This document discusses a thermosensitive micelle-hydrogel hybrid system for localized delivery of paclitaxel (PTX). Poloxamer 407 (P407) and carboxymethyl chitosan (CMCS) were used to form an injectable hydrogel. PTX-loaded micelles (PTX-M) were incorporated into the hydrogel (PTX-M-MG). The addition of CMCS and glutaraldehyde crosslinking transformed P407 into a non-thermoresponsive hydrogel with improved mechanical strength and drug release. In vitro and in vivo tests found PTX-M-MG had extended drug retention, reduced metabolism, and improved antitumor efficacy compared to Tax

1. Thermosensitive Micelles–Hydrogel
Hybrid System Based on
Poloxamer 407 for Localized Delivery of
Paclitaxel
SURAJ CHODHARY
Dept. of Pharmaceutics
AL AMEEN COLLEGE OF PHARMACY
Bangalore

2. REFERENCE JOURNAL
 Volume : 102
 Issue : 8
 Page : 2409 - 88
 Year : Aug. 2013
 Edited By : Dr. Ronald T.B.
 Impact Factor : 3.13
 Caoyun J, Juan S, Peng Z, Xiang J,
Can Z.
 Center of Drug Discovery,
 China Pharmaceutical University,
 Nanjing 210009, People’s Republic of
China.

3. INTRODUCTION

4. HYDROGEL

5. HYDROGEL
• Widely used for tissue scaffolds, and drug and cell delivery systems in
regenerative medicine because of its unique characteristics.
• There into, in situ gel has attracted more attention because of……..
“its transition of sol to a gel state by the variation of environment”
• Thereby, hydrogel as
accurate dosage,
ready to use,
good compliance for patients in sol state;
prolonged drug action,
reduced side effects, and
Low frequency

6. HYDROGEL
• In particular,
“Thermosensitive hydrogel is one of the most frequently applied
carriers because of the conveniently controllable adjustment of
temperature.
• In addition, in situ gel via intratumoral (i.t.) injection has been
explored in cancer therapy because of the distinguished advantages
of……
Longer exposure time in tumor mass and
Less systemic exposure

7. POLOXAMER – 407
P407

8. POLYMER (P-407)
• Hydrophilic linear tri-block polymer.
• Can be transformed from sol to a gel through micellization and
gelation at a certain concentration and temperature.
• P407 gels have been widely used in drug delivery systems, especially
the local delivery, because ……
“Can be administered in liquid form and serve on sustained
release depot at body temperature”

9. POLYMER (P-407)
• Drawbacks:
rapid erosion in the physiological environment.
low loading capacity for poor soluble drugs.

10. Carboxymethyl Chitosan
CMCS

11. POLYMER (CMCS)
• Water soluble derivative of chitosan (CS) with excellent
biocompatibility.
• Investigated for a wide range of biomedical applications, such as tissue
engineering scaffolds and drug-delivery carriers.
• Has abundant 2-NH2 groups that can be cross-linked by
glutaraldehyde (GA) to generate a network, which promotes ……
the mechanical intensities &
prevents it from the fast erosion of P407 gels.

12. PACLITAXEL
PTX

13. DRUG (PACLITAXEL)
• Chemotherapeutic agent in Tumors.
• Acts by stabilizing microtubules and imparting mitotic arrest to the
tumorous cells..
• Broad-spectrum activity in several solid tumors.
• DRAWBACKS:
low therapeutic index (extremely hydrophobic property)

14. OBJECTIVE

15. OBJECTIVE
To overcome the fast dissolution,
Improve the mechanical strength of P407 gels,
Enlarge the loading capability &
Extend the release period of PTX.

16. RAW MATERIALS

17. MATERIALS REQUIRED
• Poloxamer 407 (P407, Mw = 12, 600, PEO99-PPO67-PEO99)
• CMCS (carboxylation degree of 45%)
• Drug - Paclitaxel
• Human hepatocyte cell line L02
• Mice hepatoma solidity cell (Heps)
• RPMI-1640 medium (Hyclone®),
• Fetal bovine serum,
• Penicillin–streptomycin solution (Hyclone®),
• Phosphate buffered saline (PBS, Hyclone®),
• 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazoliumbromide (MTT)
• Trypsin (Gibco®)
NOTE: All other chemicals and reagents were of analytical grade

18. METHOD

19. METHODS
• PREPARATION:
Unloaded CMCS/GA-Modified P407 Hydrogels (P407-CMCS/GA)
Preparation of PTX-M-MG

20. Unloaded CMCS/GA-Modified
P407 Hydrogels

21. Unloaded CMCS/GA-Modified P407 Hydrogels
P407 dispersed in CMCS
solution
Mixed solution
Complete dissolution of P407
0.1 mL of GA (of diff. conc.)
added
To 4.9 mL of p407-CMCS
solution
Eddied to initiate cross
linking
(P407 19%, CMCS 1.5%, w/v),
Overnight at 4oC

22. Lower Critical Solution Temperature (LCST)
and Gelation Time
• Method : Tube Inverting (for sol-gel transition temp. & time)
 LCST:
“The temperature when the liquid did not flow for 30 s was
recorded as LCST.”
1 mL
sample
5mL tubes
at 4oC
Heated in
temp.
controlled
water bath
added
18 to 40◦C at a heating
rate of 1◦C/min

23. Lower Critical Solution Temperature (LCST)
and Gelation Time
• Method : Tube Inverting (for sol-gel transition temp. & time)
 Gelation time:
“The time at which the liquid did not flow for 30 s was recorded as
gelation time.”
1 mL
sample
Water bath
Inverting the
tube every
0.5 min
incubate
Constant temp.

