Ultrasound triggered release of anticancer agents from alginate chitosan hydrogels

1. ULTRASOUND TRIGGERED RELEASE OF
ANTICANCER AGENTS FROM ALGINATE-CHITOSAN
HYDROGELS
MSc. Antonio Di Martino, Ph.D.
dimartino@utb.cz / dimartino@tpu.ru
MSDT 2017, 1-3 November 2017, Tomsk

2. Triggered Drug Delivery
 Advanced drug delivery systems (DDS) enhance efficacy of different therapeutics in a dosage, spatial, and/or
temporal way
 Numerous chemical, physical and biological-based stimuli-responsive formulations or devices for
controlled drug release have been developed
Theranostics 2013; 3(3):141-151

3. Physical stimuli for local drug delivery
X-Rays
 Good penetration
 Precise
 Easily tuned
 High cost
 Ionizing radiation
Magnetic field
 Localized accumulation
 Energy modulation
 Accumulation – embolism, increased cytotoxicity
Light
 Precise
 Low cost
 Easily tuned
 Limited tissue penetration (can be enhanced
with NIR light) Invasive for deep zone
Ultrasound
 Good penetration (Dependent on frequency)
 Easily tuned
 Low cost
 Difficulty to target moving organs
 Not homogeneous exposure of large zones
Radiofrequency  Invasive, temperature gradient from the heated zone Easily tuned

4. Ultrasounds –general definition
-Ultrasounds are mechanical longitudinal waves propagating in a medium through changes in pressure-
Ultrasound technology is already well implanted in the medical field
 Frequency modulation of the depth of penetration and spatial resolution
 Intensity amount of energy delivered at the target site
 Focused
 Not focused
diagnosis (ultrasound imaging)
therapeutic
More interesting in drug delivery – large amount of energy in a small area
Physical therapy

5. Medical US-imaging
http://www.thejournal.ie
http://ultrasourcemedical.com
Data flow in an artery of transplanted kidneys
Doppler Effect in the observation of vein–
Acta Inform Med. 2011 Sep; 19(3): 168–171.

6. US-therapeutic
“any type of procedure that uses ultrasound for therapeutic benefit”
Applied Physics Letters 106(2):021902 · January 2015
http://www.trust-biosonics.com/technology/1
Ultrasound probe
Blood vessel
4. Local nanoparticle
delivery
3. Targeted sonoporation at
the region of interest
2. Microbubbles and nanoparticles reach
target tissue
1. IV injection of microbubbles
and nanoparticles
Microbubble Nanoparticle
Microbubble
undergoing inertial
cavitation
Therapeutic
ultrasound

7. c
US in Drug Delivery
US-induced effects
Thermal
Mechanical
Acoustic radiation
forces
Journal of Controlled Release 241 (2016) 144-163
Theranostics 2014; 4(4):432-444.

8. Drug Delivery Carriers designed for use with US
 Using carriers prevents premature and extraneous delivery of the drug
 Control the release, reduction of burst
 Targeting
 Nano or Micro-carrier in solid or liquid form
 Combination of gas-liquid form ( lipospheres = hybrid between liposomes and microbubbles)
Low sensibility to US
http://www.dataphysics.de
Liposomes
Micelles
http://www.fbs.leeds.ac.uk
Solid Lipid Nanoparticles
Di Martino et al, International Journal of Pharmaceutics, 526,2017,380-390
Hybrid-NPs

9.  Highly adsorbent, are able to swell in water or in a biological fluid while conserving their shape
 Swelling behavior is related to the Mw and nature of the polymer(s) used
 Unique properties such as flexibility, low interfacial energy with water,
highly charged structure in water
 Biocompatible materials, because of their high water content, their composition
and their mechanical behaviour which is close to that for an extracellular matrix
of natural origin
 Conventional hydrogels are not sensitive to changes in the environment, as opposed to stimuli-responsive
hydrogels which can sense an external stimulus
Hydrogels
http://dev.nsta.org

10. Stimuli responsive hydrogels
 Stimuli-responsive hydrogels are based on smart polymers
 Ability to respond to small changes in the surrounding environment by changes in volume, swelling or
shrinking
 Slight variations in parameters (pressure, temperature, concentration, solvent, etc...) induce considerable
modifications of the physical and chemical properties of the macromolecules (solubility, structure, shape,
size)
Soft Matter, 2011,7, 4414-4424

11. Development of biopolymer hydrogel beads for multidrug encapsulation
Control the release of the drugs, individually
Deliver drugs at the right place and the right time
Trigger the release by application of US = pulsatile release
No constant release over time = more effective
Control the biodegradation rate
Self-healing
Aims

