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J Pharm Pharm Sci (www.cspsCanada.org) 16(3) 470 - 485, 2013
470
Design and Pharmaceutical Evaluation of a Nano-Enabled Crosslinked
Multipolymeric Scaffold for Prolonged Intracranial Release of
Zidovudine
Sheri-lee Harilall1, Yahya E. Choonara1, Girish Modi2, Lomas K. Tomar1, Charu Tyagi1, Pradeep Kumar1, Lisa C. du
Toit1, Sunny E. Iyuke3, Michael P. Danckwerts1 and Viness Pillay1
1 University of the Witwatersrand, Faculty of Health Sciences, Department of Pharmacy and Pharmacology, 7 York
Road, Parktown, Johannesburg, South Africa. 2 University of the Witwatersrand, Department of Neurology, Division of
Neurosciences, Johannesburg, South Africa. 3 University of the Witwatersrand, School of Chemical and Metallurgical
Engineering, Johannesburg, South Africa.
Received, May 9, 2013; Revised, June 18, 2013; Accepted, July 9, 2013; Published, July 11, 2013.
Abstract – Purpose. Nanomedicine explores and allows for the development of drug delivery devices with
superior drug uptake, controlled release and fewer drug side-effects. This study explored the use of
nanosystems to formulate an implantable drug delivery device capable of sustained zidovudine release over
a prolonged period. Methods. Pectin and alginate nanoparticles were prepared by applying a salting out and
controlled gelification approach, respectively. The nanoparticles were characterized by attenuated total
reflectance-fourier transform infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM),
transmission electron microscopy (TEM) and dynamic light scattering (DLS) and were further evaluated for
zidovudine (AZT) entrapment efficiency. Multipolymeric scaffolds were prepared by crosslinking
carboxymethyl cellulose, polyethylene oxide and epsilon caprolactone for entrapment of zidovudine-loaded
alginate nanoparticles to impart enhanced controlled release of zidovudine over the time period. Swelling
and textural analysis were conducted on the scaffolds. Prepared scaffolds were treated with hydrochloric
acid (HCl) to reduce the swelling of matrix in the hydrated environment thereby further controlling the drug
release. Drug release studies in phosphate buffered saline (pH 7.4, 37°C) were undertaken on both
zidovudine-loaded nanoparticles and native scaffolds containing alginate nanoparticles. Results. A higher
AZT entrapment efficiency was observed in alginate nanoparticles. Biphasic release was observed with both
nanoparticle formulations, exhibiting an initial burst release of drug within hours of exposure to PBS,
followed by a constant release rate of AZT over the remaining 30 days of nanoparticle analysis. Exposure of
the scaffolds to HCl served to reduce the drug release rate from the entrapped alginate nanoparticles and
extended the AZT release up to 30 days. Conclusions. The crosslinked multipolymeric scaffold loaded with
alginate nanoparticles and treated with 1% HCl showed the potential for prolonged delivery of zidovudine
over a period of 30 days and therefore may be a potential candidate for use as an implantable device in
treating Aids Dementia Complex.
This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For
Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.
_______________________________________________________________________________________
INTRODUCTION
The National Science Foundation in the United
States of America (USA) estimates that by 2015,
the annual global market for nanotechnological-
related goods and services will reach $1 trillion. It
has also been estimated that by 2014, 16% of
healthcare and life sciences related commodities
will incorporate nanotechnology, making it one of
the fastest growing industries in history, and an
exceptionally large economic force (1).
Nanotechnology offers much promise for the
enhancement of disease diagnosis, management
and prevention (2,3). Drug incorporation into
nanosystems is used to achieve site-specific drug
delivery, providing improved control of drug
release which augments the bioavailability,
efficacy, pharmacokinetics and
pharmacodynamics of the drug. Nanostructures
offer drug release for desired time periods,
thereby improving therapeutic efficacy and
reducing drug toxicity (2,4,5). Nanoparticles can
even be used as carriers for poorly soluble drugs,
acting to augment bioavailability of such drugs
(6,7,8).
________________________________________
Corresponding Author: Professor Viness Pillay; University
of the Witwatersrand, Faculty of Health Sciences,
Department of Pharmacy and Pharmacology, 7 York Road,
Parktown, Johannesburg, South Africa; Email:
viness.pillay@wits.ac.za
J Pharm Pharm Sci (www.cspsCanada.org) 16(3) 470 - 485, 2013
471
Nanocarriers are made from an assortment of
organic and inorganic materials including non-
degradable and biodegradable polymers, lipids,
self-assembling amphiphilic molecules,
dendrimers, metal, and inorganic semiconductor
nanocrystals (2). Biocompatible and
biodegradable polymers with desirable
physicochemical and physicomechanical
properties have been researched extensively for
the formulation of novel drug delivery systems.
Alginate is one such hydrophillic natural
polysaccharide of algal origin, which is
biocompatible, biodegradable and non-toxic in
nature. It comprises of linear chains of α-1-
glucuronic acid (G) and (1-4)-linked β-D-
mannuronic acid (M) residues, which vary in
composition and sequence (9,10,11). Alginate
forms insoluble gels in the presence of bivalent
calcium (Ca2+) ions and other multivalent metals
by binding to the carboxylic groups of adjacent G
units in the polymer. This Ca2+-alginate gel is
frequently used as drug delivery vehicle (10,11).
