invited talk: nanotechnology strategy in medicine on nanotech and bioresource 2015 vfinal-p… ·...
TRANSCRIPT
Invited Talk:
Nanotechnology Strategy in Medicine Shahidan Radiman
School of Applied Physics , Faculty of Science and Technology UKM Bangi 43600 , Selangor DE. E-mail: [email protected]
w w w . u k m . m y / f s t
Conference on Nano- and Bioresource Technology 2015 (NBT2015) , 28 -29
March 2015, Universiti Kebangsaan Malaysia
Content of Talk 1. Background
2. Brief History of Nanoparticles in Medicines
3. Drug delivery mechanism for passing the Blood- Brain
Barrier
4. Some of our work in this area
5. New strategies to be developed
6. Conclusions
Possible Applications of Gold Nanorods
By combining terminal protection of small molecule (folate)-capped
DNA probes, exonuclease III signal amplification and gold
nanoparticles, a simple and label-free colorimetric assay were
develpoed for highly sensitive detection of folate receptor (FR). A
detection limit of 50 fM FR was obtained using UV-vis spectrometry and
10 pM FR could be visualized by the naked eye.
1.Background
Use of nanotechnologies in medicine is not new- has been
around for 30 years.
Today 44 nano-delivery products have received marhet
approval in the USA already ( it includes 18 pharmaceutical
products and 15 field imaging and diagnostics)
According to BCC Research Market report (2012) the global
nanomedicine market is expected to grow from $63.6 billion
in 2010 to $130.9 billion by 2016.
Intake of Nanoparticles
Petros and DeSimone , 2010
In general , NP are used for therapeutics applications
to achieve:
(i) precise delivery
(ii) improve solubility
(iii) extend half-life
(iv) improve therapeutic index ,
(v) reduce immunogenicity
(vi) enhanced multifunctionality
The first liposome- based therapeutic is liposome-
encapsulated doxorubicin (Doxil) approved by US FDA in
1995 for the treatment of HIV-related Kaposi sarcoma – it
showed reduced cardiotoxicity compared to free drug.
PEG (polyethylene glycol) can enhance the solubility and
plasma stability of proteind and reduce immunogenicity .
PEG- L-asparaginase was approved by FDA in 1994 for
treatment of acute lymphocytic leukemia. Other PEGlated
drugs follow suits namely PEG-interferon –alpha2a and
2b for hepatitis C and PEG-granulocyte colony-simulating
factor for neutropania.
In general PEG and other polymers act as steric brush and
prevent protein absorption on NP surface immediately after
contact in plasma called opsonization.
Later albumin-coated liposomes were shown to decrease
accumulation in liver, spleen and heart with increased
accumulation in tumour as well as longer retention time and
faster cell uptake using elongated particles.
Many types of cancer cells overexpress transferrin, folate
receptors making conjugation of transferrin , folic acid or
antibodies to these receptors successfully targeting approach.
For cell uptake ,ligands such as folic acids , albumin,
cholesterol have been shown to facilitate uptake through
caveolin-mediated endocytosis whereas ligands for
glycoreceptors promote clathrin-mediated endocytosis
destined for lysosomal compartment ( cytosol,
mitochondria , nucleus).
Another approach is to use macropinocytosis , a non-
caveolin-mediated nor clathrin-mediated process by using
cell-penetrating peptides such as a trans-activating
transcriptional activator (TaT) peptide into the design of
engineered nanoparticles.
Tumour tissue in lungs contain high concentration of
some proteases which are enzymes that breakdown
and cut specific proteins. By modifying nanocarriers
with a protective layer than only these proteases can
breakdown , the process then releases the drug within
the nanocarriers.
The reducing nature of cytosol has been used extensively in
protein-conjugate chemistry to trigger release of payload on
cellular internalisation with cargos ranging from
oligonucleotides to toxins and chemotherapeutics.
In cellular targeting , most current methods almost
exclusively target some type of membrane-bound protein
with one exception i.e targetting carbohydrates on the
surface of cancer cell with lectins.
