Download Link - University of California, Berkeley

Nano-Vehicular Targeted
Drug Delivery
Gabriel Lavella, Jaehyun Park, Jaeseok Jeon
EE235 Final Project
May 11, 2009
Electrical Engineering and Computer Sciences
University of California, Berkeley
• Overview of Drug Delivery
• Targeted Drug Delivery
• Current Anti-Cancer Strategies
• U.S. Cancer Statistics
• Nano-Engineered Cancer Treatment Solution
Drug Delivery
• In vast majority cases, drugs are delivered throughout the body via circulatory system
• Example : Intravascular injection or oral ingestion
Tablet of Chitosan
and Enteric-coated
layers
Stomach : where coated
tablet remain intact
Small intestine : where
enteric-coated layer dissolves
Large intestine : where
Chitosan-coated layer
disintegrates
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
3
Targeted Drug Delivery
• Method of delivering medication preferentially
– To the right place in a body
– At the right time, the right dose
– For the right time period
– NOT to the other places where drug side-effects may arise
Active Receptor
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
4
Surgery
• Surgical excision of cancer tissues
• Accompany pain, risk of infection, poor wound healing
Radiation
• Method of completely killing cancer cells or shrinking tumors
or relieving symptoms using high-energy radiation
• Harm health tissues and damage nearby normal cells
• Accompany pain, nausea, hair loss, damage to normal cells
Chemotherapy
• Chemical method of killing cells that divide rapidly (cancer cells)
• Kills normal cells that divide rapidly under normal conditions
• Accompany pain, nausea, memory loss, malnutrition
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
5
About 0.5 Million Expected to Die of Cancer in 2008
Overall Costs of Cancer in 2007 = $219.2 Billion
• $89 billion for health expenditures
• $18.2 billion for productivity loss due to illness
• $112 billion for productivity loss due to premature death
Lack of Health Care and Other Barriers
34%
Leading causes of death in 1999, adults ages > 25
Cancer : 1 of every 4 deaths!
42%
Cardiovascular Disease
24%
Other Causes
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
6
Estimated Number of NEW Cancer Cases for 2008
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
7
Blood
stream
Targeted Drug Delivery Systems
• Nano-particle that carries attached drug to the site of action,
with its path monitored by attached fluorescent detecting agent
Tumor
Cell
Target to
tumor cell
Technical Advantages
• Higher efficacy resulting from selectively targeting
and killing cancer cells
• Reduced toxicity and lower side-effects
Monitor
targeting
• Potentially more cost-effective
• Prospect for shorter treatments times
Signaling
Agent
Targeting
Agent
Drug
Released inside
tumor cell
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
Smuggle attached
drug into tumor
cell
8
• Dendrimers : Introduction / Characteristics
• Multifunctional Dendrimers as Drug Delivery
• Targeting to Cancer Cells
• DNA-Linked Dendrimers
• Fabrication Process
• Experiments
Dendrimers : Repeatedly Branched Molecules
• Consist of a series of chemical shells built on a small core molecule
• Each shell (generation) consists of monomer layers, made by repeating chemical-linking
• Beyond G5 : Begin to become spherical and 3-D structure
Surface Groups
• Can be variously functionalized
• Cationic / Anionic / Neutral
• Targeting groups
• Dyes & Biomarkers
Similarity to Protein
• Size / Weight
• Very well-defined chemical structure
• Ease of cellular uptake
Dendrimers : Ideal Building Block
for Creating a Biologically Active Nano-material
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
10
Possible Tasks Can Be Performed
• Diseased cell recognition
• Reporting detection
• Diagnosis of disease state
• Reporting outcome of therapy
• Drug delivery
Multifunctional Dendrimers
• Multiple functions can be bound onto dendrimers
Targeting
Agent
Detecting
Agent
Therapeutic
Agent
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
11
To Target Specifically to Cancer Cells
• Cells have on their surfaces receptors for specific molecules
• Specific receptors for specific molecules are targeted
• Specificity can be precisely controlled by targeting active receptor
• Taken up into the cell through specific receptor on cancer cell
Fluorescent tagged nanodevice are taken up through
the receptors then spread into cytosolic area
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
12
Targets : Folic Acid (FA)
• FAR (high affinity receptor for FA) : overexpressed in several human cancers, even
up to a 100-fold
• Easily available and inexpensive / small molecular size
Therapeutic Agent : Methotrexate (MTX)
• Widely used chemotherapeutic drug for the treatment of a variety of malignancies
• Inhibits cytosolic enzyme dihydrofolate reductase (DHFR)
• Results in depletion of reduced FA required for nucleotide synthesis
• Thus leading to the inhibition of DNA replication and subsequent cell death
Fluorescence Tag
• Various chemicals : Fluorescein, AlexaFluor
• Used for monitoring and tracking
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
13
Difficult to Assemble Multiple Functions onto One Dendrimer
• Complex chemistry needed for self-assembly of core and shell dendrimers
• No specificity between the coupling of dendrimers
Self-Assembly using DNA
• Each of two dendrimers carries single-stranded DNA with the same length
• DNA strands are complementary
• Self-assembly with forming double-stranded DNA
• Barbell-shaped, two-dendrimer complexes
Fluorescent Dye
• Fluorescence can be separated to the other
Folate (to receptor)
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
14
Preparation of Each DNA-Dendrimer Conjugate
1. Control surface charge density of amines
- By substituting with acetyl groups to prevent infinite network formation due to
electrostatic interaction
- Acetylation limited to 90% of amines due to densely packed structure
- G5 : 12 amine groups / G7 : 108 amine groups
2. Prepare DNA strand
- 16-32 nucleotides for spacer from dendrimer / 34 for complementary base pairing
3. EDC/imidazole (0.1M) chemistry used : to activate DNAs for 10mins
4. Slowly mix with LiCl (0.5M) used to weaken electrostatic interactions
5. Allow to react overnight at RT
6. Remove small molecules with membrane filter
7. Purify non-conjugated DNA from using gel electrophoresis
8. Extract purified DNA-dendrimer conjugate from gel
9. Each dendrimer is functionalized : Target, drug and Fluorescence
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
15
Self-assembly
1. Mix two DNA-conjugated G7 and G5 dendrimers in equimolar ratio
- to prevent crosslinking and formation of very large complexes
2. Annealing : Heated at 90°C for 10 mins with hybridization buffer
3. Cool at RT for 3 hours
G7
G7
G5
G5
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
16
Does Target Only Cancer Cells, NOT Others?