Rajaonarivony et al., in 1993 developed a method
for the preparation of alginate nanoparticles using
a novel cation induced pre-gelation of alginate
approach (12). This method has since been
adapted by many scientists for the preparation of
alginate nanoparticles, with chitosan being used
instead of poly-L-lysine which is a cationic
natural polymer but toxic and immunogenic if
injected. These nanoparticles have been used as
carrier for a variety of drugs, including insulin,
chemotherapeutics, as well as the hydrophobic
antihypertensive agent, Nifedipine (9,10,13-17).
Pectin is another linear polysaccharide of plant
origin, derived by aqeuos extraction of edible
plants, mainly apples and citrus fruits (10,18,19).
It is a biocompatible, biodegradable, stable, non-
toxic and hydrophillic natural polymer mainly
consisting of α-(1→4)-D-galacturonic acid
residues arranged in a linear fashion (10,18-20).
Like alginate pectin forms gel in the presence of
Ca2+ ions, sugars and acids and is therefore
utilised in the preparartion of various food and
pharmaceutical products. Sodium carboxymethyl
cellulose (NaCMC) is a carboxymethyl ether of
cellulose, a naturally occurring plant
polysaccharides present in the fibrous tissue of all
plants. The polymer exhibits high water solubility
attributed to the introduction of sodium
carboxymethyl groups into the cellulose
molecule, by etherification of the hydroxyl groups
of glucopyranose unit (18, 21-24). NaCMC is
often utilised pharmaceutically as a viscosity
enhancer in oral, parental and topical formulations
(18,23).
Epsilon caprolactone (ECL) is synthetic,
semi-crystalline aliphatic polyester, whose
properties can be altered by catalysed ring-
opening copolymerisation. ECL has been
employed for the preparation of many sustained
release drug delivery formulations due to its slow
rate of degradation, as well as superior drug
permeability (25-28). Polyethylene oxide (PEO)
is a semi-crystalline water soluble polymer. The
hydrophilic hydroxyl (OH) groups present in the
molecule, allows for bioadhesivity and
mucoadhesivity of the polymer. PEO is frequently
used for oral drug delivery due to its ability to
swell and form a gel layer upon exposure to
water, thereby controlling drug release as a result
of retarded erosion of the formulation (29,30).
The novelty of this study involves the use of
the polymers described above to prepare
nanoparticles and a crosslinked multipolymeric
scaffold for prolonged drug delivery of the model
drug zidovudine (AZT) as a biodegradable,
biocompatible and non-cytogenic device for the
potential intracranial treatment of Aids Dementia
Complex (ADC). The by-products of these
polymers are non-toxic and readily excreted from
the body (7,27,31). They possess desirable
mechanical properties and superior drug
permeability and are therefore safe for use in the
human body. (7,27,28,31,32). Nanoparticles were
prepared using two methods, the salting-out
approach and the controlled gelification of
alginate approach. Suitable nanoparticles were
then dispersed within a multipolymeric scaffold,
prepared by means of polymer crosslinking to
obtain a drug delivery device capable of sustained
drug release over a period of at least 30 days.
METHODS
Materials
Alginate, pectin, polyethylene oxide (PEO),
polyvinyl alcohol (PVA), sodium carboxymethyl
cellulose (NaCMC) and epsilon caprolactone
(ECL) were purchased from Sigma Aldrich (St
Louis, MO, USA). These polymers were utilized
to prepare nanoparticles and the polymer scaffold.
Calcium chloride (CaCl2), zinc sulphate solution
(ZnSO4), aluminium chloride (AlCl3) and sodium
thiosulphate (Na2S2O3) were also purchased from
Sigma Aldrich (St Louis, MO, USA) and were
used as the crosslinking agents for the synthesis
of the nanoparticles and the multipolymeric
scaffold. Zidovudine (AZT) used as the model
drug and was purchased from Evershine Ind.,
(Naejar Malad, Mumbai, India). All buffers were
prepared using Milli-Q water.
J Pharm Pharm Sci (www.cspsCanada.org) 16(3) 470 - 485, 2013
472
Preparation of the polymeric nanoparticles
Pectin nanoparticles prepared by the salting-
out approach
Pectin nanoparticles were prepared by employing
a salting-out approach whereby a polymeric
dispersion was formed by agitating 0.8% w/v
pectin (50mL) with a 24% w/v ZnSO4 solution
containing 0.6g PVA (75mL) that was used as the
salting-out reagent. The rapid addition of
deionized water to the polymeric dispersion
induced the formation of nanoparticles which
were subsequently lyophilized for 24 hours. In
situ drug-loading of AZT was accomplished by
dissolving the drug in the pectin solution prior to
the addition of the crosslinking solution.
Alginate nanoparticles prepared by controlled
gelification
Nanoparticles were also prepared by ionotropic
gelification of anionic sodium alginate (0.6%w/v)
(200mL) with cationic CaCl2 (10mL) after which
a 0.5%w/v pectin solution (40mL) was added to
the Ca2+-alginate gel to form a stable nanoparticle
colloidal system. The crosslinked solution was
then rapidly stirred for 1 hour and thereafter
centrifuged (Optima® LE-80K, Beckman, USA)
at 20,000rpm for 15 minutes. The supernatant was
then removed and lyophilized for 24 hours at
25mtorrs (Virtis, Gardiner, NY, USA) to obtain a
free flowing powder after having refrigerated the
sample at -70°C. AZT-loaded nanoparticles were
prepared similarly with 0.3g of AZT dissolved in
the alginate solution prior to CaCl2 addition to
enable encapsulation of the drug within a
polymeric core.