24. Rheological Studies
• Apparatus: By a Rheometer (with plate geometry in oscillation mode).
• PROCESS:
Measurements were performed at a fixed frequency of 1 Hz and a
strain of 0.1% with a gap size of 0.5 mm.
NOTE: To prevent the evaporation of samples, a solvent trap was used
in conjunction with liquid paraffin.
The viscosities of P407 and P407-CMCS solutions + storage modulus
(G’) + the loss modulus (G’’)
NOTE: The gelation temperature was got when G’ and G’’ were
equivalent in value.

25. SWELLING BEHAVIOUR
• Swelling Ratio (SR):
P407-CMCS solutions
Different
amounts of GA
Placed in 37oC water bath
(Mixed)
For 2 mins.
To form gel
Gel weighed
20 mM phosphate buffer
solutions (PB, pH 6.8)
after removing the surface
solution with a filter paper
Fresh medium+ Fresh container
(immersed)
Weighed at predetermined
time
Reloaded in a

26. MECHANICAL PROPERTY
• Apparatus: Texture analyzer TA-XT2 (SMS, Stable Micro Systems)
NOTE: At least five replicate analyses of each sample were performed.
Hydrogels were
transferred into glass bottles
placed in a 37◦Cwater
bath
Analytical probe (D – 5mm)
is pushed in each sample
To allow complete gelation
At defined rate (1 mm/s) &
defined depth (3 mm)

27. INVITRO CYTOTOXICITY
• Performed on human hepatocyte cell line L02 as normal cells by MTT
assay.
• About 1 × 105 cells per well were seeded in 96-well plates.
• After 24 h culturing, the cells were treated with diluted P407-CMCS/GA
solution.
(for 24 h, 48 h, and 72 h, respectively)
• Subsequently, 20 μL of MTT PBS solution (5 mg/mL) was added into
each well
• After incubating for 4 h at 37◦C, the medium was removed, and 150 μL
of dimethyl sulfoxidewas added and shaken thrice.
• Readings were taken at 570 nm using Eliasa (Thermo Scientific)

28. PTX-M

29. PTX-M
• Preparation of PTX-M
Mixed Solution
subjected to dialysis against deionized water overnight
micelle solution was filtrated through a 0.45 μm pore-sized membrane
lyophilized by a freeze dryer system to obtain
the dried powers of PTX-M
PTX (16 mg)
0.355 mL of dehydrated
ethanol and 4 mL of distilled
water
NOSC (16 mg).
0.355 mL of dehydrated
ethanol and 4 mL of distilled
water

30. PTX-M Evaluation
• The drug-loading content (LC%) was calculated using the following
equations:
LC% = (WPTX in PTX-M/WPTX-M) × 100%
Where, WPTX in PTX-M : the weight of PTX loaded in the micelles,
WPTX-M : the weight of micelles.
• The particle size and polydispersity index (PDI) of the PTX-M were
assayed by Dynamic Light Scattering.
 PTX-M was dispersed in deionized water with 0.05% (w/v) GA or
1.5% (w/v) CMCS for different times, respectively.

31. PTX-M-MG

32. PTX-M-MG
• Preparation of PTX-M-MG
Mixed Solution (in a solution of P407)
GA was added in (0.05% GA in final, w/v)
Agitated intensely
certain concentration of
CMCS (1.5%, w/v)
PTX-M solutions
(4 mg/mL, 5 mL)

33. PTX-M-MG EVALUATION
1. Dispersibility of PTX-M in PX-M-MG
• “Cross-sectional images” of hydrogels were examined with Field
Emission Scanning Electron Microscope (FE-SEM, Sirion 200,
Holland).
• Samples obtained by freeze drying, and the cross-sectional area was
sputter coated with platinum before examination.
• P407-modified hydrogels loaded with same amount of PTX (PTX-MG)
were used as a control.

