Controlling the Characteristics of Polymeric Nanoarticles as Carriers for the Model Peptide Capreomycin

1
Controlling the Characteristics of Polymeric
Nanoarticles as Carriers for the Model Peptide
Capreomycin
MOHSEN A. BAYOMI, Ph.D
2
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3
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  Hyderabad, Bengaluru and Mumbai.

4
Peptides

• 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
Stability of Peptides

• 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).
• 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.
• Decompose on applying high shear energy or
  pressure.
• Stable only at narrow range of pH (4-8).
• Degradation pathways include hydrolysis,
  deamidation, oxidation etc.

6
Capreomycin
7
Capreomycin cont.

• 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

8
Capreomycin Sulfate
Molecular Weight 750.786
Formula C24 H44 N14 O12 S
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
OBJECTIVES

• Formulation of nanoparticles as carrier for
  peptides (Capreomycin as a model drug) with the
  following considerations
• 1- Particle size lt 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

• 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.

• Chitosan is a linear polysaccharide composed of
  randomly distributed ß-(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.

12
Chitosan Cont.

• 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.

13
Poly D,L lactide-co-glycolic acid (PLGA)
Carboxyl terminus
Hydroxyl terminus
Lactide
Glycolide
14
Properties of PLGA
Hydroxyl terminus
Carboxyl terminus
Lactide
Glycolide

• PLGA undergoes acid catalyzed hydrolysis to
  release cellular metabolites of lactic and
  glycolic acid.

• 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.

• 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.

16
Double Emulsion (W/O/W) Solvent EVap.
Aqueous Phase with Stabilizer (PVA)
Aqueous Phase
Sonication or Homogenization
W/O/W Emulsion
W/O Emulsion
Nanoparticles

• Organic Phase

Solvent Evaporation
17

• All nanoencapsulation formulation techniques
  including double emulsion create
• Mechanical.
• Thermal
• Chemical
• STRESS

18
Capreomycin Chitosan
ionotropic gelation with CLA
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.
19
Controlling Parameters for Chitosan NPs

• Mechanical stress of preparation (probe
  sonication, probe homogenization, stirring)
• Chitosan concentration.
• Capreomycinchitosan (HMW) ratio.
• Effect of chitosan pH.
• Cross linking agent (Type and concentration).
• Amount of surfactant.

20
Over Head Michanical Stirrer
Probe Homogenizer
Probe Ultrasound Sonicator
21
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.

Liposomes
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
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.

Liposomes
Drug Remaining () Homogenization Speed (rpm)
98.5 5000
96.2 7600
93.9 10000
89.8 15000
81.8 24000
23
Homogenization time120 sec in ice bath. Using
High-Pressure Homogenizer T 25 digital
ULTRA-TURRAX ( IKA-Werke GmbH Company G,
Staufen, Germany).
24
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
Chitosancapreomycin 11 0.8 Span 80TPP 0.05
M 1 PVA stirring speed 500 rpm.
25
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
26
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 Chitosancapreomycin 11 0.8 Span 80
TPP 0.05 M 1 PVA .
27

• Effect of Crosslinking Agents (CLA) 0n Chitosan
  NPs Properties.

Liposomes
Notes (Entrapment Efficiency (EE) Polydispersity Particle Size SD (nm) Concentration of CLA (M) Type of CLA
Interaction with capreomycin -------- --------- ------- 0.5 0 Gluteraldehyde
No distinct particles --------- --------- --------- 0.007 Sodium Sulfate
Particles aggregations -------- 0.066 1095.9 185.8 0.013 Sodium Sulfate
Particles aggregations --------- 0.012 721.3 120 0.025 Sodium Sulfate
distinct particles 23.5 0.005 615.7 50.4 0.05 Sodium Sulfate
distinct particles 24.1 0.005 617.1 31.1 0.065 Sodium Sulfate
No distinct particles ---------- ------- ------- 0.005 Tripolyphosphate (TPP)
distinct particles 42.71 0.005 712.7 73.2 0.025 Tripolyphosphate (TPP)
distinct particles 33.35 0.005 397.9 36.8 0.050 Tripolyphosphate (TPP)
gel --------- -------- ------- 0.07 Tripolyphosphate (TPP)
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 (HMW) concentration 0.5
Chitosancapreomycin 11 0.8 Span 80 1 PVA
stirring speed 500 rpm.
28
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
Chitosancapreomycin 11 TPP 0.05 M 1 PVA .
29
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. Chitosancap 11 0.8 Span 80 TPP 0.05
M 1 PVA stirring speed 500 rpm.
30
Figure1 TEM photograph of capreomycin loaded
chitosan nanoparticles before lyophilization.Exp.
Conditions Homogenization speed 15000 and
time 120 sec. Chitosan (HMW) conc. 0.5
Chitosancapreomycin 11 0.8 Span 80TPP 0.05
M 1 PVA stirring speed 500 rpm.
31
Figure2 TEM photograph of capreomycin loaded
chitosan nanoparticles after lyophilization.Exp.
Conditions Homogenization speed 15000 and time
120 sec. Chitosan (HMW) conc. 0.5
Chitosancapreomycin 11 0.8 Span 80TPP 0.05
M 1 PVA stirring speed 500 rpm.
32
Figure3 TEM photograph of capreomycin loaded
chitosan nanoparticles after lyophilization.Exp.
Conditions Homogenization speed 15000 and
time 120 sec. Chitosan (HMW) conc. 0.5
Chitosancapreomycin 11 0.8 Span 80TPP 0.05
M 1 PVA stirring speed 500 rpm.

33
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 Chitosancapreomycin 11 0.8 Span
80TPP 0.05 M 1 PVA stirring speed 500 rpm.

34
Capreomycin
PLGA
NPs were prepared using double emulsion-solvent
evaporation technique (w/o/w). Capreomycin was
dissolved in aqueous media while, PLGA (5050)
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.
35
Controlling Parameters for PLGA NPs

• CapreomycinPLGA (5050) ratio.
• Mechanical stress of preparation (probe
  sonication intensity and time).

36
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.

Liposomes
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
37
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 14 Stirring speed 500 rpm.

• Effect of Probe Sonication Time and Intensity
  on the Properties of PLGA NPs.

Liposomes
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
38
Liposomes

• Effect of CapreomycinPLGA 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 11
31.93 0.075 423.5 15.4 25 120 12
37.09 0.005 459.4 15.9 25 120 14
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.
39
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 11 Stirring speed 500 rpm.
40
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 14
Stirring speed 500 rpm.
41
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 14 Stirring speed 500 rpm.
42
Figure 6 Release of capreomycin from selected
Chitosan and PLGA nanoparticles in Phosphate
buffer pH 7.4 at 37 0.5 oC .
43
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
Acknowledgements

• PROJECT COWORKERS
• Ahmed Al-Anzy, MS
• Ibrahim Al-Sarra, Ph.D.

Thanks
45
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46
Let Us Meet Again

• We welcome you all to our future conferences of
  OMICS Group International
• Please Visitwww.omicsgroup.com
• www.conferenceseries.com
• www.pharmaceuticalconferences.com