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Artificial Cells, Nanomedicine, and Biotechnology Comparative in vitro transportation of pentamidine across the blood-brain barrier using polycaprolactone nanoparticles and phosphatidylcholine liposomes



Nanoparticles (NPs) have gained importance in addressing drug delivery challenges across biological barriers. Here, we reformulated pentamidine, a drug used to treat Human African Trypanosomiasis (HAT) in polymer based nanoparticles and liposomes and compared their capability to enhance pent-amidine penetration across blood brain barrier (BBB). Size, polydispersity index, zeta potential, morphology , pentamidine loading and drug release profiles were determined by various methods. Cytotoxicity was tested against the immortalized mouse brain endothelioma cells over 96 h. Moreover, cells monolayer integrity and transportation ability were examined for 24 h. Pentamidine-loaded poly-caprolactone (PCL) nanoparticles had a mean size of 267.58, PDI of 0.25 and zeta potential of-28.1 mV and pentamidine-loaded liposomes had a mean size of 119.61 nm, PDI of 0.25 and zeta potential 11.78. Pentamidine loading was 0.16 mg/mg (w/w) and 0.17 mg/mg (w/w) in PCL NPs and liposomes respectively. PCL nanoparticles and liposomes released 12.13% and 22.21% of pentamidine respectively after 24 h. Liposomes transported 87% of the dose, PCL NPs 66% of the dose and free pentamidine penetration was 63% of the dose. These results suggest that liposomes are comparatively promising nanocarriers for transportation of pentamidine across BBB. ARTICLE HISTORY
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Artificial Cells, Nanomedicine, and Biotechnology
An International Journal
ISSN: 2169-1401 (Print) 2169-141X (Online) Journal homepage:
Comparative in vitro transportation of
pentamidine across the blood-brain barrier
using polycaprolactone nanoparticles and
phosphatidylcholine liposomes
Geofrey Omarch, Yunus Kippie, Shireen Mentor, Naushaad Ebrahim, David
Fisher, Grace Murilla, Hulda Swai & Admire Dube
To cite this article: Geofrey Omarch, Yunus Kippie, Shireen Mentor, Naushaad Ebrahim, David
Fisher, Grace Murilla, Hulda Swai & Admire Dube (2019) Comparative in�vitro transportation
of pentamidine across the blood-brain barrier using polycaprolactone nanoparticles and
phosphatidylcholine liposomes, Artificial Cells, Nanomedicine, and Biotechnology, 47:1, 1428-1436,
DOI: 10.1080/21691401.2019.1596923
To link to this article:
© 2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Published online: 22 Apr 2019.
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Comparative in vitro transportation of pentamidine across the blood-brain
barrier using polycaprolactone nanoparticles and phosphatidylcholine liposomes
Geofrey Omarch
, Yunus Kippie
, Shireen Mentor
, Naushaad Ebrahim
, David Fisher
, Grace Murilla
, Hulda
and Admire Dube
School of Life Sciences, The Nelson Mandela African Institution of Science and Technology, Tengeru, Arusha, Tanzania;
Tanzania Veterinary
Laboratory Agency, Temeke, Dar es Salaam, Tanzania;
School of Pharmacy, University of the Western Cape, Bellville, South Africa;
of Life Sciences, University of the Western Cape, Bellville, South Africa;
Biotechnology Research Institute, Kenya Agricultural and Livestock
Research Organization, Kikuyu, Nairobi, Kenya
Nanoparticles (NPs) have gained importance in addressing drug delivery challenges across biological
barriers. Here, we reformulated pentamidine, a drug used to treat Human African Trypanosomiasis
(HAT) in polymer based nanoparticles and liposomes and compared their capability to enhance pent-
amidine penetration across blood brain barrier (BBB). Size, polydispersity index, zeta potential, morph-
ology, pentamidine loading and drug release profiles were determined by various methods.
Cytotoxicity was tested against the immortalized mouse brain endothelioma cells over 96 h. Moreover,
cells monolayer integrity and transportation ability were examined for 24 h. Pentamidine-loaded poly-
caprolactone (PCL) nanoparticles had a mean size of 267.58, PDI of 0.25 and zeta potential of 28.1 mV
and pentamidine-loaded liposomes had a mean size of 119.61 nm, PDI of 0.25 and zeta potential
11.78. Pentamidine loading was 0.16 mg/mg (w/w) and 0.17 mg/mg (w/w) in PCL NPs and liposomes
respectively. PCL nanoparticles and liposomes released 12.13% and 22.21% of pentamidine respectively
after 24 h. Liposomes transported 87% of the dose, PCL NPs 66% of the dose and free pentamidine
penetration was 63% of the dose. These results suggest that liposomes are comparatively promising
nanocarriers for transportation of pentamidine across BBB.
Received 9 October 2018
Revised 27 December 2018
Accepted 29 December 2018
Pentamidine; PCL
nanoparticles; liposomes;
blood brain barrier;
transendothelial electrical
resistance; Human African
Human African Trypanosomiasis (HAT) is a fatal vector borne
trypanosomal infection characterized by wasting condition in
which there is a slow progressive loss of condition accompa-
nied by increasing anaemia and weakness to the point of
extreme emaciation, collapse and death often due to heart
failure [1,2]. The disease is endemic in 36 countries of Sub-
Saharan Africa and impacts about 70 million people [35].
Two distinct subspecies of the Trypanosoma brucei are the
causative parasites of the disease [6]. Trypanosoma brucei
gambiense which is confined to the Central and West Africa
causes a chronic infection and Trypanosoma brucei rhode-
sience which is found in the East and South of Africa causes
an acute form of the disease [3].
The disease occurs in two stages and control relies on
both therapeutics and vector control. In the first stage
(haemo-lymphatic), pentamidine isethionate is the first line
drug of choice for treating T.b. gambiense and Suramin for
treating T.b. rhodesiense [7]. In the second stage, (neuro-
logical) melarsoprol is the main choice for the treatment of
both T.b. gambiense and T.b. rhodesiense infections;
Eflornithine against T.b. gambiense; Nifurtimox against
T.b. gambiense; and eflornithine in combination with nifurti-
mox against T.b. gambiense [8,9].
The major problems and challenges in treatment of HAT
are the level of toxicities exhibited by the drugs [1,10].
