Acknowledgements :  I would like to
express my deepest appreciation to all those who provided me the possibility to
complete this report. A special gratitude I give to our final year project
manager, Ass. Prof. Hamza Maswadeh and Dr. Ahmed A. H. Abdellatif , which
contributes in stimulating suggestions and encouragement, helped me to
coordinate my project . I want thank Dr. Abdulwadood khyri for
assistance with optical microscope technique. I want to thank my
colleagues Waleed Al-Harbi , Meshari AL-dhafiri and Waleed AL-Mutiri .                          2.List of figure and tables
Page Number  Table 1                                                                                                
10 Figure 1                                                                                                  9                                                                   
Figure 2                                                                                                 
9                                               Figure
Figure 4                                                                                                 12                                          
Figure 5                                                                                                
Figure 6                                                                                                 13                                                                                                                                             
    3. List of abbreviations :ethyl cellulose (EC) , Etoposide(ETP),
polymeric microparticles
(PoMPs)  , encapsulation efficienct (EE%) ,            4. Abstract Background :The purpose of this study was to study the effect of Ethylcellulose
(EC) on the physicochemical characteristics on Etoposide(ETP), the effects on
the size and loading of drugs from microsphere, polymer matrix and surface
morphology, and the release of the ETP from microsphere. EC was used in this
study as a targeting polymer in the form of microparticles. Objective:                                                                                                                                         This
study was defended forprotecting the decomposition of ETP in acidic media of
stomach by protecting it using EC as a potential coating and to use
microspheres loaded with ETP for the treatment of colon cancer.                                                                                                                        
     Method :Ethyl cellulose EC and
the (Etoposide) was being dissolved in a mixture of chloroform and methanol
(3:1). Then the mixture was beingdispersed in theaqueous solution to 5% methylcellulose
with the aid of mechanical homogenization at 10000 rpm for one hour. The emulsion was being stirred at room
temperature till the evaporation of the organic solvents. The formed PoMPs were
being filtered, washed and dried for further treatment.ResultsThe results showed that encapsulation efficienct EE% for all
forms of microparticles were decreased with the decrease in drug: polymer ratios. The microparticles had a mean
diameter of (….) µm, confirming that the structure was in micro-size. The release rate of Etoposide from microparticles was strikingly
higher than that of thedrug itself, confirming that the release
was  enhanced with controlling for
The here
presented microparticle formulations serve as apromising
platform to improve the solubility, absorption and sustained release of Etoposide.   5. Keywords:Etoposide,Ethylcellulose,Methylcellulose, polymeric microparticle, Microencapsulation.   6.Background
and Review of literatureEthyl
cellulose(EC) is a derivative of cellulose which converts some hydroxyl groups
on repeated glucose units into ethyl ether groups. EC polymer has been used as a coating material for oral
sustained-release dosage forms since 1985 (1).EC aqueous dispersion
provides a stable, reproducible, pH-independent drug-release profile with
similar dissolution profiles in both fed and fasted states (2). EC is thermoplastic, with a
softening point of 140° -170°C and a melting point of 160 °-210°C; it
decomposes intotemperatures above 200°C. Since the melts of ethyl cellulose are
rapidly oxidized in the air, stabilizers-such as derivatives of aromatic
alcohol or amines-must be added (3). EC was used in this study
as a targeting polymer in the form of microparticles.Microencapsulation may be defined as the packaging
technology of solids, liquid or gaseous material with thin polymeric coatings,
forming small particles called microcapsules (4).The polymer acts as a protective film, isolating the core
and avoiding the effect of its inadequate exposure. This membrane dissolves
itself through a specific stimulus, releasing the core of the ideal place or at
the ideal time (5).Microencapsulation has numerous applications for areas
such as the pharmaceutical, agricultural, medical and food industries, iswidely used in the encapsulation of essential
oils, colorings, flavorings, sweeteners, microorganisms, among others(6).Etoposide(ETP) used for the treatments of a number of
types of cancer. This includes testicular cancer, lung cancer, lymphoma, leukemia, neuroblastoma, and ovarian cancer. It is used by mouth or injection into a
vein. ETP was approved for medical use for the United States in 1983 (7).  It is on the World Health
Organization’s List of Essential Medicines,
the most effective and safe medicines needed in a health system(8). The lyophilization
(Freez drying ) mean dehydration, used in the food, chemical and
pharmaceutical. Lyophilizatation used to improve the stability of product and
make them easy to store and transport(9).  Freez drying is process that remove the water
by freezing the material within lyophilizer and convert material into solid
phase ( powder).Aims
and ObjectivesThe
objective of this project: is to deliver polymeric microparticles (PoMPs) loaded with anti-cancer drugs
in the form of enteric tablet or capsules to the colon and GIT. Thus, will take
the benefits of both anti-cancer effect and the efficient targeting through
targeting to its region in the GIT.   7.
Material and Method1.1  Materials and
equipment­All chemicals and reagents used were of pharmaceutical
reagent grades and some of them were used as such without any further
purification. Etoposide standard powder was
purchased from (Boots Co., Nottingham, U.K.).Ethyl cellulose was purchased from
(Sigma Chemical Co., Ca, U.S.A). Methylcellulosewas
purchased from (Dow Chemical Fluka, Greifensee, Switzerland).
Potassium chloride, hydrochloric acid, sodium hydroxide, acetic acid, and
potassium dihydrogen orthophosphate were
purchased from (Elnasr pharmaceutical chemical Co., Abu­Zaabal., Cairo, Egypt).
U.S.P.dissolution tester: Validata –
Hansen research Chatsworth, Ca, USA.
Ultraviolet double beam Spectrophotometer 1601: Shimadzu Co., u.v­ 240, Jasco,
Tokyo, Japan. Magnetic Stirrer (type MMS), U. K. Electric Stirrer (V. E. B),
Germany. Electrical digital balance: Metler­toledo, Ag, CH­8606, Greifensee, Switzerland. Shaker: Julabo, India. pH meter: Gateway, Ltd. U.K. Optical microscope with
camera lucida (power 3.2, 10, 40, and 100), 60­500 HZ, 230 V, Type B: Carl Zeiss Co., Germany. Freeze drying, (Germany).
Optical microscope (LEICA DMD 108, Located in Wetzlar and Mannheim ( Germany).1.1
Preparation of microparticles;Ethyl cellulose ECand
the (Etoposide) was being dissolved in a mixture of chloroform and methanol
(3:1) (10). Then the mixture was beingdispersed
in theaqueous solution to 5% methylcellulose with the aid of mechanical
homogenization at 10000 rpm for one hour.
The emulsion was being stirred at room temperature till the evaporation of the
organic solvents. The formed PoMPs were being filtered, washed and dried for
further treatment as shown in figure 1. The microparticles were prepared in
according to thedrug to polymer ratios as
shown in table 1.The
first stage was involved the following
steps; simultaneously, different Etoposide(ETP)drugs were be entrapped within different PoMPs
preparations. Other need for formulation of PoMPs from (EC), as to protect the Etoposide PoMPs from
gastric pH and concentrate the PoMPs in GIT. Etoposide PoMPs was be released
very slowly as the pH in GIT is not suitable for sensitive drugs. Freeze Drying is a
water removal process typically used to preserve perishable materials, to
extend shelf life or make the material more convenient for transport. Freeze-drying
works by freezing the material, then reducing the pressure and adding heat to
allow the frozen water in the material to sublimate. Briefly, the samples(1:1)
were frozen in therefrigerator overnight.
The samples were put in the Frieze drier for drying. Frieze drier was operated
for 2 hours until the temperature
decreased to -55 oC. The pressure was applied overnight until the
samples were completely dried. The samples were removed and kept in 4 oC. 

