Journal of Pharmaceutical Research and Opinion 2: 2 (2012) 28 – 41.
Contents lists available at www.innovativejournal.in
JOURNAL OF PHARMACEUTICAL RESEARCH AND OPINION
Journal homepage:http://www.innovativejournal.in/index.php/jpro
RESEARCH
PREPARATION AND CHARACTERIZATION OF BENZOPHENONE-3 LOADED
POLYMERIC NANOPARTICLES OF LACTIDE-CO- Ɛ - CAPROLACTONE AS DRUG
CARRIERS
Amin M. M. ∗1, Sheta N. M.2, Abd El Gawad N. A. 1,2, Badawi A. A. 1
1Pharmaceutics
2Pharmaceutics
ARTICLE INFO
Received 10 Feb 2012
Accepted 25 Feb 2012
Corresponding Author:
Amin M.M*
Department of Pharmaceutics
and
Industrial
Pharmacy,
Faculty of Pharmacy, Cairo
University, Kasr El Aini Street,
11562, Cairo, Egypt.
[email protected]
KeyWords
Benzophenone-3,
Polymeric nanoparticles, SPF,
EUVA-PF, UV blocking activity
using E.coli.
Department, Faculty of Pharmacy, Cairo University. Cairo, Egypt
Department, Faculty of Pharmacy, October 6th University. Cairo, Egypt
ABSTRACT
Polymeric nanoparticles (PNPs) have been developed as carriers for
sunscreens being widely used for protecting the skin from adverse effects of UV
radiations. The aim of this study is to formulate PNPs using Lactide Co-ƐCaprolactone (PLCL) polymer containing Benzophenone-3 (Bp-3) as chemical
sunscreening agent aiming to decrease the possibility of transdermal
permeation, and adverse systemic side effects, as well as protecting it against
photodegradation to prevent toxic and allergic degradation products. In this
study, the effects of stabilizer type Tween 80 (T80) & poloxamer 188 (P188),
stabilizer concentration (1% and 5% w/v) in the external aqueous phase and
drug to polymer ratio (D/P) (1:1 and 2:1 w/w) were studied on entrapment
efficiency percent (EE%), and particle size. Incorporation of the prepared Bp-3
PNPs into emulgels (EGs) as vehicles with highly favorable cosmetic properties
were further evaluated regarding rheological properties, spreadability test, in
vitro SPF, and EUVA-PF, further evaluation involving in vitro release and in vitro
permeation tests were also carried out. Finally, an in vitro microbiological assay
was adopted to compare the survival percent of E.coli against UV exposure for
200 minutes which is the decimal reduction time (DRT) before and after
application of the selected formula. Results show that F6T80 has the highest
EE% of 98.80%±0.04. F6T80EG showing convenient spreadability with
pseudoplastic thixotropic flow, higher SPF and EUVA-PF values, with lower
amount of Bp-3 permeated through the skin. In addition the survival percent of
E.coli against UV exposure reached 31% ± 2.92 after the application of the
selected formula F6T80. This study illustrated the potential use of PNPs to
improve the UV blocking activity and SPF of Bp3 compared to traditional EGs
containing free unencapsulated drug.
©2011, JPRO, All Right Reserved.
INTRODUCTION
Sun exhibition can be beneficial (Vitamin D synthesis) [1, 2], but it can have serious consequences on skin according
to the proportion and exhibition length. Thus, it is very important to use sunscreen agents in order to protect the skin from
the harmful effects of sun exposure [3].
It is well recognized that the UV portion (290-400 nm) of the sun light spectrum reaching the earth surface is
responsible for skin photodamage [4]. Adverse reactions to the sun’s UV rays include short-term inflammatory responses
(i.e. erythema, oedema) and long-term effects such as cutaneous photoageing, immunosuppression and skin cancers which
are increasing throughout the world [4-8].
Sunscreen preparations are usually applied superficially to large skin areas. Therefore, effectiveness implies that
sunscreen filters adhere to skin like a protective film. They should have a high affinity for the stratum corneum. The UV
filters are designed to remain on the uppermost layers of the skin; penetration through the skin is low [9]. For sunscreens
to be effective, the UV absorbers must remain in the outermost regions of the skin [10]. An ideal sunscreen product should
exhibit high skin accumulation of UV absorbers with minimal permeation to the systemic circulation [11, 12].
Colloidal drug carriers, including submicron emulsions, nanospheres, nanocapsules, liposomes, and lipid
complexes, have been attracting increasing interest as vehicles for the topical administration of lipophilic drugs [13, 14].
29
Amin et. Al/ Preparation And Characterization Of Benzophenone-3 Loaded Polymeric Nanoparticles Of LactideCo- Ɛ - Caprolactone As Drug Carriers
Concretely, the nanocapsules (NC) are introduced as the new generation of carriers for cosmetics, especially for UV
blockers for the use on human skin and/or hair.
Sunscreen preparations contain organic chemicals which lessen the amount of UV light reaching human skin by
absorbing the radiation. The photoactivated sunscreen molecule disposes of the excitation energy in several ways: in the
form of heat, by fluorescence, phosphorescence, interaction with neighbouring molecules or by undergoing photoinduced
decompositions [15, 16]. The latter reactions not only decrease the sunscreen UV-protective capacity during usage [1, 8, 16] but
can also produce allergic or toxic degradation products [17, 18]. Therefore a high photostability is a prerequisite for the
effectiveness of sunscreen products.
Benzophenone-3 (Bp-3) is a widely used lipophilic, broad spectrum molecular sunscreen agent, which effectively
absorbs ultraviolet B (UVB) (290–320 nm), some ultraviolet A (UVA; 320–360 nm), and some ultraviolet C (250–290) [19].
However, Bp-3 is the most common cause of photoallergic contact dermatitis [20]. In addition, systemic absorption of Bp-3
following its topical application to the skin has been reported [21]. Therefore, there is an urgent need for the development
of safer sunscreen systems. This can be achieved by formulations that penetrate less into the skin or by formulations with
a reduced amount of potentially dangerous molecular UV blocker while maintaining the sun protection factor by other
means, e.g., carriers with sunblocking characteristics [22].
The effectiveness of a chemical sunscreening agent can be related to its ability to reduce or prevent the passage of
the UV light to the biological indicator (BI) in order to reduce or prevent the biological indicator death, and can be
expressed in terms of total viable count (TVC) of colonies after the exposure to the UV light for a predetermined time. In
vitro microbiological assay was used to determine the UV blocking ability by the help of Escherichia coli (E.coli), a type of
bacteria, which will act as a model to substitute human skin [23]. Depending on the fact that upon exposing the bacterial
DNA to UV light a thymine dimer is produced. These dimers cause a distortion of the DNA molecule, and therefore cause
malfunctions in the replication of the cell, potentially leading to cell death in unicellular organisms [24-27]
The aim of this study is to formulate polymeric nanoparticles (PNPs) as carriers for sunscreens as a mean of
lowering the risk of developing skin cancer. Poly Lactide Co-ε-Caprolactone (PLCL) polymer was used to prepare Bp-3
PNPs adopting solvent evaporation technique, aiming to enhance the sunscreening efficacy and reduce skin and systemic
adverse effects of Bp-3 by decreasing its contact with the skin, as well as protecting it against photodegradation.
Introducing Bp-3 PNPs into emulgel (EG) formulae giving rise to a greaseless transparent product with high SPF, low skin
permeability, high in vitro UV blocking activity using E.coli, and good patient compliance.
