Nawkar Ganesh Mahadeo

Key Interactions Between Fields of Biology and Nanotechnology

Biology NanotechnologyModel

Tools

DNA / Gene Therapy

DNA / Gene therapy is defined as the transfer of genetic material into a cell for therapeutic benefit

A "correct copy" or "wild type" gene is inserted into the genome

The most common type of vectors are viruses

Target cells such as the patient's liver or lung cells are infected with the vector

It promises to provide new treatments for a large number of inherited and acquired diseases

(Verma and Weitzman, 2005)

DNA Vaccines

DNA vaccines consist of a DNA molecule, generally a circular plasmid, with a gene that codes for the protein against which an immune response is desired

The first demonstration of a plasmid-induced immune response was when mice inoculated with a plasmid expressing human growth hormone elicited antibodies instead of altering growth

They are capable of providing a broad, long lasting immune response

They are relatively simple, cheap and quick to produce and they are stable at room temperature

(Tang et al., 1992)

DNA Vaccination vs Gene Therapy

DNA Vaccines• The purpose of influencing the

immune system

• It aims to produce large amounts of protein in a short span of time so as to generate an immune response

• Does not requires a more targeted and finely tuned technology

• Aimed at a short-term presence of DNA in the animal is the desired result

• E.g. vaccines for HIV, herpes, hepatitis and influenza

Gene Therapy • The purpose of carrying out a

specific function

• It aimed at achieving a long lasting, physiologically matched expression of the gene, without activating the immune system

• It requires advanced technologies to target gene at specific site and its expression

• Aimed at presence of the added genetic material over a longer period of time

• E.g. Single gene defect disorders, cancer etc

Types of DNA / Gene Therapy

Germ line DNA / Gene therapy

• Germ cells - sperm or eggs, are modified by the introduction of functional genes

• Results in heritable change • Prohibited for application in human beings

Somatic cell DNA / Gene therapy

• The gene is introduced only in somatic cells• Expression of the introduced gene relieves/ eliminates symptoms of

the disorder• Effect is not heritable • Somatic cell therapy is the only feasible option

Genetic Diseases Potential Candidates for Gene Therapy

Three of the genetic diseases listed in table are presently the subject of gene therapy clinical trials

• Adenosine Deaminase deficiency using T lymphocytes, • Familial Hypercholesterolemia using hepatocytes, and • Hemophilia using fibroblasts

Defective gene Disease

1. Adenosine deaminase Severe Combined Immunodeficiency

2. Cystic fibrosis transmembrane regulator Cystic fibrosis

3. Factor IX Hemophilia B

4. Factor VIII Hemophilia A

5. Glucocerebrosidase Gaucher’s Disease

6. Low-density lipoprotein receptor Familial Hypercholesterolemia

7. 3-Globin Sickle Cell Anemia

Different Methods of Gene Delivery

Viral gene transfer Non-viral gene transfer

1. RNA virus vectorse.g. Oncoretroviruses, Lentiviruses, Spumaviruses

2. DNA virus vectorse. g. Adenoviruses, Adeno-Associated viruses, Herpesvirus

1. Electroporation 2. Microinjection3. Naked DNA4. Particle Bombardment5. Ultrasound

Novel gene transfer

Nanoparticlese.g. Liposomes, Gold Nanoparticles, Magnetic Nanoparticles

Viral Vector Construction

( Verma and Weitzman, 2005)

First Approved Gene Therapy Procedure

Ashanthi De Silva - A rare genetic disease called severe combined immunodeficiency (SCID)

Defective adenosine deaminase gene results in deficiency of ADA protein

It plays important role in deamination reaction

Causes toxicity of T lymphocytes

Lack of healthy immune system

Dr. W. French Anderson with four-year old Ashanthi De Silva at U.S. National Institutes of Health

Deoxyadenosine DeoxyinosineADA

Gene Therapy Strategy

Isolated T lymphocytes from patient and cultured in laboratory conditions

The correct copy of ADA gene was introduced into the T-cells using a retroviral vector

Following transduction, the cells ware grown in culture to attained significant number of cells

Gene engineered cells given back to the patient in procedure similar to a blood transfusion

The amount of the ADA protein in the T-cells has risen to 25% normal

Why ADA Deficiency was First Target of Gene Therapy?

