2. About OMICS Group
OMICS Group International is an amalgamation of Open Access publications and
worldwide international science conferences and events. Established in the year 2007
with the sole aim of making the information on Sciences and technology ‘Open Access’,
OMICS Group publishes 400 online open access scholarly journals in all aspects of
Science, Engineering, Management and Technology journals. OMICS Group has been
instrumental in taking the knowledge on Science & technology to the doorsteps of
ordinary men and women. Research Scholars, Students, Libraries, Educational
Institutions, Research centers and the industry are main stakeholders that benefitted
greatly from this knowledge dissemination. OMICS Group also organizes
300 International conferences annually across the globe, where knowledge transfer
takes place through debates, round table discussions, poster presentations, workshops,
symposia and exhibitions.
3. About OMICS Group International
OMICS Group International is a pioneer and leading science event organizer,
which publishes around 400 open access journals and conducts over 300
Medical, Clinical, Engineering, Life Sciences, Phrama scientific conferences all
over the globe annually with the support of more than 1000 scientific
associations and 30,000 editorial board members and 3.5 million followers to its
credit.
OMICS Group has organized 500 conferences, workshops and national
symposiums across the major cities including San Francisco, Las Vegas, San
Antonio, Omaha, Orlando, Raleigh, Santa Clara, Chicago, Philadelphia,
Baltimore, United Kingdom, Valencia, Dubai, Beijing, Hyderabad, Bengaluru
and Mumbai.
4. Peptides are short chains of amino acid monomers linked by peptide
(amide) bonds.
e:
Any number of amino acids can be joined together to form peptides of any
length.
5. 1. Solid peptides should be stored dry and frozen (-20 °C and lower).
They decompose on excessive heating and stable only at normal
temperature (room temperature and preferred lower).
2. Stability of peptides in solution is relatively low even at
temperatures lower than -20 °C. Therefore, a peptide solution once
prepared should be used as soon as possible.
3. Decompose on applying high shear energy or pressure.
4. Stable only at narrow range of pH (4-8).
5. Degradation pathways include: hydrolysis, deamidation, oxidation
etc.
6.
7. Capryomycin is a cyclic pentapeptide antibiotic similar to
viomycin; produced by Streptomyces capreolus.
Mixture of capreomycins IA, IB, IIA, and IIB in the approx
percentages, 25%, 67%, 3%, 6%, resp.
The mixture is a white solid; Sol in water; Practically
insoluble in most organic solvents.
Molecular Formula: C25H44N14O8 and Molecular
Weight: 668.71
9. Capreomycin Sulfate
Used for treatment of active (clinical)
tuberculosis.
Soluble in water.
Solution stable for up to 24 hours in refrigerator.
Stable in aqueous solution at pH 4-8 (unstable in
strongly acidic or strongly basic solutions).
10. Formulation of nanoparticles as carrier for peptides
(Capreomycin as a model drug) with the following considerations:
1- Particle size < 500 nm.
2- highest entrapment efficiency (EE%) as possible.
3- Using biodegradable polymers (chitosan and PLGA).
4- Formulation conditions compatible with peptides on using Double
Emulsion Solvent Evaporation Method.
5- Evaluation of the produced nanoparticles.
11. Chitosan is a linear polysaccharide composed of randomly distributed β-(1-4)-
linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine
(acetylated unit). It is made by treating chitin with the alkali sodium
hydroxide.
Chitosan is produced commercially by deacetylation of chitin, a naturally
occurring and abundantly available polysaccharide present in the
exoskeleton of crustaceans (such as crabs and shrimp) and cell walls of
fungi.
12. Chitosan is soluble in aqueous media at acidic pH.
Chitosan is available in a wide range of molecular
weight and degree of deacetylation.
Chitosan is receiving a lot of interest in the
encapsulation of active compounds due to its
biocompatibility, low toxicity and biodegradability.
14. Lactide Glycolide
PLGA undergoes acid catalyzed hydrolysis to release cellular metabolites of lactic
and glycolic acid.
Hydroxyl
terminus
Carboxyl
terminus
Variations in lactic acid to glycolic acid ratios affect the degradation profile of
the polymer (release rate).
Degradation rate is also affected through variations in the intrinsic viscosity
(i.v.) of the polymer.
Properties of PLGA
PLGA can be dissolved by a wide range of common organic solvents, including
tetrahydrofuran, acetone, ethyl acetate or chlorinated solvents such as
dichloromethane (DCM), .
