SELF-ASSEMBLY AMPHIPHILIC CHITOSAN NANOCARRIERS FOR ORAL CO-DELIVERY OF HYDROPHOBIC AND HYDROPHILIC DRUGS

1. SELF-ASSEMBLY AMPHIPHILIC CHITOSAN NANOCARRIERS FOR
ORAL CO-DELIVERY OF HYDROPHOBIC AND HYDROPHILIC
DRUGS
MSc.Antonio Di Martino, Ph.D
dimartino@tpu.ru
dimartino@utb.cz

2. Chitosan : Chemical structure and properties
Fungi
Crustaceans
Insects
Chitin
 Deproteinization
 Demineralization
 Decolorization
 Deacetylation
Chitosan
Deacetylation (>50%) Molecular weight
Solubility
Biocompatibility Biodegradability
Antibacterial
Hypoallergenic
Mucoadhesivity
Hemostatic
Anticholesterolemic Anticarcinogenic
Functionalization
 Solubility
 Functionalization

3. Adv Pharm Bull. 2017 Sep; 7(3): 427–432.
Chemical Modification
Self-assembly systems for Drug Delivery
Hydrophobic/ Hydrophilic balance
Chitosan Derivatives
Hydrophobic Hydrophilic
Ethylene glycol
Alkyl-
PLA PEG
PLGA
Fatty acids

4. Self-assembly mechanism
Monocomponent
Hydrophobic interactions
Hydrogen bonds
Multicomponent Electrostatic interactions
Hydrogen bonds
Major Role Minor Role
Van der Waals forces
Van der Waals forces
Polymers 2018, 10(7), 760; https://doi.org/10.3390/polym10070760
J Pharm Pharmaceut Sci 13(3) 536 - 557, 2010

5.  Chitosan was first studied as a drug delivery agent in 1990
 In the late 1990s a new form of chitosan was introduced – conjugation of hydrophobic
and additional hydrophilic units
 Self-assembly amphiphilic chitosan
 Chitosan amphiphiles to increase the oral bioavailability of hydrophobic drugs ( up to 10 times)
Linear
Claw
 Chitosan amphiphilies nanoparticles targeting delivery, gene delivery to liver, muscles and to tumours via intratumoural route
Hydrophobic moiety
 Chitosan amphiphiles formulations have been developed by companies such as Nanomerics Ltd
 Currently there are no commercial pharmaceutical applications containint chitosan as excipient
 No chitosan amphiphilies formulations are on the market up to now
Chitosan in Drug Delivery
Diazepam
Sertraline L-Thyroxine
Cisplatin

6. Chitosan Amphiphilic- Oral Drug Delivery
quaternary ammonium palmitoyl glycol chitosan
(GCPQ)
Int J Pharm. 2001 Aug 14;224(1-2):185-99.
Enhance the oral bioavailability and oral solubility of
Hydrophobic graft = self assembly in acid environment
Hydrophobic + Hydrophilic = self assembly at neutral pH
N-palmitoyl
N,N,N, trymethetyl
Glycol  Mw 13kDa,
 20.4 mol% palmytoilation
 10.5 mol% quaternary ammonium
Griseofulvin (6 times)Cyclosporine A (3 times)

7. Claw amphiphilic – 3rd generation poly(propylenimine) dendrimer – DAB-GCPQA
Polym Int 2014;63:1145-1153
Claw-shape
 To form multiple hydrophobic contacts
 CMC on picomolar range
Chemotherapeutics
Chitosan amphiphilic- Oral Drug Delivery
Quaternization
Hydrophobic
Hydrophilic
PTX DOX 5FU CPT
Rifampicin
Antibiotics
Gramicidin C
Eritromicin
Sensible molecules
Proteins Vit. C
Vit. E

8. Journal of Pharmaceutical Sciences, 99 (11), 4543-4553, 2010
N,5,b-cholanyl
6-O-glycol
CS Mw 100kDa
Increase the oral bioavailability of Paclitaxel up to 3 times
compared with the Chremophor EL-ethanol formulation Taxol
Hypoglycaemic effect in diabetic rats
Lauroyl
(PTX)
J Biomed Nanotechnology 9: 167-176 (2013)
Human Insulin
Nanospheres
Chitosan amphiphilic- Oral Drug Delivery
 Paclitaxel is a P-glycoprotein efflux pump substrate
 Chitosan Amphiphilie facilitate the adsorption via pathway not
susceptible to the P-gp efflux

