Synthesis and characterization of ligand. Preparation of NLCs using melt homogenization.

1. DESIGN OF LIPID
PARTICULATE SYSTEM FOR
INFECTIOUS DISEASE
Presented by - Vipul A. Sansare.
Bombay College Of Pharmacy, Mumbai.

2. CONTENTS
1. Tuberculosis
2. Standardization and analytical method development
for Rifampicin (RIF)
3. Synthesis and characterization of ligand
4. Development and characterization of RIF loaded
nanostructured lipid carrier (NLCs)
5. Development and characterization of dried powder
for inhalation of RIF loaded NLCs
6. Summary and conclusion
7. References
8. Acknowledgements
7/18/2018
2

3. INTRODUCTION
7/18/2018
3
Tuberculosis :Mycobacterium tuberculosis
Oral/parenteral
administration
Nonspecific distribution in
human body
Dose related side effects
(hepatotoxicity, epigastric
pain)
Less amount of drug
accumulate in AM
high drug
concentration in
the lung
Target alveolar
macrophages
Noninvasive
Reduce dose
related side
effects
Survival of
TB bacteria
in AM
Entry of TB
bacteria in
AM
Inhibit
phagosome
lysosome
fusion
Inhibit
disruption
by
lysosomal
enzymes
Adapted in
environment
of AM

4. AIM AND OBJECTIVE
7/18/2018
4
Aim of the present study was to develop ligand conjugated RIF loaded
nanostructured lipid carrier (NLCs) based dry powder for inhalation and
their characterization.
1. Fabrication and characterization of RIF loaded NLCs for passive targeting to
infected alveolar macrophages.
2. Design and evaluation of ligand conjugated NLCs containing RIF for active
targeting to infected alveolar macrophages.
Fig. 1 Receptors on AM

5. 7/18/2018
5

6. Standard plot in methanol
Regression equation y = 0.03x + 0.003
Linearity range 2.5- 20 ppm
Detection
wavelength
337nm
Correlation
coefficient
0.9999
Regression
equation
y = 0.03x + 0.0233
Linearity range 5- 25 ppm
Detection
wavelength
333.5nm
Correlation
coefficient
0.9993
Standard plot in simulated lung fluid pH 7.4
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 10 20 30
Absorbance
Concentration (ppm)
Linear (337 nm)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 10 20 30
Absorbance
Concentration (ppm)
334 nm
The developed analytical methods were validated and were found to be
precise, accurate and specific.
7/18/2018
6
Fig. 2 Linearity plot of RIF

7. 7/18/2018
7

8. 7/18/2018
8
• A 5 mmol of stearyamine was dissolved in 15ml
ethanol and heated up to 700C.
• 5 mmol of D-Mannose was added with stirring
• The solution was stirred for 15 min at 700C. After 15
min the solution was allowed to cooled down to
400C.
• The solution was diluted with 35ml hexane.
• The obtained crystals were washed with 30ml
hexane and ethanol and collected by filtration at
room temperature.
Stearylamine
D-Mannose N-Octadecylmannopyranosylamine

9. 7/18/2018
9
Fig. 3 IR spectrum of D-Mannose, Stearylamine and NODM
3398
2926
1638
1064
3331
2917
2849
1463
1606
2917
2850
3383
1465
1071

10. 7/18/2018
10
Fig. 4 NMR and Mass spectrum of synthesized NODM
432.4

11. 7/18/2018
11

12. SCREENING OF EXCIPIENTS
7/18/2018
12
Solid lipid Stearic acid
Liquid lipid Oleic acid
Surfactant Tween 20
Fig. 5 Solubility of RIF in excipients

13. OPTIMIZATION OF RATIO OF SOLID LIPID TO LIQUID LIPID
7/18/2018
13
Ratio of stearic acid:
oleic acid
Presence of oil droplets on
filter paper
Result
5:5 Yes Not selected
6:4 Yes Not selected
7:3 Yes Not selected
8:2 No selected
9:1 No Not selected
Miscibility test

14. PREPARATION OF RIF NLCS
7/18/2018
14
Melting Dispersion Sonication
Homogeniz
ation

15. OPTIMIZATION OF FORMULATION VARIABLE BY 3^3
BOX-BEHNKEN DESIGN
7/18/2018
15
Independent variables
Levels
-1 0 +1
X1 = total lipid (% w/v) 2 4 6
X2 = lipid : drug ratio 20 50 80
X3 = surfactant
concentration (%w/v)
1 1.5 2
The dependent variables were entrapment efficiency and particle size.