The ADA gene had been cloned earlier

The gene is of average size and can easily be inserted into a retroviral vector

Bone marrow transplantation vs T cell replacement

The amount of the ADA protein that needs to be produced in order to maintain a functioning immune system is only 5-10 % of normal

Limitations of Viral Mediated DNA delivery

Toxicity and immunogenicity

Restricted targeting of specific cell types

Limited DNA carrying capacity e.g. for rAAV, commonly reported as 4.7kb

Production and packaging problems

Recombination and random integration into host genome

High cost

(Flotte, 2000)

Failures of Viral Mediated Gene Therapy Retroviral vector

• Dr. Alan Fischer – Conducting gene therapy on SCID-X1 linked hereditary disorder

• Hematopoietic stem cells from patients were stimulated and transduced ex vivo with MLV-based retroviral vector

• Expressing the γc cytokine receptor subunit, and then were reinfused into the patients

• During a 10-month follow up, γ c-expressing T and NK cells counts and function were comparable to age-matched controls

• Two of the children developed T-cell leukemia(Cavazzana et al., 2000)

Contd… Adeno-Associated Virus Vector

• Patients suffering from hemophilia B were treated with AAV vectors expressing human factor IX

• Intramuscular injecting AAV factor IX vectors directly into liver, which in turn have shown some unexplained toxicity

University of Pennsylvania (1999)

• A human Phase I clinical trial for ornithine transcarbamylase deficiencies

• This trial was designed to test the safety of an E1/E4- deleted recombinant adenovirus vector

• Jessie Gelsinger received highest dose and first person to die as result of vector delivery ( Raper et al., 2003)

Nanobiotechnology

Nanobiotechnology is a rapidly advancing area of scientific and technological opportunity that applies the tools and processes of nano/microfabrication to build devices for studying biosystems

Applications of nanobiotechnology are in various fields such as predictive diagnosis, medical care, drug discovery and environment

Fig. Nanobiotechnology Interdisiplinary Integration

What is Nanoscale?

“Nano” means dwarf in Greek

Nanocsale : 1 nm = 1 x 10-9 m

Nanodevices

NanoporesDendrimersNanotubesQuantum dotsNanoshells

Tennis ballWhite blood cell

Water molecule

The Timeframe of Nanobiotechnology

Applications of Different Nanoparticles in Medicine

Liposomes

• Liposomes are phospholipid vesicles (50–100 nm) • They have a bilayer membrane structure similar to that of

biological membranes and an internal aqueous phase• Liposomes show excellent circulation, penetration and

diffusion properties

Dendrimers

• These are highly branched synthetic polymers (<15 nm)• It show layered architectures constituted of a central

core, an internal region and numerous terminal groups• Wide application in Drug Delivery System (DDS) and

gene delivery

Liposomes

Dendrimers

Contd… Carbon nanotubes• These are formed of coaxial graphite sheets (<100 nm)

rolled up into cylinders• It exhibit excellent strength and electrical properties and

are efficient heat conductors• Due to semiconductor nature of nanotubes are used as

biosensors

Magnetic nanoparticles • These are spherical nanocrystals of 10–20 nm of size

with a Fe2+ and Fe3+ core surrounded by dextran or PEG molecules

• Their magnetic properties make them excellent agents to label biomolecules in bioassays, as well as MRI contrast agents

• Useful in targeted gene delivery

Carbon nanotubes

Magnetic nanoparticles

Contd… Quantum dots

• These are colloidal fluorescent semiconductor nanocrystals (2–10 nm)

• They are resistant to photobleaching and show exceptional resistance to photo and chemical degradation

• Quantum dots excellent contrast agents for imaging and labels for bioassays

Gold nanoparticles • These are one type of metallic nanoparticle of

size <50 nm• These are prepared with different geometries,

such as nanospheres, nanoshells, nanorods or nanocages

• These are excellent labels for biosensors

Quantum dots

Goldnanoparticles

Ideal Characteristics of NP Gene Vector System

A safe and efficient NP gene vector system must fulfill the following four requirements

1) Particle sizes must be in the submicron range that facilitates the penetration of the NPs through the cellular membrane

2) The possibility of surface modification that permits binding of NPs with the pDNA and enhances the stability of the NP-DNA complex

3) Biodegradability, so that the accumulated NPs in cells could be degraded

4) High transfection efficiency

Hurdles in DNA Delivery

Fig. DNA delivery pathways with three major barriers

(A) DNA–complex formation (B) Uptake (C) Endocytosis (endosome) (D) Escape from endosome (E) Degradation (edosome) (F) Intracellular release (G) Degradation (cytosol) (H) Nuclear targeting (I) Nuclear entry and expression