Preparation of the crosslinked multipolymeric
scaffold
Polymer solutions of NaCMC (3%w/v, 100mL),
PEO (1%w/v, 100mL) and ECL (5%w/v, 100mL)
were blended until a homogenous mixture was
obtained followed by the dispersion of the AZT-
loaded alginate nanoparticles (7.265g) into the
multipolymeric solution. The polymeric mixture,
in 5mL increments, was then placed into Teflon
moulds (measuring 15mm in diameter and 20mm
in height) lubricated with liquid paraffin,
containing 2mL of a 10%w/v AlCl3, CaCl2 and
Na2S2O3 crosslinking solution. The polymeric
mixture with the dispersed nanoparticles was left
to crosslink for 30 minutes after which the
resultant scaffolds were removed and allowed to
dry at 25°C. NaCMC-PEO-ECL crosslinked
scaffolds were then agitated in a 1%v/v HCl
solution for 15 minutes. This was undertaken in
an attempt to reduce the swelling properties of the
scaffold thereby improving erosion and drug
release when potentially used intracranially.
Characterization Studies
Investigation of the chemical structural
transitions of the nanoparticles and scaffold
Fourier Transform Infrared (FTIR) spectroscopy
was utilized to assess possible structural
transitions which had occurred during the
preparation process by comparing the functional
groups of the parent compounds with that of the
formulations produced. Polymer, nanoparticle and
scaffold samples were analysed using a Perkin
Elmer FTIR Spectrometer (Spectrum 100,
Beaconsfield, United Kingdom). Spectra are
recorded over the range 4000-625cm-1, with a
resolution of 4cm-1 and 32 accumulations.
Transmission electron microscopy analysis of
the synthesized nanoparticles
Nanoparticles were dispersed in methanol and
sonicated to deflocculate the particles. The
samples were placed on carbon grids and analysed
using transmission electron microscopy (TEM)
(JEOL 1200 EX, 120keV) and photomicrographs
were obtained to study the morphology of the
individual particles produced.
Analysis of the surface morphology of the
multipolymeric scaffold
Scanning electron microscopy (SEM), (JEOL,
Tokyo, Japan) was employed for observation of
the surface morphology of the multipolymeric
scaffold. Samples were coated with carbon and
gold-palladium for 10 minutes after which they
were visualized between 5-9keV under different
magnifications. SEM micrographs were obtained
at various magnifications and analyzed for
description of the surface morphology. The
degree of polymer entanglement, network density
and porosity of the scaffold was qualitatively
determined using the photomicrographs obtained.
Determination of nanoparticle size and zeta
potential
A ZetaSizer Nano ZS (Malvern Instruments Ltd,
UK) was utilized to determine the average size
and size distribution of the prepared nanoparticles
employing non-invasive back scatter (NIBS)
technology with particle size distribution
determined using dynamic light scattering.
Brownian motion was measured and related to
particle size. The Stokes-Einstein equation
(Equation 1) was used to determine the
translational diffusion coefficient (D), which
defined the velocity of Brownian motion. D
depends on size of the particle core and the
J Pharm Pharm Sci (www.cspsCanada.org) 16(3) 470 - 485, 2013
473
surface structures on the particle and the
concentration and type of ions in the medium.
Particle size was calculated using the translational
diffusion coefficient.
d(H)=kT/3πηD (Equation 1)
Where: d(H)=hydrodynamic diameter,
D=translational diffusion coefficient
k=Boltzmann’s constant
T=absolute temperature
η=viscosity
Texture profile analysis to assess the
physicomechanical properties of the scaffold
Textural profiling of the 3D core of the
crosslinked scaffold enabled characterization of
the physicomechanical properties of the prepared
multipolymeric scaffold. A Texture Analyzer
(TA.XTplus, Stable Microsystems, Surrey, UK)
was employed to establish various stress-strain
parameters of the polymeric scaffold. Samples
were assessed in both the hydrated and
unhydrated states. Force-Distance and Force-
Time profiles were obtained and the Matrix
Resilience and Matrix Hardness were computed.
In vitro swelling studies on the multipolymeric
scaffold
The NaCMC-PEO-ECL multipolymeric scaffold
was tested for change in volume after exposure to
100mL of PBS at predetermined intervals to
assess the degree of swelling of the polymer
scaffold. Samples were removed from the PBS,
blotted on filter paper, after which the mean of
three diameters and lengths was calculated
following measurements using a callipers while
the volume (V) was calculated employing
Equation 3.
V = πr2l (Equation 2)
Where, r is the radius (mm) and l, the length
(mm). Studies were conducted in triplicate to
ensure accuracy of the results obtained.
Drug entrapment efficiency studies on the
prepared nanoparticles
Drug entrapment efficiency (DEE) was assessed
by homogenizing 100mg of the AZT-loaded
nanoparticles in 100mL of PBS, pH 7.4 (Polytron
PT2000, Kinematika AG, Switzerland) to
completely release all entrapped AZT. Free drug
molecules were removed from the nanoparticle
formulation prior to homoginization by washing
the nanoparticles in distilled water. The total
content of entrapped AZT was established in
triplicate by means of ultraviolet (UV)
spectroscopy, (Specord 40, Analytik Jena, AG,
Germany) at a wavelength of 267nm. Due to
possible interference of the polymer and
excipients used to prepare the samples, a solution
of 100mg of drug-free nanoparticles dissolved in
PBS was used as a reference. Equation 4 was
employed to calculate the DEE value.