MATERIALS AND METHODS
Materials
Benzophenone-3 (Bp-3), (ISP)(USA); Tween 80 (T80) (Merck-schuchardt, Germany); Potassium dihydrogen
phosphate and Disodium hydrogen phosphate, (Merck-schuchardt, Germany), Sodium chloride, (Merck-schuchardt,
Germany); deionized double distilled water; chloroform, methanol and methylene chloride analar (India Mumbai);
Transpore® surgical tape (3M Australia Pty Ltd.,Australia); Poly(DL-lactide-co-ε-caprolactone (PLCL) (MW 110.000 Da,
copolymer ratio 75/50) was purchased from Birmingham polymers (USA); Poloxamer 188 (P188) (Pluronic ® F-68, BASF,
Ludwigshafen,Germany); Synthetic cellulose nitrate membrane 0.45µm Tuffyrn membrane filter (Sartorius stedium,
Germany); Sodium nitroprusside & Phosphotungestic acid (PTA) (Adwic, El-Nasr pharmaceutical chemicals Co., AbuZaabal, Cairo, Egypt); Carbomer 940 (BF Good rich company, Cleveland, Ohio, USA); Triethanolamine (TEA) (Nouresh’
shark Company, 10th of Ramadan city, Egypt); newly born hairless rat skin (one week age) were brought from the animal
house of applied research center for medicinal plant ARCMP, Egypt; Dialysis bag (molecular weight cut off 12–20 KDa) was
obtained from Sartorius, Germany. All other chemicals and solvents were of analytical grade.
Study Design plan
The Study design plan involves the study of the effects of different variables namely; stabilizer type (T80 or P188),
stabilizer concentration (1% and 5% w/v) in the external aqueous phase and drug to polymer (D/P) ratio (1:1 & 2:1) w/w
on entrapment efficiency, and particle size of the different prepared Bp-3 PNPs (Table 1).
Table 2: Composition of the different prepared Bp-3 PNPs according
to the different used variables.
Drug:Polymer
Formula
Surfactant Surfactant
concentration
ratio
code
type
(%w/v)
(w/w)
P188
T80
1%
5%
1:1
2:1
F1P188
+
+
+
F2P188
+
+
+
F3P188
+
+
+
F4T80
+
+
+
F5T80
+
+
+
F6T80
+
+
+
Table 1: Variables investigated in the preparation of Bp-3 PNPs
Variables
Level
Stabilizer type
Pluronic (P188)
Tween (T80)
PVA concentration
1% w/v
5% w/v
Drug/polymer ratio
1:2 w/w
2:1 w/w
Preparation of Bp-3 PNPs
Table (2) gives the composition of the different prepared Bp-3 PNPs according to the above variables. The solvent
evaporation method was described by Niwa et al. [28] and has since been widely used to prepare PNPs [28- 31].
Bp-3 loaded PLCL PNPs were prepared by the solvent evaporation procedure as follows: Five hundred milligrams of PLCL
were dissolved in 10 mL of methylene chloride at room temprature. Then a specified quantity of Bp-3 was added to the
methylene chloride solution. The resulting organic solution was injected at once into 50 ml of distilled water containing
either 1% or 5% w/v non-ionic surfactant—P188 or T80 —used as stabilizing agent while homogenized (Homogenizer; ltra
Turrax, IKA), at 7000 rpm for 2 minutes, the aqueous phase immediately turns milky with bluish opalescence as a result of
Amin et. Al/ Preparation And Characterization Of Benzophenone-3 Loaded Polymeric Nanoparticles Of LactideCo- Ɛ - Caprolactone As Drug Carriers
the formation of nanoparticles suspension. The methylene chloride, was then removed by stirring on a magnetic stirrer
(MS-300HS, Korea) until the disappearance of the organic solvent odour (5-6 hrs). Finally, the nanoparticles suspension
was concentrated by the removal of water to specified volume (20 mL) under reduced pressure at 50–60◦ C (Rotavapor®
Heidolph VV2000, Germany) [28, 32, 33].
Evaluation of the different prepared Bp-3 PNP suspensions
Determination of drug encapsulation efficiency (E.E%)
Encapsulation efficiency (EE%) was determined indirectly from calculating the non-encapsulated drug which
remained in the PNPs suspension medium according to the following equation: [34, 35]
1 mL of the Bp-3 PNP suspension was vortexed (VM-300, Gemmy industrial corp, Taiwan) for 2 min, then this
suspension was subjected to centrifugation (Centrifuge, (PLC-012, Gemmy industrial corp, Taiwan) to separate the Bp-3
PNPs from the supernatant under cooling at 0oC and 8000 rpm for 30 minutes. Both residue and supernatant were then
stored for further investigations. The residue was washed with 1mL (1% Tween PBS solution) then, vortex for 2 min, and
recentrifuge for 30 min at the same rpm and same temperature as before, this washing was repeated two times to separate
the free non entrapped Bp-3 from the PNPs. Aliquot from the supernatant of each washing was collected.
Free Bp-3 was measured in the clear supernatant following separation of nanoparticales and washing for two
successive times. Thereafter, the concentration of the free Bp-3 in the collected aliquot was analysed
spectrophotometrically at the predetermined λmax. All experiments were run in triplicates and the results were expressed
as the mean average values ± SD. The supernatant was used to detect the free drug, then drug EE% was calculated.
Bp-3 content in the PNPs or encapsulation efficiency (EE%) was calculated by the difference between the total and free
estimated drug concentrations.
Particle size analysis
Particle size analysis was performed by Malvern Zetasizer (Malvern zetasizer version 6.20 serial number: MAL
104 4595). Exactly 0.1 ml of BP-3 PNPs samples were diluted with 10 ml of double distilled water and shaking to weak
opalescence prior to particle size analysis. The span or polydispersity index value gives an indication of the polydispersity
and the homogenity of the preparations [36].
Surface charge measurement (zeta potential - ζ)
The ζ-potential or Surface charge measurement of Bp-3 PNPs was determined using a Malvern Zetasizer. The
instrument is a laser-based multiple angle particle electrophoresis analyzer., the instrument measures the electrophoretic
mobility (or zeta potential), Bp-3 PNPs were diluted with double distilled water and placed in the electrophoretic cell
where an electric field of 80 mV was established. Measurements were carried out in triplicate at 120º angle and 25oC [33, 37].
Differential Scanning Calorimetry (DSC)
DSC study was done to investigate the crystalline status of Bp-3 within the prepared PNPs. 2-4 mg of samples of
Bp-3, PLCL and Bp-3 PNPs were sealed in a 30 µl aluminum pans and heated in the DSC instrument under dynamic
nitrogen atmosphere with the flow rate of 50 mL/min. The temperature range of 30ºC to 200ºC was used and the heating
rate was 10ºC/min. The DSC thermograms were recorded [38, 39].
Determination of morphology (TEM)
The morphology of the selected Bp-3 PNP dispersions (F6T80) was observed using transmission electron
microscope (TEM) for the freshly prepared sample. One drop of diluted sample was deposited on a film-coated 200-mesh
copper grid, stained with one drop of 2% w/v aqueous solution of Phosphotungstic acid (PTA) and allowed to dry and any
excess fluid was removed with filter paper before examination [40].
Formulation of Bp-3 PNP emulgels (EGs)
The composition of the different prepared EGs was given in Table (3). All of the six prepared Bp-3 PNP dispersions
were formulated into (EGs). Hydrogel formulations containing the corresponding free Bp-3 suspensions were prepared for
comparison. It should point out that humectants (glycerin and Triehanolamine TEA) were used to improve the visual
appearance and consistency as the hygroscopic polyhydric alcohols provides moisture balance to the end product [41]. The
specified amount of Carbapol 940 was sprinkled over the specified amount of water and soaked overnight, after swelling,
glycerin and isopropyl alcohol (IPA) were added. Then, TEA was added with stirring for neutralization to form phase (A).
The emulsifier, Tween 80 was mixed with isopropyl myristate (IPM); the oily phase after that, Bp-3 PNPs or free Bp-3 was
added to form phase (B). The EG was obtained by mixing phase (A) with phase (B) with rapid stirring. The final weight
was adjusted to 100 g with purified water and the prepared EG was left overnight for equilibration.
