The document discusses using magnetic nanoparticles for hyperthermia cancer therapy. It notes that resistance is a major challenge in cancer treatment. Mild hyperthermia between 42-45°C can induce apoptosis in cancer cells without damaging normal tissues. The document then describes a new type of nanoparticle called RA IN (resistance-free apoptosis-inducing nanoparticle) that aims to overcome resistance. The RA IN contains two subunits - one to inhibit heat shock proteins that protect cancer cells from heat-induced apoptosis, and another magnetic nanoparticle subunit to generate localized heat with an external magnetic field to kill cancer cells through apoptosis.

1. svetlana.avvakumova@unimib.it

2. “Those diseases which medicines do not cure,
the knife cures;
those which the knife cannot cure, fire cures;
and those which fire cannot cure,
are to be reckoned wholly incurable.”
—Hippocrates (460-370 BC)
A major technical problem is the difficulty in
heating the local tumor region without damaging normal tissue.
Hyperthermia (Thermotherapy)
Hyperthermia in cancer therapy is
1) heating tumor above 42˚C.
2) a physical treatment and could result in
fewer side effects than chemotherapy.
Hyperthermia in cancer therapy is
1) heating tumor above 42 °C
2) a physical treatment and could result in fewer side effects
than chemotherapy
Difficulty in heating the local tumor region WITHOUT
DAMAGING normal tissue
Hyperthermia (Thermotherapy)

3. Hyperthermia – how it works
1. Local hyperthermia – Superficial tumours are heated by
means of antennas or applicators emitting microwaves or
radiowaves placed on their surfaces
2. Interstitial and endocavitary hyperthermia - antennas or
applicators are implanted within the tumour (less than 5 cm
in diameter)
3. Regional hyperthermia and part-body hyperthermia -
Deep-seated tumours - Treatment monitoring might be
provided by magnetic resonance tomography
4. Whole-body hyperthermia
• carcinomas with methastases
• need of deep analgesia and sedation or general anaesthesia
 cardiac disorders
 changes in the coagulation system (thrombocytopenia and
disseminated intravascular coagulation)
 permeability of the capillary endothelia

4. Hyperthermia – currently…

5. Hyperthermia vs other therapies

6. Hyperthermia – mechanism of action

7. Hyperthermia – why nanoparticles?
1. Decrease side effects and pain
2. Enhance the delivery of therapeutic agents
3. Enhance the efficacy of therapeutic agents
TUMOUR
TARGETING APPLY EXTERNAL
STIMULUS
CANCER CELL
DEATH
INCREASED BLOOD
FLOW AND
PERMEABILITY

8.  Complement currently available therapies
 chemotherapy
 radiation therapy
 gene therapy
 immunotherapy
 Remove residual microtumors after surgery
Nano-hyperthermia aims to…

9. Nanoparticles used in hyperthermia

10. Hyperthermia – stimulus?
• High-intensity focussed ultrasound (HIFU)
• Magnetic fluid hyperthermia (MFH)
• Microwave/radiofrequency
• Plasmonic photothermal therapy (PPTT)

11. Plasmonic photothermal therapy

12. NANOPARTICLE CHARACTERISTICS FOR PPPT
•plasmonic band in NIR field of spectrum
•strong scattering properites (big size)
•thermal stability
•high thermal conversion efficiency
•easy functionalization for active
targeting

13. Gold nanoparticles

14. Surface Plasmon Resonance

15. Surface Plasmon Resonance
(a) nanostars, (b) nanorods
(c) nanocages, (d) nanoshells

16. Heating mechanism
Large (>80 nm) or anisotropic nanoparticles have good scattering
properties
 high extinction coefficient - large amount of absorbed energy
(compared to molecules)
 temperature increase ranges from ~10 °C to nearly 1000 °C,
depending on laser power, time of irradiation, and concentration
of gold nanoparticles
 NIR laser has good penetration through tissues (5-10 cm)