• Targets works to tumor cells
PBS (Control)
Non-targeted
Nanodevice
Targeted
Nanodevice
Only targeted nanodevices taken up
• Targets DOES NOT work to other cells
PBS (Control)
Non-targeted
Nanodevice
Targeted
Nanodevice
Spleen cells : no fluorescence
In Vivo Experiment : Drug (MTX) Study in Mice
Mice received conventional drug
Mice received drug in targeted nanodevice
• Loss hair / Loss weight (general toxicity)
• No tumor reduction unless high dose
• Retained hair / No weight loss (Non-toxic)
• Necrotic tumors : reduction in size with
low dose
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
17
Growth Inhibition to KB Cells
Dose-Dependent Inhibition
Dose-Dependent Binding of FAs
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
18
• Market Description
• Competing Technologies
• Valuation and Growth of the aggregate
Cancer Therapy Market
• Sub-Valuation of the Folate Receptor
Targeting Market
Market Divisions
• Market research into targeted drugs is typically subdivided into passive and active
delivery vehicles and cancer and non-cancerous diseases
Passive Delivery
• Preferential release that exploits structural differences of
tumor tissue
- Example: microcapsule delivery through highly fenestrated
tumor vasculature
Active Delivery
• Release agents at specific molecular docking sites
– Docking sites: Folate receptor (epithelial),
RAK1 (breast cancer)
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
20
Hybrid Delivery Systems
• Use of large nanovehicles with targeting molecules to penetrate fenestrated
vasculature and bind
Non-Cancer Targeting
• Includes multitude of disease states, where receptor is uniquely or over expressed
(Alzheimer's, Parkinson's, Cardio-pulminary diseases, etc.)
Cancer Cell Targeting
• This is currently the primary focus for targeted drug delivery
While Applicable to Four of Market Subdivisions,
Multivalent Oligonucleotide – dendrimer Based Delivery
Is Primarily Focused on Active Delivery to Tumors
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
21
General Comments on Competing Technologies
• Efficacy and reduction of side effects will primarily determine which technologies
become adapted for use in treatment
• Active targeting technologies are expected to become the next generation cancer
therapeutic agent
• Search for targets for next generation medications has been slow,
folate receptors targets are the most promising
Competing Folate Receptor Targeting Technologies
• Radiopharmaceutical
• Antisense oligonucleotides
• Plasmids
• Immunotherapeutic agents
• Liposomes with entrapped drugs
• GNPs
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
22
Overall Valuation
• Total cost in 2008 was estimated at $219.2 billion per year, this includes:
– Indirect Mortality Costs : $ 112.0 billion
– Indirect Morbidity Costs : $ 18.2 billion
– Direct Medical Costs :
$ 89.0 billion
Cancer Pharmaceutical Treatment Market Valuation
• From the $ 89.0 billion in direct costs
• $ 43.0 billion is was spent on drug therapy
Growth
• Average rate of cancer treatment market growth from 1963 to 2004 was 10.3%
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
23
Current Revenues for Individual Targeted Therapies
• As of 2007, 5 targeted therapies have annual gross revenues each exceeding $1 billion
(all of these used passive targeting)
Rituxan – Herceptin – Avastin – Eribtux – Gleevec
• Total revenues in 2007 for targeted cancer therapies totaled $17.3 billion
with a year over year growth rate of 33%. Analysts expect growth rates to slow to
10-11% as non targeted are replaced by targeted
• Eight new targeted therapies are expected to also reach blockbuster status
(revenues > 1 billion) in 2008-2009
• Typical cost of development and approval for new cancer drugs ranges from $250
million – $1.25 billion, these costs exclude marketing and sales and include failed trials.
New targeted therapies have generated extremely high ROI for pharmaceutical
companies in recent years.
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
24
Projected Market Share for Targeted Technologies
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
25
Estimated Pharmaceutical Expenditure for FR based Cancers: $ 19 billion/year
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
26
Nano-Vehicular Targeted Cancer Therapy
• Expected to Be the Next Generation Cancer Treatment
DNA-Linked Dendrimers
• Provide Versatility and Controllability to Making Them Strong Candidates for
Targeting Vehicles
Folate Receptor Mediated Molecular Recognition
• Currently the Most Promising Oncological Target
Development of Targeted Therapy
• Has Proven to Provide Large Returns on Investment with the Potential for
Extremely Large Revenue Generation
Targeted Nano-Vehicular Cancer
Therapy
May 11, 2009
EE235 Final Project
27