MOHSEN A. BAYOMI, Ph.D

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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.

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.

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

Formula C24 H44 N14 O12 S Molecular Weight 750.786

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).

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.

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.

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.

Lactide Glycolide

Hydroxyl terminus

Carboxyl terminus

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.

PLGA can be dissolved by a wide range of common organic solvents, including tetrahydrofuran, acetone, ethyl acetate or chlorinated solvents such as dichloromethane (DCM), .

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.

Organic Phase

W/O Emulsion

W/O/WEmulsion

Nanoparticles

Aqueous Phase

Sonication orHomogenization

Aqueous Phase with Stabilizer (PVA)

Solvent Evaporation

+

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

STRESS

aqueous phase

Organic phase(DCM)

Homogenization orsonication

With cooling

emulsification

& organic solvent evaporation

Stirring(PVA solution)

11 22

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 &

Chitosanionotropic gelation with CLA

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.

Probe Ultrasound Sonicator

Probe Homogenize

r

Over Head Michanical

Stirrer

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 .

Total Sonication Time (sec.)Total Sonication Time (sec.) Sonication intensitySonication intensity

(% )(% )Drug remaining (%)Drug remaining (%)

6060 2525 100100

6060 5050 96.6496.64

6060 100100 93.5293.52

120120 2525 96.596.5

120120 5050 93.493.4

120120 100100 90.490.4

180180 2525 92.2892.28

180180 5050 90.2490.24

180180 100100 87.9687.96

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.

Homogenization SpeedHomogenization Speed

(rpm)(rpm)Drug Remaining (%)Drug Remaining (%)

5000500098.598.5

7600760096.296.2

100001000093.993.9

150001500089.889.8

240002400081.881.8

LiposomesEffect of Mechanical Equipment* for w/O first emulsion on Chitosan NPS Properties*.

Mechanical Equipment

Specification Particle Size ± SD (nm)

Polydispersity EntrapmentEfficiency (EE%)

Ultrasonicator Ultrasonicator 100% intensity 936.7 ± 38.7 0.00528.5828.58

HomogenizerHomogenizer 5000 rpm 826.9 ± 19.6 0.005 62.3

HomogenizerHomogenizer 7600 rpm 783.8 ± 87.27 0.005 42.48

HomogenizerHomogenizer 10000 rpm 559.9 ± 13.2 0.005 38.04

HomogenizerHomogenizer 15000 rpm 397.9 ± 36.8 0.005 33.35

HomogenizerHomogenizer 24000 rpm 343.7 ± 52.9 0.005 17.3 )extensive Capreomycin

(degradation

*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 **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. 1:1; 0.8% Span 80;TPP 0.05 M ; 1% PVA ; stirring speed 500 rpm.

Stability of capreomycin under different stirring* rate.

Stirring Rate (rpm) Capreomycin Remaining %

500 97.93

800 97.22

1000 96.93

1200 91.72

1500 86.19

*Over head mechanical stirrer.*Over head mechanical stirrer.Stirring time = 60 minStirring time = 60 min

Effect of stirring* rate on the properties of chitosan NPs**.

Stirring Rate (rpm) Particle size ± SD (nm)

Polydispersity EE%

500 397.9 ± 36.8 0.005 33.35

800 396.45 ± ±32.732.7 0.005 32.21

1000 392.02 ± ±32.2232.22 0.005 30.23

1200 394.40 ± 21.22± 21.22 0.005 28.88

1500 391.96 ±± 27.87 27.87 0.005 26.77

*over head mechanical stirrer; stirring for 60 min.*over head mechanical stirrer; stirring for 60 min.**Exp. Conditions: Homogenization time = 120 sec. and speed = 1500 rpm; Chitosan (HMW) conc. 0.5%; **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 . Chitosan:capreomycin 1:1; 0.8% Span 80; TPP 0.05 M ; 1% PVA .

LiposomesEffect of Crosslinking Agents (CLA) 0n Chitosan NPs Properties.*

Type of CLAType of CLA Concentration Concentration

of CLA (M)of CLA (M)Particle SizeParticle Size± ± SD SD

(nm)(nm)PolydispersityPolydispersity (Entrapment (Entrapment

Efficiency (Efficiency (EE%)EE%)NotesNotes

GluteraldehydeGluteraldehyde0.5 00.5 0 -------------- ------------------ ---------------- Interaction with capreomycin

Sodium SulfateSodium Sulfate 0.0070.007 ------------------ ------------------ ------------------ No distinct particlesNo distinct particles

0.0130.013 1095.91095.9±± 185.8 185.8 0.0660.066 ---------------- Particles + aggregationsParticles + aggregations

0.0250.025 721.3 721.3 ±±120120 0.0120.012 ------------------ Particles + aggregationsParticles + aggregations

0.050.05 615.7 615.7 ±± 50.4 50.4 0.0050.005 23.523.5 distinct particlesdistinct particles

0.0650.065 617.1 617.1 ±± 31.1 31.1 0.0050.005 24.124.1 distinct particlesdistinct particles

TripolyphosphateTripolyphosphate

(TPP)(TPP)

0.0050.005 -------------- -------------- -------------------- No distinct particlesNo distinct particles

0.0250.025 712.7712.7 ± ±73.273.2 0.0050.005 42.7142.71 distinct particlesdistinct particles

0.0500.050 397.9397.9 ±36.8±36.8 0.0050.005 33.3533.35 distinct particlesdistinct particles

0.070.07 -------------- ---------------- ------------------ gelgel*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%; Exp. Conditions: Homogenization speed =15000 rpm and time = 120 sec. ; chitosan (HMW) concentration 0.5%; Chitosan:capreomycin 1:1; 0.8% Span 80; 1% PVA ; stirring speed 500 rpm. Chitosan:capreomycin 1:1; 0.8% Span 80; 1% PVA ; stirring speed 500 rpm.