17. AuroShell® nanoparticles
Naomi Halas and Jennifer West
Rice University
mid-1990s
PEGylated silica-cored Au nanoshells
In clinical trials from 2008 by
Nanospectra Biosciences, TX, USA
120 nm diameter silica core and 10 nm
thick gold shell

18. AuroShell® nanoparticles
Photothermal tumor ablation:
(A) tumor before treatment;
(B) complete ablation of tumor
in the high dose group
(a) Mean tumor size on treatment day and
day 10 for the treatment group (green),
control group (red), and sham treatment
(blue).
(b) Survival for first 60 days. Average survival
time for the nanoshell-treated group was >60
days, control group was 10.1 days, and sham
treatment group was 12.5 days.

19. AuroLase® therapy – clinical studies
 primary and/or metastatic lung tumors – currently performed
 head and neck refractory or recurrent tumors - completed
1. The NPs are delivered intravenously
2. Accumulate in the tumor by EPR effect
3. Tumor is illuminated with a NIR laser
4. The particles selectively absorb the laser
energy, converting the light into heat
5. The heat thermally destroy the tumor and the
blood vessels supplying it
6. Surrounding healthy tissue are not significantly
damaged
Determination of any adverse device effects attributable to AuroShell
particle administration

20. Gold nanorods

21. A new delivery and photothermal ablation system based on AuNRs-
laden-macrophages is described for cancer therapy
 macrophages as Trojan horses
carrying 7 nm diameter sAuNRs
 enhances tumor coverege
compared to AuNRs alone
optimization of in vivo delivery
carrier is important

23. Photothermal ablation of tumors in the
mice by intratumorally injected with PBS,
free macrophages, free BSA-coated
AuNRs and BSA-coated AuNRs-laden-
macrophages.
The use of macrophages to facilitate
AuNRs delivery can overcome the
extracelluar matrix and penetrate
more deeply into the tumor resulting
in enhanced tumor coverage
minimized tumor recurrence rates
and even distribution of heat
generation

24. Hollow gold nanoparticles – Au nanocages
Galvanic replacement reaction:

25. RBC-AuNCs exhibit superior blood retention and circulation lifetime

26. Mice injected with the RBC-
AuNCs and PVP-AuNCs and
irradiated with an 850 nm
laser for different periods of
time.
When RBC-AuNCs are
injected:
temperature rise in tumor
site is higher
tumor volume decreases
body weight remains
stable
survival ratio 100%
compared to PVP-AuNCs

27. A) Photographs of mice prior to NIR irradiation and
on the 19th day after NIR irradiation;
B) Hematoxylin and eosin (H&E) stained sections of
major organs and tumors on the 19° day after NIR
irradiation
When RBC-AuNCs are
injeced:
tumors shrunk to
negligible sizes;
no noticeable
abnormality or lesion is
noticed by histological
staining of different
organs;
tumor slices exhibit
apparent abnormality or
lesion compared to those
of the PBS- treated mice
(consistent with their
observed inhibition on
cancer growth)

30. Tumor volume grows even after
treatment with laser in absence of NPs.
On the contrary, mice treated with NPs
show a drastic tumor volume reduction
during the treatmet period
Survival rate decreases in absence
of NPs, while remain stable when
treated with NPs

31. Magnetic nanoparticles

32. Magnetic nanoparticles
BY MATERIAL
Simple nanoparticles
•Magnetite (Fe3
O4
)
•Ferrites (MeOFe2
O3;
Me = Ni,
Co, Mg, Zn, Mn)
•Maghemite (γ-Fe2
O3
)
•Greigite (Fe3
S4
)
•Iron, nickel
Hybrid nanoparticles
•Silica coated
•Au coated
BY SHAPE
• Spherical
• Cubic
• Stars

33. IDEAL NANOPARTICLES HAVE…
•small size
•narrow size distribution
•high magnetization values
•combine high magnetic susceptibility for an optimum
magnetic enrichment and loss of magnetization after
removal of the magnetic field
•optimal surface coating in order to ensure tolerance,
biocompatibility and specific localization at the biological
target site