34. PTX-M-MG EVALUATION
2. In vitro drug release:
• Membrane-less model is used to assay the release behaviour.
• SHY-2A thermostatic shaker under 37oC at 50 ± 10 rpm
PTX-M-MG
Placed in a test
tube
PBS (pH 6.8)
+
1% Tween 80
SHY shaker started
Predetermined
time
Release medium
collected
HPLC estimation
Incubated
37oC, 30
mins
Release
medium
(sink
condition)
Replaced
with fresh
release
medium
HPLC
Column : C18 column (250 × 4.6 mm, Diamonsil)
M.P. : CH3OH + H2O (75:25 v/v)
Temp. : 35oC
Flow rate : 1.0 mL/min
Detector : UV

35. PTX-M-MG EVALUATION
3. Heps Tumor-Bearing Mice Model:
• For tumor formation,
Heps cells from ascites of tumor bearing mice were harvested.
Cell suspended in PBS.
inoculated subcutaneously cell suspension in the same flank of
each mouse.
The Tumor volume (V) was calculated:
V = [length × (width)2]/2
NOTE: Once the solid tumor reached about 100mm3, used for Tissue
distribution & anti-tumor activity study in rats.

36. PTX-M-MG EVALUATION
4. Tissue Distribution:
• Animals divided in 4 grps,
Taxol ®
PTX-M,
PTX-M-P407, and
PTX-M-MG
• Blood sample collection at predefined time points by: Retro-orbital
plexus puncture into heparinized tubes.
• Plasma obtained by: Centrifugation.
• Animal sacrificed → tumor & liver harvested → HPLC estimation.
Given intratumorally,
20mg/kg
HPLC Estimation
 Conc. Of PTX determined:
100 L or 200 L of tissue homogenate
Mixed with 200 L of Acetone (5 min)
Centrifuged – 12,000 rpm, 10 min
20 L supernatant introduced in HPLC system

37. PTX-M-MG EVALUATION
5. Anti Tumor Activity:
• Selected infected six groups (n =15)
(1) Negative control group (sterile normal saline, i.t.);
(2) Positive control group one (Taxol® intravenous (i.v.), every other
day for four times);
(3) Positive control group two (Taxol® i.t.);
(4) PTX-M (i.t.);
(5) PTX-M-P407 (i.t.);
(6) PTX-M-MG (i.t.).
NOTE: Single Dose administration, 20mg/kg of PTX formulations

38. PTX-M-MG EVALUATION
5. Anti Tumor Activity:
• To evaluate the antitumor efficacy,
the change oftumor volume,
in vivo toxicity,
survival rate, and
body weight
Tumor size and body weight (day for eight days) after administration.
Sacrificed by cervical dislocation and tumors were harvested
Followed by photographing.
NOTE: The survival time of six groups treated was calculated by
GraphPad Prism 5.

39. RESULTS
&
DISCUSSION

40. Sol–Gel Transition Behaviour
• LCST: P407- (29.96± 0.25OC) and P407-CMCS (28.03± 0.15OC).
(Indicates that they are reversibly thermo sensitive.)
• LCST: P407-CMCS –GA (0.025%–0.10%, w/v)
(Converts them into non-thermo-sensitive after certain period of time –
due to cross linking.)

41. PTX-M-MG EVALUATION
5. Sol–Gel Transition Behaviour:

42. Rheology Studies
a) Without GA
b) With GA

43. Swelling Behaviour & Mechanical Properties
c) % of GA
d) Hardness/integrity based on % GA

44. In vitro Cytotoxicity
L02 cells treated with different conc. Of P407 – CMCS/GA

45. Dispersibility of Micelles in P407-M-MG Hydrogels
Effect of CMCS or GA on Micelles

46. Dispersibility of Micelles in P407-M-MG Hydrogels
FE-SEM (Cross Sectional Images)
a) PTX only b) PTX-Micelles

47. In Vitro Drug Release

48. In Vitro Drug Release
TEM - Images
Micellar drug
loaded PTX
Size: 200nm

49. Tissue Distribution
Intratumoral administration in Heps tumor bearing mice
mice

50. Tissue Distribution
Intratumoral administration in Heps tumor bearing mice

51. Tissue Distribution
Intratumoral administration in Heps tumor bearing mice

52. In Vivo Antitumor Efficacy Assay

53. In Vivo Antitumor Efficacy Assay

54. In Vivo Antitumor Efficacy Assay

55. In Vivo Antitumor Efficacy Assay
Survival Percentage

56. In Vivo Antitumor Efficacy Assay
I.V. vs Intramural route

57. CONCLUSION

58. In short………………
• New intratumoral injectable PTX-M-MG hydrogels were constructed
and characterized.
• Incorporation of CMCS-GA cross-linked networks finally transformed
the thermosensitive P407 gels into nonthermosensitive hydrogels
thereby increasing:
higher swell ratios,
stronger mechanical property, and
Improved drug-release profile.
• P407-CMCS/GA hydrogels containing 0.05% (w/v) GA could be a
remarkable drug carrier for in situ injection.

59. In short………………
• PTX-M-MG hydrogels had excellent performance as a drug depot,
including
extending retention time at tumor sites,
reducing hepatic metabolism,
improving antitumor efficiency, and
weakening side effects of PTX
NOTE: when compared with Taxol®, PTXM, and PTX-M-P407
• In a word,
“the INJECTABLE MICELLES–HYDROGEL SYSTEM may
provide a platform for the insoluble drug to achieve” -
enhanced localized delivery and
treatment efficacy with minimal side effects.

60. Thank you