Effect of Amount of Span 80 on the properties of chitosan NPs*.

Span 80 % w/v Particle size ± SD (nm) Polydispersity

0.35 1572.2 ± 106.6 0.065

0.58 519.1 ± 54.4 0.005

0.80 397.9 0 ± 36.8 0.005

*Exp. Conditions: Homogenization time = 120 sec. and speed = 1500 rpm; Chitosan (HMW) conc. 0.5%; *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 . Chitosan:capreomycin 1:1; TPP 0.05 M ; 1% PVA .

LiposomesEffect of Chitosan (HMW) concentration in 1% w/v acetic acid on NPS Properties*.

Chitosan ConcentrationChitosan Concentration

% w/v% w/vParticle SizeParticle Size± ± SD SD

(nm)(nm)PolydispersityPolydispersity Entrapment Entrapment

Efficiency (Efficiency (EE%)EE%)

0.50.5397.9 397.9 ±± 36.8 36.8 0.0050.005 33.3533.35

11669.5 669.5 ±± 75.1 75.1 0.3410.341 37.9337.93

1.51.51656.5 1656.5 ±± 215.8 215.8 0.0050.005 52.3052.30

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 ; *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. 1% PVA ; stirring speed 500 rpm.

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 .

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 .

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 .

Figure 4: Release of Capreomycin from chitosan nanoparticles in Phosphate buffer pH 7.4 at 37Figure 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%; 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. Chitosan:capreomycin 1:1; 0.8% Span 80;TPP 0.05 M ; 1% PVA ; stirring speed 500 rpm.

aqueous phase

organic phase(DCM)

SonicationWith cooling

emulsification Organic solvent evaporation

Stirring(PVA solution)

11 22

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

Capreomycin:PLGA (50:50) ratio. Mechanical stress of preparation (probe sonication intensity and time).

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 .

Total Sonication Time (sec.)Total Sonication Time (sec.) Sonication intensitySonication intensity

(% )(% )Drug remaining (%)Drug remaining (%)

6060 2525 100100

6060 5050 96.6496.64

6060 100100 93.5293.52

120120 2525 96.596.5

120120 5050 93.493.4

120120 100100 90.490.4

180180 2525 92.2892.28

180180 5050 90.2490.24

180180 100100 87.9687.96

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**.

Total Sonication Total Sonication

Time (sec.)Time (sec.)Sonication intensitySonication intensity

(% )(% )Particle SizeParticle Size± ± SD SD

(nm)(nm)PolydispersityPolydispersity Entrapment Entrapment

((Efficiency (Efficiency (EEEE%%

6060 2525 1003 1003 ±± 36.0 36.0 0.1720.172 54.8854.88

6060 5050 -------------- -------------- ----------

6060 100100 462.4 462.4 ±± 25.1 25.1 0.0050.005 40.9740.97

120120 2525 459.4 ± 15.9 0.0050.005 37.0937.09

120120 5050 -------------- ---------------- ------------

120120 100100 383.5 383.5 ±± 14.6 14.6 0.0050.005 33.2033.20

180180 2525 450.5 450.5 ±± 34.5 34.5 0.0060.006 35.1935.19

180180 5050 416.6 416.6 ±± 32.4 32.4 0.0050.005 30.8030.80

180180 100100 361.6 361.6 ±± 16.0 16.0 0.0050.005 28.4428.44

LiposomesEffect of Capreomycin:PLGA ratio on NPs Properties*.

Drug :Polymer Drug :Polymer

RatioRatioSonication Sonication

Time (sec)Time (sec)Probe Sonication* Probe Sonication*

intensityintensity(%) (%) Particle SizeParticle Size± ± SD SD

(nm)(nm)PolydispersityPolydispersity Entrapment Entrapment

Efficiency (Efficiency (EE%)EE%)

1:11:1120120 2525 401.8 401.8 ±± 8.7 8.7 0.1130.113 21.1821.18

1:21:2120120 2525 423.5 423.5 ±± 15.4 15.4 0.0750.075 31.9331.93

1:41:4120120 2525 459.4 ± 15.9 0.0050.005 37.0937.09

*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.

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.

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.

Figure 5: Release of capreomycin from PLGA nanoparticles in Phosphate buffer pH 7.4 at 37in 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.

Figure 6: Release of capreomycin from selected Chitosan and PLGA nanoparticles in Phosphate in Phosphate buffer pH 7.4 at 37buffer pH 7.4 at 37 ±0.5 oC . .

CONCLUSIONS1- 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.

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

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