%DEE = Da/Dt x 100 (Equation 3)
Where Da is the actual quantity of drug
(mg/100mL) and Dt is the theoretical quantity of
drug (mg/100mL) entrapped within the
formulation. The study was performed on five
samples, with an average DEE being calculated to
ensure accuracy of results obtained.
In vitro drug release studies
Drug release studies were performed on AZT-
loaded nanoparticle formulations and the
multipolymeric scaffold containing the AZT-
loaded alginate nanoparticles by immersing both
sets of formulations in 100mL PBS separately and
placing them in an orbital shaking incubator
(25rpm, 37°C). Samples were withdrawn at
predetermined intervals and were analysed using
UV spectroscopy at 267nm wavelength for AZT
concentration.
RESULTS
Assessment of infrared spectroscopy data for
chemical structure transition analysis
Figure 1 illustrates the FTIR spectra of prepared
nanoparticle and scaffold compared with the
parent polymeric compounds and AZT. In
Figure1b characteristic hydroxyl band between
3600 and 3200cm-1 of the alginate molecule is
observed in the spectra of alginate nanoparticle
aswell. Peaks observed at 1592 and 1406cm-1 are
attributed to asymmetric and symmetric –COO-
stretching. In the FTIR spectra of pectin (Figure
1b), the presence of hydroxyl groups was also
observed between 3600 and 3200cm-1 and -COO-
stretching occurred at 1590cm-1. The peak at
3458cm-1 in the AZT spectra is indicative of
hydroxyl substituents and the bands between 3200
and 3000cm-1 are due to amine groups. Bands
between 2100 and 1900cm-1 are attributed to
multiple bonded N2 molecules. The vibration
energies of -C=O moieties present in the AZT,
molecule was observed at 2813 and 1671cm-1.
Characteristic bands were present in the spectra of
PVA at 3274 and 2907cm-1 indicating the
presence of hydroxyl groups and –C-H stretching
vibrations respectively. The bands present
J Pharm Pharm Sci (www.cspsCanada.org) 16(3) 470 - 485, 2013
474
between 1300 and 1000cm-1 are due to the
bending vibrations of alcohol moieties present in
PVA molecule or to –C-O stretching vibrations
(Figure 1b). The alginate nanoparticle formulation
presented with bands between 3600 and 3200cm-1
indicating the presence of possible hydroxyl or
amino substituents (Figure 1b). The FTIR spectra
of pectin nanoparticles (Figure 1c) displayed a
broad band between 3600 and 3200cm-1
indicating the presence of hydroxyls groups. A
peak at 2944cm-1, attributed to –C-H stretching
vibrations observed on the spectra of PVA and
pectin is absent in nanoparticle formulation.
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
cm-1
%T
3332.59
2921.49
2110.35
1642.15
1415.94
1377.38
1273.43
1088.15
3458.56
3147.63
3021.53
2935.78
2813.65
2081.61
1671.12
1465.51
1438.03
1402.90
1381.06
1351.41
1333.76
1316.51
1279.08
1259.30
1224.98
1208.85
1189.93
1143.47
1110.33
1087.78
1057.15
1020.09
1006.47
984.68
956.25
934.54
896.97
845.52
789.41
759.58
734.47
677.03
3235.66
2944.63
1738.43
1590.27 1408. 89
1339.19
1224.28
1145.74
1102.87
1077.52
1048.68
1013.56
915.68 837. 15
773.88
3236.91 1592. 46
1406.87
1316.94 1123. 70 946.94
Alginate
Alginate nanoparticle
Pectin
AZT
(a)
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
cm-1
%T
3235.66
2944.63
1738.43
1590.27
1408.89
1339.19
1224.28
1145.74
1102.87
1077.52
1048.68
1013.56
915.68
837.15
773.88
3274.95
2907.42
1711.34
1420.45 1325.27
1141.93
1086.69
835.07
3458.56
3147.63
3021.53
2935.78
2813.65
2081.61
1671.12
1465.51
1438.03
1402.90
1381.06
1351.41
1333.76
1316.51
1279.08
1259.30
1224.98
1208.85
1189.93
1143.47
1110.33
1087.78
1057.15
1020.09
1006.47
984.68
956.25
934.54
896.97
845.52
789.41
759.58
734.47
677.03
3231.59
1622.68
1505.58
1019.24
863.58
Pectin Nanoparticles
Polyvinyl Alcohol
AZT
Pectin
(b)
J Pharm Pharm Sci (www.cspsCanada.org) 16(3) 470 - 485, 2013
475
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
cm-1
%T
3224.89
1606.91
1084.58 990.29
3294.92
3196.86
3073.84
2969.43
2928.79
2857.42
1652.03
1486.25
1467.28
1437.56
1416.96
1364.99
1351.26
1332.68
1315.50
1291.03
1256.66
1238.71
1197.67
1125.19
1088.12
1020.59
982.69
963.94
892.39
866.59
822.61
802.06
692.70
2877.96
1466.67
1359.80
1341.68
1279.16
1241.19
1144.44
1094.28
1059.08
960.80 840.93
3252.20 2878.92 1588.27
1412.54
1322.09
3306.64
1610.97
1427.82
1335.21
993.54
Epsilon caprolactone
Polyethylene oxide
HCl treated multipolymeric
scaff old
Carboxymethyl cellulose
HCl untreated multipolymeric
scaffold
(c)
Figure 1. (a) FTIR spectra of nanoparticles prepared by means of a controlled gelification of alginate compared to the
parent compounds; (b) FTIR spectra of nanoparticles prepared using the salting out approach; (c) FTIR of the prepared
multipolymeric scaffold compared to the parent polymer compounds.
