1.
Polysaccharide hydrogels: a versatile tool
for biomedical and pharmaceutical
applications
Department of Drug Chemistry and Technologies
Sapienza University of Rome
Rome, Italy
Pietro Matricardi
XVII Brazil
MRS Meeting
September,16th -20th, 2018
Natal

2.
Hydrogels for food
“molecular cuisine”

3.
Hydrogels for personal care

4.
Hydrogels for health
and wellness

5.
Hydrogels for pharma

6.
The colloid condition, the gel, is one which is easier to
recognize than to define
Dorothy Jordan Lloyd (1926)
Or, in other words, “if it looks Jello, it must be a gel”
What is a hydrogel

7.
• Like a “fourth state” of matter
ü polymer network (10-20%) and water
ü the concepts of solute and solvent from a “classical”
thermodynamic point of view are unable to describe the system
• Depending on its nature:
ü self sustaining under its own weight
ü able to shear under the action of a stress
Some characteristics

8.
Hydrogel network structures
Based on polymers
Chemical hydrogels
Physical hydrogels
reversible
crosslinks
covalent
crosslinks

9.
10
From a rheological point of view
(Ross-Murphy and Burchard):
viscoelastic behaviour
Solution
Strong gel
Weak gel

10.
Polysaccharides
ü Most of them are abundant in nature (commodities or mass-market
products)
ü Readily available from renewable sources (algal and plant kingdoms,
cultures of microbial selected strains, recombinant DNA techniques)
ü Large variety of compositions and properties
ü Biocompatible (food, medical device or pharma applications)
ü Often cheaper than synthetic polymers
ü Favourable chemical and physico-chemical properties due to the wide
variety of functional groups, macromolecular architecture and molecular
weights

11.
Dextran
Gellan Gum
Guar Gum
Locust Bean Gum
Hyaluronic acid
Alginate
Xanthan Gum
Scleroglucan
SOME POLYSACCHARIDES

12.
As “inert” drug carrier in conventional formulations:
• Filler (bulk formulation)
• Film forming (tablets or capsules)
• Viscosity agent in liquid formulations

13.
Hydrogels & Polysaccharides

14.
Ionotropic gelation
Chemical cross-linking
Calcium alginate hydrogels
Dextran methacrylate hydrogels

15.
Hydrogels & Polysaccharides

16.
Drug and Protein
Delivery
Tissue
Engineering

17.
Bulk hydrogel
Nano hydrogel

18.
GEL FOR DENTAL APPLICATIONS

19.
HYDROGEL FOR DENTAL APPLICATIONS
üComplete filling of confinedspaces, such as gum pockets
üAble to carry
q Drugs
q Biologically Active Substances
üThe gel adheres to soft tissues due to the film-formingproperties
of one of the polymer component
üObtained by means of ionotropic gelation so no chemical
reactionsare involved nor toxic substances are formed as
byproducts

20.
IONOTROPIC GELATION
Ions
Synthetic polymer
Polysaccharide

21.
To improve bone growth for implant
On the market
Ø Homologous bone - animal derived products - synthetic
bone
Ø Osteocompatible resins
Pilot study #1 - Hydroxyapatite

22.
ü Treatement of severe periodontitis
ü Cleaning of the pocket by curette
ü Applications of the drug loaded-hydrogel
ü New implant after 8 weeks
Pilot study #2 - nimesulide
NB
BM
n-MT
NB
NB
n-MT
BM
NB
Ematossilina-Eosina 4X
% n-MT (non-Mineralized Tissue): 65%
% BM (Biomaterial): 5%
% NB (New Bone): 30%

23.
üFilling effect
üPain relief (with and without drug) in all
patiences
üHydrogel resorption in 4 weeks and
promotion of new bone formation
Results

24.
Hydrogel
for heritage protection
Stone materials
and
Biodeterioration

25.
ü Cleaning and protection of stone materials
ü Bio-colonization
ü Organic solvent-free product
Why polysaccharide hydrogels
Italy possesses a huge heritage
(45% of the world cultural heritage?!?!?)

26.
Surface
Hydrogel on
the surface
After film removal

27.
Duchessa di Genova
(1900)
by Pietro Canonica

28.
Polysaccharide nanohydrogels for drug
delivery applications

29.
Drug Delivery – Controlled release
Polymeric nanoparticle
Liposome
dendrimer
Inorganic nanoparticle
“MAGIC BULLET”
A substance or therapy capable of destroying pathogenic agents
(as bacteria or cancer cells) or providing a remedy for a disease
or condition without deleterious side effects, by specifically
targeting the diseased tissue
Dr. Paul Ehrlich
Solid Lipid Nanoparticle
Nanogels
Nanoscale hydrogels