9.  Delivery of gut labile molecules to the brain remains a formidable challenge
 No oral meuropeptides available on the market
 Delivery of the opiod peptide leucine –enkephalin encapsulated in GCPQ nanoparticles to the brain
 Positively charged chitosan NPs protect the peptide from degradation in the gastrointestinal tract
 Protection of the peptide from plasma enzymes
 The particles associate with the luminal side of the brain endothelial cells which make enable the uptake by the brain capillarie
Mol. Pharmaceutics, 2012, 9 (6), pp 1764–1774
Chitosan Amphiphiles: Oral Drug Delivery
m and d –opioid receptors

10. Chitosan Amphiphiles- Oral Drug Delivery
 Liposome low stability and tend to accumulate in the liver
• Chitosan amphiphiles avoid liver and spleen – release in the intestine
Chitosan-cholesterol
Quaternized chitosan
Thiolate chitosan
N-succynil chitosan
Irinotecan
Ibuprofen
Diclofenac
Vitamin C
Vitamin E
Ciprofloxacin

11. one-pot preparation of CHC/DMC-CDDP/anti-CD133 nanoparticles.
Enhanced synergy between cisplatin and demethoxycurcumin against multidrug-resistant stem-like lung cancer cells
European Journal of Pharmaceutics and Biopharmaceutics 109,165-173, 2016.
CHC/DMC-CDDP/anti-CD133 nanoparticles enter the cells and how the drugs act in
synergy to overcome MDR
Chitosan Amphiphiles- Oral and Parenteral Drug Delivery

12. Bioactive Polymer System
Objectives
study polymer materials, which are able to interact
specifically with living cells or tissues.
Explored materials involve not only bulk materials,
modified polymer surfaces,
nanoparticles and composites with various properties
(magnetic, drug transport etc.)
various surface modifications.
Head of the group: doc. Ing. Petr Humpolíček, Ph.D.

13. Enhancement of the antioxidant activity and stability of b-carotene using amphiphilic chitosan/nucleic acid
polyplexes
Di Martino Antonio, Trusova E. Marina E., Postnikov S. Pavel, Sedlarik Vladimir
International Journal of Biological Macromolecules 117, pp 773-780 ; DOI: 10.1016/j.ijbiomac.2018.06.006
A)Variation over time of the average dimension of the polyplex stored at 4 °C and 25 °C,
over time (red: bare polyplexes; blue: polyplexes loaded with β-carotene); B) retention rate;
D–E) TEM micrograph of β-carotene loaded polyplexes in solution at N/P 2.5.
A)DPPH activities in percentage; C) FRAP value in mmol/L at different concentrations of free b-carotene(F) and β-carotene loaded in the polyplex (L).
B) DPPH activities in percentage: D) FRAP values versus time at concentration of 200 μg/mL of free (F) and loaded (L) β-carotene. N/P ratio 2.5.
CS-PLA
b-carotene
ds-DNA
PECs 120-150 nm
up to 800mg/mg
Inverted fluorescent microscopy of NIH/3T3 cells after 24 h, 48 h and 72 h of incubation with polyplexes at 100 μg/mL and N/P 2.5

14. Enhancement of 5-aminolevulinic acid phototoxicity by encapsulation in polysaccharides based nanocomplexes for
photodynamic therapy application
Di Martino Antonio, Pavelkova Alena, Postnikov S.Pavel, Sedlarik Vladimir
Journal of Photochemistry and Photobiology B: Biology, 175,2017,226-234 DOI: 10.1016/j.jphotobiol.2017.08.010
Fig. 3 The fluorescent intensity of PpIX in HeLa cells cultured in the medium A) with serum addition and
B) without serum addition. pH of the medium was 7.4. Positive control is represented by the fed
containing free 5-ALA while negative control by fed without 5-ALA.
Fig.2 HeLa cells viability A) free 5-ALA and bare NCs; B) 5-ALA free and loaded in CS-ALG and CS-PGA
NCs BI (Before irradiation) and PI (Post Irradiation); C) 5-ALA free and loaded in CS-ALG and PGA NCs
over time after initial irradiation.
 Polysaccharide nanocomplexes (NCs) for Photodynamic Therapy application (PDT)
 High stability in physiological condition
 Average dimension in the range 90–120 nm
 Up to 700 μg of 5-ALA per mg of carrier
 Improvement of 5-ALA phototoxicity up to 72 h

15. Chitosan-based nanocomplexes for simultaneous loading, burst reduction and controlled release of doxorubicin and 5-
fluorouracil
Di Martino Antonio, Kucharczyk Pavel, Capakova Zdenka, Humpolicek Petr, Sedlarik Vladimir
International Journal of Biological Macromlecules 102,2017,613-624 DOI: 10.1016/j.ijbiomac.2017.04.004
Fig. 2 MS spectra for (A) DOX, (B)5-FU, and (C) DOX + 5-FU after 24 h of release from CS-SPLA NCs in PBS.
Fig.1 Schematic representation of SPLA synthesis, and conjugation with CS
Fig. 3 Evaluation of cytotoxicity of loaded and unloaded
CS and CS-SPLA NCs in NTH/3T3 cells
by MTT assay
 CS-SPLA NCs for multidrug delivery
 NCs diameter 120–200 nm; ζ-potential 20–37 mV
 SPLA side chain improves the swelling, EE and release properties of the NCs
 CS-SPLA NCs are suitable to hold and release simultaneously two bioactive compounds
 Increase of in vitro drugs cytotoxicity
 No alteration in the chemical structure of the drugs occurred during encapsulation and 24 h after the release.