Table (3): Composition of the different prepared Bp-3 PNP EGs
Ingredients
(% w/w)
Carbopol 940
1
*IPM
2.5
*IPA
10
Glycerin
5
Tween 80
5
*TEA
Q.S
Distilled water
100 g
* (qs)
*IPM indicates isopropyl myristate;*IPA indicates isopropyl alcohol
*TEA indicates triethanolamine;*qs, quantity sufficient.
Amin et. Al/ Preparation And Characterization Of Benzophenone-3 Loaded Polymeric Nanoparticles Of LactideCo- Ɛ - Caprolactone As Drug Carriers
Evaluation tests of the different prepared Bp-3 PNP EGs
Test for spreadability
Spreadability test was carried out by pressing 0.5 g of the prepared formulae between two slides of glass and left
for about 5 min where no more spreading was expected by the help of known weight & constant pressure. The diameter of
the formed circle was measured and taken as comparative values for spreadability [42]. Needless, to say that the larger the
diameter, the better the spreadability [43].
pH measurements
The pH of 10% w/w aqueous solution was measured by pH meter (Hanna-213, Portugal). The solutions were
prepared by dissolving 1 g of each of the prepared EG in 9 g of double distilled water using magnetic stirrer [44, 45].
Rheological properties measurements
Viscosity and rheology studies are necessary for the evaluation of the different prepared Bp-3 EGs to characterize
the systems physically and to investigate their stability. Rheology of the EGs was determined using Brookfield digital
viscometer (RV-TD), Spindle (LV4). About 25 g of the gel was packed in a suitable container and the rpm was changed
from 0.5 to 100 up and down under ambient conditions, with 30 seconds between each two successive speeds, and the
rheological behavior of each EG was evaluated by plotting the shear stress versus the shear rate values obtained. The flow
behavior was studied according to the following equation: [46]
Where; D is the shear rate in sec.-1; S is the shear stress in dyne/cm2; η is the viscosity in cp; and N (Farrow’s
number) which is the slope of log S against log D plot, indicating the deviation from Newtonian flow.
If N is less than 1, dilatant flow is indicated, and if greater than 1, pseudoplastic flow is assured. When the system showed
thixotropic behavior, the hysteresis area was measured in (cm2) .The area of hysteresis loop between upward and
downward curve as well as Farrow's constant was calculated. The viscosity value was measured at minimum (ηmin) and
maximum (ηmax) rates of shear [46].
In vitro sunscreen efficacy testing:
Sodium nitroprusside solution method was used for in vitro sunscreen efficacy testing using UV lamp. Sodium
nitroprusside in aqueous solution is colourless photosensitive substance [47] where, the UV light causes its degradation and
yield Prussian blue colour and nitric oxide (NO).
A 0.05% w/w solution of sodium nitroprusside in distilled water was prepared and 40 mL of this solution was
placed in three petriplates [22]. Two petriplates were covered with cellophane membrane. One petriplate containing
sodium nitroprusside solution was not covered with cellophane membrane to expose it directly to UV lamp, the second
one was left at the dark for comparison, while, on the third, a known amount (2 g) of the preparation was spread uniformly
over the cellophane membrane as a layer, then exposed to UV lamp for 2 hrs.
After exposure to UV lamp, the samples were analyzed using the spectrophotometric measurements to determine the
stability of sodium nitroprusside with most emphasis on increase in the absorbance at 390–395 nm with degradation [22].
In-vitro measurement of SPF and EUVA-PF of Bp-3 PNP EGs
Determination of the SPF of the formulations was done in accordance with the method described by Diffey and
Robson [48]. Transpore Tape® was used for sample application which was hydrated by placing it on the shelf of a closed,
controlled-humidity chamber (containing 85% water, 15% glycerin in its bottom) for 16-24 h prior to use [49] and then
mounted on a quartz cuvette. The principle of this method is the measurement of the spectral transmission of UVR through
a sample placed on a tape with or without the sunscreen to be evaluated. By using this method, the applied radiation
provides a continuous spectral power distribution in a range between 290 and 400 and measure the transmittance every 5
nm increment using the following equation for calculation of total in vitro SPF [50-52]:
Where Eλ was the spectral irradiance of terrestrial sunlight under defined conditions, Bλ was erythemal
effectiveness, and MPFλ was the monochromatic protection factor at each wavelength increment measured as the ratio of
the detector signal intensity without sunscreen applied to the substrate to that which sunscreen applied to the substrate
[53].
A known amount of 2 mg/cm2 of the final product was used for 4 cm2 quartz cuvette surface area, the slide is
placed on an analytical balance, the samples are distributed on the sample plate by dotting the sunscreen on the slide and
noting the weight, the sunscreen-coated slide is removed from the balance and the sunscreen is distributed over the entire
surface slowly. After a drying period of 15 min, transmission measurements were done. These experiments were
performed in triplicate.
Testing of samples is performed by running a baseline on the reference media, then successively running each of
the prepared samples. Typically, three scans per spot are performed with three spots being evaluated on each sample,
Amin et. Al/ Preparation And Characterization Of Benzophenone-3 Loaded Polymeric Nanoparticles Of LactideCo- Ɛ - Caprolactone As Drug Carriers
results obtained were prossesed and SPF was obtained [48, 51]. All formulae were compared to the corresponding free Bp-3
conventional EG.
Considering the UVA wavelength range (320-400 nm) and using the terms of the above SPF equation (3), the in
vitro erythemal UVA protection factor could be calculated from the same equation but instead of substitution from 290400 nm for SPF just use wavelength from 320-400 nm to get UVA-PF [48] as follows:
In vitro release of Bp-3 PNP EGs
In vitro release of the prepared Bp-3 PNP EG formulae using cellulose nitrate membrane was carried out as
follows: 0.7g of the EG was introduced into 300 mL of receptor medium containing 1% w/v Tween 80 PBS (pH =7.4) [22].
The temperature was kept at 37 ± 1ºC and stirred at 100 rpm. 5 mL samples at different time intervals (1, 2, 3, 4, 5, 6 & 24
hrs) were withdrawn and assayed for drug content against calibration in the same medium at the predetermined λmax, all
experiments were run in triplicates and the results were expressed as the mean average values ± SD. The comparison of
the in vitro release was done at Q2hrs. For comparison the corresponding concentration of free Bp-3 EGs were prepared and
subjected for in vitro release under the same conditions.
In-vitro drug permeation studies through excised newly born rat skin
Permeation of Bp-3 from all the prepared Bp-3 PNP EG formulae were done through the newly born rat skin as
follows: 0.7 g of Bp-3 PNP EG formulae were spread over the surface of a glass of 8.03 cm2 surface area and then covered
with the newly born rat skin where the stratum corneum side was facing downward into the Bp-3 PNP EG and the dermal
side was facing upward into the receiver medium. The glass and the excised rat skin were held together by blaster and
equally spaced plastic clips. This assembly was placed at the bottom of the dissolution vessel containing 300 ml of 1%
(w/v) Tween 80 PBS (pH = 7.4).
The temperature was kept at 37 ± 1ºC and stirred at 100 rpm. Five milliliters samples at different time intervals
(1, 2, 3, 4, 5, 6 & 8 hrs) were withdrawn and from the center of the dissolution vessel and replaced immediately with equal
volume of fresh medium then, assayed for drug content against calibration in the same medium at the predetermined λmax.
All experiments were run in triplicates and the results were expressed as the mean average values ± SD. Comparison of in
vitro permeation was done at Q8hrs and compared to the corresponding free Bp-3 EG under the same conditions.
The amount of Bp-3 permeated through the skin per unit surface area 8.03 cm2 (µg/cm2) was plotted as a function of time.
The drug flux (permeation rate) at steady state (JSS) was calculated from the slope of the straight line. Permeability
coefficient (KP), correlation coefficient (r2), t50% (time to 50% drug permeated) and the amount of drug permeated after 8
hours (Q8hrs) were also determined for each formula.
