Gold nanoparticle/interferon conjugates as directed drug delivery mechanismsElizabeth Donoway

Pine Crest School

Miller School of Medicine, University of Miami

Background

• Pancreatic Cancer

• 5-year survival rate ranging from 14% to 1%

• Limited options for cancer treatment

• Low quality of life during treatment and tumorigenesis

• Current Pancreatic Cancer Treatments

• Surgery

• Whipple Procedure

• Chemotherapy

• Radiation Therapy

http://medicdaily.co/new-pancreatic-cancer-drug-trial-showing-promise/

Background

• Gold nanoparticles (GNPs)

• Excellent electrostatic properties

• Small size

• Physiologically compatible

• High binding affinity

• Interferons (IFNs)

• Three types of proteins released by host cells in response to pathogens, such as viruses, bacteria, and cancer cells

• Are broken down easily if not stabilized

• Type I interferons IFN-α and IFN-β are present in humans• Signal release of transcription factor p53 (suppresses tumors by

inducing cell apoptosis in mutated tumor cells)

• Cell-type-specific binding sites to existing cells in the body• Only bind to cells with the IFN-α receptor

• Can only bind to and upregulate p53 in cancer cells because the IFN-α receptor is a biomarker that is only present on cancer cells

• MIA PaCa-2 Cells

• Model cell line for pancreatic cancer

• Mesothelin overexpression

http://goo.gl/K232Lq

Interferon Induced Cell Apoptosis

Release of

interferon

Binding of interferon

to IFN-α/β receptor

Protein kinase R

(PKR) activation

Upregulation of p53

transcription factor

Activation of PP2A

tumor suppressor

Induced cell

apoptosis and

tumor suppression

Purpose and Hypothesis

• The goal of this research is to create a method of

treatment for pancreatic carcinomas that is more

accurate than current technologies, which targets,

as well as destroys, only malignant cancer cells but

does no harm to existing healthy pancreatic cells.

• By binding interferons IFN-α and IFN-β that induce

cell apoptosis solely in cancer cells to gold

nanoparticles, these stabilized novel drug delivery

mechanisms will be more easily localized to a single

amalgamation of cells and will initiate programmed

cell death in pancreatic carcinoma cells alone.

Materials & Methods

GNP/Interferon Conjugation

Introduction of Treatment

Data Analysis

Materials & Methods

• Gold nanoparticle formation• Turkevich method

• Standard method of GNP synthesis

• Determination of Interferon Avidity• Binding affinity of interferons to MIA PaCa-2 cells

• Interferons IFN-α and IFN-β bind to MIA PaCa-2 cells because they have the IFN-α receptor on their surface

• Drug Vector Synthesis• PEGylation of interferons IFN-α and IFN-βand binding with

biotinylated GNPs to create GNP/INF conjugates• Binding molecules are attached to the interferons and GNPs to

facilitate conjugation

• Cell culture• MIA PaCa cells cultured at 37.0 C in DMEM media• Culture split after 48 hours

• Introduction of treatment• Gold nanoparticle/interferon conjugates introduced directly to

MIA PaCa cell culture (100 mM)

• Controls• MIA PaCa cells cultured without introduction of treatment

Data Collection Methods

• UV-Vis Spectrometry• Confirm correct wavelength of GNPs

• Quantify size of particles

• Each wavelength measured corresponds to a certain size of particle

• DLS Zetasizing• Analyze zeta potential of GNP colloid

• Zeta potential is an arbitrary value that indicates the stability of a particle

• Particles with higher zeta potentials do not biodegrade or interact with physiological substrates

• Nuclear Staining via TUNEL Assay• Measure cell apoptosis beginning 48 hours after

introduction of treatment and analyze with an optical microscope to quantify apoptosis

• The TUNEL assay does not stain necrotic tissue; it only quantifies cell apoptosis. This is integral in assuring that the particles cause apoptosis and not total tissue death.

UV-Vis Spectrometry for GNPs

Above: UV-Vis Spectrometry of pure GNPs(absorption vs. wavelength) **Sample E is a

controlFigure A: UV-Vis Spectrometry of biotinylatedGNPs (absorption vs. wavelength)Figure B: UV-Vis Spectrometry ofGNP/Interferon conjugates

UV-Vis Spectrometry for GNPs

• Width of the peak indicates particle size

• Pure GNP Data Set:• Small observed peak width and more specific

wavelength interval corresponds to particles with an initial diameter of 2 nm

• Biotinylated GNP Data Set:• Larger observed peak width and broader wavelength

interval than pure GNP data indicates that the biotin binding molecules were successfully attached to the GNPs

• GNP/Interferon Data Set:• Largest observed peak width and broadest wavelength

interval of all data sets (pure GNPs and biotinylatedGNPs) indicates that the PEGylated interferon proteins IFN-α and IFN-β were successfully conjugated to the biotinylated gold nanoparticles. This peak size corresponds to particles with a final diameter of 20 nm, small enough that the particles will not impede biological functions and processes