34. What is magnetism?
 Physical phenomenon arising from orbital and spin
motions of electrons and how the electrons interact
with one another
 All materials experience magnetism, some more
strongly than others

35. Why some elements show magnetic properties?
1s 2s 2p 3s 3p
3d 4s

36. The magnetic behavior of materials
1. Diamagnetism
• atoms with filled orbital shells and with no unpaired
electrons
• magnetization is negative in the presence of field
• Pramagnetim
• a net magnetic moment due to unpaired electrons in partially
filled orbitals
• magnetization is positive in the presence of field
• Ferromagnetism
1. atomic moments strongly interact resulting in parallel
alignment
2. large net magnitization even in the absence of a magnetic
field
What is magnetism?
magnetic, paramagnetic, ferromagnetic, ferrimagnetic, and antiferromagnetic [56,57]. Figure 2
ows the net magnetic dipole arrangement for each of these types of magnetic materials. For
magnetic materials in the absence of a magnetic field, magnetic dipoles are not present. However,
on application of a field, the material produces a magnetic dipole that is oriented opposite to that of
applied field; thus, a material that has strong diamagnetic character is repelled by a magnetic field.
r paramagnetic materials, there exist magnetic dipoles as illustrated in Figure 2, but these dipoles are
gned only upon application of an external magnetic field. For the balance of the magnetic properties
ustrated in Figure 2, the magnetization in the absence of an applied field reveals their fundamental
aracter. Ferromagnetic materials have net magnetic dipole moments in the absence of an external
gnetic field. In antiferromagnetic and ferrimagnetic materials, the atomic level magnetic dipole
ments are similar to those of ferromagnetic materials, however, adjacent dipole moments exist that
not oriented in parallel and effectively cancel or reduce, respectively, the impact of neighboring
gnetic dipoles within the material in the absence of an applied field.
Figure 2. Magnetic dipoles and behavior in the presence and absence of an external
magnetic field. Based on the alignment and response of magnetic dipoles, materials are
classified as diamagnetic, paramagnetic, ferromagnetic, ferrimagnetic, antiferromagnetic.
Reproduced with permission from [57].

37. For example…
Iron Fe
Nickel Ni
Cobalt Co
FERROMAGNETIC
Quartz (SiO2)
Calcite (CaCO3)
Water
DIAMAGNETIC
Ferrimagnetism
•occurs in oxides and ionic compounds
•interaction of two different
superlattices separated by oxigens
•antiparallel alignment of spins between
the superlattices leading to net positive
magnetization
Magnetite (Fe3O4)
Maghemit (γ-Fe2
O3
)
FERRIMAGNETIC

38. Physical methods
Nanoparticle synthesis
• gas-phase deposition
• electron beam lithography
“ ” inability to control the particle size
down to the nanometer scale
Wet chemistry
• chemical coprecipitation
• polyol synthesis
• hydrothermal reactions
• oxidation method
• flow injection
• electrochemical method
• aerosol/vapor-phase method
• sonochemical decomposition
• supercritical fluid method
• synthesis using nanoreactors

39. Microbial methods
Nanoparticle synthesis
Magnetotactic bacteria
• Gram-negative prokaryotes
• Discovered by Salvatore
Bellini in 1963 (Università di
Pavia)
• high abundance in the
sediments of many
freshwater and marine
habitats
• Magnetic nanoparticles
present in magnetosomes
• passively align with the
magnetic field
• various morphological types
exist: bacillus, vibrios,
spirilla, cocci, and
multicellular

40. • composed from magnetite
(Fe3O4) or greigite (Fe3S4)
• 35-120 nm in diameter
• covered with a lipidic
membrane
• cubo- octahedral, bullet-
shaped, elongated prismatic,
and rectangular morphologies
• biocompatible character
(phospholipid bilayer)
MAGNETOSOMES