15. PLGA cont.
Extensively investigated polyester.
Numerous assets
Release profile can be controlled.
Nanoparticle size can be controlled through variations in nanoparticle
formulation conditions.
Capable of the capture of any therapeutic agent including:
Hydrophobic (ATRA, doxorubicin, 5 fluorouracil).
Hydrophilic (DNA, protein, small molecules).
Potential for development of targeted or combinational therapies.
Very low immunogenicity and cytotoxicity.
18. aqueous phase
Organic phase
(DCM)
Homogenization or
sonication
With cooling
emulsification
& organic solvent
evaporation
Stirring
(PVA solution)
1 2
Chitosan NPs
NPs were prepared using double emulsion-solvent evaporation technique (w/o/w). Chitosan
and capreomycin were dissolved in acidic aqueous solution to give w/o emulsion with
addition of Span 80 using chlorinated organic solvent such as dichloromethane (DCM) with
probe homonization and then emulsified on polyvinylalcohol (PVA). The organic solvent was
evaporated by medium stirring and suspended chitosan NPs were obtained followed by
washing and freeze drying.
Capreomycin
&
Chitosan
ionotropic gelation with CLA
19. Mechanical stress of preparation (probe sonication, probe
homogenization, stirring)
Chitosan concentration.
Capreomycin:chitosan (HMW) ratio.
Effect of chitosan pH.
Cross linking agent (Type and concentration).
Amount of surfactant.
21. Liposomes
*Ultrasound SONOPULS HD 2070 (Germany) Titanium flat tip probe of diameter 6mm and processing frequency 20 kHz of 70 W power.
Effect of Probe Sonication* Time and Intensity on the stability of capreomycin in water.
Drug remaining (%)
Sonication intensity
) %(
Total Sonication Time (sec.)
100
25
60
96.64
50
60
93.52
100
60
96.5
25
120
93.4
50
120
90.4
100
120
92.28
25
180
90.24
50
180
87.96
100
180
22. Liposomes
Homogenization time = 120 sec. in ice bath.
Using High-Pressure Homogenizer: T 25 digital ULTRA-TURRAX® ( IKA®-Werke GmbH & Company KG, Staufen, Germany).
Effect of Homogenization speed on the stability of capreomycin in 1% v/v acetic acid aqueous
solution.
Drug Remaining (%)
Homogenization Speed
(rpm)
98.5
5000
96.2
7600
93.9
10000
89.8
15000
81.8
24000
23. Liposomes
Effect of Mechanical Equipment* for w/O first emulsion on
Chitosan NPS Properties*.
Entrapment
Efficiency (EE%)
Polydispersity
Particle Size ± SD
(nm)
Specification
Mechanical
Equipment
28.58
0.005
936.7 ± 38.7
100% intensity
Ultrasonicator
62.3
0.005
826.9 ± 19.6
5000 rpm
Homogenizer
42.48
0.005
783.8 ± 87.27
7600 rpm
Homogenizer
38.04
0.005
559.9 ± 13.2
10000 rpm
Homogenizer
33.35
0.005
397.9 ± 36.8
15000 rpm
Homogenizer
17.3
(extensive Capreomycin
(
degradation
0.005
343.7 ± 52.9
24000 rpm
Homogenizer
*Ultrasound SONOPULS HD 2070 (Germany) Titanium flat tip probe of diameter 6mm and processing frequency 20 kHz of 70 W power
and High-Pressure Homogenizer: T 25 digital ULTRA-TURRAX® ( IKA®-Werke GmbH & Company KG, Staufen, Germany).
**Exp. Conditions: Sonication or Homogenization time = 120 sec. ; Chitosan (HMW) conc. 0.5%; Chitosan:capreomycin
1:1; 0.8% Span 80;TPP 0.05 M ; 1% PVA ; stirring speed 500 rpm.
24. Stability of capreomycin under different stirring* rate.
Capreomycin Remaining %
Stirring Rate (rpm)
97.93
500
97.22
800
96.93
1000
91.72
1200
86.19
1500
*Over head mechanical stirrer.
Stirring time = 60 min
25. Effect of stirring* rate on the properties of chitosan NPs**.
EE%
Polydispersity
Particle size ± SD
(nm)
Stirring Rate (rpm)
33.35
0.005
397.9 ± 36.8
500
32.21
0.005
396.45 ± 32.7
800
30.23
0.005
392.02 ± 32.22
1000
28.88
0.005
394.40 ± 21.22
1200
26.77
0.005
391.96 ± 27.87
1500
*over head mechanical stirrer; stirring for 60 min.