     Figure 1 . Schematic
diagram represents the process of ethyl
cellulose microspheres formation 

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2 .Freez drying    Table (1): Different drug to polymer ratios for preparation of
microparticles using different dispersion mediums.

Concentration of ETOPOSIDE

Concentration of Methyl cellulose

Concentration of Ethyl cellulose


Stock solution (6ml ) contain (17

Stock solution (25 ml ) contain (18.2

Stock solution (30 ml) contain (73.3


412 micro litter

384 micro litter

96 micro litter

1:1 concentration

412 micro litter

1.2 mlL

286.5 micro litter

1:3 concentration

412 micro litter

1.2 mlL

573 micro litter

1:6 concentration

412 micro litter


1.25 ml


412 micro litter


2 ml

1:20 concentration

Characterization of the formed MPs; The
formed PoMPs were be characterized byits drug
content, encapsulation efficiency and drug-loading using UV spectroscopy.
Particle size was being determined using particle size analyzer.Drug
released kinetics from PoMPs was being determined as follows; PoMPs are placed
into dialysis membrane with a molecular weight cut-off of 8000–15000. The
dialysis membranes are in 80mL release medium (phosphate-buffer, PBS; pH 6.8)
or (diluted hydrochloric acid, HCl; pH 1.0) respectively, contained in
Erlenmeyer ?ask (200mL). The Erlenmeyer ?ask is closed with aplastic membrane and stirred continuously at
160r/min in a vibrant incubator at (37±0.5) °C. Triplicate samples are run. At
predetermined time intervals, samples of 4mL are taken out of the solution and
replaced by the equal volume of the same release medium to maintain a constant
volume (11,
12). Samples are analyzed using HPLC and data are to be
presented graphically utilizing standard calibration curves constructed from
standards prepared from each antimicrobial agent.1.3
Shape and surface The morphology of
the optimized formulation wasbeing
investigated using an optical microscope
with Carl Zeiss camera connected to the
computer for saving the pictures (LEICA DMD 108, Located in Wetzlar and
Mannheim ( Germany)(13, 14).