16. Enhancement of temozolomide stability by loading in chitosan-carboxylated polylactide-based nanoparticles
Di Martino Antonio, Kucharczyk Pavel, Capakova Zdenka, Humpolicek Petr, Sedlarik Vladimir
Journal of Nanoparticles Research 19,2,2017,71 DOI: 10.1007/s11051-017-3756-3
Inverted fluorescent microscopy observation of NIH/3T3 cells after exposition to a) CS-SPLA,
b) CS-SPLA + TMZ (24 h) c) CS-SPLA +TMZ (48 h)
a Chemical structure of CS-SPLA, b FTIR-ATR spectra related to SPLA
(track A), CS (track B) and the CS-SPLA product obtained (track C)
UV spectra for a free TMZ, control solution, and b TMZ released from nanoparticles.
The pH level of the control solution and release media equals 7.4
 Amphiphilic chitosan derivative to load and improve the stability of TMZ (temozolomide)
 Hydrodinamic diameter 150-180 nm; z-pot: 28-33mV
 Good stability in physiological solution (pH 7.4) up to 1 month
 TMZ encapsulation efficiency (>80%)
 CS-SPLA enhance the stability of TMZ by preventing the hydrolysis
 Control TMZ release rate by pH
 Improved in vitro cytotoxicity of TMZ
SEM micrographs of a unloaded and b, c loaded CS-SPLA freeze-dried nanoparticles.

17. Organic-inorganic hybrid nanoparticles controlled delivery system for anticancer drugs
Di Martino Antonio, Guselnikova A.Olga, Trusova E.Marina, Postnikov S.Pavel, Sedlarik Vladimir
International Journal of Phramaceutics 526,1-2,2017,380-390 DOI:10.1016/j.ijpharm.2017.04.061
A) schematic representation of surface modified iron NPs; TEM of B) uncoated
and C) coated iron NPs
A B C
 Surface modified iron NPs coated by chitosan (CS) and its amphiphilic derivative (CS-g-PLA)
 10-30 nm iron NPs,- 100–200 nm iron NPs coated by CS and CS-g-PLA NPs
 Presence of the coat improves the stability in physiological condition
 No variation in Ms at iron to polymer w/w ratio of 5
 Presence of the coat reduce BSA absorption
 Up to 80% EE of DOX and pH dependent release rate
 Presence of the coat reduce the release rate and enhance DOX cytotoxicity overtime
HeLa viability after incubation with A) free drug and bare NPs and B) drug loaded NPs
A B
Viability(%)
2 4 h 4 8 h 7 2 h
0
1 0
2 0
3 0
4 0
5 0
Iro n N P s + D O X
Iro n -C S N P s + D O X
Iro n -C S -g -P L A N P s + D O X
Viability(%)
2 4 h 4 8 h 7 2 h
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
Iro n N P s
Iro n -C S N P s
Iro n -C S -g -P L A N P s
D O X
C o n c .m g /m L
Hemolysis(%)
5 .0 1 0 .0 2 0 .0 4 0 .0 6 0 .0 8 0 .0 1 0 0 .0
0
1 0
2 0
3 0
4 0
5 0
Iro n N P s
Iro n -C S N P s
Iro n -C S -g -P L A N P s
A) Effect of the polymeric coating on the magnetization curve. B) Evaluation of the effect of coating and NPs
concentration on the hemolysis
-1
5
0
0
0
-1
2
5
0
0
-1
0
0
0
0
-7
5
0
0
-5
0
0
0
-2
5
0
0
2
5
0
0
5
0
0
0
7
5
0
0
1
0
0
0
0
1
2
5
0
0
1
5
0
0
0
- 1 0 0
- 7 5
- 5 0
- 2 5
2 5
5 0
7 5
1 0 0
Iron N Ps
Iron-C S N P s
Iron-C S -g-P L A N P s
F ie ld (O e)
M o m en t/M a ss (em u /g)
A B
C
A B
HeLa cells micrograph after incubation with DOX loaded iron-CS-g-PLA NPs A)24h; B) 48h;C) 72h. Scale bar 100mm