**Exp. Conditions: Homogenization time = 120 sec. and speed = 1500 rpm; Chitosan (HMW) conc. 0.5%;
Chitosan:capreomycin 1:1; 0.8% Span 80; TPP 0.05 M ; 1% PVA .
27. Effect of Amount of Span 80 on the properties of chitosan NPs*.
Polydispersity
Particle size ± SD (nm)
Span 80 % w/v
0.065
1572.2 ± 106.6
0.35
0.005
519.1 ± 54.4
0.58
0.005
397.9 0 ± 36.8
0.80
*Exp. Conditions: Homogenization time = 120 sec. and speed = 1500 rpm; Chitosan (HMW) conc. 0.5%;
Chitosan:capreomycin 1:1; TPP 0.05 M ; 1% PVA .
28. Liposomes
Effect of Chitosan (HMW) concentration in 1% w/v acetic acid on NPS
Properties*.
Entrapment
Efficiency (EE%)
Polydispersity
Particle Size ± SD
(nm)
Chitosan Concentration
% w/v
33.35
0.005
397.9 ± 36.8
0.5
37.93
0.341
669.5 ± 75.1
1
52.30
0.005
1656.5 ± 215.8
1.5
Using High-Pressure Homogenizer: T 25 digital ULTRA-TURRAX® ( IKA®-Werke GmbH & Company KG, Staufen, Germany).
*Exp. Conditions: Homogenization speed =15000 rpm and time = 120 sec. ; Chitosan:cap 1:1; 0.8% Span 80; TPP 0.05 M ;
1% PVA ; stirring speed 500 rpm.
29. Figure1: TEM photograph of capreomycin loaded chitosan nanoparticles before lyophilization.
Exp. Conditions: Homogenization speed =15000 and time = 120 sec. ; Chitosan (HMW) conc. 0.5%;
Chitosan:capreomycin 1:1; 0.8% Span 80;TPP 0.05 M ; 1% PVA ; stirring speed 500 rpm.
30. Figure2: TEM photograph of capreomycin loaded chitosan nanoparticles after lyophilization.
Exp. Conditions: Homogenization speed =15000 and time = 120 sec. ; Chitosan (HMW) conc. 0.5%;
Chitosan:capreomycin 1:1; 0.8% Span 80;TPP 0.05 M ; 1% PVA ; stirring speed 500 rpm.
31. Figure3: TEM photograph of capreomycin loaded chitosan nanoparticles after lyophilization.
Exp. Conditions: Homogenization speed =15000 and time = 120 sec. ; Chitosan (HMW) conc. 0.5%;
Chitosan:capreomycin 1:1; 0.8% Span 80;TPP 0.05 M ; 1% PVA ; stirring speed 500 rpm.
32. 0
20
40
60
80
100
120
0 10 20 30 40 50 60 70 80
%
capreomycin
Released
Time (hours)
Release of capreomycin from chitosan
nanoparticles (NPs)
Figure 4: Release of Capreomycin from chitosan nanoparticles in Phosphate buffer pH 7.4 at 37 ±0.5 oC .
Exp. Conditions: Homogenization speed =15000 and time = 120 sec. ; Chitosan (HMW) conc. 0.5%;
Chitosan:capreomycin 1:1; 0.8% Span 80;TPP 0.05 M ; 1% PVA ; stirring speed 500 rpm.
33. aqueous phase
organic phase
(DCM)
Sonication
With cooling
emulsification Organic solvent
evaporation
Stirring
(PVA solution)
1 2
PLGA NPs
NPs were prepared using double emulsion-solvent evaporation technique (w/o/w).
Capreomycin was dissolved in aqueous media while, PLGA (50:50) was dissolved in
chlorinated organic solvent such as dichloromethane (DCM) to give w/o emulsion using
probe sonication and then emulsified on polyvinylalcohol (PVA). The organic solvent was
evaporated by medium stirring and suspended PLGA NPs were washed and freeze dryed.
PLGA
Capreomycin
35. Liposomes
*Ultrasound SONOPULS HD 2070 (Germany) Titanium flat tip probe of diameter 6mm and processing frequency 20 kHz of 70 W power.