All skin permeation experiments were repeated three times and data were expressed as the average mean value
±S.D. Statistical data were analyzed by one way analysis of variance (ANOVA) for a multiple comparison, a P value of <0.05
was considered significant. Unpaired student t-test for the comparison between two formulae, and compared to the
corresponding free Bp-3 EG having the same concentration.
In vitro Microbiological assay (UV blocking activity) of the selected F6T80 EG formula
Preparation of Solutions and Media:
1 mL of E. coli bacteria was transferred by pipette from the standardized bacterial culture into sterile petri dishes.
A Petri dish usually requires 15-20 mL of the sterilized agar solution which was aseptically poured into the dishes as soon
as it cools to 60ºC. Complete mixing of the bacterial dilution and the agar can be done by rotating the plate sufficiently to
disperse the standardized bacterial culture, and then the nutrient agar was allowed to solidify.
Screening of the optimum number of colonies per plate:
Commercially bought E.coli was diluted using phosphate buffer saline (PBS). Four different serial dilutions of
E.coli were made: 10-1, 10-2, 10-3 & 10-4 to select the suitable number of colonies that could be cultured per plate, after
overnight (24hrs) incubation (Titanox oven, Italy) at (37±1ºC) in the incubator without the exposure to the UV light. The
bacterial counts were carried out using standard pour plate method [26].
Screening of the Decimal Reduction Time "DRT" or (D- value):
From the previous screening tests, the dilution which will show white, uniform, clearly and freely distributed,
easily counted, not over lapped colonies will be selected for the “Decimal Reduction Time” (DRT) determination and will
be fixed for further investigations.
The plates were made as previously mentioned with the predetermined dilution. The prepared plates were then
exposed to UV lamp with a wave length of 365 nm. In order to sterilize the radiation area and allow the lamp to reach a
stable state, we suggest that the lamp operate for at least 30 minutes before irradiating the petri dishes in the lab. The
optimal distance between the UV lamp and the dish is 30-35cm.
Amin et. Al/ Preparation And Characterization Of Benzophenone-3 Loaded Polymeric Nanoparticles Of LactideCo- Ɛ - Caprolactone As Drug Carriers
Three control petri plates were kept in the dark (Cdark) while, the other plates were irradiated at different time intervals 0,
3, 5, 10, 20, 40, 60, 80, 100 and 120 minutes (three plates per UV light exposure duration), then all the plates were
incubated upside-down for (24hrs) at 37±1ºC inside the incubator.
The survival of bacterial cells and its ability to form colonies on a solid medium following irradiation assume that
every colony is founded by a single cell and that the cell must have been alive in order to grow and form a colony.
Calculating the number of colonies per plate was expressed in the form of colony forming units (CFU) and Log survivors is
plotted versus exposure time.
The resistance value (or death kinetics) of a bioburden (BI) was characterized in terms of decimal reduction time
(DRT) or D-value, which is the exposure time required, under specified set of conditions, to cause one log10 or 90%
reduction of the initial population (No, bioburden) of viable BI in the suspension [54] .
The Decimal Reduction Time (DRT) is used as the microbiological parameter for sunscreen efficacy and was estimated
from the corresponding regression equation graphs on which the logarithms of the survivors were plotted against time.
The D-value at 37±1°C was determined from the negative reciprocal of the slopes of the regression lines, using the linear
portions of the survivor curves (log10 CFU/mL versus time of exposure to the UV radiation at constant temperature [54]. The
D-value was determined from the inactivation kinetic curve given by the equation:
Where; No= bioburden; Nf = survival population; D-value = decimal reduction time; (-1/D) = slope and t = total
exposure time (min).
The D-value was used as the optimum time required for the microbiological assay to determine the UV blocking
ability of the selected sunscreen formula. Each petri plate was placed on the electrical balance and an aliquot of 2 mg/cm2
of the selected formula (F6 T80) PNP EG was distributed on it by dotting the sunscreen formula EG on the petri plates
(Experimental) lid surface. The sunscreen-coated petri plates were removed from the balance and the sunscreen was
distributed over the entire surface by slowly and deliberately rubbing the surface of the slide with a single finger-cotcoated finger [55]. The sample should then be put aside to dry before exposing them to UV light. After exposure the
experimental dishes and the control petri plates which were kept in the dark (Cdark) were placed in an incubator (37±1°C)
in an inverted position for 24 hrs.
The percent survival of the bacteria was then calculated by counting the number of colonies of E.coli that survived
after exposure to the UV light divided by total number present multiplied by 100.
All experiments were run in triplicates and the results were expressed as the average mean values ± (S.D),
statistical analysis was carried on the percent survival of the bacteria between the control and the selected formula (F6T80)
using unpaired student t-test. Statistical analysis was performed using Statview version 4.53 computer program.
RESULTS AND DISCUSSION
Evaluation of Bp-3 PNP nanosuspensions
Determination of Bp-3 PNPs (EE%)
Encapsulation efficiency percent (EE %) of the different prepared Bp-3 PNPs was determined indirectly from
calculating the non-encapsulated drug which remained free in the PNPs suspension medium as shown in Table (4).
Table 4: Entrapment efficiency (EE %) and particle size of the different prepared Bp-3 PNPs.
Entrapment Mean
Formula
efficiency
particle size
PDI
code
(EE%)
(nm)
(±S.D)
(± S.D)
(± S.D)
F1P188
96.33±0.32
325.90 ±3.11
0.35±0.00
F2P188
93.82±0.38
503.90±19.94 0.29±0.03
F3P188
96.82±0.10
331.40±28.85 0.46±0.00
F4T80
90.80±0.04
289.70±26.59 0.49±0.00
F5T80
79.64±0.38
356.20±11.74 0.42±0.06
F6T80
98.80±0.04
441.00±86.27 0.61±0.02
Data are presented as mean average ±SD, n=3.
It is obvious that a high EE% was obtained for all the prepared Bp-3 PNPs; similar finding was reported in case of
lipophilic compounds where, a high encapsulation efficiency (>70%) was observed [56, 57]. For compounds such as Bp-3, the
entrapment efficiency percent has been reported by Wissing and Müller and Almaida et al. [58, 59] who were working on the
same drug and found that the EE % was in the range of 99–100%. A successful nanoparticle system is the one which have
high encapsulation efficiency [30, 60].
Similar findings were obtained by Almeida et al (2009) [59] who was working on Bp-3 as a model drug for the
preparation of NPs with PCL by spontaneous emulsification method and found that the encapsulation efficiency which was
determined indirectly was found to be 99.98± 0.01%.
4
Amin et. Al/ Preparation And Characterization Of Benzophenone-3 Loaded Polymeric Nanoparticles Of LactideCo- Ɛ - Caprolactone As Drug Carriers
Upon comparing the effect of stabilizer type (T80 & P188) on EE%; a significant higher difference was observed in
case of P188 at D/P (1:1) while, in case of D/P (2:1), the EE % of T80 was significantly higher than that of P188 suggesting that
changing both stabilizer type and D/P ratio will give a different effect on the EE % of the prepared Bp-3 PNPs (Table 4).
Regarding the effect of stabilizer concentration (1 & 5%) w/v in the external aqueous phase on EE%, it is clear 1%
stabilizer shows a higher EE than 5% stabilizer (p<0.001) for each pair respectively. This could be explained on the basis
that upon increasing the amount of stabilizer with a constant amount of Bp-3 and PLCL, the surface of the PNPs formed
was too small to adsorb all of the surfactant molecules. This might result in the formation of micellar solutions of the drug
(Bp-3). Hence the solubility of the drug in the water phase would increase. Therefore, the drug could partition from the
PNPs into the formed micelles in the water phase, thereby reducing the final entrapment efficiency [61]. In other words, if
the concentration of stabilizer is too low, aggregation of the nanoparticles will take place, whereas, if too much stabilizer is
used, drug incorporation could be reduced as a result of the interaction between the drug and stabilizer [62].