Use of antibodies to target prostate specific membrane
antigen (PSMA), a 100kDa type II membrane glycoprotein is
highly expressed in all prostate cancel cell and not in
healthy cells.
Currently , there is a need to direct engineered NP to specific
subcellular compartments (nucleus, cytosol, mitochondria ,
peroxisomes , lysosomes/endosomes). Delivery to
mitochondria is largely based on electrostatic interactions
between NP and mitochondrial membrane which has
membrane potential ca.130-150 mV. This can be done by
grafting cationic species such as triphenylphsophonium
cations as well as peptides.
Targeting NP to the nucleus remains a significant challenge.
The nucleus is separated from the cytosol by two
membranes with pores ca. 10 nm which allows free diffusion
of macromolecules ca. 30-40 kDa. One way is to activate the
nuclear transporter , transportin .
The term theranostics was probably first used by
PharmaNetics president and CEO John Funkhouser
(1998) in describing his company’s business model
in developing diagnostic tests directly linked to the
application of a specific therapies. In the case of
PharmaNetics this takes the form of new generations
of point of care coagulation tests supporting
coagulation therapies:
Diagnostics – the ability to define a disease state.
Theranostics – the ability to affect therapy or
treatment of a disease state.
Definition of Theranostics
What do you get when you combine a molecular diagnostic
agent with a targeted therapeutic? A powerful new tool
paving the path to personalized medicine called a
Theranostic. The basic architecture of a theranostic is as
follows:
a targeting agent which directs the theranostic to a
molecular target on the surface of a cell or tumor
a chelate in the form of an imaging agent (which enables
visualization of the target) or a therapeutic drug (for
delivery of treatment to the target site)
a linker which serves to connect the two entities
The integration of multiple moieties into a single agent for
imaging and therapeutic purposes provides a powerful new
paradigm for advancing treatments against cancers and
other diseases.
The 3rd Theranostics World Congress (3TWC)
will take place on the campus of
Johns Hopkins University
on March 12-14, 2015
As an example – Upconversion Nanoparticles
Wang et al , Nanoscale 7 ,190- (2015)
M.S Muthu et al , Theranostics (2014) , 4 (6)
2. Brief History on Nanoparticles in Medicines
Petros and DeSimone (Nature Reviews 9, 2010)
2013: Glucose-coated gold nanoparticles transfer across
human brain endothelium and enter astrocytes in vitro,
similalrly also for gold NP coated with nucleic acids.
2014 : A fungal protein from Cryptococcus neoformans
called Mpr1 ( a metallo protease) can pass the blood brain
barrier. It could be attached/conjugated to NP as carrier in
drug delivery pass the BBB
2014: Model BBB from culturing both primary rat brain
endothelial cells and pericytes to support tight junction of
endothelial cells followed by NP permeability experiments
The EPR (enhanced permeability retention) effect
The EPR effect was first reported by Matsumura and Maeda in
1986 and was described in greater detail and validated by Maeda
et al. Their investigations showed that most solid tumors have
blood vessels with defective architecture and usually produce
extensive amounts of various vascular permeability factors. Most
solid tumors therefore exhibit enhanced vascular permeability,
which will ensure a sufficient supply of nutrients and oxygen to
tumor tissues for rapid growth. The EPR effect considers this
unique anatomical–pathophysiological nature of tumor blood
vessels that facilitates transport of macromolecules into tumor
tissues. Macromolecules larger than 40 kDa selectively leak out
from tumor vessels and accumulate in tumor tissues. In contrast,
this EPR effect-driven delivery does not occur in normal tissues .
This unique phenomenon in solid tumors— the EPR effect — is
thus considered to be a landmark principle in tumor-targeting
chemotherapy and is becoming an increasingly promising
paradigm for anticancer drug development.
For example, Doxil, which is a PEGylated (polyethylene
glycol-coated) liposome-encapsulated formulation of
doxorubicin, was approved for treatment of Kaposi
sarcoma and other cancers.