12. Hydrogel-mbead : preparation
Alginic acid
Chitosan
Doxorubicin Temozolomide
+
• 100 mm average diameter
• Drug loading does not affect dimension

13. Multiple Drug Loading
0
20
40
60
80
100
0.25 0.5 1 1.5 2 2.5 3 5 10
Drug conc. mg/ml
EncapsulationEfficiency(%)
Individual
0
20
40
60
80
100
0.25 0.5 1 1.5 2 2.5 3 5 10
Drug conc. mg/ml
EncapsulationEfficiency(%)
Encapsulation
Holding
Weight ratio between drugs
Multiple
 Chitosan to Alginic acid weight ratio : 2
 pH 5
 Temp : 22-25‫ﹾ‬C

14. Release investigation
Simulated body fluids
pH 2-7.4 T 37‫ﹾ‬C
US transducer
ON/OFF
Stirrer
IN
OUT
Temp. contr.
Solution
Ultrasonics Sonochemistry 16 (2009) 41–49

15. Improve TMZ stability
Control
6h
24h
Di Martino et al.Journal of Nanoparticle Research 19(2) ·2017
 TMZ undergoes pH dependent ring-opening under both
acidic and alkaline conditions
Free drug
Drug loaded
Methylating agent
 TMZ half life : 1.8h
 MTIC half life : slight longer than TMZ

16. Individual Release
 70kHz, 2W/cm2
REF
US 5x10s
US 5x30s
 US every 20 minutes (ON/OFF)
 Temperature is stable
pH 3
pH 5
pH 7.4
 T = 37‫ﹾ‬C ± 1
 120 rpm oscillatory shaker
Pulsatile Release
More effective than constant release rate

17. T im e ( m in )
mgReleased
1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0
0
2 5
5 0
7 5
1 0 0
1 2 5
1 5 0
1 7 5
2 0 0
T im e ( m in )
mgReleased
1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0
0
2 5
5 0
7 5
1 0 0
1 2 5
1 5 0
1 7 5
2 0 0
U S 5 x 1 0 s
U S 5 x 3 0 s
U S 5 x 1 0 s
U S 5 x 3 0 s
pH 3
pH 7.4
Ref : 128 mg/mg
Tot : 233 mg/mg 5x10s
Tot : 461 mg/mg 5x30s
Ref : 211 mg/mg
Tot : 400 mg/mg 5x10s
Tot : 669 mg/mg 5x30s
650 mg/mg 720 mg/mg
Ref : 276 mg/mg
Tot : 385mg/mg 5x10s
Tot : 611 mg/mg 5x30s
Ref :338 mg/mg
Tot : 460 mg/mg 5x10s
Tot : 704 mg/mg 5x30s
Individual Release

18. Simultaneous release
mgReleased
D O X T M Z
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
R E F
U S 5 x 1 0 s
U S 5 x 3 0 s
mgReleased
D O X T M Z
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
R E F
U S 5 x1 0s
U S 5 x3 0s
mgReleased
D O X T M Z
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
R E F
U S 5 x1 0s
U S 5 x3 0s

19. In vitro degradation rate
 PBS (pH 7.4)
 Lysozyme human
(0.5 mg/ml)
Evaluation of the Content of reducing sugar (GPC)
 Pectinase from A.Niger
(0.5 mg/ml)
 Cellulase from A.Niger
(0.5 mg/ml)
T im e (d a y s )
%Degradation
0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0
0
2 0
4 0
6 0
8 0
1 0 0
E n z y m e fre e
L y s o z y m e
P e c tin a se
C ellu lase
Release time ( 2h )
Up to 80% of the loaded drugs are released

20. In vitro studies
No US treatment
No Drugs
5x10s treatment
DOX
5x10s treatment
DOX+TMZ
5x10s treatment
TMZ
MCF7NIH/3T3
Viability(%)
M C F 7 N IH 3 T 3 H E K 2 9 3
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
 Flow cytometry demonstrate that US cycles does
not affect cells viability
 5x10s treatment has been chose
Drug free mbead
 Cells respond in a different way when short burst occurs

21. In vitro studies
 The decrease in cell viability is greater when combination of US + drug is used
 Three possible mechanisms
I)The release after US events create a concentration gradient across the cell membrane
promoting the transport by diffusion
II)Upregulation of endocytosys --- cells respond differently to US
III)US cause a perturbation in the cell membrane leading to the passive transport

22. Molecules investigated
Cortisone B-estradiol
5-Fluorouracil
5 aminolevulinic
acid
Lidocaine Tetracaine Human Serum Albumin Porcine Trypsin
Riboflavin Nicotinic acid

23. • Functionalization of the carrier to control the release selectively – in
serie release
• Derivatives to tune the biodegradation rate according with the
needed
• US-hydrogel relation
Perspectives