Pentamidine isethionate (see chemical structure in Figure 1)
is an aromatic dicationic diamidine molecule [11,12] which is
a white crystalline powder soluble in water and glycerine and
insoluble in acetone, ether and chloroform. It is chemically
designated as 4, 40diamidino-dephenoxypentane di (b
hydroxyethanesulfonate) [13]. It is administered daily by deep
intramuscular administration at a dose of 4 mg/kg for 710
days [8,12,14]. Patients treated with pentamidine are reported
to experience undesirable side effects including abdominal
pain, diarrhoea, nausea, vomiting [15] cardiac arrhythmias,
very high or low blood sugar, pancreas, kidney and liver
problems [11,16]. Furthermore, pentamidine treatment is
complicated by the mode of drug administration which
requires hospitalization of a patient for more than 7 days
and therefore, creates significant logistical and societal prob-
lems in the remote rural areas of Africa, where HAT is
endemic. This makes it difficult to implement with consist-
ency because of poor health facilities and few healthcare
CONTACT Geofrey Omarch School of Life Sciences, The Nelson Mandela African Institution of Science and Technology, Tengeru,
Arusha, P.O. Box 447, Tanzania
ß2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
2019, VOL. 47, NO. 1, 14281436
personnel [5,12,16,17]. Pentamidine is not well absorbed
through gastrointestinal tract (GIT) which leads to poor oral
bioavailability and has a very slow rate of diffusion across the
biological membranes and thus, making the concentrations
not sufficient enough to affect the trypanosomes which
advance to the central nervous system (CNS) [11].
Our work aimed at investigating the possibility of mitigat-
ing the toxicity of pentamidine and enhancing its transporta-
tion across the blood brain barrier (BBB) using nanoparticles.
We loaded pentamidine in both polycaprolactone (PCL)
nanoparticles and liposomes which are popular drug delivery
vehicles for sustained and targeted drug delivery due to
being relatively non-toxic, biodegradable, biocompatible and
well tolerated by tissues [18,19]. The two loaded nanocarriers
were characterized for physicochemical properties, examined
for cytotoxicity and comparatively studied for the drug trans-
portation efficiency across the endothelial monolayer tissue
of the mouse BBB (b.End5 cells), an in vitro model for BBB.
Materials and methods
Materials for preparation of PCL nanoparticles
Polycaprolactone (PCL) polymer (2-oxepanone homopolymer,
6-caprolactone polymer; d¼1.146g/ml; average molecular
weight ¼14,000; product of Japan in form of flakes) was a
product from Sigma-Aldrich, St. Louis, MO, USA). The D-
a-Tocopherol polyethylene glycol 1000 succinate (TPGS)
(BioXtra, water soluble vitamin E conjugate; solubility: H2O 1g/
10 ml; product of France) was a product from Sigma-Aldrich,
St. Louis, MO, USA). Polysorbate (TweenV
R80) (viscous liquid;
P1754-25ML; CMC: 0.012 Mm (2025OC) product of France)
was a product from Sigma-Aldrich, MO, USA. Pentamidine
isethionate salt (drug) (MW¼592.68 g/mol; P0547-250G; prod-
uct of USA) was a product from Sigma-Aldrich, St. Louis, MO,
USA). Chloroform was a product from Kimix Chemicals, Cape
Town, SA). Materials for preparation of liposomes. L-a-phos-
phatidylcholine (Soy PC -95%); MW¼775.04; a product of USA
from AvantiV
Rpolar lipids, Inc. 700 industrial Park Drive,
Alabaster, Alabama. Cholesterol (Sigma Grade 93%; C8667-
5G; MW ¼386.65g/mol; was a product from Sigma-Aldrich, St.
Louis, MO, USA. Phosphate Buffer Saline (PBS) (pH 7.4) was
sourced from Nanotechnology innovation Centre (NIC), UWC
laboratories and deionized water millipore (18.2 X) from
Thermo Scientific) was used in all experiments.
Synthesis of pentamidine-loaded PCL nanoparticles
Pentamidine loaded PCL NPs were obtained by double solv-
ent evaporation method as previously described by Ubrich
and colleagues [20] with some modifications. Briefly, a 1 mg/
ml solution of pentamidine was made in a 0.1 mg/ml aque-
ous solution of TPGS prepared. Separately, a 5 mg/ml solu-
tion of PCL in chloroform was prepared. Thereafter,
pentamidine-TPGS solution was poured to the PCL solution
to mix the aqueous and oil phases. The container was placed
on ice bath, and the primary emulsion (w/o) was formulated
by homogenization using a high speed homogenizer (IKAV
R; model: T18D) at 10 000 rpm for
3 min followed by probe tip sonication at 85% intensity for 3
min. To make the second emulsion, a 0.1 mg/ml aqueous
solution of TweenV
R80 was prepared and was added to the
primary emulsion and homogenized at a speed of 3000 rpm
for 3 min followed by probe sonication at 65% intensity for
30 s in order to form the double emulsion (w/o/w). To
remove chloroform, the nanosuspension was evaporated
under reduced pressure for 1 h at 40 C. Nanoparticles were
washed thrice with deionized water at 10,000 rpm, 10 min
and 4 C and collected after centrifugation.
Synthesis of pentamidine-loaded liposomes
Liposomes were formulated by thin lipid film hydration tech-
nique followed by extrusion as previously described by
Begun and friends [21] and Sha with colleagues [22] with
modifications. Briefly, L-phosphatidylcholine and cholesterol
were weighed and mixed at the ratio of 4:1 and dissolved in
chloroform. The formed emulsion was evaporated under vac-
uum (1 h, 55 C) to remove chloroform and form a thin lipid
film. Thereafter, 1 mg/ml solution of pentamidine isethionate
were prepared in PBS at pH of 7.4. The mixture was then
added to the flask containing the lipid film to hydrate the
lipid film and encapsulate the drug. This was left for 30 min
in a water-bath sonicator warmed at 25 C. Thereafter, the
contents were vortexed for 10 min to obtain the homoge-
neous solution. The suspension was extruded 10 times.
Thereafter, the synthesized liposomes in the syringe were
transferred to a beaker for purification. Liposomes were dou-
ble purified with PBS (pH 7.4) by centrifugation (Eppendorf;
centrifuge 5417R) at 10,000 rpm for 10 min at 4 C.
