared for
microscopic visualization. PNT-A was excited using 488nm and PNT-B with
513nm laser in a laser scanning microscope. The emitted fluorescence was
collected by appropriate filters and collected by detectors as digital images.
Their dimensions were measured by marking scale bars. These self
assembled tubes were 10um in length, 1um in width and their height varied
in nm ranges. These tubes were soluble in organic solvents like methanol,
ethanol and DMSO. These tubes were also scanned in the Transmission
Electron microscope and Scanning Electron microscope wherein the
dimensions were verified. Several nanostructures were visible like rods and
screws indicating the self assembly of these molecules. The tubes were
dissolved in 1:1 methanol water solvent and added to coverslip and allowed
to dry. This was then mounted on to the stub for Electron Microscopy. The
voltage parameters were adjusted to obtain a high resolution image. (Figure
5.1)
5.4.2 Biocompatibility in insect and mammalian cells
Microscopy
Insect and mammalian cells were used to estimate the efficiency of cellular
uptake of two different nano-tubes in vitro. Drosophila S2 cells, non-
neoplastic Human Embryonic Kidney (HEK-293) and neoplastic HeLa cells
were grown on cover glasses and incubated with the nanostructures
dissolved in 0.1% DMSO. After 24 or 48 hrs incubation, cells were fixed in
4% paraformaldehyde, followed by a few gentle washes with PBS and
mounted with 70% glycerol. The cells were viewed under laser confocal
microscope (Olympus FV1000). The reconstituted images showed that both
PNT-A and PNT-B were accumulated in the periphery of the nucleus of both
insect and human (HEK293, HeLa) cells incubated with 60mg/ml nanotubes
and 0.1% DMSO. The accumulation was increased proportionately to the
concentration of nanotubes in the media. In contrast, a negligible amount of
fluorescence signals was emitted from the cells incubated in 0.1% DMSO
alone for the same period of time. These findings demonstrate that
nanostructures are accumulated in the cultured cell after penetrating the
plasma membrane. Moreover, an identical distribution pattern of
fluorescence emitted from BAMT-A and PNT-B in the insect and human cells
further verified that similar to intrinsic green in PNT-A, embedded red
fluorophore remains coupled with PNT-B tube wall after accumulation in the
cytosol. (Figure 5.2a, 5.3a, 5.4, 5.5)
Flow cytometry
The nanotube internalized cells were collected from the flasks and fixed for
propidium iodide staining. The PI stained nanotube internalised cells were
made to a single cell suspension for fluorescence collection in flow cytometry.
Green intrinsic fluorescence of PNT-A was collected by FL-1 filter and PI
fluorescence by Fl-2 filter. DNA profile thus obtained indicated no toxicity
due to internalisation of nanotubes when compared to the DMSO controls.
Further the viability of Drosophila S2 and noncancerous HEK-293 cells in
the presence of nanotubes showed more than 95% viability relative to DMSO
treated cells. Auto fluorescence in each of the channels has been collected
and subtracted. (Figure 5.2b, 5.3b) Cytotoxicity level was also assessed by
the MTT assay which has been plotted as histogram. (Figure 5.6)
Bacterial growth assay
To estimate the activity of PABA content of these self assembled
nanostructures a bacterial growth assay was designed wherein the fact that
PABA is known to inhibit bacterial proliferation was utilised. Whether PABA
retains its own biological properties in the self-assembled tubes, growth and
viability of the wild type bacterial stains (E coli K12) was used as a criterion.
To bacterial growth media PABA/PNT-A/PNT-B/DMSO was added in serial
dilutions of 200, 400, 800, 1600ug per ml media. On these bacterial plates
was spread equal volumes of bacterial cell suspension. The plates were
observed for colony size microscopically every 24hrs. Reduction in colony
size was evident in the PABA/PNT-A/PNT-B containing plates whereas this
was not observed in the control plates. This indeed establishes the active
PABA content of the nanotubes. The colony size was plotted against the
number of days in the line graph. (Figure 5.7a&b)
5.4.3 Discussion
Nanotechnology is the engineering and manufacturing of materials at
the atomic and molecular scale. In its strictest definition from the National
Nanotechnology Initiative, nanotechnology refers to structures roughly in the
1-100nm size regime in at least one dimension. Despite this size restriction,
nanotechnology commonly refers to structures that are up to several
hundred nanometers in size and that are developed by top-down or bottom-
up engineering of individual components. Herein, we focussed on the
application of nanotechnology to drug delivery and highlighted several areas
of opportunity where current and emerging nanotechnologies could enable
entirely novel classes of therapeutics.