The FTIR spectra in Figure 1d illustrate the parent
polymer compounds compared with the prepared
multipolymeric scaffold treated and untreated
with HCl. From the FTIR spectra of NaCMC, a
broad band at 3252cm-1 can be observed due to
hydroxyl stretch vibrations and bands at 2878 and
1322cm-1 are due to -C-H groups. The peak at
1588cm-1 is typical of the carboxymethyl ether, (-
CH2-COOH), and that at 1412cm-1 is due to
symmetric stretching of the carboxylate group.
Bands present at 1018cm-1 are representative of
>CH–O–CH2 stretching. Bands present in the
FTIR spectra of PEO at 960cm-1 and between 900
and 1250cm-1 are due to –C-O group vibrations
and –C-O-C stretching respectively. Those at
2877cm-1 and between 1400 and 1300cm-1 are
due to –C-H stretching and the band` at 840cm-1
is due to –C-H bending vibrations. Bands between
1060 and 1150cm-1 can be attributed to the
aliphatic ether moieties in the structure of PEO.
The ECL spectra displays characteristic peaks
between 1050 and 1300cm-1, indicative of the
cyclic ester present in the structure of ECL
(Figure 1a) and the carbonyl –C=O stretching was
observed at 1652cm-1 and between 3400 and
3150cm-1. Bands between 2800 and 3000cm-1 are
due to –C-H stretching.
Transmission electron microscopy image
analysis of the nanoparticles
TEM conducted on the drug-loaded alginate
nanoparticles, prepared by means of the
controlled gelification of alginate approach,
revealed the presence of spherical particles,
whereas drug-free nanoparticles displayed a
tendency to agglomerate, as seen in Figure 2a-b.
Micrographs obtained from drug-free Zn-pectin
nanoparticles, prepared using the salting out
approach, also revealed agglomeration, and the
drug-loaded Zn-pectin nanoparticles showed
spherical morphology as observed in Figure 2c-d.
Surface morphological analysis of the
multipolymeric scaffold
SEM micrographs of NaCMC-PEO-ECL
multipolymeric scaffolds revealed a porous
polymer matrix, as observed in Figure 3.
Scaffolds exposed to 1%v/v HCl exhibited
smaller, more uniform pores than scaffolds
untreated with HCl (Figure 3(a)-(b)). HCl treated
scaffolds contained large areas of densely packed
polymer matrices, interspersed by porous regions
(Figure 3b), whereas HCl untreated scaffolds
contained immense craters within the matrix and
densely packed polymer regions were rarely noted
on the photomicrographs (Figure 3a). SEM was
J Pharm Pharm Sci (www.cspsCanada.org) 16(3) 470 - 485, 2013
476
also conducted on multipolymeric scaffolds
crosslinked only once. Photomicrographs
obtained for these samples contained larger pores
and more craters within the polymer matrix unlike
smaller pores present in multi-crosslinked
scaffolds. Air pockets are clearly visible in the
scaffold matrix as seen in Figure 3(c)-(d).
Nanoparticle size and zeta potential
Zeta potential and particle size distribution was
determined in deionised water. Zn-Pectin
nanoparticles displayed an average size
distribution of 1323nm (SD<0.008), with a
polydispersity index (PDI) of 0.816 for drug-free
nanoparticles and 119.6nm (SD<0.005), PDI
0.676, for AZT-loaded nanoparticles (Figure 4).
Multiple wide peaks were obtained (Figure 4a)
and microparticles observed with drug free
nanoparticles as agglomerates were formed which
could not be redispersed upon sonication of the
nanoparticles prior to analysis (Figure 5).
Drug-free pectin nanoparticles displayed an
average zeta potential of -0.012mV (SD<0.001),
whereas AZT-loaded nanoparticles displayed an
average zeta potential of -0.419mV (SD<0.004).
The average zeta potential of AZT-loaded alginate
nanoparticles was -6.39mV (SD<0.003) and that
of drug-free nanoparticles was -3.53mV
(SD<0.002) (Figure 6).
Texture profile analysis
Texture analysis performed on unhydrated
samples revealed a decrease in the resilience of
the HCl treated matrix compared to untreated
systems with a matrix resilience (MR) of 12.83%
(SD<0.004) as compared 18.12% (SD<0.004)
obtained with untreated scaffold. However,
hardness of both treated and untreated matrices
was similar, with treated scaffold having a mean
value of 3.45N/mm (SD<0.001) and untreated
scaffold having a mean value of 3.712N/mm
(SD<0.006) (Figure 7).
A decline in matrix resilience was accounted
upon scaffold hydration, both in the case of HCl
treated and untreated samples, the scaffolds
unexposed to HCl displaying a mean resilience of
8.043% (SD<0.004) and scaffolds exposed to HCl
displaying a mean resilience of 6.361%
(SD<0.004) (Figure 8).
Figure 2. TEM images of nanoparticles: (a) Drug free alginate nanoparticles displaying agglomeration, (1mm=20nm),
(b) AZT loaded alginate nanoparticles displaying the presence of spherical particles, (1mm=20nm), (c) Drug free Zn-
pectin nanoparticles displaying agglomeration, (d) AZT loaded Zn-pectin nanoparticles displaying inter-dispersed
spherical structures in the absence of agglomeration, (1mm=10nm).