Many other polymeric or micellar drugs are in clinical
stage development (phases I and II) . Compared with
conventional anticancer drugs, most of which are small
molecular drugs, these macromolecular drugs have
superior in vivo pharmacokinetics (e.g., a prolonged
plasma half-life) and, more important, greater tumor
selectivity, so that they produce improved antitumor
effects with no or less adverse reactions .
Bertrand et al Adv Drug
Delivery Rev , 2013
Theranostic mechanism of functionalised QD at
cellular level Xu et al , Frontiers in Phamacology
2013
3. Drug delivery mechanism passing the blood brain barrier A number of obstacles present substantial challenges when
attempting to treat CNS disorders. For example, systemically
delivered products must pass through the blood–brain barrier
(BBB), and substances delivered intracranially must withstand
the substantial dynamic force of cerebrospinal fluid (CSF) flow
in the brain interstitium. In addition, the complex cellular
organization of the brain and spinal cord complicates the
targeted treatment of specific cell populations.
Nanotechnology presents a potential solution to these
problems.The scale of nanoengineered materials enables the
structures to interact with biological substrates at a molecular
level, providing these materials with the potential to effect
change in biological systems in unprecedented ways.
BBB limits the brain penetration of most CNS drug
candidates
Neurological disorders such as HIV-associated
encephalopathy has significant morbidity and
mortality.
Of more than 7000 drugs in the comprehensive
medicinal chemistry database only 5 % can be used
for CNS treatment , only 12 % are active in the CNS
and only 1 % are active in the brain.
The Blood-Brain Barrier
•To be BBB permeable , molecules need to be lipid soluble and less than 400 Da in size.
•Larger biomolecules can pass if they are transported by receptor-mediated transcytosis (RMT) using ligands that bind to specific BBB receptor.
• Some of the attempted delivery systems include transcranial brain drug delivery, trans-nasal brain drug delivery , BBB disruption and small molecule lipidisation.
. One can use the circulating phagocytic cells such as monocytes or macrophages as Tojan horse to deliver drug molecules into the brain . In the human brain there are about 100 billion capillaries providing a combined length of brain capillary endothelium of approximately 650 km and total surface area of ca. 20 m**2. Any molecules entry into the brain is strictly controlled by Blood-Brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) .The chief anatomical and functional site of the BBB is the brain endothelium. Physiologically in addition to brain capillary endothelilal cells, extracellular base membrane , adjoining pericytes , astrocytes and microglia are all integral part of BBB supporting system.
A feature of the BBB is its low and selective permeabilities attributed to its unqique biological characteristics : (i) The presence of tight junction (TJ) between adjacent
endothelial cells formed by intricate complex transmembrane proteins which are linked to the actin cytoskeleton forming the most intimate cell-to-cell connection
(ii)The expression of various transporters including glucose carrier (GLUT 1) , amino acid carrier (LAT1) , transferring receptors , insulin receptors , lipoprotein receptors and ATP family of efflux transporters.
(iii)The synergistic inductive functions and upregulating of BBB features by astrocytes , astrocytic perivascular endfeet , pericytes , perivascular macrophage and neurons ( as suggested from cell culture studies) (iv)The lack of lymphatic damage and absence of major histocompatibility complex antigens. The BBB has a strict limit for the passage of immune cells especially lymphocytes and its immune barrier is made by the association between BBB endothelial cells and perivascular macrophages and mast cells. This immune barrier is reinforced by local microglial cells.
Xu et al , Frontiers in Pharmacology 4(10), 2013
Blood –brain barrier
Transport route across the blood-brain barrier
Some BBB terms :
CMT – carrier mediated transport : provide a facilitated
mechanism for certain small molecules , nutrients and
hormones to passively cross the BBB following a concentration
gradient ( mediated by CMT proteins e.g large neutral amino
acid trasnporter (LAT1))
Trojan horse drugs can be designed to target specific CMT
systems ( glucose transporter, organic anion transporting
polypeptides)
RMT- Receptor mediated transport: brain microvascular
endothelial cells express receptor –mediated transport through
transcytosis. Receptors include transferrin , insulin, lipoprotein,
peptides called angiopeps .
Efflux transport system : Efflux transporters include P-gp,
breast-cancer resistance protein (BCRP), multidrug
resistance proteins (MRPs).
