In the current research, a spray-dried
nanoparticles-in-microparticles system (NiMS) of Acetazolamide (ACZ) was
formulated. The objective behind the research was to examine the consequence of
spray-drying parameters that are inlet temperature and feed rpm on the
entrapment efficiency, loading capacity, percentage yield, and particle size on
formulating NiMS of ACZ. The prepared NiMS were evaluated for entrapment
efficiency, loading capacity, percentage yield, and particle size, and it was
found that PP-5 (formulated using an inlet temperature of 160°C and feed rpm
30) has a maximum entrapment efficiency 17.94% (w/w), loading capacity 33.2%
(w/w), percentage yield 25.8% (w/w), and smallest particle size of 763 nm out
of all the five formulations (PP-1 to PP-5). The DSC analysis of PP-5 suggested
that the entrapment of the nano and microparticles and spray-drying generate a noticeable
crystallinity of ACZ and confers a nearly amorphous state to this drug.
Infrared analysis of PP-5 showed no interaction between drug and polymer during
the formulation process. The SEM of PP-5 found that the particles are of
irregular shape, typically in the range of 2.5 to 3.5 μm. Therefore, NiMS of
ACZ have been successfully formulated and it was observed there was an effect
of inlet temperature and feed rpm of the spray dryer on the entrapment
efficiency, loading capacity, percentage yield and particle size. Thus it can
be concluded this study can be beneficial for the formulation of NiMS of ACZ by
In recent years, significant consideration
has been focused on the expansion of novel drug delivery systems (NDDS). The
reasons behind the development of NDDS include drug delivery to the site of
action without any significant immunogenicity reactions, biological inactivation,
or the potential side effect to the critical tissues, such as liver, lungs,
bone marrow, kidney, etc. The main goal in developing NDDS is to advance the
therapeutic efficacy and safety of existing drugs by altering the
biodistribution pattern of the drugs, reducing the amount and frequency of dosing.1
NiMS are the systems that reduce drug dosage frequency and increase patient
com- pliance.2 The most important purpose in designing NiMS is to
control particle size, surface properties, and make pharmacologically active
agents release to achieve site-specific drug action at the dosage regimen and
therapeutically optimal rate.3 Chitosan has excellent potential in
designing nanoparticulate drug delivery, the ability to control drug release,
and is biocompatible with living tissue. Chitosan is a natural carbohydrate
polymer and prepared by the partial N-deacetylation of chitin. Chitosan-based
NiMS have advantages, mainly for the design of novel nanoparticulate drug
delivery systems, due to their desirable properties such as biodegradability,
bio- and mucoadhesivity, biocompatibility, and hydrophilic character that
facilitate the poorly absorbable drugs administration across the various
epithelial barriers, such as intestinal, nasal, and corneal mucosa.4
Acetazolamide (ACZ) is a carbonic anhydrase inhibitor. In the eye, carbonic
anhydrase inhibition decreases the flow of sodium, bicarbonate, and water into
the posterior chamber. Suppression of this reaction in the ciliary process
reduces the aqueous humour production by almost total enzyme inhibition. Thus,
the intraocular pressure in both normal and glaucomatous eyes is reduced.5
Spray drying is a technique described as
the feed conversion into a dry particulate form by subjecting the feed into a
hot drying medium from a fluid state. Liquids of various types, such as
slurries, emulsions, and dispersions, can be converted into solid particles
with preferred size, porosity, shape, density, and distribution.6 There
are various spray-drying benefits, which include control of size and shape and
porosity and density, a rapid and simple process that produces free flowing
particles, scalability and reproducibility, cost effectiveness, and enhanced
dissolution rate of drugs.7 The spray-drying process effectiveness is
exaggerated by different factors, such as inlet temperature, outlet temperature,
flow rate, and feed concentration and rate.8 Therefore, it is
desirable to evaluate the various effects of spray-dried parameters on the
ACZ-loaded NiMS. In the present study, the goal was to formulate NiMS of ACZ by
spray drying, and the objective behind the study was to investigate the effect
of spray-drying parameters, i.e., inlet temperature
and feed rpm on the percentage yield, entrapment efficiency, loading capacity,
and particle size.
Chitosan (95% deacetylated), having a
molecular weight of 40 to 80 kDa, was purchased from Fluka Chemika,
Switzerland. Sodium tripolyphosphate (NaTPP) and glacial acetic acid were
purchased from Central Drug House, New Delhi. ACZ was obtained from Kaizen
Pharmaceuticals, Chandigarh. Tetrabutylammonium hydrogen sulphate was obtained
from Spectrochem Pvt. Ltd, Mumbai. Acetonitrile (HPLC grade) was purchased from
Rankem, New Delhi.
of ACZ-Loaded NiMS
In the current research, NiMS were prepared
using the ionic gelation method.9 In this method, chitosan (0.5%
w/v) was dissolved in 0.3% v/v glacial acetic acid. 500 mg of the ACZ was
dissolved in it. The solution of sodium tripolyphosphate (NaTPP) (0.2% w/v) was
prepared in distilled water. The NaTPP solution (32 to 50 ml) was added to the
chitosan solution (70 to 100 ml) dropwise stirring continuously. The suspension
was spray dried (JISL, Navi Mumbai) at a feed rate (30, 50, and 80 rpm) and at
specified temperature (50°C, 100°C, and 160°C) to get the free flowing powder.