Figure 3 . optical microscope 1.4
Determination of drug content of ethyl cellulose microparticles To determine the entrapping efficiency
of Etoposide in ethylcellulose microparticles, an amount equivalent to 20 mg of
Etoposide entrapped-microparticles was dissolved in 1 ml CHCl 3/Methanol. After
complete dissolution, 4 ml of Sorensen phosphate buffer pH 7.4 was added to the
CHCl 3 solution and the mixture was stirred on a magnetic stirrer at room
temperature until the odor of CHCl3/Methanol disappears. The solution
was then filtered and diluted to 5 ml in avolumetric
flask with phosphate buffer. 1 mL of this solution was diluted with
phosphate buffer to 25 ml in avolumetric flask.
The solution was then analyzed for its FLB content spectrophotometrically at 247 µm against a blank solution prepared
in the same manner using plain microparticles.1.5 In-vitro release study of
ethyl cellulose microspheresAn accurately weighed quantity of ethyl cellulose
microspheres equivalent to
….. of … was placed in the 2o mL beaker, rotating at 50 r.p.m. One ml
samples were withdrawn at predetermined intervals, the removed sample was
replaced with one ml of phosphate buffer. Each sample was diluted with anappropriate volume of phosphate buffer so that
absorbance values lie between 0.1 and 0.9 when measured spectrophotometrically at 247 nm.                                                           8- Result 
emulsions formed using5% methylcellulose as the emulsifier. Stable emulsions
and microspheres were prepared as methylcellulose
was dissolved in the aqueous phase. Methylcellulose in a concentration as low
as 5% was used in order to avoid anexcessive
increase in the aqueous phase viscosity with higher concentration. The excessively high viscosity of the aqueous
phase could have adeleterious effect on the
morphology of microspheres produced (14).Figure 4, shows the optical microscopewith CarlZeiss camera of ethyl cellulose 200 microspheres
loaded with Etoposide prepared at 1: 1 D: P ratio. The microspheres were spherical
in shape, nonsticky and free-flowing. The microspheres have a particle
size of about 30- 80µm.

 Figure 4 .Optical micrographs of ethyl cellulose microspheres
loaded with Etoposide prepared at 1: 1 D: P. The drug content of ethylcellulose microspheres showed the
Etoposide was with (……..)
it can also be found that the precipitated on the surface of
microspheres. The release profile from ethylcellulose microspheres
prepared using the 1:1 drug: polymer ratio demonstrated the lowest efficiency
in retarding release, where 100 % of the drug content of the microspheres was
released after 30 min (Figs5). Also, the
release ofmicroparticles in 0.1 N HCl
gives synergistic effect with apolymer to
retard the drug release by maintaining anacidic
environment in polymeric microspheres.

Figure 5 .  The time
for Release of  Etoposide from
Ethylcellulose .  Physicochemical characterization ofPoMPs :PoMPs were
prepared by asingle emulsion-solvent evaporation
technique. The rounded shape of PoMPs was obtain by optical
microscope  ( fig 4  ). All formulation show one layer under
microscope except 1:20 . My
sample ( 1:1) is high concentration more than (1:3, 1:6 and 1:20) because different
concentration of emulsifying agent (fig 6 ).

Figure 6 .
The drug content in different concentration9-Discussion The emulsifying agents are used to deliver
chemotherapeutic agents. In the current study, we used methylcellulose as an emulsifying agent to form an emulsion. In this section, we compare current study with another study (Toxicogenomics of nanoparticulate delivery of
etoposide: potential impact on nanotechnology in retinoblastoma therapy) that
used polyvinyl alcohol as emulsifier agent. polyvinyl alcohol and mehyl  cellulose are equal in the efficacy of microencapsulation
of Etoposide (15).The initial release of Etoposide
rapid release and deposited on the surface along with formation of water
channel in microparticle. The degradation of EC is slow , at a later stage ,
the release of Etoposide from micrparticle depends on the drug diffusion and
degradation (16). The sample (1:1) have higher drug contet more
than other sample  but slowly release
from ethylcellulose when comparison to other sample . Fig 5 shows the release
of (1:3 and 1:6) are faster than (1:1) .  I planned used tween80 as an emulsifying agent because of it more effective to form emulsion (17).Instead, we used methylcellulose as an emulsifying
agent and form an emulsion.                We want using scanning
electron microscope and transmission electron microscope but it was not
available at Qassim university instead
was used anoptical microscope.The time not enough to complete this study so we did not study on toxicity and Cellular uptake. we expect in future the student will
complete all part of the study.                              10.ConclusionThe
research outcomes: are expected to be highly valuable in terms of both
academic research and society serving. The academic outcomes of the project
will be in the form of scientific publications in high impact peer-reviewed international journals. Final outcomes: will adhere to presenting a novel formulation for thetreatment of colon cancer that can deliver
safely to the patients in a high dose to the affected tumor cells. 



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