16. 7/18/2018
16
Run
A = total lipid
(% w/v)
B = lipid : drug
ratio
C = surfactant
concentration (%w/v)
Response 1
Particle size
(nm)
Response 2
Entrapment
efficiency
(%)
1 2 80 1.5 475.8 52
2 4 80 1 490 60.2
3 4 80 2 462.5 59.6
4 6 50 1 518.1 62.35
5 4 50 1.5 487.8 55
6 2 20 1.5 468.7 43.2
7 4 50 1.5 486 55.7
8 2 50 1 477.2 47.12
9 4 20 2 461.8 48
10 4 50 1.5 486.9 54.6
11 6 50 2 485.2 60.36
12 4 50 1.5 487.2 56.2
13 4 20 1 491.2 49.2
14 6 20 1.5 490.2 54.4
15 6 80 1.5 489.8 54.6
16 4 50 1.5 488.5 54
17 2 50 2 435.8 45

17. MODEL FOR PARTICLE SIZE 7/18/2018
17
Parameter Experimental value Required value
Model F-value 16.97 -
Probability ˃ F for model 0.0001 ˂ 0.05
Probability ˃ F for factor A 0.0001 ˂ 0.1
Probability ˃ F for factor B 0.7840 ˂ 0.1
Probability ˃ F for factor C 0.0001 ˂ 0.1
Probability ˃ F for factor AC 0.5969 ˂ 0.1
Predicted R-square 0.8106 -
Adjusted R-square 0.7134 -
Adequate Precision 15.14 ˃ 4
Particle size = +481.34 +
15.72A + 0.7750B - 16.40C +
2.12AC
Fig.6 3-D, contour and
perturbation plot

18. MODEL FOR ENTRAPMENT EFFICIENCY (%) 7/18/2018
18
Parameter Experimental value Required value
Model F-value 53.50 -
Probability ˃ F for model 0.0001 ˂ 0.05
Probability ˃ F for factor A 0.0001 ˂ 0.1
Probability ˃ F for factor B 0.0001 ˂ 0.1
Probability ˃ F for factor C 0.1079 ˂ 0.1
Probability ˃ F for factor AB 0.3481 ˂ 0.1
Probability ˃ F for factor AC 0.9559 ˂ 0.1
Probability ˃ F for factor BC 0.798 ˂ 0.1
Probability ˃ F for factor A2 0.134 ˂ 0.1
Probability ˃ F for factor B2 0.4991 ˂ 0.1
Probability ˃ F for factor C2 0.4362 ˂ 0.1
Predicted R-square 0.8406 -
Adjusted R-square 0.9672 -
Adequate Precision 28.104 ˃ 4
Entrapment efficiency = +55.10 + 6.91A +5.31B + 0.0325C + 0.57AB +
0.0325AC +0.15 BC -0.9363 A² - 0.3938B2 -0.4562C2

19. 7/18/2018
19Fig. 7 3-D, contour and perturbation plot

20. VALIDATION OF MODEL FOR PARTICLE SIZE
7/18/2018
20
Checkpoint
batch
Total lipid
(% w/v)
Lipid : drug
ratio
Surfactant
concentratio
n (% w/v)
Predicted
value (nm)
Observed
value (nm)
% Error
1 3 35 1.5 473.12 477.4 0.9046330
2 4 50 1.5 481.33 487.6 1.3026406
3 5 65 1.5 489.8 496.2 1.3066557
Fig.8 Validation of model for particle size

21. VALIDATION OF MODEL FOR ENTRAPMENT EFFICIENCY
7/18/2018
21
Checkpoint
batch
Total lipid
(% w/v)
Lipid : drug
ratio
Surfactant
concentration
(% w/v)
Predicted
value (%)
Observed
value (%)
% Error
1 3 35 1.5 48.69 50.19 2.98864316
2 4 50 1.5 54.958 55.16 0.3662074
3 5 65 1.5 61.384 62.23 1.35947292
Fig.9 Validation of model for entrapment efficiency

22. CHARACTERIZATION OF RIF NLCS
7/18/2018
22
Appearance: homogeneous and red in colour,
Particle Size and Polydispersity Index (PDI): using Zetasizer Nano ZS
(Malvern) 240.9 nm (PDI=0.135)
Zeta potential: - 43.3 mV
Entrapment efficiency: 52±0.88%.
Fig.10 Zeta potential of optimize formulation