The aspirator blower capacity was 118 Nm3/hr, and the nozzle size
was 0.7 mm with auto de-blocking device. To study the effect of various
formulation variables, NiMS were prepared as shown in Table 1.
In the current research, using Agilent
Technologies 1200 series, Germany [Quaternary pump, a stainless steel column 25
cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (3 μm), UV
detector dual wavelength]. HPLC analysis was performed utilizing the parameter
as shown in Table 2.10
Initially, the column was washed with a
mixture of acetonitrile and methanol with varying flow rate for half an hour
and decreasing ratio of methanol. After that, the column was saturated with 1.0
ml/min of flow rate for 30 mins and mobile phase. After that, the standard drug
dilution samples (5 μl) were injected and run for 10 min. The drug retention
time was found between 1.5 to 2.0 min. In Table 3, HPLC of standard solution of
ACZ is shown, and the calibration curve was prepared as shown in Figure 1.
For analysis of particle size, using
Zetasizer Instrument (Beckman Coulter Desla Nano, USA) equipped with the
hydro-dispersing unit by dissolving 2 mg of sample in 5 ml of distilled water,
the dilution of sample was analyzed. In a polystyrene cuvette in hydro-dispensing
unit, the dilution of sample was filled, and the scan was carried out at 64 runs
per sample. At the end of scan, the average diameter of all the 64 runs were
taken out and recorded as Z-average.
Yield, Entrapment Efficiency & Loading Capacity
The ACZ percentage yield, entrapment efficiency,
and loading capacity were determined directly using NiMS. The analyses of HPLC
were carried out using a system (Agilent Technologies 1200 series, Germany)
that consisted of a column 25 cm x 4.6 mm made up of stainless steel packed
with octadecylsilane bonded to porous silica (3 μm). The mobile phase was a
mixture of 60 volumes of a 0.33 % w/v solution of tetrabutylammonium hydrogen sulphate
and 40 volumes of acetonitrile. The wavelength was set at 234 nm, and the flow
rate was 1.0 mL/ min.10 The percentage yield, entrapment efficiency,
and drug loading were calculated (Equations 1, 2, & 3).11
Scanning Calorimetry (DSC) Analysis
DSC was carried out on DSC Q10 (Waters
Corporation, USA) using indium as standard to calibrate the instrument. In sealed
pans of aluminum, samples were heated at a rate of 10°C/min under nitrogen atmosphere
(60 ml/min) and temperature range from 30°C to 300°C, with empty pan as
Transform Infrared (FTIR) Spectrophotometry
FT-IR is a technique of obtaining
infrared spectra using an interferometer by first collecting an interferogram
of a sample signal, and then obtaining the spec trum performing a Fourirer
transform on the interferogram. Fourier transform IR spectra were recorded on
FT-IR (Alpha, Bruker, Germany). Over the range of 500 to 3500 cm-1,
the spectra were recorded.
Electron Microscopy (SEM)
The NiMS were mounted on metal stubs using double-sided tape and
using Sputter gold coater and
visualized under scanning electron
microscope. The particles were
coated with gold to a thickness of
about 450 Å.12 The surface appearance and morphology of spray-dried PP-5 was observed via SEM (Hitachi S 3400 N, Japan).
In the current research, the
spray-drying technique was used for the conversion of suspensions into solid
particles (nanoparticles/microparticles) and no additional drying adjuvant was
needed. TPP is an anion that can form cross-linkage involving ionic interactions
between the TPP molecules (negatively charged) and amino group (positively
charged) of chitosan. The opalescence indicated the development of particles
with a size range of nanoparticles to microparticles with the incorporation of
ion TPP to chitosan solution.13 In spray drying, there are various
factors that affect the formulated product, such as inlet temperature, outlet
temperature, feed rpm, feed concentration, aspirator rate, nozzle size, etc.
Out of these factors, inlet temperature and feed rpm of spray dryer were
selected to evaluate, and the analysis of percentage yield, entrapment
efficiency, loading capacity, and particle size of all five formulations were
carried out and are tabulated in Table 4. It has been observed that the PP-5 formulations
have the highest percentage yield (25.8%) with highest entrapment efficiency
(17.94%), highest loading capacity (33.2%), and with a smallest particle size
of 763 nm.