Both pentamidine-loaded PCL nanoparticles and liposomes
were cryoprotected with 10% sucrose, and freeze-dried for 24
h. Thereafter, size, polydispersity index (PDI) and zeta poten-
tial were determined using dynamic light scattering (DLS)
techniques by a Zetasizer NanoZS90. Measurements were
performed at 25 C and 90measurement scattering angle
(10 runs). All measurements were recorded in triplicate as
Mean diameter (nm) ± Standard Error (SE). Morphology was
analyzed using Scanning Electron Microscope (LEO 1450
SEM, Zeiss).
Determination of pentamidine loading
The amount of loaded pentamidine was determined using
HPLC coupled to a photo-diode array detector at ambient
temperature. The Perkin Elmer (Shelton, CT, USA) HPLC-PDA
system comprised of the Flexar LC Autosampler (100 ul injec-
tion loop), Flexar Solvent Manager 3-channel degasser, Flexar
Figure 1. Pentamidine structure (Source [11]).
LC pump and the Flexar PDA Plus detector. Sample prepar-
ation was performed by breaking 1 mg of freeze dried pent-
amidine loaded nanoparticles in 1 ml of 0.1% formic acid
(FA) warmed in a waterbath at 25 C for 5 min. Thereafter,
the solution was centrifuged at 10,000 rpm, 4 C for 10 min
and supernatant analyzed using HPLC. Working standards of
concentration (0.01, 0.02, 0.1, 0.2, 0.5, 1 and 2) mg/ml were
diluted from a 1 mg/ml pentamidine stock solution prepared
in 0.1% FA to a final volume of 1,000 ml using 0.1% FA as
diluent. 20 ml of sample or working standard was injected in
triplicate at a flow rate of 1 ml/min onto an end-capped C8
reverse phase analytical LC column (Zorbax Eclipse XDB-C8
4.6 x 150 mm, 5 mm) with retention achieved by isocratic elu-
tion of the analyte at 10% of 60% aq. Acetonitrile and 90%
of 0.1% FA. The absorbance maximum was determined at
265 nm and 270 nm. The standard curve of pentamidine was
constructed at linearity over the range of 0.1 to 2 mg/ml (r
¼0.9984) to quantify extracted pentamidine from PCL nano-
particles and liposomes. Loading was determined by using
the following equation;
Drug loading w=w
¼Quantity of pentamidine in nanoparticles lg
=Mass of freeze dried nanoparticles mg
Determination of pentamidine release in vitro
To characterize drug release in vitro, 10 mg/ml concentrations
of pentamidine-loaded freeze-dried PCL nanoparticles and
freeze dried pentamidine-loaded liposomes were separately
prepared in PBS (pH 7.4). Samples were incubated in a water
bath at 37 C and removed at time periods of 30 min, 1, 2, 6,
12, 24 and 48 h and centrifuged at 10,000 rpm, 4 C for 10
min. Thereafter supernatants were frozen at 80 C and later
analyzed using the validated HPLCPDA assay.
In vitro cell culture and cytotoxicity of PCL
nanoparticles and liposomes
The b.End5 cells seeded at a density of 5 10
per well in
supplemented Dulbeccos Modified Eagles medium hams
F12 (DMEM:F12) were cultured in a 6-well tissue culture
plates and incubated at atmosphere of 37 C and 5% CO
Cells were incubated overnight to allow them to attach and
expand to confluence. To determine the cytotoxic effect, in
triplicate, three concentrations (1, 2.5 and 5 mg/ml) in sup-
plemented DMEM:F12 of PCL nanoparticles, liposomes and
free pentamidine were exposed to cells over a period of 96
h. After every 24 h interval, a Trypan blue exclusion assay
(Sigma Cell Culture ReagentsV
R, T-8154) was performed to
evaluate % cell viability.
Pentamidine transport across b.End5 cell line
The immortalized mouse brain endothelioma (b.End5) cell
line was purchased from the European Collection of Cell
Cultures (ECACC, Sigma-Aldrich (Cat no. 96091930)) and was
derived from brain endothelial cells of BALB/c mice. The
b.End5 cells were seeded at the concentration of 5 10
cells per insert/well in a 24- well microtiter plate, on mixed
cellulose esters MillicellV
Rinserts with an area of 0.6 cm
then incubated at 37 C and 5% CO
. Cells were allowed to
attach to the bottom of the filter insert and allowed to grow
to confluence in 24 h. Thereafter, they were removed from
the incubator and allowed to acclimatize to room tempera-
ture for 20 min in a sterile laminar flow cabinet. Highest con-
centrations of nanoparticles and free drug which did not
show effect on cells growth during cytotoxicity study of
empty nanoparticles were used. Cells were exposed for 24 h
to concentrations of 2.5 mg of PCL NPs loaded with 0.4 mg
of pentamidine (0.4 mg/ml (w/v)), 1.1 mg of liposomes loaded
with 0.2 mg of pentamidine (0.2 mg/ml (w/v)) and 0.4 mg
of free pentamidine (0.4 mg/ml (w/v)). The permeability
of the cell monolayer was assessed by measuring the
Transendothelial Electrical Resistance (TEER) using a
R- ERS voltohmmeter at 30 min, 1, 2, 3, 4, 6, 8, 12
and 24 h. All readings were taken in duplicate and the true
resistance of endothelial monolayer was computed by sub-
tracting the blank reading (insert without the cells) from the
experimental sample reading. The reading was then standar-
dized to square centimetres by multiplying the true resist-
ance with the insert area (0.6 cm
). At the same time, 500 ml
of sample were collected from the basolateral compartment
at 2, 4, 6, 8, 12 and 24 h for quantification of pentamidine
that crossed the monolayer from the apical side. Pentamidine
from the samples was later analyzed with a validated HPLC-
PDA and quantitation was carried out by extrapolating the
peak areas against the concentrations to the calibration curve
of pentamidine in the cell culture media with linearity over
the range of 0.1 to 10 ppm (r
¼0.9608). All experiments
were conducted in triplicate.
Statistical analysis was performed using One-way Analysis of
Variance (1-way ANOVA) and paired t-test with GraphPad
Prism 7.04 software. Statistical significant values were
regarded when pvalues was <.05. Data were presented as
Mean ± Standard Error (Mean ± SE).