J Pharm Pharm Sci (www.cspsCanada.org) 16(3) 470 - 485, 2013
477
Figure 3. Scanning Electron Micrograph of (a)-(b) multicrosslinked polymeric scaffolds: (a) HCl untreated, displaying
densely packed polymer matrix due to efficient crosslinking of the multipolymeric scaffold, (1mm=50μm). (b) HCl
treated, displaying uniform pores present within the polymer matrix of scaffolds which can efficiently entrap drug-
loaded nanoparticles, thereby serving to modulate drug release (1mm=125μm). (c)-(d) polymeric scaffolds crosslinked
once: (c) Craters can be seen between the pores in the matrix due to air bubbles in the polymer solution during scaffold
preparation (1mm=50μm). (d) Upon closer magnification, densely packed polymer regions and porous areas can be
observed surrounding the craters (1mm=250μm).
(a)
(b)
Figure 4. Size distribution profile of Zn-Pectin nanoparticles (a) drug-free nanoparticles, (n=3) (b) AZT-loaded
nanoparticles, (n=3).
J Pharm Pharm Sci (www.cspsCanada.org) 16(3) 470 - 485, 2013
478
(a)
(b)
Figure 5. Size distribution profile of alginate nanoparticles: (a) drug-free nanoparticles, (n=3) (b) AZT-loaded
nanoparticles, (n=3).
Figure 6. Zeta potential profile of AZT-loaded alginate nanoparticles, (n=3).
Matrix swelling analysis
The polymeric matrix exposed to HCl treatment
exhibited reduced swelling behaviour. However,
both sets of scaffolds still exhibit appreciable
swelling behaviour as seen in Figure 9. A reduced
erosion of the multipolymeric scaffolds treated
with HCl, was noticed in the first 24hours as
compared to untreated scaffolds (Figure 9).
However, upon prolonged exposure to PBS, pH
7.4, NaCMC-PEO-ECL multipolymeric scaffolds,
treated and untreated with HCl, displayed a
weight gain. Multipolymeric scaffolds exposed to
HCl displayed a larger percentage weight gain
than those unexposed to HCl.
Drug entrapment efficiency and in vitro
zidovudine release studies
A calibration curve was generated for AZT using
PBS, pH 7.4 at 25°C, employing a UV
spectrophotometry at the wavelength of maximum
absorption, 267nm. Alginate nanoparticles
exhibited superior DEE, 91.1%, as compared to
76.53% obtained with Zn-pectin nanoparticles.
Biphasic release was observed with both
nanoparticle formulations, consisting of an initial
burst release of drug within hours of exposure to
PBS, followed by a constant release rate of AZT
over the remaining 30days of nanoparticle
analysis (Figure 10a). Zn-pectin and alginate
nanoparticles displayed a burst release of 7.9%
and 14.9% respectively of the entrapped drug
after 1 hour post exposure to PBS. Incorporation
of alginate nanoparticles, chosen due to its
superior DEE, into the polymeric scaffold
significantly retarded drug release, 2.341%
(SD<0.005) obtained with NaCMC-PEO-ECL
scaffolds untreated with HCl and 1.576%
(SD<0.005) obtained with NaCMC-PEO-ECL
scaffolds treated with HCl, 30days post exposure
to PBS, pH 7.4 (Figure 10b).
J Pharm Pharm Sci (www.cspsCanada.org) 16(3) 470 - 485, 2013
479
(a)
(b)
Figure 7. Texture analysis: (a) Multipolymeric scaffold unexposed to HCl (i) hardness and (ii) resilience profiles; (b)
Multipolymeric scaffold exposed to HCl (i) hardness and (ii) resilience profiles, (n=3).
Figure 8. Percentage resilience of HCl treated and untreated scaffolds, upon exposure to PBS, pH 7.4 (n=3, SD<0.004)
(i) (ii)
(ii) (i)
J Pharm Pharm Sci (www.cspsCanada.org) 16(3) 470 - 485, 2013
480
(a)
(b)
Figure 9. Swelling behavior of HCl treated and untreated multipolymeric scaffolds (a) Volume change, (b) Percentage
weight change, (n=3).
J Pharm Pharm Sci (www.cspsCanada.org) 16(3) 470 - 485, 2013
481
(a) (b)
Figure 10. Drug release profile of (a) Zn-pectin and alginate nanoparticles and (b) alginate nanoparticles dispersed
within HCl treated and untreated multipolymeric scaffold, under cerebrospinal fluid simulated conditions (20rpm,
37ºC, 0.1M PBS, pH7.4) (n=3; SD<0.005 in all cases). Insets show release from free nanoparticles.
DISCUSSION
The analysis of prepared nanoparticles through
FTIR studies indicated that the basic polymeric
structures of the parent compounds was
maintained, however surface interactions occurred
in the formation of both Zn-pectin and alginate
nanoparticles and the scaffold, which is
represented by the shifting of the transmittance
peaks in the nanoparticle and the multipolymeric
scaffold preparations. In prepared alginate
nanoparticles the band at 3332cm-1 is more
defined than that of alginate, indicating possible
interactions of the polymers with drug AZT, as
AZT has an amino substituent present at 3458cm-1
as observed in the FTIR spectra. Polymer
interactions with AZT are further supported by
the presence of a band at 2110cm-1 which can be
attributed to N2 substitution or bonding (e.g. ionic
bonds). Shifting of the –COO- stretch to 1642cm-1
in the nanoparticle spectra indicates possible
interaction of the polymers with AZT or CaCl2.