41. Magnetic hyperthermia

42. Principle of action
 transformation of external magnetic field to heat
 heat dissipation is utilized for a thermal therapy known as thermal
ablation or hyperthermia
Brownian rotation refers to the physical rotation of the particles themselves within
the fluid. It can be characterized by a relaxation time τB, which depends on the
viscosity of the fluid.
Néel relaxation stands for the rotation of the atomic magnetic moments within each
particle. The Néel process can be characterized by a relaxation time τN, which is
determined by the magnetic anisotropy energy of the superparamagnetic
nanoparticles relative to the thermal energy.

43. Magnetism vs heat dissipation
 The heating efficiency is represented by the specific loss power
(SLP), which is defined as the initial temperature rise per unit mass of
nanoparticle-containing solution per unit mass
 Magnetic parameters of NPs are tuned by controlling their size,
composition, and shape or by constructing heterostructures
10 and 30 nm

44. MagForce - a fully operative clinical
therapy based on aminosilane-coated
Fe3O4 NPs together with a magnetic
actuator
Applying 100 kHz magnetic field, treat tumours of about 5 cm
after injecting 3 mL of a simple core–shell Fe3O4@amilosane
ferrofluid into the patient
 Glioblastoma Multiforme
 Prostate Cancer
 Eosphageal Cancer
 Pancreatic Cancer

45. 1. Hyperthermia allows for quick tumor removal, BUT the surrounding
normal tissues are possibly damaged and cannot be preserved at the
high temperatures needed to kill surrounding cancer cells
2. Tumor necrosis, which is a cell death caused by unexpected and
accidental cell damages, can be harmful because it is correlated with
inflammatory disease and metastasis
3. Nonliving cells that die through the apoptotic process are cleaned by
phagocytosis without affecting their neighboring normal cells.
Apoptotic (mild) hyperthermia
MILD HYPERTHERMIA NEEDED
(temperature window between 42 and 45 °C )

46. Apoptotic (mild) hyperthermia
Posiible solutions…
1. Affect thermotolerance of cancer cells by inhibition of Heat
Shok Proteins, which protect cells from apoptosis by
preventing the unfolding and aggregation of key proteins
when they are exposed to thermal stress
2. Presensitization of cancer cells by induction of ROS production
– ROS generation makes the cells vulnerable to mild
temperatures