Characterization of PCL nanoparticles and liposomes
All synthesized particles were within nanoscale range where
the smaller mean size was observed with pentamidine-loaded
PCL NPs which was 267.58 ± 65.63 nm (Figure 2(A)) and the
larger size was observed with unloaded PCL NPs which was
344.18 ± 27.48 nm. The PDI was lower at all steps of synthesis
with smaller mean PDI observed being 0.12 ± 0.05 (Figure
2(B)) with unloaded PCL NPs and the higher was 0.3 ± 0.09
observed with freeze dried PCL NPs. The empty PCL NPs pos-
sessed a negative zeta potential with the lower mean zeta
potential of 28.1 ± 4.19 mV (Figure 2(C))observedwith
loaded PCL NPs and the highest with freeze dried pentami-
dine-loaded PCL NPs which was 33.4 ± 8.12 mV. The mean
PDI of 0.25 ± 0.15 of pentamidine loaded PCL NPs indicated
uniformity of particle sizes produced and lower chance of
aggregation of particles. The loaded PCL nanoparticles had a
loading of pentamidine of 0.16 mg/mg (w/w). Likewise, lipo-
somes had sizes within the nanoscale range with smaller
mean size observed with pentamidine-loaded liposomes which
was 119.61 ± 14.31 nm (Figure 3(A)) and the larger mean size
was observed with unloaded liposomes which was 360.84 ±
125.93 nm. The mean size of pentamidine loaded liposomes
was 119.61 ± 14.31 nm and Agrawal [23]reportedthatlipo-
somes with the size range below 200 nm were easily able to
cross the BBB. The mean PDI of 0.25 ± 0.02 (Figure 3(B))
observed with loaded liposomes indicated monodispersion of
liposomes in suspension, unlikely to aggregate and stable.
Both positive and negative zeta potential were observed. The
mean surface charge of these liposomes was positive 11.78 ±
10.15 mV (Figure 3(C)) which suggested that some pentami-
dine was adsorbed on the surface of liposomes. Post freeze
drying we observed the zeta potential changing to 6.6 mV
which might inform that because pentamidine is water-sol-
uble, the traces might have come into contact with bound
water and dissolved in water during freezing process and then
was taken out with water during secondary drying in process
of freeze drying [24]. Loaded liposomes had a loading of 0.178
Mean sizes of liposomes
Synthesis steps
Size (nm)
A= Unloaded liposomes B= Pentamidine loaded liposomes
C= Freeze dried pentamidine loaded liposomes
Mean PDI of liposomes
Synthesis steps
Polydispersity index (PDI)
A= Unloaded liposomes B= Pentamidine loaded liposomes
C= Freeze dried pentamidine loaded liposomes
Mean Zeta potentials of liposomes
Synthesis steps
Zeta potential (mV)
A= Unloaded liposomes B= Pentamidine loaded liposomes
C= Freeze dried pentamidine loaded liposomes
Figure 3. Liposomal particle physicochemical properties of unloaded, pentami-
dine-loaded and freeze dried pentamidine-loaded liposomes. (A) Sizes, (B) PDI,
(C) Zeta potential. Data is presented as mean ± SE. Sample size (unloaded and
loaded liposomes n¼3 and freeze-dried n¼1(pooled sample)). No statistical
differences were found at p<.05.
Mean sizes of PCL NPs
Synthesis steps
Size (nm)
A=unloaded PCL-NPs B=Pentamidine loaded PCL-NPs
C= Freeze dried pentamidine loaded PCL-NPs
Mean PDIs of PCL-NPs
Synthesis steps
Polydispersity index (PDI)
A=unloaded PCL-NPs B=Pentamidine loaded PCL-NPs
C= Freeze dried pentamidine loaded PCL-NPs
Mean Zeta of PCL NPs
Synthesis steps
Zeta potential (mV)
A=unloaded PCL-NPs B=Pentamidine loaded PCL-NPs
C= Freeze dried pentamidine loaded PCL-NPs
Figure 2. Particle physicochemical properties of unloaded, pentamidine-loaded
and freeze dried pentamidine-loaded PCL nanoparticles. (A) Sizes, (B) PDIs, (C)
Zeta potentials. Data is presented as mean ± SE. Sample size (n¼6). No statis-
tical differences were found at p<.05.
mg/mg (w/w). Both PCL nanoparticles and liposomes presented
good physicochemical properties of nanomaterials and we
considered them suitable carriers of drug across the BBB.
There was no statistical significant difference between the size
means of PCL NPs and liposomes (paired t-test, p¼.5928).
This presents the double solvent evaporation and thin film
hydration techniques being suitable for synthesis of drug car-
riers for BBB transportation.
SEM revealed smooth spherical shapes for both PCL nano-
capsules and liposomes (Figure 4). Spherical shaped or
spheres are ease to synthesis and have been studied for
years. They are quickly up taken by microphages and stay for
shorter time in circulation as compared to non-spherical
nanoparticles [25,26]. However, the sizes observed also corre-
lated well with the hydrodynamic diameters measured by the
Zetasizer (Figure 4).
The amounts of pentamidine released were extrapolated
from the calibration curve of pentamidine in PBS. Results
show that over a period of 24 h, 12.13 ± 0.21% and 22.21 ±
0.53% of pentamidine was released from PCL nanoparticles
and liposomes respectively. Statistical difference between the
mean percentages of PCL NPs and liposomes pentamidine
release was significant (paired t-test, p¼.0002).
Toxicological effect
It was important to screen for the appropriate concentration
of both unloaded and loaded nanoparticles with no toxic
effect to b.End5 cells to use for transportation studies in vitro.
The survival rates were determined by using Trypan Blue
exclusion assay. The study indicate that unloaded PCL nano-
particles exposed at highest concentration of 5 mg/ml
(Figure 5)(p¼.2072) and unloaded liposomes at concentra-
tions of 5 mg/ml and 2.5 mg/ml (Figure 6)(p¼.0378) had
cytotoxic effect and consequently inhibited the growth of
the b.End5 cells over a period of 96 h. In these concentra-
tions hardly growing cells were observed at 48 h to 96 h.
However, statistically, the difference in cell viability following
exposure of three concentrations of unloaded PCL NPs was
insignificant (p¼.2072). Conversely, pentamidine-loaded PCL
nanoparticles exposed at the concentration of 2.5 mg/ml
(loading 0.4 mg pentamidine) (Figure 7)(p¼.9916), pentami-
dine-loaded liposomes exposed at higher concentrations of 1
mg/ml (loading 0.2 mg pentamidine) (Figure 8)(p¼.9023)
and free pentamidine of up to 0.4 mg(Figure 9) did not affect
cells viability. However, free pentamidine exposed at higher
level concentration of 200 mg/ml and 400 mg/ml had severe
Figure 4. These are electron micrographs of freeze dried PCL nanoparticles (A)
and freeze dried liposomes (B) generated by SEM. The legends were generated
by SEM and all bars calibrated at 1 mm.