The presence of hydroxyl band in pectin
nanoparticles is consistent with the spectra of the
parent polymers, PVA and pectin. The -C-H
stretching vibration in PVA and pectin is found
absent in the formed nanoparticle spectra
supporting interaction between the formulation
constituents. The carboxymethyl ether groups of
NaCMC can be observed in both the
multipolymeric scaffolds exposed and unexposed
to HCl at 1610 and 1606cm-1, respectively and
bands present between 3450 and 3200cm-1 are due
to hydroxyl groups. The C-O groups present in
the polymer structure allows for complexation of
PEO with other polymers and salts (33,34). A
shift in the occurrence of the hydroxyl groups can
be seen from 3306 to 3224cm-1 in the untreated
and HCl treated scaffolds respectively, indicating
possible interactions of the hydroxyl groups in
NaCMC with H+ ions upon exposure to HCl. This
is further supported by fewer bands present in the
FTIR spectra of HCl treated multipolymeric
scaffolds as compared to untreated scaffolds.
The micrographs of nanoparticles prepared
using both the salting-out and the controlled
gelification of alginate approach revealed
agglomeration in case of the drug-free
nanoparticles, whereas the drug-loaded
formulations appeared more disperse. This
observation relates to the low charge distribution
within the drug-free colloidal system. The
addition of AZT influenced the charge
distribution by interacting with the polymer as
confirmed by the FTIR data. This was further
supported by the zeta potential analysis.
The existence of craters in micrographs of
prepared scaffolds unexposed to HCl can be
explained on account of the two polymers used
for the preparation of the multipolymeric scaffold
being hydrogels, namely NaCMC and PEO.
Hydrogels, which are three-dimensional
crosslinked polymer capable of extreme swelling
due to absorption and retention of water, have a
loosely crosslinked structure due to their
hydrophilic nature, resulting in the presence of air
J Pharm Pharm Sci (www.cspsCanada.org) 16(3) 470 - 485, 2013
482
bubbles in the scaffolds unexposed to HCl (35).
Exposure of the scaffolds to HCl causes the
COONa group in CMC to change to the acidic
COOH which forms a more compact polymeric
matrix (36). The appearance of still larger pores in
scaffolds crosslinked only once can be explained
by the presence of higher concentrations of Ca2+
and Al3+ ions that form intramolecular complexes
within NaCMC, resulting in a tightly coiled
polymer matrix in multi-crosslinked scaffolds.
Such formulations with large pore size are not
ideal for nanoparticle dispersion, supporting the
need for multiple crosslinking to achieve an
optimum scaffold structure. The distinct bright
white areas visible on the multipolymeric
scaffolds are due to a high electric charge on the
surface accredited to the presence of multivalent
ions, present on the surface of the polymer matrix
(37), as well as free H+ and Cl- ions on scaffolds
exposed to 1%v/v HCl. A porous polymer matrix
is considered ideal for the dispersion of drug-
loaded nanoparticles. The drug release is
anticipated to enhance as the nanoparticles will be
released from polymer matrix when it swells or
erodes, allowing for the prolonged delivery of
drug. Additionally, premature drug degradation
will be prevented and drug toxicity reduced as
controlled drug release will be achieved (4,17).
Agglomeration of drug-free nanoparticles -
both Zn-pectin and alginate - as indicated by
TEM micrographs, nanoparticle size zeta
potential, can be attributed to poor crosslinking
and a low charge distribution within the colloidal
systems. Pectin microparticles observed are due to
nanoparticle agglomeration and poor crosslinking
of pectin with ZnSO4.The large particle size and
even the presence of microparticles obtained for
drug free nanoparticles, indicated the tendency of
the colloidal system to agglomerate and was
confirmed with zeta potential analysis. The use of
DLS for the analysis of a sample containing a
mixture of extremely large and small particles
yields large Z-averages as the contribution to the
total light scattered by the small particles will be
extremely small, Wider peaks in the graph are due
to the tendency of nanoparticles to agglomerate,
which can be noted by the peak in the 900nm
range or close to the 1000 nm in Figure 5a.
Agglomeration of the nanoparticles can be
reduced by the addition of a surface active agent
to the formulation, which will be explored in
further studies. The overall poor zeta potential
obtained can be attributed to the strong positive
surface charge from the Ca2+ ions being shielded
due to ionotropic crosslinking of the Ca2+ ions
with the carboxyl groups of the guluronic acid
residues, which makes up alginate, in which Ca2+
is held in the centre of the 3-dimensional
structure. Alginate is an anionic polymer,
resulting in the negative zeta potential obtained
(13,38,39). Although AZT in an uncharged
molecule (40), there is a possible interaction of
AZT with the polymers, as observed from the
FTIR spectra, with resultant inferior crosslinking
of Ca2+ with alginate. Alginate nanoparticles
prepared by crosslinking with CaCl2 exhibited
superior size distribution compared to the Zn-
pectin nanoparticles.
Assessment of MR provides information
regarding in vitro degradation and drug release
behaviour of the multipolymeric scaffold device.
MR also has implications for device storage. It is
therefore imperative to have a rigid polymeric
scaffold which can withstand fracture during
storage and handling of the device. A decline in
the resilience of HCl treated matrix as compared
to untreated matrix was expected as HCl exposed
scaffolds presented with less porous matrices due
to acid transition of the COONa groups in CMC
to COOH. This could possibly result in a brittle
scaffold, accounting for the low MR obtained.
Less water will therefore permeate into the
polymer matrix leading to reduced
disentanglement of the polymer matrix, allowing
for prolonged drug release. MR of both sets of
scaffold samples declines further with exposure to
PBS due to swelling and chain relaxation of the
polymer matrix. The viscoelastic property of the
scaffold resultantly increases and matrix
resilience declines.