23. 7/18/2018
23

24. 7/18/2018
24
Feed rate 1ml/min
Atomization pressure 2 bars
Inlet temperature 105-110 0C
Outlet temperature 50-60 0C
Vacuum 135-140 mm of Hg
Product temperature 40-500C
Spray drying of RIF NLCs
Spray drier: Labaultima
Carrier: Mannitol
Antiadherent: L-Leucine
Lipid: carrier ratio: (1:2)
Table: Operating conditions for spray drying
Aggregation
Exhalation
(nano size)
Drawbacks of NLCs for pulmonary delivery
Sedimentation

25. PARTICLE SIZE AND ASSAY
7/18/2018
25
Particle Size and Polydispersity Index (PDI): using Zetasizer Nano ZS
(Malvern) 409.5nm, (PDI= 0.324)
Assay: using UV spectroscopy from three different locations of container.
91 ± 2.6 %.
Fig.11 Particle size of redispered spray dried NLCs

26. SURFACE MORPHOLOGY
7/18/2018
26
Scanning Electron Microscopy (Philips XL 30)
Flow properties
Compressibility index: 16.66
Hausners ratio: 1.2
Angle of repose: 29.360
Fig.12 SEM images of RIF, spray dried RIF NLCs

27. IN-VITRO LUNG DEPOSITION STUDY
7/18/2018
27
Andersons Cascade Impactor (Copley Scientific)
Cut -off diameter
(micron)
Amount deposited
(µg)
Device - 44.583
Capsule - 34.33
Induction port - 430
Preseparator - 510
Stage 0 8.6 185.06
Stage1 6.5 330.56
Stage 2 4.4 490
Stage 3 3.3 400
Stage 4 2 200
Stage 5 1.1 80
Stage 6 0.54 32
Stage 7 0.25 9.53

28. 7/18/2018
28
Spray dried RIF NLCs
Total drug impinged (µg) 3000
Recovered dose (µg) 2746.063
Emitted dose(µg) 2667.15
FPD(µg) 1211.53
FPF (%) 44.1188
Dispersibility (%) 45.42414
MMAD (µm) 4.71
GSD 1.71
Fig.13 Comparative plot of % of RIF deposited on stages of ACI

29. IN–VITRO RIF RELEASE STUDY
7/18/2018
29
Apparatus Dissolution apparatus (Labindia)
Release medium Simulated lung fluid pH 7.4
Volume of release medium 150ml
Membrane
Dialysis membrane (13-14kD)
Temperature 37±0.50C
Stirring speed 50 rpm
Study duration 96 hours
Quantity of RIF Equivalent to 3mg of RIF (403.26mg)
Volume of aliquot 5ml
Time points
0,0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 24, 48, 72, 96
hours
0
10
20
30
40
50
60
70
80
90
0 20 40 60 80 100
%Cumulativerelease
Time (hours)
RIF NLCs
RIF
Fig.14 In-vitro release profile of RIF from NLCs
84.1±4.34% of RIF release
at the end of 96 hrs.

30. X-RAY DIFFRACTION
7/18/2018
30Fig.15 XRD diffractogram of mannitol, RIF, RIF
NLCs
2Ɵ values Intensity of peak for
RIF pure drug
Intensity of peak for
RIF NLCs with
conjugation
7 4376 1685
9.93 5939.7 No peak
11.13 13911.39 182
15.72 11176 No peak
19.94 13561.97 770
Bruker D8 Discover XRD analyzer

31. THERMAL STABILITY OF RIF 7/18/2018
31
Temp Cel
250.0200.0150.0100.050.0
DSCmW
5.00
0.00
-5.00
-10.00
-15.00
-20.00
DDSCmW/min
63.2Cel
-11.65mW
STEARIC ACID
Temp Cel
250.0200.0150.0100.050.0
4.00
2.00
0.00
-2.00
-4.00
-6.00
-8.00
-10.00
-12.00
DDSCmW/min
172.3Cel
-8.62mW
mannitol
Fig.16 DSC thermograms of stearic acid and
mannitol
Melting endotherm: 63.20C
Melting endotherm: 172.30C
Perkin-Elmer Pyris 1 DSC

32. 7/18/2018
32
Temp Cel
250.0200.0150.0100.050.0
DSCmW
1.000
0.500
0.000
-0.500
-1.000
-1.500
DDSCmW/min
197.1Cel
-0.820mW 297.8Cel
-0.949mW
RIF 50
Temp Cel
250.0200.0150.0100.050.0
DSCmW
2.000
1.000
0.000
-1.000
-2.000
-3.000
-4.000
-5.000
-6.000
-7.000
DDSCmW/min49.2Cel
-1.311mW
168.4Cel
-4.785mW
RIF NLC'S WITH CONJUGATION
Fig.17 DSC thermograms of RIF and RIF
NLCs
Melting endotherm: 197.10C
Exotherm: Degradation 260-2700C
Melting endotherm: 168.40C
49.20C

