Invited Talk:

Nanotechnology Strategy in Medicine Shahidan Radiman

School of Applied Physics , Faculty of Science and Technology UKM Bangi 43600 , Selangor DE. E-mail: shahidan@ukm.edu.my

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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