30.
POLYSACCHARIDE-BASED NANOHYDROGELS
SELF-ASSEMBLING
IN WATER
HYDROPHOBIC MOIETY
POLYSACCHARIDE
ü Nanosized hydrogels (150-300 nm)
ü Nanoparticulate drug carriers
ü High water content
ü High biocompatibility
ü Mucoadhesive properties
gellan gum
or
hyaluronan
prednisolone
cholesterol
riboflavin derivatives

31.
HYDROPHOBIC MOIETY
POLYSACCHARIDE
CRYO-TEM micrographs
Optical image
10 µm
100 nm 50 nm
* new patented method
AFM image
by several approaches
POLYSACCHARIDE-BASED NANOHYDROGELS

32.
(121°C, 1.10 bar, 20 min)
1) preparation of
sterile NHs
2) Reduction of the NHs
polydispersity
3) Simultaneous formation, loading*
and sterilization of NHs
HYDROPHOBIC MOIETY
POLYSACCHARIDE
Autoclave treatment *
* new patented method:
• WO2014199318 (A2) ― 2014-12-18 MC De Rugeriis, E. Montanari, C. Di Meo, P. Matricardi - METHOD FOR PREPARING NANOHYDROGELS
• WO2014199319 (A2) ― 2014-12-18 G. D’Arrigo, C. Cencetti, C. Di Meo, P. Matricardi - METHOD FOR THE TREATMENT OF NANOHYDROGELS
An innovative method for sterile polysaccharide NHs production
Cryo -TEM
100 nm
Autoclave
121°C
1.10 bar, 20 min
* For thermo-stable drugs
polymerdrug
Co-suspension in water Film-casting technique
drug film
polymer suspension

33.
Long-term storage
0
50
100
150
NHs before freeze-
drying
NHs + dextrose
before freeze-drying
NHs + dextrose after
freeze-drying
d (nm)
0
0.2
0.4
0.6
0.8
1
PDI
diameter PDI
E. Montanari, M.C. De Rugeriis, C. Di Meo, R. Censi, T. Coviello, F. Alhaique, P. Matricardi Journal of Materials Science: Materials in Medicine , 2015,26:32
Freeze-drying

34.
HACH
HARfv
NHs self assembly
~ 200 - 300 nm
(hydrophobic interactions)
Autoclave
20 min 121 °C
CAC = 134 µg/mL
scattering intensity
mean size
scattering intensity
mean size
CAC = 235 µg/mL
NHs properties

35.
ü Stability
Denaturating agent: urea
Dilution test
37

36.
HACH NHs
Dry conditions
SWELLING RATIO
ü Atomic Force Microscopy
Wet conditions
Dry conditions:TAPPING MODE (Substrate: mica + MgCl₂ 10mM)
Wet conditions:CONTACTMODE/ScanAsyst® (Substrate: Si 1,0,0 derivatised with APTES((3-amminopropil)-trietossisilane)

37.
NHs HACH NHs HARfv
Mean diameter 330 ± 20 nm 180 ± 24 nm
PDI 0.120 ÷ 0.250 0.100 ÷ 0.200
ζ Potential -46.7 ± 1.8 mV -40.5 ± 4.5 mV
Q: swelling ratio 6200 4400
Young Modulus 108 KPa 610 KPa
Fluorescence No Yes

38.
hydrophilic drug
hydrophobic drug
empty NHs
Loading within
hydrophilic
domains
Loading within
hydrophobic
domains
Loaded molecules
Paclitaxel
Levofloxacin
Piroxicam
Dexamethasone
DRUGS
NATURAL SUBSTANCES
Curcumin
Resveratrol
Annona extracts
PROTEINS
BSAO
by nanoprecipitation, autoclave treatment, solvent casting….
ALGINATE LYASE
Highly versatile drug carriers

39.
Gellan-prednisolone (Ge-pred) and Gellan-cholesterol (Ge-CH) NHs
for dual drugs therapy

40.
Advantages:
ü Prednisolone is already clinically used in combination with chemotherapeutics in
several anticancer protocols (e.g. prostate cancer) to prevent or reduce side
effects.
ü Inflammation contributes to tumor initiation, induces proliferation of malignant
cells, enhances their survival, and stimulates angiogenesis and metastatic spread.
ü Cancer, in turn, takes advantage of inflammatory mediators to grow, thereby
generating an inflammatory microenvironment in tumors for which there is no
underlying inflammatory condition.
Paclitaxel (PCT)
Prednisolone

41.
Mw reduction
by Ultrasonication
Sonication time
Mw/Mn
Sonication time1
3
2
4
5
1
3
2
4
5
1
3
2
4
5
1
32
4
5
6 6’
6 6’
-CH3
1.40 1.30 1.20
18
20
22
ppm
Gellan Gum
Glc Glc haGlc
GPC
ü Mw reduction of gellan by probe ultrasonication
1
H-13
C HSQC NMR map