The use of nanoscale or nanostructured materials in medicine has
gained a lot of importance recently. According to their structure, they have
unique medical effects – for example, the ability to cross biological barriers or
the passive targeting of tissues. Nanoscale particles/molecules developed to
improve the bioavailability and pharmacokinetics of therapeutics is used in
drug delivery. (Vagner et al, 2006) PABA nanotubes have shown excellent
internalization properties and good biocompatibility not only in vitro but also
in vivo. These nanotubes would now be further studied for the route of
internalization and conjugation with siRNA.
Most carrier systems are taken up by endocytosis and will probably
reach the lysosomes, where siRNA will be degraded by lysosomal enzymes.
Nonetheless, there are some possible methods of avoiding lysosomal
degradation. There are a number of different endocytic pathways to
internalize substances by eukaryotic cells. These pathways include
phagocytosis, macropinocytosis, clathrin-mediated endocytosis, non-
clathrin-mediated endocytosis, and caveolin-mediated uptake. Cationic-lipid-
DNA complexes, for example, are internalized via clathrin-mediated
endocytosis. (Zuhorn I 2002) Each of those pathways delivers siRNA to
specific cellular compartments. For an efficient RNAi activity, nucleic acid
tools have to reach their cellular targets after gaining entry into the cell. This
ability is based on their internalization pathway and it is highly dependent
on the delivery system used. Important for biological function, siRNA
requires protection from enzymatic degradation and cellular uptake without
lysosomal compartmentalization; finally, it must encounter its target mRNA
to ensure RNAi. (Chiu Y-L 2004)
RNA interference (RNAi) has been very successfully applied as a gene-
silencing technology in both plants and invertebrates, but many practical
obstacles need to be overcome before it becomes viable in mammalian
systems. Greater specificity and efficiency of RNAi in mammals is being
achieved by improving the design and selection of small interfering RNAs
(siRNA), by increasing the efficacy of their delivery to cells and organisms,
and by engineering their conditional expression. The use of RNAi may lead to
novel treatments for many diseases, yet, it appears that RNAi delivery and
specificity are the hurdles to overcome. (Manoharan et al, 2004) Novartis,
Alnylam and other leading Pharmaceuticals have started employing
nanotechnology and magnets for delivery.
Similar kind of drugs where an siRNA / shRNA is conjugated to
nanoparticle is envisaged for the future and it is already in the pipeline. The
delivery of exogenous nucleic acids to the various organs has the potential
for treatment of a wide range of acute and chronic diseases, such as acute
respiratory distress syndrome, cancer, asthma, cystic fibrosis and alpha-1
antitrypsin deficiency. The route of gene delivery is principally dictated by
the disease that is being treated, the cell types to be targeted and the gene
that is being delivered (i.e., whether a gene encoding an intracellular or
secreted protein is being delivered). Therefore an attempt has been made to
study the delivery of nanoparticles into Drosophila by the lab.
Furthermore, convenience and reproducibility of drug production, the
ability to target the desired cell type, and a lack of immune response are
desirable. Moreover multiple nonviral- based delivery methods have been
used in vivo for delivering siRNA, including hydrodynamic injection, cationic
liposome encapsulation, formation of cationic polymer complexes, and
antibody-specific targeting delivery systems. The large majority of current
nonviral methodologies have relied on nanoparticles or insoluble-complex
formation to protect the siRNA from the RNase-rich in vivo environment as
well as help siRNA cross cellular membranes. Unfortunately, nanoparticle
delivery systems have been shown to have limitations in vivo due to
insufficient biodistribution, low transfection efficiency, rapid plasma
clearance, and cellular toxicity. (Meade BR 2007)
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