Upon comparing the effect of (D/P) ratio (1:1 & 2:1) on EE%; a significant difference between each pair was observed
(p<0.001) and that the (EE %) of 2:1 (D/P) was significantly higher than that of 1:1 (D/P). Similar findings were obtained
by Alvarez-Romàn et al. [63] who prepared NPs by solvent displacement procedure with 310 mg Octyl methoxy cinnamate
(OMC) and 125 mg PCL using Tween 85 as stabilizer; and the E.E. % was found to be 99±1 %.
From the above results, it was found that the stabilizer concentration 1% w/v and (D/P) ratio (2:1) w/w were
considered as the optimum concentrations giving the highest EE%.
Particle size analysis
The mean particle size and span (which is the measure of polydispersity index "PDI") of the different prepared
PNPs are depicted in Table (4). Results reveal that the range of mean particle size of PNPs is from 289.70±26.59 to
503.90±19.94 nm for all the prepared PNPs [64]. The value of polydispersity index (PdI) is in the range of 0-1.
A characteristic parameter for the extent of particle size distribution which is the span value, ranged from 0.29±0.03 to
0.61±0.02, these values contributed to a relatively broad size distribution, this means that the polydispersed particle
dispersion is suitable for topical cosmetic application [65].
Upon comparing the effect of stabilizer type (T80 and P188) on particle size analysis (nm), a non significant difference
between each pair was observed (p>0.05), as shown in Table (4) which means that the particle size doesn’t depend on the
stabilizer type and both T80 & P188 have the same outcomes.
Regarding the effect of stabilizer concentration (1 & 5 % w/w) on particle size (nm) of the prepared Bp-3 PNPs;
the particle size of 1% stabilizer showed a significantly smaller particle size than 5% stabilizer (p<0.001). This could be
explained that when the surfactant concentration was increased, the viscosity of the diffusion phase also increased leading
to lowering in the organic solvent evaporation and resulted in larger particles [66]. Concerning the effect of (D/P) ratio
(1:1&2:1) on Particle size (nm) of the different prepared Bp-3 PNPs, a non significant difference between the two drugs:
polymer ratio (1:1&2:1) w/w of each pair (p>0.05) is observed. Similar findings were obtained by Almaida et al. [59], who
were working on the formulation of Bp-3-loaded microspheres using carnauba wax, stearic acid and cetyl alcohol as
carrier and polysorbate as stabilizer and found that upon increasing the concentration of Bp-3 from 375mg to 750mg, a
non significant difference in the mean particle size was observed.
Surface charge measurement (zeta potential - ζ)
Results revealed that all of the prepared PNPs had a considerable Zeta potential. The Zeta potential of PNPs
ranged from -27.10±0.56 mV to -41.50±0.99 mV. Values of Zeta potential (ζ) of the different prepared Bp-3 PNPs are
illustrated in Figure (1).
Figure 1: zeta potential in (mV) of the different prepared Bp-3 PNPs.
The zeta potential of a nanoparticle is commonly used
to characterise the surface charge property of nanoparticles [30].
It reflects the electrical potential of particles and is influenced
by the composition of the particle; chemical nature of the
polymer, chemical nature of the drug, chemical nature of the
stabilizing agent, pH of the medium [33] and the nature of
medium in which it is dispersed.
Zeta potential is a key factor to evaluate the stability of
colloidal dispersion [55] as it allows predicting good colloidal
stability due to the high-energy barrier between particles.
Large negative or large positive zeta potential is required for
colloidal dispersion stability. Theoretically, zeta potential
stabilizes suspensions whether its value is positive or negative
[67, 68].
Differential Scanning Calorimetry (DSC)
DSC is used to study the melting and recrystallization behavior of crystalline material. The breakdown of the
crystal lattice by heating the sample yields information on polymorphism and crystal ordering, in other words DSC gives
an indication of the extent of orderliness within each system and gives indication for the physical state of Bp-3 loaded in
the nanoparticles [69, 70].
Figure (2) shows the DSC thermograms of pure Bp-3, Placebo PNPs, and F6T80 PNPs. The DSC thermogram of Bp-3
showed a sharp characteristic endothermic peak at 63ºC corresponding to the melting point of the drug (Figure 2). The
DSC thermogram of Placebo PNPs showed a characteristic endothermic peak at 57ºC corresponding to the melting point of
the carrier (PLCL).
Amin et. Al/ Preparation And Characterization Of Benzophenone-3 Loaded Polymeric Nanoparticles Of LactideCo- Ɛ - Caprolactone As Drug Carriers
The higher melting enthalpy value of Bp-3 at 63◦ C [71] as shown in Figure (2) suggests higher ordered lattice arrangement.
The transformation of a sharp to a broad DSC peak with the decrease in the melting point of the prepared F6T80 PNPs is
associated with numerous lattice defects and the formation of amorphous regions in which the drug is located [72, 73]. The
disappearance of the characteristic peak of Bp-3, might be ascribed to the conversion of the crystalline needle crystals of
Bp-3 to an amorphous or molecularly dispersed structure of the Bp-3 within the PNPs.
The high entrapment efficiency of Bp-3 in the PNPs (>97%) can suggest a less ordered arrangement and permit
incorporation of the drug molecules into PNPs. Less ordered crystalline structure also can indicate physical stability as
little effusion of drug molecules from particles during storage time [74].
Morphology (TEM) of the selected Bp-3 PNPs
The morphology of the freshly prepared (F6T80) Bp-3 PNPs is illustrated in Figure (3). Figure (3) reveals that all droplets
after dilution possessed nanometer-size with the same spherical uniform shape [62] and no rectangular Bp-3 crystals were
visible, which could be attributed to the high entrapment efficiency (>97%).
Figure 3: TEM Photograph of fresh F6T80 Bp-3 PNPs
Table 5: pH and spreadability of the different prepared Bp-3 PNP EGs.
Formula code
pH
Spreadability
(±S.D)
(±S.D)
F1P188EG
6.91±0.03
5.26±0.04
F2P188EG
7.02±0.02
5.63±0.12
F3P188EG
6.73±0.05
5.31±0.08
F4T80EG
7.19±0.05
4.96±0.16
F5T80EG
7.22±0.04
4.30±0.21
F6T80EG
7.00±0.01
5.05±0.08
Data are presented as mean average (±S.D), n=3.
Evaluation tests for the formulated Bp-3 PNP EGs
pH and spreadability measurements
Table (5) shows both pH and spreadability values of the different prepared Bp-3 PNP EGs. The pH of all the prepared
formulae are within the physiological accepted range for dermal and transdermal preparations (4-7 pH units) in order to
be non-irritant and safe for dermal application [45].
The spreadability values for all the prepared Bp-3 PNP EG formulae varied between (4.30±0.21 to 5.63±0.12 cm).
Spreadability of the prepared Bp-3 PNP EGs was measured in terms of the average diameter of the spread circle, as shown
in Table (5). Needless, to say that the larger the diameter, the better the spreadability [43].
Sunscreen Efficacy Testing
The sunscreen efficacy test between the different prepared Bp-3 PNP EG formulae is given in Figure (4). The high
sunscreen efficacy of F6T80EG compared to other formlae and to control sample exposed directly to light, may be attributed
to the high loading of sunscreening agent (98.80±0.04), thereby, absorbing higher amount of UV light and reducing the
amount of light passing to the sodium nitroprusside solution therefore protecting it from photodegradation. It was found
that upon increasing the concentration of Bp-3, an increased level of protection was reported by increasing the
concentration of the filter [75].
Figure 4: Sunscreen efficacy test of the different prepared Bp-3 PNP EGs
Rheological properties measurements
Bp-3 PNP EG formulae showed non-Newtonian,
pseudoplastic flow with thixotropy as the viscosity
decreased upon increasing shear rates. Illing and Unruh
[76] stated that dermal dosage forms should exihibt
comparatively high viscosity and plastic or thixotropic
flow behavior to enable sticking onto the skin for a
sufficient time (Figure 5).