24 48 72 96
Time (Hours)
Viable cells/ml
0 mg/ml (only DMEM added) 1 mg/ml unloaded PCL-NPS
2.5 mg/ml unloaded PCL-NPs 5 mg/ml unloaded PCL-NPS
Figure 5. Shows the toxic effect of 5 mg/ml concentration of unloaded PCL
NPs on the growth of b.End5 cells. Data is presented as mean ± SE, n¼3. No
statistical differences were found at p<.05.
24 48 72 96
Time (Hours)
Viable cells/ml
0 mg/ml (only DMEM added) 1 mg/ml pentamidine-loaded PCL-NPS
2.5 mg/ml pentamidine-loaded PCL-NPs
Figure 6. Shows the effect of different concentrations of pentamidine-loaded
NPs on the growth b.End5 cells. Data is presented as mean ± SE, n¼3. No stat-
istical differences were found at p<.05.
cytotoxic effect on cells (p¼.0006) such that hardly any
viable cells were observed after 24 h exposure (Figure 9).
However, we anticipated that loaded nanoparticles in con-
centrations above the screened ones would result into
cytotoxic effect as observed with unloaded particles and
would not be safe to use such concentrations for transporta-
tion study.
Transportation of pentamidine across the in b.End5 cells
TEER was measured to assess the integrity of the cell mono-
layer following drug exposure to either pentamidine, or pent-
amidine loaded PCL nanoparticles or liposomes. When
pentamidine-loaded PCL NPs, pentamidine-loaded liposomes,
free pentamidine and media as control were added in wells,
TEER values dropped in the first two hours which signify loos-
ening of the tight junctions but then values raised with time
which imply increased resistance of the monolayer restricting
penetration of molecules through the tight junctions over 24
h. The TEER values raised and from the eighth hour of expos-
ure resistance was constantly sustained. TEER values for the
control were observed to raise shortly and continually
decreased with time (Figure 10). However, the TEER values dif-
ferences observed between the control and liposomes (p¼
.9458), control and PCL NPs (p¼.2972), control and free pent-
amidine (p¼.3014), Liposomes and PCL NPs (p¼.6076), lipo-
somes and free pentamidine (p¼.6131), PCL NPs and free
pentamidine (p¼.9999) were all statistically insignificant indi-
cating that the cells monolayer was intact and sensitively
restricting the penetration of any molecule across it.
The quantities of pentamidine collected from the baso-
lateral compartment were analyzed by HPLCPDA.
Comparatively, liposomes were observed to deliver more dose
of pentamidine to the basolateral compartment compared to
PCL NPs and movement of free pentamidine (Figure 11) which
correlates well with what was observed with TEER responses
as well as drug release profile of liposomes and PCL NPs. After
24 h, liposomes could transport 87 ± 0.01% of the pentami-
dine dose, while PCL NPs could transport 66 ± 0.1% of the
dose and free pentamidine solution could deliver 63 ± 0.25%
of the dose. The percent dose transport difference of lipo-
somes against PCL NPs was statistically significant (p¼.0006)
and against free drug (p¼.0001) but there was no statistical
24 48 72 96
Time (Hours)
Viable cells/ml
0mg/ml (only DMEM added) 0.55mg/ml pent-loaded liposomes
1.1mg/ml pent-loaded liposomes
Figure 8. Shows the effect of different concentrations of pentamidine- loaded
liposomes on growth of b.End5 cells. Data is presented as mean ± SE, n¼3.
No statistical difference was found at p<.05.
24 48 72 96
Time (Hours)
Viable cells/ml
g/ml (only DMEM added) 0.2
g/ml pentamidine
g/ml pentamidine 200
g/ml pentamidine
g/ml pentamidine
  
Figure 9. Shows the effect of different concentrations of free pentamidine on
the growth of b.End5 cells. 200 mg/ml and 400 mg/ml were severely toxic to
cells that no cells could grow. Both concentrations had statistical significant dif-
ference at p<.05. Data is presented as mean ± SE of mean, n¼3.
10 20 30
Time (Hours)
Resistance to control (Ω)Resistance to liposomes (Ω)
Resistance to PCL NPs (Ω)Resistance to free pentamidine (Ω)
Figure 10. The effects of pentamidine-loaded PCL nanoparticles, pentamidine
loaded liposomes and free pentamidine on the TEER of b.End5 cell monolayers. Data
is presented as mean ± SE, n¼3. No statistical difference was found at p<.05.
24 48 72 96
Time (Hours)
Viable cells/ml
0 mg/ml (only DMEM added) 1mg/ml unloaded liposomes
2.5 mg/ml unloaded liposomes 5 mg/ml unloaded liposomes
Figure 7. Shows toxic effect of 2.5 mg/ml and 5 mg/ml concentrations of pent-
amidine-loaded liposomes on the growth of b.End5 cells. Data is presented as
mean ± SE, n¼3. Statistical significant difference was found with 2.5 mg/ml
concentration at p<.05.
significant difference of percent dose transport between PCL
NPs and movement of free drug (p¼.1281). Overtime, the
delivered quantities were observed to decrease which is also a
reflection of the TEER values profile (see Figure 10) which first
dropped signifying allowed transportation of molecules
through the BBB and then slowly raised indicating strong
restriction movement of molecules across the barrier. We may
explain that free drug, liposomes and nanoparticles were
trapped within the monolayer cells as time went on but also
we think transportation of liposomes and nanoparticles uti-
lized mainly the transport mediated mechanism such as endo-
cytosis or transcytosis. Dose delivered differences were
statistically significant (p¼.0001).