42.
ü Synthesis of polymer derivatives
Same steps with cholesterol (dd = 10%)
derivatization degree (dd) = 10% m/m
yield = 50%
ü NHs formation and stability
PREDNISOLONE or CHOLESTEROL
GELLAN GUM
nanoprecipitation ultrasound treatment
or
25°C

43.
ü Cell biocompatibility
G. D'Arrigo, C. Di Meo, E. Gaucci, S. Chichiarelli, T. Coviello, D. Capitani, F. Alhaique, P. Matricardi Soft Matter 2012, 8, 11557
Fibroblast
Heart myoblast
Prostate cancer

44.
ü Anti-inflammatory activity

45.
• Paclitaxel loading into Ge-pred and Ge-CH NHs by
solvent casting technique
• Paclitaxel concentration reached in NHs suspension =
140 µg/mL (free Paclitaxel in water = 0.1 µg/mL)
Paclitaxel (PCT)
Prednisolone
ü Loading with anticancer drug
PCT release in vitro
PC-3 cells, 72h
PCT 3 nM

46.
IC50
Paclitaxel: 33.7 nM
Paclitaxel in NHs: 15.1 nM
Paclitaxel in NHs + Prednisolone: 7.7 nM
G. D'Arrigo, G. Navarro, C. Di Meo, P. Matricardi, V. Torchilin, European Journal of Pharmaceutics and Biopharmaceutics 2014, 87, 208
Skov-3 cell line
Anticancer activity of PCT-loaded NHs

47.
Hyaluronan-cholesterol (HA-CH) NHs
for antibiotic delivery

48.
A new challenge

49.
• Antibiotic resistance due to the intracellular escape (for
facultative or obligate pathogens)
• Levofloxacine: poorly effective against intracellular bacteria
(efflux pump)
Levofloxacin

50.
• Levofloxacin loading into HA-CH NHs by autoclave technique
• Levofloxacin concentration reached in NHs suspension = 12 ± 1 %
w/w
• Antibacterial activity against S. Aureus and P. Aeruginosa strains
• Cell (HeLa) infection by S. Aureus and P. Aeruginosa and treatment
with free LVF and LVF-loaded NHs
Levofloxacin
MIC values of Levoflloxacin (LVF) and NH-encapsulated
Levofloxacin (NH-LVF) on S. aureus ATCC6538P, S.aureus
USA300-0114 and P. aeruginosa PAO-1.
Comparable MIC
E. Montanari, G. D’Arrigo, C. Di Meo, A. Virga, T. Coviello, C. Passariello, P. Matricardi. Eur. J. of Pharmaceutics and Biopharmaceutics, 2014, 87, 518

51.
FREE LVF: no significant activity
on intracellular bacteria
Activity of LVF and NH-LVF (both freshly prepared and resuspended
after freeze-drying) on intracellular P. aeruginosa PAO-1 and S.
aureus USA 300-0114.
LVF-NHs: > 90% eradication
of intracellular bacteria

52.
Levofloxacin
Gentamicin
Topical applications

53.
• Levofloxacin (LVF) and gentamicin(GM) loading into HA-CH NHs
• Antiibacterial activity against S. Aureus
• Human keratinocites (HaCaT) infection by S. Aureus and treatment
with free LVF and GM drug-loaded NHs

54.
Scheme of the intracellular fate of free GM, LVF and their nanoformulations in
S. aureus-infected keratinocytes.
E. Montanari, A.Oates, C. Di Meo, J. Meade, R. Cerrone, A. Francioso, S. Devine, T. Coviello, P. Mancini, L. Mosca and P. Matricardi Adv. Healthcare Mater. 2018, 1701483

55.
Topical applications of NHs

56.
Topical applications of NHs -1
Piroxicam
Ge-CH
Ge-Rfv
U.M. Musazzi, C. Cencetti, S. Franzé, N. Zoratto, C. Di Meo, P. Procacci, P. Matricardi, F. Cilurzo, Mol. Pharmaceutics 2018, 15, 1028−1036

57.
Baicalin
Ge-CH
M. Manconi, M.L. Manca C. Caddeo, C. Cencetti, C. Di Meo, N. Zoratto, .. P. Matricardi Eur. J. Pharm. Biopharm. 2018, 127, 244–249
Topical applications of NHs -2

58.
Other applications
ü NHs in aesthetic surgery
ü Antioxidant for cardiovascular applications
ü Anti-inflammatory-loaded NHs for the treatment of primary
sclerosing cholangitis
ü NHs formulations for ocular applications
ü HA-based NHs in cosmetics

59.
Special thanks to:
Franco Alhaique
Tommasina Coviello
Chiara Di Meo
Elita Montanari
Nicole Zoratto

60.
Thank you for your kind attention