Effect of Probe Sonication* Time and Intensity on the stability of capreomycin in water.
Drug remaining (%)
Sonication intensity
) %(
Total Sonication Time (sec.)
100
25
60
96.64
50
60
93.52
100
60
96.5
25
120
93.4
50
120
90.4
100
120
92.28
25
180
90.24
50
180
87.96
100
180
36. Liposomes
*Ultrasound SONOPULS HD 2070 (Germany) Titanium flat tip probe of diameter 6mm and processing frequency 20 kHz of 70 W power.
** Sonication intensity = 25% and time = 120 sec; Capreomycin : PLGA 1:4; Stirring speed 500 rpm.
Effect of Probe Sonication* Time and Intensity on the Properties of PLGA NPs**.
Entrapment
(
Efficiency (EE%
Polydispersity
Particle Size ± SD
(nm)
Sonication intensity
) %(
Total Sonication
Time (sec.)
54.88
0.172
1003 ± 36.0
25
60
-----
-------
-------
50
60
40.97
0.005
462.4 ± 25.1
100
60
37.09
0.005
459.4 ± 15.9
25
120
------
--------
-------
50
120
33.20
0.005
383.5 ± 14.6
100
120
35.19
0.006
450.5 ± 34.5
25
180
30.80
0.005
416.6 ± 32.4
50
180
28.44
0.005
361.6 ± 16.0
100
180
37. Liposomes
Effect of Capreomycin:PLGA ratio on NPs Properties*.
Entrapment
Efficiency (EE%)
Polydispersity
Particle Size ±
SD
(nm)
Probe Sonication*
intensity (%)
Sonication
Time (sec)
Drug :Polymer
Ratio
21.18
0.113
401.8 ± 8.7
25
120
1:1
31.93
0.075
423.5 ± 15.4
25
120
1:2
37.09
0.005
459.4 ± 15.9
25
120
1:4
*Ultrasound SONOPULS HD 2070 (Germany) Titanium flat tip probe of diameter 6mm and processing frequency 20 kHz of 70 W power;
Sonication intensity = 25%; Sonication time 120 sec.; Stirring speed 500 rpm.
38. Figure 5: TEM* photograph of capreomycin loaded PLGA nanoparticles** .
*Instrument ; Jeol (Jem-1011) Tokyo, Japan.
Exp. Condition: Sonication intensity = 25% and time = 120 sec; Capreomycin : PLGA 1:1; Stirring speed 500
rpm.
39. Figure 6: TEM* photograph of capreomycin loaded PLGA nanoparticles** .
*Instrument ; Jeol (Jem-1011) Tokyo, Japan.
Exp. Condition: Sonication intensity = 25% and time = 120 sec; Capreomycin : PLGA 1:4; Stirring speed 500
rpm.
40. 0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50 60 70 80
%
capreomycin
Released
Time (hours)
Release of capreomycin from PLGA
nanoparticles (NPs)
Figure 5: Release of capreomycin from PLGA nanoparticles in Phosphate buffer pH 7.4 at 37 ±0.5 oC .
Experementral condition: Sonication intensity = 25% and time = 120 sec; Capreomycin : PLGA 1:4;
Stirring speed 500 rpm.
41. 0
20
40
60
80
100
120
0 10 20 30 40 50 60 70 80
%
capreomycin
Released
Time (hours)
Release of Capreomycin from Chitosan and PLGA NPs
Figure 6: Release of capreomycin from selected Chitosan and PLGA nanoparticles in Phosphate
buffer pH 7.4 at 37 ±0.5 oC .
42. CONCLUSIONS
1- Nanoparticles were successfully obtained with particle size less than 500
nm and good particle size distribution.
2- Stability of capreomycin (as a model peptide) should be considered for
each step during formulation.
3- EE% of water soluble capreomycin did not exceeded 40% for the
obtained particle size and used formulation conditions.
4- Chitosan nanoparticles give fast capreomycin release compared with
slow release from PLGA nanoparticles.
5- Differences of nanoparticles properties such as particle size and drug
release may allow for different applications.
44. We welcome you all to our future conferences of OMICS Group International
Please Visit:
www.omicsgroup.com
www.conferenceseries.com
www.pharmaceuticalconferences.com
45. Let Us Meet Again
We welcome you all to our future conferences of OMICS
Group International
Please Visit:
www.omicsgroup.com
www.conferenceseries.com
www.pharmaceuticalconferences.com