Several techniques have been developed and became suc-
cessful in loading drugs into the polymeric nanoparticles for
drug delivery, and the choice for the method of encapsula-
tion usually depends on the physicochemical properties of
the drug itself [27]. Using the double emulsion solvent evap-
oration as a technique to encapsulate pentamidine, a water-
soluble drug in this work was due to the fact that the double
emulsion techniques was previously shown to improve the
drug encapsulation and minimize the escape of the hydro-
philic drug from the aqueous core which was our aim
[20,27]. Furthermore, two important surfactants (Polysorbate
80 and TPGS) were used. TPGS has been shown to increase
the loading ability of hydrophilic drugs into the polymeric
nanoparticles and polysorbate 80 has been reported to help
synthesis of small sized nanoparticles and also increase the
ability of nanoparticles to cross the BBB [2831]. Hans and
Lowman [28] stated that in blood circulation, nanoparticles
coated with polysorbate 80 adsorbs apolipoprotein E on the
surface of the coating, the apolipoprotein E mimics the low
density lipoprotein (LDL) causing the particles to be trans-
ported via the LDL receptors into the brain. Our production
of nanoparticles involved two rounds of homogenization and
sonication. We successfully produced spherical polycaprolac-
tone nanoparticles with suitable characteristics. The mean
size of the dried loaded particles was 329.62 ± 121.91 nm
which was desirable and hypothesized to be able to improve
drug penetration across the BBB. The particles were also
observed to have higher negative charge of 33.4 ± 8.12
which could induce the repulsive force to the particles when
they get into contact with the biological membranes which
are negatively charged. However, we suggested that nano-
particles being coated with polysorbate 80 which adsorbs
apolipoprotein E would increase the penetration of nanopar-
ticles across the BBB [29,32].
Drug release showed that pentamidine released was slow
and constant over 24 h. Drug diffusion and degradation of
PCL polymer is usually slow and the release would be
expected to start slowly and extend as the polymer erodes
[33,34]. However, the constant release showed that PCL
hydrolyzes slowly and thus sustain slow drug release over a
longer period as observed [33]. In addition, the longer release
might be caused by poor drug loading and thus slower
release rate [28] we observe. This could be good for the
release of toxic drugs like pentamidine but also we think that
optimization of the drug loading protocols could help to
improve the encapsulation efficiency.
Liposomes are made of phospholipids and hence consid-
ered biocompatible, biodegradable and less toxic [21].
Because liposomes are amphiphilic in nature they are appro-
priate for encapsulation of drugs and delivery of both hydro-
philic and hydrophobic drugs [35]. Therefore, liposomes were
also an appropriate choice for encapsulation of pentamidine
in this study. The aim was to ascertain which of the nanocar-
riers between polymeric nanoparticles and liposomes would
be efficient in transporting pentamidine across in vitro endo-
thelial cells of the BBB. We successfully formulated liposomes
of the desirable characteristics for delivery of pentamidine.
We hypothesized that with the increased concentration of
liposomes on the luminal side of the endothelial membrane,
liposomes would be easily transported across the barrier.
This study demonstrated that higher concentrations of
both PCL nanoparticles and liposomes may inhibit cellular
growth. We observed that PCL NPS at a dose of 5 mg/ml and
liposomes at 2.5 mg/ml and 5 mg/ml were able to inhibit
multiplication of cells and caused death to cells (Figures 5
and 6). However, this study alone cannot justify the real
cause of cytotoxicity. Nevertheless, polysorbate 80 and TPGS
may be toxic if used at high concentrations.
TEER values are a good indicator when the integrity and
permeability of the biological barrier like the BBB is assessed
[36]. When TEER values rise, this typically indicates that the
barrier restricts the movement of any solute through the
tight junction. On the other hand, a drop in TEER values is
also a good indicator that the barrier allows the passive
movement of molecules through the tight junction.
The average size of pentamidine loaded liposomes was about
2 4 6 8 12 24
Time (h)
Mean % dose transported
Mean % dose transported by PCL NPs
Mean % dose transported by liposomes
Mean % dose of free pentamidine crossed
Figure 11. Shows the percentage dose of free pentamidine, pentamidine-
loaded PCL NPS and pentamidine-loaded liposomes transported across the cells
monolayer. Here, we were testing the transportation ability of liposomes and
PCL NPs nanocarriers to ferry pentamidine across the BBB as compared to free
movement of pentamidine. Data is presented as mean ± SE, n¼3. Transport
means differences between liposome and PCL NPs, and liposome and free drug
were found statistically significant at p<.05.
119 nm and we suggest that most of the liposomes together
with other factors penetrated the barrier through the para-
cellular pathway. However, liposomes are lipophilic in nature
and that might have added the advantage for them to cross
the barrier. Restriction to PCL nanoparticles and free drug as
assessed by TEER values was comparatively the same. In our
experiment, we observed that liposome nanocarriers were
better and able to transport more dose percentage of pent-
amidine compared to PCL NPs and the movement of free
drug. Nevertheless, the two nanocarriers are promising deliv-
ery systems which can improve the delivery of pentamidine
drug across the BBB. Therefore, we recommend future studies
to evaluate these nanocarriers in vivo.
The authors would like to thank the UWC, South Africa and particularly
the Schools of Pharmacy and Life Sciences for provision of facilities and
laboratory space for carrying out this research. Furthermore, authors
extend thanks to Dr. Cummings and Mr. Earl from the School of Physics
for helping the morphological characterization of nanoparticles and lipo-
somes by SEM. We also thank NMAIST for providing permission to under-
take this study at the UWC. GO acknowledges the Tanzanian Higher
Education StudentsLoan Board (HESLB) and NMAIST CREATES Program
for providing financial support that enabled undertaking of this project.
Disclosure of interest
The authors report no conflict of interest.
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... Pentamidine-loaded nanoparticles and phosphatidylcholine liposomes were compared by Geofrey [72]. The goal of this study was to see if these two formulations could cross the blood-brain barrier in vitro. ...
... When the PDI of these two formulations was measured, it was discovered that freeze-dried pentamidine loaded liposomes had a PDI greater than 0.6. Non-lyophilized pentamidine loaded PCL NPs, on the other hand, showed particle size uniformity with a low PDI (0.25 ± 0.15) and lower aggregation [72]. ...
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... Infusion-related adverse effects and nephrotoxicity are its major limiting parameters [42]. Pentamidine (PTM) therapy is often accompanied by problems such as painful necrotic injection site lesions, nephrotoxicity, and hypoglycemia; the main reasons for its poor adherence and associated lower cure rates [43,44]. The discovery of anti-leishmanial activity of MFS is, merely, a novelty because it is the first oral anti-leishmanial drug for the treatment of Leishmaniasis [45,46]. ...