Upon treatment with 1%v/v HCl, the
carboxylate COONa groups in NaCMC are
converted to the acidic COOH form. This COOH
form is less susceptible to ionisation in PBS, as
the COO- group has a greater affinity for the H+
ion resulting in a lower degree of ion exchange of
the Na+ and H+ to reform COONa. The polymeric
matrix resultantly imbibes less water, reducing the
swelling behaviour of HCl treated scaffold.
Exposure to HCl also leads to a more densely
crosslinked polymer matrix which is less prone to
swelling than the loosely packed untreated
scaffolds. Exposure to HCl also leads to a more
densely crosslinked polymer matrix which is less
prone to swelling than the loosely packed
untreated scaffolds. Still, an appreciable swelling
in both scaffold types could be attributed to the
highly hydrophilic nature of the constituent
polymers which enhances the rate of uptake and
water uptake capacity. A comparative decrease in
the amount of water penetrating into the HCl
treated scaffold reduces the disentanglement of
this polymeric matrix, attributing to the reduced
erosion observed for this scaffold compared to
J Pharm Pharm Sci (www.cspsCanada.org) 16(3) 470 - 485, 2013
483
HCl untreated scaffold, in first 24hours of PBS
exposure. However, the weight gain in both types
of scaffold on prolonged exposure to PBS is
thought to be due to sequestration of phosphate
and Na+ ions indicating degree of entrapped
moisture and ions within of the polymer matrix
accounting for the gain in weight of the scaffolds
and resultant decrease of the entanglement of the
polymer chains (36).
The comparatively high DEE recorded for the
alginate nanoparticle formulation is accredited to
the ability of alginate to form a gel in the presence
of an aqueous medium, with the hydrophilic AZT
entrapped within this gel structure along with the
nanoparticle precipitation upon crosslinking of the
alginate-AZT solution with CaCl2 (16,17,41). The
high DEE of alginate nanoparticles, compared to
the pectin nanoparticles, coupled to their even
size distribution with a comparatively stable
formulation qualified them for further analysis.
Drug release obtained with alginate nanoparticles
was more suitable than that of pectin
nanoparticles for dispersion within a
multipolymeric scaffold matrix for possible
application as an implantable drug delivery
device. Dispersing the alginate nanoparticles in
multipolymeric scaffold was aimed to prolong the
drug release. However, the burst release of drug
from both the types of nanoparticle was attributed
to association of the AZT onto the surface of the
polymers by weak interactive forces, which was
reversed upon exposure to PBS. The nanopartcle
liberation and subsequent drug release from
polymeric scaffold is governed primarily by two
factors: scaffold swelling and erosion. Erosion of
the scaffold is due to weakening of the
crosslinked bonds with exposure to PBS (pH 7.4).
ECL present in the matrix degrades by means of
hydrolysis of the ester bonds, without creating a
localised acidic environment upon degradation.
When exposed to the CaCl2 salt solution, CaCl2
either forms bonds with the ester groups on the
lactone ring or the Ca2+ surrounds the ester group
and protects it from degradation (26,28,31). PEO
degrades by means of hydrolysis of the ester
bonds. CMC and PEO imbibes large quantities of
water due to their hydrophilic nature, resulting in
rapid degradation of PEO, subsequently causing
break down of the polymer scaffold. The degree
of swelling and subsequent drug release is
determined by the availability of hydrogen bonds
in the polymer matrix for water molecules to
attach to. For scaffolds treated with 1%v/v HCl,
the availability of such bonds is decreased,
thereby resulting in reduced drug release. The
PBS penetrated the scaffold matrix, which has
small uniform pores as well as air pockets,
causing it to erode, allowing for nanoparticles to
diffuse out. Nanoparticles released drug by a
similar method, with swelling of the nanoparticles
upon exposure to PBS playing a major role in
drug release. This is consistent with the polymers
used to prepare the nanoparticles, being alginate
and pectin, which are hygroscopic in nature and
prone to swelling in the presence of water. This
swelling behaviour would allow for weakening of
the crosslinked bonds between the polymers and
the CaCl2, and release of drug from the
nanoparticles. Swelling of the nanoparticles
further facilitates surface erosion and subsequent
release of drug (38,39).
CONCLUSIONS
From the studies conducted, the controlled
gelification of alginate method of producing
nanoparticles was chosen for further analysis as
alginate nanoparticles exhibited a more stable
formulation, as determined by zeta potential
analysis and smaller particles are formed using
this approach. Pectin nanoparticles demonstrated
suitable size and drug release properties for
application as a drug carrier in the absence of a
scaffold matrix. However, further investigations
are required to enhance the stability of the
formulation and to decrease agglomeration.
Dispersing the polymeric nanoparticles within a
multipolymeric scaffold served to noticeably
decrease the drug release rate. Exposing the
prepared multipolymeric scaffolds to 1%v/v HCl
served to reduce the swelling behaviour of the
scaffolds, thereby retarding drug release and
creating a suitable device for the prolonged
release of drug over a period of one month. The
nanoparticle-loaded system can be used for the
drug delivery, where controlled drug delivery
over extended periods is required. A possible
application for such a device is as subcutaneous
implant to achieve systemic drug release which
bypasses the gastrointestinal tract and the liver.
This is useful for drugs which are sensitive to the
acidic environment of the stomach and
susceptible to first pass metabolism. The device
can even be employed as a neural implant to
manage neurodegenerative diseases, where
prolonged controlled drug delivery would serve to
significantly enhance patient quality of life.
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