In-vitro SPF and EUVA-PF of the different prepared Bp-3 PNP EGs
The SPF and EUVA-PF values of the investigated formulations compared to Placebo PNPs and free Bp-3 suspensions are
illustrated graphically in Figure (6). Results of SPF and EUVA-PF reveal that all the prepared EG formulations have better
sun protection factor compared to their corresponding dispersions where the SPF and EUVA-PF values of F6T80EG are:
12.43±1.11 and 11.83±1.18 respectively. The SPF of the prepared Bp-3 PNP EGs was at least three times higher than that
of Placebo PNPs and non-encapsulated Bp-3 suspensions respectively.
6
Amin et. Al/ Preparation And Characterization Of Benzophenone-3 Loaded Polymeric Nanoparticles Of LactideCo- Ɛ - Caprolactone As Drug Carriers
Figure 5: Rheograms of the different prepared Bp-3 EG formulae.
Figure 6: SPF and EUVA-PF values of the different prepared Bp-3 PNP EGs.
It is clear that at constant PLCL and constant surfactant concentration it was found that F6T80EG showed the
highest SPF (12.43± 1.11), this may be attributed to the high concentration of Bp-3 in this formula, an increased level of
protection was reported by increasing the concentration of the filter [75].
In vitro release of the different prepared Bp-3 PNP EGs
The in vitro release profiles of the nanoencapsulated Bp-3 from the different prepared EGs were investigated over 24 hrs
and are illustrated in Figure (7). The influence of the nanocarriers was compared to a similar EG formulation containing
non-encapsulated free Bp-3 suspension [77]. The concentration of Bp-3 was determined spectrophotometrically at the
predetermined time intervals. The in-vitro cumulative percent released outcomes were compared at (Q2hrs).
Figure 7: Cumulative release profiles of Bp-3 from the different prepared EG formulae.
Upon comparing the release of Bp-3 from the nanoencapsulated EG and the corresponding non-encapsulated Bp3 EG having the same concentration, it was found that the release of Bp-3 was greatly retained in the emulgel within the
nanocarriers at Q2hrs indicating that the diffusion of Bp-3 was highly reduced from the vehicle to the skin surface when it is
formulated into nanocarriers, this reduction in the drug diffusion through the vehicle might be attributed to the presence
of PLCL surrounding the drug [68]. (Figure 7)
7
Amin et. Al/ Preparation And Characterization Of Benzophenone-3 Loaded Polymeric Nanoparticles Of LactideCo- Ɛ - Caprolactone As Drug Carriers
In-vitro drug permeation studies through excised newly born rat skin
Results of in-vitro permeation profiles are illustrated graphically in Figure (8) and compared to the corresponding
free Bp-3 EG. The amount permeated per unit area (8.03 cm2) was plotted against time (hr).
Figure 8: Permeation profiles of Bp-3 from the different prepared EG formulae
The permeation of the non-encapsulated drug from EG formulae was significantly higher (p<0.0001) when
compared to the nanoencapsulated Bp-3 EG formulae which may be attributed to the presence of PLCL which surrounds
the drug and was reported to retard both release and permeation of drugs [68].
Ourique and co-workers [78] showed that tretinoin permeation from nanoencapsulation prepared by PCL and Tween as
stabilizer was significantly decreased when compared to samples containing non-encapsulated tretinoin. This change in
Bp-3 permeation may be due to the polymer matrix which surrounds Bp-3 and causes an increase in the diffusional path
length that the drug molecules have traversed [22]. Addition of Bp-3 PNPs into carbopol EGs will result in further decrease
due to the higher viscosity media. Similar finding was observed by Mandawgade and Patravale [79] who showed a
considerable less skin permeation after the application of tretinoin-loaded solid lipid nanoparticles-based gels compared
to a marketed formulation.
F6T80EG, shows significantly the lowest Bp-3 flux (JSS) =14.21 (µg/cm2/hr), the lowest Kp=0.304 (cm/hr), the lowest Bp-3 amount P
Figure 9: In-vitro permeation outcomes for the different EG formulae
Screening for the of Decimal Reduction
Time (DRT value)
The number of colonies survived at
the different exposure time intervals, reflects
the effect of UV light bactericidal activity
according to the duration of exposure. It was
observed that the number of colonies
decreased as the duration of UV exposure
increased. The initial number of bacteria (No)
exposed to the UV light at time zero was
around 428.00±14.14 CFU/mL (colony
forming units/mL) while at the end of the
experiment it was found to be around
67.50±3.53 CFU/mL.
Figure (10) shows the logarithms of the survivors plotted against time. The Decimal Reduction Time (DRT) was
determined from the negative reciprocal of the slopes of the regression lines, from graph at 37±1°C using the linear
portions of the survivor curves (log10 CFU/mL versus time of exposure to the UV radiation.
The Decimal Reduction Time was found to be 200 minutes, which means that the viable count will be reduced to
90% of its value after 200 minutes which means that 200 minutes were required to kill 90% of the initial bioburden. This
D-value was taken as the time at which the comparison between the tested sunscreening formula will take place.
Figure 10: Log survivor/time graph at different time intervals
The percent survival of the bacteria was calculated by counting
the number of colonies of E.coli that survived after exposure to
the UV light divided by total number present multiplied by 100.
The initial bioburden can be expected to be reduced to 10% of its
value after 200 minutes without the application of sunscreening
product, while upon applying the sunscreening product, this
percentage increases according to its UV light blocking activity &
the barrier protective properties of the sunscreening agent. The
higher the blocking activity, the higher the survival percent. In
Amin et. Al/ Preparation And Characterization Of Benzophenone-3 Loaded Polymeric Nanoparticles Of LactideCo- Ɛ - Caprolactone As Drug Carriers
case of F6T80 PNPs EG (SPF12) an increase in the survival percent up to 31±2.92 CFU/mL was observed (Figure 11).
Figure 11: Plate showing the survival percent between control plate and F6T80
CONCLUSION
In the present work, Bp-3 loaded PNPs were
successfully prepared adopting solvent
evaporation technique showing high EE% of
Bp-3 within the prepared PNPs. 1% w/v
Tween 80 and 2:1 D/P w/w ratio are
considered the optimum concentrations
during PNPs preparation. Nanoencapsulation
of Bp-3 within PLCL polymer succeeded to
enhance the sunscreen efficacy and decrease
the transdermal permeation of Bp-3 through
the skin, allowing the preparation of a safer
sunscreen product with less systemic side
effects than the traditional non-encapsulated
BP-3 one.
REFERENCES
1. Berset G, Gonzenbach H, Christ R, Martin R, Deflandre A, Mascotto RE, Jolley JDR, Lowell W, Pelzer R, Stiehm T.
Proposed protocol for determination of photostability. Part I: cosmetic UV filters. Int J Cosmet 1996; 18:167–177.
2. Vanquerp V, Rodriguez C, Coiffard C, Coiffard LJM. High-performance liquid chromatographic method for the
comparison of the photostability of five sunscreen agents. J Chromatogr A 1999;832:273–277.
3. Godwin DA, Kim NH, Felton LA. Influence of Transcutol® CG on the skin accumulation and transdermal permeation of
ultraviolet absorbers. Eur J Pharm Biopharm 2002;53:23–27.
4. National Institute of Health. National Institute of Health Consens Statement Online, Sunlight, Ultraviolet Radiation, and
the Skin. 1989;7:1 –29.
5. Ziegler A, Jonason AS, Leffell DJ, Simon JA, Sharma HW, Kimmelman J, Remington L, Jacks T, Brash DE. Sunburn in the
onset of skin cancer. Nature 1994;372:773/–776.
6. Urbach F. Ultraviolet radiation and skin cancer of human. J Photochem Photobiol B 1997;40:3 –/7.
7. Hochberg M, Enk CD. Partial protection against epidermal IL-10 transcription and Langerhans cell depletion by
sunscreens after exposure of human skin to UVB. Photochem Photobiol 1999;70:766/–772.