Polyion Complex (PIC) Micelles for Plasmid DNA Delivery

Fig. Polymeric micelles as intelligent nanocarriers for drug and gene delivery

Development of Polyion Complex (PIC) Micelle

Biocompatibility of the polyplexes improved by using PEG -b- polycation copolymers which electrostatically interact with pDNA to protect DNA from enzymatic and hydrolytic degradation

pDNA /PEG -b-PLL micelles intravenously injected intact pDNA observed in blood circulation after 3 hr

PIC micelles stabilized by disulfide cross linking

The intravenous injection of cross linked PIC micelles into mice resulted in a uniform gene expression in the liver

To achieve a site-specific gene delivery, polyplex micelles might be modified with targetable ligands such as peptides and antibodies

(Miyata et al., 2005)

(Harada-Shiba et al., 2002)

(Merdan et al., 2003)

A-B-C type Triblock Copolymer

(Fukushima et al., 2005)

A) PEG SegmentB) poly[(3-morpholinopropyl) aspartamide] (PMPA) as a low pKa polycationC) PLL segment

Dendritic Photosensitizer for Light-induced Gene Transfer

pDNA condensation = quadruplicated cationic peptide (CP4) + nuclear localization signal (NLS) Anionic DPc (Dendritic phthalocya- nine) = photosensitizer Mechanism 1.Cellular uptake of the ternary complexes via endocytosis,2.Dissociation of DPc from the complexes in acidic vesicles due to the protonation of the carboxyl groups on the dendrimer periphery 3.Endosomal escape of the pDNA/CP4 complexes to the cytoplasm upon photo irradiation

Fig. pDNA/CP4/DPc ternary complexes

(Nishiyama et al., 2005)

Advantages of Nanocarriers over Viral Vector

They are easy to prepare and to scale-up

They are more flexible with regard to the size of the DNA being transferred e.g. DNA compacted nanoparticles can contain plasmids up to 20 kb

They do not elicit a specific immune response and can therefore be administered repeatedly

They are better for delivering cytokine genes

They show little to no toxicity in the targeted tissues, and modest immune response when high concentration

Targeted gene delivery is possible

(Fink et al., 2006)

(Cooper, 2007; Farjo et al., 2006)

Future Challenges in Nanoparticle Application

It is not yet possible to predict nanoparticle biodistribution according to their physicochemical properties

Once nanoparticles reach their target site, and despite their small size, they do not enter into biological systems, such as cells or organelles, easily

Inside the cell, nanoparticles can remain structurally unaltered, can be modified or can be metabolized

More study is required about toxic effects of nanoparticles

Case Study

Objectives

To develop the preparation protocol of PBCA-CTAB NPs

To study its characteristics

To develop the AFP-positive hepatocellular carcinoma gene therapy using the PBCA-CTAB NP–pAFP-TK complex

To study the expression of pAFP-TK in vitro

Materials and Methods HepG2, HeLa, and 3T3 cells (American Type Culture Collection)

Escherichia coli DH5 α and pAFP-TK plasmid

Enhanced green fluorescent protein plasmid N1 (pEGFP-N1) from Clonetech

Herpes simplex virus thymidine kinase (HSV-TK) primers

A-butyl-ester cyanoacrylic-acid (BCA) from Baiyun Limited Co.

Cetyltrimethylammonium bromide (CTAB) from Sigma

3-(4,5-dimethyl-2-thiazolyl)-2,5–diphenyl-2H- tetrazolium bromide (MTT) and DNase I from Sigma

Equipment used

• Zetasizer 3000 and AJ-III Atomic Force Microscope (AFM)

Contd… Amplification and purification of plasmid DNA

Preparation of PBCA NPs

• PBCA NPs were prepared by an emulsion polymerization method

• Tween 80 was dissolved in distilled water (pH 2.8)

• PBCA was added in slowly and mixed by magnetic stirring at room temperature (22 °C-25 °C) for 5 hours

• Centrifuged at room temperature at 5000 rpm for 15 minutes

• The suspension was filtered by polyethylene terephthalate nuclear membrane filter (diameter of pores = 0.22 µm)

AFP Photograph

Contd… Surface modification of PBCA-CTAB NPs

• 16.6 ml of PBCA NP solution (0.625%, w/v)

+ 33.33 ml of CTAB solution (0.25%, w/v)

• Precipitation washed with dd H2O and resuspended in 150 mM NaCl

• Mixture lyophilized to steady state

Characterization of PBCA NPs and PBCA-CTAB NPs

• PBCA-CTAB NPs were uniform and that the average diameters were between 80 and 200 nm