33. 7/18/2018
33
1. In-vitro anti-microbial activity of RIF NLCs using Bacillus
subtilis strain ATCC 6633.
2. In-vitro cytotoxicity study and cellular uptake study using
alveolar macrophages cell line.
3. Particle size analysis of spray dried RIF NLCs.
4. Atomic force microscopy: spray dried RIF NLCs.
5. % moisture content: Karl Fischer titration.
6. New conjugation of D-Mannose and solid fatty acid.
7. Stability studies

34. 7/18/2018
34

35. 7/18/2018
35
• RIF NLCs for active targeting to AM were prepared using
stearic acid, oleic acid and tween 20 using melt
homogenization ultrasonication method. RIF NLCs were
converted into dry powder by spray drying.
• Spray dried RIF NLCs showed good redispersibility,
morphology, and flow properties.
• In-vitro lung deposition study showed RIF NLCs are suitable
for pulmonary drug delivery. In-vitro release study showed
sustained drug release of RIF from spray dried NLCs.
• Cell internalization studies are required to conform efficacy of
ligand conjugated RIF NLCs over non conjugated RIF NLCs.

36. 7/18/2018
36

37. 7/18/2018
37
1. Lawlor, C., et al., Cellular targeting and trafficking of drug delivery systems
for the prevention and treatment of MTb. Tuberculosis (Edinb), 2011.
91(1): p. 93-7.
2. Maretti, E., et al., Inhaled Solid Lipid Microparticles to target alveolar
macrophages for tuberculosis. Int J Pharm, 2014. 462(1-2): p. 74-82.
3. Pham, D.-D., E. Fattal, and N. Tsapis, Pulmonary drug delivery systems
for tuberculosis treatment. International Journal of Pharmaceutics, 2015.
478(2): p. 517-529.
4. Witoonsaridsilp, W., et al., Development of mannosylated liposomes using
synthesized N-octadecyl-D-mannopyranosylamine to enhance
gastrointestinal permeability for protein delivery. AAPS PharmSciTech,
2012. 13(2): p. 699-706.
5. Negi, L.M., M. Jaggi, and S. Talegaonkar, Development of protocol for
screening the formulation components and the assessment of common
quality problems of nano-structured lipid carriers. Int J Pharm, 2014.
461(1-2): p. 403-10.
6. Kasongo, K.W., et al., Selection and characterization of suitable lipid
excipients for use in the manufacture of didanosine-loaded solid lipid
nanoparticles and nanostructured lipid carriers. J Pharm Sci, 2011.
100(12): p. 5185-96.

38. 7/18/2018
38
6. Pilcer, G. and K. Amighi, Formulation strategy and use of excipients in
pulmonary drug delivery. Int J Pharm, 2010. 392(1-2): p. 1-19.
7. Tran, T.H., et al., Preparation and characterization of fenofibrate-loaded
nanostructured lipid carriers for oral bioavailability enhancement. AAPS
PharmSciTech, 2014. 15(6): p. 1509-15.
8. Ali, M.E. and A. Lamprecht, Spray freeze drying for dry powder inhalation
of nanoparticles. European Journal of Pharmaceutics and
Biopharmaceutics, 2014. 87(3): p. 510-517.
9. Reverchon, E., I. De Marco, and G. Della Porta, Rifampicin microparticles
production by supercritical antisolvent precipitation. Int J Pharm, 2002.
243(1-2): p. 83-91.
10. Xia, D., et al., Spray drying of fenofibrate loaded nanostructured lipid
carriers. Asian Journal of Pharmaceutical Sciences, 2016. 11(4): p. 507-515.

39. 7/18/2018
39

40. 7/18/2018
40
I am grateful to my research guide Dr. (Mrs) Ujwala A. Shinde, Associate
Professor of Pharmaceutics for her invaluable guidance, encouragement and
advice during the research work.
I express my gratitude to Dr. (Mrs) Mangal S. Nagarsenker, Dr.
(Mrs) Mala D. Menon, Dr. (Mrs) Namita D. Desai for allowing use of various
instruments and apparatuses.
I am grateful to Lupin Ltd. (Mumbai), CIRCOT (Mumbai), SAIF Punjab
University (Chandigarh), Kelkar Education Trust's Scientific Research
Centre (Mumbai), Dept. of Nanoscience (University of Mumbai), Bharti
Vidyapreeth College of Pharmacy, Diya Lab, Ambernath Organics Pvt. Ltd.
(Mumbai), MKR Laboratories

41. 7/18/2018
41
End of presentation