BBB disruption : breaking down of tight-junctions, osmotic
disruptions etc. Very risky even though transient (e.g of
disruptors are short-chain alkylglycerols and medium
chain fatty acid salt sodium caprate ). Phamacologic
disruptors include sildenafil (Viagra) and vardenafil (Levitra)
Cell-penetrating peptides (CPP) : They are also known as
protein-transduction domains . CPPs are oligocationic or
amphiphilic peptides sequence of 10-30 amino acids that
can transverse mammalian plasma membrane and BBB.
The mechanism of CPP transport is currently believed to be
through endocytotic uptake.
Yan Chen and Lihong Liu , Advaced Drug Delivery Reviews 64(2012), 640-665
Transport route across the blood-brain-barrier
Use of quantum dot-bioconjugates to pass the BBB
(Xu et al , Frontiers in Pharmacology 2013)
Yan Chen and Lihong Liu , Advaced Drug Delivery
Reviews 64(2012), 640-665
Problems Formulation of water soluble drug with lipidisation is difficult to execute successfully. Poor solubility and stability on physiological medium Possible solutions- Nanotech approach Improved bioavailability and site specific biotargeting Drug delivery nanoplatform can also be used for diagnostics at the same time (theranostics) e.g transferrin (an Fe-binding glycoprotein) are easily conjugated to NP for targeting BBB and facilitate the RMT process.
Srikanth and Kessler (2014)
Srikanth and Kessler (2014)
4. Some of our own work in drug delivery
Materials and Methods
Materials: - N-dimethylglycine Betaine with 35% active substance in H2O
(classifies as zwitterionic surfactant)
- 5-cholesten-3β-ol (Cholesterol, purity≥ 99%)
- 1-Decanol with purity ≥ 99%
- Deionized water
Materials was purchased from Fulka Co. and used as received without any further purification.
a) b)
Figure3: a) N-dimethylglycine betaine structure. b) Cholesterol
R. Elenaizi et al, JCIS submitted
Figure 1. Partial-Ternary phase diagram of Betaine/ Cholesterol/water system for short time period (~ 1 month).
Figure 2. Partial-Ternary phase diagram of Betaine/ Cholesterol/water system for long time period (~ 6 month).
Akter et al , Nature Sc. Report 1 (2011)
For topical application
Carrageenans are large, highly flexible molecules that curl forming helical
structures. This gives them the ability to form a variety of different gels at room
temperature. They are widely used in the food and other industries as
thickening and stabilizing agents.
All carrageenans are high-molecular-weight polysaccharides made up of
repeating galactose units and 3,6 anhydrogalactose (3,6-AG), both sulfated
and nonsulfated. The units are joined by alternating α-1,3 and β-1,4 glycosidic
linkages.
There are three main commercial classes of carrageenan:
Kappa forms strong, rigid gels in the presence of potassium ions; it reacts with
dairy proteins. It is sourced mainly from Kappaphycus alvarezii.
Iota forms soft gels in the presence of calcium ions. It is produced mainly from
Eucheuma denticulatum
Lambda does not gel, and is used to thicken dairy products.
The primary differences that influence the properties of kappa, iota, and
lambda carrageenan are the number and position of the ester sulfate groups
on the repeating galactose units. Higher levels of ester sulfate lower the
solubility temperature of the carrageenan and produce lower strength gels, or
contribute to gel inhibition (lambda carrageenan).
Thermo-
reversible gels
of kappa-
carrageenan
suitable for
topical delivery
Drug delivery
using
colloidal
lyotropic
liquid
crystal
X.Mulet et al ,
JCIS 393
(2013),1-20
X.Mulet et al , 2013
X.Mulet et al, 2013
Bioresource-based nanocellulose for drug
delivery ?
Lin and Dufresne , Eur. Poly.J 59 (2014)
Can we use it for passing the BBB?