• Zeta potential of the NPs revealed a positive surface charge of +15.6 mV

1 hr incubation @ 4000 rpm for 30 min

Results

Cell viability• Cytotoxicity of PBCA NPs and

PBCA-CTAB NPs to HepG2 cells and 3T3 cells was estimated by MTT assay

• The toxicity of NPs would suddenly strengthen with increasing concentration

Cytotoxicity of PBCA NPs

Cytotoxicity of CTAB PBCA NPs

No. Cell Type Concentration of NPs

1. HepG2 100 ng/µl

2. 3T3 200 ng/µl

DNA Loading Efficiency of PBCA-CTAB NPs

The difference between the total amount of pDNA added in the NP preparation buffer and the amount of non-entrapped pDNA remaining in the aqueous suspension

PBCA CTAB NP solution + pDNA in 50-µL reaction system (pH = 7)

Results in different kinds of PBCA

CTAB- pDNA NPs in which the ratio of PBCA-CTAB NPs to pDNA was 1:1, 5:1, 10:1, 15:1, 20:1, 30:1, and 50:1, respectively

Fig. The change in DNA loading efficiency of various NPs

Gel Retarding Analysis and Protection Effect of NPs to pDNA

PBCA-CTAB-pDNA complexes (containing 2 µg pDNA) were incubated with 2 µl of RNase-free DNase I solution (1 µg/µl) in 50 µl of reaction buffer for 15 minutes at 37 ° C

The reaction was stopped by adding 2 µL of RQ1 DNase stop solution

The integrity of the pDNA was analyzed by gel electrophoresis (1% agarose)

Fig. Electrophoretic mobility analyses of PBCA-CTAB NP–pDNA complexes

1:1 5:1 10:1 15:1 30:1 50:1 10:1

In vitro Gene Transfection Efficiency

The transfection efficiency of PBCA-CTAB NPs was evaluated in HepG2 cells and 3T3 cells using the enhanced green fluorescent protein (EGFP) gene as a reporter

Super- Fect Transfection Reagent was used as a positive control

Naked pDNA was used as negative control

The results, observed by inversion fluorescence microscope after transfection

Fig. The expression of the EGFP gene loaded by A) PBCA-CTAB NPs expressed in HepG2 cells B) SuperFect Transfection Reagent expressed in HepG2 cells C) PBCA-CTAB NPs expressed in 3T3 cells D) SuperFect Transfection Reagent expressed in 3T3 cells.

RT-PCR Analysis

To detect the expression of the HSV-TK gene

Expression of β-actin mRNA was detected as an internal standard

PBCA NPs modified with CTAB can enter the cells effectively with exogenous genes, which can also normally express in cells

Fig. Expression of the TK gene in three kinds of cells transfected by different pDNAs

*(pAFP-TK gene, p3.1-TK and pcDNA3.1)

The expression of the TK gene in the AFP-positive cells was controlled by AFP enhancer more strongly than cytomegalovirus

Sensitivity of Transfected Cells to GCV

MTT assay was used to examine the sensitivity of GCV to transfected HepG2 cells

The concentration of GCV was 10 µg/ml then the cell viability was not influenced

The concentration of GCV was 50 µg/ml then 50% of cells were killed

GCV had a dose-dependent effect on survival of AFP-positive cells

Fig. Examination by MTT assay of sensitivity to GCV of transfected HepG2 cells

Apoptosis Induced by PBCA-CTAB NP - Mediated pAFP - TK/ GCV System

HepG2 cells transfected by pAFP-TK loaded by PBCA-CTAB NPs

Stained using Hoechst 33258 stain

After treatment of GCV, the nucleus of cells was condensed

It confirmed one of the mechanisms of the lethal effect of the PBCA-CTAB NP-mediated pAFP-TK/GCV system

Fig. Apoptosis of cells induced by GCV after transfection by pAFP-TK plasmid

A)Fluorescence staining of transfected HepG2 cells not treated by GCVB)Fluorescence staining of transfected HepG2 cells treated by GCV

Summary

Developed a novel transfection vector, PBCA-CTAB NPs - a non-viral vector that can deliver DNA into targeted cells

The pAFP-TK/GCV suicide gene therapy system have a high transfection efficiency in AFP-positive cells and potent antitumoral activities in vitro

The system may be an effective candidate vector for treatment of AFP-positive tumors