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... NLB-SLN brain targeting was confirmed by noninvasive scintigraphy, reaching its maximum permeability eighth h after intranasal administration. Omarch et al. [145] conducted a comparative study; the authors developed polymeric PCL NPs and phosphatidylcholine LP to evaluate pentamidine in vitro transport through the BBB. The pentamidine-loaded PCL NPs had a mean size of 267.58 nm, PDI of 0.25, and zeta potential of -28.1 mV, while pentamidine-loaded LP had a mean size of 119.61 nm, PDI of 0.25, and zeta potential 11.78 mV. ...
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... Sortilin mAb, in the case of brain tumors), which act as a great therapeutic agent in highly-selective drug delivery, are also unable to cross BBB. Thus, researchers are focusing on different strategies executable for delivery of small therapeutic medications across BBB and more specifically to the target brain cells [89,90]. In this context, efficient nanoscale drug delivery systems are being developed to overcome the limitations related to the BBB (Fig. 6). ...
Obviously, delivery of the medications to the brain is more difficult than other tissues due to the existence of a strong obstacle, which is called blood-brain barrier (BBB). Because of the lipophilic nature of this barrier, it would be a complex (and in many cases impossible) process to cross the medications with hydrophilic behavior from BBB and deliver them to the brain. Thus, novel intricate drug-carriers in nano scales have been recently developed and suitably applied for this purpose. One of the most important categories of these hydrophilic medications, are reactivators for acetyl cholinesterase (AChE) enzyme that facilitates the breakdown of acetylcholine (as a neurotransmitter). The AChE function is inhibited by organophosphorus (OP) nerve agents that are extremely used in military conflicts. In this review, the abilities of the nanosized drug delivery systems to perform as suitable vehicles for AChE reactivators are comprehensively discussed.
... In this field, among the different drug delivery nanosystems, liposomes and nanoparticles have been proposed to carry PTM 14,15 and even a recent study using both polycaprolactone nanoparticles and phosphatidylcholine liposomes was performed to compare the in vitro transportation of PTM across the blood-brain barrier. 16 On the contrary, only a minor effort has been devoted to the use of nanocarriers to deliver PTM in anticancer therapy. Concerning liposomes in this field, M erian et al. tested some liposomal PTM formulations in order to enhance tumor drug accumulation and lower drug exposure to other tissues. ...
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Sleeping sickness, also known as human African trypanosomiasis (HAT), is a neglected disease that impacts 70 million people living in 1.55 million km² in sub-Saharan Africa. Since the beginning of the 20th century, there have been multiple HAT epidemics in sub-Saharan Africa, with the most recent epidemic in the 1990s resulting in about half a million HAT cases reported between 1990 and 2015. Here we review the status of HAT disease at the current time and the toolbox available for its control. We also highlight future opportunities under development towards novel or improved interventions.
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Introduction: Vascular-targeted drug delivery is a promising approach for the treatment of atherosclerosis, due to the vast involvement of endothelium in the initiation and growth of plaque, a characteristic of atherosclerosis. One of the major challenges in carrier design for targeting cardiovascular diseases (CVD) is that carriers must be able to navigate the circulation system and efficiently marginate to the endothelium in order to interact with the target receptors. Areas covered: This review draws on studies that have focused on the role of particle size, shape, and density (along with flow hemodynamics and hemorheology) on the localization of the particles to activated endothelial cell surfaces and vascular walls under different flow conditions, especially those relevant to atherosclerosis. Expert opinion: Generally, the size, shape, and density of a particle affect its adhesion to vascular walls synergistically, and these three factors should be considered simultaneously when designing an optimal carrier for targeting CVD. Available preliminary data should encourage more studies to be conducted to investigate the use of nano-constructs, characterized by a sub-micrometer size, a non-spherical shape, and a high material density to maximize vascular wall margination and minimize capillary entrapment, as carriers for targeting CVD.
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Pralidoxime is an organophosphate antidote with poor central nervous system distribution due to a high polarity. In the present study, pralidoxime-loaded poly(lactic-co-glycolic acid) nanoparticles were prepared and evaluated as a potential delivery system of the drug into the central nervous system. The nanoparticles were prepared using double emulsion solvent evaporation method. Poly(lactic-co-glycolic acid) (PLGA) in ethyl acetate made the organic phase and pralidoxime in water made the aqueous phase. The system was stabilized by polyvinyl alcohol. Different drug/polymer ratios were used (1 : 1, 1 : 2, and 1 : 4) and the fabricated particles were characterized for encapsulation efficiency using UV-VIS Spectroscopy; particle size distribution, polydispersity index, and zeta potential using photon correlation spectroscopy; and in vitro drug release profile using UV-VIS Spectroscopy. Mean particle sizes were 386.6 nm, 304.7 nm, and 322.8 nm, encapsulation efficiency was 28.58%, 51.91%, and 68.78%, and zeta potential was 5.04 mV, 3.31 mV, and 5.98 mV for particles with drug/polymer ratios 1 : 1, 1 : 2, and 1 : 4, respectively. In vitro drug release profile changed from biphasic to monobasic as the drug/polymer ratio decreased from 1 : 1 to 1 : 4. Stable pralidoxime-loaded PLGA nanoparticles were produced using double emulsion solvent evaporation techniques.
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Transepithelial/transendothelial electrical resistance (TEER) is a widely accepted quantitative technique to measure the integrity of tight junction dynamics in cell culture models of endothelial and epithelial monolayers. TEER values are strong indicators of the integrity of the cellular barriers before they are evaluated for transport of drugs or chemicals. TEER measurements can be performed in real time without cell damage and generally are based on measuring ohmic resistance or measuring impedance across a wide spectrum of frequencies. The measurements for various cell types have been reported with commercially available measurement systems and also with custom-built microfluidic implementations. Some of the barrier models that have been widely characterized using TEER include the blood-brain barrier (BBB), gastrointestinal (GI) tract, and pulmonary models. Variations in these values can arise due to factors such as temperature, medium formulation, and passage number of cells. The aim of this article is to review the different TEER measurement techniques and analyze their strengths and weaknesses, determine the significance of TEER in drug toxicity studies, examine the various in vitro models and microfluidic organs-on-chips implementations using TEER measurements in some widely studied barrier models (BBB, GI tract, and pulmonary), and discuss the various factors that can affect TEER measurements. © 2015 Society for Laboratory Automation and Screening.