47. Cancer Hot Paper
DOI: 10.1002/anie.201306557
Magnetically Triggered Dual Functional Nanoparticles for Resistance-
Free A poptotic Hyperthermia**
Dongwon Yoo, Heeyeong Jeong, Seung-Hyun Noh, Jae-Hyun L ee, and Jinwoo Cheon*
Therapeutic resistance is one of the major clinical problems
and remains a persistent hurdle for disease treatments.[1]
For
example, both bacteria and cancer cells gradually increase
their ability to shield themselves from treatments such as
chemotherapy and radiotherapy. Some of the reported
protective mechanisms for resistance involve chemodrug
efflux and bypassing targeted signaling feedback loops.[1c]
A s a consequence of these problems, researches for circum-
venting therapy resistance are intensively pursued in the
biomedical fieldsincluding pharmaceutics and cancer biology.
Recently, innovative therapeutic approaches beyond
conventional orthodox therapies, such as chemical drugs,
have been actively developed and one of them is cancer
hyperthermia, in which thermal treatments of cancer cells at
mild temperature (40–458C) preferentially eliminate them
through an apoptotic cell death process without damaging
normal tissues.[2]
With the exceptional capability to generate
thermal energy at targeted areas, nanomaterials such as gold,
iron oxide, and graphene have been investigated for use in
hyperthermia treatment of cancer.[3]
Unique advantages of
hyperthermia using nanomaterials include spatiotemporally
controlled treatments of targeted diseases in a noninvasive
manner. Compared with other heat generation nanomaterials
such as gold and graphene which use light as the trigger,
magnetic hyperthermia can be advantageous for targets that
reside even deep inside the biological system without
penetration depth problem.[4]
In addition, the fact that
magnetic field causes no adverse effect on biological tissues
In this study, we introduce a new type of resistance-free
apoptosis-inducing magnetic nanoparticle (RA IN) that can
promote thermoresistance-free apoptosis. The RA IN consists
of two functional subunits of 1) heat shock protein (Hsp)
inhibition and 2) heat generation from magnetic nanoparticle
(MNP) in which these two functions are designed to be
triggered only by the application of an alternating magnetic
field (AMF). We demonstrate that the RA IN successfully
promotes exclusive apoptosis and obstructs cell survival by
inhibiting Hsp not only in vitro but also in vivo under low-
temperature (ca. 438C) hyperthermia conditions (Scheme 1).
Scheme 1. Resistance-free apoptosis-inducing magnetic nanoparticle
(RAIN) for effective apoptotic hyperthermia. 1) Heat-treated cancer
of two functional subunits of 1) heat shock protein (Hsp)
inhibition and 2) heat generation from magnetic nanoparticle
(MNP) in which these two functions are designed to be
triggered only by the application of an alternating magnetic
field (A MF). We demonstrate that the RA IN successfully
promotes exclusive apoptosis and obstructs cell survival by
inhibiting Hsp not only in vitro but also in vivo under low-
temperature (ca. 438C) hyperthermia conditions (Scheme 1).Resistance-free apoptosis-inducing magnetic nanoparticle (RAIN) that can
promote thermoresistance-free apoptosis
Geldanamycin -
a benzoquinone
ansamycin
known as an
inhibitor for
Hsp90
Linked via
thermally
cleavable azo
linker

48.  MDA-MB-231 breast cancer cells are
incubated with NPs
 After hyperthermia treatment for 80
minutes at 43 °C, cell death percentage is
measured by CCK-8 assay
Inhibition of Hsp90 makes the cells more
vulnerable to apoptotic hyperthermia,
showing 100% cell death at shorter time

49.  Temperature is mantained at 43 °C
during 30 minutes of AMF
application
 Over a period of 14 days post-
treatment, the tumors receiving
RAIN hyperthermia are eliminated
by day 8
 Immunofluorescence histology
shows the presence of cancer cells in
control

50. Temperature–controlled drug release

51. Release under…
Thermal energy from nanoparticles
used as a trigger to control the release
of therapeutic molecules remotely
Endogenous
stimulus
Exogenous
stimulus
Enzyme presence and pH
change inside cell
(endosome)
1. Thermolabile linkers, such as hybridized DNA strands,
carbonate, or azo groups
2. Thermosensitive polymers
3. Thermoresponsive material – molecular
4. Soft external structures - liposomes and micelles

52. Hyperthermia and immune response
Properly applied hyperthermia is immunogenic

53. 1. Iron Oxide Based Nanoparticles for Multimodal Imaging and
Magnetoresponsive Therapy. DOI: 10.1021/acs.chemrev.5b00112
Chem. Rev. 2015, 115, 10637−10689.
2. Hyperthermia in combined treatment of cancer. THE LANCET Oncology
Vol 3 August 2002.
3. Nanoshell-Enabled Photothermal Cancer Therapy: Impending Clinical
Impact. ACCOUNTS OF CHEMICAL RESEARCH 1842-1851 December
2008 Vol. 41, No. 12.
4. Emerging advances in nanomedicine with engineered gold
nanostructures. Nanoscale, 2014, 6, 2502.
5. Hyperthermia as an immunotherapy strategy for cancer. Curr Opin
Investig Drugs. 2009 June ; 10(6): 550–558.
6. Gold nanorods: Their potential for photothermal therapeutics and drug
delivery, tempered by the complexity of their biological interactions.
Advanced Drug Delivery Reviews 64 (2012) 190–199.
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