DLS Zetasizing

Sample Zeta Potential

A 69

B 68

C 72

D 68

Average of all samples 69.25

Sample Zeta Potential

A 62

B 64

C 67

D 68

Average of all

samples

65.25

Zeta potential

analysis of pure

gold nanoparticle

colloid

Zeta potential

analysis of

biotinylated gold

nanoparticle

colloid and

PEGylated

interferons IFN-α

and IFN-ß

DLS Zetasizing

• The zeta potential of a colloid indicates its stability and is based on an arbitrary scale with a baseline 0, which indicates zero stability of a colloid. Higher zeta potentials denote more stable compounds.• 0 to ±5 = Rapid coagulation or flocculation• ±10 to ±30 = Incipient stability• ±30 to ±40 = Moderate stability• ±40 to ±60 = Good stability• ±61 = Excellent stability

• The pure gold nanoparticle colloids had an average zeta potential of 69.25. None of the samples had a zeta potential of less than 68

• The GNP/Interferon conjugate colloids had an average zeta potential of 65.25, signifying excellent stability after conjugation. None of the samples had a zeta potential of less than 62. Some of the stability of the particles was lost after binding them with the interferons because the proteins have a minute charge; however, this charge is insignificant to the overall stability of the conjugates

Transfection of MIA PaCa-2 with GNP/IFN Conjugates—TUNEL Assay

Control SampleNo interferon protein

introduced to MIA PaCa-

2 cell culture.

Little to no cell apoptosis

observed in each culture

after 48 hours. All cultures

were stained using TUNEL.

Transfection with IFN-α

and IFN-βInterferon proteins IFN-α

and IFN-β introduced to

MIA PaCa-2 cell culture.

Cell apoptosis

observed in every culture

transfected after 48

hours.

Transfection withGNP/IFN-α and IFN-β

ConjugatesGold nanoparticle/ IFN-αand IFN-β protein conjugatesintroduced to MIA PaCa-2

cell culture. Significant cellapoptosis observed in everyculture transfected after 48hours.

Transfection of MIA PaCa-2 with GNP/IFN Conjugates—TUNEL Assay• Brown staining of cells indicates cell apoptosis

• Transfection of the MIA PaCa-2 cell culture with interferons IFN-α and IFN-β causes cell apoptosis within the culture; however, since the interferons are broken down before binding with all of the cells, apoptosis is less significant than when interferons are stabilized with GNPs.

• Transfection of the MIA PaCa-2 cell culture with the GNPs/interferon conjugates induces significant cell apoptosis within the culture. Since the interferonsare stabilized with the gold nanoparticles, they are not degraded before binding to the IFN-αreceptors on the surface of the cells. This allows more time for the interferons to bind to the IFN-α receptors and initiate cell apoptosis by upregulating p53 before the proteins are destroyed.

Results

• Determination of interferon avidity

• Interferons specific to MIA PaCa-2 cells

• Interferons will only bind to cancer cells (which express the IFN-α receptor on their surfaces), leaving healthy, benign cells untouched and untargeted by the conjugates because transcription factor p53 is not upregulated in cells that are not mutated (healthy pancreatic tissue).

• Observation of wavelength shift

• ~535 λ ~580 λ

• Peak width increase indicates successful formation of GNP/IFN conjugates.

• Determination of physiological suitability

• ~2 nm without interferons

• ~20 nm conjugated with interferons

• Conjugates remain below the size threshold for cellular suitability

• Size of particles does not impede necessary cellular or biological functions. Size prevents bioaccumulation of particles, which can be filtered out of the body via the kidneys

• Formation of highly stable colloid

• Zeta potential of ~65

• Particles will not biodegrade before reaching target site or interact with physiological substrates.

• Induced Cell Apoptosis

• Stable interferon conjugates directed to cells cause programmed cell death

Discussion and Application

• GNPs increase the stability of the interferon protein, allowing it to reach the target site before being deconstructed by the body

• Interferon-induced upregulation of p53• Controlled cell-specific apoptosis

• Interferon presence within the body

• Easy disposal of particles• Physiologically suitable

• Size, stability, binding affinity• Easily filtered out of the blood by the kidneys

• Cancer treatment• Pancreatic cancer• Chemotherapy and Radiation Therapy

• Interact with biological substrates

• Issues with treatment localization

• New Cancer Therapies• Interferons are easily localized, do not interact with physiological

substrates, and do not interrupt crucial biological processes, making them interesting and novel options as cures for cancer, especially for those who are not candidates for other treatments

Future Research

• Genetic analysis of treated MIA PaCa-2 cell culture and untreated MIA PaCa-2 cell culture

• Compare the effects of the interferon protein on the genetic code of pancreatic cancer cells

• Assessment of efficacy of GNP/interferon conjugates compared to that of radiation and chemotherapy

• Clinical study of biological effects of GNP/IFN drug delivery mechanism on M. musculus

• Injection of drug vector into tumor site

• Analysis of tumor suppression and effects of GNP conjugates on mouse physiology

• Additional experimentation of GNP/IFN drug delivery mechanisms in differentiated cancer cell lines (HeLa, MCF-7, a549)

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