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)