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Targeted delivery of therapeutics is an alternative approach for the selective treatment of infectious diseases. The surface of African trypanosomes, the causative agents of African trypanosomiasis, is covered by a surface coat consisting of a single variant surface glycoprotein, termed VSG. This coat is recycled by endocytosis at a very high speed, making the trypanosome surface an excellent target for the delivery of trypanocidal drugs. Here, we report the design of a drug nanocarrier based on poly ethylen glycol (PEG) covalently attached (PEGylated) to poly(D,L-lactide-co-glycolide acid) (PLGA) to generate PEGylated PLGA nanoparticles. This nanocarrier was coupled to a single domain heavy chain antibody fragment (nanobody) that specifically recognizes the surface of the protozoan pathogen Trypanosoma brucei. Nanoparticles were loaded with pentamidine, the first-line drug for T. b. gambiense acute infection. An in vitro effectiveness assay showed a 7-fold decrease in the half-inhibitory concentration (IC50) of the formulation relative to free drug. Furthermore, in vivo therapy using a murine model of African trypanosomiasis demonstrated that the formulation cured all infected mice at a 10-fold lower dose than the minimal full curative dose of free pentamidine and 60% ofmice at a 100-fold lower dose. This nanocarrier has been designedwith components approved for use in humans and loaded with a drug that is currently in use to treat the disease. Moreover, this flexible nanobody-based system can be adapted to load any compound, opening a range of new potential therapies with application to other diseases.
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Introduction: Nanoparticles have been successfully used for cancer drug delivery since 1995. In the design of commercial nanoparticles, size and surface characteristics have been exploited to achieve efficacious delivery. However, the design of optimized drug delivery platforms for efficient delivery to disease sites with minimal off-target effects remains a major research goal. One crucial element of nanoparticle design influencing both pharmacokinetics and cell uptake is nanoparticle morphology (both size and shape). In this succinct review, the authors collate the recent literature to assess the current state of understanding of the influence of nanoparticle shape on the effectiveness of drug delivery with a special emphasis on cancer therapy. Areas covered: This review draws on studies that have focused on the role of nonspherical nanoparticles used for cancer drug delivery. In particular, the authors summarize the influence of nanoparticle shape on biocirculation, biodistribution, cellular uptake and overall drug efficacy. By comparing spherical and nonspherical nanoparticles, they establish some general design principles to serve as guidelines for developing the next generation of nanocarriers for drug delivery. Expert opinion: Pioneering studies on nanoparticles show that nonspherical shapes show great promise as cancer drug delivery vectors. Filamentous or worm-like micelles together with other rare morphologies such as needles or disks may become the norm for next-generation drug carriers, though at present, traditional spherical micelles remain the dominant shape of nanocarriers described in the literature due to synthesis and testing difficulties. The few reports that do exist describing nonspherical nanoparticles show a number of favorable properties that should encourage more efforts to develop facile and versatile nanoparticle synthesis methodologies with the flexibility to create different shapes, tunable sizes and adaptable surface chemistries. In addition, the authors note that there is a current lack of understanding into the factors governing (and optimizing) the inter-relationships of size, surface characteristics and shapes of many nanoparticles proposed for use in cancer therapy.
In this modern era, with the help of various advanced technologies, medical science has overcome most of the health-related issues successfully. Though, some diseases still remain unresolved due to various physiological barriers. One such condition is Alzheimer; a neurodegenerative disorder characterized by progressive memory impairment, behavioral abnormalities, mood swing and disturbed routine activities of the person suffering from. It is well known to all that the brain is entirely covered by a protective layer commonly known as blood brain barrier (BBB) which is responsible to maintain the homeostasis of brain by restricting the entry of toxic substances, drug molecules, various proteins and peptides, small hydrophilic molecules, large lipophilic substances and so many other peripheral components to protect the brain from any harmful stimuli. This functionally essential structure creates a major hurdle for delivery of any drug into the brain. Still, there are some provisions on BBB which facilitate the entry of useful substances in the brain via specific mechanisms like passive diffusion, receptor-mediated transcytosis, carrier-mediated transcytosis etc. Another important factor for drug transport is the selection of a suitable drug delivery systems like, liposome, which is a novel drug carrier system offering a potential approach to resolving this problem. Its unique phospholipid bilayer structure (similar to physiological membrane) had made it more compatible with the lipoidal layer of BBB and helps the drug to enter the brain. The present review work focused on various surface modifications with functional ligand (like lactoferrin, transferrin etc.) and carrier molecules (such as glutathione, glucose etc.) on the liposomal structure to enhance its brain targeting ability towards the successful treatment of Alzheimer disease.
A high performance liquid chromatographic method for quantitating pentamidine in plasma has been developed. Sample clean-up involved precipitating plasma with acetonitrile containing the internal standard, hexamidine. The supernatant was passed through a C//8 Bond Elut column and eluted with a methanolic solution of sodium 1-heptanesulfonate. The eluate was then analyzed on an Altex C//8 column with a mobile phase consisting of 45% CH//3CH, 0. 02% tetramethylammonium chloride and 0. 1% H//3PO//4. Using fluorescence detection (EX: 275 nm and EM: 340 nm), the detection limit was 1. 25 ng/mL for 0. 5 mL of plasma. The coefficients of variation for interday and intraday were around 10%.
Polymeric nanoparticles have attracted growing attention because of their unique properties and extensive application. In this study, polycaprolactone (PCL) nanoparticles were prepared via double emulsion solvent evaporation-like process using power ultrasound, and the effects of various process parameters on particle size, zeta potential, and morphology were investigated and optimized. Nanoparticles (NPs) were prepared by two-step emulsification process. In the first step, the inner aqueous phase (W1) was homogenized with organic phase (PCL in dichloromethane) to obtain primary emulsion. In the second step, the primary emulsion was emulsified with outer aqueous phase (W2) containing polyvinyl alcohol (PVA) as stabilizer using power ultrasound, followed by evaporation of solvent which resulted in a particulate suspension at the end. Effects of various parameters like ultrasound exposure time and amplitude, outer aqueous phase volume, PVA concentration, and PCL content were investigated. It has been shown that, by increasing ultrasound exposure time, amplitude, and outer aqueous phase volume, the particle size decreases. Additionally, particle size was also related to amount of PCL and PVA concentration. Spherical NPs with smooth surfaces were observed by scanning electron microscopy (SEM).