Lin and Dufresne , Eur. Poly.J 59 (2014)
Bacteria-synthesised Nanoparticles for medicines
Microbial metal reduction can be a strategy for remediation of
metal contaminations and wastes. Bacteria are capable of
mobilization and immobilization of metals and in some cases,
the bacteria which can reduce metal ions show the ability to
precipitate metals at nanometer scale. Biosynthesis of
nanoparticles (NPs) using bacteria has emerged as rapidly
developing research area in green nanotechnology across the
globe with various biological entities being employed in
synthesis of NPs constantly forming an impute alternative for
conventional chemical and physical methods. Optimization of
the processes can result in synthesis of NPs with desired
morphologies and controlled sizes, fast and clean.
5. New enabling strategies to be developed
(i) MISFET (metal-insulator-semiconductor field
effect transistor) base biosensing for neuronal
electrical activities recording ( Larramendy et al,
2014)
(ii) Quantitative biology of single neurons (Eberwine et
al , 2012)
(iii) Quantitative Biophysics (see the following) e.g mean
diffusion time
Disjoining pressure between membranes
A VBIAS
time
Current
time
Current
What are nanopores good for in biotechnology?
e.g. building single-molecule sensors Nanopore technology
Recent convergence of the molecular and systems branches
of neuroscience has led to unprecedented progress towards
a systematic linkage between neuronal structure and
function, driven in part by development of genetically
encodable neural actuators and indicators.
With these biological tools, neuroscientist can now control
and image the activity of genetically and anatomically defined
specific cell types in the brains of behaving mammals and
connect this behaviorally relevant function to detailed
structure.
Recent years have seen a fairly comprehensive toolbox of
genetically encoded optical actuators from microbial
organisms. These proteins which are derived from single
component opsin genes permit fine control over the activity of
genetically or topolologically defined neutral circuit elements
embedded in a matrix of dissimilar cell types.
Optical neural control is a long -sought goal,but the microbial
single-component tools turn out to be very critical for
genetically encoded optical actuation. The first microbial
opsin succeefully employed to excite neural activity and alter
the behaviour of freely moving mammals was
channelrhodopsin-2 (ChR2) .These membrane-bound non-
selective cation channels could be activated with pulses of
blue light to depolarise expressing neurons and induce single
action potentials.
Warden et al (Annual Rev.Biomed.Eng , 2014)
Warden et al (Annual Rev.Biomed.Eng , 2014)
Halorhodopsin from Natromonas pharaonis (NpHR) is a
chloride pump that could hyperpolarise and silence
expressing neurons upon the application of yellow light as
well as spectrally shifted opsins e.g that for biochemical
control.
Bioresource - based drug delivery system
6. Conclusions and future works
■■ To be effective, therapies for CNS disorders must
overcome hurdles including the blood–brain barrier, the
complex cellular architecture of the CNS, and the
multifactorial nature of CNS disease
■■Nanotechnology—engineering of materials that measure
less than 100 nm in at least one dimension—can combat
these challenges, and enable multimodal therapeutic
targeting at the molecular level
■■The efficacy of nanoscale treatments has been
demonstrated in models of neurodegenerative disease,
neuroregeneration and brain tumours, but few of these
treatments have been successfully translated to the clinic
■■The future of nanotechnology in clinical neuroscience will
rely on our ability to interface this technology with our
burgeoning understanding of the molecular underpinnings of
CNS disease
Effect of shapes on NP-cell interactions still not clear
Betaine- cholesterol vesicles forming myelin figures.
Can artificial figures replace myelin sheath
degeneration ?
Nano-herbs formulation - boost local industry
Y-M Tsai et al , Int J of Pharmaceutic (2011)
A fusion of Eastern and Western medicinal systems integrated with nanotechnology
The Future !
Collecting Bacteria samples (for Nanoparticle production ) in Langkawi with
Environmental Biotech Group
Acknowledgements
Thanks you to all my postgrad students , my postdoc
(Dr. Nasima Akter) and co-workers – Dr. Irman
A.Rahman , Dr. Faizal K.P Mohamed , Dr. Khoo Kok
Siong, Prof. Ainon Hamzah dan Prof. Fauziah Aziz
(UPNM) who make some of the research work
interesting and successful.
Thank you very much
for your attention !
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