8. Tarras-Wahlberg N, Stenhagen G, Larko¨ O, Rose´n A, Wennberg AM, Wennerstro¨m O. Changes in ultraviolet
absorption of sunscreens after ultraviolet irradiation. J Invest Dermatol 1999;113:547/–553.
9. Jiang R, Roberts MS, Prankerd RJ, Benson HA. Percutaneous absorption of sunscreen agents from liquid paraffin: selfassociation of octyl salicylate and effects on skin flux. J Pharm Sci 1997;86:791–796.
10. Lu Z, Bei J, Wang S. A method for the preparation of polymeric nanocapsules without stabilizer. J Control Release
1999;61:107–112.
11. Gupta VK, Zatz JL, Rerek M. Percutaneous absorption of sunscreens through micro-yucatan pig skin in vitro. Pharm
Res 1999;16:1602–1607.
12. Potard G, Laugel C, Baillet A, Schaefer H, Marty JP. Quantitative HPLC analysis of sunscreens and caffeine during in
vitro percutaneous penetration studies. Int J Pharm1999;189:249–260.
13. Allémann E, Gurny R, Doelker E. Drug-loaded nanoparticles-preparation methods and drug targeting issues. Eur J
Pharm Biopharm 1993;39:173–191.
14. Santos-Magalhaes NS, Fessi H, Puisieux F, Benita S, Seiller M. An in vitro release kinetic examination and comparative
evaluation between submicron emulsion and polylactic acid nanocapsules of clofibride. J Microencapsul 1995;12:195–
205.
15. Broadbent JK, Martincigh BS, Raynor MW, Salter LF, Moulder R, Sjoberg P, Markides KE. Capillary supercritical fluid
chromatography combined with atmospheric pressure chemical ionisation mass spectrometry for the investigation of
product formation in the sunscreen absorber 2-ethylhexyl-p -methoxycinnamate. J Chromatogr A 1996;732:101/–110.
16. Stokes R, Diffey B. In vitro assessment of sunscreen photostability: the effect of radiation source, sunscreen application
thickness and substrate. Int J Cosmet Sci 1999;21:341–351.
17. Deflandre A, Lang G. Photostability assessment of sunscreens. Benzylidene camphor and dibenzoylmethane
derivatives. Int J Cosmet Sci 1988;10:53–/62.
18. Dromgoole SH, Maibach HI. Sunscreening agent intolerance: contact and photocontact sensitization and contact
urticaria. J Am Acad Dermatol 1990; 22:1068/–1078.
19. Srinivas CR. Photoprotection. Indian J Dermatol Venereol Leprol 2007; 73:73-79.
20. Wissing S, Müller R. The development of an improved carrier system for sunscreen formulations based on crystalline
lipid nanoparticles. Int J Pharm 2002; 242:373–375.
Amin et. Al/ Preparation And Characterization Of Benzophenone-3 Loaded Polymeric Nanoparticles Of LactideCo- Ɛ - Caprolactone As Drug Carriers
21. Wissing SA, Müller RH. A novel sunscreen system based on tocopherol acetate incorporated into solid lipid
nanoparticles. Int J Cosmet Sci 2001; 23:233-243.
22. Patel M, Jain SK, Yadav AK, Gogna D, Agrawal GP. Preparation and characterization of oxybenzone-loaded gelatin
microspheres for enhancement of sunscreening efficacy. Drug Deliv 2006;13:323–330.
23. Zion M, Guy D, Yarom R, Slesak M. UV radiation damage and bacterial DNA repair systems. JBE 2006; 41(1).
24. Munshi HA, Sasikumar N, Jamaluddin AT, Mohammed K. Evaluation of Ultra-Violet Radiation Disinfection on the
Bacterial growth in the SWRO Pilot Plant Al-Jubail, Seawater), Proceeding of the Fourth Gulf Water Conference,
Bahrain. Water Science and Technology Association, Manama, Bahrain, 13-18 February 1999;603-618.
25. Oguma K, Katayama H, Mitani H, Morita S, Hirata T, Ohgaki S. Determination of pyrimidine dimmers in Escherichia coli
and Cryptosporidium parvum during UV light inactivation, photoreactivation, and dark repair. Appl Env Microbiol
2001; 67:4630–4637.
26. Munshi HA, Saeed MO, Green TN, Al-Hamza AA, Farooque MA, Ismail ARA. Impact of UV irradiation on controlling
biofouling problems in NF-SWRO desalination process. International Desalination Association (IDA) World Congress
Conference held at Singapore in 2005.
27. Milosevic DMD, Solaja MM, Lj NTT, Stijepic MJ, Glusac JR. The survival of Escherichia coli upon exposure to irradiation
with non-coherent polychromatic polarized light. Veter Medicina 2011;56(10):520–527.
28. Niwa T, Takeuchi H, Hino T. Biodegradable submicron carriers for peptide drugs. Preparation of DL-lactide/glycolide
copolymer ( PLGA)nanospheres with narfelin acetate by novel emulsion-phase separation method in an oil system. Int
J Pharm 1995;121:45-54.
29. Song C, Labhasetwar V, Guzman L, Topol E, Levy RJ. Dexamethasone nanoparticles for intra-arterial localisation in
restenosis in rats. Proc Intern Symp Control Rel Bioact Mater 1995;(22).
30. Mohanraj VJ, Chen Y. Nanoparticles – A Review. Tropical J Pharm Res 2006; 5 (1):561-573.
31. Zhang H, Cui W, Bei J, Wang S. Preparation of poly(lactide-co-glycolide-co-caprolactone) nanoparticles and their
degradation behaviour in aqueous solution. Polymer Degradation and Stability 2006;91:1929-1936.
32. Hoa LTM, Chi NT, Triet NM, Nhan LNT, Chien DM. Preparation of drug nanoparticles by emulsion evaporation method.
Journal of Physics: 2009;Series (187).
33. Mora-Huertasa CE, Fessi H, Elaissari A. Polymer-based nanocapsules for drug delivery. Int J Pharm 2010; 385:113–
142.
34. Leroux J-Ch, Alle´mann E, Doelker E, Gurny R. New approach for the preparation of nanoparticles by an emulsification–
diffusion method. Eur J Pharm Biopharm 1995;41:14–18.
35. Sanyogitta Puri M.A., Ph.D. Thesis, Faculty of Pharmacy, University of Nottingham, 2007.
36. Yap SP, Yuen KH. Influence of lipolysis and droplet size on tocotrientol absorption from self emulsifing formulations.
Int J Pharm 2004; 281.
37. Cruz L, Soares LU, Costa TD, Mezzalira G, da Silveira NP, Guterres SS, Adriana RP. Diffusion and mathematical modeling
of release profiles from nanocarriers. Int J Pharm 2006;313:198–205.
38. Abdelkader H, Abdalla OY, Salem H. Formulation of controlled-release baclofen matrix tablets: Influence of some
hydrophilic polymers on the release rate and in-vitro evaluation. AAPS Pharm Sci Tech 2007;8(4):156-166.
39. Ochoa L, Igartua M, Ma R., Hernandez, Gascon AR, Pedraz JL. Preparation of sustained release hydrophilic matrices by
melt granulation in a high-shear mixer. J Pharm Pharmaceut Sci 2005;8(2):132-140.
40. Montenegro L, Carbone C, Puglisi G. Vehicle effects on in vitro release and skin permeation of octylmethoxycinnamate
from microemulsions. Int J Pharm 2011; 405:162–168.
41. El-Hawary IMA. Formulation of non-steroidal anti-inflammatory drug in different topical dosage forms. M.Sc. Faculty
of pharmacy, Cairo university (2006).
42. Lucero MJ, Vigo J, Leon MJ. A study of shear and compression deformations on hydrophilic gels of tretinoin. Int J Pharm
1994;106:125-133.