By means of laser diffractometry,
using Zetasizer instrument (Beckman Coulter Desla Nano, USA) equipped with a
hydro-dispersing unit, particle sizing experiments were carried out. The
particles size analysis of each batch (PP-1 to PP-5) have been tabulated in
Yield, Entrapment Efficiency & Loading Capacity
When we compared the results of
percentage yield, entrapment efficiency, and loading capacity of PP-1 and PP-2,
which have the same inlet temperature, PP-1 showed the better result as
compared to the PP-2, due to the PP-2 having the lower feed rate so that the
solution gets efficient time to get converted into solid particles. While the
entrapment efficiency, loading capacity, percentage yield of two formulations
(PP-3 and PP-4) were lowest because the prepared solution was not completely
converted into the solid particles due to the higher feed rate and lower
temperature, respectively. The best results showed by the PP-5 with respect of
having highest percentage yield, entrapment efficiency, and loading capacity
due to the higher inlet temperature and lower feed rpm of spray dryer. The
results of percentage yield, entrapment efficiency, and drug loading of all
five batches (PP-1 to PP-5) are tabulated in Table 4.
Scanning Calorimetry (DSC) Analysis
The ACZ was confirmed by DSC analysis,
and there was a sharp peak at 273.86°C almost corresponding to its melting point
(260.52°C) (Figure 2). During NiMS formation, the loss of endothermic peak of
PP-5 (Figure 3) showed that the drug may have been dispersed in the amorphous
form in the polymer matrix.11,14 The entire amalgamation of the drug
in the NiMS formulation indicates the molecular dispersion of the drug is
within the system. By spray drying, the peak of drug disappeared in NiMS,
indicating the drug was molecularly dispersed in the medium of chitosan as a
solid solution. These outcomes suggest the spray-drying process produces a marked
decline in crystallinity of ACZ and confer to this drug a nearly amorphous state.11
Transform Infrared (FTIR) Spectrophotometry
The spectra were recorded for pure drug
(ACZ) and ACZ-loaded PP-5 and the comparison of peaks of pure drug and PP-5 are
tabulated in Table 5. There was no major variation in the FTIR spectra of pure ACZ
and PP-5. No significant shifting of functional peaks, no overlapping of
characteristic peaks, and also no appearance of new peaks were observed upon
comparison of obtained spectra with reference spectra. The results suggest drug
stability during the entrapment process. The FTIR data suggested that molecular
interactions that could alter the chemical structure of the drug did not occur.
Therefore, no chemical interface between the functional group of drug and
Electron Microscopy (SEM)
Figure 4 shows the SEM of the PP-5 of spray-dried
powder. It was found that the particles were of irregular shapes, typically in
the range of 2.5 to 3.5 μm. The irregular particles may be attributed to the
fact that during the spray-drying process, high pump rates result in a large
volume of spraying solution to be dried, but heated air might not transform
liquid droplets into solid droplets, immediately leading to the formation of
bigger irregular-shape particles that are not completely dried and tend to form
Spray drying has been successfully applied
to prepare ACZ-loaded NiMS. The effect of spray-drying parameters that are
inlet temperature and feed rpm of the spray dryer were evaluated on the percentage
yield, entrapment efficiency, loading capacity, and particle size of ACZ-loaded
NiMS. The PP-5 was regarded as the best batch because it has the highest
entrapment efficiency (17.94%), loading capacity (33.2%), and percentage yield
(25.8%) with the smallest particle size (763 nm). The current research supports
the approach to prepare redispersible ACZ-loaded NiMS in a powdered form by
using the spray-drying technique and also marked the effect of spray-drying
parameters on the formulation of ACZ-loaded NiMS. From the research, it has
been concluded that the formulation with higher inlet temperature and lower
feed rpm resulted in maximum percentage yield along with entrapment efficiency,
loading capacity, and smallest particle size.
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Dr. Harish Dureja earned his Master’s degree
from Punjabi University, Patiala, and
his Doctorate (Gold-Medal) from M.D. University,
Rohtak. He is presently working as
Associate Professor in the Department
of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak (India). He
has 16 years of experience in teaching and Research. He has written 10 invited
book chapters/monographs in various books published by international
publishers. He is author of more than 115 publications in various national and
international journals of repute. He has been awarded two research projects for
research on anti-cancer drug delivery by the AICTE and UGC. His current research
interests include in silico ADME modelling, nanoparticulate drug delivery, and pharmaceutical
Mr. Parijat Pandey earned his Master’s degree
in Industrial Pharmacy and is presently pursuing his PhD as Research Scholar
(JRF) in the Department of Pharmaceutical Sciences, M.D. University, Rohtak. He
has published 8 manuscripts in various international and national journals and
is a life member of IPGA.