43. Desai KGH. Enhanced skin permeation of rofecoxib using topical microemulsion gel. Drug Dev Res 2004;63:33-40.
44. Soliman SMH, Abd El-Rehim AEA, El-Gazayerly ON, Abd El-Malak NS. Formulation of microemulsion gel systems for
transdermal delivery of celecoxib: invitro Permeation, anti-inflammatory activity and skin irritation tests, Drug Discov
Therap 2010;4(6):459-471.
45. Badawi AA, Nour SA, Sakran WS, El-Mancy SS. Preparation and evaluation of microemulsion systems containing
salicylic acid. AAPS Pharma Sci Tech 2009;10(4):1081-1084.
46. El Laithy HM, El-Shaboury KMF. The development of cutina lipogels and gel microemulsion for topical administration
of fluconazole. AAPS Pharm Sci Tech 2002;3(4):1-9.
47. Kaidbey KH, Kligman AM. Laboratory methods for appraising the efficacy of sunscreens. J Soc Cosmet Chem 1978;
29:525–536.
48. Diffey BL, Robson J. A new substrate to measure sunscreen protection factors throughout the ultraviolet spectrum. J
Soc Cosmet Chem 1989;40:127–33.
49. Vitro Skin®. 2009. http://www.ims-usa.com
50. Diffey BL, Robson J. The influence of pigmentation and illumination on the perception of erythema. Photodermatol
Photoimmunol Photomed 1992;9:45–47.
Amin et. Al/ Preparation And Characterization Of Benzophenone-3 Loaded Polymeric Nanoparticles Of LactideCo- Ɛ - Caprolactone As Drug Carriers
51. Vettor M, Perugini P, Scalia S, Conti B, Genta I, Modena T, Pavanetto F. Poly lactide nanoencapsulation to reduce
photoinactivation of a sunscreen agent. Int J Cosm Sci 2008;30:219–227.
52. Santa-María C, Revilla E, Miramontes E, Bautista J, García-Martínez A, Romero E, Carballo M, Parrado J. Protection
against free radicals (UVB irradiation) of a water-soluble enzymatic extract from rice bran. Study using human
keratinocyte monolayer and reconstructed human epidermis. Food Chem Toxicol 2010;48:83–88.
53. Villalobos-Hernández JR, Müller-Goymann CC. In vitro erythemal UV-A protection factors of inorganic sunscreens
distributed in aqueous media using carnauba wax-decyl oleate nanoparticles. Eur J Pharm Biopharm. 2007; 65:122125.
54. Mazzola PG, Penna TCV, da S Martins AM. Determination of decimal reduction time (D value) of chemical agents used
in hospitals for disinfection purposes,BMCInfectiousDiseases 2003, http://www.biomedcentral.com/14712334/3/24.
55. Abd El-Malak NS, Nour SA, El-Gazayerly ON, Ph.D. Thesis, Faculty of Pharmacy, Cairo University, 2006.
56. Ma J, Feng P, Ye C, Wang Y, Fan Y. An improved interfacial coacervation technique to fabricate biodegradable
nanocapsules of an aqueous peptide solution from polylactide and its block copolymers with poly (ethylene glycol).
Colloid Polym Sci 2001;279:387–392.
57. Stella B, Arpicco S, Rocco F, Marsaud V, Renoir JM, Cattel L, Couvreur P. Encapsulation of gemcitabine lipophilic
derivatives into polycyanoacrylate nanospheres and nanocapsules. Int J Pharm 2007;344:71–77.
58. Wissing SA, Müller RH. Solid lipid nanoparticles as carrier for sunscreens: in vitro release and in vivo skin penetration,
Journal of Controlled Release 2002; 81:225–233.
59. Almeida JS, Jezur L, Fontana MC, Paese K, Silva CB, Pohlmann AR, Guterres SS, Ruy Beck RCR. Oil-Based Nanoparticles
Containing Alternative Vegetable Oils (Grape Seed Oil and Almond Kernel Oil): Preparation and Characterization. Latin
Am J Pharm 2009;28(2):165-172.
60. Kumari A, Yadav SK, Yadav SC. Biodegradable polymeric nanoparticles based drug delivery systems, Colloids and
Surfaces B: Biointerfaces 2010; 75:1–18.
61. Tiyaboonchai W. Chitosan nanoparticles: a promising system for drug delivery. Naresuan Univ J 2003;11:51.
62. Ashish K. Mehta, Yadav KS., Krutika K. Sawant. Nimodipine loaded PLGA nanoparticles: formulation optimization using
factorial design, characterization and in-vitro evaluation. Curr Drug Del 2007;4:185-93
63. Alvarez-Romàn R, Barré G, Guy RH, Fessi H. Biodegradable polymer nanocapsules containing a sunscreen agent:
preparation and photoprotection. Eur J Pharm Biopharm 2001;52:191–195.
64. Decuzzi P, Ferrari M. Design maps for nanoparticles targeting the diseased microvasculature. Biomaterials
2008;29:377–384.
65. Souto EB, Müller RH, Gohla SA. Novel approach based on lipid nanoparticles (SLN) for topical delivery of α-lipoic acid. J
Microencapsul 2005;22:581-589.
66. Mainardes RM, Programa RCE. PLGA nanoparticles containing praziquantel: effect of formulation variables on size
distribution, Int J Pharm 2005; 290:137–144.
67. Thanki PN, Dellacherie E, Six JL. Surface characteristics of PLA and PLGA films. Appl Surf Sci 2006;253:2758.
68. Stevanović M, Radulović A, Jordović B, Uskoković D. Poly (DL-lactide-co-glycolide) Nanospheres for the Sustained
Release of Folic Acid. J Biomed Nanotech 2008;4:1–10.
69. Dong Y, Feng SS. Pharmaceutical Nanotechnology: Poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles prepared by
high pressure homogenization for paclitaxel. Chemother Int J Pharm 2007;342:208–214.
70. Tan SW, Billa N, Roberts CR, Burley JC. Surfactant effects on the physical characteristics of Amphotericin B-containing
nanostructured lipid carriers Colloids and Surfaces A: Physicochem Eng Aspects 2010;372:73–79.
71. Giokas DL, Salvador A, Chisvert A. UV filters: From sunscreens to human body and the environment. Trends in
Analytical Chemistry 2007;26(5).
72. Pople PV and Singh KK. Development and Evaluation of Topical Formulation Containing Solid Lipid Nanoparticles of
Vitamin A. AAPS PharmSciTech 2006;7 (4) Article 91.
73. Kuo YC, Chung JF. Physicochemical properties of nevirapine-loaded solid lipid nanoparticles and nanostructured lipid
carriers Colloids and Surfaces B: Biointerfaces 2011;83:299–306.
74. Kheradmandnia S, Farahani EV, Nosrati M, Atyabi F. Preparation and characterization of ketoprofen-loaded solid lipid
nanoparticles made from beeswax and carnauba wax. Nanomedicine: Nanotechnology, Biology, and Medicine
2010;6:753–759.
75. Forestier S. Rationale for sunscreen development. J Am Acad Dermatol 2008.
76. Illing A, Unruh T. Investigation on the flow behavior of dispersion of solid triglyceride nanoparticles. Int J Pharm
2004;284:123.
77. Alves MP, Scarrone AL, Santos M, Pohlmann AR, Guterres SS. Human skin penetration and distribution of nimesulide
from hydrophilic gels containing nanocarriers. Int J Pharm 2007;341:215–220.
78. Ourique AL, Melero A, da Silva CDB, Schaefer UF, Pohlmann AR, Guterres SS, Lehr CM, Kostk KH , Beck RCR. Improved
photostability and reduced skin permeation of tretinoin: Development of a semisolid nanomedicine. Eur J Pharm
Biopharm 2011;79:95–101.
79. Mandawgade SS, Patravale VB. Development of SLNs from natural lipids: application to topical delivery of tretinoin. Int
J Pharm 2008;363:132–138.